{"input": "BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1\nBackground\nBone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and are secreted proteins with pleiotropic roles in many different cell types. A potential role of BMP-6 in the immune system has been implied by various studies of malignant and rheumatoid diseases. In the present study, we explored the role of BMP-6 in normal human peripheral blood B cells.\nResults\nThe B cells were found to express BMP type I and type II receptors and BMP-6 rapidly induced phosphorylation of Smad1/5/8. Furthermore, Smad-phosphorylation was followed by upregulation of Id1 mRNA and Id1 protein, whereas Id2 and Id3 expression was not affected. Furthermore, we found that BMP-6 had an antiproliferative effect both in naive (CD19+CD27-) and memory B cells (CD19+CD27+) stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induced cell death in activated memory B cells. Importantly, the antiproliferative effect of BMP-6 in B-cells was completely neutralized by the natural antagonist, noggin. Furthermore, B cells were demonstrated to upregulate BMP-6 mRNA upon stimulation with anti-IgM.\nConclusion\nIn mature human B cells, BMP-6 inhibited cell growth, and rapidly induced phosphorylation of Smad1/5/8 followed by an upregulation of Id1.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 723, "end": 733}, "arguments": [{"role": "Theme", "text": "Id2", "start": 711, "end": 714}]}, {"trigger": {"text": "expression", "start": 723, "end": 733}, "arguments": [{"role": "Theme", "text": "Id3", "start": 719, "end": 722}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 581, "end": 596}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 600, "end": 605}]}, {"trigger": {"text": "phosphorylation", "start": 581, "end": 596}, "arguments": [{"role": "Theme", "text": "5", "start": 606, "end": 607}]}, {"trigger": {"text": "phosphorylation", "start": 581, "end": 596}, "arguments": [{"role": "Theme", "text": "8", "start": 608, "end": 609}]}, {"trigger": {"text": "phosphorylation", "start": 629, "end": 644}, "arguments": [{"role": "Theme", "text": "Smad", "start": 624, "end": 628}]}, {"trigger": {"text": "phosphorylation", "start": 1326, "end": 1341}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 1345, "end": 1350}]}, {"trigger": {"text": "phosphorylation", "start": 1326, "end": 1341}, "arguments": [{"role": "Theme", "text": "5", "start": 1351, "end": 1352}]}, {"trigger": {"text": "phosphorylation", "start": 1326, "end": 1341}, "arguments": [{"role": "Theme", "text": "8", "start": 1353, "end": 1354}]}], "positive regulation": [{"trigger": {"text": "upregulation", "start": 85, "end": 97}, "arguments": [{"role": "Theme", "text": "Id1", "start": 101, "end": 104}]}, {"trigger": {"text": "induced", "start": 573, "end": 580}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 559, "end": 564}, {"role": "Theme", "text": "phosphorylation", "start": 581, "end": 596}]}, {"trigger": {"text": "followed", "start": 649, "end": 657}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 629, "end": 644}, {"role": "Theme", "text": "upregulation", "start": 661, "end": 673}]}, {"trigger": {"text": "upregulation", "start": 661, "end": 673}, "arguments": [{"role": "Theme", "text": "Id1", "start": 677, "end": 680}]}, {"trigger": {"text": "upregulation", "start": 661, "end": 673}, "arguments": [{"role": "Theme", "text": "Id1", "start": 690, "end": 693}]}, {"trigger": {"text": "upregulate", "start": 1187, "end": 1197}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1198, "end": 1203}]}, {"trigger": {"text": "induced", "start": 1318, "end": 1325}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 1277, "end": 1282}, {"role": "Theme", "text": "phosphorylation", "start": 1326, "end": 1341}]}, {"trigger": {"text": "followed", "start": 1355, "end": 1363}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 1326, "end": 1341}, {"role": "Theme", "text": "upregulation", "start": 1370, "end": 1382}]}, {"trigger": {"text": "upregulation", "start": 1370, "end": 1382}, "arguments": [{"role": "Theme", "text": "Id1", "start": 1386, "end": 1389}]}], "regulation": [{"trigger": {"text": "affected", "start": 742, "end": 750}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 629, "end": 644}, {"role": "Theme", "text": "expression", "start": 723, "end": 733}]}]}}, "schema": []}
{"input": "Members of the transforming growth factor beta (TGF-beta) superfamily play central roles in controlling cellular proliferation, differentiation, migration and apoptosis [1]. These cytokines can be divided into three subgroups: TGF-beta, the activins/inhibins, and the bone morphogenetic proteins (BMPs), of which the latter constitute the largest family. BMPs are 30-38 kDa hetero- or homodimeric proteins originally identified by their ability to induce ectopic cartilage and bone formation [2,3]. Several studies have demonstrated an essential role of these proteins during embryogenesis, and more recently, also in adult tissues [1]. TGF-beta has been intensively studied in normal and malignant haematopoietic cells and is one of the most potent endogenous negative regulators known to date. [4]. In contrast, the effect of BMPs in the immune system has not been widely investigated. In that respect, BMP- 2, -4 and -7 have been found to control differentiation of hematopoietic stem cells [5] and early T cell development [6,7]. BMP-6 has been reported to reduce the number of cobblestone-area-forming cells of normal human haematopoietic cells [8]. Furthermore, BMP-2, -4, 6 and -7 had an antiproliferative and a proapoptotic effect on multiple myeloma cells [9-11]. In addition, by gene expression profiling, BMP-6 significantly increased the predictive value for a multi-gene signature test and was associated with a poor outcome in diffuse large B cell lymphomas (DLBCL) [12].\nBMP-6, like the other BMP members, signals through ligation and heterodimerzation of BMP type I [activin-like-kinase (ALK)] and type II serine-threonine kinase receptors, which subsequently propagates the signal downstream by phosphorylating Smad proteins. BMP-6 can signal through the ligation of the type I receptors Act-RIA, BMP-RIA, and BMP-RIB and the type II receptors BMP-RII, Act-RIIA and Act-RIIB, which lead to the phosphorylation of the receptor Smads (Smad-1, Smad-5, and Smad-8). The R- Smads then form complexes with the co-Smad (Smad4) and are translocated into the nucleus where they exert gene regulation [1,13].\nGiven the reported role of BMP-6 in B-cell malignancies and haematopoietic progenitor cells, we wanted to explore its potential role in normal human B cells. We studied the effects of BMP-6 on proliferation and apoptosis on resting and stimulated B cells. Furthermore, the expression of BMP receptors and BMP-6 induced activation of the Smad signalling pathway with subsequent regulation of the target genes Id1-Id4, were resolved. Finally, we investigated whether B cells also were capable of producing BMP-6.", "output": {"json_structures": {"binding": [{"trigger": {"text": "ligation", "start": 1537, "end": 1545}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1486, "end": 1491}]}, {"trigger": {"text": "heterodimerzation", "start": 1550, "end": 1567}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1486, "end": 1491}]}, {"trigger": {"text": "ligation", "start": 1772, "end": 1780}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1743, "end": 1748}, {"role": "Theme2", "text": "Act-RIA", "start": 1805, "end": 1812}]}, {"trigger": {"text": "ligation", "start": 1772, "end": 1780}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1743, "end": 1748}, {"role": "Theme2", "text": "BMP-RIA", "start": 1814, "end": 1821}]}, {"trigger": {"text": "ligation", "start": 1772, "end": 1780}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1743, "end": 1748}, {"role": "Theme2", "text": "BMP-RIB", "start": 1827, "end": 1834}]}, {"trigger": {"text": "ligation", "start": 1772, "end": 1780}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1743, "end": 1748}, {"role": "Theme2", "text": "BMP-RII", "start": 1861, "end": 1868}]}, {"trigger": {"text": "ligation", "start": 1772, "end": 1780}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1743, "end": 1748}, {"role": "Theme2", "text": "Act-RIIA", "start": 1870, "end": 1878}]}, {"trigger": {"text": "ligation", "start": 1772, "end": 1780}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1743, "end": 1748}, {"role": "Theme2", "text": "Act-RIIB", "start": 1883, "end": 1891}]}, {"trigger": {"text": "form complexes", "start": 1997, "end": 2011}, "arguments": [{"role": "Theme", "text": "Smad-1", "start": 1950, "end": 1956}, {"role": "Theme2", "text": "Smad4", "start": 2030, "end": 2035}]}, {"trigger": {"text": "form complexes", "start": 1997, "end": 2011}, "arguments": [{"role": "Theme", "text": "Smad-5", "start": 1958, "end": 1964}, {"role": "Theme2", "text": "Smad4", "start": 2030, "end": 2035}]}, {"trigger": {"text": "form complexes", "start": 1997, "end": 2011}, "arguments": [{"role": "Theme", "text": "Smad-8", "start": 1970, "end": 1976}, {"role": "Theme2", "text": "Smad4", "start": 2030, "end": 2035}]}], "gene expression": [{"trigger": {"text": "expression", "start": 2389, "end": 2399}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 2421, "end": 2426}]}, {"trigger": {"text": "producing", "start": 2610, "end": 2619}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 2620, "end": 2625}]}], "localization": [{"trigger": {"text": "translocated", "start": 2045, "end": 2057}, "arguments": [{"role": "Theme", "text": "Smad-1", "start": 1950, "end": 1956}, {"role": "ToLoc", "text": "nucleus", "start": 2067, "end": 2074}]}, {"trigger": {"text": "translocated", "start": 2045, "end": 2057}, "arguments": [{"role": "Theme", "text": "Smad-5", "start": 1958, "end": 1964}, {"role": "ToLoc", "text": "nucleus", "start": 2067, "end": 2074}]}, {"trigger": {"text": "translocated", "start": 2045, "end": 2057}, "arguments": [{"role": "Theme", "text": "Smad-8", "start": 1970, "end": 1976}, {"role": "ToLoc", "text": "nucleus", "start": 2067, "end": 2074}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1911, "end": 1926}, "arguments": [{"role": "Theme", "text": "Smad-1", "start": 1950, "end": 1956}]}, {"trigger": {"text": "phosphorylation", "start": 1911, "end": 1926}, "arguments": [{"role": "Theme", "text": "Smad-5", "start": 1958, "end": 1964}]}, {"trigger": {"text": "phosphorylation", "start": 1911, "end": 1926}, "arguments": [{"role": "Theme", "text": "Smad-8", "start": 1970, "end": 1976}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 1336, "end": 1345}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1316, "end": 1321}]}, {"trigger": {"text": "lead", "start": 1899, "end": 1903}, "arguments": [{"role": "Cause", "text": "ligation", "start": 1772, "end": 1780}, {"role": "Theme", "text": "phosphorylation", "start": 1911, "end": 1926}]}, {"trigger": {"text": "induced", "start": 2427, "end": 2434}, "arguments": [{"role": "Cause", "text": "expression", "start": 2389, "end": 2399}, {"role": "Theme", "text": "regulation", "start": 2493, "end": 2503}]}], "regulation": [{"trigger": {"text": "regulation", "start": 2493, "end": 2503}, "arguments": [{"role": "Theme", "text": "Id1", "start": 2524, "end": 2527}]}, {"trigger": {"text": "regulation", "start": 2493, "end": 2503}, "arguments": [{"role": "Theme", "text": "Id4", "start": 2528, "end": 2531}]}]}}, "schema": []}
{"input": "BMP-6 inhibits anti-IgM induced proliferation of human B cells\nThe effects of BMP-6 on normal and neoplastic hematopoietic cells prompted us to investigate the effects of BMP-6 on normal human B cells. All experiments in this study were performed under serum-free conditions as FCS has been shown to interfere with BMP-signalling [14](own observations). To study the effect of BMP-6 on proliferation, B-cells from healthy volunteers were stimulated with anti-IgM and/or CD40L in the presence or absence of BMP-6 for three days. We found that BMP-6 led to a 35% mean reduction of anti-IgM- induced DNA synthesis (n = 8; p <= 0.0002, Figure 1A). Similar results were obtained for B cells treated with anti-IgM and CD40L (26% mean reduction, n = 6; p <= 0.023). The BMP-6-induced inhibition of proliferation was dose-dependent in both peripheral B cells (Figure 1B) and the Burkitt lymphoma cell line Ramos (40% reduction of DNA synthesis, Figure 1C). The BMP-6 effects could be reversed by addition of the extracellular inhibitor Noggin (Figure 1D). Similarly, a combination of the soluble BMP receptors BMP-RIB-Fc and BMP-RII-Fc also neutralized the effects of BMP-6 (data not shown). Next, we wanted to test whether BMP-6 had different effect on naive and memory B cells. Naive (CD19+CD27-) and memory (CD19+CD27+) B cells were isolated from peripheral blood by cell sorting of immunobead-isolated CD19+ B cells [15], and tested for their capacity to proliferate in the presence of BMP-6. However, BMP-6 inhibited anti-IgM induced DNA synthesis in the two subpopulations to a similar extent, with a mean reduction of DNA-synthesis of 45% (n = 5; p <= 0,004) for naive B cells and 48% (n = 5; p <= 0,001) for memory B cells (Figure 1E).", "output": {"json_structures": {}}, "schema": []}
{"input": "BMP-6 induces cell death in human memory B cells and Ramos cells\nNext, we wanted to establish whether BMP-6 also could affect the viability of normal B cells. Cell viability was determined by propidium iodide (PI) staining after culture with or without BMP-6 for 48 hours. Interestingly, BMP-6 showed a small, but reproducible mean increase of cell death from 17 to 23% (n = 5; p <= 0,003) in anti-IgM stimulated CD27+ memory B cells. Furthermore, Ramos cells showed a mean increase in cell death from 20 to 50% (n = 3, p < 0,001, figure 3) after BMP-6 treatment. In contrast, cell death of total CD19+ cells (n = 6; p <= 0,32; data not shown) or CD27-IgG- naive B cells was not significantly affected (n = 5; p <= 0,65, figure 2).", "output": {"json_structures": {}}, "schema": []}
{"input": "Human B cells express BMP-6 receptors\nDetailed knowledge regarding expression of different BMP receptors in B cells is currently not available. To further elucidate the role of BMPs in human B cells, we performed western blot analysis for type I and type II BMP receptors. This analysis revealed that the type I receptors Act-RIA, BMP-RIB and the type II receptors BMP-RII and Act-RIIb are expressed on resting human B-cells (Figure 4). Ramos cells expressed the type I receptors Act-RIA, weakly BMP-RIB and the type II receptor BMP-RII, but more weakly than normal B cells (Figure 4). HL60 cells were used for comparison and weakly expressed Act-RIA and BMP-RII.\nTaken together, these data show that normal human B cells and Ramos cells express a set of BMP receptors, previously shown to bind BMP-6 [16].", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 790, "end": 794}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 795, "end": 800}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 390, "end": 399}, "arguments": [{"role": "Theme", "text": "Act-RIA", "start": 322, "end": 329}]}, {"trigger": {"text": "expressed", "start": 390, "end": 399}, "arguments": [{"role": "Theme", "text": "BMP-RIB", "start": 331, "end": 338}]}, {"trigger": {"text": "expressed", "start": 390, "end": 399}, "arguments": [{"role": "Theme", "text": "BMP-RII", "start": 365, "end": 372}]}, {"trigger": {"text": "expressed", "start": 390, "end": 399}, "arguments": [{"role": "Theme", "text": "Act-RIIb", "start": 377, "end": 385}]}, {"trigger": {"text": "expressed", "start": 449, "end": 458}, "arguments": [{"role": "Theme", "text": "Act-RIA", "start": 480, "end": 487}]}, {"trigger": {"text": "expressed", "start": 449, "end": 458}, "arguments": [{"role": "Theme", "text": "BMP-RIB", "start": 496, "end": 503}]}, {"trigger": {"text": "expressed", "start": 449, "end": 458}, "arguments": [{"role": "Theme", "text": "BMP-RII", "start": 529, "end": 536}]}, {"trigger": {"text": "expressed", "start": 633, "end": 642}, "arguments": [{"role": "Theme", "text": "Act-RIA", "start": 643, "end": 650}]}, {"trigger": {"text": "expressed", "start": 633, "end": 642}, "arguments": [{"role": "Theme", "text": "BMP-RII", "start": 655, "end": 662}]}]}}, "schema": []}
{"input": "BMP-6 induces phosphorylation of Smad1/5/8\nUpon ligand binding, the type II receptor transphosphorylates and activates the type I receptor. Type I receptors can signal via several pathways. We examined the effect of BMP-6 on Smad phosphorylation, as the activation of Smad is considered to be a major signalling pathway for BMPs [17]. B cells were cultured in serum-free media over night and then treated with BMP-6 for various time points. Total protein lysates were prepared, and the amounts of the phosphorylated forms of Smad1/5/8 were determined by western blot analysis. Interestingly, treatment with 500 ng/ml BMP-6 induced phosphorylation of Smad. The BMP-6 induced phosphorylation was high at the earliest time point tested (15 minutes), and remained high for at least 48 hours (Figure 5). A similar phosphorylation was observed in Ramos cells, but not in HL60 cells (Figure 6). Furthermore, we also tested whether other known downstream signalling pathways of BMP-6 could be triggered by BMP-6 in human B cells. However, we did not observe any significant changes in the level of phospho-STAT3 or phospho-p38 upon BMP-6 treatment of B cells (data not shown).", "output": {"json_structures": {"phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 14, "end": 29}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 33, "end": 38}]}, {"trigger": {"text": "phosphorylation", "start": 14, "end": 29}, "arguments": [{"role": "Theme", "text": "5", "start": 39, "end": 40}]}, {"trigger": {"text": "phosphorylation", "start": 14, "end": 29}, "arguments": [{"role": "Theme", "text": "8", "start": 41, "end": 42}]}, {"trigger": {"text": "phosphorylated", "start": 501, "end": 515}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 525, "end": 530}]}, {"trigger": {"text": "phosphorylated", "start": 501, "end": 515}, "arguments": [{"role": "Theme", "text": "5", "start": 531, "end": 532}]}, {"trigger": {"text": "phosphorylated", "start": 501, "end": 515}, "arguments": [{"role": "Theme", "text": "8", "start": 533, "end": 534}]}, {"trigger": {"text": "phosphorylation", "start": 631, "end": 646}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 525, "end": 530}]}, {"trigger": {"text": "phosphorylation", "start": 631, "end": 646}, "arguments": [{"role": "Theme", "text": "5", "start": 531, "end": 532}]}, {"trigger": {"text": "phosphorylation", "start": 631, "end": 646}, "arguments": [{"role": "Theme", "text": "8", "start": 533, "end": 534}]}, {"trigger": {"text": "phosphorylation", "start": 674, "end": 689}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 525, "end": 530}]}, {"trigger": {"text": "phosphorylation", "start": 674, "end": 689}, "arguments": [{"role": "Theme", "text": "5", "start": 531, "end": 532}]}, {"trigger": {"text": "phosphorylation", "start": 674, "end": 689}, "arguments": [{"role": "Theme", "text": "8", "start": 533, "end": 534}]}, {"trigger": {"text": "phospho", "start": 1090, "end": 1097}, "arguments": [{"role": "Theme", "text": "STAT3", "start": 1098, "end": 1103}]}, {"trigger": {"text": "phospho", "start": 1107, "end": 1114}, "arguments": [{"role": "Theme", "text": "p38", "start": 1115, "end": 1118}]}], "positive regulation": [{"trigger": {"text": "induces", "start": 6, "end": 13}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 0, "end": 5}, {"role": "Theme", "text": "phosphorylation", "start": 14, "end": 29}]}, {"trigger": {"text": "induced", "start": 623, "end": 630}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 617, "end": 622}, {"role": "Theme", "text": "phosphorylation", "start": 631, "end": 646}]}, {"trigger": {"text": "induced", "start": 666, "end": 673}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 660, "end": 665}, {"role": "Theme", "text": "phosphorylation", "start": 674, "end": 689}]}], "regulation": [{"trigger": {"text": "changes", "start": 1066, "end": 1073}, "arguments": [{"role": "Theme", "text": "phospho", "start": 1090, "end": 1097}, {"role": "Cause", "text": "BMP-6", "start": 1124, "end": 1129}]}, {"trigger": {"text": "changes", "start": 1066, "end": 1073}, "arguments": [{"role": "Theme", "text": "phospho", "start": 1107, "end": 1114}, {"role": "Cause", "text": "BMP-6", "start": 1124, "end": 1129}]}]}}, "schema": []}
{"input": "BMP-6 induces upregulation of Id1\nNext, we wanted to explore whether the BMP-6 induced phosphorylation of Smad 1/5/8 also could induce transcriptional changes of target genes. In this regard, the inhibitors of DNA binding proteins (Ids) are considered to be some of the major target genes for Smad-signalling [17]. Thus, B cells were pre-incubated over night in X-VIVO 15, and then cultured in medium alone or in the presence of BMP-6 for various time points before preparation of total RNA. The amount of Id1-Id4 mRNA was quantified by real-time RT-PCR. Interestingly, we observed a specific four-fold upregulation of Id1 mRNA in BMP-6-treated B cells (Figure 7). The up-regulation of Id1 mRNA was characteristic of an early inducible gene, with maximal upregulation two hours after the addition of BMP-6 and returned to baseline after 24 hours. In contrast, no significant changes were observed for Id2 and Id3 mRNA, whereas Id4-transcripts were not detectable (Figure 7, data not shown).\nWestern blot analysis revealed that the BMP-6-induced upregulation of Id1 mRNA also was correlated with upregulation of Id1 protein as well. The increase in Id1 protein level was detectable after one hour and increased until 24 hours after BMP-6 addition, showing a 16-fold upregulation compared with t0 (p <= 0.020, n = 4) (Figure 8 and 9). In line with the mRNA data, no consistent change in the amounts of Id2 and Id3 protein could be observed (Figure 8 and 9). We were able to block the Id1 specific band with a blocking peptide (data not shown). Taken together, these data suggest that Id1 could be a possible target gene for mediating the effects of BMP-6 in human B cells, whereas Id2 and Id3 not seem to be involved.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "amounts", "start": 1389, "end": 1396}, "arguments": [{"role": "Theme", "text": "Id2", "start": 1400, "end": 1403}]}, {"trigger": {"text": "amounts", "start": 1389, "end": 1396}, "arguments": [{"role": "Theme", "text": "Id3", "start": 1408, "end": 1411}]}], "negative regulation": [{"trigger": {"text": "block", "start": 1472, "end": 1477}, "arguments": [{"role": "Theme", "text": "Id1", "start": 1482, "end": 1485}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 87, "end": 102}, "arguments": [{"role": "Theme", "text": "Smad 1", "start": 106, "end": 112}]}, {"trigger": {"text": "phosphorylation", "start": 87, "end": 102}, "arguments": [{"role": "Theme", "text": "5", "start": 113, "end": 114}]}, {"trigger": {"text": "phosphorylation", "start": 87, "end": 102}, "arguments": [{"role": "Theme", "text": "8", "start": 115, "end": 116}]}], "positive regulation": [{"trigger": {"text": "induces", "start": 6, "end": 13}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 0, "end": 5}, {"role": "Theme", "text": "upregulation", "start": 14, "end": 26}]}, {"trigger": {"text": "upregulation", "start": 14, "end": 26}, "arguments": [{"role": "Theme", "text": "Id1", "start": 30, "end": 33}]}, {"trigger": {"text": "induced", "start": 79, "end": 86}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 73, "end": 78}, {"role": "Theme", "text": "phosphorylation", "start": 87, "end": 102}]}, {"trigger": {"text": "upregulation", "start": 603, "end": 615}, "arguments": [{"role": "Theme", "text": "Id1", "start": 619, "end": 622}, {"role": "Cause", "text": "BMP-6", "start": 631, "end": 636}]}, {"trigger": {"text": "up-regulation", "start": 669, "end": 682}, "arguments": [{"role": "Theme", "text": "Id1", "start": 686, "end": 689}, {"role": "Cause", "text": "BMP-6", "start": 800, "end": 805}]}, {"trigger": {"text": "upregulation", "start": 755, "end": 767}, "arguments": [{"role": "Theme", "text": "Id1", "start": 686, "end": 689}, {"role": "Cause", "text": "BMP-6", "start": 800, "end": 805}]}, {"trigger": {"text": "detectable", "start": 952, "end": 962}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 800, "end": 805}, {"role": "Theme", "text": "Id4", "start": 927, "end": 930}]}, {"trigger": {"text": "induced", "start": 1037, "end": 1044}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 1031, "end": 1036}, {"role": "Theme", "text": "upregulation", "start": 1045, "end": 1057}]}, {"trigger": {"text": "upregulation", "start": 1045, "end": 1057}, "arguments": [{"role": "Theme", "text": "Id1", "start": 1061, "end": 1064}]}, {"trigger": {"text": "correlated", "start": 1079, "end": 1089}, "arguments": [{"role": "Cause", "text": "upregulation", "start": 1045, "end": 1057}, {"role": "Theme", "text": "upregulation", "start": 1095, "end": 1107}]}, {"trigger": {"text": "upregulation", "start": 1095, "end": 1107}, "arguments": [{"role": "Theme", "text": "Id1", "start": 1111, "end": 1114}]}, {"trigger": {"text": "increase", "start": 1136, "end": 1144}, "arguments": [{"role": "Theme", "text": "Id1", "start": 1148, "end": 1151}, {"role": "Cause", "text": "BMP-6", "start": 1231, "end": 1236}]}, {"trigger": {"text": "increased", "start": 1200, "end": 1209}, "arguments": [{"role": "Theme", "text": "Id1", "start": 1148, "end": 1151}, {"role": "Cause", "text": "BMP-6", "start": 1231, "end": 1236}]}, {"trigger": {"text": "upregulation", "start": 1265, "end": 1277}, "arguments": [{"role": "Theme", "text": "Id1", "start": 1148, "end": 1151}, {"role": "Cause", "text": "BMP-6", "start": 1231, "end": 1236}]}], "regulation": [{"trigger": {"text": "changes", "start": 875, "end": 882}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 800, "end": 805}, {"role": "Theme", "text": "Id2", "start": 901, "end": 904}]}, {"trigger": {"text": "changes", "start": 875, "end": 882}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 800, "end": 805}, {"role": "Theme", "text": "Id3", "start": 909, "end": 912}]}, {"trigger": {"text": "change", "start": 1375, "end": 1381}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 1231, "end": 1236}, {"role": "Theme", "text": "amounts", "start": 1389, "end": 1396}]}]}}, "schema": []}
{"input": "BMP-6 production in B cells\nThe fact that BMP-6 has been reported to act as an autocrine stimulator in chondrocytes [18] and ovarium [19], prompted us to investigate whether normal human B cells could produce BMP-6 upon stimulation. Ramos cells, which have been described to express BMP-6 mRNA endogenously [20], and the T cell line Jurkat, served as positive and negative controls, respectively. Endogenous BMP-6 mRNA levels in normal B cells were quantified by real-time RT-PCR after stimulation with anti-IgM for different time points. Interestingly, the up-regulation of BMP-6 mRNA was characteristic of an early-to intermediate inducible gene with maximal upregulation four hours after the addition of anti-IgM. The level of BMP-6 mRNA was back to baseline after 24 hours upon stimulation (Figure 10). Furthermore, both FCS and human AB-serum induced significant upregulation of BMP-6 mRNA (Figure 11). Interestingly, in a separate study we have found that normal human T cells do not express BMP-6 mRNA after activation (Sivertsen et al, manuscript in preparation). Next, we wanted to detect BMP-6 protein in normal B-cells and tested various commercially available antibodies. However, in our hands these anti-BMP-6 antibodies did only recognize the recombinant BMP-6 protein and not the native protein.", "output": {"json_structures": {"binding": [{"trigger": {"text": "recognize", "start": 1243, "end": 1252}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1269, "end": 1274}]}], "gene expression": [{"trigger": {"text": "production", "start": 6, "end": 16}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 0, "end": 5}]}, {"trigger": {"text": "produce", "start": 201, "end": 208}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 209, "end": 214}]}], "negative regulation": [{"trigger": {"text": "back to baseline", "start": 745, "end": 761}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 730, "end": 735}]}], "positive regulation": [{"trigger": {"text": "up-regulation", "start": 558, "end": 571}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 575, "end": 580}]}, {"trigger": {"text": "upregulation", "start": 661, "end": 673}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 575, "end": 580}]}, {"trigger": {"text": "induced", "start": 848, "end": 855}, "arguments": [{"role": "Theme", "text": "upregulation", "start": 868, "end": 880}]}, {"trigger": {"text": "upregulation", "start": 868, "end": 880}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 884, "end": 889}]}], "transcription": [{"trigger": {"text": "express", "start": 275, "end": 282}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 283, "end": 288}]}, {"trigger": {"text": "express", "start": 990, "end": 997}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 998, "end": 1003}]}]}}, "schema": []}
{"input": "Recent studies have demonstrated an important role for BMP superfamily members in hematopoietic stem cells, early thymocytes [6,7] and B-cell malignancies [8,11,12], but a role for BMPs in normal human B cells has previously not been reported. The present study demonstrated a significant antiproliferative effect of BMP-6 in peripheral blood CD19+ B cells. Additionally, BMP-6 induced cell death in CD27+ memory B cells as well as in a Burkitt lymphoma cell line (Ramos). Importantly, BMP-6 induced a rapid and marked increase in Smad-1/5/8 phosphorylation. Furthermore, the BMP-6 induced Smad phosphorylation was followed by a selective upregulation of Id1 mRNA and subsequent Id1 protein.\nIn the present study, the demonstrated antiproliferative effect of BMP-6 in anti-IgM treated B cells was significant and dose-dependent. Importantly, the anti-proliferative effect of BMP-6 could be completely neutralized by the use of a natural inhibitor, Noggin. This is in line with others, showing that Noggin can function as a BMP-6 antagonist [21,22]. In addition, the combination of soluble BMP-RIB-Fc and BMP-RII-Fc fusion proteins also neutralized the anti-proliferative effect of BMP-6 in human B cells. Interestingly, as for other TGF family members, bifunctional effects have also been demonstrated for BMPs. Whereas several of the BMPs have been shown to promote proliferation in various cell types including condrocytes [23], liver [24] and granulosa cells [25], antiproliferative effects and induction of apoptosis has been reported for B and T lineage cells. Similar effects as demonstrated for BMP-6 on human B cells in the present study, were demonstrated for BMP-2, 4, 6 and -7 in human myeloma cells [9-11]. Other members of the BMP-family have also been reported to induce apoptosis, including in mouse B lineage cells [26]. Additionally, BMP-4 inhibits thymocyte proliferation [6]. Taken together, these data suggest that the role of BMPs in the regulation of proliferation and apoptosis is highly cell type dependent.\nTo examine how BMP-6 exerts its functional effects in B cells, we analysed BMP receptor expression by western blot analysis. Human peripheral B cells were found to express the BMP type I receptors Act-RIA and BMP-RIB, and the type II-receptors BMP-RII and Act-RIIb, which signal after binding of several BMPs, including BMP-6 [16,13]. To further explore BMP-6 induced signalling, activation of several pathways is possible. The major signalling pathway known to date, is activation of R-Smads [13,27]. In that respect, BMPs have been shown to exert antiproliferative effects in B lineage cells via phosphorylation of R-Smad [11,28]. Furthermore, BMP-2 has been shown to induce activation of STAT3 in myeloma cells [9]. However, phosphorylation of R-Smad was not investigated in that study. BMP-2 has also been shown to induce phosphorylation of p38 [29]. Thus, phosphorylation of p38, STAT3 and Smad1/5/8 represent important BMP-signalling pathways that mediated the effects of BMPs and even cross-talk between these pathways has been reported [29,30]. In the present study, we were not able to detect BMP-6-induced changes in the phosphorylation status of STAT3 or p38 in human peripheral B cells. Instead, a rapid and marked phosphorylation of Smad1/5/8 was revealed. In a parallel study, we have found that other BMPs also induced phosphorylation of Smad1/5/8 in peripheral B cells (data not shown). We are currently pursuing microarray studies to identify the signalling pathways and target genes that are differently regulated by the various BMPs in human B cells.\nUpregulation of Id1 via Smad1/5/8 phosphorylation is a known mechanism for BMP-6 signalling in other cell systems [31,32] and regulation of Id-proteins is thought to be an important mechanism for Smad-signalling [17]. In the present study, real-time RT-PCR experiments revealed a specific four-fold upregulation for Id1 in BMP-6-treated B cells, while the amount of Id2-Id4 remained unchanged. In agreement with this, western blot analysis demonstrated an upregulation of Id1 protein, while the amount of Id2 and Id3 protein levels remained unchanged. Previously, Id1 has been considered not to be expressed in later developmental stages than pro-B cells [33,34], and its constitutive expression has been reported to impair mouse B cell development [35]. Therefore, our demonstration of the time-dependent upregulation of Id1 mRNA and protein in mature normal human B cells is of particular interest. In that respect, it is noteworthy that TGF-beta signalling in early and mature B cells induces both Id2 and Id3 expression [36,37], but not Id1 (data not shown). Interestingly, these results show that various members of the TGF-beta family regulate Id proteins differently. Id2 and Id3 are considered to be the Id proteins mainly expressed in mature B cells [38]. The present study also found Id2 and Id3 protein in B cells to be more highly expressed than Id1 in resting B cells. However, BMP-6 did not induce significant changes in the protein expression of Id2 and Id3. It is believed that Id proteins block differentiation and promote proliferation in various cell types [39,33]. Id proteins act as dominant-negative inhibitors of E-proteins and Pax5 function by forming dimers with these proteins, making them unable to bind DNA. It has been proposed that the balance among E-proteins, Pax5 and Id proteins might have an important role in activated B cells [38]. In that respect, E-proteins have been implicated in both the promotion and inhibition of cell survival and growth at different points in lymphocyte development [40]. The antiproliferative and death inducing effect of BMP-6 in B cells with concomitant upregulation of Id1 protein is therefore in line with the view that Id proteins are required for the induction of growth arrest and apoptosis in B-lymphocyte progenitors by TGF-beta [40].\nFurthermore, Id proteins are known as important parts of signalling pathways involved in development, cell cycle and tumorigenesis [32]. It is well established that various members of the Id family are overexpressed in a range of human tumours and generally, Id1 appears to be the family member most widely overexpressed in a variety of human malignancies [41], including multiple myeloma [42,32]. Additionally, our findings that BMP-6 activates intracellular signalling pathways in human B cells might be of potential pathophysiological significance in lymphoma and inflammation. High BMP-6 mRNA expression in DLBCL has been shown to correlate to unfavourable outcome [12]. In this respect, it is of interest that targeted expression of Id1 to B-lymphocytes resulted in aberrant B cell development, massive apoptosis, and subsequent development of B cell lymphomas [35]. Moreover, BMP-6 has been suggested to play a role in rheumatoid arthritis (RA) [43,44] and elevated levels of Id1 and Id3 have been found in the synovia of RA-patients [45]. Altogether, these results point to an important role for Id proteins in the regulation of normal B cell homeostasis and in diseases, where B cells are involved. It will therefore be important to further elucidate the role of Id-1 in human B cells by selective over expression or inhibition of Id-1 gene expression.\nGiven the role of BMP-6 in mature human B cells demonstrated here, identification of BMP-6 producing cells in vivo with possibility of interaction with naive and memory B cells might contribute to the understanding of mature B cell biology. High BMP-6 mRNA expression in DLBCL has been detected by gene expression profiling [12]. Furthermore, production of BMP-6 transcripts in normal activated B cells was detected in the same study. Of note, an autocrine BMP-6 loop has been reported by others in chondrocytes and in the ovarium [46,19,18]. Therefore, we wanted to explore the possibility for an autocrine BMP-6 loop in human B cells. We analysed the expression BMP-6 mRNA in peripheral blood B cells by real-time PCR, and report here the upregulation of endogenous BMP-6 transcripts after stimulation with FCS, human AB-serum and, most importantly, anti-IgM. However, our attempts to study BMP-6 protein levels were unsuccessful due to problems with unspecific binding of the anti-BMP-6 antibodies tested, and lack of specific staining in control cells known to express BMP-6 mRNA. In contrast, the recombinant protein was readily detected. In that respect, few investigators have detected BMP-6 protein in humans, especially in non-pathogenic tissue. The possibility of BMP-6 production in human B-cells is in line with a recent work that reported the production of BMP-6 in mouse B cells, infiltrating the bone marrow of mice with inflammatory arthritis [43]. In this study, a role for BMPs in the inflammatoric process of arthritis was suggested. The upregulation of the BMP-6-transcripts after IgM-crosslinking is of pathophysiologic interest [12]. A loss of TGF-beta-responsiveness has been suggested to be a critical contribution to malignant transformation [47,48] and similar oncogenic mechanisms have been postulated for BMPs. Lines of evidence suggest [49] that at early stages of carcinogenesis, BMP-6 is not a tumour promoter, but suppresses benign and malignant tumour outgrowth. These findings are in good agreement with previous findings for other TGF-beta family members, including TGF-beta1 and BMP-4 [50], indicating that cellular context of the BMP target cell might define the various observed effects. In contrast to the upregulation of BMP-6 transcript in B cells, we were not able to detect BMP-6 transcripts in human peripheral blood CD4+ or CD8+ T cells (resting or stimulated with anti-CD3 and anti-CD28; data not shown), consistent with the findings in T cell lines [20] and T cells in mice [43]. Other potential BMP-6 sources for mature B cells in vivo might be other cells of the immune system or tissue with contact to the hematopoietic system. One well recognized source for BMP-6 production is the human bone and bone marrow stroma [51,8]. Furthermore, it is noteworthy that human umbilical vein endothelial cells (HUVEC) highly express BMP-6 mRNA [52], and vascular endothelium has been reported to produce BMP-6 [53]. These studies might imply a role for BMP-6 in transendothelial migration of B cells. BMP-6 mRNA has been demonstrated in murine macrophage cell lines, but not in humans [54]. In accordance with these findings, other human cell lines of neutrophil and monocytic origin have been described to be negative for the BMP-6 transcript [20]. To our knowledge, there is currently no report about BMP-6 production of human dendritic cells.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 2317, "end": 2324}, "arguments": [{"role": "Theme", "text": "Act-RIA", "start": 2229, "end": 2236}, {"role": "Theme2", "text": "BMP-6", "start": 2352, "end": 2357}]}, {"trigger": {"text": "binding", "start": 2317, "end": 2324}, "arguments": [{"role": "Theme", "text": "BMP-RIB", "start": 2241, "end": 2248}, {"role": "Theme2", "text": "BMP-6", "start": 2352, "end": 2357}]}, {"trigger": {"text": "binding", "start": 2317, "end": 2324}, "arguments": [{"role": "Theme", "text": "BMP-RII", "start": 2276, "end": 2283}, {"role": "Theme2", "text": "BMP-6", "start": 2352, "end": 2357}]}, {"trigger": {"text": "binding", "start": 2317, "end": 2324}, "arguments": [{"role": "Theme", "text": "Act-RIIb", "start": 2288, "end": 2296}, {"role": "Theme2", "text": "BMP-6", "start": 2352, "end": 2357}]}, {"trigger": {"text": 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"arguments": [{"role": "Theme", "text": "Pax5", "start": 5253, "end": 5257}, {"role": "Cause", "text": "forming dimers", "start": 5270, "end": 5284}]}, {"trigger": {"text": "inhibition", "start": 7235, "end": 7245}, "arguments": [{"role": "Theme", "text": "expression", "start": 7259, "end": 7269}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 542, "end": 557}, "arguments": [{"role": "Theme", "text": "Smad-1", "start": 531, "end": 537}]}, {"trigger": {"text": "phosphorylation", "start": 542, "end": 557}, "arguments": [{"role": "Theme", "text": "5", "start": 538, "end": 539}]}, {"trigger": {"text": "phosphorylation", "start": 542, "end": 557}, "arguments": [{"role": "Theme", "text": "8", "start": 540, "end": 541}]}, {"trigger": {"text": "phosphorylation", "start": 595, "end": 610}, "arguments": [{"role": "Theme", "text": "Smad", "start": 590, "end": 594}]}, {"trigger": {"text": "phosphorylation", "start": 2893, "end": 2908}, "arguments": [{"role": "Theme", "text": "STAT3", "start": 2917, "end": 2922}]}, {"trigger": {"text": "phosphorylation", "start": 2893, "end": 2908}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 2927, "end": 2932}]}, {"trigger": {"text": "phosphorylation", "start": 2893, "end": 2908}, "arguments": [{"role": "Theme", "text": "5", "start": 2933, "end": 2934}]}, {"trigger": {"text": "phosphorylation", "start": 2893, "end": 2908}, "arguments": [{"role": "Theme", "text": "8", "start": 2935, "end": 2936}]}, {"trigger": {"text": "phosphorylation", "start": 3163, "end": 3178}, "arguments": [{"role": "Theme", "text": "STAT3", "start": 3189, "end": 3194}]}, {"trigger": {"text": "phosphorylation", "start": 3259, "end": 3274}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 3278, "end": 3283}]}, {"trigger": {"text": "phosphorylation", "start": 3259, "end": 3274}, "arguments": [{"role": "Theme", "text": "5", "start": 3284, "end": 3285}]}, {"trigger": {"text": "phosphorylation", "start": 3259, "end": 3274}, "arguments": [{"role": "Theme", "text": "8", "start": 3286, "end": 3287}]}, {"trigger": {"text": "phosphorylation", "start": 3366, "end": 3381}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 3385, "end": 3390}]}, {"trigger": {"text": "phosphorylation", "start": 3366, "end": 3381}, "arguments": [{"role": "Theme", "text": "5", "start": 3391, "end": 3392}]}, {"trigger": {"text": "phosphorylation", "start": 3366, "end": 3381}, "arguments": [{"role": "Theme", "text": "8", "start": 3393, "end": 3394}]}, {"trigger": {"text": "phosphorylation", "start": 3636, "end": 3651}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 3626, "end": 3631}]}, {"trigger": {"text": "phosphorylation", "start": 3636, "end": 3651}, "arguments": [{"role": "Theme", "text": "5", "start": 3632, "end": 3633}]}, {"trigger": {"text": "phosphorylation", "start": 3636, "end": 3651}, "arguments": [{"role": "Theme", "text": "8", "start": 3634, "end": 3635}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 492, "end": 499}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 486, "end": 491}, {"role": "Theme", "text": "increase", "start": 519, "end": 527}]}, {"trigger": {"text": "increase", "start": 519, "end": 527}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 542, "end": 557}]}, {"trigger": {"text": "induced", "start": 582, "end": 589}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 576, "end": 581}, {"role": "Theme", "text": "phosphorylation", "start": 595, "end": 610}]}, {"trigger": {"text": "followed", "start": 615, "end": 623}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 595, "end": 610}, {"role": "Theme", "text": "upregulation", "start": 639, "end": 651}]}, {"trigger": {"text": "upregulation", "start": 639, "end": 651}, "arguments": [{"role": "Theme", "text": "Id1", "start": 655, "end": 658}]}, {"trigger": {"text": "upregulation", "start": 639, "end": 651}, "arguments": [{"role": "Theme", "text": "Id1", "start": 679, "end": 682}]}, {"trigger": {"text": "induce", "start": 2702, "end": 2708}, "arguments": [{"role": "Cause", "text": "BMP-2", "start": 2678, "end": 2683}, {"role": "Theme", "text": "activation", "start": 2709, "end": 2719}]}, {"trigger": {"text": "activation", "start": 2709, "end": 2719}, "arguments": [{"role": "Theme", "text": "STAT3", "start": 2723, "end": 2728}]}, {"trigger": {"text": "induced", "start": 3140, "end": 3147}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 3134, "end": 3139}, {"role": "Theme", "text": "changes", "start": 3148, "end": 3155}]}, {"trigger": {"text": "induced", "start": 3358, "end": 3365}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 3366, "end": 3381}]}, {"trigger": {"text": "Upregulation", "start": 3602, "end": 3614}, "arguments": [{"role": "Theme", "text": "Id1", "start": 3618, "end": 3621}, {"role": "Cause", "text": "phosphorylation", "start": 3636, "end": 3651}]}, {"trigger": {"text": "upregulation", "start": 3901, "end": 3913}, "arguments": [{"role": "Theme", "text": "Id1", "start": 3918, "end": 3921}, {"role": "Cause", "text": "BMP-6", "start": 3925, "end": 3930}]}, {"trigger": {"text": "remained unchanged", "start": 3976, "end": 3994}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 3925, "end": 3930}, {"role": "Theme", "text": "amount", "start": 3958, "end": 3964}]}, {"trigger": {"text": "upregulation", "start": 4058, "end": 4070}, "arguments": [{"role": "Theme", "text": "Id1", "start": 4074, "end": 4077}]}, {"trigger": {"text": "remained unchanged", "start": 4134, "end": 4152}, "arguments": [{"role": "Theme", "text": "amount", "start": 4097, "end": 4103}]}, {"trigger": {"text": "upregulation", "start": 4408, "end": 4420}, "arguments": [{"role": "Theme", "text": "Id1", "start": 4424, "end": 4427}]}, {"trigger": {"text": "induces", "start": 4590, "end": 4597}, "arguments": [{"role": "Theme", "text": "expression", "start": 4615, "end": 4625}]}, {"trigger": {"text": "induces", "start": 4590, "end": 4597}, "arguments": [{"role": "Theme", "text": "Id1", "start": 4643, "end": 4646}]}, {"trigger": {"text": "induce", "start": 5007, "end": 5013}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 4993, "end": 4998}, {"role": "Theme", "text": "changes", "start": 5026, "end": 5033}]}, {"trigger": {"text": "making", "start": 5306, "end": 5312}, "arguments": [{"role": "Cause", "text": "forming dimers", "start": 5270, "end": 5284}, {"role": "Theme", "text": "bind", "start": 5328, "end": 5332}]}, {"trigger": {"text": "upregulation", "start": 5722, "end": 5734}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 5688, "end": 5693}, {"role": "Theme", "text": "Id1", "start": 5738, "end": 5741}]}, {"trigger": {"text": "overexpressed", "start": 6217, "end": 6230}, "arguments": [{"role": "Theme", "text": "Id1", "start": 6169, "end": 6172}]}, {"trigger": {"text": "High", "start": 6491, "end": 6495}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 6502, "end": 6517}]}, {"trigger": {"text": "elevated", "start": 6873, "end": 6881}, "arguments": [{"role": "Theme", "text": "levels", "start": 6882, "end": 6888}]}, {"trigger": {"text": "over", "start": 7216, "end": 7220}, "arguments": [{"role": "Theme", "text": "expression", "start": 7221, "end": 7231}]}, {"trigger": {"text": "High", "start": 7512, "end": 7516}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 7523, "end": 7538}]}, {"trigger": {"text": "upregulation", "start": 8012, "end": 8024}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 8039, "end": 8044}]}, {"trigger": {"text": "upregulation", "start": 8828, "end": 8840}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 8848, "end": 8853}]}, {"trigger": {"text": "upregulation", "start": 9516, "end": 9528}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 9532, "end": 9537}]}, {"trigger": {"text": "highly", "start": 10128, "end": 10134}, "arguments": [{"role": "Theme", "text": "express", "start": 10135, "end": 10142}]}], "regulation": [{"trigger": {"text": "changes", "start": 3148, "end": 3155}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 3163, "end": 3178}]}, {"trigger": {"text": "mechanism", "start": 3663, "end": 3672}, "arguments": [{"role": "Theme", "text": "Upregulation", "start": 3602, "end": 3614}, {"role": "Cause", "text": "BMP-6", "start": 3677, "end": 3682}]}, {"trigger": {"text": "changes", "start": 5026, "end": 5033}, "arguments": [{"role": "Theme", "text": "expression", "start": 5049, "end": 5059}]}, {"trigger": {"text": "autocrine", "start": 7718, "end": 7727}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 7728, "end": 7733}, {"role": "Theme", "text": "BMP-6", "start": 7728, "end": 7733}]}, {"trigger": {"text": "autocrine", "start": 7869, "end": 7878}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 7879, "end": 7884}, {"role": "Theme", "text": "BMP-6", "start": 7879, "end": 7884}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 6502, "end": 6517}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 6496, "end": 6501}]}, {"trigger": {"text": "mRNA expression", "start": 7523, "end": 7538}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 7517, "end": 7522}]}, {"trigger": {"text": "production", "start": 7614, "end": 7624}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 7628, "end": 7633}]}, {"trigger": {"text": "expression", "start": 7924, "end": 7934}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 7935, "end": 7940}]}, {"trigger": {"text": "express", "start": 8336, "end": 8343}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 8344, "end": 8349}]}, {"trigger": {"text": "detect", "start": 9581, "end": 9587}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 9588, "end": 9593}]}, {"trigger": {"text": "express", "start": 10135, "end": 10142}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 10143, "end": 10148}]}, {"trigger": {"text": "demonstrated", "start": 10331, "end": 10343}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 10311, "end": 10316}]}, {"trigger": {"text": "negative", "start": 10520, "end": 10528}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 10537, "end": 10542}]}]}}, "schema": []}
{"input": "In conclusion, our results show that BMP-6 induces activation of intracellular Smad signalling in mature human B-cells with consecutive production of Id1 protein. Furthermore, we report that BMP-6 has an antiproliferative effect in B cells stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induces cell death in activated memory B cells and Ramos cells. Taken together, these results provide a rationale to further examine the role of BMP-6 signalling in normal B cell biology as well as in pathologic conditions like B cell malignancies and autoimmune disorders.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 136, "end": 146}, "arguments": [{"role": "Theme", "text": "Id1", "start": 150, "end": 153}]}], "positive regulation": [{"trigger": {"text": "consecutive", "start": 124, "end": 135}, "arguments": [{"role": "Theme", "text": "production", "start": 136, "end": 146}]}]}}, "schema": []}
{"input": "Cell culture\nIf not specified, all cells were cultured in X-VIVO 15(TM) (BioWhittaker, Verviers, Belgium) serum-free medium at 37degreesC and 5% CO2 in air.\nPeripheral blood was provided by the Blood Bank at Buskerud Regional Hospital with formal agreement by the patients, and approval by the regional ethics committee. Highly purified resting human B-lymphocytes (CD19+ cells) were isolated from the peripheral blood by rosetting with immunomagnetic beads (Dynabeads M450; Dynal, Oslo, Norway) as described [55]. This procedure yields less than 0.5% T cells, 0.1% NK cells, and 0.5% monocytes as judged by indirect immunofluorescence staining.\nThe following cell lines from human lymphoid malignancies were maintained in RPMI 1640 (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% foetal bovine serum (FCS), 100 units/ml penicillin G, and 100 units/ml of streptomycin sulphate, but serum-starved for at least four hours and cultured in X-VIVO 15(TM) when included in experiments: EBV-negative BL cell lines Ramos (ECACC 85030802), HL60 (JCRB0085).", "output": {"json_structures": {}}, "schema": []}
{"input": "Growth factors/supplements\nThe following reagents were used at indicated concentrations: recombinant human (rhu) BMP-6 (1 mug/ml, if not specified otherwise), rhu BMP-RIB/ALK-6/Fc Chimera (5 mug/ml), rhu BMPR-II/Fc Chimera (5 mug/ml), and recombinant mouse Noggin (5 mug/ml) were purchased from R&D Systems (Abingdon, UK); Anti-IgM F(ab)2 fragments of rabbit polyclonal antibodies to human IgM heavy chain (37.5 mug/ml) was obtained from Dako, Copenhagen, Denmark and rhu CD40 ligand (CD40L, 10 ng/ml) was a gift from Immunex Corp. (Seattle, WA).", "output": {"json_structures": {}}, "schema": []}
{"input": "Antibodies used for flow cytometric analysis and immunoblot analysis\nAntibodies against the human BMP-receptors Act-RIA, BMP-RIB, BMPR-II, Act-RIIA and Act-RIIb were purchased from R & D Systems (Abingdon, UK). Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).\nCharacterisation of BMP-signalling pathways was done by use of anti-phospho-Smad1, -5, -and 8 polyclonal antibody (Chemicon, Temecula, CA, USA). Expression levels of Id1-3 proteins were detected with polyclonal rabbit antibody and detection was blocked with blocking peptide from Santa Cruz Biotechnology (Santa Cruz, San Diego, CA, USA). As secondary antibodies served anti-mouse, anti-goat or anti-rabbit IgG- horseradish peroxidase (HRP) from Dakocytomation AS (Copenhagen, Denmark) for immunoblot analysis. Anti-beta-actin was from Santa-Cruz. From Becton Dickinson (San Jose, CA), we purchased anti-CD19-PE, anti-CD19-FITC. The antibodies used for cell sorting were anti-CD19 PC5 from Immunotech SA (Marseille, France) and anti-CD27 PE from Becton Dickinson, Biosciences Pharmingen (San Diego, CA, USA).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Expression levels", "start": 669, "end": 686}, "arguments": [{"role": "Theme", "text": "Id1", "start": 690, "end": 693}]}, {"trigger": {"text": "Expression levels", "start": 669, "end": 686}, "arguments": [{"role": "Theme", "text": "3", "start": 694, "end": 695}]}], "negative regulation": [{"trigger": {"text": "blocked", "start": 769, "end": 776}, "arguments": [{"role": "Theme", "text": "Expression levels", "start": 669, "end": 686}]}]}}, "schema": []}
{"input": "Cell sorting\nHighly purified CD19+CD27- or CD19+CD27+ cells were obtained by staining CD19+ cells with anti-CD27 PE and CD19 PC5 mAbs for 30 minutes at 4degreesC, followed by washing with PBS and sorting on FACS DiVa from Becton Dickinson.", "output": {"json_structures": {}}, "schema": []}
{"input": "Western-blot analysis\nB cells from peripheral blood or cultured cell-lines were lysed in lysis buffer (glycerol 10%, beta-mercaptoethanol 5%, 0.0625 M Tris-HCL [pH 6.8], sodium dodecyl sulphate [SDS] 2.5%w/vol). Total protein (30-100 mug) from each sample was run on 10% or 12% SDS/polyacrylamide (SDS/PAGE) gels and blotted onto nitrocellulose filters (Protran; Schleicher &Schuell GmbH, Dassel, Germany). Blocking, washing and incubation of the filters with primary antibodies were done according to the manufacturer's protocols at room temperature (RT). After washing with TBS/0.1% Tween-20 (TBS-T), the filters were incubated with horseradish peroxidase (HRP) coupled to relevant secondary antibodies (see above) for 60 minutes at RT. Enzyme activity was visualised by the enhanced chemiluminescence system, ECL+PLUS (Amersham, Buckinghamshire, UK). Densitometric analysis was performed by scanning hyperfilms on a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA). Quantification of Id1, Id2 and Id3 protein was calculated by normalizing the specific protein bands to beta-actin using Image Quant 5.5 software (Molecular Dynamics).", "output": {"json_structures": {}}, "schema": []}
{"input": "Analysis of BMP-6 messenger RNA (mRNA) expression\nEndogenous expression of the BMP-6 gene was examined by reverse transcription-polymerase chain reaction. Total RNA was isolated using Absolutely RNA(TM) RT-PCR Miniprep Kit (Stratagene Europe, Amsterdam, Netherland) according to the manufacturers instructions. Quantification of the isolated total RNA was achieved by using spectrophotometric OD260 measurements. Equal amounts of RNA were then reverse transcribed to cDNA with TaqMan(R) Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, U.S.A). To measure mRNA expression of BMP6, Id1-Id4 and PGK1 PCR were carried out with TaqMan(R) universal master mix. Primers and probes were provided by Assay-on-Demand (Applied Biosystems). PCR reactions were carried out in a final volume of 25 mul (BMP-6) or 20 mul (ID1). The cDNA added to each reaction was equivalent to the input of 20 ng of total RNA. The gene expression was quantified using the standard curve method (BMP6), or the comparative CT method (Id1) as described in ABI7700 User Bulletin 2 (Applied Biosystems). The expression was then normalized to the expression level of PGK1. PGK1 was chosen, because it has been shown to have low expression variability among lymphocyte specimens [56]. Expression levels in B cells were then related to the expression levels in Ramos cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 61, "end": 71}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 79, "end": 84}]}, {"trigger": {"text": "expression", "start": 925, "end": 935}, "arguments": [{"role": "Theme", "text": "Id1", "start": 1021, "end": 1024}]}, {"trigger": {"text": "expression", "start": 925, "end": 935}, "arguments": [{"role": "Theme", "text": "BMP6", "start": 984, "end": 988}]}, {"trigger": {"text": "expression", "start": 1130, "end": 1140}, "arguments": [{"role": "Theme", "text": "PGK1", "start": 1150, "end": 1154}]}, {"trigger": {"text": "have low expression", "start": 1202, "end": 1221}, "arguments": [{"role": "Theme", "text": "PGK1", "start": 1156, "end": 1160}]}], "transcription": [{"trigger": {"text": "expression", "start": 39, "end": 49}, "arguments": [{"role": "Theme", "text": "BMP6", "start": 594, "end": 598}]}, {"trigger": {"text": "expression", "start": 39, "end": 49}, "arguments": [{"role": "Theme", "text": "Id1", "start": 600, "end": 603}]}, {"trigger": {"text": "expression", "start": 39, "end": 49}, "arguments": [{"role": "Theme", "text": "Id4", "start": 604, "end": 607}]}, {"trigger": {"text": "expression", "start": 39, "end": 49}, "arguments": [{"role": "Theme", "text": "PGK1", "start": 612, "end": 616}]}, {"trigger": {"text": "mRNA expression", "start": 575, "end": 590}, "arguments": [{"role": "Theme", "text": "BMP6", "start": 594, "end": 598}]}, {"trigger": {"text": "mRNA expression", "start": 575, "end": 590}, "arguments": [{"role": "Theme", "text": "Id1", "start": 600, "end": 603}]}, {"trigger": {"text": "mRNA expression", "start": 575, "end": 590}, "arguments": [{"role": "Theme", "text": "Id4", "start": 604, "end": 607}]}, {"trigger": {"text": "mRNA expression", "start": 575, "end": 590}, "arguments": [{"role": "Theme", "text": "PGK1", "start": 612, "end": 616}]}]}}, "schema": []}
{"input": "Cell proliferation\nFor estimation of DNA synthesis, CD19+ cells (7.5 x 104 cells/0.2 ml) or Ramos cells (1 x 104 cells/0.2 ml) were cultured in triplicate in microtiter wells. The cells were pulsed with 3.7 x 104Bq [3H]thymidine (Amersham, Buckinghamshire, UK) for the last 16 h of a 72-h incubation. The cells were harvested using an automated cell harvester (Packard Instrument Company, Meriden, CT, USA) and [3H]thymidine incorporation was determined in a scintillation counter (TopCount, Packard Instrument Company Inc., Meriden, CT).", "output": {"json_structures": {}}, "schema": []}
{"input": "Determination of cell death\nCell death was measured by vital dye exclusion test by staining cells with 5 mug/ml propidium iodide ([PI]; Calbiochem Corp.; La Jolla, CA; 5 mg/ml) for one minute on ice. At least 1,000 cells per sample were run on a BD FACSCalibur flow cytometer.", "output": {"json_structures": {}}, "schema": []}
{"input": "Statistical analysis\nThe statistical significance of differences between groups was determined using the paired two-tailed Wilcoxon nonparametric test, by applying SPSS10.1 software (SPSS Inc., Chicago, IL, USA). P values less than 0.05 were considered significant.", "output": {"json_structures": {}}, "schema": []}
{"input": "Sp1 and Sp3 regulate basal transcription of the human APOBEC3G gene\nAPOBEC3G (A3G), a member of the recently discovered family of human cytidine deaminases, is expressed in peripheral blood lymphocytes and has been shown to be active against HIV-1 and other retroviruses. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G. Transcriptional start sites were identified by 5'-rapid amplification of cDNA ends analysis. Luciferase reporter assays demonstrated that a 1025 bp A3G promoter sequence (from -959 to +66 relative to the major transcriptional start site) displayed constitutive promoter activity. In T cells, the A3G promoter was not inducible by mitogenic stimulation, interferon treatment or expression of HIV-1 proteins. Using a series of 5' deletion promoter constructs in luciferase reporter assays, we identified a 180 bp region that was sufficient for full promoter activity. Transcriptional activity of this A3G core promoter was dependent on a GC-box (located at position -87/-78 relative to the major transcriptional start site) and was abolished after mutation of this DNA element. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that the identified GC-box represented a binding site for the ubiquitous transcription factors specificity protein (Sp) 1 and Sp3.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 1322, "end": 1329}, "arguments": [{"role": "Theme", "text": "A3G", "start": 709, "end": 712}, {"role": "Site", "text": "GC-box", "start": 1301, "end": 1307}, {"role": "Theme2", "text": "specificity protein (Sp) 1", "start": 1376, "end": 1402}]}, {"trigger": {"text": "binding", "start": 1322, "end": 1329}, "arguments": [{"role": "Theme", "text": "A3G", "start": 709, "end": 712}, {"role": "Site", "text": "GC-box", "start": 1301, "end": 1307}, {"role": "Theme2", "text": "Sp3", "start": 1407, "end": 1410}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 160, "end": 169}, "arguments": [{"role": "Theme", "text": "APOBEC3G", "start": 68, "end": 76}]}], "positive regulation": [{"trigger": {"text": "inducible", "start": 730, "end": 739}, "arguments": [{"role": "Theme", "text": "A3G", "start": 709, "end": 712}, {"role": "Site", "text": "promoter", "start": 713, "end": 721}]}], "regulation": [{"trigger": {"text": "regulate", "start": 12, "end": 20}, "arguments": [{"role": "Cause", "text": "Sp1", "start": 0, "end": 3}, {"role": "Theme", "text": "transcription", "start": 27, "end": 40}]}, {"trigger": {"text": "regulate", "start": 12, "end": 20}, "arguments": [{"role": "Cause", "text": "Sp3", "start": 8, "end": 11}, {"role": "Theme", "text": "transcription", "start": 27, "end": 40}]}, {"trigger": {"text": "regulation", "start": 318, "end": 328}, "arguments": [{"role": "Theme", "text": "APOBEC3G", "start": 68, "end": 76}]}], "transcription": [{"trigger": {"text": "transcription", "start": 27, "end": 40}, "arguments": [{"role": "Theme", "text": "APOBEC3G", "start": 54, "end": 62}]}]}}, "schema": []}
{"input": "The recently discovered APOBEC3 family of cytidine deaminases is considered to play an important role in antiviral intrinsic immunity (1,2). In primates, the seven paralogs APOBEC3A, B, C, DE, F, G, H (A3A-H) have been described (3), and they appear to fulfill individual functions. Human APOBEC3G (A3G), the most prominent member of the APOBEC3 family has been identified as the cellular restriction factor that is responsible for inhibition of virion infectivity factor (Vif)-deleted human immunodeficiency virus-1 (HIV-1) replication in non-permissive cells (4). A3G is packaged into HIV-1deltavif particles and causes C-to-U deaminations on the single-stranded viral DNA during reverse transcription (5-8). This leads to degradation of the uracile-containing DNA by cellular repair mechanisms or to hypermutation of the viral genome (5,6). As a result, only a marginal fraction of the A3G-containing HIV-1 particles is able to complete the replication cycle. In addition to the inhibition of HIV-1, A3G restricts replication of other lentiviruses, gammaretroviruses, deltaretroviruses, spumaviruses, long-terminal-repeat (LTR)-retrotransposons, orthohepadnaviruses and avihepadnaviruses (9-21). Interestingly, deamination seems not to be the only A3G-mediated antiviral mechanism; in the case of hepatitis B virus (HBV) and human T cell leukemia virus type 1 (HTLV-1), A3G was shown to restrict virus replication by deamination-independent mechanisms (12, 13, 19, 22-25). Another member of the APOBEC3 family, APOBEC3F (A3F), appears to have similar activities like A3G (26,27). A3F is also packaged into HIV-1deltavif particles and induces similar C-to-U deaminations, although the proteins differ in their target sequences specificity (26,28). Furthermore, A3F proteins were detected in many tissues that express A3G and are able to form heteromultimers with A3G (26,29,30). Both proteins localize to mRNA processing (P) bodies, cytoplasmic compartments involved in the degradation and storage of non-translating mRNAs (30,31).\nA3G has been shown to be expressed in T cells, a relevant cell target for HIV-1 in vivo, but little is known about its regulation (4,29,32). There is a report describing that mitogenic stimulation of T cells upregulates A3G mRNA levels, but this was not analyzed on the transcriptional level (33). Since the A3G promoter has not been systematically analyzed so far, our aim was to clone the A3G promoter and characterize its regulation in T cells. In our study, we observed that A3G uses multiple transcriptional start sites (TSS). By generating a series of 5' deletions of the A3G promoter, we identified a 180 bp region that mediated basal transcription. In T cells, transcriptional activity of this core promoter was not inducible by mitogenic stimulation or interferon treatment, but was dependent on a GC-box which was recognized by Sp (specificity protein) 1 and Sp3 transcription factors.", "output": {"json_structures": {"binding": [{"trigger": {"text": "form heteromultimers", "start": 1839, "end": 1859}, "arguments": [{"role": "Theme", "text": "A3F", "start": 1763, "end": 1766}, {"role": "Theme2", "text": "A3G", "start": 1865, "end": 1868}]}, {"trigger": {"text": "recognized", "start": 2858, "end": 2868}, "arguments": [{"role": "Theme", "text": "A3G", "start": 2612, "end": 2615}, {"role": "Site", "text": "GC-box", "start": 2841, "end": 2847}, {"role": "Theme2", "text": "Sp (specificity protein) 1", "start": 2872, "end": 2898}]}, {"trigger": {"text": "recognized", "start": 2858, "end": 2868}, "arguments": [{"role": "Theme", "text": "A3G", "start": 2612, "end": 2615}, {"role": "Site", "text": "GC-box", "start": 2841, "end": 2847}, {"role": "Theme2", "text": "Sp3", "start": 2903, "end": 2906}]}], "gene expression": [{"trigger": {"text": "express", "start": 1811, "end": 1818}, "arguments": [{"role": "Theme", "text": "A3F", "start": 1763, "end": 1766}]}, {"trigger": {"text": "express", "start": 1811, "end": 1818}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1819, "end": 1822}]}, {"trigger": {"text": "expressed", "start": 2059, "end": 2068}, "arguments": [{"role": "Theme", "text": "A3G", "start": 2034, "end": 2037}]}, {"trigger": {"text": "mRNA levels", "start": 2258, "end": 2269}, "arguments": [{"role": "Theme", "text": "A3G", "start": 2254, "end": 2257}]}], "localization": [{"trigger": {"text": "localize", "start": 1895, "end": 1903}, "arguments": [{"role": "Theme", "text": "A3F", "start": 1763, "end": 1766}, {"role": "ToLoc", "text": "mRNA processing (P) bodies", "start": 1907, "end": 1933}]}, {"trigger": {"text": "localize", "start": 1895, "end": 1903}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1819, "end": 1822}, {"role": "ToLoc", "text": "mRNA processing (P) bodies", "start": 1907, "end": 1933}]}], "negative regulation": [{"trigger": {"text": "deleted", "start": 478, "end": 485}, "arguments": [{"role": "Theme", "text": "virion infectivity factor", "start": 446, "end": 471}]}], "positive regulation": [{"trigger": {"text": "upregulates", "start": 2242, "end": 2253}, "arguments": [{"role": "Theme", "text": "mRNA levels", "start": 2258, "end": 2269}]}], "regulation": [{"trigger": {"text": "regulation", "start": 2153, "end": 2163}, "arguments": [{"role": "Theme", "text": "A3G", "start": 2034, "end": 2037}]}, {"trigger": {"text": "regulation", "start": 2459, "end": 2469}, "arguments": [{"role": "Theme", "text": "A3G", "start": 2425, "end": 2428}, {"role": "Site", "text": "promoter", "start": 2429, "end": 2437}]}]}}, "schema": []}
{"input": "Cell culture\nThe human T cell lines A3.01 and PM1 (NIBSC, UK) and the human myeloid cell line U937 (NIBSC, UK) were grown in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine and 100 U/ml penicillin-streptomycin. The human hepatic cell lines HepG2 and Huh7 (kindly provided by Dr Thomas Pietschmann, Department of Molecular Virology, University of Heidelberg) as well as HeLa cells were maintained in Dulbecco's high glucose modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM l-glutamine and 100 U/ml penicillin-streptomycin. Cells lines were incubated at 37degreesC with 100% humidity in 5-7% CO2 and passaged using standard cell culture techniques.", "output": {"json_structures": {}}, "schema": []}
{"input": "Plasmids\nFor cloning of an APOBEC3G promoter-driven reporter plasmid, genomic DNA was prepared from the T cell line PM1 using the DNeasy Kit (Qiagen). The DNA sequence ranging from positions -959 to +66 relative to the identified transcription start was amplified via PCR using the primers 3Gprom1025 (5'-TGTGAACGCGTTGCTGCAGGCCATCTGGATGTATATG-3') and 3Gpromreverse (5'-ACAGCAGATCTAGGGACCTCTGATAAAGACAGG-3'). PCR reactions were performed with Pwo DNA Polymerase (Roche) using the following cycle conditions: one cycle 94degreesC for 2 min; 30 cycles 94degreesC for 30 s, 58degreesC for 60 s, 72degreesC for 60 s; one cycle 72degreesC for 7 min. The amplicon was ligated into the promoterless luciferase reporter plasmid pGL3-Basic (Promega) via MluI and BglII restriction sites, which were introduced by the primers. The resulting construct contained 1025 bp of the A3G promoter and was designated pGL3-APOprom1025. Reporter plasmids containing shorter fragments of the APOBEC3G promoter were constructed using pGL3-APOprom1025 as template and the following forward primers: for plasmid pGL3-APOprom502 (containing sequence -436/+66): 3Gprom502 (5'-TGTGAACGCGTTCCATAACATGGGGACAAGA-3'); for plasmid pGL3-APOprom225 (containing sequence -159/+66): 3Gprom225 (5'-TGTGAACGCGTCGAGGGCAGGATCCGGGAGT-3'); for plasmid pGL3-APOprom180 (containing sequence -114/+66): 3Gprom180 (5'-TGTGAACGCGTTCTTGATGGTGGAGAGGAGG-3'); for plasmid pGL3-APOprom150 (containing sequence -84/+66): 3Gprom150 (5'-TGTGAACGCGTGCGGGACCACCAGGGGAGGGGCTT-3'); for plasmid pGL3-APOprom120 (containing sequence -54/+66): 3Gprom120 (5'-TGTGAACGCGTTGCTGGCTCAGCCTGGTGTG-3'); for plasmid pGL3-APOprom60 (containing sequence +7/+66): 3Gprom60 (5'-TGTGAACGCGTCCCTTTGCAATTGCCTTG-3'); each in combination with the reverse primer 3Gpromreverse (described above). PCR reactions were performed with Pfu Ultra Hotstart (Stratagene) using the following cycle conditions: one cycle 94degreesC for 2 min; 30 cycles 94degreesC for 45 s, 58degreesC for 45 s, 72degreesC for 60 s; one cycle 72degreesC for 7 min. As for pGL3-APOprom1025, MluI and BglII restriction sites were introduced via the primers and PCR products were ligated into pGL3-Basic (Promega) via these restriction sites. pGL3-APOprom180mut carries two point mutations (bold) and was generated using the primer 3GProm180mut (5'-TGTGAACGCGTTCTTGATGGTGGAGAGGAGGCTCCAGCTGTTCGGGACCACCAG-3') in combination with primer 3Gpromreverse. This PCR was performed with an annealing temperature of 65degreesC. pGL3promE1 (containing nucleotides -114/-85) and pGL3promE2 (containing nucleotides -92/-63) were constructed by annealing the following single-stranded oligonucleotides: 114_85Plus (5'- CGCGTTCTTGATGGTGGAGAGGAGGCTCCAGCTGGA-3') and 114_85Minus (5'- GATCTCCAGCTGGAGCCTCCTCTCCACCATCAAGAA-3') or 92-63Plus (5'-CGCGTCCAGCTGGGCGGGACCACCAGGGGAGGGGCA-3') and 92_63Minus (5'-GATCTGCCCCTCCCCTGGTGGTCCCGCCCAGCTGGA-3'). After annealing, the double-stranded oligonucleotides which contained the respective 30 bp of the APOBEC3G promoter and sticky ends compatible with MluI and BglII restriction sites were ligated into the pGL3-Promoter (Promega) vector. The sequences of all constructed plasmids were verified by sequence analysis. Nucleotide -219 of the cloned APOBEC3G promoter differs from the sequence in the database (GenBank(TM) accession number DQ147772). An A-to-C substitution is present at this position. Numbering is relative to the major transcriptional start site we identified.\nThe reporter plasmids pGL3-Control and phRG-TK were purchased from Promega. pGL2-CVX contains two repeats of the IFN-responsive GAS (gamma activated sequence) elements (GATCTGGATTTAGAGTAATATGAAACTGAAAGTACTTCG) of the guanylate-binding protein (GBP) gene in front of a CMV minimal promoter and was kindly provided by Ute Pagelow and Mario Koster from the Helmholtz-Zentrum fur Infektionsforschung. Plasmid pNL4-3 (NIBSC, UK) contains the full-length HIV-1NL4-3 genome and has been described previously (34). pcDNA3.1Vif was generously provided by Nathaniel R. Landau from the Salk Institute, La Jolla. It was generated by amplifying the Vif gene from pNL4-3 and ligating it into the pcDNA3.1 vector via BamHI and XhoI restriction sites. A 3'-WPRE element was included into the XhoI site. pBS-kRSPA-TatHIV-1(NL4-3) was constructed by amplifying the two exons of Tat via PCR reaction using the molecular clone pNL4-3 as template and the following primer sets: exon1, 5UXho1HIV-Tat1Plus (5'-GCATGCTCGAGATGGAGCCAGTAGATCCTAG-3') and HIV-Tat1Minus (5'-TGCTTTGATAGAGAAGCTTGATG-3'); exon2, 15FHIV-Tat2Plus (5'-TTCTCTATCAAAGCAACCCACCTCCCAATCCCG-3') and 5USpe1HIV-Tat2Minus (5'-GACGTACTAGTCTATTCCTTCGGGCCTGTC-3'). XhoI and SpeI restriction sites were introduced via the primers. The sense-primer of exon2 starts with a 15-mer which is homologous to the 3' end of exon1 and necessary for fusion of both exons. PCRs were performed with Expand High Fidelity PCR System (Roche) using the following conditions: one cycle 94degreesC for 3 min; 35 cycles 94degreesC for 45 s, 55degreesC for 45 s, 68degreesC for 45 s; one cycle 68degreesC for 7 min. For fusion of both exons, the following PCR conditions were applied: one cycle 94degreesC for 3 min; 35 cycles 94degreesC for 45 s, 58degreesC for 45 s, 68degreesC for 60 s. After 10 cycles without primers, the sense-primer of exon1 and the antisense-primer of exon2 were added for the remaining cycles. The resulting amplicon was ligated into the pBS-kRSPA vector (35) via XhoI and SpeI restriction sites.", "output": {"json_structures": {}}, "schema": []}
{"input": "5'-Rapid amplification of cDNA ends analysis (RACE)\nTotal RNA was isolated from A3.01 T cells using RNeasy mini kit (Qiagen). The transcriptional start sites of A3G were identified using the 5'/3' RACE Kit, 2nd Generation (Roche) according to the manufacturer's instructions. The following primers were used: RACE-APO3G1 (5'-TATCCCTTGTACACTTTGT-3') for cDNA synthesis, RACE-APO3G2 (5'-CATACTCCTGGTCACGAT-3') for the first PCR and RACE-APO3Gnest (5'-GAATACACCTGGCCTCGAA-3') for the nested PCR. Reaction products were analyzed by agarose gel electrophoresis, purified using QIAquick gel extraction kit (Qiagen), T/A-cloned into vector pCR4-TOPO (Invitrogen) and sequenced.", "output": {"json_structures": {}}, "schema": []}
{"input": "Luciferase assay\nFor transient transfection of A3.01 and U937 cells, DMRIE-C transfection reagent (Life Technologies) was used (36). Cells were seeded in 6-well tissue culture plates (5 x 105 cells per well) in 1.5 ml Opti-MEM (Life Technologies) containing 0.5 microg firefly luciferase reporter plasmid and 3.5 microl DMRIE-C. After 4-5 h of incubation, 1.5 ml complete RPMI medium were added. HepG2 and Huh7 cell lines were transfected using LipofectAMINE Plus as recommended by the manufacturer (Life Technologies). Briefly, exponential growing cells (1.5 x 106) were transfected with 5 microl LipofectAMINE, 6 microl PLUS reagent and the required amount of plasmid DNA in a final volume of 1 ml Opti-MEM. Following 4 h of incubation, cells were washed in PBS and 3 ml of complete DMEM medium were added.\nFor cotransfection of reporter plasmids and siRNA into HeLa cells, HiPerfect transfection reagent (Qiagen) was used according to the manufacturer's protocol for cotransfection of adherent cells with siRNA and plasmid DNA.\nTwo days after transfection of the respective cell lines, cells were harvested in 100 microl (suspension cells) or 300 microl (adherent cells) of Passive Lysis Buffer (Promega) and luciferase assay was performed using the Dual Luciferase Assay System (Promega) according to the manufacturer's instructions. As an internal control, 50 ng (adherent cells) or 100 ng (suspension cells) of ph-RG-TK plasmid (Promega), which constitutively expresses renilla luciferase was cotransfected in every sample and firefly luciferase activities were normalized to renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown in the figures. For stimulation of cells, final concentrations of 20 ng/ml TPA (Sigma) or 30 ng/ml IFN-alpha or 30 ng/ml IFN-gamma (Tebu-Bio) were applied approximately 15 h before harvesting for luciferase assay.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expresses", "start": 1466, "end": 1475}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1484, "end": 1494}]}]}}, "schema": []}
{"input": "Electrophoretic mobility shift assay (EMSA)\nFor preparation of nuclear extracts, 5 x 106 A3.01 T cells were washed in cold PBS and resuspended in 500 microl buffer A (10 mM HEPES pH7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). After incubation for 15 min on ice, swollen cells were pressed 10 times through a syringe with a 26G needle and centrifuged at 5000 r.p.m. for 5 min. Pellets contained the nuclei and were washed in buffer A for two times and resuspended in 50 microl buffer C (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA 1 mM DTT, 1 mM PMSF). After shaking for 30 min at 4degreesC and centrifugation for 10 min at 13 000 r.p.m., supernatants were used as nuclear extracts.\nEMSA probes were generated by annealing the following complementary oligonucleotides: APO-Sp1/3, 5'- CCAGCTGGGCGGGACCACCAGGGGAGGGGC-3' and 5'-GCCCCTCCCCTGGTGGTCCCGCCCAGCTGG-3'; APO-Sp1/3mut, 5'- CCAGCTGTTCGGGACCACCAGGGGAGGGGC-3' and 5'- GCCCCTCCCCTGGTGGTCCCGAACAGCTGG-3' according to standard procedures. Nucleotides differing from the original promoter sequence are shown in bold type. A commercially available Sp1 probe (sc-2502, referred to as Sp1cons) was purchased from Santa Cruz Biotechnology. The double-stranded oligonucleotides were 5' end-labeled using T4 polynucleotide kinase (New England Biolabs) and [gamma-32P]ATP (3000 Ci/mmol, Amersham) and purified by using Nick G50 columns (Amersham).\nFor EMSA, 5 microg of nuclear proteins were preincubated on ice with 2 microg of poly(dI-dC) (Roche) as an unspecific competitor and 1 microg of bovine serum albumin in band shift buffer (50 mM Tris, 150 mM KCl, 5 mM EDTA, 2.5 mM dithiothreitol, 20% Ficoll) for 15 min. 32P-labeled oligonucleotides (50 000 c.p.m.) were added in a total volume of 20 microl, incubated on ice for 20 min and loaded onto 5% native polyacrylamide gels in 0.5xTris-borate-EDTA buffer. Upon fractionation, gels were dried and exposed for autoradiography. For competition experiments, 1- or 30-fold molar excess of the unlabeled APO-Sp1/3 or APO-Sp1/3mut oligonucleotides was added to the preincubation mixture. For supershift experiments, 2 microg Sp1 antibody (sc-59x, Santa Cruz Biotechnology) or Sp3 antibody (sc-644x, Santa Cruz Biotechnology) were added to the preincubation mixture and preincubation time was extended to 30 min.", "output": {"json_structures": {}}, "schema": []}
{"input": "Chromatin immunoprecipitation (ChIP) assay\nA3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37degreesC. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 x 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 microl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 x 107 cells and 2 microg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 microl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 microl of the resolved DNA in the first PCR, and 1 microl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5'-ccacggtggcctccgagggtga-3' and ChIP3Gminus: 5'-ctctccaccatcaagacagac-3' (1. PCR); ChIP3G2plus: 5'-tactctccctccctgtcccca-3' and ChIP3G nested minus: 5'-aggctgatgcctccgcag-3' (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94degreesC for 2 min; 30 cycles 94degreesC for 30 s, 60degreesC for 60 s, 72degreesC for 2 min; one cycle 72degreesC for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5'-taagtaccacccagagatgag-3' and ChIP3Gneg_minus: 5'-catgatcttcatggtggcacg-3' for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55degreesC.", "output": {"json_structures": {}}, "schema": []}
{"input": "RNA interference and western blot analysis\nSp1 and Sp3 translation was silenced in HeLa cells using the siRNA duplexes Hs_SP1_1_HP and Hs_SP3_1_HP (Qiagen). A nonspecific siRNA (Qiagen) was used as control. HeLa cells were transfected with 150 or 300 ng siRNA per 6-well, using the HiPerfect transfection reagent (Qiagen) according to the manufacturer's protocol for reverse transfection of adherent cells in 6-well plates.\nForty-eight hours after transfection, HeLa cells were harvested for detection of Sp1 and Sp3 proteins. Cells were washed in PBS, lysed in RIPA (25 mM Tris pH 8.0, 137 mM NaCl, 1% Glycerol, 0.5% sodium deoxycholate, 1% NP-40, 2 mM EDTA pH 8, 0.1% SDS and protease inhibitors) and lysates were cleared by centrifugation. After boiling with Laemmli's buffer, samples were subjected to SDS-polyacrylamide gel electrophoresis followed by transfer to a nitrocellulose membrane. Sp1 and Sp3 proteins were detected using alpha-Sp1(Pep2) antibody (sc-59, Santa Cruz) or alpha-Sp3(D-20) antibody (sc-644, Santa Cruz) followed by incubation with alpha-rabbit-HRP (Amersham Biosciences). For detection of tubulin, alpha-tubulin (B5-1-2, Sigma) and alpha-mouse-HRP (Amersham Biosciences) antibodies were used. Signals were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "detection", "start": 492, "end": 501}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 505, "end": 508}]}, {"trigger": {"text": "detection", "start": 492, "end": 501}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 513, "end": 516}]}, {"trigger": {"text": "detected", "start": 922, "end": 930}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 896, "end": 899}]}, {"trigger": {"text": "detected", "start": 922, "end": 930}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 904, "end": 907}]}]}}, "schema": []}
{"input": "Characterization of the transcriptional start sites of APOBEC3G by 5'-RACE\nTo identify the transcriptional start sites (TSS) of APOBEC3G (A3G) in A3.01 T cells, we performed 5'-rapid amplification of cDNA ends analysis (RACE) with A3G-specific primers (see Figure 1). Agarose gel electrophoresis resolved the nested PCR products into three bands of different electrophoretic mobility with a dominant middle band (Figure 2). For each band, the DNA was cloned and sequence analysis of six or seven individual transformants was performed. We observed that the transcriptional start sites of the A3G gene were located between 58 and 361 nt upstream of the ATG start codon (Figure 1). Although TSS were variable and most sites were only detected once among the 19 clones analyzed, one TSS was identified in six individual clones. This TSS was located 66 nt upstream of the start of the published A3G mRNA sequence (GenBank(TM) accession number NM021822) and we defined this position as the major transcriptional start site of the A3G gene.", "output": {"json_structures": {}}, "schema": []}
{"input": "The core promoter of A3G is located within the region -114/+66 relative to the TSS\nFor characterization of the A3G promoter, we cloned the 1025 bp located at position -959/+66 relative to the identified transcription start into the promoterless pGL3-Basic luciferase reporter plasmid and designated the plasmid pGL3-Basic-APOprom1025. Similarly, pGL3-APOprom502 (containing sequence -436/+66), pGL3-APOprom225 (containing sequence -159/+66) and further 5' deletion reporter constructs containing 180, 150, 120 or 60 bp upstream of position +65 were generated (Figure 3A). In order to analyze transcriptional activity, the luciferase reporter plasmids were transiently transfected into A3.01 T cells. Luciferase assays revealed a ~20-fold increased transcriptional activity of the 1025 bp sequence as compared to the empty vector, indicating that we had identified an active A3G promoter sequence (Figure 3B). This transcription rate was not significantly altered by the 5' deletions leading to the 502, 225 and 180 bp fragments (Figure 3B). In contrast, a drop in luciferase activity was observed in the case of the 150 bp fragment. This construct only retained 28% of the transcriptional activity of the 180 bp promoter in A3.01 T cells and activity of the 120 bp fragment was further reduced. Comparable reductions relative to the activity of 180 bp fragment were observed in the myeloid cell line U937 and the hepatic cell lines HepG2 and Huh7 (Figure 3C), indicating that the core promoter of A3G is located within the region -114/+66 relative to the TSS.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "drop", "start": 1056, "end": 1060}, "arguments": [{"role": "Theme", "text": "transcriptional activity", "start": 748, "end": 772}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 738, "end": 747}, "arguments": [{"role": "Theme", "text": "transcriptional activity", "start": 748, "end": 772}, {"role": "CSite", "text": "1025 bp sequence", "start": 780, "end": 796}, {"role": "Cause", "text": "A3G", "start": 874, "end": 877}]}], "regulation": [{"trigger": {"text": "altered", "start": 955, "end": 962}, "arguments": [{"role": "Theme", "text": "increased", "start": 738, "end": 747}]}], "transcription": [{"trigger": {"text": "transcriptional activity", "start": 748, "end": 772}, "arguments": [{"role": "Theme", "text": "Luciferase", "start": 700, "end": 710}]}]}}, "schema": []}
{"input": "The A3G promoter is not inducible in T cells\nAccording to the current knowledge, APOBEC3G plays a role in the innate defence against pathogens like HIV-1. The latter has evolved a mechanism to counteract A3G activity by expressing the regulatory protein Vif (4). Vif induces proteasomal degradation of A3G and additionally inhibits A3G activity by further mechanisms (8, 37-43). To investigate whether the overexpression of HIV-1 proteins also influences A3G promoter activity, we either expressed HIV-1 Vif or the HIV-1 Tat protein, the latter has been shown to activate different viral and cellular promoters (44,45). In addition, increasing amounts of the plasmid pNL4-3, containing the full-length genome of HIV-1, were transfected. These constructs or the empty vector pcDNA3.1 were cotransfected with the 1025 bp construct into A3.01 T cells. Luciferase assays showed that the transcriptional activity of the A3G promoter was not significantly altered in the presence of Vif, Tat or HIV-1NL4-3 (Figure 4A), suggesting that HIV-1 is not modulating A3G expression on the transcriptional level.\nIt has been shown that the amount of A3G mRNA in T cells is increased in response to mitogenic stimulation with phorbol ester (46-48). In addition, interferons have been described to upregulate A3G expression in hepatocytes and macrophages (46,48). To investigate whether these stimuli interfere with A3G promoter activity in T cells, we transfected A3.01 T cells with the 1025 bp promoter or the empty vector pGL3-Basic (vector) and treated the cells with phorbol ester (TPA). The luciferase assay showed that the ~15-fold increased transcriptional activity of the 1025 bp promoter relative to the empty vector was not further enhanced by TPA treatment (Figure 4B), although the functional activity of TPA was confirmed by induction of the SV40 promoter-containing reporter plasmid pGL3-Control (data not shown). Similarly, treatment of A3.01 T cells transfected with the A3G promoter deletion constructs with IFN-alpha or IFN-gamma showed no effect (Figure 4C). Interestingly, a control plasmid (pGL2-CVX) containing two IFN-responsive GAS (gamma activated sequence) elements upstream of the luciferase reporter gene was only induced by IFN-alpha in these cells (Figure 4C). Since two reports describe A3G upregulation by interferons in hepatocytes (47,48), we additionally performed the experiment in the hepatic cell line HepG2. In line with these publications, we observed an induction of the A3G promoter by approximately 2-fold after IFN-alpha or IFN-gamma stimulation (Figure 4D) with IFN-gamma being slightly more potent. For both interferon types, induction of A3G promoter activity was observed for all deletion constructs except for the 60 bp fragment, indicating that the responsible region is located within the 60 nt present in the 120 bp, but not in the 60 bp fragment.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressing", "start": 220, "end": 230}, "arguments": [{"role": "Theme", "text": "Vif", "start": 254, "end": 257}]}, {"trigger": {"text": "expressed", "start": 488, "end": 497}, "arguments": [{"role": "Theme", "text": "Vif", "start": 504, "end": 507}]}, {"trigger": {"text": "expressed", "start": 488, "end": 497}, "arguments": [{"role": "Theme", "text": "Tat", "start": 521, "end": 524}]}, {"trigger": {"text": "expression", "start": 1057, "end": 1067}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1053, "end": 1056}]}, {"trigger": {"text": "expression", "start": 1296, "end": 1306}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1292, "end": 1295}]}], "negative regulation": [{"trigger": {"text": "counteract", "start": 193, "end": 203}, "arguments": [{"role": "Theme", "text": "A3G", "start": 204, "end": 207}, {"role": "Cause", "text": "expressing", "start": 220, "end": 230}]}, {"trigger": {"text": "inhibits", "start": 323, "end": 331}, "arguments": [{"role": "Cause", "text": "Vif", "start": 263, "end": 266}, {"role": "Theme", "text": "A3G", "start": 332, "end": 335}]}], "positive regulation": [{"trigger": {"text": "inducible", "start": 24, "end": 33}, "arguments": [{"role": "Theme", "text": "A3G", "start": 4, "end": 7}, {"role": "Site", "text": "promoter", "start": 8, "end": 16}]}, {"trigger": {"text": "induces", "start": 267, "end": 274}, "arguments": [{"role": "Cause", "text": "Vif", "start": 263, "end": 266}, {"role": "Theme", "text": "degradation", "start": 287, "end": 298}]}, {"trigger": {"text": "increased", "start": 1158, "end": 1167}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1135, "end": 1138}]}, {"trigger": {"text": "upregulate", "start": 1281, "end": 1291}, "arguments": [{"role": "Theme", "text": "expression", "start": 1296, "end": 1306}]}, {"trigger": {"text": "increased", "start": 1622, "end": 1631}, "arguments": [{"role": "Theme", "text": "transcriptional", "start": 1632, "end": 1647}]}, {"trigger": {"text": "activity", "start": 1648, "end": 1656}, "arguments": [{"role": "Theme", "text": "transcriptional", "start": 1632, "end": 1647}]}, {"trigger": {"text": "enhanced", "start": 1726, "end": 1734}, "arguments": [{"role": "Theme", "text": "increased", "start": 1622, "end": 1631}]}, {"trigger": {"text": "upregulation", "start": 2306, "end": 2318}, "arguments": [{"role": "Theme", "text": "A3G", "start": 2302, "end": 2305}]}, {"trigger": {"text": "induction", "start": 2479, "end": 2488}, "arguments": [{"role": "Theme", "text": "A3G", "start": 2496, "end": 2499}, {"role": "Site", "text": "promoter", "start": 2500, "end": 2508}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 287, "end": 298}, "arguments": [{"role": "Theme", "text": "A3G", "start": 302, "end": 305}]}], "regulation": [{"trigger": {"text": "modulating", "start": 1042, "end": 1052}, "arguments": [{"role": "Theme", "text": "expression", "start": 1057, "end": 1067}]}], "transcription": [{"trigger": {"text": "transcriptional", "start": 1632, "end": 1647}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1580, "end": 1590}]}]}}, "schema": []}
{"input": "A GC-box located at position -87/-78 of the A3G promoter is important for transcriptional activity\nThe reporter studies shown in Figure 3 had demonstrated a drop in luciferase activity after deletion of the 30 nt at the 5' end of the 180 bp core promoter. We therefore inspected the 30 bp sequence deleted in the 150 bp fragment and identified a GC-box at position -87/-78 (see Figure 1). The sequence TGGGCGGGAC, which is interrupted in the 150 bp fragment, represents a variant of the (G/T)GGGCGG(G/A)(G/A)(C/T) consensus motif recognized by Sp1 and Sp3 transcription factors. To analyze whether this putative Sp1/Sp3-binding site mediates transcriptional activity of the 180 bp core promoter, we introduced two point mutations which changed the sequence from TGGGCGGGAC to TGTTCGGGAC (mutations shown in bold). This resulted in a 71% reduction of the transcriptional activity compared to the unmodified 180 bp promoter (Figure 5A) and this value was only marginally higher than the luciferase activity of the 150 bp fragment, indicating that the identified motif is essential for basal activity of the A3G core promoter. To further examine the transcriptional potency of the 30 nt present in the 180 bp promoter, we cloned the region -114/-85 (containing all nucleotides which are deleted in the 150 bp fragment, designated E1) or the region -92/-63 (containing the putative Sp1/Sp3 motif, designated E2) into the vector pGL3-Promoter (see Figure 1). This vector contains a luciferase reporter gene under the control of an SV40 promoter without enhancer sequences, and putative transcriptionally active sequences can be cloned upstream of the SV40 promoter. Luciferase assays showed that the E1 element increased SV40 promoter activity only by ~2-fold (Figure 5B). In contrast, the 30 nt of E2 enhanced the transcriptional activity of the SV40 promoter by ~4.3-fold, indicating that the intact GC-box present in the E2 element was responsible for the strongly enhanced transcriptional activity of the SV40 promoter.", "output": {"json_structures": {"binding": [{"trigger": {"text": "recognized", "start": 530, "end": 540}, "arguments": [{"role": "Theme", "text": "A3G", "start": 44, "end": 47}, {"role": "Site", "text": "the (G/T)GGGCGG(G/A)(G/A)(C/T) consensus motif", "start": 483, "end": 529}, {"role": "Theme2", "text": "Sp1", "start": 544, "end": 547}]}, {"trigger": {"text": "recognized", "start": 530, "end": 540}, "arguments": [{"role": "Theme", "text": "A3G", "start": 44, "end": 47}, {"role": "Site", "text": "the (G/T)GGGCGG(G/A)(G/A)(C/T) consensus motif", "start": 483, "end": 529}, {"role": "Theme2", "text": "Sp3", "start": 552, "end": 555}]}], "positive regulation": [{"trigger": {"text": "enhanced", "start": 1797, "end": 1805}, "arguments": [{"role": "Cause", "text": "A3G", "start": 1105, "end": 1108}, {"role": "CSite", "text": "30 nt", "start": 1785, "end": 1790}, {"role": "Theme", "text": "transcriptional activity", "start": 1810, "end": 1834}]}, {"trigger": {"text": "responsible", "start": 1934, "end": 1945}, "arguments": [{"role": "Theme", "text": "enhanced", "start": 1963, "end": 1971}]}, {"trigger": {"text": "enhanced", "start": 1963, "end": 1971}, "arguments": [{"role": "Theme", "text": "transcriptional activity", "start": 1972, "end": 1996}]}, {"trigger": {"text": "transcriptional activity", "start": 1972, "end": 1996}, "arguments": [{"role": "Theme", "text": "Luciferase", "start": 1661, "end": 1671}]}], "regulation": [{"trigger": {"text": "under the control", "start": 1502, "end": 1519}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1477, "end": 1487}]}], "transcription": [{"trigger": {"text": "transcriptional activity", "start": 1810, "end": 1834}, "arguments": [{"role": "Theme", "text": "Luciferase", "start": 1661, "end": 1671}]}]}}, "schema": []}
{"input": "Sp1 and Sp3 transcription factors bind to the GC-box at position -87/-78 of the A3G promoter\nTo investigate whether the GC-box represents a binding site for the transcription factors Sp1 and Sp3, we performed EMSA analyses with nuclear extracts isolated from A3.01 T cells. As probes, we radioactively labeled the unmodified E2 sequence (nucleotides -92/-63 of the A3G promoter, probe designated APO-Sp1/3) or the same region carrying the two point mutations described above (probe designated APO-Sp1/3mut). As control, we used a commercially available Sp1 consensus oligonucleotide (Sp1cons), which is known to be recognized by Sp1 transcription factors. Four DNA-protein complexes were observed in the presence of the APO-Sp1/3 probe (Figure 6A). The upper two complexes were specific since they disappeared in the presence of a 30-fold molar excess of unlabeled APO-Sp1/3 probe (Figure 6A, lane 7). Further confirmation of the specificity of these DNA-protein complexes was demonstrated by the inability of the unlabeled APO-Sp1/3mut probe to abolish binding (Figure 6A, lanes 12 and 13). As expected, the two specific complexes were also present in the case of the Sp1cons control probe (Figure 6A, lanes 2 and 3). In contrast, none of these specific complexes was observed when the mutated probe was used (Figure 6A, lanes 10 and 11), confirming that protein binding was dependent on the identified GC-box. In order to characterize the complexes, we performed supershift experiments using Sp1- and Sp3-specific antibodies. The upper of the complexes that appeared in combination with the Sp1cons or APO-Sp1/3 probes, shifted in the presence of the Sp1 antibody (Figure 6B, lanes 3 and 7), whereas the lower complex shifted in the presence of the Sp3 antibody (Figure 6B, lanes 4 and 8). Taken together, the EMSA demonstrated that the GC-box located on the A3G core promoter serves as a binding site for Sp1 and Sp3.\nTo show binding of Sp1 and Sp3 factors also in the context of the endogenous A3G promoter, a chromatin immunoprecipitation (ChIP) assay was performed. Sp1 and Sp3 antibodies, but not an actin antibody, immunoprecipitated the A3G promoter in A3.01 T cells (Figure 6C, upper panel). In contrast, no PCR signal was received with a primer pair recognizing a region in the A3G gene ~4000 bp downstream of the Sp1/Sp3-binding site (Figure 6C, lower panel). Only the positive controls showed a DNA band, with an A3G expression plasmid or the sheared and cross-linked input DNA used as template. This demonstrates the binding of Sp1 and Sp3 transcription factors to the endogenous A3G promoter.", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 34, "end": 38}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 0, "end": 3}, {"role": "Site2", "text": "GC-box", "start": 46, "end": 52}, {"role": "Theme2", "text": "A3G", "start": 80, "end": 83}]}, {"trigger": {"text": "bind", "start": 34, "end": 38}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 8, "end": 11}, {"role": "Site2", "text": "GC-box", "start": 46, "end": 52}, {"role": "Theme2", "text": "A3G", "start": 80, "end": 83}]}, {"trigger": {"text": "represents a binding site", "start": 127, "end": 152}, "arguments": [{"role": "Theme", "text": "A3G", "start": 80, "end": 83}, {"role": "Site", "text": "GC-box", "start": 120, "end": 126}, {"role": "Theme2", "text": "Sp1", "start": 183, "end": 186}]}, {"trigger": {"text": "represents a binding site", "start": 127, "end": 152}, "arguments": [{"role": "Theme", "text": "A3G", "start": 80, "end": 83}, {"role": "Site", "text": "GC-box", "start": 120, "end": 126}, {"role": "Theme2", "text": "Sp3", "start": 191, "end": 194}]}, {"trigger": {"text": "recognized", "start": 615, "end": 625}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 629, "end": 632}]}, {"trigger": {"text": "serves as a binding site", "start": 1879, "end": 1903}, "arguments": [{"role": "Site", "text": "GC-box", "start": 1839, "end": 1845}, {"role": "Theme", "text": "A3G", "start": 1861, "end": 1864}, {"role": "Theme2", "text": "Sp1", "start": 1908, "end": 1911}]}, {"trigger": {"text": "serves as a binding site", "start": 1879, "end": 1903}, "arguments": [{"role": "Site", "text": "GC-box", "start": 1839, "end": 1845}, {"role": "Theme", "text": "A3G", "start": 1861, "end": 1864}, {"role": "Theme2", "text": "Sp3", "start": 1916, "end": 1919}]}, {"trigger": {"text": "binding", "start": 1929, "end": 1936}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 1940, "end": 1943}, {"role": "Theme2", "text": "A3G", "start": 1998, "end": 2001}, {"role": "Site2", "text": "promoter", "start": 2002, "end": 2010}]}, {"trigger": {"text": "binding", "start": 1929, "end": 1936}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 1948, "end": 1951}, {"role": "Theme2", "text": "A3G", "start": 1998, "end": 2001}, {"role": "Site2", "text": "promoter", "start": 2002, "end": 2010}]}, {"trigger": {"text": "binding", "start": 2531, "end": 2538}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 2542, "end": 2545}, {"role": "Theme2", "text": "A3G", "start": 2594, "end": 2597}, {"role": "Site2", "text": "promoter", "start": 2598, "end": 2606}]}, {"trigger": {"text": "binding", "start": 2531, "end": 2538}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 2550, "end": 2553}, {"role": "Theme2", "text": "A3G", "start": 2594, "end": 2597}, {"role": "Site2", "text": "promoter", "start": 2598, "end": 2606}]}], "gene expression": [{"trigger": {"text": "expression", "start": 2430, "end": 2440}, "arguments": [{"role": "Theme", "text": "A3G", "start": 2426, "end": 2429}]}]}}, "schema": []}
{"input": "Silencing of Sp1 and Sp3 reduces A3G promoter activity\nTo confirm the role of Sp1 and Sp3 in regulation of A3G promoter activity, we silenced their translation via RNA interference. Functionality of the siRNAs directed against Sp1 or Sp3 was confirmed by western blot analysis: protein levels of Sp1 as well as the long and short isoforms of Sp3 were strongly reduced in the presence of 150 or 300 ng specific siRNA (Figure 7A). An unspecific control siRNA had no influence (Figure 7A). We then cotransfected the luciferase reporter plasmid containing the 180 bp A3G promoter together with 100 ng siRNA using an optimized protocol for the cotransfection of plasmid plus siRNA. This resulted in a 31-43% reduction of luciferase activity in the presence of Sp1- or Sp3-specific siRNA compared to the control siRNA. In contrast, no influence on transcriptional activity of the 150 bp fragment, which does not contain the Sp1/Sp3-binding motif, was observed. Thus, both Sp1 and Sp3 factors are mediating transcriptional activity of the A3G promoter.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "protein levels", "start": 278, "end": 292}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 296, "end": 299}]}, {"trigger": {"text": "protein levels", "start": 278, "end": 292}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 342, "end": 345}]}, {"trigger": {"text": "cotransfected", "start": 495, "end": 508}, "arguments": [{"role": "Theme", "text": "luciferase reporter plasmid containing the 180 bp A3G promoter", "start": 513, "end": 575}]}], "negative regulation": [{"trigger": {"text": "Silencing", "start": 0, "end": 9}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 13, "end": 16}]}, {"trigger": {"text": "Silencing", "start": 0, "end": 9}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 21, "end": 24}]}, {"trigger": {"text": "reduced", "start": 360, "end": 367}, "arguments": [{"role": "Theme", "text": "protein levels", "start": 278, "end": 292}]}, {"trigger": {"text": "reduction", "start": 703, "end": 712}, "arguments": [{"role": "Theme", "text": "luciferase reporter plasmid containing the 180 bp A3G promoter", "start": 513, "end": 575}, {"role": "Cause", "text": "siRNA", "start": 776, "end": 781}]}, {"trigger": {"text": "siRNA", "start": 776, "end": 781}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 755, "end": 758}]}, {"trigger": {"text": "siRNA", "start": 776, "end": 781}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 763, "end": 766}]}]}}, "schema": []}
{"input": "Shortly after the discovery of APOBEC3G, it became clear that this human gene plays an important role in antiretroviral defense (4). Originally identified as a restriction factor of HIV-1 infection, expression of APOBEC3G was found in human peripheral blood lymphocytes and macrophages, which represent the main target cells for HIV-1 (32). In addition, the protein was detected in lung, liver, spleen, testis and ovary, but little is known about its transcriptional regulation (29,32,47). To address this question, we cloned the human APOBEC3G promoter and analyzed its regulation in T cells. Applying 5'-RACE, we identified multiple start sites for transcription of the A3G gene. This observation is consistent with the fact that the gene appears to lack canonical CCAAT and TATA boxes (32), a condition often associated with multiple transcriptional start sites (49-51). However, one single start site was detected in 6 of 19 clones and was designated as +1. This TSS is located 66 bp upstream of the start of the published mRNA sequence (GenBank(TM) NM021822).\nWe cloned a 1025 bp promoter, which ranges from position -959/+66 relative to the identified TSS. Luciferase reporter assays showed that this promoter was transcriptionally active in A3.01 T cells. Treatment with the phorbol ester TPA did not further enhance transcriptional activity, although upregulation of A3G mRNA levels in T cells by TPA has been described (33). Rose et al. used actinomycin D to block further transcription and did not find evidence for enhanced mRNA stability. Therefore, the authors suggested that an enhanced transcription rate could be responsible for the increased amount of A3G mRNA after TPA treatment. However, the promoter analysis we performed indicates a different mechanism that is independent of transcriptional regulation. In addition, we observed that the A3G promoter was not inducible by IFN-alpha or IFN-gamma in A3.01 T cells, whereas in the hepatic cell line HepG2, a moderate induction was measured. It has been described previously that A3G gene expression is upregulated by interferons in hepatocytes and macrophages (46-48, 52). In this context, two interferon-responsive elements have been identified at the positions -6/+9 and +1/+15 relative to the TSS (48). Consistent with this data, we observed an interferon induction in hepatocytes for the fragments ranging between 1025 and 120 bp length. The activity of the 60 bp fragment that does not contain these motifs was not affected. Thus, the induction we observed was most likely mediated by the described interferon-responsive elements. However, according to our results, these motifs can enhance transcription in hepatic cells, but not in T cells. This is surprising, since IFN-alpha-inducible signaling cascades are present in A3.01 T cells: we showed that the control plasmid harboring interferon-responsive GAS elements was markedly induced by IFN-alpha treatment. In contrast, the GAS element was not responsive to IFN-gamma treatment in these cells, suggesting that our T cell line was not able to mediate IFN-gamma-induced signals. So far, enhanced A3G promoter activity by interferons is clearly described in hepatocytes and macrophages, whereas the situation in T cells is still unclear. A recent study found no influence of IFN-alpha or IFN-gamma on A3G expression in resting primary blood lymphocytes (52). Another publication describes an enhanced expression of A3G after IFN-alpha treatment in resting primary CD4 T cells, but not in activated T cells (53). This observation is not contradictory to our results since T cell lines are mitotically active and therefore rather in an activated than in a resting state. In conclusion, the current data suggests that regulation of the A3G promoter activity by interferons is dependent on the cell type and possibly also from the cellular activation status.\nWe additionally analyzed the influence of HIV-1 proteins on A3G promoter activity. The HIV-1 Vif protein is neutralizing the antiviral function of A3G by a multitude of ways. Targeting the A3G protein for proteasomal degradation is considered to be the main mode of action (37-41), but it becomes more and more clear that other mechanisms are also involved. There is evidence that Vif also promotes exclusion of A3G from the virus particles and an influence on the A3G translation process is discussed (8,42,43). The question whether Vif alters transcription controlled by the A3G promoter has not been analyzed so far. Our analysis indicates that transcription from the A3G promoter is unaffected by Vif or other HIV-1 proteins. Taken together, in T cell lines, the A3G promoter appears constitutively active.\nBy generating a series of 5' deletions, we showed that the core promoter is located within the region -114/+66 relative to the TSS. This 180 bp promoter was transcriptionally active in the lymphoid, myeloid and hepatic cell lines we tested, indicating that ubiquitous transcription factors are involved in regulation of A3G gene transcription. A GC-box with the sequence TGGGCGGGAC was identified at position -87/-78 of the A3G promoter. This GC-box is essential for basal promoter activity, since changing the motif to TGTTCGGGAC by introducing two point mutations strongly reduced A3G promoter activity. The transcriptional potency of the GC-box was further demonstrated by cloning it upstream of an SV40 promoter. This resulted in a 4-fold enhanced transcription rate. The hypothesis that a general transcription factor is involved in regulation of A3G transcription was confirmed by EMSA. Supershift analysis showed that the transcription factors Sp1 and Sp3 which are ubiquitously expressed in mammalian cells, bind specifically to the identified motif. In addition, the binding of Sp1 and Sp3 to the endogenous A3G promoter was demonstrated by a chromatin immunoprecipitation assay. This observation is consistent with the results of the 5'-RACE, since Sp1 is known to play an important role for RNA polymerase II to bind to the transcription initiation site in TATA-boxless promoters and is associated with multiple transcription initiation sites (54-56). We also tested whether overexpression of Sp1 or Sp3 proteins had an influence on A3G promoter activity. Luciferase assays revealed that A3G promoter activities remained unchanged (data not shown), most probably due to the high basal expression level of the endogenous Sp1 and/or Sp3 proteins. The Sp family of transcription factors is involved in transcriptional regulation of many housekeeping, tissue-specific, viral and inducible genes (57). Whereas Sp1 typically acts as an activator, Sp3 can serve as a repressor or activator (57). However, in the context of the A3G promoter, both transcription factors serve as activators, as shown by siRNA-mediated silencing of the single factors. Although siRNAs directed against Sp1 or Sp3 significantly reduced transcription controlled by the 180 bp promoter, transcriptional activity was not reduced to the level of the 150 bp fragment. This can be explained by the remaining low amounts of Sp1/Sp3 proteins which can be seen in the western blot analysis. Alternatively, other transcription factors could be involved in A3G regulation.\nOur results also provide information about the regulation of APOBEC3F (A3F), another member of the human APOBEC3 family. A3F is expressed in many human tissues that also express A3G and both proteins have been shown to form heteromultimers, which are most likely generated through binding to an RNA intermediate (26,29,30). A3F only differs in 16 nt from the 560 nt upstream of the TSS of A3G (GenBank(TM) DQ146365 and DQ147772). The 180-bp core promoter region, which we identified for A3G has 100% identity with the A3F sequence. Therefore, Sp1/Sp3 transcription factors are most likely also mediating basal transcription of the A3F gene.\nOur study revealed that the human A3G gene is controlled by a promoter with multiple transcriptional start sites. In A3.01 T cells, the A3G promoter appears constitutively active and is not inducible by TPA, type I or II interferons or by HIV-1 proteins. The core promoter is located within a region of 180 bp at position -114/+66 relative to the TSS. A GC-box is crucial to the function of the core promoter and represents a binding site for Sp1 and Sp3 transcription factors. Our results can serve as a basis for future studies aimed at understanding how A3G and A3F expression is controlled in different tissues and how these restriction factors can be used to develop novel therapeutic strategies against HIV-1 infection.", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 5709, "end": 5713}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 5644, "end": 5647}]}, {"trigger": {"text": "bind", "start": 5709, "end": 5713}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 5652, "end": 5655}]}, {"trigger": {"text": "binding", "start": 5769, "end": 5776}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 5780, "end": 5783}, {"role": "Theme2", "text": "A3G", "start": 5810, "end": 5813}, {"role": "Site2", "text": "promoter", "start": 5814, "end": 5822}]}, {"trigger": {"text": "binding", "start": 5769, "end": 5776}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 5788, "end": 5791}, {"role": "Theme2", "text": 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"end": 8333}]}], "gene expression": [{"trigger": {"text": "expression", "start": 199, "end": 209}, "arguments": [{"role": "Theme", "text": "APOBEC3G", "start": 213, "end": 221}]}, {"trigger": {"text": "detected", "start": 370, "end": 378}, "arguments": [{"role": "Theme", "text": "APOBEC3G", "start": 213, "end": 221}]}, {"trigger": {"text": "expression", "start": 2057, "end": 2067}, "arguments": [{"role": "Theme", "text": "A3G", "start": 2048, "end": 2051}]}, {"trigger": {"text": "expression", "start": 3332, "end": 3342}, "arguments": [{"role": "Theme", "text": "A3G", "start": 3328, "end": 3331}]}, {"trigger": {"text": "expression", "start": 3428, "end": 3438}, "arguments": [{"role": "Theme", "text": "A3G", "start": 3442, "end": 3445}]}, {"trigger": {"text": "expressed", "start": 5679, "end": 5688}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 5644, "end": 5647}]}, {"trigger": {"text": "expressed", "start": 5679, "end": 5688}, "arguments": [{"role": "Theme", "text": "Sp3", 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"start": 8436, "end": 8439}]}, {"trigger": {"text": "expression", "start": 8448, "end": 8458}, "arguments": [{"role": "Theme", "text": "A3F", "start": 8444, "end": 8447}]}], "negative regulation": [{"trigger": {"text": "block", "start": 1468, "end": 1473}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1482, "end": 1495}]}, {"trigger": {"text": "silencing", "start": 6813, "end": 6822}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 6609, "end": 6612}]}, {"trigger": {"text": "silencing", "start": 6813, "end": 6822}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 6645, "end": 6648}]}, {"trigger": {"text": "reduced", "start": 6904, "end": 6911}, "arguments": [{"role": "Theme", "text": "transcription", "start": 6912, "end": 6925}]}, {"trigger": {"text": "reduced", "start": 6994, "end": 7001}, "arguments": [{"role": "Theme", "text": "transcriptional", "start": 6961, "end": 6976}]}], "positive regulation": [{"trigger": {"text": "enhance", "start": 1316, "end": 1323}, "arguments": [{"role": "Theme", "text": "transcriptional", "start": 1324, "end": 1339}]}, {"trigger": {"text": "upregulation", "start": 1359, "end": 1371}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1375, "end": 1378}]}, {"trigger": {"text": "enhanced", "start": 1592, "end": 1600}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1601, "end": 1614}]}, {"trigger": {"text": "increased", "start": 1649, "end": 1658}, "arguments": [{"role": "Cause", "text": "enhanced", "start": 1592, "end": 1600}, {"role": "Theme", "text": "A3G", "start": 1669, "end": 1672}]}, {"trigger": {"text": "inducible", "start": 1881, "end": 1890}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1860, "end": 1863}, {"role": "Site", "text": "promoter", "start": 1864, "end": 1872}, {"role": "Cause", "text": "IFN-alpha", "start": 1894, "end": 1903}]}, {"trigger": {"text": "inducible", "start": 1881, "end": 1890}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1860, "end": 1863}, {"role": "Site", "text": "promoter", "start": 1864, "end": 1872}, {"role": "Cause", "text": "IFN-gamma", "start": 1907, "end": 1916}]}, {"trigger": {"text": "upregulated", "start": 2071, "end": 2082}, "arguments": [{"role": "Cause", "text": "IFN-alpha", "start": 1894, "end": 1903}, {"role": "Theme", "text": "expression", "start": 2057, "end": 2067}]}, {"trigger": {"text": "upregulated", "start": 2071, "end": 2082}, "arguments": [{"role": "Cause", "text": "IFN-gamma", "start": 1907, "end": 1916}, {"role": "Theme", "text": "expression", "start": 2057, "end": 2067}]}, {"trigger": {"text": "enhance", "start": 2657, "end": 2664}, "arguments": [{"role": "Cause", "text": "A3G", "start": 1860, "end": 1863}, {"role": "CSite", "text": "the described interferon-responsive elements", "start": 2559, "end": 2603}, {"role": "Theme", "text": "transcription", "start": 2665, "end": 2678}]}, {"trigger": {"text": "enhanced", "start": 3419, "end": 3427}, "arguments": [{"role": "Theme", "text": "expression", "start": 3428, "end": 3438}, {"role": "Cause", "text": "IFN-alpha", "start": 3452, "end": 3461}]}, {"trigger": {"text": "Targeting", "start": 4057, "end": 4066}, "arguments": [{"role": "Cause", "text": "Vif", "start": 3975, "end": 3978}, {"role": "Theme", "text": "proteasomal degradation", "start": 4087, "end": 4110}]}, {"trigger": {"text": "from", "start": 4544, "end": 4548}, "arguments": [{"role": "Theme", "text": "transcription", "start": 4530, "end": 4543}, {"role": "Cause", "text": "A3G", "start": 4553, "end": 4556}, {"role": "CSite", "text": "promoter", "start": 4557, "end": 4565}]}, {"trigger": {"text": "active", "start": 4685, "end": 4691}, "arguments": [{"role": "Theme", "text": "A3G", "start": 4649, "end": 4652}, {"role": "Site", "text": "promoter", "start": 4653, "end": 4661}]}, {"trigger": {"text": "play an important role", "start": 5968, "end": 5990}, "arguments": [{"role": "Cause", "text": "Sp1", "start": 5952, "end": 5955}, {"role": "Theme", "text": "bind", "start": 6016, "end": 6020}]}, {"trigger": {"text": "serve as activators,", "start": 6765, "end": 6785}, "arguments": [{"role": "Cause", "text": "Sp1", "start": 6609, "end": 6612}, {"role": "Theme", "text": "A3G", "start": 6724, "end": 6727}, {"role": "Site", "text": "promoter", "start": 6728, "end": 6736}]}, {"trigger": {"text": "serve as activators,", "start": 6765, "end": 6785}, "arguments": [{"role": "Cause", "text": "Sp3", "start": 6645, "end": 6648}, {"role": "Theme", "text": "A3G", "start": 6724, "end": 6727}, {"role": "Site", "text": "promoter", "start": 6728, "end": 6736}]}, {"trigger": {"text": "mediating", "start": 7832, "end": 7841}, "arguments": [{"role": "Cause", "text": "Sp1", "start": 7781, "end": 7784}, {"role": "Theme", "text": "transcription", "start": 7848, "end": 7861}]}, {"trigger": {"text": "mediating", "start": 7832, "end": 7841}, "arguments": [{"role": "Cause", "text": "Sp3", "start": 7785, "end": 7788}, {"role": "Theme", "text": "transcription", "start": 7848, "end": 7861}]}, {"trigger": {"text": "active", "start": 8051, "end": 8057}, "arguments": [{"role": "Theme", "text": "A3G", "start": 8015, "end": 8018}, {"role": "Site", "text": "promoter", "start": 8019, "end": 8027}]}, {"trigger": {"text": "inducible", "start": 8069, "end": 8078}, "arguments": [{"role": "Theme", "text": "A3G", "start": 8015, "end": 8018}, {"role": "Site", "text": "promoter", "start": 8019, "end": 8027}]}, {"trigger": {"text": "crucial", "start": 8243, "end": 8250}, "arguments": [{"role": "Cause", "text": "A3G", "start": 8015, "end": 8018}, {"role": "CSite", "text": "GC-box", "start": 8233, "end": 8239}, {"role": "Theme", "text": "binding", "start": 8305, "end": 8312}]}], "protein catabolism": [{"trigger": {"text": "proteasomal degradation", "start": 4087, "end": 4110}, "arguments": [{"role": "Theme", "text": "A3G", "start": 4071, "end": 4074}]}], "regulation": [{"trigger": {"text": "regulation", "start": 571, "end": 581}, "arguments": [{"role": "Theme", "text": "APOBEC3G", "start": 536, "end": 544}, {"role": "Site", "text": "promoter", "start": 545, "end": 553}]}, {"trigger": {"text": "affected", "start": 2489, "end": 2497}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1860, "end": 1863}, {"role": "Cause", "text": "IFN-alpha", "start": 1894, "end": 1903}, {"role": "Site", "text": "60 bp fragment", "start": 2431, "end": 2445}]}, {"trigger": {"text": "affected", "start": 2489, "end": 2497}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1860, "end": 1863}, {"role": "Cause", "text": "IFN-gamma", "start": 1907, "end": 1916}, {"role": "Site", "text": "60 bp fragment", "start": 2431, "end": 2445}]}, {"trigger": {"text": "influence", "start": 3289, "end": 3298}, "arguments": [{"role": "Cause", "text": "IFN-alpha", "start": 3302, "end": 3311}, {"role": "Theme", "text": "expression", "start": 3332, "end": 3342}]}, {"trigger": {"text": "influence", "start": 3289, "end": 3298}, "arguments": [{"role": "Cause", "text": "IFN-gamma", "start": 3315, "end": 3324}, {"role": "Theme", "text": "expression", "start": 3332, "end": 3342}]}, {"trigger": {"text": "alters", "start": 4420, "end": 4426}, "arguments": [{"role": "Cause", "text": "Vif", "start": 4416, "end": 4419}, {"role": "Theme", "text": "controlled", "start": 4441, "end": 4451}]}, {"trigger": {"text": "controlled", "start": 4441, "end": 4451}, "arguments": [{"role": "Theme", "text": "transcription", "start": 4427, "end": 4440}, {"role": "Cause", "text": "A3G", "start": 4459, "end": 4462}, {"role": "CSite", "text": "promoter", "start": 4463, "end": 4471}]}, {"trigger": {"text": "unaffected", "start": 4569, "end": 4579}, "arguments": [{"role": "Theme", "text": "transcription", "start": 4530, "end": 4543}, {"role": "Cause", "text": "Vif", "start": 4583, "end": 4586}]}, {"trigger": {"text": "regulation", "start": 4999, "end": 5009}, "arguments": [{"role": "Theme", "text": "transcription", "start": 5022, "end": 5035}]}, {"trigger": {"text": "regulation", "start": 5531, "end": 5541}, "arguments": [{"role": "Theme", "text": "transcription", "start": 5549, "end": 5562}]}, {"trigger": {"text": "controlled", "start": 6926, "end": 6936}, "arguments": [{"role": "Cause", "text": "A3G", "start": 6724, "end": 6727}, {"role": "Theme", "text": "transcription", "start": 6912, "end": 6925}, {"role": "CSite", "text": "180 bp promoter", "start": 6944, "end": 6959}]}, {"trigger": {"text": "regulation", "start": 7226, "end": 7236}, "arguments": [{"role": "Theme", "text": "A3G", "start": 7222, "end": 7225}]}, {"trigger": {"text": "regulation", "start": 7285, "end": 7295}, "arguments": [{"role": "Theme", "text": "APOBEC3F", "start": 7299, "end": 7307}]}, {"trigger": {"text": "controlled", "start": 7925, "end": 7935}, "arguments": [{"role": "Cause", "text": "A3G", "start": 7913, "end": 7916}, {"role": "Theme", "text": "A3G", "start": 7913, "end": 7916}, {"role": "CSite", "text": "promoter with multiple transcriptional start sites", "start": 7941, "end": 7991}]}, {"trigger": {"text": "controlled", "start": 8462, "end": 8472}, "arguments": [{"role": "Theme", "text": "expression", "start": 8448, "end": 8458}]}], "transcription": [{"trigger": {"text": "transcription", "start": 651, "end": 664}, "arguments": [{"role": "Theme", "text": "A3G", "start": 672, "end": 675}]}, {"trigger": {"text": "transcriptional", "start": 1324, "end": 1339}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1375, "end": 1378}]}, {"trigger": {"text": "transcription", "start": 1482, "end": 1495}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1375, "end": 1378}]}, {"trigger": {"text": "transcription", "start": 1601, "end": 1614}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1669, "end": 1672}]}, {"trigger": {"text": "transcription", "start": 2665, "end": 2678}, "arguments": [{"role": "Theme", "text": "A3G", "start": 1860, "end": 1863}]}, {"trigger": {"text": "transcription", "start": 4427, "end": 4440}, "arguments": [{"role": "Theme", "text": "A3G", "start": 4459, "end": 4462}]}, {"trigger": {"text": "transcription", "start": 4530, "end": 4543}, "arguments": [{"role": "Theme", "text": "A3G", "start": 4553, "end": 4556}]}, {"trigger": {"text": "transcription", "start": 5022, "end": 5035}, "arguments": [{"role": "Theme", "text": "A3G", "start": 5013, "end": 5016}]}, {"trigger": {"text": "transcription", "start": 5549, "end": 5562}, "arguments": [{"role": "Theme", "text": "A3G", "start": 5545, "end": 5548}]}, {"trigger": {"text": "transcription", "start": 6912, "end": 6925}, "arguments": [{"role": "Theme", "text": "A3G", "start": 6724, "end": 6727}]}, {"trigger": {"text": "transcriptional", "start": 6961, "end": 6976}, "arguments": [{"role": "Theme", "text": "A3G", "start": 6724, "end": 6727}]}, {"trigger": {"text": "transcription", "start": 7848, "end": 7861}, "arguments": [{"role": "Theme", "text": "A3F", "start": 7869, "end": 7872}]}]}}, "schema": []}
{"input": "Sequence of the A3G promoter and the downstream region. The first 1000 bp upstream of the major TSS are shown in lower case, the first 800 bp of the transcribed sequence are shown in upper case. Introns are removed, but their positions are indicated. Arrows refer to transcriptional start sites and their observed frequency is given by the numbers above. The primer binding sites for 5'-RACE analysis and cloning of the luciferase reporter constructs are underlined and the names of the primers are annotated. Gray and black arrowheads define the regions designated E1 and E2 which were cloned into vector pGL3-Promoter and used as EMSA probes. The ATG start codon and the identified Sp1/Sp3 transcription factor binding site are shown in bold.", "output": {"json_structures": {}}, "schema": []}
{"input": "5'-RACE analysis of the A3G cDNA. Agarose gel electrophoresis of size marker and A3G 5'-RACE products after nested PCR with primer RACE-APO3Gnest (see Figure 1 for primer details). Arrowheads indicate the three resulting DNA bands which were cloned and sequenced.", "output": {"json_structures": {}}, "schema": []}
{"input": "Luciferase activities of A3G promoter constructs in different cell lines. (A) A3G promoter 5' deletion constructs of different sizes were cloned into pGL3-Basic luciferase reporter plasmids. Numbering is relative to the major TSS. A putative Sp1/Sp3 consensus site (gray square) is depicted. (B) A3.01 T cells were transiently transfected with the A3G promoter deletion constructs. Numbers on the x-axis refer to the length of the A3G promoter fragments in bp. (C) A3G promoter plasmids were transfected into U937, HepG2 and Huh7 cell lines. Numbers in the legends refer to the length of the A3G promoter fragments in bp. After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "coexpressed", "start": 726, "end": 737}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 685, "end": 695}]}, {"trigger": {"text": "coexpressed", "start": 726, "end": 737}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 746, "end": 756}]}], "positive regulation": [{"trigger": {"text": "activities", "start": 11, "end": 21}, "arguments": [{"role": "Theme", "text": "Luciferase", "start": 0, "end": 10}, {"role": "Cause", "text": "A3G", "start": 25, "end": 28}, {"role": "CSite", "text": "promoter", "start": 29, "end": 37}]}]}}, "schema": []}
{"input": "A3G promoter activities after coexpression of HIV-1 proteins or treatment with TPA or interferons. (A) A3.01 T cells were cotransfected with pGL3-Basic reporter plasmid containing the 1025 bp A3G promoter and 1 microg of Vif expression plasmid, 1 microg Tat expression plasmid or increasing amounts of HIV-1NL4-3 (0.1, 0.5 and 1 microg). After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. (B) A3.01 T cells were transiently transfected with pGL3-Basic reporter plasmid containing the 1025 bp A3G promoter or with empty vector. Fifteen hour before harvesting for luciferase assay, a subset of the cell culture was stimulated with 20 ng/ml TPA. Forty-eight hour after transfection, luciferase assay was performed. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities and the values for the empty vectors (untreated and TPA-stimulated) were set as 1. (C) A3.01 T cells were transiently transfected with the A3G promoter constructs or with the interferon-responsive reporter plasmid pGL2-CVX (GAS). Fifteen hour before harvesting for luciferase assay, a subset of the cell culture was stimulated with 30 ng/ml IFN-alpha or IFN-gamma. Forty-eight hour after transfection, luciferase assay was performed. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. (D) HepG2 cells were used for transfection. The experiment was performed as described in (C). Mean values (+/-SD) of representative experiments performed in triplicate are shown.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "coexpressed", "start": 442, "end": 453}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 401, "end": 411}]}, {"trigger": {"text": "coexpressed", "start": 442, "end": 453}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 462, "end": 472}]}, {"trigger": {"text": "coexpressed", "start": 857, "end": 868}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 816, "end": 826}]}, {"trigger": {"text": "coexpressed", "start": 857, "end": 868}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 877, "end": 887}]}, {"trigger": {"text": "coexpressed", "start": 1382, "end": 1393}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1341, "end": 1351}]}, {"trigger": {"text": "coexpressed", "start": 1382, "end": 1393}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1402, "end": 1412}]}]}}, "schema": []}
{"input": "A GC-box mediates transcriptional activity of the 180 bp core promoter. (A) A3.01 T cells were transfected with reporter plasmid pGL3-Basic containing 180, 150 or 120 bp of the A3G promoter. The two G-to-T substitutions introduced into the GC-box of the 180 bp fragment (180mut) are specified. After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown. (B) pGL3-Promoter reporter plasmids containing the regions E1 or E2 (see Figure 1) upstream of the SV40 promoter were transfected into A3.01 T cells. Firefly luciferase activities after 48 h were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "coexpressed", "start": 398, "end": 409}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 357, "end": 367}]}, {"trigger": {"text": "coexpressed", "start": 398, "end": 409}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 418, "end": 428}]}, {"trigger": {"text": "coexpressed", "start": 737, "end": 748}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 685, "end": 695}]}, {"trigger": {"text": "coexpressed", "start": 737, "end": 748}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 757, "end": 767}]}]}}, "schema": []}
{"input": "Sp1 and Sp3 bind to the GC-box present in the A3G promoter. (A) Nuclear extracts of A3.01 T cells were incubated with a 32P-labeled commercial Sp1 oligonucleotide probe (Sp1cons) or labeled probes homologous to the unmodified or mutated E2 region (see Figure 1) of the A3G promoter (APO-Sp1/3 and APO-Sp1/3mut). Protein-DNA complexes were separated by polyacrylamide electrophoresis and detected by autoradiography. EMSA was performed with a 1- or 30-fold molar excess of unlabeled APO-Sp1/3 probe (competitor, lanes 6 and 7) or APO-Sp1/3mut probe (competitor mut., lanes 12 and 13). (B) Sp1- and Sp3-specific antibodies were added to the EMSA reactions resulting in a supershift (ss) of the respective antibody-protein-oligo complexes (lanes 3, 4, 7, 8, 11, 12). (C) ChIP assay was performed with DNA from A3.01 T cells. Immunoprecipitation was performed with antibodies against Sp1, Sp3 or actin. PCR primer pairs specific for the A3G promoter (upper panel) or the A3G gene (lower panel) were used. As positive controls, the sheared and cross-linked DNA before the immunoprecipitation step (input) or a plasmid carrying the target sequence (plasmid) was used as template.", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 12, "end": 16}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 0, "end": 3}, {"role": "Site2", "text": "GC-box", "start": 24, "end": 30}, {"role": "Theme2", "text": "A3G", "start": 46, "end": 49}]}, {"trigger": {"text": "bind", "start": 12, "end": 16}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 8, "end": 11}, {"role": "Site2", "text": "GC-box", "start": 24, "end": 30}, {"role": "Theme2", "text": "A3G", "start": 46, "end": 49}]}]}}, "schema": []}
{"input": "Silencing of Sp1 and Sp3 reduces A3G promoter activity. (A) HeLa cells were transfected with 150 and 300 ng of unspecific, Sp1-specific or Sp3-specific siRNA. After 48 h, cells were harvested and Sp1 and Sp3 proteins were detected by western blot analysis. As loading control, protein levels of tubulin are shown. (B) HeLa cells were cotransfected with reporter plasmid pGL3-Basic containing 180 or 150 bp of the A3G promoter and siRNA. Hundred nanogram of unspecific siRNA (control), Sp1-specific siRNA (Sp1), Sp3-specific siRNA (Sp3) or a mixture of 50 ng Sp1-specific plus 50 ng Sp3-specific siRNA (Sp1+Sp3) were used. Firefly luciferase activities after 48 h were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "coexpressed", "start": 682, "end": 693}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 630, "end": 640}]}, {"trigger": {"text": "coexpressed", "start": 682, "end": 693}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 702, "end": 712}]}], "negative regulation": [{"trigger": {"text": "Silencing", "start": 0, "end": 9}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 13, "end": 16}]}, {"trigger": {"text": "Silencing", "start": 0, "end": 9}, "arguments": [{"role": "Theme", "text": "Sp3", "start": 21, "end": 24}]}]}}, "schema": []}
{"input": "Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFkappaB activity in vertebrate B-cells\nTo investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569-1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFkappaB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology.", "output": {"json_structures": {"phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 698, "end": 713}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 692, "end": 697}]}], "positive regulation": [{"trigger": {"text": "required", "start": 671, "end": 679}, "arguments": [{"role": "Theme", "text": "regulate", "start": 683, "end": 691}]}], "regulation": [{"trigger": {"text": "regulate", "start": 683, "end": 691}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 698, "end": 713}]}]}}, "schema": []}
{"input": "1The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCgamma and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3-6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8-12].\nPKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13-16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFkappaB transcription factor and in regulating cell survival during oxidative stress [17,21-23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26-28].\nTo investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFkappaB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 706, "end": 713}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 741, "end": 745}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 2172, "end": 2187}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 2166, "end": 2171}]}], "positive regulation": [{"trigger": {"text": "contributes", "start": 726, "end": 737}, "arguments": [{"role": "Cause", "text": "binding", "start": 706, "end": 713}, {"role": "Theme", "text": "activation", "start": 746, "end": 756}]}, {"trigger": {"text": "activation", "start": 746, "end": 756}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 741, "end": 745}]}, {"trigger": {"text": "indispensable", "start": 2148, "end": 2161}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 2172, "end": 2187}]}]}}, "schema": []}
{"input": "2.1Cell culture, transient transfections and cell stimulation\nThe generation, culture and activation of PKD1-/-, PKD3-/- and PKD1/3-/- knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29].", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "knockout", "start": 135, "end": 143}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 104, "end": 108}]}, {"trigger": {"text": "knockout", "start": 135, "end": 143}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 113, "end": 117}]}, {"trigger": {"text": "knockout", "start": 135, "end": 143}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 125, "end": 129}]}, {"trigger": {"text": "knockout", "start": 135, "end": 143}, "arguments": [{"role": "Theme", "text": "3", "start": 130, "end": 131}]}]}}, "schema": []}
{"input": "2.2sIgM staining\nDT40 B cells (2 x 106 cells per point) were resuspended in 200 mul buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry.\nAll results shown are representative of at two to four independent experiments unless otherwise indicated.", "output": {"json_structures": {}}, "schema": []}
{"input": "3.1Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases\nDT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3-/- cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A).\nPreviously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3-/- B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "express", "start": 103, "end": 110}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 129, "end": 133}]}, {"trigger": {"text": "express", "start": 103, "end": 110}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 138, "end": 142}]}, {"trigger": {"text": "expression", "start": 227, "end": 237}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 248, "end": 252}]}, {"trigger": {"text": "expression", "start": 227, "end": 237}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 256, "end": 260}]}, {"trigger": {"text": "expressed", "start": 381, "end": 390}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 398, "end": 402}]}, {"trigger": {"text": "expression", "start": 517, "end": 527}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 512, "end": 516}]}, {"trigger": {"text": "expression", "start": 1811, "end": 1821}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 1834, "end": 1838}]}, {"trigger": {"text": "expression", "start": 1971, "end": 1981}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 2004, "end": 2008}]}], "negative regulation": [{"trigger": {"text": "Loss", "start": 3, "end": 7}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 17, "end": 32}]}, {"trigger": {"text": "lack", "start": 222, "end": 226}, "arguments": [{"role": "Theme", "text": "expression", "start": 227, "end": 237}]}, {"trigger": {"text": "double knockout", "start": 538, "end": 553}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 531, "end": 535}]}, {"trigger": {"text": "double knockout", "start": 538, "end": 553}, "arguments": [{"role": "Theme", "text": "3", "start": 536, "end": 537}]}, {"trigger": {"text": "defective", "start": 1126, "end": 1135}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1136, "end": 1151}]}, {"trigger": {"text": "lacking", "start": 1311, "end": 1318}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 1326, "end": 1330}]}, {"trigger": {"text": "lacking", "start": 1311, "end": 1318}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 1334, "end": 1338}]}, {"trigger": {"text": "knockout", "start": 1604, "end": 1612}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 1584, "end": 1588}]}, {"trigger": {"text": "knockout", "start": 1604, "end": 1612}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 1592, "end": 1596}]}, {"trigger": {"text": "abolished", "start": 1713, "end": 1722}, "arguments": [{"role": "Theme", "text": "induced", "start": 1669, "end": 1676}, {"role": "Cause", "text": "lacked", "start": 1739, "end": 1745}]}, {"trigger": {"text": "lacked", "start": 1739, "end": 1745}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 1751, "end": 1755}]}, {"trigger": {"text": "lacked", "start": 1739, "end": 1745}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 1760, "end": 1764}]}, {"trigger": {"text": "deficient", "start": 1994, "end": 2003}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 2004, "end": 2008}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 17, "end": 32}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 11, "end": 16}]}, {"trigger": {"text": "phosphorylation", "start": 1136, "end": 1151}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 1155, "end": 1160}, {"role": "Site", "text": "serine 82", "start": 1164, "end": 1173}]}, {"trigger": {"text": "phosphorylation", "start": 1263, "end": 1278}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 1257, "end": 1262}]}, {"trigger": {"text": "phosphorylation", "start": 1452, "end": 1467}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 1446, "end": 1451}, {"role": "Site", "text": "S82", "start": 1471, "end": 1474}]}, {"trigger": {"text": "phosphorylation", "start": 1565, "end": 1580}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 1559, "end": 1564}]}, {"trigger": {"text": "phosphorylation", "start": 1677, "end": 1692}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 1696, "end": 1701}, {"role": "Site", "text": "S82", "start": 1705, "end": 1708}]}, {"trigger": {"text": "phosphorylation", "start": 1931, "end": 1946}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 1925, "end": 1930}]}, {"trigger": {"text": "phosphorylation", "start": 2113, "end": 2128}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 2107, "end": 2112}]}, {"trigger": {"text": "phosphorylation", "start": 2187, "end": 2202}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 2181, "end": 2186}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 1422, "end": 1431}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1452, "end": 1467}]}, {"trigger": {"text": "increase", "start": 1550, "end": 1558}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1565, "end": 1580}]}, {"trigger": {"text": "induced", "start": 1669, "end": 1676}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1677, "end": 1692}]}, {"trigger": {"text": "induced", "start": 1803, "end": 1810}, "arguments": [{"role": "Theme", "text": "expression", "start": 1811, "end": 1821}]}, {"trigger": {"text": "sufficient to restore", "start": 1882, "end": 1903}, "arguments": [{"role": "Cause", "text": "induced", "start": 1803, "end": 1810}, {"role": "Theme", "text": "regulation", "start": 1911, "end": 1921}]}, {"trigger": {"text": "restore", "start": 2069, "end": 2076}, "arguments": [{"role": "Cause", "text": "expression", "start": 1971, "end": 1981}, {"role": "Theme", "text": "induced", "start": 2099, "end": 2106}]}, {"trigger": {"text": "induced", "start": 2099, "end": 2106}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 2113, "end": 2128}]}], "regulation": [{"trigger": {"text": "regulation", "start": 1243, "end": 1253}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1263, "end": 1278}]}, {"trigger": {"text": "regulation", "start": 1911, "end": 1921}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1931, "end": 1946}]}, {"trigger": {"text": "regulate", "start": 2172, "end": 2180}, "arguments": [{"role": "Cause", "text": "PKD3", "start": 2147, "end": 2151}, {"role": "Theme", "text": "phosphorylation", "start": 2187, "end": 2202}]}, {"trigger": {"text": "regulate", "start": 2172, "end": 2180}, "arguments": [{"role": "Cause", "text": "PKD1", "start": 2163, "end": 2167}, {"role": "Theme", "text": "phosphorylation", "start": 2187, "end": 2202}]}]}}, "schema": []}
{"input": "3.2Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases\nPKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3-/-: Flag-PKD3+ve) or absence (PKD1/3-/-) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3-/- cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3-/- B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3-/- cells was slightly slower than that of wild type DT40 cells (12.7 +/- 2.8 h versus 10.2 +/- 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3-/- cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells.\nPKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30-32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "re-expression", "start": 527, "end": 540}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 549, "end": 553}]}, {"trigger": {"text": "re-expression", "start": 950, "end": 963}, "arguments": [{"role": "Theme", "text": "PKD3", "start": 945, "end": 949}]}, {"trigger": {"text": "expression", "start": 1637, "end": 1647}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 1630, "end": 1634}]}, {"trigger": {"text": "expression", "start": 1637, "end": 1647}, "arguments": [{"role": "Theme", "text": "3", "start": 1635, "end": 1636}]}], "negative regulation": [{"trigger": {"text": "loss", "start": 1622, "end": 1626}, "arguments": [{"role": "Theme", "text": "expression", "start": 1637, "end": 1647}]}]}}, "schema": []}
{"input": "3.3Antigen receptor regulated signalling pathways in PKD-null DT40 B cells\nTo further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3-/- (and in PKD1/3-/-:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1/3-/- cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3-/- B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33-35]. However, BCR-induced Erk phosphorylation was also reduced in PKD1/3-/--Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3-/- (and PKD1/3-/--Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway.\nTo search for other potential PKD targets that may show defective regulation in PKD1/3-/- DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3-/- cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1/3-/- DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs. Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs. This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera.\nBCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFkappaB transcription factor. Thus, PKD-mediated activation of NFkappaB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFkappaB activation [38]. In B cells, NFkappaB is known to be regulated via DAG and PKCbeta [39,40] but whether PKDs are key intermediaries for NFkappaB regulation has not been explored.\nThe data (Fig. 4A) show that NFkappaB transcriptional activity was strongly induced in both wild-type and PKD1/3-/- DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NFkappaB transcriptional activity was abolished in PKCbeta-/- DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCbeta-/- cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFkappaB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFkappaB.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "detectable", "start": 780, "end": 790}, "arguments": [{"role": "Theme", "text": "induced", "start": 638, "end": 645}, {"role": "Cause", "text": "null", "start": 801, "end": 805}]}, {"trigger": {"text": "null", "start": 801, "end": 805}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 794, "end": 798}]}, {"trigger": {"text": "null", "start": 801, "end": 805}, "arguments": [{"role": "Theme", "text": "3", "start": 799, "end": 800}]}, {"trigger": {"text": "loss", "start": 2438, "end": 2442}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 2446, "end": 2450}]}, {"trigger": {"text": "loss", "start": 2438, "end": 2442}, "arguments": [{"role": "Theme", "text": "3", "start": 2451, "end": 2452}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 722, "end": 737}, "arguments": [{"role": "Theme", "text": "S6 ribosomal protein", "start": 701, "end": 721}]}, {"trigger": {"text": "autophosphorylation", "start": 3988, "end": 4007}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 4011, "end": 4015}, {"role": "Site", "text": "S916", "start": 4019, "end": 4023}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 638, "end": 645}, "arguments": [{"role": "Theme", "text": "activation", "start": 646, "end": 656}]}, {"trigger": {"text": "activation", "start": 646, "end": 656}, "arguments": [{"role": "Theme", "text": "Akt", "start": 664, "end": 667}]}, {"trigger": {"text": "activation", "start": 646, "end": 656}, "arguments": [{"role": "Theme", "text": "mTOR", "start": 669, "end": 673}]}, {"trigger": {"text": "activation", "start": 646, "end": 656}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 722, "end": 737}]}, {"trigger": {"text": "expression of an activated", "start": 3367, "end": 3393}, "arguments": [{"role": "Theme", "text": "PKD1", "start": 3394, "end": 3398}]}, {"trigger": {"text": "observed", "start": 4029, "end": 4037}, "arguments": [{"role": "Theme", "text": "autophosphorylation", "start": 3988, "end": 4007}]}]}}, "schema": []}
{"input": "4Protein kinase D serine kinases have been proposed to regulate diverse cellular functions including the phosphorylation and nuclear localisation of class II HDACs and the phosphorylation of HSP27. It has also been suggested that PKDs act as mitochondrial sensors for oxidative stress and play a role in regulating NFkappaB transcription factors [41]. Most of the data about the function of PKDs has come from experiments that ectopically express active or inhibitory PKD mutants or that use RNAi to reduce PKD expression. We have used gene targeting to specifically delete PKD alleles in DT40 chicken B cells and can thus use PKD-null DT40 cells to assess the relative contribution of individual PKD isoforms in class II HDAC control versus oxidative stress responses and NFkappaB regulation in lymphocytes. We have previously used these PKD-null DT40 cells to define an essential role for PKDs in regulation of class II HDACs, the present report now describes an indispensable role for PKDs in regulating the phosphorylation of HSP27 on serine 82, a site previously identified as a target for the p38-MAPKAPK2 signalling cascade [42]. However, studies of PKD-null DT40 cells reveal that PKD family kinases are not essential for oxidative stress survival responses nor are they required for activation of NFkappaB transcription factors. These latter findings are in striking contrast to previous observations in HeLa and epithelial cell lines where overexpression/RNAi approaches have implicated PKD1/2 in the control of proliferation, survival and NFkappaB activation [20,23]. Hence, the present report shows that the proposed roles for PKDs as key sensors that modulate survival pathways in response to oxidative stress and regulate cell survival and proliferation are not ubiquitous and may be restricted to certain cell lineages. Taken together, these data indicate that loss of expression of PKD family members does not globally impact on early BCR-regulated signalling pathways.", "output": {"json_structures": {"phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 172, "end": 187}, "arguments": [{"role": "Site", "text": "serine", "start": 18, "end": 24}, {"role": "Theme", "text": "HSP27", "start": 191, "end": 196}]}, {"trigger": {"text": "phosphorylation", "start": 1011, "end": 1026}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 1030, "end": 1035}, {"role": "Site", "text": "serine 82", "start": 1039, "end": 1048}]}], "regulation": [{"trigger": {"text": "regulate", "start": 55, "end": 63}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 172, "end": 187}]}, {"trigger": {"text": "regulating", "start": 996, "end": 1006}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1011, "end": 1026}]}, {"trigger": {"text": "target", "start": 1084, "end": 1090}, "arguments": [{"role": "Theme", "text": "HSP27", "start": 1030, "end": 1035}, {"role": "Site", "text": "serine 82", "start": 1039, "end": 1048}]}]}}, "schema": []}
{"input": "GATA3-Driven Th2 Responses Inhibit TGF-beta1-Induced FOXP3 Expression and the Formation of Regulatory T Cells\nTranscription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes. FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown. We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway. We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3. This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-beta-mediated FOXP3 induction. This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process. Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response. IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 997, "end": 1004}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1008, "end": 1013}, {"role": "Theme2", "text": "FOXP3", "start": 1021, "end": 1026}, {"role": "Site2", "text": "promoter", "start": 1027, "end": 1035}]}], "gene expression": [{"trigger": {"text": "Expression", "start": 59, "end": 69}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 53, "end": 58}]}, {"trigger": {"text": "expression", "start": 496, "end": 506}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 339, "end": 344}]}, {"trigger": {"text": "overexpressing", "start": 826, "end": 840}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 841, "end": 847}]}], "negative regulation": [{"trigger": {"text": "Inhibit", "start": 27, "end": 34}, "arguments": [{"role": "Theme", "text": "Induced", "start": 45, "end": 52}]}, {"trigger": {"text": "inhibits", "start": 718, "end": 726}, "arguments": [{"role": "Cause", "text": "interleukin 4", "start": 659, "end": 672}, {"role": "Theme", "text": "FOXP3", "start": 727, "end": 732}]}, {"trigger": {"text": "deficient", "start": 882, "end": 891}, "arguments": [{"role": "Theme", "text": "mediated", "start": 933, "end": 941}]}], "positive regulation": [{"trigger": {"text": "Induced", "start": 45, "end": 52}, "arguments": [{"role": "Cause", "text": "TGF-beta1", "start": 35, "end": 44}, {"role": "Theme", "text": "Expression", "start": 59, "end": 69}]}, {"trigger": {"text": "mediated", "start": 933, "end": 941}, "arguments": [{"role": "Cause", "text": "transforming growth factor (TGF)-beta", "start": 895, "end": 932}, {"role": "Theme", "text": "induction", "start": 948, "end": 957}]}, {"trigger": {"text": "induction", "start": 948, "end": 957}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 942, "end": 947}]}, {"trigger": {"text": "mediated", "start": 978, "end": 986}, "arguments": [{"role": "Theme", "text": "deficient", "start": 882, "end": 891}, {"role": "Cause", "text": "binding", "start": 997, "end": 1004}]}], "regulation": [{"trigger": {"text": "regulating", "start": 481, "end": 491}, "arguments": [{"role": "Theme", "text": "expression", "start": 496, "end": 506}]}, {"trigger": {"text": "target", "start": 1298, "end": 1304}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 1305, "end": 1309}]}, {"trigger": {"text": "target", "start": 1298, "end": 1304}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1313, "end": 1318}]}]}}, "schema": []}
{"input": "Effective immune responses are characterized by T cell activation, which directs adaptive and innate immune responses to kill pathogens efficiently. Dependent on the pathogen, T cells differentiate into different subtypes, such as Th1 or Th2 cells, which are most efficient in defeating microbial or parasitic invaders respectively. A hallmark of Th1 and Th2 differentiation pathways is the exclusiveness of the individual phenotype leading to either Th1 or Th2, but not to mixed populations. The exclusiveness of this mechanism is provided by a polarization process, where Th2 differentiation inhibits Th1 commitment and vice versa. Specifically interleukin 4 (IL-4)-induced STAT6 and GATA3 inhibit differentiation into Th1 cells in the early phase of commitment [1,2]. GATA3 is sufficient to induce a Th2 phenotype [3] and acts not only through the induction of IL-4, IL-5 and IL-13, the Th2 cytokines, but also through the inhibition of Th1 cell-specific factors [3]. Recently, it was shown that T-bet directly modulates GATA3 function, suggesting that transcription factors compete in the early differentiation phase of T cells, potentially integrating environmental signals to finally imprint the T cell phenotype [4,5]. A GATA3-dominated immune response has been shown to be essential in airway hyperresonsiveness [6] and IL-4-dominated responses can break antigen-specific immune tolerance [7]. Overexpression of a dominant negative form of GATA3 [8] or treatment with antisense-mediated GATA3 blockade [9] decreased the severity of the allergic airway hyper-responsiveness.\nThe discovery of regulatory T (Treg) cells highlights another phenotype of T cells, which is essential for tolerance against self-antigens. Naturally occurring, thymus-derived Treg (nTreg) cells are generated in the thymus and are assumed to protect against the activity of autoreactive T cells in the periphery. These cells express the forkhead transcription factor FOXP3 and constitutively express CD25 on their surface, but they lack expression of Th1 or Th2 cytokines. Particularly interesting are those Treg cells that are generated in the periphery and thus are potential targets for therapeutic interventions. These induced Treg (iTreg) cells were reported to express FOXP3 [10]. The exact mechanisms of iTreg generation are unclear, but T cell receptor (TCR) triggering has been shown to induce FOXP3 expression and suppressive cells in human [11,12], however the phenotype appears to be of transient nature [13,14]. TGF-beta has been demonstrated to be important for the persistent induction of these cells in vitro and in vivo, since animals lacking the TGF-betaRII on T cells have fewer peripherally iTreg cells [15] and suffer from a T cell-dependent multiorgan inflammatory disease [16]. Although the effect of TGF-beta on natural and inducible Treg cell induction has been demonstrated repeatedly [15,17], its molecular mechanisms remain to be identified.\nThe current study provides evidence that GATA3 and FOXP3 play a competitive role in iTreg cell commitment as T-bet and GATA3 for Th1 and Th2 differentiation, respectively. We show that GATA3 inhibits FOXP3 induction and that IL-4 limits FOXP3 expression in a GATA3-mediated way, both in vitro and in vivo. We also show that GATA3 directly binds to the FOXP3 promoter and thereby prevents the induction of this gene, demonstrating that Th2 differentiation overrules iTreg induction.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 3291, "end": 3296}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 3276, "end": 3281}, {"role": "Theme2", "text": "FOXP3", "start": 3304, "end": 3309}, {"role": "Site2", "text": "promoter", "start": 3310, "end": 3318}]}], "gene expression": [{"trigger": {"text": "express", "start": 1907, "end": 1914}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1949, "end": 1954}]}, {"trigger": {"text": "express", "start": 1974, "end": 1981}, "arguments": [{"role": "Theme", "text": "CD25", "start": 1982, "end": 1986}]}, {"trigger": {"text": "express", "start": 2249, "end": 2256}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2257, "end": 2262}]}, {"trigger": {"text": "expression", "start": 2391, "end": 2401}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2385, "end": 2390}]}, {"trigger": {"text": "expression", "start": 3195, "end": 3205}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 3189, "end": 3194}]}], "negative regulation": [{"trigger": {"text": "Overexpression of a dominant negative form", "start": 1402, "end": 1444}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1448, "end": 1453}]}, {"trigger": {"text": "blockade", "start": 1501, "end": 1509}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1495, "end": 1500}]}, {"trigger": {"text": "lacking", "start": 2634, "end": 2641}, "arguments": [{"role": "Theme", "text": "TGF-betaRII", "start": 2646, "end": 2657}]}, {"trigger": {"text": "inhibits", "start": 3143, "end": 3151}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 3137, "end": 3142}, {"role": "Theme", "text": "induction", "start": 3158, "end": 3167}]}, {"trigger": {"text": "limits", "start": 3182, "end": 3188}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 3177, "end": 3181}, {"role": "Theme", "text": "expression", "start": 3195, "end": 3205}]}, {"trigger": {"text": "prevents", "start": 3331, "end": 3339}, "arguments": [{"role": "Cause", "text": "binds", "start": 3291, "end": 3296}, {"role": "Theme", "text": "induction", "start": 3344, "end": 3353}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 668, "end": 675}, "arguments": [{"role": "Cause", "text": "interleukin 4", "start": 647, "end": 660}, {"role": "Theme", "text": "STAT6", "start": 676, "end": 681}]}, {"trigger": {"text": "induced", "start": 668, "end": 675}, "arguments": [{"role": "Cause", "text": "interleukin 4", "start": 647, "end": 660}, {"role": "Theme", "text": "GATA3", "start": 686, "end": 691}]}, {"trigger": {"text": "induction", "start": 851, "end": 860}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 771, "end": 776}, {"role": "Theme", "text": "IL-4", "start": 864, "end": 868}]}, {"trigger": {"text": "induction", "start": 851, "end": 860}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 771, "end": 776}, {"role": "Theme", "text": "IL-5", "start": 870, "end": 874}]}, {"trigger": {"text": "induction", "start": 851, "end": 860}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 771, "end": 776}, {"role": "Theme", "text": "IL-13", "start": 879, "end": 884}]}, {"trigger": {"text": "induce", "start": 2378, "end": 2384}, "arguments": [{"role": "Theme", "text": "expression", "start": 2391, "end": 2401}]}, {"trigger": {"text": "induction", "start": 3158, "end": 3167}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 3152, "end": 3157}]}, {"trigger": {"text": "mediated way", "start": 3217, "end": 3229}, "arguments": [{"role": "Theme", "text": "limits", "start": 3182, "end": 3188}, {"role": "Cause", "text": "GATA3", "start": 3211, "end": 3216}]}, {"trigger": {"text": "induction", "start": 3344, "end": 3353}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 3304, "end": 3309}]}], "regulation": [{"trigger": {"text": "modulates", "start": 1014, "end": 1023}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 999, "end": 1004}, {"role": "Theme", "text": "GATA3", "start": 1024, "end": 1029}]}]}}, "schema": []}
{"input": "FOXP3 Induction in T Cell Subsets\nIt is assumed that FOXP3 expression can be induced in nonregulatory T cells, which is an important step in iTreg cell differentiation. However, it is not known if all CD4+ T cells have the same capacity to express FOXP3. To investigate whether FOXP3 can be expressed by any T cell subset or if expression is restricted to a distinct lineage, FOXP3 mRNA expression was analyzed in freshly isolated T cells such as CD25-depleted CD4+ cells, CD45RA+ naive or CD45RO+ memory T cells (Figure 1A), as well as T cells driven in vitro toward Th1, Th2, or iTreg phenotypes (Figure 1B; phenotype on Figure S1). The CD4+CD25-, CD45RA+, CD45RO+, and CD4+CD45RO+CD25- were able to significantly induce FOXP3 mRNA up to 30-fold upon TCR activation and addition of TGF-beta. Th1 cells showed only a 10-fold increase. In contrast, Th2 cells stimulated under the same conditions did not increase FOXP3 expression. The in vitro generated iTreg cells were unable to further up-regulate FOXP3, which was already at high levels under the resting conditions (Figure 1B, right panel).\nTh2 cells are known to produce IL-4 upon activation, which may interact with TGF-beta signaling and thus prevent FOXP3 induction. However, the neutralization of IL-4 with a blocking IL-4 antibody did not rescue FOXP3 expression in the differentiated Th2 cells (unpublished data). These data demonstrated that Th2 cells have a limited capacity to express FOXP3 (Figure 1B). The inability of Th2 cells to express FOXP3 was also documented at the single-cell level, confirming that Th2 cells lack FOXP3 expression (Figure 2A). Only iTreg cells expressed FOXP3 in resting conditions. Interestingly, we observed that resting iTreg cells express FOXP3 but show low CD25 surface expression. Repeated exposure to TGF-beta did not further increase the FOXP3 expression in the iTreg lineage but transiently induced FOXP3 expression in Th1 cells. Naturally occurring Th2 cells such as CRTH2+ T cells, T cells isolated according to their IL-4 secretion, or an IL-4-producing T cell clone (BR8) were also lacking FOXP3 expression (Figure 2B). Furthermore TGF-beta-mediated FOXP3 induction failed in these cells in contrast to the naive T cells (Figure 2B). Because IL-4 is the key Th2 cytokine, the expression of IL-4 and FOXP3 in freshly isolated CD4+ T cells was analyzed by fluorescence activated cell sorting (FACS). IL-4-expressing cells were most abundant among CD45RO+CD25- cells, which did not co-express FOXP3 (Figure 3A, left panel). In contrast, CD45RO+CD25+ cells abundantly expressed FOXP3, while lacking IL-4 (Figure 3A, right panel). As shown in Figure 3B, the frequency of the IL-4+ cells was always below 1% in the FOXP3+ cells close to the background. The IL-4+ cells were confined to the FOXP3- cells, as shown for the CD45RO+CD25-, CD45RO+CD25+, and CD45RO+CD25+high cells (Figure 3B). In addition, neither the Th2 clone (BR8) nor CRTH2 cells significantly expressed FOXP3 (Figure 3C). Cells enriched for their IL-4 secretion using the magnetic cell isolation technology contained some FOXP3-expressing cells, but importantly, the expression did not overlap. Taken together, these data indicate that FOXP3 was not expressed by Th2 cells and was not inducible in those cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 59, "end": 69}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 53, "end": 58}]}, {"trigger": {"text": "express", "start": 240, "end": 247}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 248, "end": 253}]}, {"trigger": {"text": "expressed", "start": 291, "end": 300}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 278, "end": 283}]}, {"trigger": {"text": "expression", "start": 328, "end": 338}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 278, "end": 283}]}, {"trigger": {"text": "expression", "start": 919, "end": 929}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 913, "end": 918}]}, {"trigger": {"text": "produce", "start": 1119, "end": 1126}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 1127, "end": 1131}]}, {"trigger": {"text": "expression", "start": 1313, "end": 1323}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1307, "end": 1312}]}, {"trigger": {"text": "express", "start": 1442, "end": 1449}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1450, "end": 1455}]}, {"trigger": {"text": "express", "start": 1499, "end": 1506}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1507, "end": 1512}]}, {"trigger": {"text": "expression", "start": 1596, "end": 1606}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1590, "end": 1595}]}, {"trigger": {"text": "expressed", "start": 1637, "end": 1646}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1647, "end": 1652}]}, {"trigger": {"text": "express", "start": 1728, "end": 1735}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1736, "end": 1741}]}, {"trigger": {"text": "expression", "start": 1768, "end": 1778}, "arguments": [{"role": "Theme", "text": "CD25", "start": 1755, "end": 1759}]}, {"trigger": {"text": "expression", "start": 1845, "end": 1855}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1839, "end": 1844}]}, {"trigger": {"text": "expression", "start": 1907, "end": 1917}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1901, "end": 1906}]}, {"trigger": {"text": "secretion", "start": 2027, "end": 2036}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 2022, "end": 2026}]}, {"trigger": {"text": "producing", "start": 2049, "end": 2058}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 2044, "end": 2048}]}, {"trigger": {"text": "expression", "start": 2102, "end": 2112}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2096, "end": 2101}]}, {"trigger": {"text": "expression", "start": 2282, "end": 2292}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 2296, "end": 2300}]}, {"trigger": {"text": "expression", "start": 2282, "end": 2292}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2305, "end": 2310}]}, {"trigger": {"text": "expressing", "start": 2409, "end": 2419}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 2404, "end": 2408}]}, {"trigger": {"text": "co-express", "start": 2485, "end": 2495}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2496, "end": 2501}]}, {"trigger": {"text": "expressed", "start": 2570, "end": 2579}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2580, "end": 2585}]}, {"trigger": {"text": "expressed", "start": 2960, "end": 2969}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2970, "end": 2975}]}, {"trigger": {"text": "secretion", "start": 3019, "end": 3028}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 3014, "end": 3018}]}, {"trigger": {"text": "expressing", "start": 3095, "end": 3105}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 3089, "end": 3094}]}, {"trigger": {"text": "expressed", "start": 3217, "end": 3226}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 3203, "end": 3208}]}], "negative regulation": [{"trigger": {"text": "prevent", "start": 1201, "end": 1208}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 1127, "end": 1131}, {"role": "Theme", "text": "induction", "start": 1215, "end": 1224}]}, {"trigger": {"text": "neutralization", "start": 1239, "end": 1253}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 1257, "end": 1261}]}, {"trigger": {"text": "inability", "start": 1473, "end": 1482}, "arguments": [{"role": "Theme", "text": "express", "start": 1499, "end": 1506}]}, {"trigger": {"text": "lack", "start": 1585, "end": 1589}, "arguments": [{"role": "Theme", "text": "expression", "start": 1596, "end": 1606}]}, {"trigger": {"text": "lacking", "start": 2088, "end": 2095}, "arguments": [{"role": "Theme", "text": "expression", "start": 2102, "end": 2112}]}, {"trigger": {"text": "failed", "start": 2172, "end": 2178}, "arguments": [{"role": "Theme", "text": "induction", "start": 2162, "end": 2171}]}, {"trigger": {"text": "lacking", "start": 2593, "end": 2600}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 2601, "end": 2605}]}], "positive regulation": [{"trigger": {"text": "Induction", "start": 6, "end": 15}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 0, "end": 5}]}, {"trigger": {"text": "induced", "start": 77, "end": 84}, "arguments": [{"role": "Theme", "text": "expression", "start": 59, "end": 69}]}, {"trigger": {"text": "induce", "start": 716, "end": 722}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 723, "end": 728}, {"role": "Cause", "text": "TGF-beta", "start": 784, "end": 792}]}, {"trigger": {"text": "increase", "start": 904, "end": 912}, "arguments": [{"role": "Theme", "text": "expression", "start": 919, "end": 929}]}, {"trigger": {"text": "up-regulate", "start": 989, "end": 1000}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1001, "end": 1006}]}, {"trigger": {"text": "induction", "start": 1215, "end": 1224}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1209, "end": 1214}]}, {"trigger": {"text": "rescue", "start": 1300, "end": 1306}, "arguments": [{"role": "Cause", "text": "neutralization", "start": 1239, "end": 1253}, {"role": "Theme", "text": "expression", "start": 1313, "end": 1323}]}, {"trigger": {"text": "low", "start": 1751, "end": 1754}, "arguments": [{"role": "Theme", "text": "expression", "start": 1768, "end": 1778}]}, {"trigger": {"text": "increase", "start": 1826, "end": 1834}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 1801, "end": 1809}, {"role": "Theme", "text": "expression", "start": 1845, "end": 1855}]}, {"trigger": {"text": "induced", "start": 1893, "end": 1900}, "arguments": [{"role": "Theme", "text": "expression", "start": 1907, "end": 1917}]}, {"trigger": {"text": "induction", "start": 2162, "end": 2171}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 2138, "end": 2146}, {"role": "Theme", "text": "FOXP3", "start": 2156, "end": 2161}]}, {"trigger": {"text": "inducible", "start": 3252, "end": 3261}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 3203, "end": 3208}]}], "regulation": [{"trigger": {"text": "restricted", "start": 342, "end": 352}, "arguments": [{"role": "Theme", "text": "expression", "start": 328, "end": 338}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 382, "end": 397}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 376, "end": 381}]}]}}, "schema": []}
{"input": "FOXP3 and GATA3 Kinetics in Differentiating Cells\nThe limited capacity of differentiated effector cells to induce FOXP3 expression suggests that iTreg induction has to occur before effector T cell differentiation occurs. Therefore, we analyzed the expression of FOXP3 and GATA3 during the differentiation of naive CD4 T cells into Th0 (neutral, anti-IL-4, anti-IFN-gamma, and anti IL-12), Th2, and iTreg phenotypes. After initiation of the differentiation process, FOXP3 and GATA3 showed a similar expression kinetic within the first 3 d, which are considered to be critical in T cell commitment [18]. Under Th2 differentiation conditions, FOXP3 mRNA expression increased only marginally (Figure 4A, left panel). Thus, although GATA3 and FOXP3 showed similar kinetics, their expression polarizes at the end of the differentiation process when cells were cultured towards Th2 or iTreg cells, respectively (Figure 4A and 4B). Interestingly, the Th0 cells were expressing more FOXP3 than the Th2 cells, but expressed low levels of GATA3; however, the protein expression slightly differed from mRNA expression, suggesting also posttranslational regulation and degradation as potential additional mechanisms in the differentiation process. The phenotype of iTreg cells included an anergic phenotype upon anti-CD3 re-stimulation (Figure S1A), CD103, CTLA-4, GITR, and PD-1 surface expression (Figure S1B). On the single-cell level, it can be seen that cells progress through a transition phase, where GATA3 and FOXP3 expression coexist to some degree in the same cells, which is resolved in iTreg cells after 7 d (Figure 4B). Taken together, these data demonstrated that Th2 cells have lost their capacity to express FOXP3 and showed that Th2 and iTreg cells arise from two different differentiation pathways.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 120, "end": 130}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 114, "end": 119}]}, {"trigger": {"text": "expression", "start": 248, "end": 258}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 262, "end": 267}]}, {"trigger": {"text": "expression", "start": 248, "end": 258}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 272, "end": 277}]}, {"trigger": {"text": "expression", "start": 498, "end": 508}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 465, "end": 470}]}, {"trigger": {"text": "expression", "start": 498, "end": 508}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 475, "end": 480}]}, {"trigger": {"text": "expression", "start": 775, "end": 785}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 728, "end": 733}]}, {"trigger": {"text": "expression", "start": 775, "end": 785}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 738, "end": 743}]}, {"trigger": {"text": "expressing", "start": 958, "end": 968}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 974, "end": 979}]}, {"trigger": {"text": "expressed", "start": 1004, "end": 1013}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1028, "end": 1033}]}, {"trigger": {"text": "expression", "start": 1375, "end": 1385}, "arguments": [{"role": "Theme", "text": "CD103", "start": 1337, "end": 1342}]}, {"trigger": {"text": "expression", "start": 1375, "end": 1385}, "arguments": [{"role": "Theme", "text": "CTLA-4", "start": 1344, "end": 1350}]}, {"trigger": {"text": "expression", "start": 1375, "end": 1385}, "arguments": [{"role": "Theme", "text": "GITR", "start": 1352, "end": 1356}]}, {"trigger": {"text": "expression", "start": 1375, "end": 1385}, "arguments": [{"role": "Theme", "text": "PD-1", "start": 1362, "end": 1366}]}, {"trigger": {"text": "expression", "start": 1511, "end": 1521}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1495, "end": 1500}]}, {"trigger": {"text": "expression", "start": 1511, "end": 1521}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1505, "end": 1510}]}, {"trigger": {"text": "express", "start": 1703, "end": 1710}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1711, "end": 1716}]}], "negative regulation": [{"trigger": {"text": "resolved", "start": 1573, "end": 1581}, "arguments": [{"role": "Theme", "text": "expression", "start": 1511, "end": 1521}]}, {"trigger": {"text": "lost", "start": 1680, "end": 1684}, "arguments": [{"role": "Theme", "text": "express", "start": 1703, "end": 1710}]}], "positive regulation": [{"trigger": {"text": "induce", "start": 107, "end": 113}, "arguments": [{"role": "Theme", "text": "expression", "start": 120, "end": 130}]}, {"trigger": {"text": "increased", "start": 662, "end": 671}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 646, "end": 661}]}, {"trigger": {"text": "more", "start": 969, "end": 973}, "arguments": [{"role": "Theme", "text": "expressing", "start": 958, "end": 968}]}, {"trigger": {"text": "low", "start": 1014, "end": 1017}, "arguments": [{"role": "Theme", "text": "expressed", "start": 1004, "end": 1013}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 646, "end": 661}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 640, "end": 645}]}]}}, "schema": []}
{"input": "IL-4 Inhibits TGF-beta-Mediated iTreg Commitment\nIL-4 induces differentiation of naive T cells, upon antigen encounter, into the Th2 cell lineage. We therefore asked whether IL-4 is able to inhibit TGF-beta induction of FOXP3 during the priming of naive T cells. Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-beta and/or IL-4 and harvested after 5 d. IL-4 efficiently repressed the TGF-beta-mediated induction of FOXP3 expression (Figure 5A) in a concentration-dependent manner (Figure 5B). Low levels of GATA3 were induced also in the absence of IL-4, as it was previously observed [3]. However, at low concentration, IL-4 was able to marginally induce FOXP3 expression. Of note, GATA3 was also induced in the presence of TGF-beta at high IL-4 concentration (Figure 5A and 5B). The IL-4-mediated prevention of FOXP3 expression was not caused by interferences of the receptor signaling, because the phosphorylation of SMAD2 or STAT6 was not affected by the addition of IL-4 and/or TGF-beta, which demonstrates that IL-4 as well as TGF-beta signaling were functional under these conditions (Figure 5C). Increasing amounts of IL-4 increase intracellular GATA3, whereas FOXP3 decreased, which is consistent with the mRNA analysis (Figure 5D). Furthermore, injection of IL-4 into wild-type B6 mice decreased the inducible or natural Treg number in vivo. A distinction of the Treg subsets is not possible, because recently activated iTreg cells also express surface CD25. We used complexes of recombinant mouse IL-4 (rmIL-4) plus anti-IL-4 monoclonal antibodies (mAbs), which have been shown to dramatically increase the potency of the cytokine in vivo [19]. In these mice, the percentage of CD4+CD25+ and FOXP3+ T cells dramatically decreased when the antibody-cytokine immune complexes were injected (Figure S2). Upon administration of rmIL-4 plus anti-IL-4 mAb complexes, the total number of CD4+CD25+ T cell, as well as the Foxp3+ T cells diminished by half (Figure S2G and S2H), confirming that the lower percentage was not due to an increase in the CD4+CD25- cells, but a real decrease of CD4+CD25+ T cells. In conclusion, IL-4 negatively regulates the natural or inducible Treg cell turnover not only in vitro but also in vivo. To study the effects of IL-4 on already-existing human natural or inducible Treg cells, we exposed sorted CD25+ T cells (nTreg cells) to IL-4 and analyzed FOXP3 expression and suppressive capacity. In already-existing Treg cells, IL-4 failed to inhibit FOXP3 expression (Figure S3A), and the suppressive capacity was not altered (Figure S3C). Similarly pre-existing iTreg cells did not decrease FOXP3 expression upon IL-4 exposure (Figure S3B).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 468, "end": 478}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 462, "end": 467}]}, {"trigger": {"text": "expression", "start": 709, "end": 719}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 703, "end": 708}]}, {"trigger": {"text": "expression", "start": 866, "end": 876}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 860, "end": 865}]}, {"trigger": {"text": "express", "start": 1494, "end": 1501}, "arguments": [{"role": "Theme", "text": "CD25", "start": 1510, "end": 1514}]}, {"trigger": {"text": "expression", "start": 2440, "end": 2450}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2434, "end": 2439}]}, {"trigger": {"text": "expression", "start": 2538, "end": 2548}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2532, "end": 2537}]}, {"trigger": {"text": "expression", "start": 2680, "end": 2690}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2674, "end": 2679}]}], "negative regulation": [{"trigger": {"text": "inhibit", "start": 190, "end": 197}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 174, "end": 178}, {"role": "Theme", "text": "induction", "start": 207, "end": 216}]}, {"trigger": {"text": "repressed", "start": 417, "end": 426}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 400, "end": 404}, {"role": "Theme", "text": "induction", "start": 449, "end": 458}]}, {"trigger": {"text": "induced", "start": 565, "end": 572}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 554, "end": 559}, {"role": "Cause", "text": "absence", "start": 585, "end": 592}]}, {"trigger": {"text": "absence", "start": 585, "end": 592}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 596, "end": 600}]}, {"trigger": {"text": "prevention", "start": 846, "end": 856}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 832, "end": 836}, {"role": "Theme", "text": "expression", "start": 866, "end": 876}]}, {"trigger": {"text": "decreased", "start": 1222, "end": 1231}, "arguments": [{"role": "Cause", "text": "Increasing amounts", "start": 1151, "end": 1169}, {"role": "Theme", "text": "FOXP3", "start": 1216, "end": 1221}]}, {"trigger": {"text": "suppressive capacity", "start": 2455, "end": 2475}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 2416, "end": 2420}, {"role": "Theme", "text": "expression", "start": 2440, "end": 2450}]}, {"trigger": {"text": "inhibit", "start": 2524, "end": 2531}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 2509, "end": 2513}, {"role": "Theme", "text": "expression", "start": 2538, "end": 2548}]}, {"trigger": {"text": "decrease", "start": 2665, "end": 2673}, "arguments": [{"role": "Theme", "text": "expression", "start": 2680, "end": 2690}, {"role": "Cause", "text": "IL-4", "start": 2696, "end": 2700}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 948, "end": 963}, "arguments": [{"role": "Theme", "text": "SMAD2", "start": 967, "end": 972}]}, {"trigger": {"text": "phosphorylation", "start": 948, "end": 963}, "arguments": [{"role": "Theme", "text": "STAT6", "start": 976, "end": 981}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 207, "end": 216}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 198, "end": 206}, {"role": "Theme", "text": "FOXP3", "start": 220, "end": 225}]}, {"trigger": {"text": "induction", "start": 449, "end": 458}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 431, "end": 439}, {"role": "Theme", "text": "expression", "start": 468, "end": 478}]}, {"trigger": {"text": "induce", "start": 696, "end": 702}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 668, "end": 672}, {"role": "Theme", "text": "expression", "start": 709, "end": 719}]}, {"trigger": {"text": "induced", "start": 745, "end": 752}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 730, "end": 735}, {"role": "Cause", "text": "TGF-beta", "start": 772, "end": 780}]}, {"trigger": {"text": "induced", "start": 745, "end": 752}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 730, "end": 735}, {"role": "Cause", "text": "IL-4", "start": 789, "end": 793}]}, {"trigger": {"text": "caused", "start": 885, "end": 891}, "arguments": [{"role": "Theme", "text": "prevention", "start": 846, "end": 856}]}, {"trigger": {"text": "Increasing amounts", "start": 1151, "end": 1169}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 1173, "end": 1177}]}, {"trigger": {"text": "increase", "start": 1178, "end": 1186}, "arguments": [{"role": "Cause", "text": "Increasing amounts", "start": 1151, "end": 1169}, {"role": "Theme", "text": "GATA3", "start": 1201, "end": 1206}]}], "regulation": [{"trigger": {"text": "affected", "start": 990, "end": 998}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 948, "end": 963}, {"role": "Cause", "text": "IL-4", "start": 1018, "end": 1022}]}, {"trigger": {"text": "affected", "start": 990, "end": 998}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 948, "end": 963}, {"role": "Cause", "text": "TGF-beta", "start": 1030, "end": 1038}]}, {"trigger": {"text": "altered", "start": 2600, "end": 2607}, "arguments": [{"role": "Theme", "text": "inhibit", "start": 2524, "end": 2531}]}]}}, "schema": []}
{"input": "The Role of TGF-beta in T Cell Differentiation\nAlthough TGF-beta-reduced CD25, IL-4 expression, and CD25 expression (Figure 6B), IL-4 significantly inhibited TGF-beta-mediated induction of FOXP3 in naive T cells driven toward FOXP3+ T cells, as shown by FACS analysis (Figure 6A). It is known that IL-4 is a potent growth factor and may therefore favor the proliferation of FOXP3- cells and thus decrease the relative percentage of FOXP3+ cells. However, analysis of cell division kinetics by CFSE-labeling demonstrated that IL-4 did not differentially promote cell growth of FOXP3+ over that of FOXP3-. In fact both populations showed similarly enhanced proliferation (Figure 6A). Furthermore the TGF-beta-mediated induction of FOXP3 expression was not caused by overgrowth of a CD25-FOXP3+ minority, since the number of FOXP3+ cells was low/absent in the purified CD4+CD45RA+ T cells (between 0% and 1%), and the FOXP3+ cells were not confined to the highly divided cells. CD25 was down-regulated in TGF-beta-treated cells compare to activated T cells, which was even more pronounced in cells cultured with TGF-beta and IL-4.\nThe addition of IL-4 to iTreg-driving conditions decreased the number of FOXP3+ cells (Figure 6B). In line with the previous findings, the IL-4-producing cells and the FOXP3 expressing cells are nonoverlapping populations. Since FOXP3 is known to act as a repressor of cytokine expression [20], we therefore analyzed GATA3 and FOXP3 expression. The expression kinetic of naive T cells exposed to IL-4 and TGF-beta demonstrated that GATA3 and FOXP3 are initially found in separate populations (day 2), but transiently co-express both factors (days 4-8), before establishing separate populations at the end of the differentiation process (day 10; Figure 6C), suggesting that GATA3 inhibits the development of iTreg cells by repressing FOXP3.\nThese results showed that IL-4 acts in vitro as an inhibitor of FOXP3 expression, without interfering with TGF-beta signaling, probably acting at the level of transcription factors, and possibly by a GATA3-dependent mechanism.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 84, "end": 94}, "arguments": [{"role": "Theme", "text": "CD25", "start": 73, "end": 77}]}, {"trigger": {"text": "expression", "start": 84, "end": 94}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 79, "end": 83}]}, {"trigger": {"text": "expression", "start": 105, "end": 115}, "arguments": [{"role": "Theme", "text": "CD25", "start": 100, "end": 104}]}, {"trigger": {"text": "expression", "start": 735, "end": 745}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 729, "end": 734}]}, {"trigger": {"text": "producing", "start": 1272, "end": 1281}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 1267, "end": 1271}]}, {"trigger": {"text": "expressing", "start": 1302, "end": 1312}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1296, "end": 1301}]}, {"trigger": {"text": "expression", "start": 1461, "end": 1471}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1445, "end": 1450}]}, {"trigger": {"text": "expression", "start": 1461, "end": 1471}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1455, "end": 1460}]}, {"trigger": {"text": "co-express", "start": 1645, "end": 1655}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1560, "end": 1565}]}, {"trigger": {"text": "co-express", "start": 1645, "end": 1655}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1570, "end": 1575}]}, {"trigger": {"text": "expression", "start": 1938, "end": 1948}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1932, "end": 1937}]}], "negative regulation": [{"trigger": {"text": "reduced", "start": 65, "end": 72}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 56, "end": 64}, {"role": "Theme", "text": "expression", "start": 105, "end": 115}]}, {"trigger": {"text": "reduced", "start": 65, "end": 72}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 56, "end": 64}, {"role": "Theme", "text": "expression", "start": 84, "end": 94}]}, {"trigger": {"text": "inhibited", "start": 148, "end": 157}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 129, "end": 133}, {"role": "Theme", "text": "induction", "start": 176, "end": 185}]}, {"trigger": {"text": "down-regulated", "start": 984, "end": 998}, "arguments": [{"role": "Theme", "text": "CD25", "start": 975, "end": 979}, {"role": "Cause", "text": "TGF-b", "start": 1002, "end": 1007}]}, {"trigger": {"text": "repressing", "start": 1850, "end": 1860}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 1801, "end": 1806}, {"role": "Theme", "text": "FOXP3", "start": 1861, "end": 1866}]}, {"trigger": {"text": "acts in vitro as an inhibitor", "start": 1899, "end": 1928}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 1894, "end": 1898}, {"role": "Theme", "text": "expression", "start": 1938, "end": 1948}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 176, "end": 185}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 158, "end": 166}, {"role": "Theme", "text": "FOXP3", "start": 189, "end": 194}]}, {"trigger": {"text": "induction", "start": 716, "end": 725}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 698, "end": 706}, {"role": "Theme", "text": "expression", "start": 735, "end": 745}]}, {"trigger": {"text": "caused", "start": 754, "end": 760}, "arguments": [{"role": "Theme", "text": "induction", "start": 716, "end": 725}]}, {"trigger": {"text": "pronounced", "start": 1075, "end": 1085}, "arguments": [{"role": "Theme", "text": "down-regulated", "start": 984, "end": 998}, {"role": "Cause", "text": "IL-4", "start": 1122, "end": 1126}]}, {"trigger": {"text": "demonstrated", "start": 1542, "end": 1554}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 1524, "end": 1528}, {"role": "Theme", "text": "co-express", "start": 1645, "end": 1655}]}, {"trigger": {"text": "demonstrated", "start": 1542, "end": 1554}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 1533, "end": 1541}, {"role": "Theme", "text": "co-express", "start": 1645, "end": 1655}]}, {"trigger": {"text": "acting", "start": 2004, "end": 2010}, "arguments": [{"role": "Theme", "text": "acts in vitro as an inhibitor", "start": 1899, "end": 1928}, {"role": "Cause", "text": "dependent", "start": 2074, "end": 2083}]}], "regulation": [{"trigger": {"text": "dependent", "start": 2074, "end": 2083}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 1894, "end": 1898}, {"role": "Theme", "text": "GATA3", "start": 2068, "end": 2073}]}]}}, "schema": []}
{"input": "GATA3 Is a Negative Regulator of FOXP3 Expression\nFOXP3 expression decreased once GATA3 expression is high; therefore, we hypothesized a potential role for GATA3 in repressing FOXP3. Besides GATA3's well-known positive effect on gene regulation, GATA3's repressive capabilities were previously shown to restrict Th1 commitment by inhibiting STAT4 expression [2,21], and therefore GATA3 prevents differentiation into Th1 cells. To investigate whether GATA3 can directly inhibit FOXP3 induction, we transduced GATA3 or a truncated GATA3 lacking the DNA-binding domain in human primary CD4+CD45RA+ T cells using a TAT-fused, recombinantly expressed GATA3. After transduction, the cells were activated with soluble anti-CD3/CD28 in the presence or absence of TGF-beta. TAT-GATA3 was successfully transduced in a homogeneous and dose-dependent manner into human CD4+ T cells (Figure 7A, upper panel). TAT-GATA3 reduced FOXP3 expression in a dose-dependent manner, whereas a DNA-binding domain truncated version (TAT-deltaDBD-GATA3) did not affect FOXP3 expression as compared with expression in untransduced cells (Figure 7A). In addition, we analyzed the inhibitory effect of GATA3 on FOXP3 in transgenic DO11.10 mice, constitutively overexpressing GATA3 under the control of the CD2 locus control region (DO11.10xCD2-GATA3). The thymic selection into the CD4 lineage is largely intact in DO11.10xCD2-GATA3 (RW Hendriks, unpublished data). These mice develop lymphomas at an older age, but signs of autoimmune disease were not described [22]. To investigate the effect of GATA3 on iTreg, CD4+CD62L+CD25- cells were isolated, activated with OVA in the presence or absence of TGF-beta, and Foxp3 expression was analyzed after 4 d. The naive CD4+CD25- cells were Foxp3- (unpublished data). As described for the human cells, TGF-beta dramatically up-regulated Foxp3 in the DO11.10 littermate control mice. In contrast, cells from the CD2-GATA3xDO11.10 mice showed dramatically reduced Foxp3 expression when activated with TGF-beta and OVA (Figure 7B). All mice produced similar amounts of TGF-beta; in addition, Smad7 was equally expressed [23] in T cells of both mice strains (Figure 7C), indicating intact TGF-beta signaling.\nTaken together, these results demonstrated a repressive role of IL-4-induced GATA3 transcription factor in the generation of iTreg cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Expression", "start": 39, "end": 49}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 33, "end": 38}]}, {"trigger": {"text": "expression", "start": 56, "end": 66}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 50, "end": 55}]}, {"trigger": {"text": "expression", "start": 88, "end": 98}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 82, "end": 87}]}, {"trigger": {"text": "expression", "start": 347, "end": 357}, "arguments": [{"role": "Theme", "text": "STAT4", "start": 341, "end": 346}]}, {"trigger": {"text": "expression", "start": 920, "end": 930}, "arguments": [{"role": 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49}]}, {"trigger": {"text": "decreased", "start": 67, "end": 76}, "arguments": [{"role": "Theme", "text": "expression", "start": 56, "end": 66}, {"role": "Cause", "text": "expression", "start": 88, "end": 98}]}, {"trigger": {"text": "repressing", "start": 165, "end": 175}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 176, "end": 181}]}, {"trigger": {"text": "inhibiting", "start": 330, "end": 340}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 246, "end": 251}, {"role": "Theme", "text": "expression", "start": 347, "end": 357}]}, {"trigger": {"text": "inhibit", "start": 469, "end": 476}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 450, "end": 455}, {"role": "Theme", "text": "induction", "start": 483, "end": 492}]}, {"trigger": {"text": "lacking", "start": 535, "end": 542}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 529, "end": 534}, {"role": "Site", "text": "DNA-binding domain", "start": 547, "end": 565}]}, {"trigger": {"text": "reduced", "start": 906, "end": 913}, "arguments": [{"role": "Cause", "text": "TAT-GATA3", "start": 896, "end": 905}, {"role": "Theme", "text": "expression", "start": 920, "end": 930}]}, {"trigger": {"text": "truncated", "start": 988, "end": 997}, "arguments": [{"role": "Site", "text": "DNA-binding domain", "start": 969, "end": 987}, {"role": "Theme", "text": "GATA3", "start": 1020, "end": 1025}]}, {"trigger": {"text": "inhibitory effect", "start": 1151, "end": 1168}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 1172, "end": 1177}, {"role": "Theme", "text": "FOXP3", "start": 1181, "end": 1186}]}, {"trigger": {"text": "reduced", "start": 1969, "end": 1976}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 1930, "end": 1935}, {"role": "Theme", "text": "expression", "start": 1983, "end": 1993}]}], "positive regulation": [{"trigger": {"text": "high", "start": 102, "end": 106}, "arguments": [{"role": "Theme", "text": "expression", "start": 88, "end": 98}]}, {"trigger": {"text": "induction", "start": 483, "end": 492}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 477, "end": 482}]}, {"trigger": {"text": "overexpressing", "start": 1230, "end": 1244}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1245, "end": 1250}]}, {"trigger": {"text": "up-regulated", "start": 1839, "end": 1851}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 1817, "end": 1825}, {"role": "Theme", "text": "Foxp3", "start": 1852, "end": 1857}]}, {"trigger": {"text": "activated", "start": 1999, "end": 2008}, "arguments": [{"role": "Theme", "text": "expression", "start": 1983, "end": 1993}, {"role": "Cause", "text": "TGF-beta", "start": 2014, "end": 2022}]}, {"trigger": {"text": "induced", "start": 2289, "end": 2296}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 2284, "end": 2288}, {"role": "Theme", "text": "GATA3", "start": 2297, "end": 2302}]}], "regulation": [{"trigger": {"text": "role", "start": 147, "end": 151}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 156, "end": 161}, {"role": "Theme", "text": "repressing", "start": 165, "end": 175}]}, {"trigger": {"text": "affect", "start": 1035, "end": 1041}, "arguments": [{"role": "Cause", "text": "truncated", "start": 988, "end": 997}, {"role": "Theme", "text": "expression", "start": 1048, "end": 1058}]}, {"trigger": {"text": "effect", "start": 1558, "end": 1564}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 1568, "end": 1573}, {"role": "Theme", "text": "expression", "start": 1690, "end": 1700}]}]}}, "schema": []}
{"input": "GATA3 Represses the FOXP3 Promoter\nTo investigate the molecular mechanism of GATA3-mediated repression of human FOXP3, the human FOXP3 promoter was studied and a palindromic binding site for GATA3 was discovered. The GATA-binding site is located 303 bp upstream from the transcription start site (TSS) [24]. This site is highly conserved between humans, mice, and rats (Figure S4) and may therefore play an important role in FOXP3 regulation. The functional relevance of this site was studied using a FOXP3-promoter construct [24]. We transfected human primary CD4+ T cells, in vitro differentiated Th2 cells, and Jurkat cells (Jurkat cells are known to constitutively express GATA3 [25,26]), and we measured FOXP3 promoter activity. The promoter was not active in the GATA3-expressing cell line Jurkat or in the in vitro-differentiated Th2 cells, whereas the construct was active in the CD4 cells, which express a lower amount of GATA3 (Figure 8A). Overexpression of GATA3 in naive T cells diminished luciferase activity of the FOXP3 promoter compared with the control vector (Figure 8B). To further address the function of the GATA3 site, we inserted a site-specific mutation deleting the GATA3-binding site. This mutation increased luciferase activity by 3-fold in memory CD4+CD45RO+ T cells, whereas no difference was observed in naive (GATA3-) CD4+CD45RA+ T cells, revealing a repressor activity of GATA3 on the FOXP3 promoter (Figure 8C). Furthermore GATA3 binds directly to the FOXP3 promoter as investigated by pull-down assay. HEK cells were transiently transfected with GATA3 or a control vector, and increasing amounts of lysates were incubated with oligonucleotides containing the GATA3 site of the FOXP3 promoter or a control oligonucleotide with a mutated GATA3-binding site. After the pull-down, GATA3 binding was detected by Western blot. Similarly, GATA3-expressing Th2 cells and iTreg cells were subjected to this approach. Only HEK cells overexpressing GATA3 and Th2 cells showed GATA3-binding activity (Figure 8D and 8E). These experiments demonstrated that GATA3 binds the palindromic FOXP3 promoter. To gain insights into the in vivo situation, we performed a chromatin immunoprecipitation (ChIP) using an anti-GATA3 antibody and showed that GATA3 binds to the FOXP3 promoter region in Th2 cells, but not in iTreg cells (Figure 7F). Taken together these data demonstrate that the GATA3-binding to the FOXP3 promoter is repressing FOXP3 expression.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 1463, "end": 1468}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1457, "end": 1462}, {"role": "Theme2", "text": "FOXP3", "start": 1485, "end": 1490}, {"role": "Site2", "text": "promoter", "start": 1491, "end": 1499}]}, {"trigger": {"text": "binding", "start": 1817, "end": 1824}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1811, "end": 1816}]}, {"trigger": {"text": "binding", "start": 2005, "end": 2012}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1999, "end": 2004}]}, {"trigger": {"text": "binds", "start": 2084, "end": 2089}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 2078, "end": 2083}, {"role": "Theme2", "text": "FOXP3", "start": 2106, "end": 2111}, {"role": "Site2", "text": "promoter", "start": 2112, "end": 2120}]}, {"trigger": {"text": "binds", "start": 2270, "end": 2275}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 2264, "end": 2269}, {"role": "Theme2", "text": "FOXP3", "start": 2283, "end": 2288}, {"role": "Site2", "text": "promoter region", "start": 2289, "end": 2304}]}, {"trigger": {"text": "binding", "start": 2408, "end": 2415}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 2402, "end": 2407}, {"role": "Theme2", "text": "FOXP3", "start": 2423, "end": 2428}]}], "gene expression": [{"trigger": {"text": "express", "start": 669, "end": 676}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 677, "end": 682}]}, {"trigger": {"text": "expressing", "start": 775, "end": 785}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 769, "end": 774}]}, {"trigger": {"text": "express", "start": 905, "end": 912}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 931, "end": 936}]}, {"trigger": {"text": "expressing", "start": 1872, "end": 1882}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1866, "end": 1871}]}, {"trigger": {"text": "overexpressing", "start": 1957, "end": 1971}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1972, "end": 1977}]}, {"trigger": {"text": "expression", "start": 2458, "end": 2468}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2452, "end": 2457}]}], "negative regulation": [{"trigger": {"text": "Represses", "start": 6, "end": 15}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 0, "end": 5}, {"role": "Theme", "text": "FOXP3", "start": 20, "end": 25}, {"role": "Site", "text": "Promoter", "start": 26, "end": 34}]}, {"trigger": {"text": "repression", "start": 92, "end": 102}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 77, "end": 82}, {"role": "Theme", "text": "FOXP3", "start": 112, "end": 117}]}, {"trigger": {"text": "deleting", "start": 1178, "end": 1186}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1029, "end": 1034}, {"role": "Site", "text": "GATA3-binding site", "start": 1191, "end": 1209}]}, {"trigger": {"text": "repressor", "start": 1382, "end": 1391}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 1404, "end": 1409}, {"role": "Theme", "text": "FOXP3", "start": 1417, "end": 1422}, {"role": "Site", "text": "promoter", "start": 1423, "end": 1431}]}, {"trigger": {"text": "repressing", "start": 2441, "end": 2451}, "arguments": [{"role": "Cause", "text": "binding", "start": 2408, "end": 2415}, {"role": "Theme", "text": "expression", "start": 2458, "end": 2468}]}], "positive regulation": [{"trigger": {"text": "active", "start": 874, "end": 880}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 709, "end": 714}, {"role": "Site", "text": "promoter", "start": 715, "end": 723}, {"role": "Cause", "text": "express", "start": 905, "end": 912}]}, {"trigger": {"text": "Overexpression", "start": 950, "end": 964}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 968, "end": 973}]}], "regulation": [{"trigger": {"text": "play an important role", "start": 399, "end": 421}, "arguments": [{"role": "Cause", "text": "FOXP3", "start": 129, "end": 134}, {"role": "CSite", "text": "GATA-binding site", "start": 217, "end": 234}, {"role": "Theme", "text": "regulation", "start": 431, "end": 441}]}, {"trigger": {"text": "regulation", "start": 431, "end": 441}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 425, "end": 430}]}]}}, "schema": []}
{"input": "The current study reveals that FOXP3 induction, an important step in iTreg commitment, is inhibited by GATA3, which is the key regulator for polarization toward Th2 cells. After differentiation, the effector Th2 cells become refractory to conversion into a FOXP3+ phenotype.\nIn accordance with other studies, we found that CD4+CD25- cells were able to up-regulate FOXP3 [12,27]. Already-committed cells such as memory T cells and Th1 cells showed only moderate and transient FOXP3 induction, which is not sufficient to change the phenotype toward a regulatory T cell profile. In contrast, naive T cells could efficiently up-regulate FOXP3 when treated with TGF-beta to induce iTreg cells [10,28-34], suggesting that FOXP3 plays an important role in the early differentiation process and may act in a way similar to that known for the Th1/Th2 decision factors T-bet and GATA3. This commitment is characterized by competitive expression of these factors [35,36], which we also observed in differentiating FOXP3+ iTreg cells including a phase of co-expression, which turns into nonoverlapping expression upon completed differentiation. In this competitive process TGF-beta appeared to be mandatory for the induction of FOXP3, possibly by keeping the expression of GATA3 and T-bet low [37,38]. In contrast, differentiating naive T cells in the absence of polarization factors (Th0) such as IL-4, IL-12, or TGF-beta showed only a transient FOXP3 expression and failed to generate a population of FOXP3-expressing cells, but GATA3 and T-bet were up-regulated (unpublished data). Interestingly, as we and others previously described, FOXP3-promoting factors, such as dexamethasone [39], CTLA-4 [40], and estrogens [41], are also known as inhibitors of GATA3 expression [42-45]. Therefore GATA3 not only induces differentiation into Th2 cells but also inhibits FOXP3 expression and commitment into iTreg cells.\nThe Th2 cytokine IL-4 but not IL-13 (unpublished data) was able to inhibit TGF-beta-mediated FOXP3 induction and therefore prevented conversion into the regulatory phenotype. To prove the inhibitory effect of IL-4 on inducible or natural Treg commitment in vivo, we treated mice with IL-4 and anti-IL-4. This has been shown to increase the effect of the cytokine in vivo [19]. Only the IL-4/IL-4 mAb complex resulted in a decrease of the amount of natural or inducible Treg (CD25+ and Foxp3+) cells 7 d after treatment. Our results suggest that IL-4 is only interfering with the differentiation of naive T cells into iTreg cells. But since a distinction of nTreg and iTreg cells is currently not possible, because iTreg cells also transiently express CD25 after activation, we cannot exclude that IL-4 may also inhibit Foxp3 expression in nTreg cells in vivo or that additional effects may contribute to the observed drop in Foxp3 expression. IL-4 has already been shown to negatively regulate the development of naive T cells into Th1 or the IL-17-producing T cells (Th17) [46,47]. Similar effects have been recently described for IL-6, which, combined with TGF-beta, inhibits the generation of iTreg cells and induces differentiation into the Th17 cells by an unknown mechanism [48,49]. Thus the polarization into iTreg cells is negatively regulated by the effector cytokines IL-4 and IL-6.\nIL-4 has been previously shown to induce the generation of FOXP3+ Treg cells out of CD4+CD25- [50]. In those experiments, the concentrations of IL-4 used were low, and as we also observed, IL-4 at low concentration slightly enhanced FOXP3 expression. Importantly, these concentrations were not sufficient to induce GATA3 expression. IL-4 may favor proliferation of nTreg cells [47] or directly regulate FOXP3 expression in a STAT-dependent fashion [51].\nSince IL-13 does not effectively reduce FOXP3 and fails to induce GATA3, we hypothesized that the IL-4-dependent inhibition of FOXP3 could be mediated by GATA3. In fact, GATA3-inducing IL-4 concentrations repressed TGF-beta-mediated FOXP3 expression, whereas IL-4 as well as TGF-beta signaling were intact. This result suggested a competitive mechanism between GATA3 and FOXP3 transcription factors in determining lineage commitment during the early phase of differentiation. Accordingly, we investigated naturally high GATA3-expressing cells and confirmed the absence of FOXP3. Protein transduction of GATA3 into naive T cells inhibited FOXP3 induction in human, differentiating, naive T cells. This inhibitory effect of GATA3 was further confirmed in BALB/c transgenic mice, expressing GATA3 in T cells (DO11.10:CD2-GATA3 transgenic mice). In line with the transient overexpression of GATA3 in human T cells, cells of these mice failed to induce FOXP3 expression upon exposure with antigen in the presence of TGF-beta. Strikingly, the DO11.10:CD2-GATA3 mice do have peripheral FOXP3+ cells, which however displayed a 10%-25% lower frequency compared to wild-type DO11.10 mice. Thus GATA3 restrains the development of certain Treg subsets, presumably the inducible, peripheral population and not those of thymic origin. Thymic T cells undergo a different maturation process, which may explain the insensitivity of nTreg to GATA3 overexpression [52]. In contrast to the Th2-differentiating and iTreg-inhibiting function of GATA3 in peripheral T cells, GATA3 acts in the thymus together with other transcription factors such as the Repressor of GATA3 (ROG) in the differentiation process toward CD8 cells [53,54] or participates in complex transcriptional feedback network to regulate sympathoadrenal differentiation [55]. Therefore, the role of GATA3 appears to be tissue specific and cannot be generalized.\nOur study demonstrates that GATA3 repressed FOXP3 expression directly by binding to the FOXP3 promoter region. A palindromic GATA-site is located 303 bp upstream of the TSS in a highly conserved region, which we have previously identified as the FOXP3 promoter [24]. Site-specific mutation of this site increased the activity of the promoter constructs, thus revealing the repressive nature of this GATA element in memory T cells, which naturally express GATA3, whereas no difference was seen in naive T cells, which do not express GATA3. This palindromic GATA element is bound by GATA3 protein as proven with pull-down experiments. Furthermore, it is shown by ChIP that GATA3 binds this element also in intact cells. It is known that GATA3 can induce transcription by chromatin remodeling [56], by directly transactivating promoters [36], or, as shown in the current study, acts as a repressor of gene expression [21,57-59]. Therefore keeping GATA3 expression low might be required to induce efficient FOXP3+ iTreg cell generation.\nThe molecular interactions enabling GATA3 to inhibit FOXP3 are not identified yet, but the GATA-binding site is located adjacent to positive, inducing sites, composed of AP-1-NFATc2 sites [24], and GATA3 may compete with the binding of AP-1/NFAT to the promoter (unpublished observations).\nIn summary, we demonstrated that FOXP3 is negatively regulated by cytokines such as IL-4. GATA3 acts as an inhibitor of FOXP3 expression in early T cell differentiation, as well as in differentiated Th2 cells by directly binding and repressing the FOXP3 promoter. We therefore describe a new mechanism of how Il-4 avoids tolerance induction by repressing FOXP3 expression. These findings will give new perspectives toward understanding molecular mechanisms of iTreg induction and thus pathways of peripheral tolerance induction, particularly in allergy and asthma.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 5731, "end": 5738}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 5686, "end": 5691}, {"role": "Theme2", "text": "FOXP3", "start": 5746, "end": 5751}, {"role": "Site2", "text": "promoter region", "start": 5752, "end": 5767}]}, {"trigger": {"text": "bound", "start": 6230, "end": 6235}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 6239, "end": 6244}]}, {"trigger": {"text": "binds", "start": 6335, "end": 6340}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 6329, "end": 6334}]}, {"trigger": {"text": "binding", "start": 6916, "end": 6923}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 6744, "end": 6749}, {"role": "Theme2", "text": "AP-1", "start": 6927, "end": 6931}, 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"arguments": [{"role": "Theme", "text": "FOXP3", "start": 1491, "end": 1496}]}, {"trigger": {"text": "expression", "start": 1751, "end": 1761}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1745, "end": 1750}]}, {"trigger": {"text": "expression", "start": 1859, "end": 1869}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1853, "end": 1858}]}, {"trigger": {"text": "express", "start": 2646, "end": 2653}, "arguments": [{"role": "Theme", "text": "CD25", "start": 2654, "end": 2658}]}, {"trigger": {"text": "expression", "start": 2728, "end": 2738}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2722, "end": 2727}]}, {"trigger": {"text": "expression", "start": 2834, "end": 2844}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2828, "end": 2833}]}, {"trigger": {"text": "producing", "start": 2952, "end": 2961}, "arguments": [{"role": "Theme", "text": "IL-17", "start": 2946, "end": 2951}]}, {"trigger": {"text": "expression", "start": 3535, "end": 3545}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 3529, "end": 3534}]}, {"trigger": {"text": "expression", "start": 3617, "end": 3627}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 3611, "end": 3616}]}, {"trigger": {"text": "expression", "start": 3705, "end": 3715}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 3699, "end": 3704}]}, {"trigger": {"text": "expression", "start": 3989, "end": 3999}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 3983, "end": 3988}]}, {"trigger": {"text": "expressing", "start": 4276, "end": 4286}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 4270, "end": 4275}]}, {"trigger": {"text": "expressing", "start": 4527, "end": 4537}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 4538, "end": 4543}]}, {"trigger": {"text": "expression", "start": 4704, "end": 4714}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 4698, "end": 4703}]}, {"trigger": {"text": "expression", "start": 5708, "end": 5718}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 5702, "end": 5707}]}, {"trigger": {"text": "express", "start": 6105, "end": 6112}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 6113, "end": 6118}]}, {"trigger": {"text": "express", "start": 6182, "end": 6189}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 6190, "end": 6195}]}, {"trigger": {"text": "expression", "start": 6608, "end": 6618}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 6602, "end": 6607}]}, {"trigger": {"text": "expression", "start": 7107, "end": 7117}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 7101, "end": 7106}]}, {"trigger": {"text": "expression", "start": 7342, "end": 7352}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 7336, "end": 7341}]}], "negative regulation": [{"trigger": {"text": "inhibited", "start": 90, "end": 99}, "arguments": [{"role": "Theme", "text": "induction", "start": 37, "end": 46}, {"role": "Cause", "text": "GATA3", "start": 103, "end": 108}]}, {"trigger": {"text": "low", "start": 1277, "end": 1280}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 1161, "end": 1169}, {"role": "Theme", "text": "expression", "start": 1247, "end": 1257}]}, {"trigger": {"text": "absence", "start": 1340, "end": 1347}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 1386, "end": 1390}]}, {"trigger": {"text": "absence", "start": 1340, "end": 1347}, "arguments": [{"role": "Theme", "text": "IL-12", "start": 1392, "end": 1397}]}, {"trigger": {"text": "absence", "start": 1340, "end": 1347}, "arguments": [{"role": "Theme", "text": "TGF-beta", "start": 1402, "end": 1410}]}, {"trigger": {"text": "inhibitors", "start": 1731, "end": 1741}, "arguments": [{"role": "Cause", "text": "CTLA-4", "start": 1680, "end": 1686}, {"role": "Theme", "text": "expression", "start": 1751, "end": 1761}]}, {"trigger": {"text": "inhibits", "start": 1844, "end": 1852}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 1781, "end": 1786}, {"role": "Theme", "text": "expression", "start": 1859, "end": 1869}]}, {"trigger": {"text": "inhibit", "start": 1970, "end": 1977}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 1920, "end": 1924}, {"role": "Theme", "text": "induction", "start": 2002, "end": 2011}]}, {"trigger": {"text": "inhibit", "start": 1970, "end": 1977}, "arguments": [{"role": "Cause", "text": "IL-13", "start": 1933, "end": 1938}, {"role": "Theme", "text": "induction", "start": 2002, "end": 2011}]}, {"trigger": {"text": "inhibit", "start": 2714, "end": 2721}, "arguments": [{"role": "Cause", "text": "express", "start": 2646, "end": 2653}, {"role": "Theme", "text": "expression", "start": 2728, "end": 2738}]}, {"trigger": {"text": "drop", "start": 2820, "end": 2824}, "arguments": [{"role": "Theme", "text": "expression", "start": 2834, "end": 2844}]}, {"trigger": {"text": "reduce", "start": 3783, "end": 3789}, "arguments": [{"role": "Cause", "text": "IL-13", "start": 3756, "end": 3761}, {"role": "Theme", "text": "FOXP3", "start": 3790, "end": 3795}]}, {"trigger": {"text": "inhibition", "start": 3863, "end": 3873}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 3848, "end": 3852}, {"role": "Theme", "text": "FOXP3", "start": 3877, "end": 3882}]}, {"trigger": {"text": "repressed", "start": 3955, "end": 3964}, "arguments": [{"role": "Cause", "text": "inducing", "start": 3926, "end": 3934}, {"role": "Theme", "text": "mediated", "start": 3974, "end": 3982}]}, {"trigger": {"text": "absence", "start": 4311, "end": 4318}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 4322, "end": 4327}]}, {"trigger": {"text": "inhibited", "start": 4378, "end": 4387}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 4353, "end": 4358}, {"role": "Theme", "text": "induction", "start": 4394, "end": 4403}]}, {"trigger": {"text": "failed", "start": 4681, "end": 4687}, "arguments": [{"role": "Cause", "text": "overexpression", "start": 4619, "end": 4633}, {"role": "Theme", "text": "induce", "start": 4691, "end": 4697}]}, {"trigger": {"text": "repressed", "start": 5692, "end": 5701}, "arguments": [{"role": "Theme", "text": "expression", "start": 5708, "end": 5718}, {"role": "Cause", "text": "binding", "start": 5731, "end": 5738}]}, {"trigger": {"text": "low", "start": 6619, "end": 6622}, "arguments": [{"role": "Theme", "text": "expression", "start": 6608, "end": 6618}]}, {"trigger": {"text": "inhibit", "start": 6736, "end": 6743}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 6727, "end": 6732}, {"role": "Theme", "text": "FOXP3", "start": 6744, "end": 6749}]}, {"trigger": {"text": "compete", "start": 6899, "end": 6906}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 6889, "end": 6894}, {"role": "Theme", "text": "binding", "start": 6916, "end": 6923}]}, {"trigger": {"text": "negatively regulated", "start": 7023, "end": 7043}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 7014, "end": 7019}, {"role": "Cause", "text": "IL-4", "start": 7065, "end": 7069}]}, {"trigger": {"text": "acts as an inhibitor", "start": 7077, "end": 7097}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 7071, "end": 7076}, {"role": "Theme", "text": "expression", "start": 7107, "end": 7117}]}, {"trigger": {"text": "repressing", "start": 7214, "end": 7224}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 7071, "end": 7076}, {"role": "Theme", "text": "FOXP3", "start": 7229, "end": 7234}, {"role": "Site", "text": "promoter", "start": 7235, "end": 7243}]}, {"trigger": {"text": "repressing", "start": 7325, "end": 7335}, "arguments": [{"role": "Cause", "text": "Il-4", "start": 7290, "end": 7294}, {"role": "Theme", "text": "expression", "start": 7342, "end": 7352}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 37, "end": 46}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 31, "end": 36}]}, {"trigger": {"text": "up-regulate", "start": 352, "end": 363}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 364, "end": 369}]}, {"trigger": {"text": "induction", "start": 481, "end": 490}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 475, "end": 480}]}, {"trigger": {"text": "up-regulate", "start": 621, "end": 632}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 633, "end": 638}, {"role": "Cause", "text": "TGF-beta", "start": 657, "end": 665}]}, {"trigger": {"text": "up-regulated", "start": 1540, "end": 1552}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1519, "end": 1524}]}, {"trigger": {"text": "up-regulated", "start": 1540, "end": 1552}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 1529, "end": 1534}]}, {"trigger": {"text": "promoting", "start": 1633, "end": 1642}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1627, "end": 1632}, {"role": "Cause", "text": "CTLA-4", "start": 1680, "end": 1686}]}, {"trigger": {"text": "induction", "start": 2002, "end": 2011}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 1978, "end": 1986}, {"role": "Theme", "text": "FOXP3", "start": 1996, "end": 2001}]}, {"trigger": {"text": "contribute", "start": 2793, "end": 2803}, "arguments": [{"role": "Theme", "text": "drop", "start": 2820, "end": 2824}]}, {"trigger": {"text": "enhanced", "start": 3520, "end": 3528}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 3485, "end": 3489}, {"role": "Theme", "text": "expression", "start": 3535, "end": 3545}]}, {"trigger": {"text": "induce", "start": 3604, "end": 3610}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 3485, "end": 3489}, {"role": "Theme", "text": "expression", "start": 3617, "end": 3627}]}, {"trigger": {"text": "induce", "start": 3809, "end": 3815}, "arguments": [{"role": "Cause", "text": "IL-13", "start": 3756, "end": 3761}, {"role": "Theme", "text": "GATA3", "start": 3816, "end": 3821}]}, {"trigger": {"text": "mediated", "start": 3892, "end": 3900}, "arguments": [{"role": "Theme", "text": "inhibition", "start": 3863, "end": 3873}, {"role": "Cause", "text": "GATA3", "start": 3904, "end": 3909}]}, {"trigger": {"text": "inducing", "start": 3926, "end": 3934}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 3920, "end": 3925}, {"role": "Theme", "text": "IL-4", "start": 3935, "end": 3939}]}, {"trigger": {"text": "mediated", "start": 3974, "end": 3982}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 3965, "end": 3973}, {"role": "Theme", "text": "expression", "start": 3989, "end": 3999}]}, {"trigger": {"text": "high", "start": 4265, "end": 4269}, "arguments": [{"role": "Theme", "text": "expressing", "start": 4276, "end": 4286}]}, {"trigger": {"text": "induction", "start": 4394, "end": 4403}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 4388, "end": 4393}]}, {"trigger": {"text": "overexpression", "start": 4619, "end": 4633}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 4637, "end": 4642}]}, {"trigger": {"text": "induce", "start": 4691, "end": 4697}, "arguments": [{"role": "Theme", "text": "expression", "start": 4704, "end": 4714}]}, {"trigger": {"text": "overexpression", "start": 5180, "end": 5194}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 5174, "end": 5179}]}], "regulation": [{"trigger": {"text": "regulate", "start": 3690, "end": 3698}, "arguments": [{"role": "Theme", "text": "expression", "start": 3705, "end": 3715}]}]}}, "schema": []}
{"input": "Mice.\nNormal B6 mice were purchased from the Jackson Laboratories (Bar Harbor, Maine). Transgenic DO11.10 mice, expressing a T cell receptor for OVA323-339 peptide in the context of H-2d, were backcrossed with mice expressing GATA-3, driven by the human CD2 locus control region (CD2-GATA3) [22], resulting in DO11.10xCD2-GATA3 mice. Mice used for experiments were backcrossed on a BALB/c background for a minimum of eight generations and used at an age of 8-12 wk. Mice were housed under specific pathogen-free conditions and all animal studies were performed according to institutional and state guidelines.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressing", "start": 215, "end": 225}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 226, "end": 232}]}]}}, "schema": []}
{"input": "Isolation of CD4+ T cells.\nCD4+ T cells were isolated from blood of healthy human volunteers using the anti-CD4 magnetic beads (Dynal, Hamburg, Germany) as previously described [60]. The purity of CD4+ T cells was initially tested by FACS and was >= 95%. Monoclonality of T cell clones was confirmed by TCR-chain mapping and was identified to be Vbeta8 positive. The clones were characterized by high IL-4 secretion.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "secretion", "start": 406, "end": 415}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 401, "end": 405}]}], "positive regulation": [{"trigger": {"text": "high", "start": 396, "end": 400}, "arguments": [{"role": "Theme", "text": "secretion", "start": 406, "end": 415}]}]}}, "schema": []}
{"input": "Quantitative real-time PCR.\nThe PCR primers and probes were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems) as follows: FOXP3 forward primer 5'-GAA ACA GCA CAT TCC CAG AGT TC-3'; FOXP3 reverse primer 5'-ATG GCC CAG CGG ATG AG-3'; EF-1alpha forward primer and reverse primer as described [61]; GATA3 forward primer 5'-GCG GGC TCT ATC ACA AAA TGA-3' and rwd 5'-GCT CTC CTG GCT GCA GAC AGC-3'. The prepared cDNAs were amplified using SYBR-PCR mastermix (Biorad) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).\nQuantitative PCR of murine samples was performed with Brilliant SYBR Green QPCR master mix (Stratagene) and the following primers: Ubiquitin C, 5'- AGG TCA AAC AGG AAG ACA GAC GTA-3' and 5'-TCACACCCAAGAACAAGCACA-3'; Smad-7, 5'-GAA ACC GGG GGA ACG AAT TAT-3' and 5'-CGC GAG TCT TCT CCT CCC A-3'; TGF-ss1, 5'-TGA CGT CAC TGG AGT TGT ACG G-3' and 5'-GGT TCA TGT CAT GGA TGG TGC-3'. Primer pairs were evaluated for integrity by analysis of the amplification plot, dissociation curves, and efficiency of PCR amplification. PCR conditions were 10 min at 95 degreesC, followed by 40 cycles of 15 s at 95 degreesC and 60 degreesC for 1 min using an 7300 real-time PCR system (Applied Biosystems). PCR amplification of the housekeeping gene encoding ubiquitin C was performed during each run for each sample to allow normalization between samples. Relative quantification and calculation of the range of confidence was performed using the comparative deltadeltaCT method.", "output": {"json_structures": {}}, "schema": []}
{"input": "Inducible murine Treg culture.\nNaive CD4+ T cells (CD4+, CD62L+, and CD25-) were isolated from pooled lymph nodes and spleens by FACS (FACS Aria, BD Biosciences). 5 x 105 T cells were co-cultured with 2.5 x 104 bone marrow-derived dendritic cells [62] and 0.01 mug/ml OVA323-339 peptide (Ansynth) in the presence or absence of 20-ng/mul rhTGF-ss1 (Peprotech) in 48-well plates. After 4 d, cells were harvested and analyzed for intracellular FOXP3 expression by FACS or gene expression by quantitative RT-PCR.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 447, "end": 457}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 441, "end": 446}]}]}}, "schema": []}
{"input": "In vitro T cell differentiation.\nCD4+ CD45RA+ magnetically-sorted (CD45RO depletion, MACS, according to the protocol of the manufacturer) cells were stimulated with immobilized plate-bound anti-CD3 (1 mug/ml, Okt3, IgG1) and anti-CD28 (2 mug/ml) in Th1 conditions: 25 ng/ml IL-12, 5 mug/ml anti-IL-4 (R&D systems); in Th2 conditions: 25 ng/ml IL-4, 5 mug/ml anti-IFN-gamma, 5 mug/ml anti-IL-12 (R&D systems); or in Treg conditions: 10 ng/ml TGF-beta, 5 mug/ml anti-IFN-gamma, 5 mug/ml anti-IL-12, 5 mug/ml anti-IL-4. Proliferating cells were expanded in medium containing IL-2 (30 ng/ml).", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "depletion", "start": 74, "end": 83}, "arguments": [{"role": "Theme", "text": "CD45RO", "start": 67, "end": 73}]}]}}, "schema": []}
{"input": "Cloning of the FOXP3 promoter and construction of mutant constructs.\nThe FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate the pGL3 FOXP3 -511/+176 [24]. Site-directed mutagenesis in the FOXP3 promoter region were introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions. The following primer and its complementary strand were used: GTT TCT CAT GAG CCC TAT TAA GTC ATT CTT ACC TCT CAC CTC TGT GGT GA.", "output": {"json_structures": {}}, "schema": []}
{"input": "Transfections and reporter gene assays.\nT cells were rested in serum-free AIM-V medium (Life Technologies) overnight. 3.5 mug of the FOXP3 promoter luciferase reporter vector and 0.5 mug phRL-TK were added to 3 x 106 CD4+ T cells resuspended in 100 muL of Nucleofector solution (Amaxa Biosystems) and electroporated using the U-15 program of the Nucleofector. After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega Biotech) according to the manufacturer's instructions. Data were normalized by the activity of renilla luciferase.", "output": {"json_structures": {}}, "schema": []}
{"input": "RNA isolation and cDNA synthesis.\nRNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Reverse transcription of human samples was performed with TaqMan reverse-transcription reagents (Applied Biosystems) with random hexamers according to the manufacturer's protocol.", "output": {"json_structures": {}}, "schema": []}
{"input": "Recombinant TAT proteins.\nThe cDNAs encoding GATA3 protein or the truncated GATA3 (lacking the two zinc fingers) were cloned in frame into an expression vector along with the TAT sequence as previously described [63]. Proteins were expressed in BL21 Star (DE3)pLysS (Invitrogen) and lysates were purified by Ni2+-chelate column chromatography. Both TAT-linked proteins were more than 95% pure, based on Coomassie blue staining of sodium disulfate acrylamide gels.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "lacking", "start": 83, "end": 90}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 76, "end": 81}, {"role": "Site", "text": "two zinc fingers", "start": 95, "end": 111}]}]}}, "schema": []}
{"input": "TAT-GATA3 transduction.\nCD4+CD45RA+ cells were cultured in AIMV medium and transduced with 20 nM, 10 nM, or 500 nM of full-length or truncated GATA3 over the course of 4 h. After 4 h, the cells were washed and activated with soluble anti-CD3 and anti-CD28 and TGF-beta (10 ng/ml). Each day, the TAT proteins were freshly added to the medium. FOXP3 expression was measured after 5 d by intracellular staining.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 348, "end": 358}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 342, "end": 347}]}]}}, "schema": []}
{"input": "Intracellular cytokine staining.\nT cells were stimulated with 2 x 10-7 M PMA and 1 mug/ml of ionomycin (Sigma Chemicals) for 4 h. The following mAb was used: anti-IL-4-PE (8D4-8, BD). Matched isotype controls were used at the same protein concentration as the respective antibodies. Four-color FACS was performed using an EPICS XL-MCL (Beckman Coulter) using the software Expo32 version for data acquisition and evaluation.", "output": {"json_structures": {}}, "schema": []}
{"input": "Flow cytometry.\nFor analysis of FOXP3 expression at the single-cell level, cells were first stained with the monoclonal antibody CD25 (Beckman Coulter), and after fixation and permeabilization, cells were incubated with PE-conjugated monoclonal antibody PCH101 (anti-human FOXP3; eBioscience) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL). For cell surface marker staining, cells were incubated for 20 min at 4degreesC in staining buffer with the following antibodies: anti-CD152-PE (CTLA-4; BD), anti-PD-1 (eBiosciences), anti-GITR (R & D Systems), anti-CD69 (Beckman Coulter), anti-CD103 (DakoCytomation), anti-CD62L (Beckman Coulter), or anti-HLA-DR (Beckman Coulter). The controls were FITC, PE, or ECD-conjugated mouse IgG1 or rat IgG2a. For staining of mouse cells, the following mAbs from BD Biosciences were used following standard techniques as described above: anti-CD3, anti-CD4, and anti-CD25. Anti-FcgammaRII/III antibody (2.4G2, ATCC) was included in all stainings to reduce nonspecific antibody binding. To isolate naive murine CD4 T cells from murine DO11.10 or DO11.10xCD2-GATA3 T cells, cells were stained with anti-CD25-FITC, anti-CD62L-PE, and anti-CD4-APC prior to sorting. Dead cells were excluded with 4'',6-Diamidino-2-phenylindole (DAPI). To analyze murine Foxp3 expression in inducible Treg cultures, cells were stained intracellularly with anti-Foxp3-PE according to manufacturer's instruction, in conjunction with anti-CD4-APC and LIVE/DEAD fixable dead cell stain kit (Invitrogen) to discriminate live cells. All monoclonal antibodies for murine cell stainings were purchased from eBioscience or BD Biosciences.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 38, "end": 48}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 32, "end": 37}]}, {"trigger": {"text": "expression", "start": 1323, "end": 1333}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1317, "end": 1322}]}]}}, "schema": []}
{"input": "Western blotting.\nFor FOXP3 analysis on the protein level, 1 x 106 CD4+CD25- cells were lysed and loaded next to a protein-mass ladder (Magicmark, Invitrogen) on a NuPAGE 4-12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Life Science). Unspecific binding was blocked with BSA, and the membranes were subsequently incubated with an 1:200 dilution of goat anti-FOXP3 in blocking buffer (Abcam) overnight at 4 degreesC. The blots were developed using an anti-goat HRP-labeled mAb (Amersham Biosciences) and visualized with a LAS 1000 camera (Fuji). Membranes were incubated in stripping buffer and re-blocked for 1 h. The membranes were re-probed using anti-GATA3 (HG3-31; Santa Cruz Biotechnology), anti-T-bet (4B10, Santa Cruz Biotechnology), anti-GAPDH (6C5, Ambion), anti-phospho-SMAD2 (138D4), anti-phospho-STAT6 (5A4), and anti-STAT6 (Cell Signaling Technology),", "output": {"json_structures": {}}, "schema": []}
{"input": "Administration of cytokines and antibodies in vivo.\nAge- and gender-matched normal B6 mice received every other day intraperitoneal (ip) injections of PBS, 1.5 mug rmIL-4, 50 mug anti-IL-4 mAb (11B11 or MAB404), or a mixture of 1.5 mug rmIL-4 plus 50 mug anti-IL-4 mAb (11B11 or MAB404) for 7 d. Thereafter, spleen and lymph node cells were analyzed by flow cytometry for CD3, CD4, and CD25 expression. The anti-mouse IL-4 mAb MAB404 was obtained from R&D Systems, the second anti-mouse IL-4 mAb 11B11 was purchased from eBioscience.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 391, "end": 401}, "arguments": [{"role": "Theme", "text": "CD3", "start": 372, "end": 375}]}, {"trigger": {"text": "expression", "start": 391, "end": 401}, "arguments": [{"role": "Theme", "text": "CD4", "start": 377, "end": 380}]}, {"trigger": {"text": "expression", "start": 391, "end": 401}, "arguments": [{"role": "Theme", "text": "CD25", "start": 386, "end": 390}]}]}}, "schema": []}
{"input": "ChIP.\nChIP analysis was performed according to the manufacturer's protocol (Upstate Biotechnology) with the following modifications. iTreg and Th2 cells were fixed with 1% formaldehyde for 10 min at room temperature. The chromatin was sheared to 200-1000 bp of length by sonication with five pulses of 10 s at 30% power (Bandelin). The chromatin was pre-cleared for 2 h with normal mouse IgG beads and then incubated with anti-GATA3-agarose beads (HG3-31; Santa Cruz Biotechnology) for 2 h. Washing and elution buffers were used according to the protocol of Upstate Biotechnology. Crosslinks were reversed by incubation at 65 degreesC for 4 h in the presence of 0.2 M NaCl, and the DNA was purified by phenol/chloroform extraction. The amount of DNA was determined by conventional PCR. The PCR addressed for the FOXP3 promoter region -246 to -511 and was performed using the following primers: 5'-gtgccctttacgagt catctg-3' and 5'-gtgccctttacgagtcatctg-3'. The PCR products were visualized using an ethidium bromide gel.", "output": {"json_structures": {}}, "schema": []}
{"input": "Pull-down assay.\nCD4+ T cells were stimulated with PMA and ionomycin for 2 h at 37degreesC. The cells were pelleted, resuspended in buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, protease inhibitors [Sigma]. and 0.1% NP-40) and lysed on ice for 15 min. Insoluble material was removed by centrifugation. The supernatant was diluted 1:3 with buffer D (as buffer C, but without NaCl). The lysates were incubated with 10 mug of poly(dI-dC) (Sigma) and 70 mul of streptavidin-agarose (Amersham Biosciences) carrying biotinylated oligonucleotides, for 3 h at 4 degreesC. The beads were washed twice with buffer C:D (1:3) and resuspended in DTT-containing loading buffer (NuPAGE; Invitrogen), heated to 70 degreesC for 10 min, and the eluants on a NuPAGE 4-12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Biosciences) and detected using an anti-GATA3 mAb (Santa Cruz Biotechnology). Accumulated signals were analyzed using AIDA software (Raytest).", "output": {"json_structures": {}}, "schema": []}
{"input": "Th2 Cells Cannot Induce FOXP3 Expression\n(A) Human T cells were activated with plate-bound anti-CD3/CD28 with or without TGF-beta as indicated on the x-axis of (B). Cells were harvested after 5 days and FOXP3 mRNA was quantified by real-time PCR. Bars show the mean +/- SD of 4 independent experiments.\n(B) In vitro differentiated Th1, Th2, or iTreg cells were activated with anti-CD3/CD28, TGF-beta, or anti-IL-4 as indicated. The phenotype of these cells was confirmed by FACS and proliferation analysis (Figure S1). Bars show the mean +/- SD of four independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Expression", "start": 30, "end": 40}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 24, "end": 29}]}], "positive regulation": [{"trigger": {"text": "Induce", "start": 17, "end": 23}, "arguments": [{"role": "Theme", "text": "Expression", "start": 30, "end": 40}]}]}}, "schema": []}
{"input": "Th2 Cells Do Not Express FOXP3\n(A) Intracellular FACS analysis of FOXP3 expression in Th1, Th2, or iTreg-differentiated cells (two rounds, phenotype see Figure S1), rested or activated, with or without TGF-beta. FOXP3 expression was measured after 5 d in culture. The dot blots are representative of three independent experiments.\n(B) Shows the same experimental setup, but naturally occurring Th2 cells were analyzed. Data are representative of three independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Express", "start": 17, "end": 24}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 25, "end": 30}]}, {"trigger": {"text": "expression", "start": 72, "end": 82}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 66, "end": 71}]}, {"trigger": {"text": "expression", "start": 218, "end": 228}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 212, "end": 217}]}]}}, "schema": []}
{"input": "Th2- or IL-4-Producing Cells Lack FOXP3\n(A) FACS analysis of intracellular FOXP3 and IL-4 expression following PMA/Ionomycin stimulation. CD4+ T cells were gated on the basis of CD45RO and CD25 surface expression (upper panel), and gated cells are shown below for the CD45RO+CD25- (A, left panel), the CD45RO+CD25+ (right panel), and the CD45RO-CD25- subsets (central panel). A statistical analysis of eight independent donors after subtraction of the isotype control are shown in (B). The dotted gray line indicates the IC background level. The error bars show the error of the mean. (C) Similarly, a Th2 clone (BR8), CRTH2+ Th2 cells, IL-4-secreting cells, and memory T cells (CD45RO) were stained for FOXP3 and IL-4. Data are representative of three independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Producing", "start": 13, "end": 22}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 8, "end": 12}]}, {"trigger": {"text": "expression", "start": 90, "end": 100}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 75, "end": 80}]}, {"trigger": {"text": "expression", "start": 90, "end": 100}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 85, "end": 89}]}, {"trigger": {"text": "expression", "start": 202, "end": 212}, "arguments": [{"role": "Theme", "text": "CD45RO", "start": 178, "end": 184}]}, {"trigger": {"text": "expression", "start": 202, "end": 212}, "arguments": [{"role": "Theme", "text": "CD25", "start": 189, "end": 193}]}, {"trigger": {"text": "secreting", "start": 642, "end": 651}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 637, "end": 641}]}, {"trigger": {"text": "stained", "start": 692, "end": 699}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 704, "end": 709}]}, {"trigger": {"text": "stained", "start": 692, "end": 699}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 714, "end": 718}]}], "negative regulation": [{"trigger": {"text": "Lack", "start": 29, "end": 33}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 34, "end": 39}]}]}}, "schema": []}
{"input": "FOXP3 Induction During the Differentiation Process\n(A) Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-beta (5 ng/ml) or IL-4 (25 ng/ml). The cells were harvested at different time points, and mRNA was quantified by real-time PCR for FOXP3 and GATA3 expression. Bars show the mean +/- SD of three independent experiments.\n(B) Intracellular GATA3 and FOXP3 staining is shown after exposure of CD4+CD45RA+ T cells to differentiating conditions as in part A of the figure. Data are representative of three independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 297, "end": 307}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 281, "end": 286}]}, {"trigger": {"text": "expression", "start": 297, "end": 307}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 291, "end": 296}]}, {"trigger": {"text": "staining", "start": 403, "end": 411}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 387, "end": 392}]}, {"trigger": {"text": "staining", "start": 403, "end": 411}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 397, "end": 402}]}], "positive regulation": [{"trigger": {"text": "Induction", "start": 6, "end": 15}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 0, "end": 5}]}]}}, "schema": []}
{"input": "Effect of IL-4 on FOXP3 Induction\n(A) A statistical analysis was performed with six donors on day 5 (TGF-beta (10 ng/ml) and with or without IL-4 (100 ng/ml)); Shown is the mean, and error bars indicated the SD of six donors. Statistical analysis was performed using the Dunnett test. Statistical significance is indicated by asterisks (*p <= 0.05, **p <= 0.01, Dunnett).\n(B) CD4+CD45RA+ cells were activated in the presence of a constant concentration of TGF-beta (5 ng/ml) with an increasing concentration of IL-4, as indicated. Cells were harvested for mRNA quantification after 5 d.\n(C) CD4+CD45RA+ cells were stimulated in vitro with plate-bound anti-CD3/CD28, TGF-beta (10ng/ml), and IL-4 (100 ng/ml) as indicated. After 1 h, cell lysates were prepared and analyzed by Western blot for phosphorylated SMAD2 and STAT6. Total STAT6 and GAPDH served as internal control.\n(D) Intracellular GATA3 and FOXP3 staining are shown after exposure of CD4+CD45RA+ T cells to IL-4 as described for panel B. Data are representative of three independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "staining", "start": 908, "end": 916}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 892, "end": 897}]}, {"trigger": {"text": "staining", "start": 908, "end": 916}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 902, "end": 907}]}], "phosphorylation": [{"trigger": {"text": "phosphorylated", "start": 792, "end": 806}, "arguments": [{"role": "Theme", "text": "SMAD2", "start": 807, "end": 812}]}, {"trigger": {"text": "phosphorylated", "start": 792, "end": 806}, "arguments": [{"role": "Theme", "text": "STAT6", "start": 817, "end": 822}]}], "positive regulation": [{"trigger": {"text": "Induction", "start": 24, "end": 33}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 18, "end": 23}]}, {"trigger": {"text": "increasing", "start": 483, "end": 493}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 511, "end": 515}]}], "regulation": [{"trigger": {"text": "Effect", "start": 0, "end": 6}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 10, "end": 14}, {"role": "Theme", "text": "Induction", "start": 24, "end": 33}]}]}}, "schema": []}
{"input": "IL-4 Inhibits TGF-beta-Mediated iTreg Commitment\nCFSE-labeled CD4+CD45RA+ cells were activated with plate-bound anti-CD3/CD28, TGF-beta, and IL-4, as indicated. After 5 d, cells were analyzed by flow cytometry (A) and results of six independent experiments are shown in the bar graph below (B). Statistical significance (one-way Anova, Newman-Keuls) is indicated by asterisks (**p <= 0.01, ***p <= 0.001). (C) Kinetic analysis of intracellular GATA3 and FOXP3 staining is shown in panel C following exposure of CD4+CD45RA+ T cells to anti-CD3/28, IL-4 and TGF-beta. Data are representative of three independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "staining", "start": 460, "end": 468}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 444, "end": 449}]}, {"trigger": {"text": "staining", "start": 460, "end": 468}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 454, "end": 459}]}]}}, "schema": []}
{"input": "GATA3 Acts as a Negative Regulator of FOXP3 Expression\n(A) Human naive CD4+CDRA+ T cells were transduced with 0, 20, 100, and 500 nM of TAT-GATA3 protein, and intracellular presence of GATA3 was analyzed using FACS following anti-CD3/CD28 activation of the cells. GFP-positive cells were gated and analyzed for intracellular FOXP3 expression following a 2-d incubation period (lower panel). Data are representative of four independent experiments.\n(B) CD4+CD25- T cells were isolated from D011.10 and D011.10xCD2-GATA3 mice and treated with OVA and TGF-beta for 96 h. Surface CD4 and intracellular FOXP3 were measured by FACS. These data are representative of three independent experiments.\n(C) The cells treated as in (B) were harvested and mRNA was quantified by real-time PCR for SMAD7 and TGF-beta expression. Bars show the mean +/- SD of three independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "presence", "start": 173, "end": 181}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 185, "end": 190}]}, {"trigger": {"text": "expression", "start": 331, "end": 341}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 325, "end": 330}]}, {"trigger": {"text": "expression", "start": 802, "end": 812}, "arguments": [{"role": "Theme", "text": "SMAD7", "start": 783, "end": 788}]}, {"trigger": {"text": "expression", "start": 802, "end": 812}, "arguments": [{"role": "Theme", "text": "TGF-beta", "start": 793, "end": 801}]}]}}, "schema": []}
{"input": "GATA3 Represses the Human FOXP3 Promoter\n(A) Jurkat, Th2 cells, and human primary CD4 cells were transfected with an empty vector (pGL3 basic) or a vector containing the putative FOXP3 promoter region fused to the luciferase reporter gene. Bars show the mean +/- SD of arbitrary light units normalized for renilla luciferase of four independent experiments; samples were measured in triplicates.\n(B) Naive CD4 T cells were transfected with the FOXP3 promoter reporter construct together with a GATA3 expression vector or an empty vector. Bars show the mean +/- SD of three independent experiments.\n(C) Naive (left panel) or memory (right panel) CD4 T cells were transfected with wild-type or a GATA3 mutated 511-FOXP3 promoter reporter construct and activated with PMA and ionomycin. Bars show the mean +/- SD of arbitrary light units normalized for renilla luciferase of eight independent experiments; samples were measured in triplicates.\n(D) Nuclear extracts were prepared from HEK cells transfected with GATA3 or an empty vector, (E) Th1, Th2, or iTreg cells and binding factors precipitated using biotinylated oligonucleotides. The oligonucleotides-transcription factor complexes were separated on a SDS-PAGE gel. The amounts of GATA3 protein in the precipitates were assessed by immunoblotting with anti-GATA3 mAb. Total nuclear extracts were also run as controls. Data are representative of three different experiments.\n(F) iTreg or Th2 cells were analyzed by ChIP for GATA3 binding to the FOXP3 promoter. The \"input\" represents PCR amplification of the total sample, which was not subjected to any precipitation. Results are representative of three independent experiments.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 1482, "end": 1489}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1476, "end": 1481}, {"role": "Theme2", "text": "FOXP3", "start": 1497, "end": 1502}, {"role": "Site2", "text": "promoter", "start": 1503, "end": 1511}]}], "gene expression": [{"trigger": {"text": "expression", "start": 500, "end": 510}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 494, "end": 499}]}, {"trigger": {"text": "amounts", "start": 1223, "end": 1230}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1234, "end": 1239}]}], "negative regulation": [{"trigger": {"text": "Represses", "start": 6, "end": 15}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 0, "end": 5}, {"role": "Theme", "text": "FOXP3", "start": 26, "end": 31}, {"role": "Site", "text": "Promoter", "start": 32, "end": 40}]}]}}, "schema": []}
{"input": "Suppression of GATA-3 Nuclear Import and Phosphorylation: A Novel Mechanism of Corticosteroid Action in Allergic Disease\nBackground\nGATA-3 plays a critical role in regulating the expression of the cytokines interleukin (IL)-4, IL-5, and IL-13 from T helper-2 (Th2) cells and therefore is a key mediator of allergic diseases. Corticosteroids are highly effective in suppressing allergic inflammation, but their effects on GATA-3 are unknown. We investigated the effect of the corticosteroid fluticasone propionate on GATA-3 regulation in human T-lymphocytes in vitro and in vivo.\nMethods and Findings\nIn a T lymphocyte cell line (HuT-78) and peripheral blood mononuclear cells stimulated by anti-CD3 and anti-CD28 in vitro we demonstrated that fluticasone inhibits nuclear translocation of GATA-3 and expression of Th2 cytokines via a mechanism independent of nuclear factor-kappaB and is due, in part, to competition between GATA-3 and the ligand-activated glucocorticoid receptor for nuclear transport through the nuclear importer importin-alpha. In addition, fluticasone induces the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), the endogenous inhibitor of p38 MAPK, which is necessary for GATA-3 nuclear translocation. These inhibitory effects of fluticasone are rapid, potent, and prolonged. We also demonstrated that inhaled fluticasone inhibits GATA-3 nuclear translocation in peripheral blood lymphocytes of patients with asthma in vivo.\nConclusions\nCorticosteroids have a potent inhibitory effect on GATA-3 via two interacting mechanisms that potently suppress Th2 cytokine expression. This novel mechanism of action of corticosteroids may account for the striking clinical efficacy of corticosteroids in the treatment of allergic diseases.\nPlease see later in the article for Editors' Summary", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 179, "end": 189}, "arguments": [{"role": "Theme", "text": "interleukin (IL)-4", "start": 207, "end": 225}]}, {"trigger": {"text": "expression", "start": 179, "end": 189}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 227, "end": 231}]}, {"trigger": {"text": "expression", "start": 179, "end": 189}, "arguments": [{"role": "Theme", "text": "IL-13", "start": 237, "end": 242}]}, {"trigger": {"text": "expression", "start": 1085, "end": 1095}, "arguments": [{"role": "Theme", "text": "mitogen-activated protein kinase (MAPK) phosphatase-1", "start": 1099, "end": 1152}]}], "localization": [{"trigger": {"text": "Import", "start": 30, "end": 36}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 15, "end": 21}, {"role": "ToLoc", "text": "Nuclear", "start": 22, "end": 29}]}, {"trigger": {"text": "translocation", "start": 772, "end": 785}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 764, "end": 771}, {"role": "Theme", "text": "GATA-3", "start": 789, "end": 795}]}, {"trigger": {"text": "transport", "start": 993, "end": 1002}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 925, "end": 931}, {"role": "ToLoc", "text": "nuclear", "start": 985, "end": 992}]}, {"trigger": {"text": "transport", "start": 993, "end": 1002}, "arguments": [{"role": "Theme", "text": "glucocorticoid receptor", "start": 957, "end": 980}, {"role": "ToLoc", "text": "nuclear", "start": 985, "end": 992}]}, {"trigger": {"text": "translocation", "start": 1238, "end": 1251}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1223, "end": 1229}, {"role": "ToLoc", "text": "nuclear", "start": 1230, "end": 1237}]}, {"trigger": {"text": "translocation", "start": 1397, "end": 1410}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1382, "end": 1388}, {"role": "ToLoc", "text": "nuclear", "start": 1389, "end": 1396}]}], "negative regulation": [{"trigger": {"text": "Suppression", "start": 0, "end": 11}, "arguments": [{"role": "Theme", "text": "Import", "start": 30, "end": 36}]}, {"trigger": {"text": "Suppression", "start": 0, "end": 11}, "arguments": [{"role": "Theme", "text": "Phosphorylation", "start": 41, "end": 56}]}, {"trigger": {"text": "inhibits", "start": 755, "end": 763}, "arguments": [{"role": "Theme", "text": "translocation", "start": 772, "end": 785}]}, {"trigger": {"text": "competition", "start": 905, "end": 916}, "arguments": [{"role": "Theme", "text": "transport", "start": 993, "end": 1002}]}, {"trigger": {"text": "inhibits", "start": 1373, "end": 1381}, "arguments": [{"role": "Theme", "text": "translocation", "start": 1397, "end": 1410}]}, {"trigger": {"text": "inhibitory effect", "start": 1518, "end": 1535}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1539, "end": 1545}]}], "phosphorylation": [{"trigger": {"text": "Phosphorylation", "start": 41, "end": 56}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 15, "end": 21}]}], "positive regulation": [{"trigger": {"text": "activated", "start": 947, "end": 956}, "arguments": [{"role": "Theme", "text": "glucocorticoid receptor", "start": 957, "end": 980}]}, {"trigger": {"text": "induces", "start": 1073, "end": 1080}, "arguments": [{"role": "Theme", "text": "expression", "start": 1085, "end": 1095}]}, {"trigger": {"text": "necessary", "start": 1209, "end": 1218}, "arguments": [{"role": "Theme", "text": "translocation", "start": 1238, "end": 1251}]}], "regulation": [{"trigger": {"text": "regulating", "start": 164, "end": 174}, "arguments": [{"role": "Cause", "text": "GATA-3", "start": 132, "end": 138}, {"role": "Theme", "text": "expression", "start": 179, "end": 189}]}, {"trigger": {"text": "effect", "start": 461, "end": 467}, "arguments": [{"role": "Theme", "text": "regulation", "start": 523, "end": 533}]}, {"trigger": {"text": "regulation", "start": 523, "end": 533}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 516, "end": 522}]}]}}, "schema": []}
{"input": "Inflammation in allergic diseases such as asthma, rhinitis, and atopic dermatitis is mediated via expression of the cytokines interleukin (IL)-4, IL-5, and IL-13 from T helper-2 (Th2) cells. IL-4 and IL-13 regulate the expression of IgE from B lymphocytes, whereas IL-5 plays a key role in eosinophilic inflammation [1]. Th2 cytokines are regulated by the zinc finger transcription factor GATA-3, which is predominantly expressed in Th2 cells [2],[3]. GATA-3 determines Th2 cell differentiation and selectively activates the promoters of IL-4, IL-5, and IL-13 through chromatin remodelling [4]-[7]. The key role of GATA-3 in allergic airway inflammation has been demonstrated in mice by the reduced release of Th2 cytokines in animals treated with dominant-negative mutants of GATA-3 and by local application of antisense oligonucleotides to GATA-3 [8],[9]. Furthermore, conditional knock-out of the Gata3 gene in mice reduces expression of Th2 cytokines in vitro and in vivo [10], and similar results have been reported in isolated murine CD4+ lymphocytes [11]. Finally, knockdown of GATA-3 expression using siRNA in human T cells results in loss of anti-CD3/CD28-mediated Th2 cytokine expression [12].\nIn order for GATA-3 to regulate gene expression, it must translocate from the cytoplasm into the nucleus to access its target genes. Enhanced nuclear expression of GATA-3 following T cell receptor activation was first demonstrated in murine T cells [13] and was recently confirmed in human T cells following T cell receptor and co-receptor stimulation [12]. GATA-3 contains a classical nuclear import signal [14] and is transported into the nucleus by the nuclear import protein importin-alpha (also known as karyopherin-alpha) [12]. Deletion of a region encompassing the GATA-3 nuclear localisation sequence (NLS) region in murine and human cells prevents its nuclear localisation [12],[14]. The affinity of the importin-alpha-NLS interaction is regulated by phosphorylation [15], and we have shown that p38 mitogen-activated protein kinase (MAPK) plays a critical role in phosphorylating GATA-3 to enhance its interaction with importin-alpha and subsequent transport into the nucleus [12].\nCorticosteroids are highly effective in the treatment of allergic inflammation, with marked suppression of Th2 cytokines in airways of patients with asthma [16]. Corticosteroids mediate their anti-inflammatory effects through binding to glucocorticoid receptors (GRs), which then translocate to the nucleus where they interact with glucocorticoid response elements (GREs) in the promoter regions of steroid-sensitive genes. Alternatively, activated GR interacts with coactivator molecules to suppress the expression of inflammatory genes by inhibiting the action of proinflammatory transcription factors such as nuclear factor-kappaB (NF-kappaB) through the recruitment of co-repressor molecules such as histone deacetylase-2 [17],[18].\nNuclear localisation and retention of GR is mediated through the nuclear localisation sequences NL1 and NL2 [19], by nuclear retention signals [20], and by control of nuclear export via a chromosomal region maintenance 1 (CRM-1) dependent pathway [21]. NL1, which is similar to the SV40 NLS, binds to importin-alpha [22]. NL1 is activated both by glucocorticoid agonists such as dexamethasone and fluticasone propionate (FP) and by glucocorticoid antagonists such as mifepristone (RU486) [23]. NL1 can be mutated, and the resulting GR still translocates to the nucleus in response to ligands, but via interaction with importin 7, an event that requires an as-yet unknown component [23]. NL2 is poorly defined, residing in the ligand-binding domain, and much less is known about its mechanism of GR import [24]. A variety of other factors are also important for the regulation of GR activation and nuclear import including chaperones such as Hsp90 and other immunophilins [24]-[26] and FK506-binding proteins that may be linked to dynein and/or peptidylprolyl isomerase [27],[28].\nHowever, the molecular basis for the inhibition of Th2 cytokines by corticosteroids is not well understood, because the genes encoding IL-4, IL-5, and IL-13 do not have any recognisable GRE sequence [29] and are only partly regulated by NF-kappaB in human cells [30]-[32]. Using overexpression and CAT-reporter genes, Lavender and colleagues [33] have shown that GR reduced GATA-3-mediated IL-5 and -13 promoter activity in human CD4+ T cells. The authors postulated that local recruitment of GR may alter the ability of GATA-3 either to bind to its target site, to cause transcriptional up-regulation, or to maintain an environment that is permissive for transcription.\nWe therefore investigated the effects of a synthetic corticosteroid, FP, on GATA-3 phosphorylation and nuclear translocation in a T lymphocyte cell line (HuT-78) and in peripheral blood mononuclear cells activated by anti-CD3 and anti-CD28 antibodies in vitro. We also studied the effects of inhaled fluticasone therapy on GATA-3 subcellular localization in peripheral blood mononuclear cells (PBMCs) from patients with asthma.", "output": {"json_structures": {"binding": [{"trigger": {"text": "access", "start": 1312, "end": 1318}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1217, "end": 1223}]}, {"trigger": {"text": "target", "start": 1323, "end": 1329}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1217, "end": 1223}]}, {"trigger": {"text": "interaction", "start": 2116, "end": 2127}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 2094, "end": 2100}]}, {"trigger": {"text": "binding", "start": 2422, "end": 2429}, "arguments": [{"role": "Theme", "text": "glucocorticoid receptors", "start": 2433, "end": 2457}]}, {"trigger": {"text": "interact", "start": 2514, "end": 2522}, "arguments": [{"role": "Theme", "text": "glucocorticoid receptors", "start": 2433, "end": 2457}]}, {"trigger": {"text": "interacts", 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"GATA-3", "start": 389, "end": 395}]}, {"trigger": {"text": "expression", "start": 1092, "end": 1102}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1085, "end": 1091}]}, {"trigger": {"text": "activity", "start": 4425, "end": 4433}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 4403, "end": 4407}]}, {"trigger": {"text": "activity", "start": 4425, "end": 4433}, "arguments": [{"role": "Theme", "text": "-13", "start": 4412, "end": 4415}]}], "localization": [{"trigger": {"text": "translocate", "start": 1261, "end": 1272}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1217, "end": 1223}, {"role": "ToLoc", "text": "nucleus", "start": 1301, "end": 1308}]}, {"trigger": {"text": "expression", "start": 1354, "end": 1364}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 1346, "end": 1353}, {"role": "Theme", "text": "GATA-3", "start": 1368, "end": 1374}]}, {"trigger": {"text": "transported", "start": 1624, "end": 1635}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1562, "end": 1568}, {"role": "ToLoc", "text": "nucleus", "start": 1645, "end": 1652}]}, {"trigger": {"text": "translocate", "start": 2476, "end": 2487}, "arguments": [{"role": "Theme", "text": "glucocorticoid receptors", "start": 2433, "end": 2457}, {"role": "ToLoc", "text": "nucleus", "start": 2495, "end": 2502}]}, {"trigger": {"text": "localisation", "start": 2941, "end": 2953}, "arguments": [{"role": "ToLoc", "text": "Nuclear", "start": 2933, "end": 2940}, {"role": "Theme", "text": "GR", "start": 2971, "end": 2973}]}, {"trigger": {"text": "retention", "start": 2958, "end": 2967}, "arguments": [{"role": "Theme", "text": "GR", "start": 2971, "end": 2973}]}, {"trigger": {"text": "export", "start": 3108, "end": 3114}, "arguments": [{"role": "Theme", "text": "GR", "start": 2971, "end": 2973}, {"role": "ToLoc", "text": "nuclear", "start": 3100, "end": 3107}]}, {"trigger": {"text": "translocates", "start": 3474, "end": 3486}, "arguments": [{"role": "Theme", "text": "GR", "start": 3465, "end": 3467}, {"role": "ToLoc", "text": "nucleus", "start": 3494, "end": 3501}]}, {"trigger": {"text": "import", "start": 3731, "end": 3737}, "arguments": [{"role": "Theme", "text": "GR", "start": 3728, "end": 3730}]}, {"trigger": {"text": "recruitment", "start": 4491, "end": 4502}, "arguments": [{"role": "Theme", "text": "GR", "start": 4506, "end": 4508}]}, {"trigger": {"text": "translocation", "start": 4795, "end": 4808}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 4760, "end": 4766}, {"role": "ToLoc", "text": "nuclear", "start": 4787, "end": 4794}]}, {"trigger": {"text": "localization", "start": 5026, "end": 5038}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 5007, "end": 5013}]}], "negative regulation": [{"trigger": {"text": "knockdown", "start": 1072, "end": 1081}, "arguments": [{"role": "Theme", "text": "expression", "start": 1092, "end": 1102}]}, {"trigger": {"text": "reduced", "start": 4379, "end": 4386}, "arguments": [{"role": "Cause", "text": "GR", "start": 4376, "end": 4378}, {"role": "Theme", "text": "mediated", "start": 4394, "end": 4402}]}], "phosphorylation": [{"trigger": {"text": "phosphorylating", "start": 2078, "end": 2093}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 2094, "end": 2100}]}, {"trigger": {"text": "phosphorylation", "start": 4767, "end": 4782}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 4760, "end": 4766}]}], "positive regulation": [{"trigger": {"text": "activates", "start": 511, "end": 520}, "arguments": [{"role": "Cause", "text": "GATA-3", "start": 452, "end": 458}, {"role": "Site", "text": "promoters", "start": 525, "end": 534}, {"role": "Theme", "text": "IL-4", "start": 538, "end": 542}]}, {"trigger": {"text": "activates", "start": 511, "end": 520}, "arguments": [{"role": "Cause", "text": "GATA-3", "start": 452, "end": 458}, {"role": "Site", "text": "promoters", "start": 525, "end": 534}, {"role": "Theme", "text": "IL-5", "start": 544, "end": 548}]}, {"trigger": {"text": "activates", "start": 511, "end": 520}, "arguments": [{"role": "Cause", "text": "GATA-3", "start": 452, "end": 458}, {"role": "Site", "text": "promoters", "start": 525, "end": 534}, {"role": "Theme", "text": "IL-13", "start": 554, "end": 559}]}, {"trigger": {"text": "following", "start": 1375, "end": 1384}, "arguments": [{"role": "Theme", "text": "expression", "start": 1354, "end": 1364}]}, {"trigger": {"text": "following", "start": 1502, "end": 1511}, "arguments": [{"role": "Theme", "text": "expression", "start": 1354, "end": 1364}]}, {"trigger": {"text": "enhance", "start": 2104, "end": 2111}, "arguments": [{"role": "Cause", "text": "phosphorylating", "start": 2078, "end": 2093}, {"role": "Theme", "text": "interaction", "start": 2116, "end": 2127}]}, {"trigger": {"text": "activated", "start": 2635, "end": 2644}, "arguments": [{"role": "Theme", "text": "GR", "start": 2645, "end": 2647}]}, {"trigger": {"text": "mediated", "start": 2977, "end": 2985}, "arguments": [{"role": "Theme", "text": "localisation", "start": 2941, "end": 2953}, {"role": "Cause", "text": "control", "start": 3089, "end": 3096}]}, {"trigger": {"text": "mediated", "start": 2977, "end": 2985}, "arguments": [{"role": "Theme", "text": "retention", "start": 2958, "end": 2967}, {"role": "Cause", "text": "control", "start": 3089, "end": 3096}]}, {"trigger": {"text": "in response to", "start": 3502, "end": 3516}, "arguments": [{"role": "Theme", "text": "translocates", "start": 3474, "end": 3486}, {"role": "Cause", "text": "interaction", "start": 3534, "end": 3545}]}, {"trigger": {"text": "requires", "start": 3577, "end": 3585}, "arguments": [{"role": "Theme", "text": "interaction", "start": 3534, "end": 3545}]}, {"trigger": {"text": "activation", "start": 3815, "end": 3825}, "arguments": [{"role": "Theme", "text": "GR", "start": 3812, "end": 3814}]}, {"trigger": {"text": "mediated", "start": 4394, "end": 4402}, "arguments": [{"role": "Cause", "text": "GATA-3", "start": 4387, "end": 4393}, {"role": "Theme", "text": "activity", "start": 4425, "end": 4433}]}], "regulation": [{"trigger": {"text": "role", "start": 2070, "end": 2074}, "arguments": [{"role": "Theme", "text": "phosphorylating", "start": 2078, "end": 2093}]}, {"trigger": {"text": "control", "start": 3089, "end": 3096}, "arguments": [{"role": "Theme", "text": "export", "start": 3108, "end": 3114}]}, {"trigger": {"text": "dependent", "start": 3162, "end": 3171}, "arguments": [{"role": "Theme", "text": "control", "start": 3089, "end": 3096}, {"role": "Cause", "text": "chromosomal region maintenance 1", "start": 3121, "end": 3153}]}, {"trigger": {"text": "resulting", "start": 3455, "end": 3464}, "arguments": [{"role": "Theme", "text": "translocates", "start": 3474, "end": 3486}]}, {"trigger": {"text": "important", "start": 3780, "end": 3789}, "arguments": [{"role": "Theme", "text": "regulation", "start": 3798, "end": 3808}]}, {"trigger": {"text": "regulation", "start": 3798, "end": 3808}, "arguments": [{"role": "Theme", "text": "activation", "start": 3815, "end": 3825}]}, {"trigger": {"text": "regulated", "start": 4237, "end": 4246}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 4148, "end": 4152}]}, {"trigger": {"text": "regulated", "start": 4237, "end": 4246}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 4154, "end": 4158}]}, {"trigger": {"text": "regulated", "start": 4237, "end": 4246}, "arguments": [{"role": "Theme", "text": "IL-13", "start": 4164, "end": 4169}]}, {"trigger": {"text": "alter", "start": 4513, "end": 4518}, "arguments": [{"role": "Cause", "text": "recruitment", "start": 4491, "end": 4502}, {"role": "Theme", "text": "bind", "start": 4551, "end": 4555}]}, {"trigger": {"text": "effects", "start": 4714, "end": 4721}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 4767, "end": 4782}]}, {"trigger": {"text": "effects", "start": 4714, "end": 4721}, "arguments": [{"role": "Theme", "text": "translocation", "start": 4795, "end": 4808}]}, {"trigger": {"text": "effects", "start": 4965, "end": 4972}, "arguments": [{"role": "Theme", "text": "localization", "start": 5026, "end": 5038}]}]}}, "schema": []}
{"input": "Participants and Study Design\nWe studied patients with mild asthma who were not treated with inhaled corticosteroids who had been included in a previously reported double-blind, placebo-controlled, crossover study with FP [34]. Seven patients with mild asthma entered the study and were randomized to receive a single inhalation of FP (100 and 500 microg) or a matched placebo control via a spacer chamber, and the other treatment was given after a wash-out period of at least 6 d. Blood was taken for preparation of PBMCs at 1 and 2 h after drug administration. All patients gave informed consent and the study was approved by the Ethics Committee of the Royal Brompton and Harefield Hospitals NHS Trust. The clinical study was conducted before the requirement for Clinical Trial Registration.", "output": {"json_structures": {}}, "schema": []}
{"input": "Antibodies and Reagents\nThe monoclonal antibodies against human CD3, CD28, GR, and importin-alpha were purchased from BD Biosciences (Oxford, United Kingdom). Rabbit antibodies against human GATA-3 (H-48) and GR (E-20, sc-1003) were obtained from Santa Cruz Biotechnology (Santa Cruz, California, United States), polyclonal rabbit antibodies against phospho-p38 MAP kinase and phospho-ATF-2 from Cell Signaling Technology (New England Biolabs, Hertford, UK), monoclonal antibody against phosphoserine (clone 4H4) from Affiniti Research Products (Exeter, UK), and antibodies against rabbit IgG-conjugated TRITC from Dako Cytomation (Cambridge, UK). The anti-GR antibody (Clone 41) from BD Transduction Laboratories (Oxford, UK) was used for Western blot analysis. All other reagents were purchased from Sigma (Poole, UK).", "output": {"json_structures": {}}, "schema": []}
{"input": "Cell Culture and PBMC Isolation\nA human T cell line (HuT-78) was purchased from ECACC European Collection of Cell Culture (Wiltshire, UK) were cultured as previously described [12]. PBMCs were isolated by density centrifugation over Ficoll-Hypaque (density, 1.077 g/ml; Amersham Biosciences, Amersham, UK) as previously described [34]. Cells were stimulated with anti-CD3/CD28 (1 microg/ml each) for 1 h at 37degreesC to stimulate Th2 cytokine release in the presence or absence of FP (10-12 to 10-8M). Cytospins were prepared and GATA-3 localization determined by confocal microscopy as previously described [12].", "output": {"json_structures": {"localization": [{"trigger": {"text": "localization", "start": 538, "end": 550}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 531, "end": 537}]}]}}, "schema": []}
{"input": "Reverse Transcription PCR\nTotal RNA was extracted using lysis buffer (RNeasy kit; Qiagen, Crawley, UK). During RNA purification, genomic DNA was digested with RNase-free DNase (Amersham Biosciences). Next, 0.5 microg of total RNA was reversed transcribed using the avian myeloblastosis virus RT (Promega, Southampton, UK). For relative quantification, RT-PCR was carried out using cDNA probes. Primers for IL-4 were from Sigma-Genosys (Cambridge, UK). Sequences of GADPH used are as follows: forward 5'-CCACCCATGGCAAATTCCATGGC, reverse 3'-TCTAGACGGCAGGTCAGGTCCAC.", "output": {"json_structures": {}}, "schema": []}
{"input": "Cell Fractionation, Immunoprecipitation, and Western Blot Analysis\nNuclear and cytoplasmic fractions were prepared as previously described [35]. Whole cell lysates were prepared in NP-40 lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl) in the presence of complete protease cocktail inhibitor. Lysates were centrifuged at 4degreesC for 10 min at 12,000 rpm in an Eppendorf microcentrifuge to remove cellular debris. Samples were then immunoprecipitated with either 10 microl of antibody against GATA-3 or importin-alpha using A/G agarose slurry in the presence of protease inhibitor using the Catch and Release methodology (Upstate Biotechnology, Lake Placid, New York, USA). Western blot analysis was performed using anti-GATA-3, anti-importin-alpha, anti-GR, anti-p-p38 MAP kinase, anti-p-ATF-2, and anti-p-serine. Immunoreactive proteins were detected using an enhanced chemiluminescence ECL kit (Amersham Biosciences).", "output": {"json_structures": {}}, "schema": []}
{"input": "Chromatin Immunoprecipitation\nChromatin immunoprecipitation (IP) was performed as previously described [12] in HuT-78 T-cells with 2 microg of anti-GATA-3 (Santa Cruz Biotechnology), or isotypic immunoglobulin G as a non-specific control (Santa Cruz Biotechnology) overnight at 4degreesC. Promoter sequences were detected with PCR primers for the IL-5 promoter (-445 to +4): forward 5'-TTAATCTAGCCACAGTCATAG-3' and reverse: 5'-TCATGGCTCTGAAACGTTCTG-3'. PCR was performed using a Hybaid Omnigene thermal cycler (Hybaid, Ashford, UK) with cycling parameters of 72degreesC for 10 min, 35 cycles at 94degreesC for 45 s, 52degreesC for 45 s, 72degreesC for 45 s.", "output": {"json_structures": {}}, "schema": []}
{"input": "GR-GATA-3 In Vitro Competition Assay for Importin-alpha (Far-Western ELISA)\nImmunoprecipitated importin-alpha (anti-importin-alpha, Santa Cruz Biotechnology) from HuT-78 cells was separated by SDS-PAGE and purified from the excised gel by electroelution [36]. Similarly, GATA-3 was isolated from nonstimulated HuT-78 cells or cells stimulated for 30 min with anti-CD3/CD28 and GR was isolated from FP (10-8 M, 30 min) stimulated HuT-78 cells. These proteins were subsequently refolded in glycine solution. 100 microl of importin-alpha solution (100 ng/ml in TBS) was added to 96-well plates coated with goat anti-importin-alpha antibody. After 1 h incubation, GATA-3 (100 ng/ml in TBS) from stimulated or unstimulated cells were added to the importin-alpha-coated wells. GR (10 or 100 ng/ml in TBS) from stimulated or unstimulated cells were added to some wells. After a further 1 h incubation, the plate was washed and incubated with primary antibodies (a mixture of rabbit anti-GATA-3 and mouse anti-GR, Santa Cruz Biotechnology) for 1.5 h, and then incubated with secondary antibodies. FITC swine anti-rabbit IgG (Dako, Cambridge, UK) was used for the detection of GATA-3, and rhodamine donkey anti-mouse IgG (Novus, Littleton, Colorado, USA) was used for the detection of GR. FITC and rhodamine levels were measured with a fluorescent micro-plate reader (Bioline, London, UK).", "output": {"json_structures": {}}, "schema": []}
{"input": "NF-kappaB Activation\nNF-kappaB binding activity in nuclear extracts was determined using an ELISA-based kit (Trans-AM p65, Active Motif, Rixensart, Belgium). In brief, 5 microg of nuclear extracts were incubated with a plate coated with an NF-kappaB consensus oligonucleotide. Plates were washed before addition of an anti-p65 antibody. Antibody binding was detected with a secondary HRP-conjugated antibody and developed with TMB substrate. The intensity of the reaction was measured at 450 nm.", "output": {"json_structures": {}}, "schema": []}
{"input": "Immunofluorescence Staining\nImmunofluorescence staining was performed as previously described [34]. All staining was performed at room temperature and under humidification. Cells were collected and cytospins prepared in a cytocentrifuge (Shandon II, Shandon, Runcorn, UK). Cells were permeabilized, blocked, and then incubated with GR (1:50 E-20 Santa Cruz Biotechnology) or anti-GATA-3 (H-48; Santa Cruz Biotechnology) antibody for 1 h at room temperature. After three washes in phosphate-buffered saline (PBS), cells were incubated with tetrarhodamine isothiocyanate-conjugated goat anti-rabbit antibody (Dako). Cytospins were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), a fluorescent blue nuclear indole chromatin stain, and mounted in PBS:glycerol (50:50). The immunopositive signal was characterised using laser scanning confocal microscopy on a Leica TCS NT/SP interactive laser cytometer equipped with confocal optics (Leica Microsystems, Wetzlar, Germany). To determine the specificity of the antibodies, rabbit serum immunoglobulin (Dako) and secondary antibodies without the primary were used as controls. Positively stained nuclei and total cells were counted (500) on each slide with the observer blinded to the treatment.", "output": {"json_structures": {}}, "schema": []}
{"input": "GATA-3-GFP Construct\nThe GATA-3 clone (BC003070) complete cDNA was obtained from Invitrogen Life Technologies as a 5'- EcoRI/3'-XhoI insert of GATA-3 in the pOTB7 vector. GATA-3 was excised from pOTB7 using XhoI digestion and pEGFP-C2 (Clontech, Saint-Germain-en-Laye, France) was digested with BamHI. DNA was recovered by phenol extraction and ethanol precipitation, and both the GATA-3 fragment and the pEGFP-C2 vector blunt-ended by incubation with Klenow (Bioline Bio-27029) for 30 min at 37degreesC. Klenow was inactivated by incubation for 10 min at 75degreesC. DNA was recovered by phenol extraction and ethanol precipitation, and both the blunt-ended GATA-3 fragment and the GFP vector were subsequently digested with EcoRI before the 5'-EcoRI/3'-blunt end GATA-3 fragment was inserted into the 5'-EcoRI/3'-blunt ended GFP vector. Positive clones were confirmed by digestion and size analysis by 1% agarose gel electrophoresis and by sequencing.", "output": {"json_structures": {}}, "schema": []}
{"input": "Transfection\nHuT-78 cells were transfected with either EP8 or GFP vector only DNA using solution R, programme V-001 at a ratio of 3x106 cells/4 microg DNA for 7-8 h in complete medium (10% bovine serum in RPMI1640+15 L-glutamine) according to the general Amaxa protocol for nucleofection. The medium was subsequently changed to 1% RPMI for 24 h before transfected cells were added to anti-CD3/CD28 treated wells and live cell videomicroscopy performed.", "output": {"json_structures": {}}, "schema": []}
{"input": "Time-Lapse Microscopy\nHuT-78 cells expressing GATA-3-GFP were maintained at 37degreesC in growth medium in a closed FCS2 perfusion chamber (Bioptechs, Butler, Pennsylvania, USA) combined with an objective heater (Bioptechs) on the stage a Zeiss Axiovert 200 microscope (Thornwood, New York, USA). Observations were made by 40x1.0 NA oil-immersion objective lens, and fluorescence and phase contrast images were gathered using a Hamamatsu ORCA-ER charged coupled device camera (Bridgewater, New Jersey, USA) driven by Openlab software (Improvision, Coventry, UK). Photographs were taken at 0, 30, 60, 120, and 240 min.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressing", "start": 35, "end": 45}, "arguments": [{"role": "Theme", "text": "GATA-3-GFP", "start": 46, "end": 56}]}]}}, "schema": []}
{"input": "Densitometric Analysis\nDensitometry of ECL immunoblots was performed using Gelworks ID intermediate software (Ultraviolet Products, Cambridgeshire, UK). Briefly, immunoblots were scanned and gates were drawn tightly around each band. Background values from each lane were subtracted to normalize each measurement. The bands were quantified using the Gelworks software. All Western blots were exposed to film for varying lengths of time, and only films generating subsaturating levels of intensity were selected for densitometric evaluation.", "output": {"json_structures": {}}, "schema": []}
{"input": "Statistical Analysis\nData from three or more independent experiments are presented as the mean+/-standard error of the mean (SEM), except where stated and were compared using GraphPad Prism 4 (GraphPad Software, http://www.graphpad.com). Results were analysed using one-way ANOVA with Newman-Keuls post test except for the data from the in vivo inhaled FP study, which was analysed by Friedman's test with subsequent Wilcoxson matched pair signed rank sum test. Data from this analysis are presented as a box-and-whiskers plot. Friedman's test was used as three matched measures were obtained using placebo, 100 microg, and 500 microg of inhaled FP, which had variable baseline levels. We did not assume a Gaussian distribution of the data due to the limited numbers of participants analysed (seven).The null hypothesis was rejected at p<0.05.", "output": {"json_structures": {}}, "schema": []}
{"input": "The Effect of Corticosteroids on GATA-3 Nuclear Translocation and IL-4 mRNA\nCorticosteroids are effective in inhibiting GATA-3-regulated IL-4 gene expression in vitro and in vivo [32]. We therefore investigated whether corticosteroids affect anti-CD3/CD28-stimulated nuclear import of GATA-3. Stimulation of cells with anti-CD3/CD28 resulted in a rapid cytoplasmic/nuclear GATA-3 translocation (Figure 1A), confirming our previous results [12]. We also confirmed a clear separation of nuclear and cytosolic fractions as indicated by histone H1 and MEK-1 markers (Figure 1B). The potent topical corticosteroid FP caused sustained loss of nuclear GATA-3 expression and cytoplasmic retention of GATA-3 at concentrations ranging from 10-12 to 10-8 M, which cover the therapeutic range [37]. This effect was concentration- and time-dependent, with a peak effect of 11.6-fold at 30 min at a concentration of 10-8 M (Figure 1C) and was associated with marked reductions in anti-CD3/CD28-stimulated IL-4 and IL-5 mRNA expression (Figure 1D) and a loss of GATA-3 binding to the native IL-5 promoter (Figure 1E).", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 1054, "end": 1061}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1047, "end": 1053}, {"role": "Theme2", "text": "IL-5", "start": 1076, "end": 1080}, {"role": "Site2", "text": "promoter", "start": 1081, "end": 1089}]}], "gene expression": [{"trigger": {"text": "expression", "start": 147, "end": 157}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 137, "end": 141}]}], "localization": [{"trigger": {"text": "Translocation", "start": 48, "end": 61}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 33, "end": 39}, {"role": "ToLoc", "text": "Nuclear", "start": 40, "end": 47}]}, {"trigger": {"text": "import", "start": 275, "end": 281}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 267, "end": 274}, {"role": "Theme", "text": "GATA-3", "start": 285, "end": 291}]}, {"trigger": {"text": "translocation", "start": 380, "end": 393}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 365, "end": 372}, {"role": "Theme", "text": "GATA-3", "start": 373, "end": 379}]}, {"trigger": {"text": "expression", "start": 652, "end": 662}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 637, "end": 644}, {"role": "Theme", "text": "GATA-3", "start": 645, "end": 651}]}, {"trigger": {"text": "retention", "start": 679, "end": 688}, "arguments": [{"role": "ToLoc", "text": "cytoplasmic", "start": 667, "end": 678}, {"role": "Theme", "text": "GATA-3", "start": 692, "end": 698}]}], "negative regulation": [{"trigger": {"text": "inhibiting", "start": 109, "end": 119}, "arguments": [{"role": "Theme", "text": "regulated", "start": 127, "end": 136}]}, {"trigger": {"text": "loss", "start": 629, "end": 633}, "arguments": [{"role": "Theme", "text": "expression", "start": 652, "end": 662}]}, {"trigger": {"text": "reductions", "start": 952, "end": 962}, "arguments": [{"role": "Theme", "text": "stimulated", "start": 980, "end": 990}]}, {"trigger": {"text": "loss", "start": 1039, "end": 1043}, "arguments": [{"role": "Theme", "text": "binding", "start": 1054, "end": 1061}]}], "positive regulation": [{"trigger": {"text": "stimulated", "start": 256, "end": 266}, "arguments": [{"role": "Theme", "text": "import", "start": 275, "end": 281}]}, {"trigger": {"text": "resulted", "start": 333, "end": 341}, "arguments": [{"role": "Theme", "text": "translocation", "start": 380, "end": 393}]}, {"trigger": {"text": "caused", "start": 612, "end": 618}, "arguments": [{"role": "Theme", "text": "loss", "start": 629, "end": 633}]}, {"trigger": {"text": "caused", "start": 612, "end": 618}, "arguments": [{"role": "Theme", "text": "retention", "start": 679, "end": 688}]}, {"trigger": {"text": "stimulated", "start": 980, "end": 990}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 1005, "end": 1020}]}], "regulation": [{"trigger": {"text": "Effect", "start": 4, "end": 10}, "arguments": [{"role": "Theme", "text": "Translocation", "start": 48, "end": 61}]}, {"trigger": {"text": "Effect", "start": 4, "end": 10}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 66, "end": 70}]}, {"trigger": {"text": "effective", "start": 96, "end": 105}, "arguments": [{"role": "Theme", "text": "inhibiting", "start": 109, "end": 119}]}, {"trigger": {"text": "regulated", "start": 127, "end": 136}, "arguments": [{"role": "Cause", "text": "GATA-3", "start": 120, "end": 126}, {"role": "Theme", "text": "expression", "start": 147, "end": 157}]}, {"trigger": {"text": "affect", "start": 235, "end": 241}, "arguments": [{"role": "Theme", "text": "stimulated", "start": 256, "end": 266}]}, {"trigger": {"text": "This effect", "start": 787, "end": 798}, "arguments": [{"role": "Theme", "text": "associated", "start": 929, "end": 939}]}, {"trigger": {"text": "dependent", "start": 827, "end": 836}, "arguments": [{"role": "Theme", "text": "This effect", "start": 787, "end": 798}]}, {"trigger": {"text": "associated", "start": 929, "end": 939}, "arguments": [{"role": "Theme", "text": "loss", "start": 1039, "end": 1043}]}, {"trigger": {"text": "associated", "start": 929, "end": 939}, "arguments": [{"role": "Theme", "text": "reductions", "start": 952, "end": 962}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 1005, "end": 1020}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 1000, "end": 1004}]}, {"trigger": {"text": "mRNA expression", "start": 1005, "end": 1020}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 991, "end": 995}]}]}}, "schema": []}
{"input": "Ligand-Activated GR Competes with GATA-3 for Importin-alpha\nWe confirmed and extended previous data [20] to show that ligand-activated GR as well as GATA-3 uses importin-alpha for its nuclear import (Figure 2A and 2B). This interaction between GR and importin-alpha was significant at concentrations as low as 10-12 M and was maximal with 10-8 M FP. Subsequent GR nuclear translocation was rapid and sustained at significant levels for at least 14 h (Figure 2B). Using IP-Western blotting we showed that FP at 10-12-10-8 M decreased the association between GATA-3 and importin-alpha induced by anti-CD3/CD28 stimulation in a concentration-dependent manner (Figure 2C). In addition, using GFP-labelled GATA-3 and confocal microscopy we demonstrated that GATA-3 nuclear import following anti-CD3/CD28 stimulation for 30 min was attenuated by pretreatment with FP (10-8 M) (Figure 2D).", "output": {"json_structures": {"binding": [{"trigger": {"text": "for", "start": 41, "end": 44}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 34, "end": 40}]}, {"trigger": {"text": "association", "start": 537, "end": 548}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 557, "end": 563}]}], "localization": [{"trigger": {"text": "import", "start": 192, "end": 198}, "arguments": [{"role": "Theme", "text": "GR", "start": 135, "end": 137}, {"role": "ToLoc", "text": "nuclear", "start": 184, "end": 191}]}, {"trigger": {"text": "import", "start": 192, "end": 198}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 149, "end": 155}, {"role": "ToLoc", "text": "nuclear", "start": 184, "end": 191}]}, {"trigger": {"text": "translocation", "start": 372, "end": 385}, "arguments": [{"role": "Theme", "text": "GR", "start": 361, "end": 363}, {"role": "ToLoc", "text": "nuclear", "start": 364, "end": 371}]}, {"trigger": {"text": "import", "start": 768, "end": 774}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 753, "end": 759}, {"role": "ToLoc", "text": "nuclear", "start": 760, "end": 767}]}], "negative regulation": [{"trigger": {"text": "decreased", "start": 523, "end": 532}, "arguments": [{"role": "Theme", "text": "association", "start": 537, "end": 548}]}, {"trigger": {"text": "attenuated", "start": 826, "end": 836}, "arguments": [{"role": "Theme", "text": "import", "start": 768, "end": 774}]}], "positive regulation": [{"trigger": {"text": "activated", "start": 125, "end": 134}, "arguments": [{"role": "Theme", "text": "GR", "start": 135, "end": 137}]}, {"trigger": {"text": "uses", "start": 156, "end": 160}, "arguments": [{"role": "Theme", "text": "import", "start": 192, "end": 198}]}, {"trigger": {"text": "induced", "start": 583, "end": 590}, "arguments": [{"role": "Theme", "text": "association", "start": 537, "end": 548}]}, {"trigger": {"text": "following", "start": 775, "end": 784}, "arguments": [{"role": "Theme", "text": "import", "start": 768, "end": 774}]}], "regulation": [{"trigger": {"text": "dependent", "start": 639, "end": 648}, "arguments": [{"role": "Theme", "text": "decreased", "start": 523, "end": 532}]}]}}, "schema": []}
{"input": "Effect on MKP-1\nDexamethasone inhibits p38 MAPK function in a cell type-specific manner through the rapid induction of the dual kinase phosphatase MKP-1 (MAPK phosphatase-1), and this effect lasts for up to 24 h [28]. FP (10-8 M) treatment of HuT-78 cells activated by anti-CD3/CD28 in vitro significantly decreased p38 MAPK phosphorylation (Figure 3A) and activity measured by phosphorylation of the downstream target ATF-2 (Figure 3B). This effect was detected at 30 min and lasted for at least 14 h (Figure 3B). FP (10-8 M) also significantly reduced GATA-3 serine phosphorylation induced by anti-CD3/CD28 stimulation in both a time- and concentration-dependent manner (Figure 3C). This reduction in GATA-3 phosphorylation was also seen with lower concentrations of FP. We found that FP significantly induced MKP-1 mRNA in both a time- and concentration-dependent manner, reaching a plateau at 10-8 M after 10 min (Figure 3D and 3E). However, the effects of FP on GATA-3 nuclear import, importin-alpha association and IL-4 mRNA expression are seen at 10,000-fold lower concentrations (10-12 M, see Figure 2).\nUsing an in vitro competition assay (Figure 4A) utilizing purified activated GATA-3, importin-alpha, and activated GR, we demonstrated that activated GR significantly increased GR-importin-alpha association in the presence and absence of activated GATA-3 (Figure 4B). This effect is not mutual, since activated GATA-3 did not block GR-importin-alpha association (Figure 4C). These data also suggest that both activated GR and phospho-GATA-3 can directly associate with importin-alpha (Figure 4D) and that activated GR attenuates the phospho-GATA-3/importin-alpha interaction in a concentration-dependent manner (Figure 4E). Together, this suggests that ligand-activated GR may compete with phospho-GATA-3 for importin-alpha and thereby limit GATA-3 nuclear import.\nOther possible interpretations of our results could include an effect of FP on GATA-3 nuclear export and/or degradation. Leptomycin B, which inhibits nuclear export, did not affect the ability of FP to block GATA-3 nuclear localization (Figure 5A). Additionally, FP had no effect on whole cell GATA-3 expression during the time course of these experiments (Figure 5B). Nor did addition of FP subsequent to anti-CD3/CD28 nuclear translocation affect GATA-3 nuclear residency (Figure 5C), suggesting that activated GR does not enhance GATA-3 nuclear export. Finally, the effect of FP on GATA-3 nuclear import was not nonspecific, since FP (10-8 M) had no effect on p65 nuclear translocation measured at 60 min (Figure 5D).", "output": {"json_structures": {"binding": [{"trigger": {"text": "target", "start": 412, "end": 418}, "arguments": [{"role": "Theme", "text": "ATF-2", "start": 419, "end": 424}]}, {"trigger": {"text": "association", "start": 1005, "end": 1016}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 967, "end": 973}]}, {"trigger": {"text": "association", "start": 1307, "end": 1318}, "arguments": [{"role": "Theme", "text": "GR", "start": 1289, "end": 1291}]}, {"trigger": {"text": "association", "start": 1462, "end": 1473}, "arguments": [{"role": "Theme", "text": "GR", "start": 1444, "end": 1446}]}, {"trigger": {"text": "associate", "start": 1566, "end": 1575}, "arguments": [{"role": "Theme", "text": "GR", "start": 1531, "end": 1533}]}, {"trigger": {"text": "associate", "start": 1566, "end": 1575}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1546, "end": 1552}]}, {"trigger": {"text": "interaction", "start": 1675, "end": 1686}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1653, "end": 1659}]}, {"trigger": {"text": "for", "start": 1817, "end": 1820}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1810, "end": 1816}]}], "gene expression": [{"trigger": {"text": "induction", "start": 106, "end": 115}, "arguments": [{"role": "Theme", "text": "MKP-1", "start": 147, "end": 152}]}, {"trigger": {"text": "expression", "start": 2178, "end": 2188}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 2171, "end": 2177}]}], "localization": [{"trigger": {"text": "import", "start": 982, "end": 988}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 967, "end": 973}, {"role": "ToLoc", "text": "nuclear", "start": 974, "end": 981}]}, {"trigger": {"text": "import", "start": 1869, "end": 1875}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1854, "end": 1860}, {"role": "ToLoc", "text": "nuclear", "start": 1861, "end": 1868}]}, {"trigger": {"text": "export", "start": 1971, "end": 1977}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1956, "end": 1962}, {"role": "ToLoc", "text": "nuclear", "start": 1963, "end": 1970}]}, {"trigger": {"text": "localization", "start": 2100, "end": 2112}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 2085, "end": 2091}, {"role": "ToLoc", "text": "nuclear", "start": 2092, "end": 2099}]}, {"trigger": {"text": "residency", "start": 2341, "end": 2350}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 2326, "end": 2332}, {"role": "ToLoc", "text": "nuclear", "start": 2333, "end": 2340}]}, {"trigger": {"text": "export", "start": 2425, "end": 2431}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 2410, "end": 2416}, {"role": "ToLoc", "text": "nuclear", "start": 2417, "end": 2424}]}, {"trigger": {"text": "import", "start": 2477, "end": 2483}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 2462, "end": 2468}, {"role": "ToLoc", "text": "nuclear", "start": 2469, "end": 2476}]}, {"trigger": {"text": "translocation", "start": 2552, "end": 2565}, "arguments": [{"role": "Theme", "text": "p65", "start": 2540, "end": 2543}, {"role": "ToLoc", "text": "nuclear", "start": 2544, "end": 2551}]}], "negative regulation": [{"trigger": {"text": "decreased", "start": 306, "end": 315}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 378, "end": 393}]}, {"trigger": {"text": "reduced", "start": 546, "end": 553}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 568, "end": 583}]}, {"trigger": {"text": "This reduction", "start": 685, "end": 699}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 710, "end": 725}]}, {"trigger": {"text": "competition", "start": 1130, "end": 1141}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1189, "end": 1195}]}, {"trigger": {"text": "competition", "start": 1130, "end": 1141}, "arguments": [{"role": "Theme", "text": "GR", "start": 1227, "end": 1229}]}, {"trigger": {"text": "block", "start": 1438, "end": 1443}, "arguments": [{"role": "Cause", "text": "GATA-3", "start": 1423, "end": 1429}, {"role": "Theme", "text": "association", "start": 1462, "end": 1473}]}, {"trigger": {"text": "attenuates", "start": 1630, "end": 1640}, "arguments": [{"role": "Cause", "text": "GR", "start": 1627, "end": 1629}, {"role": "Theme", "text": "interaction", "start": 1675, "end": 1686}]}, {"trigger": {"text": "compete", "start": 1789, "end": 1796}, "arguments": [{"role": "Cause", "text": "GR", "start": 1782, "end": 1784}, {"role": "Theme", "text": "for", "start": 1817, "end": 1820}]}, {"trigger": {"text": "limit", "start": 1848, "end": 1853}, "arguments": [{"role": "Cause", "text": "compete", "start": 1789, "end": 1796}, {"role": "Theme", "text": "import", "start": 1869, "end": 1875}]}, {"trigger": {"text": "block", "start": 2079, "end": 2084}, "arguments": [{"role": "Theme", "text": "localization", "start": 2100, "end": 2112}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 378, "end": 393}, "arguments": [{"role": "Theme", "text": "ATF-2", "start": 419, "end": 424}]}, {"trigger": {"text": "phosphorylation", "start": 568, "end": 583}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 554, "end": 560}, {"role": "Site", "text": "serine", "start": 561, "end": 567}]}, {"trigger": {"text": "phosphorylation", "start": 710, "end": 725}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 703, "end": 709}]}, {"trigger": {"text": "phospho", "start": 1645, "end": 1652}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1653, "end": 1659}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 584, "end": 591}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 568, "end": 583}]}, {"trigger": {"text": "induced", "start": 804, "end": 811}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 818, "end": 822}]}, {"trigger": {"text": "activated", "start": 1179, "end": 1188}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1189, "end": 1195}]}, {"trigger": {"text": "activated", "start": 1217, "end": 1226}, "arguments": [{"role": "Theme", "text": "GR", "start": 1227, "end": 1229}]}, {"trigger": {"text": "activated", "start": 1252, "end": 1261}, "arguments": [{"role": "Theme", "text": "GR", "start": 1262, "end": 1264}]}, {"trigger": {"text": "increased", "start": 1279, "end": 1288}, "arguments": [{"role": "Cause", "text": "GR", "start": 1262, "end": 1264}, {"role": "Theme", "text": "association", "start": 1307, "end": 1318}]}, {"trigger": {"text": "activated", "start": 1350, "end": 1359}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1360, "end": 1366}]}, {"trigger": {"text": "activated", "start": 1413, "end": 1422}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1423, "end": 1429}]}, {"trigger": {"text": "activated", "start": 1521, "end": 1530}, "arguments": [{"role": "Theme", "text": "GR", "start": 1531, "end": 1533}]}, {"trigger": {"text": "activated", "start": 1617, "end": 1626}, "arguments": [{"role": "Theme", "text": "GR", "start": 1627, "end": 1629}]}, {"trigger": {"text": "activated", "start": 1772, "end": 1781}, "arguments": [{"role": "Theme", "text": "GR", "start": 1782, "end": 1784}]}, {"trigger": {"text": "activated", "start": 2380, "end": 2389}, "arguments": [{"role": "Theme", "text": "GR", "start": 2390, "end": 2392}]}, {"trigger": {"text": "enhance", "start": 2402, "end": 2409}, "arguments": [{"role": "Cause", "text": "GR", "start": 2390, "end": 2392}, {"role": "Theme", "text": "export", "start": 2425, "end": 2431}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 1985, "end": 1996}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1956, "end": 1962}]}], "regulation": [{"trigger": {"text": "dependent", "start": 655, "end": 664}, "arguments": [{"role": "Theme", "text": "reduced", "start": 546, "end": 553}]}, {"trigger": {"text": "dependent", "start": 857, "end": 866}, "arguments": [{"role": "Theme", "text": "induced", "start": 804, "end": 811}]}, {"trigger": {"text": "effects", "start": 950, "end": 957}, "arguments": [{"role": "Theme", "text": "association", "start": 1005, "end": 1016}]}, {"trigger": {"text": "effects", "start": 950, "end": 957}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 1026, "end": 1041}]}, {"trigger": {"text": "effects", "start": 950, "end": 957}, "arguments": [{"role": "Theme", "text": "import", "start": 982, "end": 988}]}, {"trigger": {"text": "in the presence and absence of", "start": 1319, "end": 1349}, "arguments": [{"role": "Theme", "text": "increased", "start": 1279, "end": 1288}, {"role": "Cause", "text": "activated", "start": 1350, "end": 1359}]}, {"trigger": {"text": "dependent", "start": 1706, "end": 1715}, "arguments": [{"role": "Theme", "text": "attenuates", "start": 1630, "end": 1640}]}, {"trigger": {"text": "effect", "start": 1940, "end": 1946}, "arguments": [{"role": "Theme", "text": "export", "start": 1971, "end": 1977}]}, {"trigger": {"text": "effect", "start": 1940, "end": 1946}, "arguments": [{"role": "Theme", "text": "degradation", "start": 1985, "end": 1996}]}, {"trigger": {"text": "affect", "start": 2051, "end": 2057}, "arguments": [{"role": "Theme", "text": "block", "start": 2079, "end": 2084}]}, {"trigger": {"text": "effect", "start": 2150, "end": 2156}, "arguments": [{"role": "Theme", "text": "expression", "start": 2178, "end": 2188}]}, {"trigger": {"text": "affect", "start": 2319, "end": 2325}, "arguments": [{"role": "Theme", "text": "residency", "start": 2341, "end": 2350}]}, {"trigger": {"text": "effect", "start": 2446, "end": 2452}, "arguments": [{"role": "Theme", "text": "import", "start": 2477, "end": 2483}]}, {"trigger": {"text": "effect", "start": 2530, "end": 2536}, "arguments": [{"role": "Theme", "text": "translocation", "start": 2552, "end": 2565}]}], "transcription": [{"trigger": {"text": "mRNA", "start": 818, "end": 822}, "arguments": [{"role": "Theme", "text": "MKP-1", "start": 812, "end": 817}]}, {"trigger": {"text": "mRNA expression", "start": 1026, "end": 1041}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 1021, "end": 1025}]}]}}, "schema": []}
{"input": "The Inhibitory Effect of Corticosteroids on GATA-3 Nuclear Localization in Primary T Lymphocytes Ex Vivo and In Vivo\nTreatment with FP ex vivo demonstrated a concentration-dependent decrease in the direct interaction between phospho-GATA-3 and importin-alpha in PBMCs from patients with asthma (Figure 6A and 6B), which was significantly inhibited at 10-12 M FP (p<0.001, ANOVA and Newman-Keuls test) and completely attenuated by 10-8 M FP (p<0.001, ANOVA and Newman-Keuls test).\nOur previous T cell line studies indicated that 10-12 M FP suppresses IL-4 and -5 gene expression and attenuated the interaction of GATA-3 with importin-alpha (see Figures 1D and 2). This concentration is close to peak plasma levels obtained from asthmatic patients treated with inhaled FP (500 microg) [27]. Inhaled FP (500 microg) treatment of seven steroid-naive asthma patients significantly reduced GATA-3-importin-alpha interaction in vivo in a time-dependent manner. This produced a >90% decrease in GATA-3-importin-alpha association at 2 h (median [95% CI], 13,494 [6,828-17,829] versus 879 [597-1,165]; p<0.05 Friedman's analysis). However, this did not reach significance using Wilcoxon's post-test analysis (W = 6.00) probably due to low numbers of participants. Similar results were observed when GATA-3-importin-alpha association was measured (Figure 6C and 6D). The lower dose of FP (100 microg) was not effective. The attenuated interaction of GATA-3 did not result from the defective recycling of importin-alpha, as a significant decrease in the abundance of importin-alpha in the cytoplasmic pool was not detected (Figure 6E).\nWe further examined whether inhaled FP could affect cellular localization of GATA-3 in peripheral blood T cells. Treatment with inhaled FP (500 microg) for 2 h significantly increased GR nuclear translocation (Figure 7A) and concomitantly decreased the number of nuclear GATA-3 immunoreactive peripheral blood T cells (37%+/-4.2% versus 58.2%+/-4.95%, p = 0.016, W = 28.0, Wilcoxon's rank test) compared with placebo as measured by immunocytochemistry (Figure 7A and 7B). This was confirmed by Western blotting, which also indicated that this effect was both time- and dose-dependent (Figure 7C and 7D). Thus, inhaled FP (500 microg) induced significant loss in nuclear GATA-3 at 2 h (median [95% CI], 0.40 [0.27-0.53] versus 0.14 [0.11-0.19], p<0.05, W = 21.00, Wilcoxon's rank test) (Figure 7C) and cytoplasmic GATA-3 levels were enhanced by inhaled FP in a dose-dependent manner (median [95% CI], 0.0032 [0.0026-0.0039] versus 0.658 [0.592-0.720], p<0.05, W = -21.00, Wilcoxon's rank test) (Figure 7D). In addition, FP (500 microg) inhibited p38 MAPK phosphorylation in primary T cells in vivo at 2 h in samples from two patients (Figure 7E).\nTaken together, our data suggest that inhaled FP reduces nuclear localization of GATA-3 in vivo by acutely inhibiting phospho-GATA-3-importin association. This effect may be direct, through competition for importin-alpha or associated molecules, or secondary to an effect on p38 MAPK-mediated GATA-3 phosphorylation via rapid induction of MKP-1. The combination of these two interacting effects can result in complete suppression of GATA-3 nuclear import and thus Th2 cytokine gene expression.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 205, "end": 216}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 233, "end": 239}]}, {"trigger": {"text": "interaction", "start": 597, "end": 608}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 612, "end": 618}]}, {"trigger": {"text": "interaction", "start": 906, "end": 917}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 884, "end": 890}]}, {"trigger": {"text": "association", "start": 1009, "end": 1020}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 987, "end": 993}]}, {"trigger": {"text": "association", "start": 1311, "end": 1322}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1289, "end": 1295}]}, {"trigger": {"text": "interaction", "start": 1424, "end": 1435}, "arguments": [{"role": 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{"input": "Here we have demonstrated, to our knowledge for the first time, that corticosteroids may inhibit GATA-3 function and therefore the transcription of Th2 genes via two distinct but interacting molecular mechanisms. Firstly, corticosteroid-activated GR appears to compete with activated GATA-3 for nuclear import via importin-alpha, which is required for the nuclear transport of both GATA-3 and GR. Secondly, corticosteroids at higher concentrations increase the expression of MKP-1, a potent inhibitor of p38 MAPK activity and thereby prevent T cell receptor/co-receptor activation of p38 MAPK to prevent the phosphorylation of GATA-3 that is necessary for interaction with importin-alpha and subsequent nuclear import.\nWe have previously shown that translocation of GATA-3 from the cytoplasm to the nucleus involves the nuclear transporter protein importin-alpha, which interacts with phosphorylated GATA-3 [12]. We have also previously reported that GATA-3 knockdown using siRNA results in suppression of anti-CD3/CD28-stimulated IL-4/IL-5 mRNA induction, thus implicating an essential role for GATA-3 in the transcription of these genes [12]. We now confirm, in human T cells, that GR also uses the same nuclear import mechanism as GATA-3 [22]. We therefore propose that there is competition between ligand-activated GR and phospho-GATA-3 for nuclear import. Furthermore, we have shown that there is preferential binding of importin-alpha to activated GR over phospho-GATA-3, so that corticosteroids would preferentially reduce GATA-3 entry and thus rapidly switch off Th2 gene transcription without any need for any intermediate steps. Furthermore, there was some degree of specificity for GATA-3, as nuclear translocation of the p65 subunit of NF-kappaB was not affected by corticosteroid exposure.\nWe tested some alternative explanations for this effect of FP on GATA-3 nuclear exclusion and failed to show that FP either enhances GATA-3 nuclear export directly or induces GATA-3 degradation. The evidence from the in vitro competition assays does, however, suggest that purified activated GR can clearly attenuate purified phospho-GATA-3-importin-alpha association and that the converse does not occur. Furthermore, we have shown that only phospho-GATA-3 can associate with importin-alpha. This mechanism is sensitive to very low concentrations of corticosteroid and would be rapid in onset as no changes in protein synthesis are required. This acute mechanism may also contribute to the reduction in GATA-3 nuclear import and may play a major role at low corticosteroid concentrations and/or at early time points prior to MKP-1 induction.\nCorticosteroids can modulate p38 MAPK activity through the induction of MKP-1, a potent endogenous inhibitor of MAPK function [38],[39]. We report here a rapid induction of MKP-1 mRNA following stimulation of cells with relatively high concentrations of FP. We hypothesize that this rapid induction of MKP-1 can reduce GATA-3 nuclear import by attenuating p38 MAPK activity and subsequent GATA-3 phosphorylation, thus preventing nuclear translocation. The location of the serine residue(s) of GATA-3 that are phosphorylated by p38 MAPK are currently unknown, but a bioinformatics search (Motif Scanner, http://scansite.mit.edu/motifscan_seq.phtml) indicates at least three potential p38 MAPK-sensitive serine residues.\nAs predicted from these in vitro data, impairment of GATA-3 nuclear import by FP may, at least in part, underlie the efficacy of corticosteroids in suppressing allergic inflammation. Although we did not assess the acute inhibitory effect of FP on the expression of IL-4 mRNA in vivo, a single inhalation of FP (500 microg) may have comparable effects, as it provides plasma levels within a relevant range of concentrations used to suppress IL-4 transcription in our in vitro system [37]. A lower dose of inhaled FP (100 microg) was not effective, but plasma concentrations may be below those required for GATA-3 inhibition. However, it is likely that the higher concentrations of FP in the airways after inhaled administration would be effective in inhibiting GATA-3 in airway T cells of asthma patients. The study of PBMCs from asthma patients treated with inhaled corticosteroid therapy clearly demonstrates that these molecular mechanisms are likely to also occur in patients at therapeutic doses of inhaled corticosteroids. In addition, previous studies have shown that corticosteroids can suppress IL-4 and IL-5 release from peripheral blood cells of asthma patients in vitro and in vivo [40]-[42].\nIn summary, our data provide evidence for a novel action of corticosteroids: suppression of allergic inflammation through a rapid inhibitory effect on GATA-3 nuclear translocation by preferential binding to the shared nuclear import protein importin-alpha and by a second mechanism involving increased synthesis of MKP-1, which inhibits p38 MAPK, thus preventing the phosphorylation of GATA-3 that is necessary for nuclear translocation of GATA-3. These two mechanisms are likely to be synergistic, accounting for the rapid and potent effect of corticosteroids on allergic inflammation. This is exemplified by the rapid inhibitory effect of topical corticosteroids on nasal Th2 cytokine release after allergen provocation in individuals with seasonal allergic rhinitis (hay fever) [43]. Prevention of phospho-GATA-3 interaction with importin-alpha may provide a new approach for the development of novel therapies for the treatment of allergic diseases.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 656, "end": 667}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 627, "end": 633}]}, {"trigger": {"text": "interacts", "start": 870, "end": 879}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 900, "end": 906}]}, {"trigger": {"text": "binding", "start": 1415, "end": 1422}, "arguments": [{"role": "Theme", "text": "GR", "start": 1454, "end": 1456}]}, {"trigger": {"text": "binding", "start": 1415, "end": 1422}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1470, "end": 1476}]}, {"trigger": {"text": "association", "start": 2159, "end": 2170}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 2137, "end": 2143}]}, {"trigger": {"text": "associate", "start": 2265, "end": 2274}, "arguments": 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1996}]}, {"trigger": {"text": "activated", "start": 2085, "end": 2094}, "arguments": [{"role": "Theme", "text": "GR", "start": 2095, "end": 2097}]}, {"trigger": {"text": "contribute", "start": 2476, "end": 2486}, "arguments": [{"role": "Theme", "text": "reduction", "start": 2494, "end": 2503}]}, {"trigger": {"text": "induction", "start": 2705, "end": 2714}, "arguments": [{"role": "Theme", "text": "MKP-1", "start": 2718, "end": 2723}]}, {"trigger": {"text": "following", "start": 2830, "end": 2839}, "arguments": [{"role": "Theme", "text": "induction", "start": 2806, "end": 2815}]}, {"trigger": {"text": "increased", "start": 4861, "end": 4870}, "arguments": [{"role": "Theme", "text": "synthesis", "start": 4871, "end": 4880}]}, {"trigger": {"text": "necessary", "start": 4970, "end": 4979}, "arguments": [{"role": "Theme", "text": "translocation", "start": 4992, "end": 5005}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 1985, "end": 1996}, "arguments": [{"role": 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{"trigger": {"text": "subsequent", "start": 3024, "end": 3034}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 3042, "end": 3057}]}], "transcription": [{"trigger": {"text": "induction", "start": 1046, "end": 1055}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 1031, "end": 1035}]}, {"trigger": {"text": "induction", "start": 1046, "end": 1055}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 1036, "end": 1040}]}, {"trigger": {"text": "transcription", "start": 1110, "end": 1123}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 1031, "end": 1035}]}, {"trigger": {"text": "transcription", "start": 1110, "end": 1123}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 1036, "end": 1040}]}, {"trigger": {"text": "expression", "start": 3616, "end": 3626}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 3630, "end": 3634}]}, {"trigger": {"text": "transcription", "start": 3810, "end": 3823}, "arguments": [{"role": "Theme", "text": 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{"input": "Fluticasone propionate down-regulates Th2 cytokine gene expression and inhibits GATA-3 nuclear import.\n(A) Anti-CD3/CD28 treatment of HuT-78 cells results in translocation of GATA-3 from the cytoplasm to the nucleus within 30 min. (B) Histone H1 and MEK-1 were used to confirm distinct separation of cytoplasmic and nuclear extracts in three separate experiments. (C) Western blot analysis of FP-treated HuT-78 cells demonstrated impaired nuclear localization of GATA-3 induced by anti-CD3/CD28 co-stimulation in a time- (at 10-8 M FP) and concentration- (at 60 min after stimulation) dependent manner. Cells were pretreated with FP for 30 min prior to stimulation. MEK1 and histone H1 were used to demonstrate equal cytoplasmic and nuclear loading respectively. Results are presented graphically below as mean+/-SEM of at least three independent experiments. *** p<0.001 compared to t = 0. (D) RT-PCR showing that FP inhibits IL-4 and IL-5 mRNA expression in CD3/CD28-costimulated cells. GAPDH was used as a loading control. Lower panels show graphical analysis of results presented as mean+/-SEM of at least three independent experiments. ###p<0.001 compared to control, ***p<0.001 compared to anti-CD3/CD28-stimulated. (E) FP (10 nM) reduces the ability of anti-CD3/CD28-stimulated GATA-3 to associate with the native IL-5 promoter 60 min after stimulation. Data are also shown graphically as mean+/-SEM of three independent experiments. All data were analysed by ANOVA followed by Newman-Keuls post-test.", "output": {"json_structures": {"binding": [{"trigger": {"text": "associate", "start": 1295, "end": 1304}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1285, "end": 1291}, {"role": "Theme2", "text": "IL-5", "start": 1321, "end": 1325}, {"role": "Site2", "text": "promoter", "start": 1326, "end": 1334}]}], "localization": [{"trigger": {"text": "import", "start": 95, "end": 101}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 80, "end": 86}, {"role": "ToLoc", "text": "nuclear", "start": 87, "end": 94}]}, {"trigger": {"text": "translocation", "start": 158, "end": 171}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 175, "end": 181}, {"role": "ToLoc", "text": "nucleus", "start": 208, "end": 215}]}, {"trigger": {"text": "localization", "start": 447, "end": 459}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 439, "end": 446}, {"role": "Theme", "text": "GATA-3", "start": 463, "end": 469}]}], "negative regulation": [{"trigger": {"text": "inhibits", "start": 71, "end": 79}, "arguments": [{"role": "Theme", "text": "import", "start": 95, "end": 101}]}, {"trigger": {"text": "inhibits", "start": 918, "end": 926}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 941, "end": 956}]}, {"trigger": {"text": "reduces", "start": 1237, "end": 1244}, "arguments": [{"role": "Theme", "text": "associate", "start": 1295, "end": 1304}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 470, "end": 477}, "arguments": [{"role": "Theme", "text": "localization", "start": 447, "end": 459}]}, {"trigger": {"text": "stimulated", "start": 1274, "end": 1284}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1285, "end": 1291}]}], "regulation": [{"trigger": {"text": "costimulated", "start": 969, "end": 981}, "arguments": [{"role": "Theme", "text": "CD3", "start": 960, "end": 963}]}, {"trigger": {"text": "costimulated", "start": 969, "end": 981}, "arguments": [{"role": "Theme", "text": "CD28", "start": 964, "end": 968}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 941, "end": 956}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 927, "end": 931}]}, {"trigger": {"text": "mRNA expression", "start": 941, "end": 956}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 936, "end": 940}]}]}}, "schema": []}
{"input": "Fluticasone propionate reduces GATA-3 association with importin-alpha and GATA-3 nuclear import.\n(A) Western blot analysis demonstrates a time- (at 10-8 M FP) and concentration- (at 60 min after stimulation) dependent induction of FP-activated GR interaction with importin-alpha (Imp-alpha). A positive control for GR association with importin is shown. Quantification of the densitometry data is shown below. Each bar represents mean+/-SEM of at least three independent experiments. *** p<0.001 compared to control, ### p<0.001. (B) Western blot analysis demonstrated a time- (at 10-8 M FP) and concentration- (at 60 min after stimulation) dependent induction of FP-activated GR nuclear translocation measured by IP. Quantification of the densitometry data is shown below. Each bar represents mean+/-SEM of at least three independent experiments. ***p<0.001 compared to control. (C) Western blot analysis of HuT-78 cells treated with FP and anti-CD3/CD28 co-stimulation demonstrated a concentration-dependent decrease in GATA-3-importin-alpha association at 20 min. Quantification of the densitometry data is shown below. Each bar represents mean+/-SEM of at least three independent experiments. ###p<0.001 compared to control, ***p<0.001 compared to alphaCD3/CD28-stimulated cells. (D) GFP-tagged GATA-3 was overexpressed and cells stimulated (b, c) or not (a) for 30 min with anti-CD3/CD28. The effect of 30 min pretreatment of cells with FP (10-8 M, c) is also shown. All data were analysed by ANOVA followed by Newman-Keuls post-test.", "output": {"json_structures": {"binding": [{"trigger": {"text": "association", "start": 38, "end": 49}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 31, "end": 37}]}, {"trigger": {"text": "interaction", "start": 247, "end": 258}, "arguments": [{"role": "Theme", "text": "GR", "start": 244, "end": 246}]}, {"trigger": {"text": "association", "start": 318, "end": 329}, "arguments": [{"role": "Theme", "text": "GR", "start": 315, "end": 317}]}, {"trigger": {"text": "association", "start": 1044, "end": 1055}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1022, "end": 1028}]}], "gene expression": [{"trigger": {"text": "overexpressed", "start": 1310, "end": 1323}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 1299, "end": 1305}]}], "localization": [{"trigger": {"text": "import", "start": 89, "end": 95}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 74, "end": 80}, {"role": "ToLoc", "text": "nuclear", "start": 81, "end": 88}]}, {"trigger": {"text": "translocation", "start": 688, "end": 701}, "arguments": [{"role": "Theme", "text": "GR", "start": 677, "end": 679}, {"role": "ToLoc", "text": "nuclear", "start": 680, "end": 687}]}], "negative regulation": [{"trigger": {"text": "reduces", "start": 23, "end": 30}, "arguments": [{"role": "Theme", "text": "association", "start": 38, "end": 49}]}, {"trigger": {"text": "reduces", "start": 23, "end": 30}, "arguments": [{"role": "Theme", "text": "import", "start": 89, "end": 95}]}, {"trigger": {"text": "decrease", "start": 1010, "end": 1018}, "arguments": [{"role": "Theme", "text": "association", "start": 1044, "end": 1055}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 218, "end": 227}, "arguments": [{"role": "Theme", "text": "interaction", "start": 247, "end": 258}]}, {"trigger": {"text": "activated", "start": 234, "end": 243}, "arguments": [{"role": "Theme", "text": "GR", "start": 244, "end": 246}]}, {"trigger": {"text": "induction", "start": 651, "end": 660}, "arguments": [{"role": "Theme", "text": "translocation", "start": 688, "end": 701}]}, {"trigger": {"text": "activated", "start": 667, "end": 676}, "arguments": [{"role": "Theme", "text": "GR", "start": 677, "end": 679}]}], "regulation": [{"trigger": {"text": "dependent", "start": 208, "end": 217}, "arguments": [{"role": "Theme", "text": "induction", "start": 218, "end": 227}]}, {"trigger": {"text": "dependent", "start": 641, "end": 650}, "arguments": [{"role": "Theme", "text": "induction", "start": 651, "end": 660}]}, {"trigger": {"text": "dependent", "start": 1000, "end": 1009}, "arguments": [{"role": "Theme", "text": "decrease", "start": 1010, "end": 1018}]}]}}, "schema": []}
{"input": "Fluticasone propionate-mediated inhibition of p38 MAP kinase phosphorylation and activation is associated with a marked down-regulation of GATA-3 serine phosphorylation.\n(A) Western blot analysis shows that FP (10-8 M, 30 min) treatment reduced dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK in anti-CD3/CD28-co-stimulated HuT-78 cells. (B) Time course of the effect of FP (10-8 M) on phosphorylation of activated transcription factor 2 (ATF-2), a measure of p38 MAPK activity. (C) FP-induced inhibition of p38 MAPK activity is associated with the decrease of anti-CD3/CD28 co-stimulation-induced serine phosphorylation (P-Ser) of GATA-3. For (A-C), quantification of the densitometry data is also shown. Each bar represents mean+/-SEM of at least three independent experiments. ###p<0.001 compared to control, ***p<0.001 compared to alphaCD3/CD28-stimulated cells. (D) FP induced MKP-1 mRNA in a concentration-dependent manner. All results are representative of at least three independent experiments and where appropriate expressed as means+/-SEM, *p<0.05. (E) FP induces MKP-1 mRNA in a time-dependent manner. Results are representative of two independent experiments. All data except (E) were analysed by ANOVA followed by Newman-Keuls post-test.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "down-regulation", "start": 120, "end": 135}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 153, "end": 168}]}, {"trigger": {"text": "decrease", "start": 567, "end": 575}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 623, "end": 638}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 153, "end": 168}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 139, "end": 145}, {"role": "Site", "text": "serine", "start": 146, "end": 152}]}, {"trigger": {"text": "phosphorylation", "start": 404, "end": 419}, "arguments": [{"role": "Theme", "text": "activated transcription factor 2", "start": 423, "end": 455}]}, {"trigger": {"text": "phosphorylation", "start": 623, "end": 638}, "arguments": [{"role": "Site", "text": "serine", "start": 616, "end": 622}, {"role": "Theme", "text": "GATA-3", "start": 650, "end": 656}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 608, "end": 615}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 623, "end": 638}]}, {"trigger": {"text": "induced", "start": 892, "end": 899}, "arguments": [{"role": "Theme", "text": "MKP-1", "start": 900, "end": 905}]}, {"trigger": {"text": "induces", "start": 1085, "end": 1092}, "arguments": [{"role": "Theme", "text": "MKP-1", "start": 1093, "end": 1098}]}], "regulation": [{"trigger": {"text": "associated", "start": 95, "end": 105}, "arguments": [{"role": "Theme", "text": "down-regulation", "start": 120, "end": 135}]}, {"trigger": {"text": "effect", "start": 379, "end": 385}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 404, "end": 419}]}, {"trigger": {"text": "dependent", "start": 930, "end": 939}, "arguments": [{"role": "Theme", "text": "induced", "start": 892, "end": 899}]}, {"trigger": {"text": "dependent", "start": 1114, "end": 1123}, "arguments": [{"role": "Theme", "text": "induces", "start": 1085, "end": 1092}]}]}}, "schema": []}
{"input": "Fluticasone propionate competes with phospho-GATA-3 for importin-alpha.\n(A) schematic representation of the in vitro binding competition assay. (B) GR isolated from FP (10-8 M) stimulated cells enhances GR-importin-alpha binding in the presence (*) and absence (==blacksquare, square, filled==) of activated GATA-3. * p<0.05 compared to no activated GR. (C) GATA-3 isolated from anti-CD3/CD28-stimulated cells does not attenuate GR-importin-alpha association. *p<0.05 compared to control. (D) Activated GR blocks the ability of purified phospho-GATA-3 isolated from anti-CD3/CD28-stimulated cells interacting with immobilised importin-alpha in an in vitro binding assay. *p<0.05 compared to GATA-3 isolated from unstimulated cells. #p<0.05 compared to stimulated GATA-3-importin binding. (E) The effect of activated (*) versus unstimulated (o) GR on attenuation of GATA-3-importin-alpha association was concentration-dependent. *p<0.05, **p<0.01 between groups. All results are expressed as mean+/-SEM of three independent experiments and analysed by ANOVA followed by Newman-Keuls post-test.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 221, "end": 228}, "arguments": [{"role": "Theme", "text": "GR", "start": 203, "end": 205}]}, {"trigger": {"text": "association", "start": 447, "end": 458}, "arguments": [{"role": "Theme", "text": "GR", "start": 429, "end": 431}]}, {"trigger": {"text": "interacting", "start": 597, "end": 608}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 545, "end": 551}]}, {"trigger": {"text": "binding", "start": 779, "end": 786}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 763, "end": 769}]}, {"trigger": {"text": "association", "start": 887, "end": 898}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 865, "end": 871}]}], "negative regulation": [{"trigger": {"text": "attenuate", "start": 419, "end": 428}, "arguments": [{"role": "Cause", "text": "GATA-3", "start": 358, "end": 364}, {"role": "Theme", "text": "association", "start": 447, "end": 458}]}, {"trigger": {"text": "blocks", "start": 506, "end": 512}, "arguments": [{"role": "Cause", "text": "GR", "start": 503, "end": 505}, {"role": "Theme", "text": "interacting", "start": 597, "end": 608}]}, {"trigger": {"text": "attenuation", "start": 850, "end": 861}, "arguments": [{"role": "Cause", "text": "GR", "start": 844, "end": 846}, {"role": "Theme", "text": "association", "start": 887, "end": 898}]}], "phosphorylation": [{"trigger": {"text": "phospho", "start": 37, "end": 44}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 45, "end": 51}]}, {"trigger": {"text": "phospho", "start": 537, "end": 544}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 545, "end": 551}]}], "positive regulation": [{"trigger": {"text": "enhances", "start": 194, "end": 202}, "arguments": [{"role": "Cause", "text": "GR", "start": 148, "end": 150}, {"role": "Theme", "text": "binding", "start": 221, "end": 228}]}, {"trigger": {"text": "activated", "start": 340, "end": 349}, "arguments": [{"role": "Theme", "text": "GR", "start": 350, "end": 352}]}, {"trigger": {"text": "Activated", "start": 493, "end": 502}, "arguments": [{"role": "Theme", "text": "GR", "start": 503, "end": 505}]}, {"trigger": {"text": "stimulated", "start": 752, "end": 762}, "arguments": [{"role": "Theme", "text": "binding", "start": 779, "end": 786}]}, {"trigger": {"text": "activated", "start": 806, "end": 815}, "arguments": [{"role": "Theme", "text": "GR", "start": 844, "end": 846}]}, {"trigger": {"text": "unstimulated", "start": 827, "end": 839}, "arguments": [{"role": "Theme", "text": "GR", "start": 844, "end": 846}]}], "regulation": [{"trigger": {"text": "dependent", "start": 917, "end": 926}, "arguments": [{"role": "Theme", "text": "attenuation", "start": 850, "end": 861}]}]}}, "schema": []}
{"input": "Fluticasone propionate does not affect GATA-3 nuclear export.\n(A) Western blot analysis showing that the nuclear export inhibitor leptomycin B (2 nM) does not affect the ability of FP (10-8 M) to prevent anti-CD3/CD28-stimulated GATA-3 nuclear localization measured at 60 min. ***p<0.001 compared to unstimulated cells, ###p<0.001 compared to anti-CD3/CD28-stimulated cells. (B) Western blot analysis showing that FP (10-8 M) does not affect whole-cell GATA-3 degradation over 17 h. (C) GFP-tagged GATA-3 is overexpressed and cells stimulated (b-j) or not (a) with anti-CD3/CD28. The effect of treating cells with FP (10-8 M, f-j) after 30 min stimulation with anti-CD3/CD28 is also shown. (D) FP (10-8 M) does not prevent anti-CD3/CD28-stimulated p65 nuclear translocation at 60 min after stimulation. **p<0.01 compared to unstimulated cells. All results are representative of at least four independent experiments and are shown as mean+/-SEM. Results were analysed by ANOVA followed by Newman-Keuls test.", "output": {"json_structures": {"localization": [{"trigger": {"text": "export", "start": 54, "end": 60}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 39, "end": 45}]}, {"trigger": {"text": "localization", "start": 244, "end": 256}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 229, "end": 235}, {"role": "ToLoc", "text": "nuclear", "start": 236, "end": 243}]}, {"trigger": {"text": "translocation", "start": 760, "end": 773}, "arguments": [{"role": "Theme", "text": "p65", "start": 748, "end": 751}, {"role": "ToLoc", "text": "nuclear", "start": 752, "end": 759}]}], "negative regulation": [{"trigger": {"text": "prevent", "start": 196, "end": 203}, "arguments": [{"role": "Theme", "text": "localization", "start": 244, "end": 256}]}, {"trigger": {"text": "prevent", "start": 715, "end": 722}, "arguments": [{"role": "Theme", "text": "translocation", "start": 760, "end": 773}]}], "positive regulation": [{"trigger": {"text": "stimulated", "start": 218, "end": 228}, "arguments": [{"role": "Theme", "text": "localization", "start": 244, "end": 256}]}, {"trigger": {"text": "stimulated", "start": 737, "end": 747}, "arguments": [{"role": "Theme", "text": "translocation", "start": 760, "end": 773}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 460, "end": 471}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 453, "end": 459}]}], "regulation": [{"trigger": {"text": "affect", "start": 32, "end": 38}, "arguments": [{"role": "Theme", "text": "export", "start": 54, "end": 60}]}, {"trigger": {"text": "affect", "start": 159, "end": 165}, "arguments": [{"role": "Theme", "text": "prevent", "start": 196, "end": 203}]}, {"trigger": {"text": "affect", "start": 435, "end": 441}, "arguments": [{"role": "Theme", "text": "degradation", "start": 460, "end": 471}]}]}}, "schema": []}
{"input": "Fluticasone propionate impairs GATA-3 interaction with importin-alpha and GATA-3 nuclear localization in vivo and ex vivo.\n(A and B) Co-immunoprecipitation analysis of PBMCs from steroid-naive asthma patients treated with FP in vitro demonstrated impaired interaction between GATA-3 and importin-alpha measured at 60 min. Each bar represents the mean+/-SEM of at least three independent experiments; *** p<0.001 compared with control as determined by ANOVA/Newman-Keuls analysis. (C and D) Co-immunoprecipitation analyses of PBMCs from steroid-naive asthma patients treated with inhaled FP (500 microg via a spacer) in vivo demonstrated decreased association between GATA-3 and importin-alpha. The individual values for each treatment are presented graphically. (E) Representative Western blot showing that importin-alpha expression was unaffected by inhalation of FP. Blot is representative of gels from three participants.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 38, "end": 49}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 31, "end": 37}]}, {"trigger": {"text": "interaction", "start": 256, "end": 267}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 276, "end": 282}]}, {"trigger": {"text": "association", "start": 647, "end": 658}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 667, "end": 673}]}], "localization": [{"trigger": {"text": "localization", "start": 89, "end": 101}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 74, "end": 80}, {"role": "ToLoc", "text": "nuclear", "start": 81, "end": 88}]}], "negative regulation": [{"trigger": {"text": "impairs", "start": 23, "end": 30}, "arguments": [{"role": "Theme", "text": "interaction", "start": 38, "end": 49}]}, {"trigger": {"text": "impairs", "start": 23, "end": 30}, "arguments": [{"role": "Theme", "text": "localization", "start": 89, "end": 101}]}, {"trigger": {"text": "impaired", "start": 247, "end": 255}, "arguments": [{"role": "Theme", "text": "interaction", "start": 256, "end": 267}]}, {"trigger": {"text": "decreased", "start": 637, "end": 646}, "arguments": [{"role": "Theme", "text": "association", "start": 647, "end": 658}]}]}}, "schema": []}
{"input": "Inhaled fluticasone propionate impairs GATA-3 nuclear localization in PBMCs.\n(A) Representative immunocytochemistry of showing the effect of inhaled FP (500 microg) on GR and GATA-3 nuclear localisation. (B) Nuclear GATA-3 immunoreactivity in PBMCs from seven steroid-naive asthma patients 2 h following inhaled FP treatment (100 or 500 microg via spacer). The median and interquartile ranges for each treatment are presented as a box-and-whiskers plot (n = 7); * p<0.05 Wilcoxon's rank test compared with placebo. (C) Immunoblotting analyses of PBMCs demonstrated a time-dependent decrease in nuclear expression of GATA-3, and increased cytoplasmic GATA-3 expression after inhalation of FP. (D) Immunoblotting analyses of PBMCs demonstrated a dose-dependent decrease in nuclear expression of GATA-3, and increased cytoplasmic GATA-3 expression 2 h after inhalation of FP. Histone H1 and MEK-1 immunoblotting confirmed equivalent total protein loading for the nuclear and cytoplasmic fractions respectively. Quantification of the densitometry data in (C) and (D) is shown as a box-and-whiskers plot of results from n = 6 participants for which data were available. *p<0.05 compared to control. (E) Western blot analyses of PBMCs demonstrated a time-dependent decrease in dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK after inhalation of FP (500 microg). The results shown in (E) are representative of samples from two participants.", "output": {"json_structures": {"localization": [{"trigger": {"text": "localization", "start": 54, "end": 66}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 39, "end": 45}, {"role": "ToLoc", "text": "nuclear", "start": 46, "end": 53}]}, {"trigger": {"text": "localisation", "start": 190, "end": 202}, "arguments": [{"role": "Theme", "text": "GR", "start": 168, "end": 170}, {"role": "ToLoc", "text": "nuclear", "start": 182, "end": 189}]}, {"trigger": {"text": "localisation", "start": 190, "end": 202}, "arguments": [{"role": "Theme", "text": "GATA-3", "start": 175, "end": 181}, {"role": "ToLoc", "text": "nuclear", "start": 182, "end": 189}]}, {"trigger": {"text": "expression", "start": 602, "end": 612}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 594, "end": 601}, {"role": "Theme", "text": "GATA-3", "start": 616, "end": 622}]}, {"trigger": {"text": "expression", "start": 657, "end": 667}, "arguments": [{"role": "ToLoc", "text": "cytoplasmic", "start": 638, "end": 649}, {"role": "Theme", "text": "GATA-3", "start": 650, "end": 656}]}, {"trigger": {"text": "expression", "start": 779, "end": 789}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 771, "end": 778}, {"role": "Theme", "text": "GATA-3", "start": 793, "end": 799}]}, {"trigger": {"text": "expression", "start": 834, "end": 844}, "arguments": [{"role": "ToLoc", "text": "cytoplasmic", "start": 815, "end": 826}, {"role": "Theme", "text": "GATA-3", "start": 827, "end": 833}]}], "negative regulation": [{"trigger": {"text": "impairs", "start": 31, "end": 38}, "arguments": [{"role": "Theme", "text": "localization", "start": 54, "end": 66}]}, {"trigger": {"text": "decrease", "start": 582, "end": 590}, "arguments": [{"role": "Theme", "text": "expression", "start": 602, "end": 612}]}, {"trigger": {"text": "decrease", "start": 759, "end": 767}, "arguments": [{"role": "Theme", "text": "expression", "start": 779, "end": 789}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 628, "end": 637}, "arguments": [{"role": "Theme", "text": "expression", "start": 657, "end": 667}]}], "regulation": [{"trigger": {"text": "dependent", "start": 572, "end": 581}, "arguments": [{"role": "Theme", "text": "decrease", "start": 582, "end": 590}]}, {"trigger": {"text": "dependent", "start": 572, "end": 581}, "arguments": [{"role": "Theme", "text": "increased", "start": 628, "end": 637}]}]}}, "schema": []}
{"input": "Degraded Carrageenan Causing Colitis in Rats Induces TNF Secretion and ICAM-1 Upregulation in Monocytes through NF-kappaB Activation\nCarrageenan (CGN) is a high molecular weight sulphated polysaccharide derived from red seaweeds. In rodents, its degraded forms (dCGN) can induce intestinal inflammation associated with macrophage recruitment and activation. The aim of this study was: 1) to analyze the size-dependent effects of dCGN on colon inflammation in vivo, and 2) to correlate these effects with monocyte/macrophage proliferation, cytokine production and expression of various cell surface antigens including ICAM-1 adhesion molecule. Peripheral blood monocytes (PBM) and THP-1 monocytic cells were cultured in the presence of either 10 or 40 kDa, dCGN. The 40 kDa, but not the 10 kDa dCGN, induced colitis in in vivo. Degraded CGN inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-alpha expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-kappaB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 953, "end": 963}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 946, "end": 952}]}, {"trigger": {"text": "expression", "start": 1192, "end": 1202}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1182, "end": 1191}]}], "localization": [{"trigger": {"text": "Secretion", "start": 57, "end": 66}, "arguments": [{"role": "Theme", "text": "TNF", "start": 53, "end": 56}]}, {"trigger": {"text": "secretion", "start": 1207, "end": 1216}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1182, "end": 1191}]}], "positive regulation": [{"trigger": {"text": "Induces", "start": 45, "end": 52}, "arguments": [{"role": "Theme", "text": "Secretion", "start": 57, "end": 66}]}, {"trigger": {"text": "Induces", "start": 45, "end": 52}, "arguments": [{"role": "Theme", "text": "Upregulation", "start": 78, "end": 90}]}, {"trigger": {"text": "Upregulation", "start": 78, "end": 90}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 71, "end": 77}]}, {"trigger": {"text": "through", "start": 104, "end": 111}, "arguments": [{"role": "Theme", "text": "Induces", "start": 45, "end": 52}]}, {"trigger": {"text": "increased", "start": 936, "end": 945}, "arguments": [{"role": "Theme", "text": "expression", "start": 953, "end": 963}]}, {"trigger": {"text": "stimulated", "start": 1171, "end": 1181}, "arguments": [{"role": "Theme", "text": "expression", "start": 1192, "end": 1202}]}, {"trigger": {"text": "stimulated", "start": 1171, "end": 1181}, "arguments": [{"role": "Theme", "text": "secretion", "start": 1207, "end": 1216}]}]}}, "schema": []}
{"input": "Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80degreesC) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]-[8].\nAlthough the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-kappaB (NF-kappaB) activation [13], [14]. NF-kappaB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16].\nMacrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-alpha expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-kappaB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 1292, "end": 1302}, "arguments": [{"role": "Theme", "text": "IL-8", "start": 1287, "end": 1291}]}, {"trigger": {"text": "expression", "start": 2676, "end": 2686}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 2669, "end": 2675}]}, {"trigger": {"text": "expression", "start": 2763, "end": 2773}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 2753, "end": 2762}]}], "localization": [{"trigger": {"text": "secretion", "start": 2778, "end": 2787}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 2753, "end": 2762}]}], "positive regulation": [{"trigger": {"text": "via", "start": 1357, "end": 1360}, "arguments": [{"role": "Theme", "text": "production", "start": 1292, "end": 1302}]}, {"trigger": {"text": "increased", "start": 2659, "end": 2668}, "arguments": [{"role": "Theme", "text": "expression", "start": 2676, "end": 2686}]}, {"trigger": {"text": "stimulated", "start": 2742, "end": 2752}, "arguments": [{"role": "Theme", "text": "expression", "start": 2763, "end": 2773}]}, {"trigger": {"text": "stimulated", "start": 2742, "end": 2752}, "arguments": [{"role": "Theme", "text": "secretion", "start": 2778, "end": 2787}]}]}}, "schema": []}
{"input": "Preparation of Degraded Carrageenan\nTwo preparations of degraded carrageenan with low, (~10 kDa; C10), and medium, (~40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60degreesC. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80degreesC. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60degreesC. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate(R) kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56degreesC.", "output": {"json_structures": {}}, "schema": []}
{"input": "Animals, Chemicals and Diet\nMale Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily.", "output": {"json_structures": {}}, "schema": []}
{"input": "Evaluation of Colitis\nBody weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4degreesC until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 micromol of peroxide per minute at 25degreesC.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressed", "start": 1135, "end": 1144}, "arguments": [{"role": "Theme", "text": "MPO", "start": 1123, "end": 1126}]}]}}, "schema": []}
{"input": "Cell Culture\nAll tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37degreesC in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 microg/ml) (Tebu, Le Perray en Yvelines, France) was added.", "output": {"json_structures": {}}, "schema": []}
{"input": "Cell Cycle Analysis\nTHP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis.", "output": {"json_structures": {}}, "schema": []}
{"input": "Cell Surface Antigen Expression Analysis\nPeripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5x105 cells were incubated with primary antibodies at 4degreesC for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4degreesC for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells.", "output": {"json_structures": {}}, "schema": []}
{"input": "TNF Activity Bioassay\nMonocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-alpha/beta in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml.", "output": {"json_structures": {}}, "schema": []}
{"input": "RT-PCR Analysis\nTotal RNA from monocytes was isolated using TRIzol Reagent(TM) (Invitrogen). cDNA was generated on 1 microg of total RNA in a reaction volume of 20 microl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 microl of the cDNA solution, 50 microM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92degreesC, 1 min at 58degreesC and 1 min at 72degreesC; then each PCR cycle consisted of 40 sec at 92degreesC, 40 sec at 58degreesC and 50 sec at 72degreesC. cDNA for beta-actin was amplified for 28 cycles using the oligos: sense 5'-GGCATCGTGATGGACTCCG-3' and antisense 5'GCTGGAAGGTGGACAGCGA-3'. cDNA for TNF-alpha was amplified for 35 cycles using the oligos: sense 5'-AAGCCTGTAGCCCATGTTGT-3' and antisense 5'-CAGATAGATGGGCTCATACC-3'. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5'-GTAGCAGCCGCAGTCATAATGG-3' and antisense 5'-A TGCTGTTGTATCTGACTGAGG-3'.", "output": {"json_structures": {}}, "schema": []}
{"input": "NF-kB Transcription Reporter Gene Assay\nThe plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-kappaB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI).", "output": {"json_structures": {}}, "schema": []}
{"input": "Western Blot Analysis\nTHP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 microg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 microg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-kappaBalpha protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IkappaB bands were revealed using the ECL(TM) detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to alpha-Tubulin (Santa Cruz) was use as loading control.\nFor nuclear NF-kappaB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37degreesC. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100degreesC. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-kappaB p50 subunit (# sc-114) or anti-NF-kappaB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32].", "output": {"json_structures": {}}, "schema": []}
{"input": "Analysis of NF-kappaB Activation by Flow Cytometry\nNuclear activation of NF-kappabeta by flow cytometry was performed as described [31].", "output": {"json_structures": {}}, "schema": []}
{"input": "Statistical Analysis\nThe results were expressed as the mean value +/- S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA).", "output": {"json_structures": {}}, "schema": []}
{"input": "Degraded CGN Induce Colonic Inflammation\nAll rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed.", "output": {"json_structures": {}}, "schema": []}
{"input": "Degraded CGN Induced-TNF-alpha Production by Monocytes In Vitro\nIn order to study the capacity of dCGN to stimulate TNF-alpha production, peripheral blood monocytes were cultivated in the presence of dCGN (0.1 to 1 mg/ml). Very low levels of TNF-alpha were induced in PBM after stimulation with native CGN (Fig. 2A). Addition of 0.1 mg/ml 10 kDa dCGN resulted in approximately a 60-fold increase in TNF-alpha production by PBM. This was a dose-dependent effect that reached a 180-fold increase when cells were exposed to 1 mg/ml of 10 kDa dCGN (Fig. 2A). A 250-fold increase in TNF-alpha production was detected at 1 mg/ml 40 kDa dCGN (Fig. 2A). TNF-alpha production increased in time reaching a maximum level at 8 hours of culture (Figure 2B). After 24 h, the amount of secreted TNF-alpha was still one third of the total TNF-alpha. Lipopolysaccharide (LPS), a known activator of immune cells also induced TNF-alpha production with similar kinetics as dCGN (Fig. 2B). However, the amount of TNF-alpha produced by LPS was 4-fold less than the one produced by dCGN and it was not detected after 8 hours of culture (Fig. 2B). Similarly, monocytic THP-1 cells cultivated in the presence of variable concentration of dCGN showed an increase in TNF-alpha production (Fig. 2C). This increase in TNF-alpha production was significantly smaller (about 10-fold) than the one presented by PBM (Fig. 2A). No TNF-alpha was released from THP-1 cells exposed to native CGN (not shown). TNF-alpha production by THP-1 cells was not dose dependent to the amount of dCGN used. Also there was no difference between the two forms (10 and 40 kDa) of dCGN (Fig. 2C). Interestingly, TNF-alpha release from THP-1 cells stimulated with dCGN reached a maximum level at 32 h, while stimulation with LPS reached a maximum level at 56 h (Fig. 2D).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Production", "start": 31, "end": 41}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 21, "end": 30}]}, {"trigger": {"text": "production", "start": 126, "end": 136}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 116, "end": 125}]}, {"trigger": {"text": "production", "start": 409, "end": 419}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 399, "end": 408}]}, {"trigger": {"text": "production", "start": 588, "end": 598}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 578, "end": 587}]}, {"trigger": {"text": "production", "start": 656, "end": 666}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 646, "end": 655}]}, {"trigger": {"text": "production", "start": 917, "end": 927}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 907, "end": 916}]}, {"trigger": {"text": "produced", "start": 1002, "end": 1010}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 992, "end": 1001}]}, {"trigger": {"text": "produced", "start": 1047, "end": 1055}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 992, "end": 1001}]}, {"trigger": {"text": "production", "start": 1250, "end": 1260}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1240, "end": 1249}]}, {"trigger": {"text": "production", "start": 1299, "end": 1309}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1289, "end": 1298}]}, {"trigger": {"text": "production", "start": 1481, "end": 1491}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1471, "end": 1480}]}], "localization": [{"trigger": {"text": "secreted", "start": 771, "end": 779}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 780, "end": 789}]}, {"trigger": {"text": "released", "start": 1410, "end": 1418}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1396, "end": 1405}]}, {"trigger": {"text": "release", "start": 1669, "end": 1676}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1659, "end": 1668}]}], "positive regulation": [{"trigger": {"text": "Induced", "start": 13, "end": 20}, "arguments": [{"role": "Theme", "text": "Production", "start": 31, "end": 41}]}, {"trigger": {"text": "stimulate", "start": 106, "end": 115}, "arguments": [{"role": "Theme", "text": "production", "start": 126, "end": 136}]}, {"trigger": {"text": "induced", "start": 257, "end": 264}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 242, "end": 251}]}, {"trigger": {"text": "stimulation", "start": 278, "end": 289}, "arguments": [{"role": "Theme", "text": "induced", "start": 257, "end": 264}]}, {"trigger": {"text": "resulted", "start": 351, "end": 359}, "arguments": [{"role": "Theme", "text": "increase", "start": 387, "end": 395}]}, {"trigger": {"text": "increase", "start": 387, "end": 395}, "arguments": [{"role": "Theme", "text": "production", "start": 409, "end": 419}]}, {"trigger": {"text": "increase", "start": 566, "end": 574}, "arguments": [{"role": "Theme", "text": "production", "start": 588, "end": 598}]}, {"trigger": {"text": "increased", "start": 667, "end": 676}, "arguments": [{"role": "Theme", "text": "production", "start": 656, "end": 666}]}, {"trigger": {"text": "induced", "start": 899, "end": 906}, "arguments": [{"role": "Theme", "text": "production", "start": 917, "end": 927}]}, {"trigger": {"text": "by", "start": 1011, "end": 1013}, "arguments": [{"role": "Theme", "text": "produced", "start": 1002, "end": 1010}]}, {"trigger": {"text": "by", "start": 1056, "end": 1058}, "arguments": [{"role": "Theme", "text": "produced", "start": 1047, "end": 1055}]}, {"trigger": {"text": "increase", "start": 1228, "end": 1236}, "arguments": [{"role": "Theme", "text": "production", "start": 1250, "end": 1260}]}, {"trigger": {"text": "This increase", "start": 1272, "end": 1285}, "arguments": [{"role": "Theme", "text": "production", "start": 1299, "end": 1309}]}], "regulation": [{"trigger": {"text": "dependent", "start": 444, "end": 453}, "arguments": [{"role": "Theme", "text": "resulted", "start": 351, "end": 359}]}, {"trigger": {"text": "dependent", "start": 1520, "end": 1529}, "arguments": [{"role": "Theme", "text": "production", "start": 1481, "end": 1491}]}]}}, "schema": []}
{"input": "Effect of Native and Degraded CGN on THP-1 Proliferation and Cell Cycle\nPreliminary observations by enumeration of THP-1 cells exposed to different concentrations of native and dCGN (10 and 40 kDa) during 2, 5 and 7 days, showed a decline in cell number (data not shown). This suggested that dCGN might cause an alteration in the cell cycle. Cell cycle analysis using flow cytometry showed an accumulation of THP-1 cells in G0/G1 phase, which was associated with a decrease number of cells in the S phase (Fig. 3A). The percentage of cells in the G0/G1 phase was 45.2% for control cells, 62.6% for C10 dCGN (at 2 mg/ml), and 64.2% for C40 dCGN-treated cells (at 2 mg/ml) (Fig. 3B). The effect of dCGN on cell cycle was dose-dependent (Fig. 3B). Neither native nor dCGN had an effect on the number of cells in the G2/M phase (Fig. 3). This effect is not due to cytotoxicity of dCGN even at the highest concentration (i.e. 2 mg/ml) since cell viability was not affected (data not shown).", "output": {"json_structures": {}}, "schema": []}
{"input": "ICAM-1 Expression Is Induced by Degraded CGN and Is Responsible for Monocytes Aggregation In Vitro\nIn order to study the effect of dCGN on the expression of cell surface antigens, PBM and THP-1 cells were incubated for 36 h in the presence and absence of dCGN. The expression of various cell surface molecules was analyzed by flow cytometry as described in materials and methods. Both forms of dCGN clearly stimulated expression of ICAM-1 (CD54) on PBM and THP-1 cells (Fig. 4A). The increase in ICAM-1 expression was higher on THP-1 cells treated with 40 kDa dCGN (Fig. 4B). Another surface antigen, the lymphocyte function-associated antigen 3 (CD58) was slightly reduced on PBM after treatment with 40 kDa dCGN (Fig. 4B). Interestingly, expression of major histocompatibility complex molecules of class I (HLA-ABC) and of class II (HLA-DR), as well as the monocyte marker CD14, seemed to be reduced by treatment with dCGN (Fig. 4B). However, these differences were not statistically significant.\nTreatment with dCGN also induced a strong aggregation of monocytes, detected by phase contrast inverse microscopy (Fig. 5). Although this effect was easily observed in monocytes incubated with the 10 kDa dCGN (Fig. 5B), a more robust cell aggregation was observed in monocytes incubated with the 40 kDa dCGN (Fig. 5C). ICAM-1 has been proposed to be the main adhesion molecule responsible for monocyte aggregation. To confirm this, monocytes were incubated with both types of dCGN in the presence of an anti-ICAM-1 antibody, an anti-CD58 antibody and an isotype control IgG1 antibody. The anti-ICAM-1 antibody effectively blocked the cell aggregates induced by dCGN (Fig. 5D, 5E), strongly suggesting that indeed ICAM-1 is responsible for monocyte aggregation. Both the control IgG1 and the anti-CD58 antibody did not modify monocyte aggregation (data not shown).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Expression", "start": 7, "end": 17}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 0, "end": 6}]}, {"trigger": {"text": "expression", "start": 418, "end": 428}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 432, "end": 438}]}, {"trigger": {"text": "expression", "start": 503, "end": 513}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 496, "end": 502}]}, {"trigger": {"text": "expression", "start": 740, "end": 750}, "arguments": [{"role": "Theme", "text": "CD14", "start": 875, "end": 879}]}], "negative regulation": [{"trigger": {"text": "reduced", "start": 666, "end": 673}, "arguments": [{"role": "Theme", "text": "lymphocyte function-associated antigen 3", "start": 605, "end": 645}]}, {"trigger": {"text": "reduced", "start": 894, "end": 901}, "arguments": [{"role": "Theme", "text": "expression", "start": 740, "end": 750}]}], "positive regulation": [{"trigger": {"text": "Induced", "start": 21, "end": 28}, "arguments": [{"role": "Theme", "text": "Expression", "start": 7, "end": 17}]}, {"trigger": {"text": "stimulated", "start": 407, "end": 417}, "arguments": [{"role": "Theme", "text": "expression", "start": 418, "end": 428}]}, {"trigger": {"text": "increase", "start": 484, "end": 492}, "arguments": [{"role": "Theme", "text": "expression", "start": 503, "end": 513}]}]}}, "schema": []}
{"input": "Degraded CGN Induce an Increase in ICAM-1 and TNF-alpha mRNA Expression\nThe increase in surface ICAM-1 expression and TNF-alpha production by monocytes correlated with an upregulation of mRNA for these molecules. Both 10 kDa and 40 kDa dCGN induced a robust increase in mRNA for both ICAM-1 and TNF-alpha (Fig. 6). beta-actin mRNA levels were not affected by dCGN treatment.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 103, "end": 113}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 96, "end": 102}]}, {"trigger": {"text": "production", "start": 128, "end": 138}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 118, "end": 127}]}], "positive regulation": [{"trigger": {"text": "Induce", "start": 13, "end": 19}, "arguments": [{"role": "Theme", "text": "Increase", "start": 23, "end": 31}]}, {"trigger": {"text": "Increase", "start": 23, "end": 31}, "arguments": [{"role": "Theme", "text": "mRNA Expression", "start": 56, "end": 71}]}, {"trigger": {"text": "increase", "start": 76, "end": 84}, "arguments": [{"role": "Theme", "text": "expression", "start": 103, "end": 113}]}, {"trigger": {"text": "increase", "start": 76, "end": 84}, "arguments": [{"role": "Theme", "text": "production", "start": 128, "end": 138}]}, {"trigger": {"text": "upregulation", "start": 171, "end": 183}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 118, "end": 127}]}, {"trigger": {"text": "upregulation", "start": 171, "end": 183}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 96, "end": 102}]}, {"trigger": {"text": "induced", "start": 241, "end": 248}, "arguments": [{"role": "Theme", "text": "increase", "start": 258, "end": 266}]}, {"trigger": {"text": "increase", "start": 258, "end": 266}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 284, "end": 290}]}, {"trigger": {"text": "increase", "start": 258, "end": 266}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 295, "end": 304}]}], "regulation": [{"trigger": {"text": "affected", "start": 347, "end": 355}, "arguments": [{"role": "Theme", "text": "beta-actin", "start": 315, "end": 325}]}], "transcription": [{"trigger": {"text": "mRNA Expression", "start": 56, "end": 71}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 35, "end": 41}]}, {"trigger": {"text": "mRNA Expression", "start": 56, "end": 71}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 46, "end": 55}]}]}}, "schema": []}
{"input": "Degraded CGN Induce IkappaB Degradation and NF-kappaB Activation\nThe expression of genes encoding for ICAM-1 and TNF-alpha is controlled by the nuclear factor NF-kappaB. Site-specific phosphorylation of the inhibitor IkappaB leads to its degradation by proteasome and to a consequential activation of the NF-kappaB pathway. Using a reporter plasmid for NF-kappaB activation, it was confirmed that dCGN induced a strong activation of NF-kappaB, as reflected by an increase in luciferase activity (Fig. 7A). Both forms of dCGN used induced NF-kappaB activation in a dose dependent manner. However, the effect was more strongly induced by the 40 kDa dCGN (Fig. 7A). These results were further confirmed by directly detecting NF-kappaB in the cell nucleus by Western blotting (Fig. 7C) and by FACS (Fig. 7D). These assays also allowed us to determine what NF-kappaB subunits were activated by dCGN. Both forms (10 or 40 kDa) of dCGN induced activation of the p50 and p65 subunits of NF-kappaB. This nuclear factor was present in low levels in the cell nucleus and increased considerably after treatment with dCGN. Western blots suggested the the 40 kDa form of dCGN induced a stronger activation of NF-kappaB (Fig. 7C). A more sentive assay for nuclear factor activation is flow cytometry of nuclei stained with specific antibodies for the nuclear factor of interest. In agreement with the previous data, FACS analysis of nuclei from THP-1 cells showed that there was a basal level of nuclear NF-kappaB (Fig. 7D). Again, both forms (10 or 40 kDa) of dCGN induced an increase of the p50 and p65 subunits of NF-kappaB in the nucleus of these cells. The 40 kDa degraded CGN gave a stronger increase of NF-kappaB (Fig. 7D). These data strongly suggest that the heterodimer p50/p65 is the NF-kappaB isoform activated by degraded CGN in monocytes. In addition, degradation of the inhibitor IkappaBalpha was also observed in cells treated with dCGN (Fig. 7B). No significant IkappaBalpha degradation was detected within two hours of dCGN treatment, but IkappaBalpha was markedly degraded by four hours of dCGN treatment (Fig. 7B). We focused on IkappaBalpha subunit, since it masks the nuclear localisation sequence of p65, it is the most rapidly degraded subunit and the most studied one.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 69, "end": 79}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 102, "end": 108}]}, {"trigger": {"text": "expression", "start": 69, "end": 79}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 113, "end": 122}]}], "negative regulation": [{"trigger": {"text": "masks", "start": 2165, "end": 2170}, "arguments": [{"role": "Cause", "text": "IkappaBalpha", "start": 2134, "end": 2146}, {"role": "Site", "text": "nuclear localisation sequence", "start": 2175, "end": 2204}, {"role": "Theme", "text": "p65", "start": 2208, "end": 2211}]}], "positive regulation": [{"trigger": {"text": "increase", "start": 463, "end": 471}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 475, "end": 485}]}, {"trigger": {"text": "induced", "start": 929, "end": 936}, "arguments": [{"role": "Theme", "text": "activation", "start": 937, "end": 947}]}, {"trigger": {"text": "activation", "start": 937, "end": 947}, "arguments": [{"role": "Theme", "text": "p50", "start": 955, "end": 958}]}, {"trigger": {"text": "activation", "start": 937, "end": 947}, "arguments": [{"role": "Theme", "text": "p65", "start": 963, "end": 966}]}, {"trigger": {"text": "induced", "start": 1551, "end": 1558}, "arguments": [{"role": "Theme", "text": "increase", "start": 1562, "end": 1570}]}, {"trigger": {"text": "increase", "start": 1562, "end": 1570}, "arguments": [{"role": "Theme", "text": "p50", "start": 1578, "end": 1581}]}, {"trigger": {"text": "increase", "start": 1562, "end": 1570}, "arguments": [{"role": "Theme", "text": "p65", "start": 1586, "end": 1589}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 1851, "end": 1862}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1880, "end": 1892}]}, {"trigger": {"text": "degradation", "start": 1977, "end": 1988}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1964, "end": 1976}]}, {"trigger": {"text": "degraded", "start": 2068, "end": 2076}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 2042, "end": 2054}]}, {"trigger": {"text": "degraded", "start": 2236, "end": 2244}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 2134, "end": 2146}]}], "regulation": [{"trigger": {"text": "controlled", "start": 126, "end": 136}, "arguments": [{"role": "Theme", "text": "expression", "start": 69, "end": 79}]}]}}, "schema": []}
{"input": "Inflammation of the intestinal tract is usually associated with infiltration and activation of intestinal macrophages [17]. These macrophages are able to initiate immune responses and can be induced to differentiate into cells that either exacerbate or inhibit the inflammation. Accumulation of different types of leukocytes, including monocytes/macrophages, neutrophils, and lymphocytes in the intestinal mucosa during inflammation, is normally followed by secretion of pro-inflammatory cytokines [33]. Several stimuli can induce these leukocytes to produce and secrete cytokines during inflammation. One of the most potent and known stimuli for leukocyte activation is LPS from Gram-negative bacteria [34]. In addition, other factors are also able to stimulate cytokine secretion from various leukocytes [35], [36]. One such factor is CGN, a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae) [1], [2]. Native CGN is widely used as a food additive (E 407) to improve texture. It is also used in cosmetics and pharmaceuticals. Although native form CGN (200-800 kDa) has been declared harmless to humans [8], its degraded forms (<50 kDa), also known as poligeenan, are widely used to induce colitis in rodents [3]-[5]. These degraded CGN may also have a possible carcinogenic effect [4], [6]-[8]; however this is still controversial.\nAlthough acid treatment at high temperature (80degreesC) is required for CGN hydrolysis in vitro to lower molecular weight dCGN, it is probable that some dCGN are produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Thus, understanding the mechanisms of dCGN-induced bowel inflammation is of great importance. In this report, we have analyzed the role of human monocytes (PBM and THP-1) in dCGN-induced inflammation.\nPreliminary in vivo studies in rats treated with dCGN revealed significant shortening of the large intestine associated with an inflammatory state, i.e. strong infiltration of macrophages to the intestinal mucosa similar to DSS-induced inflammation [20]. Using two fractions of dCGN (10 and 40 kDa), we observed a strong correlation between the severity of the inflammation and the dCGN molecular size, thus confirming the size related inflammation in vivo. This macrophage accumulation was not due to cell proliferation because dCGN inhibited THP-1 monocytes proliferation in vitro. These results are similar to those obtained with human colonic epithelial cells (NCM460 cell line) exposed to native CGN for 1-8 days [37]. Thus, it seems that dCGN promote macrophage infiltration by recruiting new cells to the inflamed intestinal mucosa and not by inducing cell proliferation. In addition, very few polymorphonuclear cells were detected in the mucosa at the time point analyzed as demonstrated by a very low level of MPO in the intestinal tissue.\nThese results suggest that monocytes might produce cytokines associated with activation into macrophages in response to dCGN. Thus, we analyzed the production of TNF by both PBM and THP-1 cells in response to dCGN. Degraded CGN induced a robust production of TNF by monocytes. The 40 kDa form of dCGN was more potent for monocyte stimulation than the 10 kDa or the native ones. Surprisingly, monocyte activation by dCGN to produce TNF was much stronger than the activation induced by LPS, an inflammatory factor considered to be among the most potent stimuli for leukocyte activation. These results underline the fact that partially degraded forms of CGN have important cellular effects. The amount of TNF secreted by PBM induced with LPS was much larger than the one secreted by THP-1 monocytes. Since monocyte activation by LPS is associated with the presence of the CD14 and TLR4 receptors [38]-[40], the different response observed could be due to the different expression of these receptors in PBM and THP-1 cells. CD14 is not expressed by THP-1 monocytes, but it is expressed by PBM. This fact could explain why THP-1 cells produced a much smaller response to LPS than PBM and also the difference of kinetics in the LPS induced TNF secretion. The peak of TNF response was observed after 8 h stimulation followed by a rapid decrease to baseline at 10 h, whereas on THP-1 cells the peak was not reached until after 56 h. Moreover, the amount of TNF secreted by monocytes induced with dCGN was much larger than the one induced by LPS. On the other hand, LPS and dCGN displayed a very different TNF secretion curve in THP-1 cells. These differences suggest that dCGN and LPS could use different activation mechanisms. It is noteworthy that neutralizing antibody to CD14 only partially (<40%) inhibited dCGN-induced TNF secretion (not shown). TLR4 has been recently identified as a surface membrane receptor for CGN in human colonic epithelial cells [41]. Thus, it is possible that TLR4 is activated by dCGN to induce cytokine secretion by monocytes. We can only speculate that TLR4 may have a higher affinity-binding site for dCGN than for LPS, however, this hypothesis remains to be tested.\nAnother indicator that dCGN stimulate monocytes leading to a more active phenotype is the fact that surface expression of the adhesion molecule ICAM-1 was enhanced in both PBM and THP-1 cells. The over expression of ICAM-1 caused the activated monocytes to form cell aggregates that were more abundant among cells treated with the 40 kDa dCGN. This correlates with the higher expression of ICAM-1 induced by the 40 kDa dCGN, suggesting that the partially degraded CGN is more biologically active. In addition, the cell aggregates are reminiscent of monocyte aggregates, which form multinucleated giant cells (MGC) in patients with Crohn's disease [42]. These giant cells were not observed in healthy individuals or in patients with ulcerative colitis [42]. Moreover, increased expression levels of ICAM-1 and LFA-3 (CD58) were also detected in monocytes from patients with Crohn's disease [43], [44]. Thus, degraded CGN clearly can activate monocytes to express an increased number of ICAM-1 adhesion molecules, therefore being capable of creating the conditions characteristic of Crohn's disease symptomatology, i.e. PBM accumulation and MGC formation [45], [46].\nThe NF-kappaB pathway regulates genes responsible for ICAM-1 and TNF-alpha expression. NF-kappaB activation is associated with the degradation of the inhibitor protein IkappaB [47]. Indeed, dCGN induced NF-kappaB activation as shown by degradation of IkappaBalpha, translocation of p65 and p50 sub-units to the nucleus and by activation of an NF-kappaB-responsive luciferase reporter plasmid. Again, a stronger activation of NF-kappaB was induced by the 40 kDa dCGN compared to 10 kDa dCGN, suggesting that the partially degraded CGN is more biologically active. Our data are in agreement with a previous study showing that native CGN also induced activation of NF-kappaB in human colonic epithelial cells [48].\nNF-kappaB pathway is often associated to promote cell survival and cancer cell growth, however it has been sometimes reported to behave as a tumor suppressor, arresting cell proliferation [49]. Such relationship between NF-kB activation and cell cycle regulation has been reported in normal human epidermal cells [50]. Indeed, it has been shown that NF-kappaB activation suppressed cdk4 expression, which is necessary for the transition to S phase. Finally, NF-kappaB activation was reported to induce growth arrest in normal human keratinocytes by a mechanims involving the cdk inhibitor p21 [51]. The effects of dCGN on p21 and cdk4 expression remain to be studied.\nIn these studies, we have demonstrated a direct action of dCGN on monocytes. Monocytes exposed to dCGN acquired an inflammatory phenotype that included higher expression of the adhesion molecule ICAM-1 and TNF-alpha production, via the NF-kappaB pathway. This higher expression of ICAM-1 resulted in formation of cell aggregates similar to those observed in patients with Crohn's disease. We presume that the differential effects of 10 and 40 kDa dCGN to induce these effects on monocytes are tightly linked to their capacity to induce inflammation in vivo. However, in vivo, macrophages do not come in direct contact with the intestinal lumen and are separated by the epithelial barrier. The way by which dCGN may leave the intestinal lumen and cross the epithelial barrier to reach the macrophages is an intriguing open question. One possible explanation resides in the potential of dCGN to \"induce\" cellular and paracellular injurious effects at the intestinal epithelial cell monolayer [48].\nIn conclusion, dCGN inhibited THP-1 cell proliferation in vitro, accumulating the cells in the G1 phase of the cell cycle, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation in vitro, and stimulated TNF-alpha expression and secretion. These responses were more pronounced following 40 kDa dCGN, and were all linked to NF-kappaB activation. 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{"input": "Degraded CGN induced colon inflammation in rats.\nHistograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F).", "output": {"json_structures": {}}, "schema": []}
{"input": "Degraded CGN stimulated TNF secretion from monocytes.\nLevels of TNF released from peripheral blood monocytes (A-B) and THP-1 cells (C-D) after stimulation with dCGN. A: TNF release induced by native CGN (open bars), 10 kDa dCGN (grey bars), or 40 kDa dCGN (black bars). B: Kinetics of TNF release induced by nothing (control; black diamonds), 0.1 mg/ml 10 kDa dCGN (black triangles), 0.1 mg/ml 40 kDa dCGN (black squares), or 10 microg/ml LPS (open squares). C: TNF release induced by nothing (control; open bars), 10 microg/ml LPS (hatched bars), or increasing concentrations of 10 kDa dCGN (grey bars), or 40 kDa dCGN (black bars). D: Kinetics of TNF release induced by nothing (control; black diamonds), 0.1 mg/ml 10 kDa dCGN (black triangles), 0.1 mg/ml 40 kDa dCGN (black squares), or 10 microg/ml LPS (open squares).", "output": {"json_structures": {"localization": [{"trigger": {"text": "secretion", "start": 28, "end": 37}, "arguments": [{"role": "Theme", "text": "TNF", "start": 24, "end": 27}]}, {"trigger": {"text": "released", "start": 68, "end": 76}, "arguments": [{"role": "Theme", "text": "TNF", "start": 64, "end": 67}]}, {"trigger": {"text": "release", "start": 173, "end": 180}, "arguments": [{"role": "Theme", "text": "TNF", "start": 169, "end": 172}]}, {"trigger": {"text": "release", "start": 289, "end": 296}, "arguments": [{"role": "Theme", "text": "TNF", "start": 285, "end": 288}]}, {"trigger": {"text": "release", "start": 466, "end": 473}, "arguments": [{"role": "Theme", "text": "TNF", "start": 462, "end": 465}]}, {"trigger": {"text": "release", "start": 653, "end": 660}, "arguments": [{"role": "Theme", "text": "TNF", "start": 649, "end": 652}]}], "positive regulation": [{"trigger": {"text": "stimulated", "start": 13, "end": 23}, "arguments": [{"role": "Theme", "text": "secretion", "start": 28, "end": 37}]}, {"trigger": {"text": "induced", "start": 181, "end": 188}, "arguments": [{"role": "Theme", "text": "release", "start": 173, "end": 180}]}, {"trigger": {"text": "induced", "start": 297, "end": 304}, "arguments": [{"role": "Theme", "text": "release", "start": 289, "end": 296}]}, {"trigger": {"text": "induced", "start": 474, "end": 481}, "arguments": [{"role": "Theme", "text": "release", "start": 466, "end": 473}]}, {"trigger": {"text": "induced", "start": 661, "end": 668}, "arguments": [{"role": "Theme", "text": "release", "start": 653, "end": 660}]}]}}, "schema": []}
{"input": "Degraded CGN induced THP1 cell cycle arrest in G1 phase.\nTHP-1 cells in exponential growth phase were incubated in the presence or absence of carrageenan for 24 h before being stained with propidium iodide. Cell DNA content was then analyzed by flow cytometry. A: Histograms of cells treated with medium only (control), 10 kDa dCGN (C10), or 40 kDa dCGN (C40). B: Percentage of cells in each phase of the cell cycle when treated with medium only (control), different concentrations of 10 kDa dCGN (C10), of 40 kDa dCGN (C40), or of native CGN (Native).", "output": {"json_structures": {}}, "schema": []}
{"input": "Degraded CGN stimulated ICAM-1 expression in monocytes.\nPeripheral blood monocytes (PBM) or THP-1 cells were incubated in the presence or absence of carrageenan for 24 h before being stained for various cell surface antigens. Antigen expression was then analyzed by flow cytometry. A: Histograms of ICAM-1 expression in cells treated with medium only (control), 10 kDa dCGN (C10), or 40 kDa dCGN (C40). B: Fluorescence intensity for expression of the antigens HLA-ABC, HLA-DR, CD14, ICAM-1, and CD58 in cells treated with medium only (control), 10 kDa dCGN (C10), or 40 kDa dCGN (C40). Data are mean +/- SEM.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 31, "end": 41}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 24, "end": 30}]}, {"trigger": {"text": "expression", "start": 306, "end": 316}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 299, "end": 305}]}, {"trigger": {"text": "expression", "start": 433, "end": 443}, "arguments": [{"role": "Theme", "text": "CD14", "start": 477, "end": 481}]}, {"trigger": {"text": "expression", "start": 433, "end": 443}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 483, "end": 489}]}, {"trigger": {"text": "expression", "start": 433, "end": 443}, "arguments": [{"role": "Theme", "text": "CD58", "start": 495, "end": 499}]}], "positive regulation": [{"trigger": {"text": "stimulated", "start": 13, "end": 23}, "arguments": [{"role": "Theme", "text": "expression", "start": 31, "end": 41}]}]}}, "schema": []}
{"input": "Degraded CGN induced monocytes aggregation in vitro.\nMonocytes were incubated in the absence (A) or the presence of 1 mg/ml 10 kDa dCGN (B), 1 mg/ml 40 kDa dCGN (C), or 1 mg/ml 40 kDa dCGN plus 2.5 microg/ml anti-ICAM-1 antibody (D). Cells were observed by phase contrast inverse microscopy at 150X magnification. Inserts in A and B show a close up of cells at 300X magnification. E: Number of monocyte aggregates in 24 wells plate of PBM cell cultured with nothing (control), with 10 kDa degraded CGN (C10), or with 40 kDa degraded CGN (C40). Some cultures had also 2.5 microg/ml anti-ICAM-1 antibody. Data are mean +/- SEM.", "output": {"json_structures": {}}, "schema": []}
{"input": "Degraded CGN stimulated ICAM-1 and TNF-alpha gene expression in monocytes.\nRepresentative samples of RT-PCR analysis showing over expression of ICAM-1 and TNF-alpha after stimulation of monocytes with 1 g/l of degraded CGN. beta-actin expression was used as normalization gene.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 50, "end": 60}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 24, "end": 30}]}, {"trigger": {"text": "expression", "start": 50, "end": 60}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 35, "end": 44}]}, {"trigger": {"text": "expression", "start": 130, "end": 140}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 144, "end": 150}]}, {"trigger": {"text": "expression", "start": 130, "end": 140}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 155, "end": 164}]}, {"trigger": {"text": "expression", "start": 235, "end": 245}, "arguments": [{"role": "Theme", "text": "beta-actin", "start": 224, "end": 234}]}], "positive regulation": [{"trigger": {"text": "stimulated", "start": 13, "end": 23}, "arguments": [{"role": "Theme", "text": "expression", "start": 50, "end": 60}]}]}}, "schema": []}
{"input": "Degraded CGN activated the NF-kB pathway in monocytes.\nA: THP-1 cells were transfected with a NF-kappaB reporter plasmid driving expression of luciferase. Cells were then treated with various concentrations of 10 kDa (triangles), or 40 kDa dCGN (squares). B: THP-1 cells treated with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) were lysed after various periods of time. Proteins in cell extracts were resolved by SDS-PAGE and then Western blotted for IkappaBalpha or alpha-tubulin as loading control. C: Degraded carrageenans (dCGN) induced activation of NF-kappaB. THP-1 cells were treated with nothing (control), or with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) for 30 minutes at 37degreesC. Nuclei were isolated and lysed. Proteins in nuclear extracts were resolved by SDS-PAGE and then Western blotted for NF-kappaB p50 subunit (p50) or NF-kappaB p65 subunit (p65). Lower panels show Western blots of nuclear ERK revealing equivalent amount of protein in each sample. Data are representative of three separate experiments. D: Degraded carrageenan (dCGN) induced activation of NF-kappaB. Nuclei isolated from THP-1 cells were fluorescence-stained for NF-kappaB p50 subunit or NF-kappaB p65 subunit before (filled area) or after cells were treated with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) for 30 minutes at 37degreesC. Dashed line corresponds to nuclei stained only with secondary fluorescence antibody. Fluorescence intensity was analyzed by flow cytometry as described.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 129, "end": 139}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 143, "end": 153}]}], "positive regulation": [{"trigger": {"text": "driving", "start": 121, "end": 128}, "arguments": [{"role": "Theme", "text": "expression", "start": 129, "end": 139}]}]}}, "schema": []}
{"input": "Transcription factors RUNX1 and RUNX3 in the induction and suppressive function of Foxp3+inducible regulatory T cells\nForkhead box P3 (FOXP3)+CD4+CD25+ inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis. In this study, we show that the transforming growth factor-beta (TGF-beta) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4+ T cells. This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter. Inactivation of the gene encoding RUNX cofactor core-binding factor-beta (CBFbeta) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3. The in vivo conversion of naive CD4+ T cells into Foxp3+ iT reg cells was significantly decreased in adoptively transferred CbfbF/F CD4-cre naive T cells into Rag2-/- mice. Both RUNX1 and RUNX3 siRNA silenced human T reg cells and CbfbF/F CD4-cre mouse T reg cells showed diminished suppressive function in vitro. Circulating human CD4+ CD25high CD127- T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-beta mRNA compared with CD4+CD25- cells. Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells. These data demonstrate Runx transcription factors as a molecular link in TGF-beta-induced Foxp3 expression in iT reg cell differentiation and function.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 473, "end": 480}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 484, "end": 489}, {"role": "Site2", "text": "RUNX binding sites", "start": 518, "end": 536}, {"role": "Theme2", "text": "FOXP3", "start": 544, "end": 549}]}, {"trigger": {"text": "binding", "start": 473, "end": 480}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 494, "end": 499}, {"role": "Site2", "text": "RUNX binding sites", "start": 518, "end": 536}, {"role": "Theme2", "text": "FOXP3", "start": 544, "end": 549}]}], "gene expression": [{"trigger": {"text": "expression", "start": 337, "end": 347}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 390, "end": 395}]}, {"trigger": {"text": "expression", "start": 337, "end": 347}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 400, "end": 405}]}, {"trigger": {"text": "expression", "start": 762, "end": 772}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 776, "end": 781}]}, {"trigger": {"text": "expression", "start": 1423, "end": 1433}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1417, "end": 1422}]}], "localization": [{"trigger": {"text": "colocalized", "start": 1286, "end": 1297}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1265, "end": 1270}, {"role": "ToLoc", "text": "T reg cells", "start": 1314, "end": 1325}]}, {"trigger": {"text": "colocalized", "start": 1286, "end": 1297}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 1275, "end": 1280}, {"role": "ToLoc", "text": "T reg cells", "start": 1314, "end": 1325}]}], "negative regulation": [{"trigger": {"text": "Inactivation", "start": 560, "end": 572}, "arguments": [{"role": "Theme", "text": "core-binding factor-beta", "start": 608, "end": 632}]}, {"trigger": {"text": "suppression", "start": 694, "end": 705}, "arguments": [{"role": "Theme", "text": "core-binding factor-beta", "start": 608, "end": 632}]}, {"trigger": {"text": "suppression", "start": 694, "end": 705}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 719, "end": 724}]}, {"trigger": {"text": "reduced", "start": 754, "end": 761}, "arguments": [{"role": "Cause", "text": "Inactivation", "start": 560, "end": 572}, {"role": "Theme", "text": "expression", "start": 762, "end": 772}]}, {"trigger": {"text": "reduced", "start": 754, "end": 761}, "arguments": [{"role": "Cause", "text": "suppression", "start": 694, "end": 705}, {"role": "Theme", "text": "expression", "start": 762, "end": 772}]}, {"trigger": {"text": "siRNA", "start": 977, "end": 982}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 961, "end": 966}]}, {"trigger": {"text": "siRNA", "start": 977, "end": 982}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 971, "end": 976}]}], "positive regulation": [{"trigger": {"text": "induces", "start": 325, "end": 332}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 315, "end": 323}, {"role": "Theme", "text": "expression", "start": 337, "end": 347}]}, {"trigger": {"text": "prerequisite", "start": 452, "end": 464}, "arguments": [{"role": "Theme", "text": "binding", "start": 473, "end": 480}]}, {"trigger": {"text": "mediated", "start": 685, "end": 693}, "arguments": [{"role": "Theme", "text": "suppression", "start": 694, "end": 705}]}, {"trigger": {"text": "induced", "start": 1409, "end": 1416}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 1400, "end": 1408}, {"role": "Theme", "text": "expression", "start": 1423, "end": 1433}]}], "regulation": [{"trigger": {"text": "link", "start": 1392, "end": 1396}, "arguments": [{"role": "Cause", "text": "Runx", "start": 1350, "end": 1354}, {"role": "Theme", "text": "induced", "start": 1409, "end": 1416}]}], "transcription": [{"trigger": {"text": "expressed", "start": 1162, "end": 1171}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 1189, "end": 1194}]}, {"trigger": {"text": "expressed", "start": 1162, "end": 1171}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1196, "end": 1201}]}, {"trigger": {"text": "expressed", "start": 1162, "end": 1171}, "arguments": [{"role": "Theme", "text": "TGF-beta", "start": 1207, "end": 1215}]}]}}, "schema": []}
{"input": "Regulatory T (T reg) cells expressing the transcription factor forkhead box P3 (FOXP3, human; Foxp3, mouse) play an essential role in controlling immune responses to autoantigens, allergens, tumor antigens, transplantation antigens, and infectious agents (Hori et al., 2003;Akdis, 2006). Foxp3 is a member of the forkhead/winged-helix family of transcriptional regulators, and its expression in T reg cells is essential for their development and function (Fontenot et al., 2003;Williams and Rudensky, 2007). A spontaneous mutation of the X-linkedFoxp3gene inscurfymice causes an autoimmune-like disease, whereas the mutation in humans leads to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome that is also a severe multiorgan autoimmune disease with hyper-IgE (Ziegler, 2006).Although the essential role of Foxp3 in central and peripheral tolerance has been extensively studied, its regulation, cooperation with other transcription factors, and how it functions in inducible T reg (iT reg) cells to suppress various target genes is mostly not yet understood. It is known that Foxp3 cooperates with the nuclear factor of activated T cells (NFAT) or nuclear factor-kappa B (NF-kappaB) to regulate the transcription of different target genes (Schubert et al., 2001;Bettelli et al., 2005;Wu et al., 2006). The Th2 cytokine IL-4 inhibits FOXP3 expression during T cell priming. GATA3 binds to theFOXP3promoter and can repress the FOXP3 trans-activation process directly in Th2 cells (Mantel et al., 2007). It was further demonstrated that both Th1 and Th2 transcription factors T-bet and GATA3 oppose peripheral induction of Foxp3+T reg cells in mice through STAT1-, STAT4-, and STAT6-dependent pathways (Wei et al., 2007). Although natural T reg (nT reg) cells that differentiate in the thymus are characterized by their stable Foxp3 expression, the generation of iT reg cells specific for allergens, alloantigens, and autoantigens in the periphery has been associated with a transient Foxp3+phenotype (Fontenot et al., 2003;Hori et al., 2003). The crucial role of TGF-beta in their generation has been demonstrated.The RUNX gene family (Runt-related transcription factor, acute myeloid leukemia [AML], core-binding factor-alpha [CBFalpha], and polyoma enhancer-binding protein-2alpha [PEBP2alpha]) contains three members, RUNX1 (AML1/CBFA2/PEBP2alphaB), RUNX2 (AML3/CBFA1/ PEBP2alphaA), and RUNX3 (AML2/CBFA3/PEBP2alphaC). They are essential transcriptional regulators of different developmental pathways. RUNX2 is mostly important for bone development and osteoblast differentiation (Komori et al., 1997). RUNX1 plays an important role in hematopoiesis during development, and RUNX3 has important functions in thymogenesis and neurogenesis (Wang et al., 1996;Inoue et al., 2002;Levanon et al., 2002). RUNX1 and RUNX3 also work together in the establishment of lineage specification of T lymphocytes (Taniuchi et al., 2002;Egawa et al., 2007).RUNX1is a frequent target for chromosomal translocations associated with leukemias (Look, 1997), andRUNX3methylation and silencing is observed in various human epithelial cancers (Blyth et al., 2005).RUNX family members share the Runt domain, which is responsible for DNA binding (Ito, 1999). The Runt domain-containing protein constitutes the alpha-chain partner of the heterodimeric CBF complex. RUNX proteins heterodimerize with the non-DNA-binding partner, CBFbeta, which increases the affinity for DNA binding and stabilizes the complex by preventing ubiquitin-dependent degradation (Wang et al., 1993). The CBF complexes regulate the expression of cellular genes through binding to promoters or enhancer elements. The effects of the RUNX-CBFbeta complex regulation are clearly cell lineage and stage specific. They include the crucial choices between cell-cycle exit and continued proliferation, as well as between cell differentiation and self-renewal (Blyth et al., 2005).Because of the involvement of RUNX mutations in different autoimmune diseases and the known interaction with TGF-beta, we investigated the impact of RUNX1 and RUNX3 on the expression of FOXP3 and subsequently on the development and function of iT reg cells. This study demonstrates that RUNX1 and RUNX3 induced by TGF-beta are involved in the development and suppressive function of Foxp3+iT reg cells.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 1406, "end": 1411}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1400, "end": 1405}, {"role": "Theme2", "text": "FOXP3", "start": 1418, "end": 1423}, {"role": "Site2", "text": "promoter", "start": 1423, "end": 1431}]}, {"trigger": {"text": "heterodimerize", "start": 3379, "end": 3393}, "arguments": [{"role": "Theme", "text": "CBFbeta", "start": 3428, "end": 3435}]}, {"trigger": {"text": "binding", "start": 3644, "end": 3651}, "arguments": [{"role": "Theme", "text": "CBF", "start": 3580, "end": 3583}]}, {"trigger": {"text": "interaction", "start": 4039, "end": 4050}, "arguments": [{"role": "Theme", "text": "RUNX", "start": 3977, "end": 3981}, {"role": "Theme2", "text": "TGF-beta", "start": 4056, "end": 4064}]}], "gene expression": [{"trigger": {"text": "expressing", "start": 27, "end": 37}, "arguments": [{"role": "Theme", "text": "forkhead box P3", "start": 63, "end": 78}]}, {"trigger": {"text": "expression", "start": 381, "end": 391}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 288, "end": 293}]}, {"trigger": {"text": "expression", "start": 1366, "end": 1376}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1360, "end": 1365}]}, {"trigger": {"text": "expression", "start": 1857, "end": 1867}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1851, "end": 1856}]}, {"trigger": {"text": "expression", "start": 4119, "end": 4129}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 4133, "end": 4138}]}], "negative regulation": [{"trigger": {"text": "inhibits", "start": 1351, "end": 1359}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 1346, "end": 1350}, {"role": "Theme", "text": "expression", "start": 1366, "end": 1376}]}, {"trigger": {"text": "repress", "start": 1440, "end": 1447}, "arguments": [{"role": "Cause", "text": "binds", "start": 1406, "end": 1411}, {"role": "Theme", "text": "trans-activation", "start": 1458, "end": 1474}]}, {"trigger": {"text": "oppose", "start": 1616, "end": 1622}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 1600, "end": 1605}, {"role": "Theme", "text": "induction", "start": 1634, "end": 1643}]}, {"trigger": {"text": "oppose", "start": 1616, "end": 1622}, "arguments": [{"role": "Cause", "text": "GATA3", "start": 1610, "end": 1615}, {"role": "Theme", "text": "induction", "start": 1634, "end": 1643}]}], "positive regulation": [{"trigger": {"text": "trans-activation", "start": 1458, "end": 1474}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1452, "end": 1457}]}, {"trigger": {"text": "induction", "start": 1634, "end": 1643}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1647, "end": 1652}]}, {"trigger": {"text": "dependent", "start": 1707, "end": 1716}, "arguments": [{"role": "Theme", "text": "oppose", "start": 1616, "end": 1622}, {"role": "Cause", "text": "STAT1", "start": 1681, "end": 1686}]}, {"trigger": {"text": "dependent", "start": 1707, "end": 1716}, "arguments": [{"role": "Theme", "text": "oppose", "start": 1616, "end": 1622}, {"role": "Cause", "text": "STAT4", "start": 1689, "end": 1694}]}, {"trigger": {"text": "dependent", "start": 1707, "end": 1716}, "arguments": [{"role": "Theme", "text": "oppose", "start": 1616, "end": 1622}, {"role": "Cause", "text": "STAT6", "start": 1701, "end": 1706}]}, {"trigger": {"text": "induced", "start": 4250, "end": 4257}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 4234, "end": 4239}, {"role": "Cause", "text": "TGF-beta", "start": 4261, "end": 4269}]}, {"trigger": {"text": "induced", "start": 4250, "end": 4257}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 4244, "end": 4249}, {"role": "Cause", "text": "TGF-beta", "start": 4261, "end": 4269}]}], "regulation": [{"trigger": {"text": "regulation", "start": 910, "end": 920}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 834, "end": 839}]}, {"trigger": {"text": "impact", "start": 4086, "end": 4092}, "arguments": [{"role": "Cause", "text": "RUNX1", "start": 4096, "end": 4101}, {"role": "Theme", "text": "expression", "start": 4119, "end": 4129}]}, {"trigger": {"text": "impact", "start": 4086, "end": 4092}, "arguments": [{"role": "Cause", "text": "RUNX3", "start": 4106, "end": 4111}, {"role": "Theme", "text": "expression", "start": 4119, "end": 4129}]}]}}, "schema": []}
{"input": "The role of RUNX1 and RUNX3 transcription factors in TGF-beta-mediated iT reg cell generation\nTo investigate the role of RUNX transcription factors in the development of iT reg cells, we cultured naive CD4+ T cells, isolated from human PBMCs, in conditions that enable the development of iT reg cells. Stimulation with anti-CD2/3/28 mAbs or TGF-beta alone resulted in a minimal up-regulation of RUNX1 and RUNX3 mRNA (Fig. 1 A). In contrast, the combination of both TGF-beta and anti-CD2/3/28 mAbs induced RUNX1 and RUNX3 mRNAs, as well as FOXP3 mRNA, within 48 h in naive CD4+ T cells. This result suggested further experiments to investigate whether the up-regulation of RUNX1 and RUNX3 might be a feature of iT reg cells during their development or even a prerequisite for their induction.\nTo test this hypothesis, RUNX1 and RUNX3 expression was knocked down in human naive CD4+ T cells by transfection of small interfering RNAs (siRNAs; Fig. 1 B). Deficiency of RUNX1 or RUNX3 resulted in markedly reduced TGF-beta-mediated induction of FOXP3 mRNA in naive CD4+ T cells compared with control cells transfected with scrambled siRNA. The level of FOXP3 mRNA was further reduced when both RUNX1 and RUNX3 were knocked down in naive CD4+ T cells during their differentiation to iT reg cells (Fig. 1 B).\nThe influence of RUNX1 and RUNX3 on the development of other T cell subsets and their specific transcription factor expression was further investigated. Naive CD4+ T cells were cultured under Th1, Th2, T reg cell, and Th17 differentiation conditions and the mRNA expression of the predominant transcription factor for each cell type was subsequently analyzed. We observed no change in GATA3 expression in Th2 cells, T-bet expression in Th1 cells, or RORC2 mRNA expression in Th17 cells in which RUNX1 and RUNX3 were knocked down compared with control cells. On the contrary, FOXP3 mRNA was significantly decreased in RUNX1- and RUNX3-deficient T reg cells compared with control cells (Fig. 1 C).\nThe effect of RUNX silencing on the expression level of intracellular FOXP3 during naive CD4+ T cell differentiation to iT reg was evaluated by flow cytometry. FOXP3 was only slightly reduced after RUNX1 silencing. Transfection of siRNA for RUNX3 had a stronger effect. The most striking FOXP3 reduction was observed when RUNX1 and RUNX3 were silenced together (Fig. 1 D). Similar results were obtained in total CD4+ T cells (Fig. S1 and Fig. S2). The increased impact of combined RUNX1 and RUNX3 knockdown implies that RUNX1 and RUNX3 might have redundant functions in the induction of FOXP3. In addition, the levels of IL-4, IL-5, IL-10, IL-13, and IFN-gamma in control siRNA-transfected or RUNX1 and RUNX3 siRNA-transfected CD4+ T cells that were cultured with or without anti-CD2/3/28 mAb and TGF-beta did not show any significant difference (Fig. S3).\nTo determine whether RUNX1 and RUNX3 are also expressed in human T reg cells in vivo, we isolated peripheral blood CD4+ CD127- CD25high T reg cells and compared them with CD4+ CD127+ CD25- T cells. Circulating T reg cells expressed significantly higher levels of RUNX3 mRNA compared with CD4+CD25- cells. As expected, IL-10, TGF-beta, and FOXP3 mRNAs are also expressed in circulating T reg cells (Fig. 2 A). There was no difference in RUNX1 mRNA expression between these two cell subsets. We also performed an analysis of human tonsils, which contain high numbers of FOXP3+ T reg cells (Verhagen et al., 2006). Staining of tonsil sections for FOXP3 and RUNX3 demonstrated in vivo coexpression of these two molecules in a subset of T reg cells, whereas there was low RUNX1 expression in all tonsil cells (Fig. 2 B).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 833, "end": 843}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 817, "end": 822}]}, {"trigger": {"text": "expression", "start": 833, "end": 843}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 827, "end": 832}]}, {"trigger": {"text": "expression", "start": 2034, "end": 2044}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2068, "end": 2073}]}, {"trigger": {"text": "levels", "start": 2609, "end": 2615}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 2619, "end": 2623}]}, {"trigger": {"text": "levels", "start": 2609, "end": 2615}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 2625, "end": 2629}]}, {"trigger": {"text": "levels", "start": 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"start": 2701, "end": 2706}]}, {"trigger": {"text": "low", "start": 3618, "end": 3621}, "arguments": [{"role": "Theme", "text": "expression", "start": 3628, "end": 3638}]}], "positive regulation": [{"trigger": {"text": "up-regulation", "start": 378, "end": 391}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 341, "end": 349}, {"role": "Theme", "text": "mRNA", "start": 411, "end": 415}]}, {"trigger": {"text": "induced", "start": 497, "end": 504}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 465, "end": 473}, {"role": "Theme", "text": "mRNAs", "start": 521, "end": 526}]}, {"trigger": {"text": "induced", "start": 497, "end": 504}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 465, "end": 473}, {"role": "Theme", "text": "mRNA", "start": 545, "end": 549}]}, {"trigger": {"text": "up-regulation", "start": 655, "end": 668}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 672, "end": 677}]}, {"trigger": {"text": "up-regulation", "start": 655, "end": 668}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 682, "end": 687}]}, {"trigger": {"text": "mediated", "start": 1018, "end": 1026}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 1009, "end": 1017}, {"role": "Theme", "text": "induction", "start": 1027, "end": 1036}]}, {"trigger": {"text": "induction", "start": 2572, "end": 2581}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2585, "end": 2590}]}], "regulation": [{"trigger": {"text": "change", "start": 1677, "end": 1683}, "arguments": [{"role": "Theme", "text": "expression", "start": 1693, "end": 1703}, {"role": "Cause", "text": "knocked down", "start": 1818, "end": 1830}]}, {"trigger": {"text": "change", "start": 1677, "end": 1683}, "arguments": [{"role": "Theme", "text": "expression", "start": 1724, "end": 1734}, {"role": "Cause", "text": "knocked down", "start": 1818, "end": 1830}]}, {"trigger": {"text": "change", "start": 1677, "end": 1683}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 1758, "end": 1773}, {"role": "Cause", "text": "knocked down", "start": 1818, "end": 1830}]}, {"trigger": {"text": "effect", "start": 2002, "end": 2008}, "arguments": [{"role": "Cause", "text": "silencing", "start": 2017, "end": 2026}, {"role": "Theme", "text": "expression", "start": 2034, "end": 2044}]}, {"trigger": {"text": "functions", "start": 2555, "end": 2564}, "arguments": [{"role": "Cause", "text": "RUNX1", "start": 2518, "end": 2523}, {"role": "Theme", "text": "induction", "start": 2572, "end": 2581}]}, {"trigger": {"text": "functions", "start": 2555, "end": 2564}, "arguments": [{"role": "Cause", "text": "RUNX3", "start": 2528, "end": 2533}, {"role": "Theme", "text": "induction", "start": 2572, "end": 2581}]}, {"trigger": {"text": "show any significant difference", "start": 2812, "end": 2843}, "arguments": [{"role": "Theme", "text": "levels", "start": 2609, "end": 2615}, {"role": "Cause", "text": "siRNA-transfected", "start": 2707, "end": 2724}]}, {"trigger": {"text": "show any significant difference", "start": 2812, "end": 2843}, "arguments": [{"role": "Theme", "text": "levels", "start": 2609, "end": 2615}, {"role": "Cause", "text": "TGF-beta", "start": 2795, "end": 2803}]}, {"trigger": {"text": "difference", "start": 3277, "end": 3287}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 3297, "end": 3312}]}], "transcription": [{"trigger": {"text": "mRNA", "start": 411, "end": 415}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 395, "end": 400}]}, {"trigger": {"text": "mRNA", "start": 411, "end": 415}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 405, "end": 410}]}, {"trigger": {"text": "mRNAs", "start": 521, "end": 526}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 505, "end": 510}]}, {"trigger": {"text": "mRNAs", "start": 521, "end": 526}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 515, "end": 520}]}, {"trigger": {"text": "mRNA", "start": 545, "end": 549}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 539, "end": 544}]}, {"trigger": {"text": "induction", "start": 1027, "end": 1036}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1040, "end": 1045}]}, {"trigger": {"text": "mRNA", "start": 1154, "end": 1158}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1148, "end": 1153}]}, {"trigger": {"text": "expression", "start": 1693, "end": 1703}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 1687, "end": 1692}]}, {"trigger": {"text": "expression", "start": 1724, "end": 1734}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 1718, "end": 1723}]}, {"trigger": {"text": "mRNA expression", "start": 1758, "end": 1773}, "arguments": [{"role": "Theme", "text": "RORC2", "start": 1752, "end": 1757}]}, {"trigger": {"text": "mRNA", "start": 1883, "end": 1887}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1877, "end": 1882}]}, {"trigger": {"text": "expressed", "start": 3077, "end": 3086}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 3118, "end": 3123}]}, {"trigger": {"text": "expressed", "start": 3215, "end": 3224}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 3173, "end": 3178}]}, {"trigger": {"text": "expressed", "start": 3215, "end": 3224}, "arguments": [{"role": "Theme", "text": "TGF-beta", "start": 3180, "end": 3188}]}, {"trigger": {"text": "expressed", "start": 3215, "end": 3224}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 3194, "end": 3199}]}, {"trigger": {"text": "mRNA expression", "start": 3297, "end": 3312}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 3291, "end": 3296}]}]}}, "schema": []}
{"input": "RUNX1 and RUNX3 bind to the FOXP3 promoter\nTranscription element search system analysis of the human FOXP3 promoter predicted 3 putative RUNX binding sites at 333, 287, and 53 bp upstream of the transcription start site (TSS). All three binding sites are conserved between human, mouse, and rat (Fig. S4). To verify the putative binding sites in the FOXP3 promoter, we transiently transfected HEK293T cells with RUNX1 and RUNX3. After the pull-down with oligonucleotides containing the wild-type binding sequences, but not mutant sequences, RUNX binding to the FOXP3 promoter oligonucleotides was detected by Western blot (Fig. 3 A). To confirm these results and test the ability of single binding site sequences to bind either RUNX1 or RUNX3, we used the promoter enzyme immunoassay. Cell lysates were obtained from HEK293T cells that had been transiently transfected with RUNX1 or RUNX3 expression vectors. The biotinylated FOXP3 promoter oligonucleotides were linked to a streptavidin-coated microtiter plate, and bound RUNX1 or RUNX3 was detected by using anti-RUNX antibodies and a peroxidase-labeled secondary antibody. We showed binding of RUNX1 and RUNX3 to the mixture of all three oligonucleotides containing the binding sites, whereas there was no binding detectable when a combination of the mutated oligonucleotides was used in the assay (Fig. 3 B). Although there was a similar and high degree of binding to the -333 and -287 sites, a lower degree of binding was detected when the oligonucleotide containing the -53 site in the Foxp3 promoter was used. This effect was observed both for binding to RUNX1 and RUNX3 (Fig. 3 B). The binding to the two single binding sites at -333 and -287 was comparable to the mixture of all three oligonucleotides. Chromatin immunoprecipitation (ChIP) assay results confirmed the binding of RUNX1 and RUNX3 complexes containing CBFbeta to FOXP3 promoter during the differentiation of naive T cells toward T reg cells. Here, naive CD4+ T cells were cultured with IL-2, anti-CD2/3/28 mAb, and TGF-beta as a Foxp3-inducing stimulation. Amplification of PCR products from the FOXP3 promoter region with the predicted RUNX binding sites showed that RUNX1, RUNX3, and CBFbeta were immunoprecipitated together with the FOXP3 promoter (Fig. 3 C). Negative control primer targeting open reading frame-free intergenic DNA, IGX1A did not show any significant change in site occupancy.", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 16, "end": 20}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 0, "end": 5}, {"role": "Theme2", "text": "FOXP3", "start": 28, "end": 33}, {"role": "Site2", "text": "promoter", "start": 34, "end": 42}]}, {"trigger": {"text": "bind", "start": 16, "end": 20}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 10, "end": 15}, {"role": "Theme2", "text": "FOXP3", "start": 28, "end": 33}, {"role": "Site2", "text": "promoter", "start": 34, "end": 42}]}, {"trigger": {"text": "binding", "start": 546, "end": 553}, "arguments": [{"role": "Theme", "text": "RUNX", "start": 541, "end": 545}, {"role": "Theme2", "text": "FOXP3", "start": 561, "end": 566}, {"role": "Site2", "text": "promoter", "start": 567, "end": 575}]}, {"trigger": {"text": "bind", "start": 716, "end": 720}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 561, "end": 566}, {"role": "Site", "text": "binding site", "start": 690, "end": 702}, {"role": "Theme2", "text": "RUNX1", "start": 728, "end": 733}]}, {"trigger": {"text": "bind", "start": 716, "end": 720}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 561, "end": 566}, {"role": "Site", "text": "binding site", "start": 690, "end": 702}, {"role": "Theme2", "text": "RUNX3", "start": 737, "end": 742}]}, {"trigger": {"text": "bound", "start": 1017, "end": 1022}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 926, "end": 931}, {"role": "Site", "text": "promoter", "start": 932, "end": 940}, {"role": "Theme2", "text": "RUNX1", "start": 1023, "end": 1028}]}, {"trigger": {"text": "bound", "start": 1017, "end": 1022}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 926, "end": 931}, {"role": "Site", "text": "promoter", "start": 932, "end": 940}, {"role": "Theme2", "text": "RUNX3", "start": 1032, 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"Theme", "text": "Foxp3", "start": 1542, "end": 1547}]}, {"trigger": {"text": "binding", "start": 1411, "end": 1418}, "arguments": [{"role": "Site", "text": "-287 sites", "start": 1435, "end": 1445}, {"role": "Theme", "text": "Foxp3", "start": 1542, "end": 1547}]}, {"trigger": {"text": "binding", "start": 1465, "end": 1472}, "arguments": [{"role": "Site", "text": "-53 site", "start": 1526, "end": 1534}, {"role": "Theme", "text": "Foxp3", "start": 1542, "end": 1547}]}, {"trigger": {"text": "binding", "start": 1601, "end": 1608}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 1612, "end": 1617}]}, {"trigger": {"text": "binding", "start": 1601, "end": 1608}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 1622, "end": 1627}]}, {"trigger": {"text": "binding", "start": 1644, "end": 1651}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1542, "end": 1547}, {"role": "Site", "text": "binding sites", "start": 1670, "end": 1683}]}, {"trigger": {"text": "binding", "start": 1827, "end": 1834}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 1838, "end": 1843}, {"role": "Theme2", "text": "FOXP3", "start": 1886, "end": 1891}, {"role": "Site2", "text": "promoter", "start": 1892, "end": 1900}]}, {"trigger": {"text": "binding", "start": 1827, "end": 1834}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 1848, "end": 1853}, {"role": "Theme2", "text": "FOXP3", "start": 1886, "end": 1891}, {"role": "Site2", "text": "promoter", "start": 1892, "end": 1900}]}, {"trigger": {"text": "binding", "start": 1827, "end": 1834}, "arguments": [{"role": "Theme", "text": "CBFbeta", "start": 1875, "end": 1882}, {"role": "Theme2", "text": "FOXP3", "start": 1886, "end": 1891}, {"role": "Site2", "text": "promoter", "start": 1892, "end": 1900}]}, {"trigger": {"text": "immunoprecipitated together", "start": 2222, "end": 2249}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 2191, "end": 2196}, {"role": "Theme2", "text": "FOXP3", "start": 2259, 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"expression", "start": 889, "end": 899}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 874, "end": 879}]}, {"trigger": {"text": "expression", "start": 889, "end": 899}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 883, "end": 888}]}], "positive regulation": [{"trigger": {"text": "transfected", "start": 857, "end": 868}, "arguments": [{"role": "Theme", "text": "expression", "start": 889, "end": 899}]}, {"trigger": {"text": "inducing", "start": 2058, "end": 2066}, "arguments": [{"role": "Cause", "text": "IL-2", "start": 2009, "end": 2013}, {"role": "Theme", "text": "Foxp3", "start": 2052, "end": 2057}]}, {"trigger": {"text": "inducing", "start": 2058, "end": 2066}, "arguments": [{"role": "Cause", "text": "TGF-beta", "start": 2038, "end": 2046}, {"role": "Theme", "text": "Foxp3", "start": 2052, "end": 2057}]}]}}, "schema": []}
{"input": "Regulation of FOXP3 promoter activity and FOXP3 protein expression by RUNX1 and RUNX3\nTo investigate the effect of RUNX1 and RUNX3 binding to the RUNX binding sites in the FOXP3 promoter, we transfected human peripheral blood CD4+ T cells with a FOXP3 promoter luciferase reporter vector and RUNX1 or RUNX3 expression vectors. An increase in luciferase activity was observed only when the FOXP3 promoter (-511 to +176) luciferase construct was cotransfected with RUNX1 or RUNX3 expression vectors (Fig. 4 A). The increase in promoter activity was greater upon cotransfection of RUNX3 compared with RUNX1. Luciferase expression was abrogated when the Runx binding sites in the FOXP3 promoter (-511 to +176) luciferase construct were mutated (Fig. 4 A). In these experiments, the overexpression of RUNX1 and RUNX3 eliminated the need of TGF-beta for FOXP3 promoter activation and PMA/ionomycin stimulation was sufficient.\nTo examine the role of each of the three RUNX binding sites for the FOXP3 promoter activity, we mutated each individually or in combination. No reduction in luciferase activity was observed when the -53 site was mutated and only a slight reduction when either the -287 or -333 site was mutated (Fig. 4 B). However, mutating the -53 site in combination with one of the other two sites led to a significant decrease in luciferase activity, with the greatest reduction observed when all three binding sites were mutated (Fig. 4 B), suggesting that the identified binding sites have redundant functions and RUNX binding to more than one site is necessary for the full activation of the FOXP3 promoter. Supporting these findings, the overexpression of RUNX1 in human primary CD4+ T cells resulted in significantly elevated levels of FOXP3 protein measured by flow cytometry after 48 h. This was achieved without any requirement for anti-CD3, anti-CD28 stimulation, or the presence of TGF-beta. Although there was a trend, the transfection of CD4+ T cells with RUNX3 did not lead to statistically significant increase in FOXP3 (Fig. S5).", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 131, "end": 138}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 115, "end": 120}, {"role": "Site2", "text": "RUNX binding sites", "start": 146, "end": 164}, {"role": "Theme2", "text": "FOXP3", "start": 172, "end": 177}]}, {"trigger": {"text": "binding", "start": 131, "end": 138}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 125, "end": 130}, {"role": "Site2", "text": "RUNX binding sites", "start": 146, "end": 164}, {"role": "Theme2", "text": "FOXP3", "start": 172, "end": 177}]}, {"trigger": {"text": "binding", "start": 1528, "end": 1535}, "arguments": [{"role": "Site2", "text": "binding sites", "start": 1480, "end": 1493}, {"role": "Theme", "text": "RUNX", "start": 1523, "end": 1527}, {"role": "Theme2", "text": "FOXP3", "start": 1602, "end": 1607}]}], "gene expression": [{"trigger": {"text": "expression", "start": 56, "end": 66}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 42, "end": 47}]}, {"trigger": {"text": "expression", "start": 307, "end": 317}, "arguments": [{"role": "Theme", "text": "FOXP3 promoter luciferase", "start": 246, "end": 271}]}, {"trigger": {"text": "expression", "start": 307, "end": 317}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 292, "end": 297}]}, {"trigger": {"text": "expression", "start": 307, "end": 317}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 301, "end": 306}]}, {"trigger": {"text": "expression", "start": 478, "end": 488}, "arguments": [{"role": "Theme", "text": "FOXP3 promoter (-511 to +176) luciferase", "start": 389, "end": 429}]}, {"trigger": {"text": "expression", "start": 478, "end": 488}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 463, "end": 468}]}, {"trigger": {"text": "expression", "start": 478, "end": 488}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 472, "end": 477}]}, {"trigger": {"text": "cotransfection", "start": 560, "end": 574}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 578, "end": 583}]}, {"trigger": {"text": "cotransfection", "start": 560, "end": 574}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 598, "end": 603}]}, {"trigger": {"text": "expression", "start": 616, "end": 626}, "arguments": [{"role": "Theme", "text": "Luciferase", "start": 605, "end": 615}]}, {"trigger": {"text": "overexpression", "start": 778, "end": 792}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 796, "end": 801}]}, {"trigger": {"text": "overexpression", "start": 778, "end": 792}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 806, "end": 811}]}, {"trigger": {"text": "overexpression", "start": 1649, "end": 1663}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 1667, "end": 1672}]}, {"trigger": {"text": "transfection", "start": 1941, "end": 1953}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 1975, "end": 1980}]}], "negative regulation": [{"trigger": {"text": "abrogated", "start": 631, "end": 640}, "arguments": [{"role": "Theme", "text": "expression", "start": 616, "end": 626}]}, {"trigger": {"text": "reduction", "start": 1064, "end": 1073}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1077, "end": 1087}]}, {"trigger": {"text": "reduction", "start": 1158, "end": 1167}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1077, "end": 1087}]}, {"trigger": {"text": "decrease", "start": 1325, "end": 1333}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1337, "end": 1347}]}, {"trigger": {"text": "reduction", "start": 1376, "end": 1385}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1337, "end": 1347}]}], "positive regulation": [{"trigger": {"text": "activity", "start": 29, "end": 37}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 14, 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{"input": "Role of CBFbeta in the induction of Foxp3\nCBFbeta, a common cofactor of all RUNX proteins, stabilizes and increases the binding of the runt domain to target DNA sequences. To target all Runx proteins that might be involved in the induction of Foxp3, we used mice in which loxP-flanked Cbfb alleles were inactivated in T cells through expression of a CD4-cre transgene. Retinoic acid and TGF-beta synergize in the induction of Foxp3 in naive T cells (Kang et al., 2007). To investigate whether Runx-mediated induction of Foxp3 is dependent on the expression of CBFbeta, naive CD4+ CD8- T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were stimulated with anti-CD3/28 mAbs, retinoic acid, and increasing concentrations of TGF-beta. After 3 d in culture, the cells were restimulated with PMA and ionomycin and analyzed for intracellular Foxp3 and IFN-gamma expression. TGF-beta induced Foxp3 in CbfbF/+ CD4-cre cells in a dose dependent manner, and this was significantly reduced in CbfbF/F CD4-cre cells. Retinoic acid enhanced Foxp3 expression even in 20 pg/ml of TGF-beta and more than 95% of the CD4+ T cells from CbfbF/+ CD4-cre mice became Foxp3+ in 100 and 500 pg/ml TGF-beta doses. The induction of Foxp3 was again significantly lower in CbfbF/F CD4-cre CD4+ T cells even in the presence of retinoic acid, demonstrating that deficiency in Runx binding to DNA affects the TGF-beta induction of Foxp3 in T reg cells (Fig. 5 A). There was no difference in the induction of Foxp3 when endogenous IL-4 and IFN-gamma were neutralized (Fig. S6).\nThe importance of RUNX transcription factors for the in vivo conversion of naive CD4+ T cells into iT reg cells was examined. Control CbfbF/F or CbfbF/F CD4-cre naive T cells, harboring a Foxp3-IRES-GFP allele were adoptively transferred into Rag2-/- mice. 6 wk later, CD4+ T cells in spleen, mesenteric lymph node, and lamina propria of the small intestine were analyzed for Foxp3-GFP expression (Fig. 5 B). There was a consistently lower percentage of CD4+ T cells that had developed Foxp3 expression in the mesenteric lymph node and lamina propria of mice transferred with CbfbF/F CD4-cre cells compared with control cells (Fig. 5, B and C). These data affirm the significance of RUNX proteins for the in vivo generation of CD4+ Foxp3+ T cells.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 1363, "end": 1370}, "arguments": [{"role": "Theme", "text": "Runx", "start": 1358, "end": 1362}]}], "gene expression": [{"trigger": {"text": "induction", "start": 23, "end": 32}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 36, "end": 41}]}, {"trigger": {"text": "expression", "start": 334, "end": 344}, "arguments": [{"role": "Theme", "text": "CD4-cre", "start": 350, "end": 357}]}, {"trigger": {"text": "induction", "start": 413, "end": 422}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 426, "end": 431}]}, {"trigger": {"text": "induction", "start": 507, "end": 516}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 520, "end": 525}]}, {"trigger": {"text": "expression", "start": 546, "end": 556}, "arguments": [{"role": "Theme", "text": "CBFbeta", "start": 560, "end": 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"start": 186, "end": 190}, {"role": "Theme", "text": "induction", "start": 230, "end": 239}]}, {"trigger": {"text": "affects", "start": 1378, "end": 1385}, "arguments": [{"role": "Cause", "text": "binding", "start": 1363, "end": 1370}, {"role": "Theme", "text": "induction", "start": 1399, "end": 1408}]}, {"trigger": {"text": "difference", "start": 1458, "end": 1468}, "arguments": [{"role": "Theme", "text": "induction", "start": 1476, "end": 1485}, {"role": "Cause", "text": "neutralized", "start": 1535, "end": 1546}]}]}}, "schema": []}
{"input": "CBFbeta, RUNX1, and RUNX3 are important for the suppressive activity of CD25+ Foxp3+ T reg cells\nEven though Cbfb-deficient CD4+ T cells had impaired induction of Foxp3 after stimulation with anti-CD3/28 mAbs and TGF-beta, sufficient numbers of Foxp3+ cells could be generated to permit analysis of their suppressive activity. Purified naive CD4+ T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice (Cd45.2) harboring a Foxp3-ires-GFP allele (Bettelli et al., 2006) were stimulated in vitro with anti-CD3/28 mAbs, IL-2, and TGF-beta. After 3 d, Foxp3+-GFP+ cells were sorted by flow cytometry and mixed with CFSE-labeled naive CD45.1+ CD4+ cells at ratios of 1:4, 1:2, and 1:1. The cells were then incubated with inactivated splenocytes and stimulated with anti-CD3 mAb for four more days. CD45.1+ cells were analyzed for CFSE dilution (Fig. 6 A). CbfbF/+ CD4-cre CD4+ T cells activated in the presence of TGF-beta showed a clear suppression of T cell proliferation that became even more apparent when an increased ratio of FOXP3+/CD25- cells was used (Fig. 6 B). The suppression was significantly reduced when cells from CbfbF/F CD4-cre mice were used, demonstrating that TGF-beta-induced Runx complexes are important for the suppressive activity of Foxp3+ T reg cells (Fig. 6 B). As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-beta were mixed with CFSE-labeled naive CD45.1+ CD4+ cells. No suppression could be observed in all control groups without TGF-beta at all tested ratios (Fig. 6 C). The decreased suppression capacity of CbfbF/F CD4-cre iT reg cells was unlikely to be caused by decreased survival or proliferation. Foxp3+ and Foxp3- cells generated from both CbfbF/F CD4-cre and control cells all displayed similar proliferation rates and cell death as measured by CFSE dilution and annexin V staining, respectively (Fig. S7, A and B).\nIn addition, we tested the requirement of RUNX1 and RUNX3 for the development of the suppressive capacity in human iT reg cells. We isolated human naive CD4+ T cells and transfected them with a combination of RUNX1 and RUNX3 siRNA, or with a scrambled control siRNA. Cells were cultured under T reg conditions, and then mixed with CFSE-labeled autologous CD4+ T cells and stimulated with anti-CD3 mAb. Cells in which RUNX1 and RUNX3 were knocked down showed markedly lower suppressive activity compared with control iT reg cells at a T reg/CD4+ T responder cell ratio of 1:20, but not when the T reg/CD4+ T responder cell ratio was increased to 1:5 (Fig. 6 D). These results demonstrate the important role of RUNX1 and RUNX3 not only for the induction of FOXP3, but also for the suppressive capacity of iT reg cells both in humans and in mice. The data suggest both quantity and quality of T reg cells are hampered. Reduced intrinsic suppressive capacity of iT reg cells was demonstrated in mice, because Foxp3-GFP+ cells were FACS sorted and same numbers of iT reg cells are included in control experiments. In the suppression experiment with human cells, reduced suppressive activity was caused by reduced FOXP3 expression in T cells by siRNA inhibition of RUNX1 and RUNX3.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "induction", "start": 2624, "end": 2633}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 2637, "end": 2642}]}, {"trigger": {"text": "expression", "start": 3096, "end": 3106}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 3090, "end": 3095}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 114, "end": 123}, "arguments": [{"role": "Theme", "text": "Cbfb", "start": 109, "end": 113}]}, {"trigger": {"text": "impaired", "start": 141, "end": 149}, "arguments": [{"role": "Cause", "text": "deficient", "start": 114, "end": 123}, {"role": "Theme", "text": "induction", "start": 150, "end": 159}]}, {"trigger": {"text": "siRNA", "start": 2107, "end": 2112}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 2091, "end": 2096}]}, {"trigger": {"text": "siRNA", "start": 2107, "end": 2112}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 2101, "end": 2106}]}, {"trigger": {"text": "knocked down", "start": 2320, "end": 2332}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 2299, "end": 2304}]}, {"trigger": {"text": "knocked down", "start": 2320, "end": 2332}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 2309, "end": 2314}]}, {"trigger": {"text": "reduced", "start": 3082, "end": 3089}, "arguments": [{"role": "Theme", "text": "expression", "start": 3096, "end": 3106}, {"role": "Cause", "text": "inhibition", "start": 3127, "end": 3137}]}, {"trigger": {"text": "inhibition", "start": 3127, "end": 3137}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 3141, "end": 3146}]}, {"trigger": {"text": "inhibition", "start": 3127, "end": 3137}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 3151, "end": 3156}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 150, "end": 159}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 163, "end": 168}]}, {"trigger": {"text": "important", "start": 2573, "end": 2582}, "arguments": [{"role": "Cause", "text": "RUNX1", "start": 2591, "end": 2596}, {"role": "Theme", "text": "induction", "start": 2624, "end": 2633}]}, {"trigger": {"text": "important", "start": 2573, "end": 2582}, "arguments": [{"role": "Cause", "text": "RUNX3", "start": 2601, "end": 2606}, {"role": "Theme", "text": "induction", "start": 2624, "end": 2633}]}]}}, "schema": []}
{"input": "This study demonstrates that RUNX transcription factors 1 and 3 play an important role in the generation of FOXP3+ iT reg cells by TGF-beta. TGF-beta mediates RUNX induction and FOXP3 is efficiently up-regulated by RUNX1 and RUNX3 in human CD4+ T cells. There are three putative RUNX binding sites in the proximal FOXP3 promoter. One binding site was predicted as a binding site for RUNX2. Promoter enzyme immunoassay results showed that binding of RUNX1 and RUNX3 also occurred at this site (as well as at the other two), which were initially identified as RUNX1 binding sites. This finding is not surprising because RUNX proteins bind to promoter or enhancer elements of their target genes via the runt domain, which is conserved between members of the RUNX family. The RUNX protein that actually induces the expression of FOXP3 might therefore be dependent on the availability of the specific RUNX family member at certain stages of T cell development. RUNX proteins are able to increase or inhibit transcriptional activity of their target genes depending on the cell type and the target gene (Otto et al., 2003). Mutation of only one of the three binding sites had only a little effect on the promoter activity; however, when two binding sites were mutated, the FOXP3 promoter activity dropped to a greater extent. The most striking effect was observed when all three binding sites were mutated. We therefore assume that these binding sites have partially redundant functions, but binding to at least two sites seems to be necessary for full promoter activation.\nTGF-beta promotes or inhibits the proliferation, differentiation, and survival of a wide array of different cells. It is also produced in activated T cells and it inhibits T cell proliferation (Kehrl et al., 1986; Siegel and Massague, 2003). It was shown that TGF-beta is mandatory for the maintenance of peripheral T reg cells and their expression of Foxp3 (Marie et al., 2005; Rubtsov and Rudensky, 2007). RUNX transcription factors are targets of the TGF-beta superfamily and they are involved in the TGF-beta pathway. They interact directly with regulatory SMADs (Miyazawa et al., 2002; Ito and Miyazono, 2003). TGF-beta can activate RUNX genes at the transcriptional level, and at the posttranscriptional level through activation or stabilization of RUNX proteins (Jin et al., 2004). It was shown that RUNX2 regulates the expression of TGF-beta type I receptor (Ji et al., 2001), suggesting that other mechanisms for their function could be involved. The fusion proteins RUNX1-EVI1 and RUNX1-ETO block TGF-beta inhibition of leukemic cell growth. RUNX3 plays an important role in TGF-beta-mediated growth control in epithelial cells, as loss of RUNX3 leads to decreased sensitivity to TGF-beta and hyperproliferation of the gastric mucosa (Blyth et al., 2005). The present study demonstrates that RUNX3 expression is more dominant in circulating human T reg cells and tonsil T reg cells compared with RUNX1. This could be dependent on the stage of the cells and organ from which they were isolated.\nWe observed that single siRNA interference of either RUNX1 or RUNX3 alone shows a slight decrease in Foxp3+ T reg cell induction, which could be caused by redundancy of these proteins. For this reason, we decided to use CbfbetaF/F CD4-cre mice. Foxp3 induction by TGF-beta is reduced in CD4+ T cells of CbfbF/F CD4-cre mice compared with CbfbF/+ CD4-cre mice. Retinoic acid is secreted by a subset of dendritic cells in the gut-associated lymphoid tissue. It inhibits the IL-6-driven induction of Th17 cells and facilitates the differentiation of naive T cells to Foxp3+ T reg cells (Mucida et al., 2007). We observed an increased number of Foxp3+ cells by retinoic acid and TGF-beta compared with TGF-beta treatment alone in CbfbF/+ CD4-cre mice and CbfbF/F CD4-cre mice. In addition, we showed a defective in vivo generation of T reg cells from Cbfb-deficient CD4+ T cells in Rag2-/- mice. These data in mice confirm the human data that RUNX proteins play an important role for TGF-beta-dependent FOXP3 induction, as well as in the suppressive capacity of iT reg cells. As an additional support for this concept, the overexpression of RUNX1 induced increased FOXP3 protein expression without any requirement of TGF-beta and anti-CD3 and anti-CD28 stimulation in human primary CD4+ cells. In both human and mouse systems, reduced Foxp3 expression was associated with reduced T reg cell suppressive activity.\nIn a recent study, the role of Runx-CBFbeta was investigated in nT reg cell development in the thymus (Rudra et al., 2009). It was reported that Foxp3 expression in nT reg cells is unstable in the absence of Runx-CBFbeta complexes. Cbfb-deficient nT reg cells progressively lose Foxp3 upon division, and there is no evidence of increased death of Cbfb-deficient nT reg cells in that study. The experiments in Cbfb-deficient CD4-cre T cells in mice and the knockdown experiments in humans in this study suggest that the induction of Foxp3 expression is a major contributing factor in the in vivo conversion experiment. Here, we observed that there is a twofold increased Foxp3+ iT reg cell generation in vivo. This is in the same range with previously published studies targeting different mechanisms in Foxp3 induction (Maynard et al., 2007; Sun et al., 2007). Whether the diminished capacity of Cbfb-deficient CD4-cre T cells in the generation of Foxp3 may be caused by peripheral expansion of Cbfb-deficient non-T reg cells or survival problems faced by Cbfb-deficient iT reg cells after Foxp3 induction remains to be elucidated.\nThe involvement of RUNX proteins in autoimmune diseases has been previously suggested (Alarcon-Riquelme, 2003). A mutation in the RUNX1 binding site in the promoter of programmed cell death 1 gene (PDCD-1) has been implicated in systemic lupus erythematosus pathogenesis (Prokunina et al., 2002). Polymorphisms that alter RUNX1 binding to other genes have also been described in rheumatoid arthritis linkage at 5q31 in Japanese patients (Tokuhiro et al., 2003) and in a psoriasis linkage at 17q25 (Prokunina et al., 2002; Helms et al., 2003). RUNX3-deficient mice spontaneously develop inflammatory bowel disease and hyperplastic gastritis-like lesions (Brenner et al., 2004). These disease symptoms resemble those occurring after depletion of Foxp3-expressing T reg cells (Sakaguchi, 2004). Derepression of Th2 cytokines might also account for some of the observed disease symptoms, as it was shown that T-bet first induces Runx3 in Th1 cells and then partners with Runx3 to direct lineage-specific gene activation. Runx3/Cbfbeta are both required for the activation of the Ifng gene and silencing of the Il4 gene in Th1 cells (Djuretic et al., 2007; Naoe et al., 2007). Runx proteins also play an essential role during T lymphocyte differentiation in the thymus (Taniuchi et al., 2002). Runx1 regulates the transitions of developing thymocytes from the CD4- CD8- double-negative stage to the CD4+ CD8+ double-positive stage and from the DP stage to the mature single-positive stage (Egawa et al., 2007). Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4+ helper and CD8+ cytotoxic T cell lineages. In addition, inactivation of both Runx1 and Runx3 at the double-positive stages resulted in a severe blockage in the development of CD8+ mature thymocytes. These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells, and suggest that they may play a role in nT reg cell development, which remains to be elucidated. Furthermore, it was shown that Runx1 activates IL-2 and IFN-gamma gene expression in conventional CD4+ T cells by binding to their respective promoter. RUNX1 interacts physically with Foxp3 protein, and it was demonstrated that this interaction might be responsible for the suppression of IL-2 and IFN-gamma production and up-regulation of T reg cell-associated molecules (Ono et al., 2007).\nIt has been shown that Foxp3 also influences Th17 differentiation. Specifically, Foxp3 physically interacts with RORgammat, and this interaction inhibits RORgammat function (Zhou et al., 2008). This relationship of RORgammat and Foxp3 and probably yet unknown mechanisms might be the basis of the observation that the differentiation of Th17 cells and T reg cells is often reciprocal (Bettelli et al., 2006). Recently, data suggests that Runx1 may also be involved in regulating Il17 transcription, functioning in complex with RORgammat to activate transcription (Zhang et al., 2008).\nThe Runx3-deficient mice develop spontaneous Th2-dominated autoimmune colitis and asthma (Brenner et al., 2004; Fainaru et al., 2005). Cbfbf/f Cd4 mice also show a spontaneous Th2 dominated disease, with increased serum IgA, IgG1, and IgE titers and lymphocyte and eosinophil infiltration of the lung (Naoe et al., 2007). All these phenotypes were previously attributed to a loss of Th2 silencing whereas our findings additionally suggest that loss of T reg function plays a role. We have shown a link between Foxp3 induction in iT reg cells and RUNX1 and RUNX3. RUNX proteins play a central role in pathways regulating cell growth and differentiation, and their interaction with the TGF-beta pathway is of particular interest.\nFoxp3 protein interacts not only with RUNX proteins but also with several other transcriptional partners, such as NFAT and possibly NF-kappaB; with histone acetyl transferases, such as TIP60; and histone deacetyl transferase (HDAC) complexes, such as HDAC7 and HDAC9 (Wu et al., 2006; Sakaguchi et al., 2008). NFAT forms a complex with AP-1 and NF-kappaB and regulates the expression of IL-2, IL-4, IFN-gamma, and CTLA4 in conventional T cells, which leads to the activation and differentiation to effector T cells (Dolganov et al., 1996; Hu et al., 2007). The NFAT-AP-1 complex also binds to the Foxp3 promoter after TCR triggering and regulates its gene expression positively (Mantel et al., 2006). It was shown that NFAT and Smad3 cooperate to induce Foxp3 expression through its enhancer (Tone et al., 2008), but no TGF-beta response element was identified in the Foxp3 gene or in the surrounding regions. The initial induction of RUNX1 and RUNX3 and the subsequent binding of these transcription factors to the Foxp3 promoter that we showed here might explain the relatively late induction of Foxp3 mRNA that peaks 24-48 h after stimulation. The interaction of Foxp3 and NFAT is dependent on their cooperative binding to DNA (Wu et al., 2006). RUNX1 alone, or together with its interacting partners p300 and CREB-binding protein, may cooperate with the NFAT transcription complex to activate the IL-2 promoter (Sakaguchi et al., 2008). Similar to this interaction, NFAT may also cooperate with RUNX1 or RUNX3 to activate Foxp3, but further studies are necessary to elaborate on this concept.\nIn conclusion, our findings elucidate the role of RUNX proteins in iT reg cell development and function. 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"arguments": [{"role": "Theme", "text": "binding", "start": 5940, "end": 5947}]}, {"trigger": {"text": "regulating", "start": 8520, "end": 8530}, "arguments": [{"role": "Cause", "text": "Runx1", "start": 8490, "end": 8495}, {"role": "Theme", "text": "transcription", "start": 8536, "end": 8549}]}, {"trigger": {"text": "regulates", "start": 9724, "end": 9733}, "arguments": [{"role": "Cause", "text": "forms a complex", "start": 9680, "end": 9695}, {"role": "Theme", "text": "expression", "start": 9738, "end": 9748}]}, {"trigger": {"text": "dependent", "start": 10549, "end": 10558}, "arguments": [{"role": "Theme", "text": "interaction", "start": 10516, "end": 10527}, {"role": "Cause", "text": "binding", "start": 10580, "end": 10587}]}], "transcription": [{"trigger": {"text": "transcriptional", "start": 2223, "end": 2238}, "arguments": [{"role": "Theme", "text": "RUNX", "start": 2205, "end": 2209}]}, {"trigger": {"text": "transcription", "start": 8536, "end": 8549}, "arguments": [{"role": "Theme", "text": "Il17", "start": 8531, "end": 8535}]}, {"trigger": {"text": "induction", "start": 10450, "end": 10459}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 10463, "end": 10468}]}]}}, "schema": []}
{"input": "Mice.\nCbfbF/F CD4-cre and Foxp3GFP mice have previously been described (Bettelli et al., 2006; Naoe et al., 2007). Cd45.1 and Rag2-/- mice were purchased from Jackson ImmunoResearch Laboratories and Taconic, respectively. For the in vivo Foxp3 conversion assay, naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice. 5 x 106 cells were used per transfer. 6 wk later, TCRbeta+CD4+ gated cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine were analyzed for Foxp3-GFP expression. All analyses and experiments were performed on animals at 6-8 wk of age. Animals were housed under specific pathogen-free conditions at the animal facility of the Skirball Institute, and experiments were performed in accordance with approved protocols for the New York University Institutional Animal Care and Usage Committee.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 595, "end": 605}, "arguments": [{"role": "Theme", "text": "Foxp3-GFP", "start": 585, "end": 594}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 388, "end": 397}, "arguments": [{"role": "Theme", "text": "Rag", "start": 384, "end": 387}]}]}}, "schema": []}
{"input": "Isolation of PBMCs, CD4+ T cells, and culture conditions.\nHuman PBMCs were isolated by Ficoll (Biochrom) density gradient centrifugation and CD4+ T cells were then isolated using the Dynal CD4+ Isolation kit (Invitrogen) according to the manufacturer's instructions. The purity of CD4+ T cells was initially tested by flow cytometry and was >=95%. Cells were stimulated with the following combination of mAbs to T cell surface molecules (Meiler et al., 2008): anti-CD2 (clone 4B2 and 6G4; 0.5 microg/ml), anti-CD3 (clone OKT3; 0.5 microg/ml), and anti-CD28 mAb (clone B7G5; 0.5 microg/ml; all from Sanquin) and cultured in serum-free AIM-V medium (Life Technologies) with the addition of 1 nmol/liter IL-2 (Roche). TGF-beta (R&D Systems) was used at 5 ng/ml, if not stated otherwise. A combination of PMA (25 ng/ml) and ionomycin (1 mg/ml; Sigma-Aldrich) was used.\nHuman CD4+ CD127- CD25high and CD4+ CD127+ CD25neg cells were purified by flow cytometry using anti-CD127, anti-CD25-PC5 (Beckman Coulter), and anti-CD4-FITC antibodies (Dako).\nMouse naive (CD62Lhi44lo25-) CD4+ T cells were purified by flow cytometry and activated in vitro with 5 microg/ml plate-bound anti-CD3 and 1 microg/ml soluble anti-CD28 antibodies (eBioscience) in RPMI supplemented with 10% FCS, 5 mM beta-mercaptoethanol, and antibiotics. Neutralizing anti-IFN-gamma and anti-IL-4 mAbs (BD) were used at 1 microg/ml concentrations when indicated.", "output": {"json_structures": {}}, "schema": []}
{"input": "In vitro T cell differentiation.\nCD4+ CD45RA+ magnetically sorted (CD45RO depletion with AutoMACS; Miltenyi Biotec) cells were stimulated with immobilized plate-bound anti-CD3 (1 microg/ml; OKT3; IgG1) and anti-CD28 (2 microg/ml). For Th1 differentiation conditions, cells were stimulated with the following: 40 ng/ml IL-2, 5 microg/ml anti-IL-4, and 25 ng/ml IL-12 (R&D Systems). For Th2 conditions, cells were stimulated with the following: 40 ng/ml IL-2, 25 ng/ml IL-4, and 5 microg/ml anti-IL-12 (R&D Systems). For T reg cell conditions, cells were stimulated with the following: 40 ng/ml IL-2, 5 ng/ml TGF-beta, 5 microg/ml anti-IL-12, 5 microg/ml anti-IL-4. For Th17 conditions, cells were stimulated with the following: 40 ng/ml IL-2, 20 ng/ml IL-6, 5 ng/ml TGF-beta, 20 ng/ml IL-23 (Alexis Biochemicals Corp.), 10 ng/ml IL-1beta, 5 microg/ml anti-IL-4, and 5 microg/ml anti-IL-12 were used. Proliferating cells were expanded in medium containing IL-2. The cytokine profile of these cells demonstrated that IFN-gamma is the predominant cytokine in Th1 cells, IL-4 and IL-13 in Th2 cells, and IL-17 in Th17 cells (Akdis et al., 2000; Burgler et al., 2009).", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "depletion", "start": 74, "end": 83}, "arguments": [{"role": "Theme", "text": "CD45RO", "start": 67, "end": 73}]}]}}, "schema": []}
{"input": "Immunohistochemistry.\nHuman tonsils were obtained from tonsillectomy samples of hypertrophic and obstructive tonsils without a current infection. Ethical permission was obtained from Cantonal Ethics Commission, and informed consent was obtained from patients. Paraformaldehyde-fixed tonsil cryosections were stained with unconjugated rabbit IgG polyclonal antibody to human RUNX1 (Santa Cruz Biotechnology, Inc.) or unconjugated mouse IgG1 mAb to human RUNX3 (Abcam). After a washing step, the sections were stained with the corresponding secondary antibodies. RUNX1-binding antibodies were detected by using Alexa Fluor 633-conjugated goat anti-rabbit IgG and RUNX3-binding antibodies were detected by using Alexa Fluor 532-conjugated goat anti-mouse IgG1. Afterward, the sections were washed and in the case of RUNX3 staining a blocking step with an unconjugated mouse IgG1 mAb was used. Finally, the sections were stained with Alexa Fluor 488-conjugated mouse IgG1 mAb to human FOXP3 (eBioscience) or the corresponding isotype control. Tissue sections were stained with DAPI for the demonstration of nuclei and mounted with Prolong antifade (Invitrogen). Images were acquired and analyzed using the confocal microscope DMI 4000B and the TCS SPE system (both from Leica).", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 567, "end": 574}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 561, "end": 566}]}, {"trigger": {"text": "binding", "start": 667, "end": 674}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 661, "end": 666}]}]}}, "schema": []}
{"input": "In vitro suppression assays.\nMouse Foxp3+ CD4+ CD8- T cells were FACS purified based on GFP expressed from a Foxp3-IRES-GFP knock-in allele (Foxp3GFP). Naive (CD62Lhi44lo25-) CD4+ T cells (effectors) were FACS-purified from Cd45.1 mice, then loaded with 5 microM CFSE (Invitrogen). Total splenocytes from C57BL/6 mice inactivated with 50 microg/ml mitomycin C (Sigma-Aldrich) for 45 min were used as APCs. A total of 4 x 105 CD4+ cells (CFSE-loaded CD25- plus Foxp3-GFP+) was mixed with 105 APCs + 1 microg/ml anti-CD3 mAb per well of a 96 well round bottom plate. Proliferation of the effector cells was analyzed by CFSE dilution. Apoptosis of the cells was investigated by annexin V staining and flow cytometry. Positive and negative control gates were made according to T cells cultured only in the presence of IL-2 without anti-CD3/28 stimulation.\nHuman naive CD4+ T cells were isolated by negative selection by MACS from PBMCs and either transfected with a scrambled siRNA or with a combination of RUNX1 and RUNX3 siRNA. Cells were then cultured under iT reg cell differentiating conditions and mixed with 2 x 105 autologous irradiated PBMCs that were used as APCs and autologous CFSE-labeled CD4+ T cells. T reg cell to responder cell ratio was 1:20, 1:10, and 1:5. To check the proliferation of the CD4+ T cells without suppression, no T reg cells were added in a control group. Cells were stimulated with 2.5 microg/ml anti-CD3 mAb, cultured in a 96-well plate and the proliferation of the effector cells was determined by analyzing the CFSE dilution by flow cytometry after 5 d of culture. Gating on the CD4+CFSE+ T cells enabled the exclusion of APCs and T reg cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressed", "start": 92, "end": 101}, "arguments": [{"role": "Theme", "text": "GFP", "start": 88, "end": 91}]}], "negative regulation": [{"trigger": {"text": "siRNA", "start": 1019, "end": 1024}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 1003, "end": 1008}]}, {"trigger": {"text": "siRNA", "start": 1019, "end": 1024}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 1013, "end": 1018}]}]}}, "schema": []}
{"input": "Cloning of the FOXP3 promoter, construction of mutant FOXP3 promoter, and RUNX expression plasmids.\nThe FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate pGL3 FOXP3 -511/+176 (Mantel et al., 2006). Site-directed mutagenesis for the three putative RUNX binding sites in the FOXP3 promoter region was introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions and confirmed by sequencing the DNA. The following primers and their complementary strands were used: foxp runx-333, forward 5'-CACTTTTGTTTTAAAAACTGTCCTTTCTCATGAGCCCTATTATC-3'; foxp runx-333 reverse 5'-GATAATAGGGCTCATGAGAAAGGACAGTTTTTAAAACAAAAGTG-3'; foxp runx-287 forward 5'-CCTCTCACCTCTGTCCTGAGGGGAAGAAATC-3'; foxp runx-287 reverse 5'-GATTTCTTCCCCTCAGGACAGAGGTGAGAGG-3'; foxp runx-53 forward 5'-GCTTCCACACCGTACAGCGTCCTTTTTCTTCTCGGTATAAAAG-3'; foxp runx-53 reverse 5'-CTTTTATACCGAGAAGAAAAAGGACGCTGTACGGTGTGGAAGC-3'.\nThe human RUNX1 fragment from the Addgene plasmid 12504 (Biggs et al., 2006) pFlagCMV2-AML1B was sub-cloned in the pEGFPN1 vector (Clontech Laboratories). The RUNX3 vector pCMV human RUNX3, which was a gift from K. Ito (Institute of Molecular and Cell Biology, Proteos, Singapore) was subcloned into pEGFPN1 vector (Clontech Laboratories; Yamamura et al., 2006).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 79, "end": 89}, "arguments": [{"role": "Theme", "text": "RUNX", "start": 74, "end": 78}]}]}}, "schema": []}
{"input": "Transfections and reporter gene assays.\nT cells were rested in serum-free AIM-V medium overnight. 3.5 microg of the FOXP3 promoter luciferase reporter vector or a combination together with the RUNX1, RUNX3 pEGFPN1 vector, and 0.5 microg phRL-TK were added to 3 x 106 CD4+ T cells resuspended in 100 microl of Nucleofector solution (Lonza) and electroporated using the program U-15. After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega) according to the manufacturer's instructions. PMA/ionomycin was used to stimulate the cells, because the transfection was only transient and the luciferase assay required a strong and fast stimulation of the cells. To evaluate the effect of overexpression of RUNX1 or RUNX3 on FOXP3 protein levels, CD4+ T cells were preactivated with 2 microg/ml phytohemagglutinin (Sigma-Aldrich) in serum-free AIM-V medium in the presence of 1 nmol/liter IL-2 (Roche) for 12 h, and then transfected with the vector pEGFPN1 containing the RUNX1 or RUNX3 fragment using the Nucleofector system (Amaxa Biosystems) and the program T-23. FOXP3 expression was evaluated by flow cytometry after 48 h of culture in AIM-V medium containing 1 nmol/liter IL-2.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "overexpression", "start": 789, "end": 803}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 807, "end": 812}]}, {"trigger": {"text": "overexpression", "start": 789, "end": 803}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 816, "end": 821}]}, {"trigger": {"text": "transfected", "start": 1021, "end": 1032}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 1072, "end": 1077}]}, {"trigger": {"text": "transfected", "start": 1021, "end": 1032}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 1081, "end": 1086}]}, {"trigger": {"text": "expression", "start": 1173, "end": 1183}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1167, "end": 1172}]}], "regulation": [{"trigger": {"text": "effect", "start": 779, "end": 785}, "arguments": [{"role": "Cause", "text": "overexpression", "start": 789, "end": 803}, {"role": "Theme", "text": "FOXP3", "start": 825, "end": 830}]}]}}, "schema": []}
{"input": "RNA interference.\nCD4+ or naive CD4+ T cells were resuspended in 100 microl of Nucleofector solution (Lonza) and electroporated with 2 microM siRNA using the Nucleofector technology program U-14 (Lonza). Five different Silencer or Silencer Select Pre-designed siRNAs for RUNX1 (Applied Biosystems) and three Silencer Pre-designed siRNAs for RUNX3 (Applied Biosystems) were tested, and the best was selected for all further experiments. The Silencer Negative Control #1 siRNA (Applied Biosystems) was used for normalization. Cells were then left unstimulated or were stimulated after 12 h with anti-CD2, anti-CD3, and anti-CD28. Cells were cultured in serum-free AIM-V medium with the addition of 1 nmol/l IL-2 (Roche). Cells were harvested for mRNA detection of the target genes after 24 h and for protein detection after 48 h.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "siRNAs", "start": 260, "end": 266}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 271, "end": 276}]}, {"trigger": {"text": "siRNAs", "start": 330, "end": 336}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 341, "end": 346}]}]}}, "schema": []}
{"input": "RNA isolation and cDNA synthesis.\nRNA was isolated using the RNeasy Mini kit (QIAGEN) according to the manufacturer's protocol. Reverse transcription of human samples was performed with reverse-transcription reagents (Fermentas) with random hexamers according to the manufacturer's protocol.", "output": {"json_structures": {}}, "schema": []}
{"input": "Real-time PCR.\nPCR primers and probes were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems) as follows: FOXP3 forward primer, 5'-GAAACAGCACATTCCCAGAGTTC-3'; FOXP3 reverse primer, 5'-ATGGCCCAGCGGATGAG-3'; EF-1a forward primer, 5'-CTGAACCATCCAGGCCAAAT-3'; and EF-1a reverse primer, 5'-GCCGTGTGGCAATCCAAT-3', as previously described (Mantel et al., 2007). GATA3 forward primer, 5'-GCGGGCTCTATCACAAAATGA-3'; and GATA3 reverse primer 5'-GCTCTCCTGGCTGCAGACAGC-3' (Mantel et al., 2007). T-bet forward primer, 5'-GATGCGCCAGGAAGTTTCAT-3'; T-bet reverse primer, 5'-GCACAATCATCTGGGTCACATT-3'; RORC2 forward primer, 5'-CAGTCATGAGAACACAAATTGAAGTG-3'; and RORC2 reverse primer 5'-CAGGTGATAACCCCGTAGTGGAT-3'. The prepared cDNAs were amplified using SYBR green PCR master mix (Fermentas) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).\nRUNX1 and RUNX3 mRNA was detected by using TaqMan Gene Expression Assays from Applied Biosystems and used according to the manufacturer's instruction using TaqMan master mix using a 7000 real-time PCR system (Applied Biosystems). PCR amplification of the housekeeping gene encoding elongation factor (EF)-1alpha or by using the 18S rRNA Gene Expression Assay (Applied Biosystems) was performed to allow normalization between samples. Relative quantification and calculation of the range of confidence was performed using the comparative deltadeltaCT method (Applied Biosystems). The percentage of FOXP3 mRNA in siRNA-mediated RUNX knockdown cells was calculated in relation to cells, which were transfected with scrambled control siRNA. Arbitrary units show the 2-(deltact) values multiplied by 10,000 incorporating the ct values of the gene of interest and the housekeeping gene.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "knockdown", "start": 1594, "end": 1603}, "arguments": [{"role": "Theme", "text": "RUNX", "start": 1589, "end": 1593}]}], "positive regulation": [{"trigger": {"text": "mediated", "start": 1580, "end": 1588}, "arguments": [{"role": "Theme", "text": "knockdown", "start": 1594, "end": 1603}]}], "transcription": [{"trigger": {"text": "mRNA", "start": 979, "end": 983}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 963, "end": 968}]}, {"trigger": {"text": "mRNA", "start": 979, "end": 983}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 973, "end": 978}]}, {"trigger": {"text": "mRNA", "start": 1566, "end": 1570}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1560, "end": 1565}]}]}}, "schema": []}
{"input": "Flow cytometry.\nFor analysis of human FOXP3 expression on the single-cell level, cells were first stained with the monoclonal CD4 mAb (Beckman Coulter), and after fixation and permeabilization, they were incubated with anti-human Foxp3-Alexa Fluor 488 antibody (BioLegend) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL; Beckman Coulter). A mouse IgG1 antibody (BioLegend) was used as an isotype control. Data were analyzed with the CXP software (Beckman Coulter). Cells were cultured with IL-2, and then left unstimulated or stimulated with anti-CD2/-CD3/-CD28 mAb.\nFlow cytometry analyses of the mouse cells were performed on an LSRII (BD) and cell sorting was performed on a FACSAria (BD). All antibodies for these experiments were purchased from eBioscience or BD.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 44, "end": 54}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 38, "end": 43}]}]}}, "schema": []}
{"input": "Western blotting.\nFor human RUNX1 and RUNX3 analysis on the protein level, 106 cells were lysed and loaded next to a protein-mass ladder (Invitrogen) on a NuPAGE 4-12% Bis-Tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (GE Healthcare). Unspecific binding was blocked with 3% milk in TBS Tween, and the membranes were subsequently incubated with a 1:1,000 dilution of rabbit anti-RUNX1 (ab11903; Abcam) or 1:200 dilution of rabbit anti-RUNX3 (H-50; Santa Cruz Biotechnology, Inc.) in blocking buffer containing 3% milk in TBS Tween overnight at 4degreesC. The blots were developed using an anti-rabbit IgG HRP-labeled mAb (Cell Signaling Technology) and visualized with a LAS-1000 gel documentation system (Fujifilm). To confirm sample loading and transfer membranes were incubated in stripping buffer and reblocked for 1 h and reprobed using anti-GAPDH (6C5; Ambion) and developed using an anti-mouse IgG HRP-labeled mAb (Cell Signaling Technology).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "protein level", "start": 60, "end": 73}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 28, "end": 33}]}, {"trigger": {"text": "protein level", "start": 60, "end": 73}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 38, "end": 43}]}]}}, "schema": []}
{"input": "Pull-down assay.\nHEK293T cells were transfected with RUNX1 or RUNX3 using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. Cells were lysed by sonication in HKMG buffer (10 mM Hepes, pH 7.9, 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM DTT, 0.5% Nonidet P-40) containing a protease inhibitor cocktail (Roche Diagnostics). The cell lysate was precleared using streptavidin-agarose beads (GE Healthcare), incubated with biotinylated double-stranded oligonucleotides containing the wild-type or mutated RUNX binding sites, and polydeoxyinosinicdeoxycytidylic acid (Sigma-Aldrich). A combination of all three oligonucleotides containing the mutated or the wild-type binding sites was used in the assay. DNA-bound proteins were collected with streptavidin-agarose beads, washed with HKMG buffer, and finally resuspended in NuPAGE loading buffer (Invitrogen Life Technologies), heated to 70degreesC for 10 min, and separated on a NuPAGE 4-12% Bis-Tris gel (Invitrogen Life Technologies). The proteins were electroblotted onto a PVDF membrane (GE Healthcare) and detected using RUNX1 or RUNX3 antibodies described in the previous section.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "transfected", "start": 36, "end": 47}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 53, "end": 58}]}, {"trigger": {"text": "transfected", "start": 36, "end": 47}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 62, "end": 67}]}]}}, "schema": []}
{"input": "Promoter enzyme immuno assay.\nAs performed in the pull-down assay, HEK293T cells were transfected with RUNX1 or RUNX3 and subsequently lysed. Insoluble material was removed by centrifugation. 384-well plates, precoated with streptavidin (Thermo Fisher Scientific) were washed 3 times with washing buffer (PBS and 0.05% Tween 20). Biotinylated FOXP3 promoter/oligonucleotides probes containing the RUNX binding sites were added (1 pmol per well; 50 fmol/microl) and incubated for 1 h at room temperature. Either a combination of all three oligonucleotides containing the mutated or the wild-type binding sites was used or single oligonucleotides were used in the assay. After 3 washing steps with washing buffer, the nuclear extract was added (concentration > 0.2 microg/microl) and incubated overnight at 4degreesC. The lysates were incubated with 10 microg of poly-deoxyinosinic-deoxycytidylic acid (Sigma-Aldrich). The plate was washed with HKMG buffer and incubated with a 1:1000 dilution of rabbit anti-RUNX1 (ab11903, Abcam) or 1:200 dilution of rabbit anti-RUNX3 (H-50, Santa Cruz Biotechnology, Inc.) at 4degreesC for 2 h. After three washing steps with HKMG buffer, a secondary antibody (anti-rabbit IgG-HRP, 1:3,000 in HKMG buffer, Cell Signaling Technology) was added, and the plate was incubated for 1 h at 4degreesC. The wells were washed 4 times with HKMG buffer before adding the substrate reagent (R&D Systems). The colorimetric reaction was stopped by adding 2 M H2SO4. Absorbance at 450 nm was measured using a microplate reader (Berthold Technologies).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "transfected", "start": 86, "end": 97}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 103, "end": 108}]}, {"trigger": {"text": "transfected", "start": 86, "end": 97}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 112, "end": 117}]}]}}, "schema": []}
{"input": "ChIP.\nHuman naive CD4+ T cells were cultured either with IL-2 only or with IL-2, anti-CD2/3/28, and TGF-beta for 72 h, and protein-DNA complexes were fixed by cross-linking with formaldehyde in a final concentration of 1.42% for 15 min. Formaldehyde was quenched with 125 mM glycine for 5 min, and cells were subsequently harvested. The ChIP assay was performed as described in the fast chromatin immunoprecipitation method (Nelson et al., 2006). Cells were lysed with immunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, NP-40 [0.5% vol/vol]) containing phosphatase (Roche) and protease inhibitors cocktails (Roche), the nuclear pellet was washed, the chromatin was sheared by sonication and incubated with antibodies for RUNX1 (H-65 X; Santa Cruz Biotechnology, Inc.), RUNX3 (H-50 X; Santa Cruz Biotechnology, Inc.), CBFbeta (PEBP2beta; FL-182 X; Santa Cruz Biotechnology, Inc.), and as controls normal rabbit IgG (Santa Cruz Biotechnology, Inc.), anti-human RNA polymerase II antibody, and mouse control IgG (both from SA Biosciences). The cleared chromatin was incubated with protein A agarose beads and, after several washing steps, DNA was isolated with 10% (wt/vol) Chelex 100 resin. Samples were treated with proteinase K at 55degreesC for 30 min. The proteinase K was then inactivated by boiling the samples for 10 min. The purified DNA was used in a real-time PCR reaction. Specific primers for the FOXP3 promoter, spanning the region from -87 to -3, FOXP3 promoter forward primer 5'-AGAGGTCTGCGGCTTCCA-3', FOXP3 promoter reverse primer 5'-GGAAACTGTCACGTATCAAAAACAA-3', or control GAPDH primer (SA Biosciences) for the RNA polymerase II were used. A negative control PCR for each immunoprecipitation using IGX1A negative control primer targeting ORF-free intergenic DNA (SA Biosciences) was used. The fold enrichment in site occupancy was calculated incorporating IgG control values and input DNA values using the ChampionChIP qPCR data analysis file (SA Biosciences).", "output": {"json_structures": {}}, "schema": []}
{"input": "Quantification of cytokine levels.\nIL-4, IL-5, IL-6, IL-10, IL-13, IL-17, and IFN-gamma secretion was assessed using fluorescent bead-based technology. The Bio-Plex-hu Cytokine Panel, 17-Plex Group 1 was used according to the manufacturer's instructions (Bio-Rad Laboratories). Fluorescent signals were read and analyzed using the Bio-Plex 200 System (Bio-Rad Laboratories).", "output": {"json_structures": {"localization": [{"trigger": {"text": "secretion", "start": 88, "end": 97}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 35, "end": 39}]}, {"trigger": {"text": "secretion", "start": 88, "end": 97}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 41, "end": 45}]}, {"trigger": {"text": "secretion", "start": 88, "end": 97}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 47, "end": 51}]}, {"trigger": {"text": "secretion", "start": 88, "end": 97}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 53, "end": 58}]}, {"trigger": {"text": "secretion", "start": 88, "end": 97}, "arguments": [{"role": "Theme", "text": "IL-13", "start": 60, "end": 65}]}, {"trigger": {"text": "secretion", "start": 88, "end": 97}, "arguments": [{"role": "Theme", "text": "IL-17", "start": 67, "end": 72}]}, {"trigger": {"text": "secretion", "start": 88, "end": 97}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 78, "end": 87}]}]}}, "schema": []}
{"input": "Online supplemental material.\nFig. S1 shows the induction of RUNX1, RUNX3, and FOXP3 mRNA in human CD4+ T cells after anti-CD2/3/28 mAb and TGF-beta stimulation. Fig. S2 shows decreased RUNX1 and RUNX3 mRNA and protein expression after siRNA-mediated knockdown and decreased FOXP3 expression in human CD4+ T cells after RUNX1 and RUNX3 knockdown. Fig. S3 shows the quantification of IL-4, IL-5, IL-10, IL-13, and IFN-gamma levels in control siRNA transfected or RUNX1 and RUNX3 siRNA transfected human CD4+ T cells. Fig. S4 shows the putative RUNX binding sites in the FOXP3 core promoter sequence of human, mouse, and rat. Fig. S5 shows the induction of FOXP3 protein after overexpression of RUNX1 and RUNX3 in human CD4+ T cells. Fig. S6 shows that endogenous IL-4 and IFN-gamma do not effect Foxp3 expression in naive CD4+ T cells of CbfbF/F CD4-cre and CbfbF/F control mice, which were stimulated with anti-CD3 and anti-CD28 mAbs, IL-2 and TGF-beta in the absence or presence of anti-IL-4 and anti-IFN-gamma neutralizing mAbs. Fig. S7 shows similar cell death (A) and proliferation (B) of Foxp3+ and Foxp3- cells in CbfbF/F CD4-cre and CbfbF/F control mice cultures. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20090596/DC1.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 281, "end": 291}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 275, "end": 280}]}, {"trigger": {"text": "overexpression", "start": 675, "end": 689}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 693, "end": 698}]}, {"trigger": {"text": "overexpression", "start": 675, "end": 689}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 703, "end": 708}]}, {"trigger": {"text": "expression", "start": 801, "end": 811}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 795, "end": 800}]}], "negative regulation": [{"trigger": {"text": "decreased", "start": 176, "end": 185}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 202, "end": 206}, {"role": "Cause", "text": "knockdown", "start": 251, "end": 260}]}, {"trigger": {"text": "decreased", "start": 176, "end": 185}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 202, "end": 206}, {"role": "Cause", "text": "decreased", "start": 265, "end": 274}]}, {"trigger": {"text": "knockdown", "start": 251, "end": 260}, "arguments": [{"role": "Theme", "text": "expression", "start": 281, "end": 291}]}, {"trigger": {"text": "decreased", "start": 265, "end": 274}, "arguments": [{"role": "Theme", "text": "expression", "start": 281, "end": 291}, {"role": "Cause", "text": "knockdown", "start": 336, "end": 345}]}, {"trigger": {"text": "knockdown", "start": 336, "end": 345}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 320, "end": 325}]}, {"trigger": {"text": "knockdown", "start": 336, "end": 345}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 330, "end": 335}]}, {"trigger": {"text": "siRNA", "start": 478, "end": 483}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 462, "end": 467}]}, {"trigger": {"text": "siRNA", "start": 478, "end": 483}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 472, "end": 477}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 48, "end": 57}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 85, "end": 89}]}, {"trigger": {"text": "mediated", "start": 242, "end": 250}, "arguments": [{"role": "Theme", "text": "knockdown", "start": 251, "end": 260}]}, {"trigger": {"text": "induction", "start": 642, "end": 651}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 655, "end": 660}, {"role": "Cause", "text": "overexpression", "start": 675, "end": 689}]}], "regulation": [{"trigger": {"text": "effect", "start": 788, "end": 794}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 762, "end": 766}, {"role": "Theme", "text": "expression", "start": 801, "end": 811}]}, {"trigger": {"text": "effect", "start": 788, "end": 794}, "arguments": [{"role": "Cause", "text": "IFN-gamma", "start": 771, "end": 780}, {"role": "Theme", "text": "expression", "start": 801, "end": 811}]}], "transcription": [{"trigger": {"text": "mRNA", "start": 85, "end": 89}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 61, "end": 66}]}, {"trigger": {"text": "mRNA", "start": 85, "end": 89}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 68, "end": 73}]}, {"trigger": {"text": "mRNA", "start": 85, "end": 89}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 79, "end": 84}]}, {"trigger": {"text": "mRNA", "start": 202, "end": 206}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 186, "end": 191}]}, {"trigger": {"text": "mRNA", "start": 202, "end": 206}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 196, "end": 201}]}]}}, "schema": []}
{"input": "RUNX1 and RUNX3 are involved in the induction of Foxp3 in iT reg cells. (A) RUNX1, RUNX3, and FOXP3 mRNA induction in human naive CD4+ T cells after alone or combined anti-CD2/3/28 mAb and TGF-beta stimulation in the presence of IL-2. Real-time PCR of human naive CD4+ T cells after 48 h of culture. Bars show the mean +/- SE of three independent experiments. (B) FOXP3 mRNA induction by anti-CD2/3/28 mAb and TGF-beta in human naive CD4+ T cells is reduced after siRNA-mediated RUNX1/3 knockdown. Real-time PCR of RNA from human naive CD4+ T cells, transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and cultured with anti-CD2/3/28, TGF-beta and IL-2. Bars show the mean +/- SD of three independent experiments. (C) FOXP3 mRNA is down-regulated in iT reg cells after siRNA-mediated knockdown of RUNX1 and RUNX3. Real-time PCR for FOXP3, T-bet, GATA3, and RORC2 from human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA or with scrambled siRNA (control) and cultured under iT reg, Th1, Th2, or Th17-driving conditions for 12 d. Bars show the mean +/- SD of three independent experiments. (D) FOXP3 protein induction in iT reg cells is reduced after siRNA-mediated RUNX1/3 knockdown. Human naive CD4+ T cells were transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and stimulated with anti-CD2/3/28 and TGF-beta in the presence of IL-2. CD4 and intracellular FOXP3 analysis by flow cytometry after 72 h. One of three independent experiments is shown. Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "induction", "start": 36, "end": 45}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 49, "end": 54}]}, {"trigger": {"text": "induction", "start": 1133, "end": 1142}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1119, "end": 1124}]}], "negative regulation": [{"trigger": {"text": "reduced", "start": 450, "end": 457}, "arguments": [{"role": "Theme", "text": "induction", "start": 375, "end": 384}, {"role": "Cause", "text": "knockdown", "start": 487, "end": 496}]}, {"trigger": {"text": "knockdown", "start": 487, "end": 496}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 479, "end": 484}]}, {"trigger": {"text": "knockdown", "start": 487, "end": 496}, "arguments": [{"role": "Theme", "text": "3", "start": 485, "end": 486}]}, {"trigger": {"text": "siRNA", "start": 586, "end": 591}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 567, "end": 572}]}, {"trigger": {"text": "siRNA", "start": 586, "end": 591}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 580, "end": 585}]}, {"trigger": {"text": "down-regulated", "start": 746, "end": 760}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 738, "end": 742}, {"role": "Cause", "text": "knockdown", "start": 798, "end": 807}]}, {"trigger": {"text": "knockdown", "start": 798, "end": 807}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 811, "end": 816}]}, {"trigger": {"text": "knockdown", "start": 798, "end": 807}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 821, "end": 826}]}, {"trigger": {"text": "siRNA", "start": 941, "end": 946}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 925, "end": 930}]}, {"trigger": {"text": "siRNA", "start": 941, "end": 946}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 935, "end": 940}]}, {"trigger": {"text": "reduced", "start": 1162, "end": 1169}, "arguments": [{"role": "Theme", "text": "induction", "start": 1133, "end": 1142}, {"role": "Cause", "text": "knockdown", "start": 1199, "end": 1208}]}, {"trigger": {"text": "knockdown", "start": 1199, "end": 1208}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 1191, "end": 1196}]}, {"trigger": {"text": "knockdown", "start": 1199, "end": 1208}, "arguments": [{"role": "Theme", "text": "3", "start": 1197, "end": 1198}]}, {"trigger": {"text": "siRNA", "start": 1276, "end": 1281}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 1257, "end": 1262}]}, {"trigger": {"text": "siRNA", "start": 1276, "end": 1281}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 1270, "end": 1275}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 105, "end": 114}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 100, "end": 104}, {"role": "Cause", "text": "IL-2", "start": 229, "end": 233}]}, {"trigger": {"text": "induction", "start": 375, "end": 384}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 370, "end": 374}, {"role": "Cause", "text": "TGF-beta", "start": 410, "end": 418}]}, {"trigger": {"text": "mediated", "start": 470, "end": 478}, "arguments": [{"role": "Theme", "text": "knockdown", "start": 487, "end": 496}]}, {"trigger": {"text": "mediated", "start": 789, "end": 797}, "arguments": [{"role": "Theme", "text": "knockdown", "start": 798, "end": 807}]}, {"trigger": {"text": "mediated", "start": 1182, "end": 1190}, "arguments": [{"role": "Theme", "text": "knockdown", "start": 1199, "end": 1208}]}], "regulation": [{"trigger": {"text": "involved", "start": 20, "end": 28}, "arguments": [{"role": "Cause", "text": "RUNX1", "start": 0, "end": 5}, {"role": "Theme", "text": "induction", "start": 36, "end": 45}]}, {"trigger": {"text": "involved", "start": 20, "end": 28}, "arguments": [{"role": "Cause", "text": "RUNX3", "start": 10, "end": 15}, {"role": "Theme", "text": "induction", "start": 36, "end": 45}]}], "transcription": [{"trigger": {"text": "mRNA", "start": 100, "end": 104}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 76, "end": 81}]}, {"trigger": {"text": "mRNA", "start": 100, "end": 104}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 83, "end": 88}]}, {"trigger": {"text": "mRNA", "start": 100, "end": 104}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 94, "end": 99}]}, {"trigger": {"text": "mRNA", "start": 370, "end": 374}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 364, "end": 369}]}, {"trigger": {"text": "mRNA", "start": 738, "end": 742}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 732, "end": 737}]}]}}, "schema": []}
{"input": "RUNX1 and RUNX3 expression in Foxp3+ T reg cells and in human CD4+, CD127-, CD25high cells. (A) Real-time PCR analysis of CD4+, CD127-, and CD25high T reg cells and CD4+, CD127+, and CD25neg T cells isolated from human peripheral blood showed an increased expression of IL-10, TGF-beta, FOXP3, and RUNX3 mRNA in CD25+ compared with CD25- cells. Bars show the mean +/- SE of three independent experiments. (B) Human tonsil sections were analyzed by confocal microscopy. Tissue sections were stained for FOXP3, RUNX1, RUNX3, and DAPI or isotype controls. HEK cells RUNX1-transfected or not transfected served as additional control for RUNX1 staining. Data shown are representative from one of the three tissue samples with similar results. Bars, 5 microm. Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 16, "end": 26}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 0, "end": 5}]}, {"trigger": {"text": "expression", "start": 16, "end": 26}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 10, "end": 15}]}, {"trigger": {"text": "transfected", "start": 569, "end": 580}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 563, "end": 568}]}, {"trigger": {"text": "transfected", "start": 588, "end": 599}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 563, "end": 568}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 246, "end": 255}, "arguments": [{"role": "Theme", "text": "expression", "start": 256, "end": 266}]}], "transcription": [{"trigger": {"text": "expression", "start": 256, "end": 266}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 270, "end": 275}]}, {"trigger": {"text": "expression", "start": 256, "end": 266}, "arguments": [{"role": "Theme", "text": "TGF-beta", "start": 277, "end": 285}]}, {"trigger": {"text": "expression", "start": 256, "end": 266}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 287, "end": 292}]}, {"trigger": {"text": "expression", "start": 256, "end": 266}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 298, "end": 303}]}]}}, "schema": []}
{"input": "Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown. The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides. Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides. The precipitated oligonucleotide-transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies. A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used. Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter. Bars show mean +/- SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFbeta to the human FOXP3 promoter in naive CD4+ T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-beta. There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used. The results are normalized to input and isotype control antibody. Bars show mean +/- SE of three independent experiments. Statistical differences were verified by the paired Student's t test. *, P < 0.05", "output": {"json_structures": {"binding": [{"trigger": {"text": "Binding", "start": 0, "end": 7}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 11, "end": 16}, {"role": "Site2", "text": "binding sites", "start": 53, "end": 66}, {"role": "Theme2", "text": "FOXP3", "start": 74, "end": 79}]}, {"trigger": {"text": "Binding", "start": 0, "end": 7}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 21, "end": 26}, {"role": "Site2", "text": "binding sites", "start": 53, "end": 66}, {"role": "Theme2", "text": "FOXP3", "start": 74, "end": 79}]}, {"trigger": {"text": "binding", "start": 1009, "end": 1016}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 1020, "end": 1025}, {"role": "Theme2", "text": "FOXP3", "start": 1078, "end": 1083}, {"role": "Site2", "text": "promoter", "start": 1084, "end": 1092}]}, {"trigger": {"text": "binding", "start": 1009, "end": 1016}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 1030, "end": 1035}, {"role": "Theme2", "text": "FOXP3", "start": 1078, "end": 1083}, {"role": "Site2", "text": "promoter", "start": 1084, "end": 1092}]}, {"trigger": {"text": "binding", "start": 1009, "end": 1016}, "arguments": [{"role": "Theme", "text": "CBFbeta", "start": 1057, "end": 1064}, {"role": "Theme2", "text": "FOXP3", "start": 1078, "end": 1083}, {"role": "Site2", "text": "promoter", "start": 1084, "end": 1092}]}]}}, "schema": []}
{"input": "Regulation of FOXP3 promoter activity by RUNX1 and RUNX3. (A) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the wild-type or mutated FOXP3 promoter region (FOXP3 -511/+176) fused to the luciferase reporter gene together with a GFP, RUNX1, or RUNX3 expression vector. Bars show the mean luciferase activity +/- SE measured as arbitrary light units of three independent experiments. (B) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the putative FOXP3 promoter region (FOXP3 -511/+176) fused to the luciferase reporter gene, or with a vector containing the putative FOXP3 promoter region (FOXP3 -511/+176) with single RUNX binding sites mutated (53, 287, or 333) or with the combination of two or three RUNX binding sites mutated (53, 287, or 333) fused to the luciferase reporter gene. Bars show the mean luciferase activity +/- SD measured as arbitrary light units of three independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "transfected", "start": 92, "end": 103}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 237, "end": 247}]}, {"trigger": {"text": "transfected", "start": 92, "end": 103}, "arguments": [{"role": "Theme", "text": "GFP", "start": 278, "end": 281}]}, {"trigger": {"text": "transfected", "start": 92, "end": 103}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 283, "end": 288}]}, {"trigger": {"text": "transfected", "start": 92, "end": 103}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 293, "end": 298}]}, {"trigger": {"text": "transfected", "start": 466, "end": 477}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 599, "end": 609}]}, {"trigger": {"text": "transfected", "start": 466, "end": 477}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 861, "end": 871}]}]}}, "schema": []}
{"input": "Diminished capacity of Cbfb-deficient CD4-cre mice T cells in the generation of Foxp3+ CD4+ T cells. (A) FACS-purified naive CD4+ CD8- T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, +/- 10 nM retinoic acid (RA), and increasing concentrations of TGF-beta. After 3 d in culture, the cells were restimulated with PMA + ionomycin, and then analyzed for intracellular Foxp3 and IFN-gamma expression. One of five experiments is shown. (B) Naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice (5 x 106 cells per transfer). 6 wk later, TCRbeta+CD4+ cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine (LP) were analyzed for Foxp3-GFP expression. Results from one of four CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice with same findings are shown. The data from four sets of mice is shown in C. Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05 between groups.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 458, "end": 468}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 438, "end": 443}]}, {"trigger": {"text": "expression", "start": 458, "end": 468}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 448, "end": 457}]}, {"trigger": {"text": "expression", "start": 831, "end": 841}, "arguments": [{"role": "Theme", "text": "Foxp3-GFP", "start": 821, "end": 830}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 28, "end": 37}, "arguments": [{"role": "Theme", "text": "Cbfb", "start": 23, "end": 27}]}, {"trigger": {"text": "deficient", "start": 634, "end": 643}, "arguments": [{"role": "Theme", "text": "Rag", "start": 630, "end": 633}]}]}}, "schema": []}
{"input": "CbfbF/F CD4-cre mouse cells and iT reg cells generated from human naive CD4+ T cells undergoing siRNA-mediated RUNX1 and RUNX3 knock down show a diminished suppressive activity. Experimental setup (A) and results of the mouse suppression assay (B), FACS-purified naive CD4+8- T cells from CbfbF/F CD4-cre (left) and control CbfbF/+ CD4-cre mice (Cd45.2; right) were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, and 2.5 ng/ml TGF-beta. After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 mAb. After a further 4 d, CD45.1+ cells were analyzed for CFSE dilution. (C) As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-beta were mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. Four days later CD45.1+ cells were analyzed for CFSE dilution. The median division number (of the naive CD45.1+ CD4+ cells in the cultures containing Foxp3-GFP+ cells) is indicated in each of the histograms. One of three experiments is shown. (D) Human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA and cultured under iT reg differentiating conditions were used in an in vitro suppression assay, cultured together with autologous CFSE-labeled CD4+ T cells, and stimulated with anti-CD3 mAb. The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry. The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5. One of two experiments is shown.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "knock down", "start": 127, "end": 137}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 111, "end": 116}]}, {"trigger": {"text": "knock down", "start": 127, "end": 137}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 121, "end": 126}]}, {"trigger": {"text": "siRNA", "start": 1172, "end": 1177}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 1156, "end": 1161}]}, {"trigger": {"text": "siRNA", "start": 1172, "end": 1177}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 1166, "end": 1171}]}], "positive regulation": [{"trigger": {"text": "mediated", "start": 102, "end": 110}, "arguments": [{"role": "Theme", "text": "knock down", "start": 127, "end": 137}]}]}}, "schema": []}
{"input": "IKKbeta phosphorylation regulates RPS3 nuclear translocation and NF-kappaB function duringEscherichia coliO157:H7 infection\nNF-kappaB is a major gene regulator in immune responses and ribosomal protein S3 (RPS3) is an NF-kappaB subunit that directs specific gene transcription. However, it is unknown how RPS3 nuclear translocation is regulated. Here we report that IKKbeta phosphorylation of serine 209 (S209) was crucial for RPS3 nuclear localization in response to activating stimuli. Moreover, the foodborne pathogen Escherichia coli O157:H7 virulence protein NleH1 specifically inhibited RPS3 S209 phosphorylation and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but decreasing mortality in a gnotobiotic piglet infection model. Thus, the IKKbeta-dependent modification of a specific amino acid in RPS3 promotes specific NF-kappaB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7.", "output": {"json_structures": {"localization": [{"trigger": {"text": "translocation", "start": 47, "end": 60}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 34, "end": 38}, {"role": "ToLoc", "text": "nuclear", "start": 39, "end": 46}]}, {"trigger": {"text": "translocation", "start": 318, "end": 331}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 305, "end": 309}, {"role": "ToLoc", "text": "nuclear", "start": 310, "end": 317}]}, {"trigger": {"text": "localization", "start": 440, "end": 452}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 427, "end": 431}, {"role": "ToLoc", "text": "nuclear", "start": 432, "end": 439}]}], "negative regulation": [{"trigger": {"text": "inhibited", "start": 583, "end": 592}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 564, "end": 569}, {"role": "Theme", "text": "phosphorylation", "start": 603, "end": 618}]}, {"trigger": {"text": "blocked", "start": 623, "end": 630}, "arguments": [{"role": "Cause", "text": "inhibited", "start": 583, "end": 592}, {"role": "Theme", "text": "RPS3", "start": 631, "end": 635}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 8, "end": 23}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 0, "end": 7}]}, {"trigger": {"text": "phosphorylation", "start": 374, "end": 389}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 366, "end": 373}, {"role": "Site", "text": "serine 209", "start": 393, "end": 403}]}, {"trigger": {"text": "phosphorylation", "start": 603, "end": 618}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 593, "end": 597}, {"role": "Site", "text": "S209", "start": 598, "end": 602}]}], "positive regulation": [{"trigger": {"text": "crucial", "start": 415, "end": 422}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 374, "end": 389}, {"role": "Theme", "text": "in response to", "start": 453, "end": 467}]}, {"trigger": {"text": "in response to", "start": 453, "end": 467}, "arguments": [{"role": "Theme", "text": "localization", "start": 440, "end": 452}, {"role": "Cause", "text": "activating", "start": 468, "end": 478}]}, {"trigger": {"text": "activating", "start": 468, "end": 478}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 427, "end": 431}]}], "protein modification": [{"trigger": {"text": "modification", "start": 794, "end": 806}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 835, "end": 839}]}], "regulation": [{"trigger": {"text": "regulates", "start": 24, "end": 33}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 8, "end": 23}, {"role": "Theme", "text": "translocation", "start": 47, "end": 60}]}, {"trigger": {"text": "regulated", "start": 335, "end": 344}, "arguments": [{"role": "Theme", "text": "translocation", "start": 318, "end": 331}]}, {"trigger": {"text": "dependent", "start": 784, "end": 793}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 776, "end": 783}, {"role": "Theme", "text": "modification", "start": 794, "end": 806}]}]}}, "schema": []}
{"input": "Nuclear Factor-kappa B (NF-kappaB) regulates crucial cellular functions and diverse stimuli activate this pleiotropic transcription factor, which in turn regulates a vast array of genetic targets1-3. The best-known mammalian NF-kappaB subunits are Rel proteins, including RelA (p65), RelB, c-Rel, p50, and p52 (refs. 4,5). However, we recently demonstrated that ribosomal protein S3 (RPS3) is a key non-Rel subunit of certain native NF-kappaB complexes6. RPS3 is defined as a \"specifier\" subunit of NF-kappaB, because it facilitates high affinity DNA binding thus determining the regulatory specificity of NF-kappaB for selected target genes7. RPS3 regulation of NF-kappaB governs key physiological processes, including immunoglobulin kappa light chain gene expression and receptor editing in B cells6,8, cytokine production in T cells6, and in host defense against enterohemorrhagicEscherichia coli(EHEC)9. In particular, theE. coliO157:H7 type III secretion system (T3SS) effector protein NleH1 selectively blocks NF-kappaB target gene transcription by attenuating RPS3 nuclear translocation, without affecting p65 localization9. Nonetheless, how specific NF-kappaB activating signals induce RPS3 nuclear translocation is unknown.Extra-ribosomal functions have been ascribed to ribosomal proteins10. Besides binding RNA within the 40S ribosomal subunit, RPS3 participates in transcription6, DNA repair11,12, and apoptosis13. Whether or not RPS3 is phosphorylated had been controversial14-18. Since kinase cascades play a critical role in NF-kappaB regulation, we tested whether RPS3 is phosphorylated in the context of NF-kappaB activation and sought to identify the responsible kinase19. Additionally, we aimed to define a regulatory role for the C-terminal tail of RPS3 whose function was unknown.Here we show that the Inhibitor of kappaB (IkappaB) kinase beta (IKKbeta) phosphorylated RPS3 at serine 209 (S209). RPS3 S209 phosphorylation enhanced its association with importin-alpha, mediating RPS3 entry into the karyopherin pathway for nuclear translocation. Furthermore, theE. coliNleH1 effector specifically inhibited RPS3 S209 revealing howE. coliO157:H7 inhibits this important innate immune response mechanism.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 1310, "end": 1317}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1356, "end": 1360}]}, {"trigger": {"text": "association", "start": 1956, "end": 1967}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1917, "end": 1921}]}], "gene expression": [{"trigger": {"text": "expression", "start": 758, "end": 768}, "arguments": [{"role": "Theme", "text": "immunoglobulin kappa light chain", "start": 720, "end": 752}]}], "localization": [{"trigger": {"text": "translocation", "start": 1080, "end": 1093}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1067, "end": 1071}, {"role": "ToLoc", "text": "nuclear", "start": 1072, "end": 1079}]}, {"trigger": {"text": "localization", "start": 1117, "end": 1129}, "arguments": [{"role": "Theme", "text": "p65", "start": 1113, "end": 1116}]}, {"trigger": {"text": "translocation", "start": 1207, "end": 1220}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1194, "end": 1198}, {"role": "ToLoc", "text": "nuclear", "start": 1199, "end": 1206}]}, {"trigger": {"text": "translocation", "start": 2051, "end": 2064}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1999, "end": 2003}, {"role": "ToLoc", "text": "nuclear", "start": 2043, "end": 2050}]}], "negative regulation": [{"trigger": {"text": "attenuating", "start": 1055, "end": 1066}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 991, "end": 996}, {"role": "Theme", "text": "translocation", "start": 1080, "end": 1093}]}, {"trigger": {"text": "inhibited", "start": 2117, "end": 2126}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 2089, "end": 2094}, {"role": "Theme", "text": "RPS3", "start": 2127, "end": 2131}, {"role": "Site", "text": "S209", "start": 2132, "end": 2136}]}], "phosphorylation": [{"trigger": {"text": "phosphorylated", "start": 1450, "end": 1464}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1442, "end": 1446}]}, {"trigger": {"text": "phosphorylated", "start": 1588, "end": 1602}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1580, "end": 1584}]}, {"trigger": {"text": "phosphorylated", "start": 1875, "end": 1889}, "arguments": [{"role": "Cause", "text": "Inhibitor of kappaB (IkappaB) kinase beta", "start": 1823, "end": 1864}, {"role": "Theme", "text": "RPS3", "start": 1890, "end": 1894}, {"role": "Site", "text": "serine 209", "start": 1898, "end": 1908}]}, {"trigger": {"text": "phosphorylation", "start": 1927, "end": 1942}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1917, "end": 1921}, {"role": "Site", "text": "S209", "start": 1922, "end": 1926}]}], "positive regulation": [{"trigger": {"text": "induce", "start": 1187, "end": 1193}, "arguments": [{"role": "Theme", "text": "translocation", "start": 1207, "end": 1220}]}, {"trigger": {"text": "enhanced", "start": 1943, "end": 1951}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 1927, "end": 1942}, {"role": "Theme", "text": "association", "start": 1956, "end": 1967}]}, {"trigger": {"text": "mediating", "start": 1989, "end": 1998}, "arguments": [{"role": "Cause", "text": "enhanced", "start": 1943, "end": 1951}, {"role": "Theme", "text": "translocation", "start": 2051, "end": 2064}]}], "regulation": [{"trigger": {"text": "affecting", "start": 1103, "end": 1112}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 991, "end": 996}, {"role": "Theme", "text": "localization", "start": 1117, "end": 1129}]}]}}, "schema": []}
{"input": "RPS3 phosphorylation in response to NF-kappaB activation\nTo test whether RPS3 is phosphorylated during NF-kappaB activation, we performed 32P-labeling experiments in tumor necrosis factor (TNF)-stimulated HEK 293T cells. While RPS3 was scarcely phosphorylated in unstimulated cells, we observed a marked increase in 32P-incorporation after TNF stimulation despite no increase in RPS3 protein (Fig. 1a). To determine which RPS3 residues were phosphorylated, we immunoprecipitated RPS3 from either resting or stimulated cells and performed immunoblotting with phosphorylation-specific antibodies. Both TNF and phorbol myristate acetate/ionomycin (PMA+I) stimulated rapid phosphorylation and degradaion of IkappaBalpha within 5 min which was accompanied by RPS3 phosphorylation on serine residues (Fig. 1b and data not shown), similar to the in vivo labeling. We did not detect tyrosine- or threonine-phosphorylation of RPS3 (Fig. 1b).", "output": {"json_structures": {"phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 5, "end": 20}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 0, "end": 4}]}, {"trigger": {"text": "phosphorylated", "start": 81, "end": 95}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 73, "end": 77}]}, {"trigger": {"text": "phosphorylated", "start": 245, "end": 259}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 227, "end": 231}]}, {"trigger": {"text": "32P-incorporation", "start": 316, "end": 333}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 379, "end": 383}]}, {"trigger": {"text": "phosphorylated", "start": 441, "end": 455}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 422, "end": 426}, {"role": "Site", "text": "residues", "start": 427, "end": 435}]}, {"trigger": {"text": "phosphorylation", "start": 669, "end": 684}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 703, "end": 715}]}, {"trigger": {"text": "phosphorylation", "start": 759, "end": 774}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 754, "end": 758}, {"role": "Site", "text": "serine residues", "start": 778, "end": 793}]}, {"trigger": {"text": "phosphorylation", "start": 898, "end": 913}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 875, "end": 883}, {"role": "Theme", "text": "RPS3", "start": 917, "end": 921}]}, {"trigger": {"text": "phosphorylation", "start": 898, "end": 913}, "arguments": [{"role": "Site", "text": "threonine", "start": 888, "end": 897}, {"role": "Theme", "text": "RPS3", "start": 917, "end": 921}]}], "positive regulation": [{"trigger": {"text": "in response to", "start": 21, "end": 35}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 5, "end": 20}]}, {"trigger": {"text": "increase", "start": 304, "end": 312}, "arguments": [{"role": "Theme", "text": "32P-incorporation", "start": 316, "end": 333}]}, {"trigger": {"text": "increase", "start": 367, "end": 375}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 379, "end": 383}]}, {"trigger": {"text": "stimulated", "start": 652, "end": 662}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 669, "end": 684}]}, {"trigger": {"text": "stimulated", "start": 652, "end": 662}, "arguments": [{"role": "Theme", "text": "degradaion", "start": 689, "end": 699}]}], "protein catabolism": [{"trigger": {"text": "degradaion", "start": 689, "end": 699}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 703, "end": 715}]}]}}, "schema": []}
{"input": "RPS3 and IKKbeta interaction\nThe activation of the inhibitor of kappaB kinase (IKK), consisting of a regulatory subunit IKKgamma and two catalytic subunits, IKKalpha and IKKbeta, is critical for the phosphorylation and dispatch of the inhibitory IkappaBs and the liberation of NF-kappaB20-22. Given that RPS3 can be found in the cytoplasmic p65-p50-IkappaBalpha inhibitory complex in resting cells6, we hypothesized that activated IKKbeta might also bind to and phosphorylate RPS3. First, we found that ectopically expressed IKKbeta and RPS3 interacted (Fig. 1c). We next examined resting Jurkat cells and detected a modest endogenous IKKbeta-RPS3 interaction (Fig. 1d), potentially accounting for the basal NF-kappaB transcription required for cell proliferation and survival. RPS3-IKKbeta association was clearly augmented upon TNF stimulation, peaking at 10 min. (Fig. 1d), following similar kinetics to RPS3 serine phosphorylation (Fig. 1b). By contrast, there was no detectable interaction between RPS3 and IKKalpha (Fig. 1d).", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 17, "end": 28}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 0, "end": 4}, {"role": "Theme2", "text": "IKKbeta", "start": 9, "end": 16}]}, {"trigger": {"text": "bind", "start": 450, "end": 454}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 431, "end": 438}, {"role": "Theme2", "text": "RPS3", "start": 476, "end": 480}]}, {"trigger": {"text": "interacted", "start": 542, "end": 552}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 525, "end": 532}, {"role": "Theme2", "text": "RPS3", "start": 537, "end": 541}]}, {"trigger": {"text": "interaction", "start": 648, "end": 659}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 635, "end": 642}, {"role": "Theme2", "text": "RPS3", "start": 643, "end": 647}]}, {"trigger": {"text": "association", "start": 791, "end": 802}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 778, "end": 782}, {"role": "Theme2", "text": "IKKbeta", "start": 783, "end": 790}]}, {"trigger": {"text": "interaction", "start": 983, "end": 994}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1003, "end": 1007}, {"role": "Theme2", "text": "IKKalpha", "start": 1012, "end": 1020}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 515, "end": 524}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 525, "end": 532}]}], "phosphorylation": [{"trigger": {"text": "phosphorylate", "start": 462, "end": 475}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 476, "end": 480}]}, {"trigger": {"text": "phosphorylation", "start": 919, "end": 934}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 907, "end": 911}, {"role": "Site", "text": "serine", "start": 912, "end": 918}]}], "positive regulation": [{"trigger": {"text": "activated", "start": 421, "end": 430}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 431, "end": 438}]}, {"trigger": {"text": "augmented", "start": 815, "end": 824}, "arguments": [{"role": "Theme", "text": "association", "start": 791, "end": 802}]}, {"trigger": {"text": "following", "start": 877, "end": 886}, "arguments": [{"role": "Cause", "text": "augmented", "start": 815, "end": 824}, {"role": "Theme", "text": "phosphorylation", "start": 919, "end": 934}]}]}}, "schema": []}
{"input": "IKKbeta is required for RPS3 nuclear translocation\nTo examine whether the RPS3-IKKbeta interaction is required for RPS3 nuclear translocation, we knocked down IKKalpha or IKKbeta expression with siRNAs (Supplementary Fig. 1) and then observed stimulation-induced RPS3 nuclear migration by confocal microscopy. Both TNF and PMA+I triggered RPS3 nuclear translocation in Jurkat cells transfected with a scrambled nonspecific (NS) siRNA (Fig. 2a)6. RPS3 nuclear translocation was only slightly, if at all, impaired by IKKalpha-silencing. Conversely, knockdown of IKKbeta attenuated 60-70% of RPS3 nuclear accumulation following stimulation (Fig. 2a). Immunoblotting of nuclear fractions confirmed that full expression of IKKbeta, but not IKKalpha, was necessary for activation-induced RPS3 nuclear translocation (Fig. 2b). Control immunoblots revealed that p65 nuclear translocation was blocked under the same conditions (Fig. 2b).\nWe next examined the nuclear translocation of RPS3 in cells ectopically expressing either kinase-dead (SSAA) or constitutively-active (SSEE) mutant IKKbeta proteins. As expected, the SSEE, but not SSAA, mutant of IKKbeta induced NF-kappaB-dependent luciferase reporter activity (Fig. 2c, left). Whereas RPS3 remained cytosolic in IKKbeta (SSAA)-expressing cells (Fig. 2c, right), a substantial proportion of RPS3 translocated to the nucleus in cells expressing IKKbeta (SSEE) (Fig. 2c, right). The percentage of cells containing detectable nuclear RPS3 increased 5-fold in IKKbeta (SSEE)-expressing cells, but not in IKKbeta (SSAA)-expressing ones (Fig. 2d and Supplementary Fig. 2). Thus, IKKbeta activity is necessary and sufficient for RPS3 nuclear translocation in response to NF-kappaB activating stimuli.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 87, "end": 98}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 74, "end": 78}, {"role": "Theme2", "text": "IKKbeta", "start": 79, "end": 86}]}], "gene expression": [{"trigger": {"text": "expression", "start": 179, "end": 189}, "arguments": [{"role": "Theme", "text": "IKKalpha", "start": 159, "end": 167}]}, {"trigger": {"text": "expression", "start": 179, "end": 189}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 171, "end": 178}]}, {"trigger": {"text": "expression", "start": 704, "end": 714}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 718, "end": 725}]}, {"trigger": {"text": "expression", "start": 704, "end": 714}, "arguments": [{"role": "Theme", "text": "IKKalpha", "start": 735, "end": 743}]}, {"trigger": {"text": "expressing", "start": 1001, "end": 1011}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1077, "end": 1084}]}], "localization": [{"trigger": {"text": "translocation", "start": 37, "end": 50}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 24, "end": 28}, {"role": "ToLoc", "text": "nuclear", "start": 29, "end": 36}]}, {"trigger": {"text": "translocation", "start": 128, "end": 141}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 115, "end": 119}, {"role": "ToLoc", "text": "nuclear", "start": 120, "end": 127}]}, {"trigger": {"text": "migration", "start": 276, "end": 285}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 263, "end": 267}, {"role": "ToLoc", "text": "nuclear", "start": 268, "end": 275}]}, {"trigger": {"text": "translocation", "start": 352, "end": 365}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 339, "end": 343}, {"role": "ToLoc", "text": "nuclear", "start": 344, "end": 351}]}, {"trigger": {"text": "translocation", "start": 459, "end": 472}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 446, "end": 450}, {"role": "ToLoc", "text": "nuclear", "start": 451, "end": 458}]}, {"trigger": {"text": "accumulation", "start": 602, "end": 614}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 589, "end": 593}, {"role": "ToLoc", "text": "nuclear", "start": 594, "end": 601}]}, {"trigger": {"text": "translocation", "start": 795, "end": 808}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 782, "end": 786}, {"role": "ToLoc", "text": "nuclear", "start": 787, "end": 794}]}, {"trigger": {"text": "translocation", "start": 866, "end": 879}, "arguments": [{"role": "Theme", "text": "p65", "start": 854, "end": 857}, {"role": "ToLoc", "text": "nuclear", "start": 858, "end": 865}]}, {"trigger": {"text": "translocation", "start": 958, "end": 971}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 950, "end": 957}, {"role": "Theme", "text": "RPS3", "start": 975, "end": 979}]}, {"trigger": {"text": "remained", "start": 1237, "end": 1245}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1232, "end": 1236}, {"role": "ToLoc", "text": "cytosolic", "start": 1246, "end": 1255}]}, {"trigger": {"text": "translocated", "start": 1342, "end": 1354}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1337, "end": 1341}, {"role": "ToLoc", "text": "nucleus", "start": 1362, "end": 1369}]}, {"trigger": {"text": "detectable", "start": 1458, "end": 1468}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 1469, "end": 1476}, {"role": "Theme", "text": "RPS3", "start": 1477, "end": 1481}]}, {"trigger": {"text": "translocation", "start": 1681, "end": 1694}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1668, "end": 1672}, {"role": "ToLoc", "text": "nuclear", "start": 1673, "end": 1680}]}], "negative regulation": [{"trigger": {"text": "knocked down", "start": 146, "end": 158}, "arguments": [{"role": "Theme", "text": "expression", "start": 179, "end": 189}]}, {"trigger": {"text": "impaired", "start": 503, "end": 511}, "arguments": [{"role": "Theme", "text": "translocation", "start": 459, "end": 472}, {"role": "Cause", "text": "silencing", "start": 524, "end": 533}]}, {"trigger": {"text": "silencing", "start": 524, "end": 533}, "arguments": [{"role": "Theme", "text": "IKKalpha", "start": 515, "end": 523}]}, {"trigger": {"text": "knockdown", "start": 547, "end": 556}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 560, "end": 567}]}, {"trigger": {"text": "attenuated", "start": 568, "end": 578}, "arguments": [{"role": "Cause", "text": "knockdown", "start": 547, "end": 556}, {"role": "Theme", "text": "following", "start": 615, "end": 624}]}, {"trigger": {"text": "blocked", "start": 884, "end": 891}, "arguments": [{"role": "Theme", "text": "translocation", "start": 866, "end": 879}]}, {"trigger": {"text": "kinase-dead", "start": 1019, "end": 1030}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1077, "end": 1084}]}, {"trigger": {"text": "SSAA", "start": 1126, "end": 1130}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1142, "end": 1149}]}, {"trigger": {"text": "SSAA", "start": 1268, "end": 1272}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1259, "end": 1266}]}, {"trigger": {"text": "SSAA", "start": 1555, "end": 1559}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1546, "end": 1553}]}], "positive regulation": [{"trigger": {"text": "required", "start": 11, "end": 19}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 0, "end": 7}, {"role": "Theme", "text": "translocation", "start": 37, "end": 50}]}, {"trigger": {"text": "required", "start": 102, "end": 110}, "arguments": [{"role": "Cause", "text": "interaction", "start": 87, "end": 98}, {"role": "Theme", "text": "translocation", "start": 128, "end": 141}]}, {"trigger": {"text": "induced", "start": 255, "end": 262}, "arguments": [{"role": "Theme", "text": "migration", "start": 276, "end": 285}]}, {"trigger": {"text": "triggered", "start": 329, "end": 338}, "arguments": [{"role": "Theme", "text": "translocation", "start": 352, "end": 365}]}, {"trigger": {"text": "following", "start": 615, "end": 624}, "arguments": [{"role": "Theme", "text": "accumulation", "start": 602, "end": 614}]}, {"trigger": {"text": "necessary", "start": 749, "end": 758}, "arguments": [{"role": "Cause", "text": "expression", "start": 704, "end": 714}, {"role": "Theme", "text": "induced", "start": 774, "end": 781}]}, {"trigger": {"text": "induced", "start": 774, "end": 781}, "arguments": [{"role": "Theme", "text": "translocation", "start": 795, "end": 808}]}, {"trigger": {"text": "constitutively-active", "start": 1041, "end": 1062}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1077, "end": 1084}]}, {"trigger": {"text": "SSEE", "start": 1112, "end": 1116}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1142, "end": 1149}]}, {"trigger": {"text": "induced", "start": 1150, "end": 1157}, "arguments": [{"role": "Cause", "text": "SSEE", "start": 1112, "end": 1116}, {"role": "Theme", "text": "dependent", "start": 1168, "end": 1177}]}, {"trigger": {"text": "induced", "start": 1150, "end": 1157}, "arguments": [{"role": "Cause", "text": "SSAA", "start": 1126, "end": 1130}, {"role": "Theme", "text": "dependent", "start": 1168, "end": 1177}]}, {"trigger": {"text": "in", "start": 1256, "end": 1258}, "arguments": [{"role": "Theme", "text": "remained", "start": 1237, "end": 1245}, {"role": "Cause", "text": "SSAA", "start": 1268, "end": 1272}]}, {"trigger": {"text": "in", "start": 1370, "end": 1372}, "arguments": [{"role": "Theme", "text": "translocated", "start": 1342, "end": 1354}, {"role": "Cause", "text": "SSEE", "start": 1399, "end": 1403}]}, {"trigger": {"text": "SSEE", "start": 1399, "end": 1403}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1390, "end": 1397}]}, {"trigger": {"text": "increased", "start": 1482, "end": 1491}, "arguments": [{"role": "Theme", "text": "detectable", "start": 1458, "end": 1468}, {"role": "Cause", "text": "SSEE", "start": 1511, "end": 1515}]}, {"trigger": {"text": "increased", "start": 1482, "end": 1491}, "arguments": [{"role": "Theme", "text": "detectable", "start": 1458, "end": 1468}, {"role": "Cause", "text": "SSAA", "start": 1555, "end": 1559}]}, {"trigger": {"text": "SSEE", "start": 1511, "end": 1515}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1502, "end": 1509}]}, {"trigger": {"text": "necessary", "start": 1639, "end": 1648}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 1619, "end": 1626}, {"role": "Theme", "text": "in response to", "start": 1695, "end": 1709}]}, {"trigger": {"text": "in response to", "start": 1695, "end": 1709}, "arguments": [{"role": "Theme", "text": "translocation", "start": 1681, "end": 1694}]}], "regulation": [{"trigger": {"text": "dependent", "start": 1168, "end": 1177}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1178, "end": 1188}]}]}}, "schema": []}
{"input": "IkappaBalpha degradation and RPS3 nuclear translocation\nImportin-alpha regulates the nuclear import of NF-kappaB Rel subunits23, 24. RPS3 harbors a nuclear localization signal (NLS) sequence and its nuclear translocation occurs in parallel to, but independently of, p65 translocation6. We envisioned that RPS3 could also utilize the importin-alpha/beta pathway. Consistent with this notion, RPS3 association with importin-alpha, but not importin-beta, was enhanced in TNF-stimulated cells (Fig. 3a). Therefore, we examined whether RPS3 binding to importin-alpha is essential for nuclear translocation during NF-kappaB activation.\nSince IkappaBalpha degradation is a prerequisite to unmask the NLS of p65, and both RPS3 and IkappaBalpha bind to p65 in the cytoplasmic inhibitory complex, we tested whether IkappaBalpha degradation is required for the liberation of RPS3. We measured the association of RPS3 with importin-alpha in 293T cells overexpressing wild-type IkappaBalpha or an IkappaBalpha mutant (SSAA) resistant to IKKbeta-induced phosphorylation and degradation. In cells transfected with wild-type IkappaBalpha, TNF stimulation augmented the interaction of RPS3 and importin-alpha to a similar degree as in non-transfected cells. By contrast, we observed that the RPS3-importin-alpha association was abolished by the presence of non-degradable IkappaBalpha (Fig. 3b).\nTo examine whether IkappaBalpha is the only cytoplasmic barrier precluding RPS3 nuclear translocation, we measured both RPS3-importin-alpha association and nuclear RPS3 after reducing IkappaBalpha expression. Compared with nonspecific siRNA, siRNA targeting of IkappaBalpha completely depleted IkappaBalpha in Jurkat cells (Fig. 3c, input). Nevertheless, the RPS3-importin-alpha association was not augmented (Fig. 3c), nor was significant nuclear RPS3 detected (Fig. 3d). Moreover, cells treated with sodium pervanadate (Pv) to induce IkappaBalpha degradation through an IKK-independent mechanism25-27 did not show increased association between RPS3 and importin-alpha (Fig. 3e and Supplementary Fig. 3b) or nuclear accumulation of RPS3, despite complete IkappaBalpha degradation (Supplementary Fig. 3c). We further examined whether a subsequent NF-kappaB activation signal independently promotes the importin-alpha association and nuclear transport of RPS3 after IkappaBalpha degradation. We found that TNF stimulation following Pv treatment was required for the RPS3-importin-alpha association, comparable to TNF stimulation alone (Fig. 3e). Thus, IkappaBalpha phosphorylation and degradation itself is required but not sufficient to cause RPS3 association with importin-alpha followed by nuclear translocation. Rather, an additional signal, potentially IKKbeta phosphorylation of RPS3, is required.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 536, "end": 543}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 531, "end": 535}]}, {"trigger": {"text": "bind", "start": 736, "end": 740}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 714, "end": 718}, {"role": "Theme2", "text": "p65", "start": 744, "end": 747}]}, {"trigger": {"text": "bind", "start": 736, "end": 740}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 723, "end": 735}, {"role": "Theme2", "text": "p65", "start": 744, "end": 747}]}, {"trigger": {"text": "association", "start": 886, "end": 897}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 901, "end": 905}]}, {"trigger": {"text": "interaction", "start": 1153, "end": 1164}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1168, "end": 1172}]}, {"trigger": {"text": "association", "start": 1295, "end": 1306}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1275, "end": 1279}]}, {"trigger": {"text": "association", "start": 1519, "end": 1530}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1499, "end": 1503}]}, {"trigger": {"text": "association", "start": 1758, "end": 1769}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1738, "end": 1742}]}, {"trigger": {"text": "association", "start": 2005, "end": 2016}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2025, "end": 2029}]}, {"trigger": {"text": "association", "start": 2296, "end": 2307}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2333, "end": 2337}]}, {"trigger": {"text": "association", "start": 2464, "end": 2475}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2444, "end": 2448}]}, {"trigger": {"text": "association", "start": 2627, "end": 2638}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2622, "end": 2626}]}], "gene expression": [{"trigger": {"text": "expression", "start": 1576, "end": 1586}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1563, "end": 1575}]}], "localization": [{"trigger": {"text": "translocation", "start": 42, "end": 55}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 29, "end": 33}, {"role": "ToLoc", "text": "nuclear", "start": 34, "end": 41}]}, {"trigger": {"text": "import", "start": 93, "end": 99}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 85, "end": 92}, {"role": "Theme", "text": "Rel", "start": 113, "end": 116}]}, {"trigger": {"text": "translocation", "start": 207, "end": 220}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 133, "end": 137}, {"role": "ToLoc", "text": "nuclear", "start": 199, "end": 206}]}, {"trigger": {"text": "translocation", "start": 270, "end": 283}, "arguments": [{"role": "Theme", "text": "p65", "start": 266, "end": 269}]}, {"trigger": {"text": "translocation", "start": 587, "end": 600}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 531, "end": 535}, {"role": "ToLoc", "text": "nuclear", "start": 579, "end": 586}]}, {"trigger": {"text": "liberation", "start": 850, "end": 860}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 864, "end": 868}]}, {"trigger": {"text": "translocation", "start": 1467, "end": 1480}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1454, "end": 1458}, {"role": "ToLoc", "text": "nuclear", "start": 1459, "end": 1466}]}, {"trigger": {"text": "detected", "start": 1832, "end": 1840}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 1819, "end": 1826}, {"role": "Theme", "text": "RPS3", "start": 1827, "end": 1831}]}, {"trigger": {"text": "accumulation", "start": 2096, "end": 2108}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 2088, "end": 2095}, {"role": "Theme", "text": "RPS3", "start": 2112, "end": 2116}]}, {"trigger": {"text": "transport", "start": 2320, "end": 2329}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 2312, 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"IkappaBalpha", "start": 1398, "end": 1410}, {"role": "Theme", "text": "translocation", "start": 1467, "end": 1480}]}, {"trigger": {"text": "reducing", "start": 1554, "end": 1562}, "arguments": [{"role": "Theme", "text": "expression", "start": 1576, "end": 1586}]}, {"trigger": {"text": "depleted", "start": 1664, "end": 1672}, "arguments": [{"role": "Cause", "text": "siRNA", "start": 1621, "end": 1626}, {"role": "Theme", "text": "IkappaBalpha", "start": 1673, "end": 1685}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1040, "end": 1055}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 984, "end": 996}]}, {"trigger": {"text": "phosphorylation", "start": 2543, "end": 2558}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 2530, "end": 2542}]}, {"trigger": {"text": "phosphorylation", "start": 2744, "end": 2759}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2763, "end": 2767}]}], "positive regulation": [{"trigger": 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"text": "degradation", "start": 1060, "end": 1071}]}, {"trigger": {"text": "augmented", "start": 1139, "end": 1148}, "arguments": [{"role": "Theme", "text": "interaction", "start": 1153, "end": 1164}]}, {"trigger": {"text": "augmented", "start": 1778, "end": 1787}, "arguments": [{"role": "Cause", "text": "depleted", "start": 1664, "end": 1672}, {"role": "Theme", "text": "association", "start": 1758, "end": 1769}]}, {"trigger": {"text": "induce", "start": 1908, "end": 1914}, "arguments": [{"role": "Theme", "text": "degradation", "start": 1928, "end": 1939}]}, {"trigger": {"text": "increased", "start": 1995, "end": 2004}, "arguments": [{"role": "Cause", "text": "induce", "start": 1908, "end": 1914}, {"role": "Theme", "text": "association", "start": 2005, "end": 2016}]}, {"trigger": {"text": "increased", "start": 1995, "end": 2004}, "arguments": [{"role": "Cause", "text": "induce", "start": 1908, "end": 1914}, {"role": "Theme", "text": "accumulation", "start": 2096, "end": 2108}]}, {"trigger": {"text": "promotes", "start": 2268, "end": 2276}, "arguments": [{"role": "Theme", "text": "association", "start": 2296, "end": 2307}]}, {"trigger": {"text": "promotes", "start": 2268, "end": 2276}, "arguments": [{"role": "Theme", "text": "transport", "start": 2320, "end": 2329}]}, {"trigger": {"text": "required", "start": 2427, "end": 2435}, "arguments": [{"role": "Theme", "text": "association", "start": 2464, "end": 2475}]}, {"trigger": {"text": "cause", "start": 2616, "end": 2621}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 2543, "end": 2558}, {"role": "Theme", "text": "followed", "start": 2659, "end": 2667}]}, {"trigger": {"text": "cause", "start": 2616, "end": 2621}, "arguments": [{"role": "Cause", "text": "degradation", "start": 2563, "end": 2574}, {"role": "Theme", "text": "followed", "start": 2659, "end": 2667}]}, {"trigger": {"text": "followed", "start": 2659, "end": 2667}, "arguments": [{"role": "Cause", "text": "association", "start": 2627, "end": 2638}, {"role": "Theme", "text": "translocation", "start": 2679, "end": 2692}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 13, "end": 24}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 0, "end": 12}]}, {"trigger": {"text": "degradation", "start": 649, "end": 660}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 636, "end": 648}]}, {"trigger": {"text": "degradation", "start": 818, "end": 829}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 805, "end": 817}]}, {"trigger": {"text": "degradation", "start": 1060, "end": 1071}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 984, "end": 996}]}, {"trigger": {"text": "degradable", "start": 1344, "end": 1354}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1355, "end": 1367}]}, {"trigger": {"text": "degradation", "start": 1928, "end": 1939}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1915, "end": 1927}]}, {"trigger": {"text": "degradation", "start": 2148, "end": 2159}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 2135, "end": 2147}]}, {"trigger": {"text": "degradation", "start": 2357, "end": 2368}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 2344, "end": 2356}]}, {"trigger": {"text": "degradation", "start": 2563, "end": 2574}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 2530, "end": 2542}]}], "regulation": [{"trigger": {"text": "regulates", "start": 71, "end": 80}, "arguments": [{"role": "Theme", "text": "import", "start": 93, "end": 99}]}, {"trigger": {"text": "unmask", "start": 682, "end": 688}, "arguments": [{"role": "Site", "text": "NLS", "start": 693, "end": 696}, {"role": "Theme", "text": "p65", "start": 700, "end": 703}]}]}}, "schema": []}
{"input": "IKKbeta phosphorylates RPS3 at serine 209\nAlthough originally defined as the kinase that phosphorylates IkappaB19, IKKbeta also phosphorylates unrelated substrates including 14-3-3beta and Bcl10, which lack the IKK consensus motif (DpSGYXpS/T)28. We therefore hypothesized that IKKbeta could directly phosphorylate RPS3. By in vitro kinase assays using recombinant IKK and RPS3 proteins, we observed strong incorporation of 32P in autophosphorylatd IKKalpha and IKKbeta (Fig. 4a, lanes 2-7) as well as phosporylated GST-IkappaBalpha (1-54) (Supplementary Fig. 4), but not the GST protein alone (Fig. 4a, lanes 3 and 6), when either IKKalpha or IKKbeta was used. We discovered that GST-RPS3 could be phosphorylated by IKKbeta, but not IKKalpha, in vitro (Fig. 4a, compare lanes 4 and 7).\nTo identify the RPS3 amino acid residue(s) phosphorylated by IKKbeta, we performed liquid chromatography-tandem mass spectrometry analyses using in vitro phosphorylated RPS3. The results indicated that IKKbeta phosphorylated S209, located in the RPS3 C-terminus (Fig. 4b). RPS3 amino acid sequence alignment revealed that S209 is conserved in many species throughout phylogeny with the exception of Caenorhabditis elegans and Schizosaccharomyces pombe, two organisms that do not possess the NF-kappaB signal pathway (Supplementary Fig. 5).\nTo verify biochemically that S209 is an IKKbeta substrate, we performed 32P-labeling in vitro kinase assays with recombinant wild-type or S209A mutant RPS3 proteins. Compared with the wild-type protein, the S209A mutation reduced IKKbeta-mediated RPS3 phosphorylation (Fig. 4c). There might be alternative phosphorylation site(s) under these conditions given modest residual phosphorylated RPS3 (Fig. 4c). RPS3 S209 does not fall within a conventional IKK recognition motif, but rather resides in a sequence motif (XXXpS/TXXE), potentially recognized by casein kinase II (CK2). Although IKKbeta kinase can display a CK2-like phosphorylation specificity29, no CK2 protein was detectable in our recombinant IKK proteins (Supplementary Fig. 6). Thus, RPS3 S209 phosphorylation was due to the alternate specificity of the IKKbeta kinase rather than any trace amount of CK2 bound to IKKs. To determine whether S209 is the critical site at which IKKbeta phosphorylates RPS3 in living cells, we transfected the wild-type or S209A mutant Flag-RPS3 alone, or together with IKKbeta into cells. Indeed, we observed that overexpressing IKKbeta enhanced Flag-RPS3 phosphorylation, but phosphorylation was effectively eliminated by alanine substitution indicating that S209 is the predominant target site for IKKbeta phosphorylation (Fig. 4d). We next generated a phospho-S209 RPS3 antibody and confirmed that endogenous RPS3 was phosphorylated at S209 in a time-dependent manner upon TNF stimulation (Fig. 4e). Thus, the RPS3 C-terminal tail potentially contains an important regulatory site.", "output": {"json_structures": {"binding": [{"trigger": {"text": "recognized", "start": 1867, "end": 1877}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1733, "end": 1737}, {"role": "Site", "text": "sequence motif", "start": 1826, "end": 1840}]}], "gene expression": [{"trigger": {"text": "transfected", "start": 2315, "end": 2326}, "arguments": [{"role": "Theme", "text": "Flag-RPS3", "start": 2357, "end": 2366}]}, {"trigger": {"text": "transfected", "start": 2315, "end": 2326}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 2391, "end": 2398}]}], "negative regulation": [{"trigger": {"text": "reduced", "start": 1549, "end": 1556}, "arguments": [{"role": "Cause", "text": "mutation", "start": 1540, "end": 1548}, {"role": "Theme", "text": "mediated", "start": 1565, "end": 1573}]}, {"trigger": {"text": "eliminated", "start": 2531, "end": 2541}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 2478, "end": 2493}, {"role": "Cause", "text": "alanine substitution", "start": 2545, "end": 2565}]}], "phosphorylation": [{"trigger": {"text": "phosphorylates", "start": 8, "end": 22}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 0, "end": 7}, {"role": "Theme", "text": "RPS3", "start": 23, "end": 27}, {"role": "Site", "text": "serine 209", "start": 31, "end": 41}]}, {"trigger": {"text": "phosphorylates", "start": 128, "end": 142}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 115, "end": 122}, {"role": "Theme", "text": "14-3-3beta", "start": 174, "end": 184}]}, {"trigger": {"text": "phosphorylates", "start": 128, "end": 142}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 115, "end": 122}, {"role": "Theme", "text": "Bcl10", "start": 189, "end": 194}]}, {"trigger": {"text": "phosphorylate", "start": 301, "end": 314}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 278, "end": 285}, {"role": "Theme", "text": "RPS3", "start": 315, "end": 319}]}, {"trigger": {"text": "incorporation of 32P", "start": 407, "end": 427}, "arguments": [{"role": "Theme", "text": "IKKalpha", "start": 449, "end": 457}]}, {"trigger": {"text": "incorporation of 32P", "start": 407, "end": 427}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 462, "end": 469}]}, {"trigger": {"text": "autophosphorylatd", "start": 431, "end": 448}, "arguments": [{"role": "Theme", "text": "IKKalpha", "start": 449, "end": 457}]}, {"trigger": {"text": "autophosphorylatd", "start": 431, "end": 448}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 462, "end": 469}]}, {"trigger": {"text": "phosporylated", "start": 502, "end": 515}, "arguments": [{"role": "Theme", "text": "GST-IkappaBalpha", "start": 516, "end": 532}]}, {"trigger": {"text": "phosporylated", "start": 502, "end": 515}, "arguments": [{"role": "Theme", "text": "GST", "start": 576, "end": 579}]}, {"trigger": {"text": "phosphorylated", "start": 699, "end": 713}, "arguments": [{"role": "Theme", "text": "GST-RPS3", "start": 681, "end": 689}, {"role": "Cause", "text": "IKKbeta", "start": 717, "end": 724}]}, {"trigger": {"text": "phosphorylated", "start": 699, "end": 713}, "arguments": [{"role": "Theme", "text": "GST-RPS3", "start": 681, "end": 689}, {"role": "Cause", "text": "IKKalpha", "start": 734, "end": 742}]}, {"trigger": {"text": "phosphorylated", "start": 830, "end": 844}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 803, "end": 807}, {"role": "Site", "text": "amino acid residue(s)", "start": 808, "end": 829}, {"role": "Cause", "text": "IKKbeta", "start": 848, "end": 855}]}, {"trigger": {"text": "phosphorylated", "start": 941, "end": 955}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 956, "end": 960}]}, {"trigger": {"text": "phosphorylated", "start": 997, "end": 1011}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 989, "end": 996}, {"role": "Site", "text": "S209", "start": 1012, "end": 1016}, {"role": "Theme", "text": "RPS3", "start": 1033, "end": 1037}]}, {"trigger": {"text": "phosphorylation", "start": 1579, "end": 1594}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1574, "end": 1578}]}, {"trigger": {"text": "phosphorylated", "start": 1702, "end": 1716}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1717, "end": 1721}]}, {"trigger": {"text": "phosphorylation", "start": 2085, "end": 2100}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2075, "end": 2079}, {"role": "Site", "text": "S209", "start": 2080, "end": 2084}]}, {"trigger": {"text": "phosphorylates", "start": 2275, "end": 2289}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 2267, "end": 2274}, {"role": "Theme", "text": "RPS3", "start": 2290, "end": 2294}]}, {"trigger": {"text": "phosphorylation", "start": 2478, "end": 2493}, "arguments": [{"role": "Theme", "text": "Flag-RPS3", "start": 2468, "end": 2477}]}, {"trigger": {"text": "phosphorylation", "start": 2630, "end": 2645}, "arguments": [{"role": "Theme", "text": "Flag-RPS3", "start": 2468, "end": 2477}, {"role": "Site", "text": "S209", "start": 2582, "end": 2586}]}, {"trigger": {"text": "phosphorylated", "start": 2743, "end": 2757}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2734, "end": 2738}, {"role": "Site", "text": "S209", "start": 2761, "end": 2765}]}], "positive regulation": [{"trigger": {"text": "mediated", "start": 1565, "end": 1573}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 1557, "end": 1564}, {"role": "Theme", "text": "phosphorylation", "start": 1579, "end": 1594}]}, {"trigger": {"text": "due", "start": 2105, "end": 2108}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 2085, "end": 2100}, {"role": "Cause", "text": "IKKbeta", "start": 2145, "end": 2152}]}, {"trigger": {"text": "critical", "start": 2244, "end": 2252}, "arguments": [{"role": "CSite", "text": "S209", "start": 2232, "end": 2236}, {"role": "Theme", "text": "phosphorylates", "start": 2275, "end": 2289}, {"role": "Cause", "text": "RPS3", "start": 2290, "end": 2294}]}, {"trigger": {"text": "overexpressing", "start": 2436, "end": 2450}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 2451, "end": 2458}]}, {"trigger": {"text": "enhanced", "start": 2459, "end": 2467}, "arguments": [{"role": "Cause", "text": "overexpressing", "start": 2436, "end": 2450}, {"role": "Theme", "text": "phosphorylation", "start": 2478, "end": 2493}]}, {"trigger": {"text": "upon", "start": 2793, "end": 2797}, "arguments": [{"role": "Theme", "text": "dependent", "start": 2776, "end": 2785}]}], "regulation": [{"trigger": {"text": "mutant", "start": 1471, "end": 1477}, "arguments": [{"role": "Site", "text": "S209A", "start": 1465, "end": 1470}, {"role": "Theme", "text": "RPS3", "start": 1478, "end": 1482}]}, {"trigger": {"text": "mutation", "start": 1540, "end": 1548}, "arguments": [{"role": "Site", "text": "S209A", "start": 1534, "end": 1539}, {"role": "Theme", "text": "RPS3", "start": 1574, "end": 1578}]}, {"trigger": {"text": "alanine substitution", "start": 2545, "end": 2565}, "arguments": [{"role": "Theme", "text": "Flag-RPS3", "start": 2468, "end": 2477}, {"role": "Site", "text": "S209", "start": 2582, "end": 2586}]}, {"trigger": {"text": "dependent", "start": 2776, "end": 2785}, "arguments": [{"role": "Theme", "text": "phosphorylated", "start": 2743, "end": 2757}]}]}}, "schema": []}
{"input": "Phosphorylation of RPS3 and its NF-kappaB function\nWe next examined whether S209 phosphorylation plays a role in the nuclear translocation of RPS3 during NF-kappaB activation. Subcellular fractions from either wild-type or S209A mutant RPS3-transfected cells were prepared and blotted for heat-shock protein 90 (hsp90), a cytoplasmic protein, and poly (ADP-ribose) polymerase (PARP), a nuclear protein, confirming a clean separation (Fig. 5a). As expected, PMA+I stimulation triggered wild-type Flag-RPS3 nuclear translocation (Fig. 5a). However, RPS3 (S209A) nuclear translocation was attenuated (Fig. 5a). We also tested the impact of activating NF-kappaB by overexpressing IKKbeta on RPS3 nuclear translocation. IKKbeta overexpression activated NF-kappaB measured by luciferase assays (Supplementary Fig. 7), and also induced the nuclear translocation of wild-type, but not S209A, RPS3 (Fig. 5b). These data suggest that S209 phosphorylation is critical for the NF-kappaB activation-induced RPS3 nuclear translocation.\nTo examine the role of S209 phosphorylation of RPS3 to its NF-kappaB function6, 7, 30, we silenced endogenous RPS3 expression using an siRNA that targets the 3' untranslated region (3' UTR) of RPS3 mRNA, followed by complementation with either wild-type or S209A mutant RPS3 via transfection. As expected, RPS3 siRNA severely reduced endogenous RPS3 abundance compared to NS siRNA, but did not affect the robust expression of Flag-tagged RPS3 from a transfected construct lacking the 3' UTR (Fig. 5c). We also found that RPS3 knockdown reduced TNF-induced expression of an Ig kappaB-driven luciferase construct6 (Fig. 5d). The impaired luciferase signal caused by RPS3 deficiency was completely restored by transfecting wild-type, but not by S209A RPS3 (Fig. 5d), despite equivalent expression (Fig. 5c). Moreover, the failure of S209A RPS3 to restore luciferase activity did not result from defective translation because the transient overexpression of green fluorescent protein (GFP) was comparable in cells complemented with wild-type or S029A RPS3 (Supplementary Fig. 8). Taken together, these data suggest that RPS3 S209 phosphorylation is critical for NF-kappaB activity involving the canonical Ig kappaB site.\nWe next used chromatin immunoprecipitation to determine whether S209 phosphorylation affects RPS3 and p65 recruitment to specific kappaB sites in intact chromatin during NF-kappaB activation. In RPS3 knockdown cells, PMA+I stimulated the recruitment of ectopically expressed, Flag-tagged wild-type, but not S209A RPS3 to the kappaB sites of the NFKBIA and IL8 promoters (Fig. 5e). While expressing RPS3 S209A had no impact on p65 nuclear translocation, it substantially attenuated p65 recruitment (Fig. 5e). Additional experiments revealed that p65 attraction to RPS3-independent NF-kappaB target gene promoters such as CD25 was increased (Supplementary Fig. 9), consistent with our previous observations6. There was no significant Flag-RPS3 or p65 recruitment to ACTB promoter lacking kappaB sites (Fig. 5e), suggesting the recruitment was kappaB site-specific. Thus, the recruitment of RPS3 as well as the contingent recruitment of p65 to key promoters depended on S209.\nInterleukin 8 (IL-8) secretion induced by either T cell receptor (TCR) agonist stimulation or PMA+I was decreased as a consequence of reduced RPS3/p65 recruitment to the IL8 kappaB sites in the presence of S209A mutant compared to wild-type RPS3 (Supplementary Fig. 10). However, cell surface CD25 expression was comparable between the wild-type and S209A RPS3 transfected cells (Supplementary Fig. 11). Therefore, RPS3 S209 phosphorylation by IKKbeta is apparently required for RPS3 in directing NF-kappaB to a specific subset of target genes.", "output": {"json_structures": {"binding": [{"trigger": {"text": "recruitment", "start": 2345, "end": 2356}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2332, "end": 2336}]}, {"trigger": {"text": "recruitment", "start": 2345, "end": 2356}, "arguments": [{"role": "Theme", "text": "p65", "start": 2341, "end": 2344}]}, {"trigger": {"text": "recruitment", "start": 2477, "end": 2488}, "arguments": [{"role": "Site", "text": "S209A", "start": 2546, "end": 2551}, {"role": "Theme", "text": "RPS3", "start": 2552, "end": 2556}, {"role": "Site2", "text": "kappaB sites", "start": 2564, "end": 2576}, {"role": "Theme2", "text": "NFKBIA", "start": 2584, "end": 2590}]}, {"trigger": {"text": "recruitment", "start": 2477, "end": 2488}, "arguments": [{"role": "Site", "text": "S209A", "start": 2546, "end": 2551}, {"role": "Theme", "text": "RPS3", "start": 2552, "end": 2556}, {"role": "Site2", "text": "kappaB sites", "start": 2564, "end": 2576}, {"role": "Theme2", "text": "IL8", "start": 2595, "end": 2598}]}, {"trigger": {"text": "recruitment", "start": 2477, "end": 2488}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2552, "end": 2556}, {"role": "Site2", "text": "kappaB sites", "start": 2564, "end": 2576}, {"role": "Theme2", "text": "NFKBIA", "start": 2584, "end": 2590}]}, {"trigger": {"text": "recruitment", "start": 2477, "end": 2488}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2552, "end": 2556}, {"role": "Site2", "text": "kappaB sites", "start": 2564, "end": 2576}, {"role": "Theme2", "text": "IL8", "start": 2595, "end": 2598}]}, {"trigger": {"text": "recruitment", "start": 2724, "end": 2735}, "arguments": [{"role": "Theme", "text": "p65", "start": 2720, "end": 2723}]}, {"trigger": {"text": "recruitment", "start": 3363, "end": 3374}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 3354, "end": 3358}, {"role": "Theme2", "text": "IL8", "start": 3382, "end": 3385}, {"role": "Site2", "text": "kappaB sites", "start": 3386, "end": 3398}]}], "gene expression": [{"trigger": {"text": "expression", "start": 1137, "end": 1147}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1132, "end": 1136}]}, {"trigger": {"text": "transfection", "start": 1301, "end": 1313}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1292, "end": 1296}]}, {"trigger": {"text": "expression", "start": 1434, "end": 1444}, "arguments": [{"role": "Theme", "text": "Flag-tagged RPS3", "start": 1448, "end": 1464}]}, {"trigger": {"text": "expression", "start": 1578, "end": 1588}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1612, "end": 1622}]}, {"trigger": {"text": "transfecting", "start": 1729, "end": 1741}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1770, "end": 1774}]}, {"trigger": {"text": "overexpression", "start": 1958, "end": 1972}, "arguments": [{"role": "Theme", "text": "green fluorescent protein", "start": 1976, "end": 2001}]}, {"trigger": {"text": "expressed", "start": 2504, "end": 2513}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2552, "end": 2556}]}, {"trigger": {"text": "expression", "start": 3510, "end": 3520}, "arguments": [{"role": "Theme", "text": "CD25", "start": 3505, "end": 3509}]}, {"trigger": {"text": "transfected", "start": 3573, "end": 3584}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 3568, "end": 3572}]}], "localization": [{"trigger": {"text": "translocation", "start": 125, "end": 138}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 117, "end": 124}, {"role": "Theme", "text": "RPS3", "start": 142, "end": 146}]}, {"trigger": {"text": "translocation", "start": 513, "end": 526}, "arguments": [{"role": "Theme", "text": "Flag-RPS3", "start": 495, "end": 504}, {"role": "ToLoc", "text": "nuclear", "start": 505, "end": 512}]}, {"trigger": {"text": "translocation", "start": 568, "end": 581}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 547, "end": 551}, {"role": "ToLoc", "text": "nuclear", "start": 560, "end": 567}]}, {"trigger": {"text": "translocation", "start": 700, "end": 713}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 687, "end": 691}, {"role": "ToLoc", "text": "nuclear", "start": 692, "end": 699}]}, {"trigger": {"text": "translocation", "start": 841, "end": 854}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 833, "end": 840}, {"role": "Theme", "text": "RPS3", "start": 884, "end": 888}]}, {"trigger": {"text": "translocation", "start": 1007, "end": 1020}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 994, "end": 998}, {"role": "ToLoc", "text": "nuclear", "start": 999, "end": 1006}]}, {"trigger": {"text": "translocation", "start": 2677, "end": 2690}, "arguments": [{"role": "Theme", "text": "p65", "start": 2665, "end": 2668}, {"role": "ToLoc", "text": "nuclear", "start": 2669, "end": 2676}]}, {"trigger": {"text": 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"promoters", "start": 3184, "end": 3193}]}, {"trigger": {"text": "secretion", "start": 3233, "end": 3242}, "arguments": [{"role": "Theme", "text": "Interleukin 8", "start": 3212, "end": 3225}]}], "negative regulation": [{"trigger": {"text": "attenuated", "start": 586, "end": 596}, "arguments": [{"role": "Cause", "text": "RPS3", "start": 547, "end": 551}, {"role": "CSite", "text": "S209A", "start": 553, "end": 558}, {"role": "Theme", "text": "translocation", "start": 568, "end": 581}]}, {"trigger": {"text": "silenced", "start": 1112, "end": 1120}, "arguments": [{"role": "Theme", "text": "expression", "start": 1137, "end": 1147}]}, {"trigger": {"text": "reduced", "start": 1348, "end": 1355}, "arguments": [{"role": "Cause", "text": "RPS3 siRNA", "start": 1328, "end": 1338}, {"role": "Theme", "text": "RPS3", "start": 1367, "end": 1371}]}, {"trigger": {"text": "knockdown", "start": 1548, "end": 1557}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1543, "end": 1547}]}, {"trigger": 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"arguments": [{"role": "Theme", "text": "impaired", "start": 1649, "end": 1657}, {"role": "Cause", "text": "deficiency", "start": 1691, "end": 1701}]}, {"trigger": {"text": "stimulated", "start": 2462, "end": 2472}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 2477, "end": 2488}]}, {"trigger": {"text": "increased", "start": 2868, "end": 2877}, "arguments": [{"role": "Theme", "text": "attraction", "start": 2788, "end": 2798}]}, {"trigger": {"text": "induced", "start": 3243, "end": 3250}, "arguments": [{"role": "Theme", "text": "secretion", "start": 3233, "end": 3242}, {"role": "Cause", "text": "stimulation", "start": 3291, "end": 3302}]}, {"trigger": {"text": "stimulation", "start": 3291, "end": 3302}, "arguments": [{"role": "Theme", "text": "Interleukin 8", "start": 3212, "end": 3225}]}, {"trigger": {"text": "by", "start": 3653, "end": 3655}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 3637, "end": 3652}, {"role": "Cause", "text": "IKKbeta", "start": 3656, "end": 3663}]}], "regulation": [{"trigger": {"text": "plays a role", "start": 97, "end": 109}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 81, "end": 96}, {"role": "Theme", "text": "translocation", "start": 125, "end": 138}]}, {"trigger": {"text": "mutant", "start": 229, "end": 235}, "arguments": [{"role": "Site", "text": "S209A", "start": 223, "end": 228}, {"role": "Theme", "text": "RPS3", "start": 236, "end": 240}]}, {"trigger": {"text": "impact", "start": 627, "end": 633}, "arguments": [{"role": "Theme", "text": "translocation", "start": 700, "end": 713}]}, {"trigger": {"text": "affect", "start": 1416, "end": 1422}, "arguments": [{"role": "Cause", "text": "RPS3 siRNA", "start": 1328, "end": 1338}, {"role": "Theme", "text": "expression", "start": 1434, "end": 1444}]}, {"trigger": {"text": "affects", "start": 2324, "end": 2331}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 2308, "end": 2323}, {"role": "Theme", "text": "recruitment", "start": 2345, "end": 2356}]}, {"trigger": {"text": "impact", "start": 2655, "end": 2661}, "arguments": [{"role": "Cause", "text": "RPS3", "start": 2637, "end": 2641}, {"role": "CSite", "text": "S209A", "start": 2642, "end": 2647}, {"role": "Theme", "text": "translocation", "start": 2677, "end": 2690}]}, {"trigger": {"text": "specific", "start": 3092, "end": 3100}, "arguments": [{"role": "Theme", "text": "attraction", "start": 2788, "end": 2798}]}, {"trigger": {"text": "depended", "start": 3194, "end": 3202}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 3112, "end": 3123}, {"role": "Cause", "text": "RPS3", "start": 3127, "end": 3131}, {"role": "CSite", "text": "S209", "start": 3206, "end": 3210}]}, {"trigger": {"text": "depended", "start": 3194, "end": 3202}, "arguments": [{"role": "Cause", "text": "RPS3", "start": 3127, "end": 3131}, {"role": "Theme", "text": "recruitment", "start": 3158, "end": 3169}, {"role": "CSite", "text": "S209", "start": 3206, 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{"input": "NleH1 inhibits RPS3 phosphorylation in vitro\nEHEC pathogens are important causative agents of both foodborne disease and pediatric renal failure31. EHEC utilize T3SS to inject effector proteins directly into intestinal epithelial cells32, a subset of which inhibit NF-kappaB-dependent innate responses9, 33-38. The E. coli O157:H7 EDL933 effector protein NleH1 binds to and attenuates RPS3 nuclear translocation, thus impairing RPS3-dependent NF-kappaB signaling9. We therefore hypothesized that NleH1 may function by inhibiting RPS3 S209 phosphorylation. As expected, transfecting increasing amounts of NleH1-HA plasmid blocked TNFalpha-induced NF-kappaB activation in a dose-dependent manner (Fig. 6a-b)9. Remarkably, NleH1 reduced both TNF-induced, as well as basal RPS3 phosphorylation to roughly 20% of vehicle control (Fig. 6c). Expressing NleH1 does not interfere with either TNF-induced IKK activation or IkappaBalpha degradation, consistent with the lack of NleH1 impact on p65 nuclear translocation9 (Fig. 6c).\nTo determine if NleH1 inhibits RPS3 phosphorylation, we infected HeLa cells with E. coli O157:H7 strains possessing or lacking either nleH1 (deltanleH1) or with a strain lacking a functional T3SS unable to inject NleH1 into mammalian cells (deltaescN). In uninfected cells, TNF-treatment stimulated a ~7-fold increase in RPS3 S209 phosphorylation, peaking at 30 minutes (Fig. 6d). By contrast, RPS3 S209 phosphorylation was substantially impaired in cells infected with wild-type E. coli O157:H7 (Fig. 6d). However, TNF-induced RPS3 S209 phosphorylation was unimpaired in cells infected with either deltanleH1 or deltaescN (Fig. 6d). We showed previously that wild-type, but not deltanleH1 or deltaescN E. coli O157:H7 significantly attenuated TNF-induced RPS3 nuclear translocation9. The parallel between RPS3 phosphorylation and its nuclear translocation during E. coli infection provides evidence in the context of an NF-kappaB-dependent disease process that RPS3 S209 phosphorylation is important for nuclear translocation.\nOur discovery that NleH1 inhibits RPS3 S209 phosphorylation suggested that it should also blocks RPS3-dependent NF-kappaB target gene transcription (e.g. IL8, NFKBIA, and TNFAIP3). Indeed, these genes were only modestly upregulated in cells infected with wild-type E. coli O157:H7, but significantly induced in cells infected with either deltanleH1 or deltaescN strains (Fig. 6e). In contrast, deleting nleH1 had no impact on the expression of RPS3-independent genes, including CD25 and TNFSF13B (Supplementary Fig. 12). Together these results demonstrate that NleH1 specifically inhibits the protective immune response by directly blocking RPS3 S209 phosphorylation and thereby impairing critical RPS3-dependent NF-kappaB target genes.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 361, "end": 366}, "arguments": [{"role": "Theme", "text": "NleH1", "start": 355, "end": 360}, {"role": "Theme2", "text": "RPS3", "start": 385, "end": 389}]}], "gene expression": [{"trigger": {"text": "Expressing", "start": 835, "end": 845}, "arguments": [{"role": "Theme", "text": "NleH1", "start": 846, "end": 851}]}], "localization": [{"trigger": {"text": "translocation", "start": 398, "end": 411}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 385, "end": 389}, {"role": "ToLoc", "text": "nuclear", "start": 390, "end": 397}]}, {"trigger": {"text": "translocation", "start": 995, "end": 1008}, "arguments": [{"role": "Theme", "text": "p65", "start": 983, "end": 986}, {"role": "ToLoc", 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"start": 2452, "end": 2457}]}, {"trigger": {"text": "blocking", "start": 2681, "end": 2689}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 2610, "end": 2615}, {"role": "Theme", "text": "phosphorylation", "start": 2700, "end": 2715}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 20, "end": 35}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 15, "end": 19}]}, {"trigger": {"text": "phosphorylation", "start": 539, "end": 554}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 529, "end": 533}, {"role": "Site", "text": "S209", "start": 534, "end": 538}]}, {"trigger": {"text": "phosphorylation", "start": 774, "end": 789}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 769, "end": 773}]}, {"trigger": {"text": "phosphorylation", "start": 1057, "end": 1072}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1052, "end": 1056}]}, {"trigger": {"text": "phosphorylation", "start": 1352, "end": 1367}, "arguments": [{"role": 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"TNF", "start": 1295, "end": 1298}, {"role": "Theme", "text": "phosphorylation", "start": 1352, "end": 1367}]}, {"trigger": {"text": "induced", "start": 1541, "end": 1548}, "arguments": [{"role": "Cause", "text": "TNF", "start": 1537, "end": 1540}, {"role": "Theme", "text": "phosphorylation", "start": 1559, "end": 1574}]}, {"trigger": {"text": "induced", "start": 1769, "end": 1776}, "arguments": [{"role": "Cause", "text": "TNF", "start": 1765, "end": 1768}, {"role": "Theme", "text": "translocation", "start": 1790, "end": 1803}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 926, "end": 937}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 913, "end": 925}]}], "regulation": [{"trigger": {"text": "impact", "start": 973, "end": 979}, "arguments": [{"role": "Cause", "text": "lack", "start": 959, "end": 963}, {"role": "Theme", "text": "translocation", "start": 995, "end": 1008}]}, {"trigger": {"text": "important", "start": 2012, "end": 2021}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 1993, "end": 2008}, {"role": "Theme", "text": "translocation", "start": 2034, "end": 2047}]}]}}, "schema": []}
{"input": "NleH1 inhibits RPS3 S209 phosphorylation in vivo\nWe previously utilized a gnotobiotic piglet infection model to determine that piglets infected with deltanleH1 mutant died more rapidly than those infected with wild-type E. coli O157:H7. Piglets infected with deltanleH1 displayed clinical disease consistent with a robust inflammatory response, but with reduced bacterial colonization and little diarrhea9. While seemingly paradoxical, based on our cell culture data (Fig. 6d), we hypothesized that NleH1 blocked RPS3 S209 phosphorylation in vivo, thereby preventing RPS3 nuclear translocation in infected piglets. We isolated piglet colons at necropsy, and subjected them to immunohistochemistry using our phospho-RPS3 antibody. Consistent with in vitro data, piglets infected with wild-type E. coli O157:H7 exhibited diffuse and low intensity phospho-RPS3 staining, whereas in piglets infected with deltanleH1 mutant, phospho-RPS3 expression was florid and intense (Fig. 6f). These data demonstrate that NleH1 inhibits RPS3 S209 phosphorylation both in vitro and in vivo, which might benefit the bacterium in colonization and transmission.", "output": {"json_structures": {"localization": [{"trigger": {"text": "translocation", "start": 580, "end": 593}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 567, "end": 571}, {"role": "ToLoc", "text": "nuclear", "start": 572, "end": 579}]}], "negative regulation": [{"trigger": {"text": "inhibits", "start": 6, "end": 14}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 0, "end": 5}, {"role": "Theme", "text": "phosphorylation", "start": 25, "end": 40}]}, {"trigger": {"text": "blocked", "start": 505, "end": 512}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 499, "end": 504}, {"role": "Theme", "text": "phosphorylation", "start": 523, "end": 538}]}, {"trigger": {"text": "preventing", "start": 556, "end": 566}, "arguments": [{"role": "Cause", "text": "blocked", "start": 505, "end": 512}, {"role": "Theme", "text": "translocation", "start": 580, "end": 593}]}, {"trigger": {"text": "low", "start": 831, "end": 834}, "arguments": [{"role": "Theme", "text": "phospho", "start": 845, "end": 852}]}, {"trigger": {"text": "inhibits", "start": 1012, "end": 1020}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 1006, "end": 1011}, {"role": "Theme", "text": "phosphorylation", "start": 1031, "end": 1046}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 25, "end": 40}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 15, "end": 19}, {"role": "Site", "text": "S209", "start": 20, "end": 24}]}, {"trigger": {"text": "phosphorylation", "start": 523, "end": 538}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 513, "end": 517}, {"role": "Site", "text": "S209", "start": 518, "end": 522}]}, {"trigger": {"text": "phospho", "start": 845, "end": 852}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 853, "end": 857}]}, {"trigger": {"text": "phospho", "start": 920, "end": 927}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 928, "end": 932}]}, {"trigger": {"text": "phosphorylation", "start": 1031, "end": 1046}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1021, "end": 1025}, {"role": "Site", "text": "S209", "start": 1026, "end": 1030}]}], "positive regulation": [{"trigger": {"text": "intense", "start": 959, "end": 966}, "arguments": [{"role": "Cause", "text": "deltanleH1", "start": 901, "end": 911}, {"role": "Theme", "text": "phospho", "start": 920, "end": 927}]}]}}, "schema": []}
{"input": "NleH1 steers the IKKbeta substrate specificities\nNleH1 is an autophosphorylated serine-threonine kinase, which depends on the lysine 159 (K159)9. To explore the mechanism by which NleH1 inhibits RPS3 S209 phosphorylation, we first performed an in vitro kinase assay with purified wild-type His-NleH1 protein and a mutant His-NleH1 (K159A) protein, confirming that NleH1 is autophosphorylated and the K159A is an NleH1 kinase-dead mutant (Fig. 7a). To examine whether the kinase activity is required for NleH1 to inhibit IKKbeta phosphorlyation of RPS3 on S209, we ectopically expressing either wild-type or K159A NleH1 in 293T cells. Wild-type NleH1 expression significantly reduced TNF-induced RPS3 S209 phosphorylation, whereas the K159A mutant failed to do so (Fig. 7b). Thus NleH1 kinase activity is required to protect RPS3 from IKKbeta-mediated phosphorylation.\nCitrobacter rodentium is a mouse pathogen that shares pathogenic strategies with E. coli 39, most notably for our investigation, C. rodentium NleH inhibited RPS3 nuclear translocation and RPS3-dependent NF-kappaB luciferase activity to an extent equivalent to E. coli NleH1 (ref. 9). We assayed RPS3 S209 phosphorylation in HeLa cells infected with different C. rodentium strains. In uninfected cells, TNF-treatment stimulated a ~3.5-fold increase in RPS3 S209 phosphorylation (Fig. 7c). Such augmentation of RPS3 phosphorylation was reduced by about 60% by wild-type C. rodentium infection (Fig. 7c). However, RPS3 phosphorylation was enhanced when cells were infected with a C. rodentium strain lacking NleH (Fig. 7c, deltanleH). We further examined the role of NleH1 kinase activity using the C. rodentium deltanleH strain as a background on which to express either wild-type or K159A E. coli NleH1. Complementing deltanleH mutant with wild-type NleH1 almost abolished TNF-induced RPS3 S209 phosphorylation whereas complementing with K159A failed to inhibit RPS3 phosphorylation (Fig. 7c). Collectively, these results demonstrate that NleH1 kinase activity is required to block RPS3 S209 phosphorylation.\nWe next examined whether the inhibitory activity of NleH1 is sufficiently robust to impair the strong nuclear translocation of RPS3 trigged by the constitutively-active IKKbeta (IKKbeta [SSEE]) (Fig. 2d). We found that ectopically expressing either wild-type or SSEE IKKbeta proteins, triggered more RPS3 nuclear translocation than the kinase-dead IKKbeta (SSAA) protein (Fig. 7d). RPS3 nuclear accumulation was substantially retarded by infecting cells with wild-type E. coli O157:H7 (Fig. 7d). In contrast, infecting with either deltanleH1 or deltaescN strains only slightly impaired RPS3 nuclear translocation in either IKKbeta- or IKKbeta (SSEE)-expressing cells (Fig. 7d). As expected, E. coli infections did not affect the RPS3 nuclear translocation in IKKbeta (SSAA)-expressing cells, where NF-kappaB signaling was low (Fig. 7d). Thus, during infection NleH1 is sufficiently potent to inhibit RPS3 nuclear translocation even in cells expressing constitutively-activated IKKbeta.\nWe examined whether NleH1 could directly phosphorylate IKKbeta thus inhibiting IKKbeta-mediated RPS3 S209 phosphorylation. We performed in vitro kinase assays using immunoprecipitated Flag-IKKbeta (K44A) as substrate and recombinant His-NleH1 as kinase, so that IKKbeta autophosphorylation would not obscure NleH1-induced phosphorylation. However, we did not observe any detectable 32P incorporation in IKKbeta (Supplementary Fig. 13), thus ruling out this possibility.\nWe then tested the hypothesis that NleH1 could alter the IKKbeta substrate specificities. To this end, we performed in vitro kinase assays using both CK2 and IKK substrates for IKKbeta. As expected, IKKbeta phosphorylated RPS3 (Fig. 7e, lane 7) and GST-IkappaBalpha (1-54) protein (Fig. 7e, lane 9), demonstrating it harbors either CK2 or IKK substrate specificity. Preincubation of IKKbeta with NleH1 reduced IKKbeta-mediated RPS3 phosphorylation, i.e. the CK2 kinase specificity, but not IKKbeta-mediated GST-IkappaBalpha phosphorylation, i.e. the IKK kinase specificity (Fig. 7e). Control experiments revealed no NleH1-mediated phosphorylation or autophosphorylation of RPS3 or GST-IkappaBalpha (Fig. 7e). Taken together, NleH1 blocks the CK2 substrate specificity of IKKbeta thus inhibiting the IKKbeta-mediated RPS3 S209 phosphorylation thus representing a novel strategy by E. coli O157:H7 to alter the host innate immune response.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 650, "end": 660}, "arguments": [{"role": "Theme", "text": "NleH1", "start": 644, "end": 649}]}, {"trigger": {"text": "express", "start": 1722, "end": 1729}, "arguments": [{"role": "Theme", "text": "NleH1", "start": 1764, "end": 1769}]}, {"trigger": {"text": "expressing", "start": 2726, "end": 2736}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 2699, "end": 2706}]}, {"trigger": {"text": "expressing", "start": 2726, "end": 2736}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 2711, "end": 2718}]}, {"trigger": {"text": "expressing", "start": 2850, "end": 2860}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 2835, "end": 2842}]}, 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"arguments": [{"role": "Theme", "text": "RPS3", "start": 2049, "end": 2053}, {"role": "Site", "text": "S209", "start": 2054, "end": 2058}]}, {"trigger": {"text": "phosphorylate", "start": 3103, "end": 3116}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 3117, "end": 3124}]}, {"trigger": {"text": "phosphorylation", "start": 3168, "end": 3183}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 3158, "end": 3162}, {"role": "Site", "text": "S209", "start": 3163, "end": 3167}]}, {"trigger": {"text": "autophosphorylation", "start": 3332, "end": 3351}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 3324, "end": 3331}]}, {"trigger": {"text": "phosphorylation", "start": 3384, "end": 3399}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 3324, "end": 3331}]}, {"trigger": {"text": "32P incorporation", "start": 3444, "end": 3461}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 3465, "end": 3472}]}, {"trigger": {"text": "phosphorylated", "start": 3739, "end": 3753}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 3731, "end": 3738}, {"role": "Theme", "text": "RPS3", "start": 3754, "end": 3758}]}, {"trigger": {"text": "phosphorylated", "start": 3739, "end": 3753}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 3731, "end": 3738}, {"role": "Theme", "text": "GST-IkappaBalpha", "start": 3781, "end": 3797}]}, {"trigger": {"text": "phosphorylation", "start": 3964, "end": 3979}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 3959, "end": 3963}]}, {"trigger": {"text": "phosphorylation", "start": 4056, "end": 4071}, "arguments": [{"role": "Theme", "text": "GST-IkappaBalpha", "start": 4039, "end": 4055}]}, {"trigger": {"text": "phosphorylation", "start": 4163, "end": 4178}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 4205, "end": 4209}]}, {"trigger": {"text": "phosphorylation", "start": 4163, "end": 4178}, "arguments": [{"role": "Theme", "text": "GST-IkappaBalpha", "start": 4213, 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"end": 2039}, "arguments": [{"role": "Theme", "text": "block", "start": 2043, "end": 2048}]}, {"trigger": {"text": "trigged", "start": 2208, "end": 2215}, "arguments": [{"role": "Theme", "text": "translocation", "start": 2186, "end": 2199}, {"role": "Cause", "text": "IKKbeta", "start": 2245, "end": 2252}]}, {"trigger": {"text": "constitutively-active", "start": 2223, "end": 2244}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 2245, "end": 2252}]}, {"trigger": {"text": "SSEE", "start": 2263, "end": 2267}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 2254, "end": 2261}]}, {"trigger": {"text": "triggered", "start": 2361, "end": 2370}, "arguments": [{"role": "Cause", "text": "SSEE IKKbeta", "start": 2338, "end": 2350}, {"role": "Theme", "text": "translocation", "start": 2389, "end": 2402}]}, {"trigger": {"text": "SSEE", "start": 2720, "end": 2724}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 2711, "end": 2718}]}, {"trigger": {"text": "activated", "start": 3043, "end": 3052}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 3053, "end": 3060}]}, {"trigger": {"text": "thus", "start": 3125, "end": 3129}, "arguments": [{"role": "Cause", "text": "phosphorylate", "start": 3103, "end": 3116}, {"role": "Theme", "text": "inhibiting", "start": 3130, "end": 3140}]}, {"trigger": {"text": "mediated", "start": 3149, "end": 3157}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 3141, "end": 3148}, {"role": "Theme", "text": "phosphorylation", "start": 3168, "end": 3183}]}, {"trigger": {"text": "mediated", "start": 3950, "end": 3958}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 3942, "end": 3949}, {"role": "Theme", "text": "phosphorylation", "start": 3964, "end": 3979}]}, {"trigger": {"text": "mediated", "start": 4030, "end": 4038}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 4022, "end": 4029}, {"role": "Theme", "text": "phosphorylation", "start": 4056, "end": 4071}]}, {"trigger": {"text": "mediated", "start": 4154, "end": 4162}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 4148, "end": 4153}, {"role": "Theme", "text": "phosphorylation", "start": 4163, "end": 4178}]}, {"trigger": {"text": "mediated", "start": 4154, "end": 4162}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 4148, "end": 4153}, {"role": "Theme", "text": "autophosphorylation", "start": 4182, "end": 4201}]}, {"trigger": {"text": "mediated", "start": 4339, "end": 4347}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 4331, "end": 4338}, {"role": "Theme", "text": "phosphorylation", "start": 4358, "end": 4373}]}], "regulation": [{"trigger": {"text": "depends", "start": 111, "end": 118}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 49, "end": 54}, {"role": "Theme", "text": "autophosphorylated", "start": 61, "end": 79}, {"role": "CSite", "text": "lysine 159", "start": 126, "end": 136}]}, {"trigger": {"text": "required", "start": 490, "end": 498}, "arguments": [{"role": "Theme", "text": "inhibit", "start": 512, "end": 519}]}, {"trigger": {"text": "dependent", "start": 1061, "end": 1070}, "arguments": [{"role": "Cause", "text": "RPS3", "start": 1056, "end": 1060}, {"role": "Theme", "text": "activity", "start": 1092, "end": 1100}]}, {"trigger": {"text": "affect", "start": 2794, "end": 2800}, "arguments": [{"role": "Theme", "text": "translocation", "start": 2818, "end": 2831}]}, {"trigger": {"text": "substrate specificity", "start": 3875, "end": 3896}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 3754, "end": 3758}]}]}}, "schema": []}
{"input": "RPS3 was previously demonstrated to function as an integral subunit conferring NF-kappaB regulatory specificity6. Here we sought to elucidate how NF-kappaB activation signaling triggers RPS3 to translocate and participate in NF-kappaB function in the nucleus. We demonstrate that IKKbeta-mediated RPS3 S209 phosphorylation represents a critical determinant in governing its nuclear import thus unveiling a novel mechanism behind NF-kappaB regulatory specificity. IKKbeta is the major kinase that phosphorylates IkappaBs in the classical NF-kappaB pathway, leading to their subsequent degradation40. Strikingly, RPS3 possesses not any consensus IKK motif; instead, S209 is centered in a consensus CK2 motif. The recent observation that human IKKbeta displayed CK2-like phosphorylation specificity29 coincides with our evidence that recombinant IKKbeta, but not IKKalpha, phosphorylated RPS3. We found this phosphorylation is a critical modulation for RPS3 nuclear translocation (via importin-alpha) and engagement in specific NF-kappaB transcription. CK2 was previously shown to phosphorylate p65 and to bind to and phosphorylate IKKbeta41-43, however, we ruled out the possibility that the IKKbeta-bound CK2 could account for the observed RPS3 phosphorylation because no CK2 was detected in the IKKbeta preparations used for the in vitro kinase assay. Because RPS3 only harbors the CK2 motif and not a traditional IKK motif, this RPS3 regulatory function could explain why IKK harbors the alternative substrate phosphorylation capability.\nMore importantly, our study has elucidated how RPS3 is biochemically integrated into NF-kappaB activation signaling in a manner that is pivotal for the pathogenesis of foodborne pathogen E. coli O157:H7. IKKbeta-mediated RPS3 S209 phosphorylation is a critical target modulated by this pathogen to subvert host NF-kappaB signaling. The bacterial effector NleH1 specifically binds to RPS3 once injected into host cells and profoundly suppresses NF-kappaB and its attendant protective immune responses9. Our data now show that NleH1 selectively inhibits RPS3 phosphorylation, thus retarding its nuclear translocation and subsequent NF-kappaB function, without altering other NF-kappaB signaling. Although NleH1 did not directly phosphorylate IKKbeta, its kinase activity was required to inhibit IKKbeta-mediated RPS3 S209 phosphorylation. Many bacteria pathogens have products that target key kinases to inactivate them in host cells, whereas E. coli O157:H7 employed NleH1 to steer the substrate specificity of IKKbeta thus specifically fine-tuning host NF-kappaB signaling. This could represent a novel strategy to fine-tune host NF-kappaB signaling that could be shared by other pathogens. These data provide new insights into the poorly understood action mechanism for most T3SS effectors.\nNleH1 attenuates the transcription of RPS3-dependent, but not all, NF-kappaB target genes, in particular those genes associated with acute proinflammatory responses, including IL8 and TNF. In contrast, NleH1 does not block NF-kappaB p65 nuclear translocation, which suggests that certain p65-dependent but RPS3-independent NF-kappaB target genes might thus be beneficial for E. coli O157:H7 to replicate and disseminate in the host. By selectively inhibiting RPS3 and its attendant NF-kappaB function with NleH1, the pathogen achieves the ability to increase colonization and diarrhea yet limiting the mortality of the host. This seemingly paradoxical combination of effects make sense when one considers that increased bacterial load and diarrhea together with survival of the infected host would promote the spreading of the bacteria among a population of susceptible individuals. Such complex and paradoxical pathological effects that influence the spread of disease are often poorly understood at the molecular level. Our data elucidate how alterations in selective NF-kappaB function, achieved by impeding RPS3, but not altering p65 nuclear translocation, can influence specific cytokines that affect bacterial colonization, diarrhea diseases and mortality. It may be fruitful in attempting to understand other infectious and autoimmune diseases involving NF-kappaB to consider selective effects of subunits such as RPS3 in addition to global NF-kappaB inhibition.", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 1103, "end": 1107}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1129, "end": 1136}]}, {"trigger": {"text": "bound", "start": 1198, "end": 1203}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1190, "end": 1197}]}, {"trigger": {"text": "binds", "start": 1913, "end": 1918}, "arguments": [{"role": "Theme", "text": "NleH1", "start": 1894, "end": 1899}, {"role": "Theme2", "text": "RPS3", "start": 1922, "end": 1926}]}], "localization": [{"trigger": {"text": "translocate", "start": 194, "end": 205}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 186, "end": 190}, {"role": "ToLoc", "text": "nucleus", "start": 251, "end": 258}]}, {"trigger": {"text": "import", "start": 382, "end": 388}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 297, "end": 301}, {"role": "ToLoc", "text": "nuclear", "start": 374, "end": 381}]}, {"trigger": {"text": "translocation", "start": 963, "end": 976}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 950, "end": 954}, {"role": "ToLoc", "text": "nuclear", "start": 955, "end": 962}]}, {"trigger": {"text": "translocation", "start": 2140, "end": 2153}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2091, "end": 2095}, {"role": "ToLoc", "text": "nuclear", "start": 2132, "end": 2139}]}, {"trigger": {"text": "translocation", "start": 3076, "end": 3089}, "arguments": [{"role": "Theme", "text": "p65", "start": 3064, "end": 3067}, {"role": "ToLoc", "text": "nuclear", "start": 3068, "end": 3075}]}, {"trigger": {"text": "translocation", "start": 3977, "end": 3990}, "arguments": [{"role": "Theme", "text": "p65", "start": 3965, "end": 3968}, {"role": "ToLoc", "text": "nuclear", "start": 3969, "end": 3976}]}], "negative regulation": [{"trigger": {"text": "inhibits", "start": 2082, "end": 2090}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 2064, "end": 2069}, {"role": "Theme", "text": "phosphorylation", "start": 2096, "end": 2111}]}, {"trigger": {"text": "retarding", "start": 2118, "end": 2127}, "arguments": [{"role": "Cause", "text": "inhibits", "start": 2082, "end": 2090}, {"role": "Theme", "text": "translocation", "start": 2140, "end": 2153}]}, {"trigger": {"text": "inhibit", "start": 2324, "end": 2331}, "arguments": [{"role": "Theme", "text": "mediated", "start": 2340, "end": 2348}]}, {"trigger": {"text": "block", "start": 3048, "end": 3053}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 3033, "end": 3038}, {"role": "Theme", "text": "translocation", "start": 3076, "end": 3089}]}, {"trigger": {"text": "inhibiting", "start": 3279, "end": 3289}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 3290, "end": 3294}, {"role": "Cause", "text": "NleH1", "start": 3337, "end": 3342}]}, {"trigger": {"text": "impeding", "start": 3933, "end": 3941}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 3942, "end": 3946}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 307, "end": 322}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 297, "end": 301}, {"role": "Site", "text": "S209", "start": 302, "end": 306}]}, {"trigger": {"text": "phosphorylated", "start": 870, "end": 884}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 843, "end": 850}, {"role": "Theme", "text": "RPS3", "start": 885, "end": 889}]}, {"trigger": {"text": "phosphorylated", "start": 870, "end": 884}, "arguments": [{"role": "Cause", "text": "IKKalpha", "start": 860, "end": 868}, {"role": "Theme", "text": "RPS3", "start": 885, "end": 889}]}, {"trigger": {"text": "phosphorylate", "start": 1078, "end": 1091}, "arguments": [{"role": "Theme", "text": "p65", "start": 1092, "end": 1095}]}, {"trigger": {"text": "phosphorylate", "start": 1115, "end": 1128}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1129, "end": 1136}]}, {"trigger": {"text": "phosphorylation", "start": 1244, "end": 1259}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1239, "end": 1243}]}, {"trigger": {"text": "phosphorylation", "start": 1770, "end": 1785}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 1760, "end": 1764}, {"role": "Site", "text": "S209", "start": 1765, "end": 1769}]}, {"trigger": {"text": "phosphorylation", "start": 2096, "end": 2111}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2091, "end": 2095}]}, {"trigger": {"text": "phosphorylate", "start": 2265, "end": 2278}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 2279, "end": 2286}]}, {"trigger": {"text": "phosphorylation", "start": 2359, "end": 2374}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 2349, "end": 2353}, {"role": "Site", "text": "S209", "start": 2354, "end": 2358}]}], "positive regulation": [{"trigger": {"text": "triggers", "start": 177, "end": 185}, "arguments": [{"role": "Theme", "text": "translocate", "start": 194, "end": 205}]}, {"trigger": {"text": "mediated", "start": 288, "end": 296}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 280, "end": 287}, {"role": "Theme", "text": "phosphorylation", "start": 307, "end": 322}]}, {"trigger": {"text": "via", "start": 978, "end": 981}, "arguments": [{"role": "Theme", "text": "translocation", "start": 963, "end": 976}]}, {"trigger": {"text": "account for", "start": 1214, "end": 1225}, "arguments": [{"role": "Cause", "text": "bound", "start": 1198, "end": 1203}, {"role": "Theme", "text": "phosphorylation", "start": 1244, "end": 1259}]}, {"trigger": {"text": "mediated", "start": 1751, "end": 1759}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 1743, "end": 1750}, {"role": "Theme", "text": "phosphorylation", "start": 1770, "end": 1785}]}, {"trigger": {"text": "mediated", "start": 2340, "end": 2348}, "arguments": [{"role": "Cause", "text": "IKKbeta", "start": 2332, "end": 2339}, {"role": "Theme", "text": "phosphorylation", "start": 2359, "end": 2374}]}], "regulation": [{"trigger": {"text": "critical", "start": 336, "end": 344}, "arguments": [{"role": "Cause", "text": "mediated", "start": 288, "end": 296}, {"role": "Theme", "text": "import", "start": 382, "end": 388}]}, {"trigger": {"text": "modulation", "start": 935, "end": 945}, "arguments": [{"role": "Cause", "text": "phosphorylated", "start": 870, "end": 884}, {"role": "Theme", "text": "translocation", "start": 963, "end": 976}]}, {"trigger": {"text": "modulated", "start": 1807, "end": 1816}, "arguments": [{"role": "Theme", "text": "mediated", "start": 1751, "end": 1759}]}, {"trigger": {"text": "steer", "start": 2514, "end": 2519}, "arguments": [{"role": "Cause", "text": "NleH1", "start": 2505, "end": 2510}, {"role": "Theme", "text": "IKKbeta", "start": 2549, "end": 2556}]}, {"trigger": {"text": "altering", "start": 3956, "end": 3964}, "arguments": [{"role": "Theme", "text": "translocation", "start": 3977, "end": 3990}]}, {"trigger": {"text": "effects", "start": 4224, "end": 4231}, "arguments": [{"role": "Theme", "text": "RPS3", "start": 4252, "end": 4256}]}]}}, "schema": []}
{"input": "Cells and Reagents\nJurkat E6.1, HEK293T and HeLa cells were cultured in RPMI 1640 and DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, and 100 U/ml each of penicillin and streptomycin, respectively. IkappaBalpha (C-21, sc-371), p65 (C-20, sc-372), and phospho-threonine (H-2, sc-5267) antibodies were from Santa Cruz Biotechnology; beta-actin (AC-15, A5441), Flag (M2, F3165), HA (HA-7, H3663), importin-alpha (IM-75, I1784), and importin-beta (31H4, I2534) antibodies were from Sigma; PARP (C2-10, 556362), IKKalpha (B78-1, 556532), and IKKbeta (24, 611254) antibodies were from BD Pharmingen; CK2alpha (31, 611610) and Hsp90 (68, 610418) antibodies were from BD Transduction Laboratories; phospho-IkappaBalpha (5A5, 9246S) and phospho-IKKalpha/beta (16A6, 2697S) antibodies were from Cell Signaling Technology; phospho-serine (AB1603) and phospho-tyrosine (4G10, 05-777) antibodies were from Millipore. The rabbit polyclonal RPS3 antiserum was as described previously6. The rabbit polyclonal antibody specific for S209 phosphorylated RPS3 was generated and affinity purified by Primm Biotech using the peptide NH2-CKPLPDHV(Sp)IVE-COOH.", "output": {"json_structures": {}}, "schema": []}
{"input": "Plasmid Constructs\nThe Flag-IKKbeta (SSEE), Flag-IKKbeta (SSAA), and HA-IkappaBalpha (SSAA) constructs were provided by C. Wu (NCI, Bethesda) and U. Siebenlist (NIAID, Bethesda), respectively. The HA-IkappaBalpha and IKKbeta (K44A)-Flag plasmids were purchased from Addgene44, 45. The Flag-RPS3, GST-RPS3, HA-RPS3, VN-HA, NleH1-HA plasmids were described previously6, 9. The point mutants of RPS3 were generated by site-directed mutagenesis using the Quick Change Kit (Stratagene) with primers forward 5'-CTGCCTGACCACGTGGCCATTGTGGAACCCAAA-3' and reverse 5'-TTTGGGTTCCACAATGGCCACGTGGTCAGGCAG-3' for S209A. All mutants were verified by DNA sequencing.", "output": {"json_structures": {}}, "schema": []}
{"input": "32P in vivo Labeling\nHEK 293T cells were labeled with 2 mCi/ml 32P-orthophosphate (Perkin Elmer) in phosphate-free medium (Invitrogen) for 2 h. Cells were then left untreated or treated with TNF (50 ng/ml, R&D Systems) for indicated periods. Cell lysates were prepared and used for immunoprecipitations with RPS3 antibody.", "output": {"json_structures": {}}, "schema": []}
{"input": "In Vitro Kinase Assay\nKinase-active recombinant IKKbeta and IKKalpha proteins were purchased from Active Motif and Millipore, respectively. Bacterially purified glutathione S-transferase (GST), GST-IkappaBalpha (1-54), wild type, mutant GST-RPS3, or RPS3 proteins were used as substrates. The in vitro kinase assay was performed as previously described29. Briefly, enzyme (100 ng) and substrate (2 mug) were co-incubated in IKK reaction buffer (25 mM Tris-HCl [pH 8.0], 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mM Na3VO4, 1 mM ATP) or NleH1 reaction buffer (50 mM Tris-HCl [pH 7.6], 5 mM MgCl2, 1 mM DTT, 1 mM ATP) with 0.5 muCi 32P-gamma-ATP (GE Healthcare) added at 37 degreesC for 30 min. The reactions were resolved by SDS-PAGE and visualized by autoradiography.", "output": {"json_structures": {}}, "schema": []}
{"input": "LC-MS/MS Analysis\nGST or GST-RPS3 was incubated with recombinant IKKbeta protein as described above in an in vitro kinase assay reaction conducted without 32P-gamma-ATP labeling. The reaction was separated by SDS-PAGE, and the protein gel was stained with Colloidal Blue (Invitrogen). The corresponding protein fragments were excised and subjected to trypsin digestion and LC-MS/MS at the Yale Cancer Center Mass Spectrometry Resource (New Haven, CT).", "output": {"json_structures": {}}, "schema": []}
{"input": "RNAi and Transfection\nThe siRNA (sense-strand sequence) IKKalpha, 5'-AUGACAGAGAAUGAUCAUGUUCUGC -3'; IKKbeta, 5'-GCAGCAAGGAGAACAGAGGUUAAUA -3'; IkappaBalpha, 5'-GAGCUCCGAGACUUUCGAGGAAAUA -3'; RPS3-3' UTR, 5'-GGAUGUUGCUCUCUAAAGACC -3' (Invitrogen). Transient transfection of siRNA and DNA constructs into Jurkat cells and 293T cells was described previously6.", "output": {"json_structures": {}}, "schema": []}
{"input": "Subcellular Fractionation\nSubcellular fractionation was performed by differential centrifugation as previously described6. Briefly, cells were resuspended in ice-cold Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.4 % NP-40, 0.5 mM PMSF, complete protease inhibitor cocktail) at 4 degreesC for 5 min. Lysates were centrifuged at 4 degreesC, 500 x g for 3 min, and supernatants were collected as cytosolic fractions. Pellets were incubated in Buffer C (20 mM HEPES pH7.9, 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 0.5 mM PMSF, 0.2 mM EDTA, 0.5 mM DTT, complete protease inhibitor cocktail) at 4 degreesC for 10 min. Supernatants were collected as nuclear fractions following a centrifuge at 4 degreesC, 13,800 x g for 10 min.", "output": {"json_structures": {}}, "schema": []}
{"input": "Luciferase Reporter Gene Assays\nLuciferase reporter gene assays were performed as previously described6. Briefly, cells were cotransfected at a ratio of 10:1 with various promoter-driven firefly luciferase constructs to the Renilla luciferase pTKRL plasmid, together with indicated plasmids. Cells were cultured for 1-2 days and then stimulated in triplicate before harvest. Lysates were analyzed using the Dual-Luciferase Kit (Promega).", "output": {"json_structures": {}}, "schema": []}
{"input": "Chromatin Immunoprecipitation (ChIP)\nChIP assays was performed as previously described6. The primers used to amplify the promoter region adjacent to the kappaB sites of IL8 and NFKBIA, as well as ACTB have been described6.", "output": {"json_structures": {}}, "schema": []}
{"input": "Immunofluorescence Microscopy\nConfocal microscopy was performed as previously described6. Briefly, cells were fixed with 4 % paraformaldehyde in PBS and then Cellspin mounted onto slides. The fixed cells were then permeabilized with 0.05 % Triton X-100 in PBS and stained with FITC-conjugated rabbit anti-RPS3 antibodies (Primm Biotech), or AlexaFluor 594-conjugated rat anti-Flag antibodies (BD) for 40 min together with 1 mug/ml of Hoechst 33342 (Sigma) for 5 min at 25 degreesC. The slides were then rinsed with PBS three times and cover mounted for fluorescence microscopy.", "output": {"json_structures": {}}, "schema": []}
{"input": "Immunoprecipitation and immunoblot\nThe cells were harvested and lysed on ice by 0.4 ml of the modified RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) supplemented with 1 x protease inhibitor cocktail (Roche) and 1 x phosphatase inhibitor cocktail set I (EMD Biosciences) for 30 min. The lysates were centrifuged at 10,000 x g at 4 degreesC for 10 min to remove insoluble material. After normalizing protein concentrations, lysates were subjected to immunoprecipitation by adding 10 mg/ml appropriate antibody plus 30 ml of protein G-agarose (Roche), and rotated for at least 2 h at 4degreesC. The precipitates were washed at least five times with cold lysis buffer followed by separation by SDS-PAGE under reduced and denaturing conditions. Nitrocellulose membranes were blocked in 5 % nonfat milk in 0.1 % PBS-Tween 20 (PBS-T), probed with specific antibodies as described previously6. For immunoblotting of phosphorylated proteins, gels were transferred to methanol-treated polyvinylidene chloride membranes, retreated with methanol, and dried for 30 min. Blots were blocked in 5 % bovine serum albumin in 0.1 % Tris buffered saline-Tween 20 (TBS-T), and probed with specific antibodies as described previously46. Bands were imaged by the Super Signaling system (Pierce) according to the manufacturer's instructions.", "output": {"json_structures": {}}, "schema": []}
{"input": "ELISA\nThe amount of IL-8 present in supernatants collected from Jurkat cell culture was measured using a Human Interleukin-8 ELISA Ready-SET-Go kit (eBioscience) according to the manufacturer's instructions.", "output": {"json_structures": {}}, "schema": []}
{"input": "Cell Infections\nHeLa cells were infected with E. coli O157:H7 or C. rodentium strains as described previously9.", "output": {"json_structures": {}}, "schema": []}
{"input": "Immunohistochemistry\nGnotobiotic piglets were infected with E. coli O157:H7 strains as described previously9. Spiral colon specimens were collected at necropsy and embedded in paraffin. Paraffin sectioning and immunohistochemical staining using phospho-RPS3 antibody were performed by Histoserv Inc.", "output": {"json_structures": {}}, "schema": []}
{"input": "Human immunodeficiency virus-1 Tat activates NF-kappaB via physical interaction with IkappaB-alpha and p65\nNuclear factor (NF)-kappaB is a master regulator of pro-inflammatory genes and is upregulated in human immunodeficiency virus 1 (HIV-1) infection. Mechanisms underlying the NF-kappaB deregulation by HIV-1 are relevant for immune dysfunction in AIDS. We report that in single round HIV-1 infection, or single-pulse PMA stimulation, the HIV-1 Tat transactivator activated NF-kappaB by hijacking the inhibitor IkappaB-alpha and by preventing the repressor binding to the NF-kappaB complex. Moreover, Tat associated with the p65 subunit of NF-kappaB and increased the p65 DNA-binding affinity and transcriptional activity. The arginine- and cysteine-rich domains of Tat were required for IkappaB-alpha and p65 association, respectively, and for sustaining the NF-kappaB activity. Among an array of NF-kappaB-responsive genes, Tat mostly activated the MIP-1alpha expression in a p65-dependent manner, and bound to the MIP-1alpha NF-kappaB enhancer thus promoting the recruitment of p65 with displacement of IkappaB-alpha; similar findings were obtained for the NF-kappaB-responsive genes CSF3, LTA, NFKBIA and TLR2. Our results support a novel mechanism of NF-kappaB activation via physical interaction of Tat with IkappaB-alpha and p65, and may contribute to further insights into the deregulation of the inflammatory response by HIV-1.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 68, "end": 79}, "arguments": [{"role": "Theme", "text": "Tat", "start": 31, "end": 34}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 85, "end": 98}]}, {"trigger": {"text": "hijacking", "start": 490, "end": 499}, "arguments": [{"role": "Theme", "text": "Tat", "start": 448, "end": 451}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 514, "end": 527}]}, {"trigger": {"text": "binding", "start": 560, "end": 567}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 514, "end": 527}]}, {"trigger": {"text": "associated", "start": 608, "end": 618}, "arguments": [{"role": "Theme", "text": "Tat", "start": 604, "end": 607}, {"role": "Theme2", "text": "p65", "start": 628, "end": 631}]}, {"trigger": {"text": "binding", "start": 679, "end": 686}, "arguments": [{"role": "Theme", "text": "p65", "start": 671, "end": 674}]}, {"trigger": {"text": "association", "start": 813, "end": 824}, "arguments": [{"role": "Theme", "text": "Tat", "start": 769, "end": 772}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 791, "end": 804}]}, {"trigger": {"text": "bound", "start": 1007, "end": 1012}, "arguments": [{"role": "Theme", "text": "Tat", "start": 929, "end": 932}, {"role": "Theme2", "text": "MIP-1alpha", "start": 1020, "end": 1030}, {"role": "Site2", "text": "NF-kappaB enhancer", "start": 1031, "end": 1049}]}, {"trigger": {"text": "recruitment", "start": 1069, "end": 1080}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 1020, "end": 1030}, {"role": "Site", "text": "NF-kappaB enhancer", "start": 1031, "end": 1049}, {"role": "Theme2", "text": "p65", "start": 1084, "end": 1087}]}, {"trigger": {"text": "displacement", "start": 1093, "end": 1105}, "arguments": [{"role": "Theme", "text": "p65", "start": 1084, "end": 1087}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 1109, "end": 1122}]}, {"trigger": {"text": "interaction", "start": 1293, "end": 1304}, "arguments": [{"role": "Theme", "text": "Tat", "start": 1308, "end": 1311}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 1317, "end": 1330}]}], "gene expression": [{"trigger": {"text": "expression", "start": 965, "end": 975}, "arguments": [{"role": "Theme", "text": " MIP-1alpha", "start": 953, "end": 964}]}], "negative regulation": [{"trigger": {"text": "preventing", "start": 535, "end": 545}, "arguments": [{"role": "Cause", "text": "Tat", "start": 448, "end": 451}, {"role": "Theme", "text": "binding", "start": 560, "end": 567}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 657, "end": 666}, "arguments": [{"role": "Cause", "text": "Tat", "start": 604, "end": 607}, {"role": "Theme", "text": "binding", "start": 679, "end": 686}]}, {"trigger": {"text": "required", "start": 778, "end": 786}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 791, "end": 804}]}, {"trigger": {"text": "required", "start": 778, "end": 786}, "arguments": [{"role": "Theme", "text": "p65", "start": 809, "end": 812}]}, {"trigger": {"text": "responsive", "start": 911, "end": 921}, "arguments": [{"role": "Theme", "text": " MIP-1alpha", "start": 953, "end": 964}]}, {"trigger": {"text": "activated", "start": 940, "end": 949}, "arguments": [{"role": "Cause", "text": "Tat", "start": 929, "end": 932}, {"role": "Theme", "text": "dependent", "start": 985, "end": 994}]}, {"trigger": {"text": "dependent", "start": 985, "end": 994}, "arguments": [{"role": "Theme", "text": "expression", "start": 965, "end": 975}, {"role": "Cause", "text": "p65", "start": 981, "end": 984}]}, {"trigger": {"text": "promoting", "start": 1055, "end": 1064}, "arguments": [{"role": "Cause", "text": "bound", "start": 1007, "end": 1012}, {"role": "Theme", "text": "recruitment", "start": 1069, "end": 1080}]}, {"trigger": {"text": "promoting", "start": 1055, "end": 1064}, "arguments": [{"role": "Cause", "text": "bound", "start": 1007, "end": 1012}, {"role": "Theme", "text": "displacement", "start": 1093, "end": 1105}]}]}}, "schema": []}
{"input": "Nuclear factor (NF)-kappaB transcription factors regulate the transcription of genes that are involved in the immune and inflammatory response (1). The NF-kappaB family includes RelA/p65, c-Rel, RelB, p50 and p52 that share a highly conserved 300-amino acid Rel homology domain (RHD) for homo- or hetero-dimerization and DNA-binding. The transcriptional activity of the NF-kappaB complex depends on dimer composition since C-terminal unrelated transcriptional activation domains are present exclusively in p65, RelB and c-Rel (2).\nInhibitors of NF-kappaB (IkappaB) associate with the NF-kappaB complex and interfere with its binding to DNA (3). In the canonical pathway of NF-kappaB activation, the activated IkappaB kinase (IKK) phosphorylates IkappaB at specific serine residues that target the protein to ubiquitination and proteasomal degradation, which releases the functional NF-kappaB complex in the nucleus. IkappaB-alpha, the most abundant inhibitor of NF-kappaB (4), is phosphorylated by IKK at Ser32 and Ser36 (5), and subsequently ubiquitylated at Lys21 and Lys22 to be degraded by the 26S proteasome (6). The NF-kappaB activity is enhanced by phosphorylation of p65 at Ser276 by PKA and MSK1 (7,8), Ser311 by PKCzeta (9) and Ser536 by IKKalpha (10,11). Acetylation of p65 at Lys218 and Lys221 increases the DNA binding and impairs the association with IkappaB-alpha, and acetylation at Lys310 enhances the p65 transcriptional activity (12,13). Post-activation turn off of NF-kappaB is regulated by negative feedback loop through inhibitors under the transcriptional control of NF-kappaB, such as IkappaB-alpha and ubiquitin-editing protein A20 (14-20). Deacetylation of p65 by histone deacetylase-3 or SIRT1, or acetylation of p65 at Lys122 and Lys123 down-regulate the NF-kappaB activity (12,21,22).\nPersistent activation of NF-kappaB occurs in human immunodeficiency virus-1 (HIV-1)-infected monocytes, macrophages and microglia, and enhances the expression of NF-kappaB-responsive genes, including pro-inflammatory cytokines, cell adhesion molecules and chemokines (23-25). Chronic inflammation is a major cause of immune and neuron dysfunction in AIDS (26,27). Consistently, non-human primate hosts for simian immunodeficiency virus, such as African green monkeys and sooty mangabey, lack aberrant immune activation and do not develop AIDS despite high virus replication (28,29). Thus, understanding the mechanisms of NF-kappaB deregulation by HIV-1 may provide further insights into AIDS pathogenesis.\nIn HIV-1 entry, the binding of the gp120 viral envelope to CD4 induces the NF-kappaB activity by activation of IKK (30) and procaspase 8 (31). Following viral integration, the early encoded HIV-1 Tat protein interacts with the HIV-1 RNA and host cell factors to sustain the viral replication. Tat binds to RNA stem-loop structures generated by the 5' end of target transcripts, including the HIV-1 transactivation-responsive element (TAR) (32), tumor necrosis factor beta (TNFbeta) (33) and interleukin-6 (IL-6) (34) to activate gene transcription. Indeed, Tat promotes the transcriptional initiation and elongation by interacting with transacting factors and cofactors, such as Sp1 (35), TFIID (36), E2F-4 (37), C/EBPbeta (38), cyclin T1/CDK9 (39,40) and the histone acetyltransferases p300/CBP and P/CAF (41-43). When released from HIV-1-infected cells, Tat deregulates the cell signaling by binding to cell receptors, such as integrins (44), Flk1/KDR receptor (45) and chemokine receptors (46).\nWe first reported that NF-kappaB was constitutively active in Jurkat cells that stably expressed the Tat gene (47). Following gene transfection or protein transduction, Tat induced the IKK activity and proteasomal degradation of IkappaB-alpha (48), and increased the p65 transcriptional activity by inhibiting the SIRT-1-mediated deacetylation of p65 Lys310 (49). These findings suggested that Tat modulates crucial enzymes involved in NF-kappaB signaling; however, it was unclear how Tat could subvert the negative feedback of NF-kappaB, which is mainly dependent on de novo synthesis of IkappaB-alpha (15,17). We previously found that IkappaB-alpha binds to Tat and promotes the nuclear export of the viral transactivator (50,51). In this study, we report that Tat counteracts the post-activation turn off of NF-kappaB through direct interaction with IkappaB-alpha and p65, which enhances the DNA binding and transcriptional activity of the NF-kappaB complex. The new mechanism of NF-kappaB deregulation here described may provide further insights into the chronic immune activation of HIV-1 infection.", "output": {"json_structures": {"acetylation": [{"trigger": {"text": "Acetylation", "start": 1266, "end": 1277}, "arguments": [{"role": "Theme", "text": "p65", "start": 1281, "end": 1284}, {"role": "Site", "text": "Lys218", "start": 1288, "end": 1294}]}, {"trigger": {"text": "Acetylation", "start": 1266, "end": 1277}, "arguments": [{"role": "Theme", "text": "p65", "start": 1281, "end": 1284}, {"role": "Site", "text": "Lys221", "start": 1299, "end": 1305}]}, {"trigger": {"text": "acetylation", "start": 1384, "end": 1395}, "arguments": [{"role": "Theme", "text": "p65", "start": 1281, "end": 1284}, {"role": "Site", "text": "Lys310", "start": 1399, "end": 1405}]}], "binding": [{"trigger": {"text": "dimerization", "start": 304, "end": 316}, "arguments": [{"role": "Theme", "text": "RelA", "start": 178, "end": 182}]}, {"trigger": {"text": "dimerization", "start": 304, "end": 316}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 188, "end": 193}]}, {"trigger": {"text": "dimerization", "start": 304, "end": 316}, "arguments": [{"role": "Theme", "text": "RelB", "start": 195, "end": 199}]}, {"trigger": {"text": "dimerization", "start": 304, "end": 316}, "arguments": [{"role": "Theme", "text": "p50", "start": 201, "end": 204}]}, {"trigger": {"text": "dimerization", "start": 304, "end": 316}, "arguments": [{"role": "Theme", "text": "p52", "start": 209, "end": 212}]}, {"trigger": {"text": "binding", "start": 325, "end": 332}, "arguments": [{"role": "Theme", "text": "RelA", "start": 178, "end": 182}]}, {"trigger": {"text": "binding", "start": 325, "end": 332}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 188, "end": 193}]}, {"trigger": {"text": "binding", "start": 325, "end": 332}, "arguments": [{"role": "Theme", "text": "RelB", "start": 195, "end": 199}]}, {"trigger": {"text": "binding", "start": 325, "end": 332}, "arguments": [{"role": "Theme", "text": "p50", "start": 201, "end": 204}]}, {"trigger": {"text": "binding", "start": 325, "end": 332}, "arguments": [{"role": "Theme", "text": "p52", "start": 209, "end": 212}]}, {"trigger": {"text": "binding", "start": 1324, "end": 1331}, "arguments": [{"role": "Theme", "text": "p65", "start": 1281, "end": 1284}]}, {"trigger": {"text": "association", "start": 1348, "end": 1359}, "arguments": [{"role": "Theme", "text": "p65", "start": 1281, "end": 1284}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 1365, "end": 1378}]}, {"trigger": {"text": "binding", "start": 2540, "end": 2547}, "arguments": [{"role": "Theme", "text": "gp120", "start": 2555, "end": 2560}, {"role": "Theme2", "text": "CD4", "start": 2579, "end": 2582}]}, {"trigger": {"text": "interacts", "start": 2728, "end": 2737}, "arguments": [{"role": "Theme", "text": "Tat", "start": 2716, "end": 2719}]}, {"trigger": {"text": "binds", "start": 2817, "end": 2822}, "arguments": [{"role": "Theme", "text": "Tat", "start": 2813, "end": 2816}]}, {"trigger": {"text": "interacting", "start": 3139, "end": 3150}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3077, "end": 3080}, {"role": "Theme2", "text": "Sp1", "start": 3199, "end": 3202}]}, {"trigger": {"text": "interacting", "start": 3139, "end": 3150}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3077, "end": 3080}, {"role": "Theme2", "text": "TFIID", "start": 3209, "end": 3214}]}, {"trigger": {"text": "interacting", "start": 3139, "end": 3150}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3077, "end": 3080}, {"role": "Theme2", "text": "E2F-4", "start": 3221, "end": 3226}]}, {"trigger": {"text": "interacting", "start": 3139, "end": 3150}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3077, "end": 3080}, {"role": "Theme2", "text": "C/EBPbeta", "start": 3233, "end": 3242}]}, {"trigger": {"text": "interacting", "start": 3139, "end": 3150}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3077, "end": 3080}, {"role": "Theme2", "text": "cyclin T1", "start": 3249, "end": 3258}]}, {"trigger": {"text": "interacting", "start": 3139, "end": 3150}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3077, "end": 3080}, {"role": "Theme2", "text": "CDK9", "start": 3259, "end": 3263}]}, {"trigger": {"text": "interacting", "start": 3139, "end": 3150}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3077, "end": 3080}, {"role": "Theme2", "text": "p300", "start": 3307, "end": 3311}]}, {"trigger": {"text": "interacting", "start": 3139, "end": 3150}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3077, "end": 3080}, {"role": "Theme2", "text": "CBP", "start": 3312, "end": 3315}]}, {"trigger": {"text": "interacting", "start": 3139, "end": 3150}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3077, "end": 3080}, {"role": "Theme2", "text": "P/CAF", "start": 3320, "end": 3325}]}, {"trigger": {"text": "binding", "start": 3414, "end": 3421}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3376, "end": 3379}]}, {"trigger": {"text": "binds", "start": 4169, "end": 4174}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 4155, "end": 4168}, {"role": "Theme2", "text": "Tat", "start": 4178, "end": 4181}]}, {"trigger": {"text": "interaction", "start": 4354, "end": 4365}, "arguments": [{"role": "Theme", "text": "Tat", "start": 4281, "end": 4284}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 4371, "end": 4384}]}], "deacetylation": [{"trigger": {"text": "Deacetylation", "start": 1666, "end": 1679}, "arguments": [{"role": "Theme", "text": "p65", "start": 1683, "end": 1686}, {"role": "Cause", "text": "histone deacetylase-3", "start": 1690, "end": 1711}]}, {"trigger": {"text": "Deacetylation", "start": 1666, "end": 1679}, "arguments": [{"role": "Theme", "text": "p65", "start": 1683, "end": 1686}, {"role": "Cause", "text": "SIRT1", "start": 1715, "end": 1720}]}, {"trigger": {"text": "acetylation", "start": 1725, "end": 1736}, "arguments": [{"role": "Theme", "text": "p65", "start": 1740, "end": 1743}, {"role": "Site", "text": "Lys122", "start": 1747, "end": 1753}]}, {"trigger": {"text": "acetylation", "start": 1725, "end": 1736}, "arguments": [{"role": "Theme", "text": "p65", "start": 1740, "end": 1743}, {"role": "Site", "text": "Lys123", "start": 1758, "end": 1764}]}, {"trigger": {"text": "deacetylation", "start": 3848, "end": 3861}, "arguments": [{"role": "Theme", "text": "p65", "start": 3865, "end": 3868}, {"role": "Site", "text": "Lys310", "start": 3869, "end": 3875}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 3605, "end": 3614}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3619, "end": 3622}]}, {"trigger": {"text": "synthesis", "start": 4094, "end": 4103}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 4107, "end": 4120}]}], "localization": [{"trigger": {"text": "released", "start": 3340, "end": 3348}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3376, "end": 3379}]}], "negative regulation": [{"trigger": {"text": "impairs", "start": 1336, "end": 1343}, "arguments": [{"role": "Cause", "text": "Acetylation", "start": 1266, "end": 1277}, {"role": "Theme", "text": "association", "start": 1348, "end": 1359}]}, {"trigger": {"text": "inhibiting", "start": 3817, "end": 3827}, "arguments": [{"role": "Cause", "text": "Tat", "start": 3687, "end": 3690}, {"role": "Theme", "text": "mediated", "start": 3839, "end": 3847}]}], "phosphorylation": [{"trigger": {"text": "phosphorylated", "start": 980, "end": 994}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 916, "end": 929}, {"role": "Site", "text": "Ser32", "start": 1005, "end": 1010}]}, {"trigger": {"text": "phosphorylated", "start": 980, "end": 994}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 916, "end": 929}, {"role": "Site", "text": "Ser36", "start": 1015, "end": 1020}]}, {"trigger": {"text": "phosphorylation", "start": 1156, "end": 1171}, "arguments": [{"role": "Theme", "text": "p65", "start": 1175, "end": 1178}, {"role": "Site", "text": "Ser276", "start": 1182, "end": 1188}]}, {"trigger": {"text": "phosphorylation", "start": 1156, "end": 1171}, "arguments": [{"role": "Theme", "text": "p65", "start": 1175, "end": 1178}, {"role": "Site", "text": "Ser311", "start": 1212, "end": 1218}]}, {"trigger": {"text": "phosphorylation", "start": 1156, "end": 1171}, "arguments": [{"role": "Theme", "text": "p65", "start": 1175, "end": 1178}, {"role": "Site", "text": "Ser536", "start": 1238, "end": 1244}]}], "positive regulation": [{"trigger": {"text": "for", "start": 284, "end": 287}, "arguments": [{"role": "Cause", "text": "RelA", "start": 178, "end": 182}, {"role": "CSite", "text": "Rel homology domain", "start": 258, "end": 277}, {"role": "Theme", "text": "dimerization", "start": 304, "end": 316}]}, {"trigger": {"text": "for", "start": 284, "end": 287}, "arguments": [{"role": "Cause", "text": "RelA", "start": 178, "end": 182}, {"role": "CSite", "text": "Rel homology domain", "start": 258, "end": 277}, {"role": "Theme", "text": "binding", "start": 325, "end": 332}]}, {"trigger": {"text": "for", "start": 284, "end": 287}, "arguments": [{"role": "Cause", "text": "c-Rel", "start": 188, "end": 193}, {"role": "CSite", "text": "Rel homology domain", "start": 258, "end": 277}, {"role": "Theme", "text": "dimerization", "start": 304, "end": 316}]}, {"trigger": {"text": "for", "start": 284, "end": 287}, "arguments": [{"role": "Cause", "text": "c-Rel", "start": 188, "end": 193}, {"role": "CSite", "text": "Rel homology domain", "start": 258, "end": 277}, {"role": "Theme", "text": "binding", "start": 325, "end": 332}]}, {"trigger": {"text": "for", "start": 284, "end": 287}, "arguments": [{"role": "Cause", "text": "RelB", "start": 195, "end": 199}, {"role": "CSite", "text": "Rel homology domain", "start": 258, "end": 277}, {"role": "Theme", "text": "dimerization", "start": 304, "end": 316}]}, {"trigger": {"text": "for", "start": 284, "end": 287}, "arguments": [{"role": "Cause", "text": "RelB", "start": 195, "end": 199}, {"role": "CSite", "text": "Rel homology domain", "start": 258, "end": 277}, {"role": "Theme", "text": "binding", "start": 325, "end": 332}]}, {"trigger": {"text": "for", "start": 284, "end": 287}, "arguments": [{"role": "Cause", "text": "p50", "start": 201, "end": 204}, {"role": "CSite", "text": "Rel homology domain", "start": 258, "end": 277}, {"role": "Theme", "text": "dimerization", "start": 304, "end": 316}]}, {"trigger": {"text": "for", "start": 284, "end": 287}, "arguments": [{"role": "Cause", "text": "p50", "start": 201, "end": 204}, {"role": "CSite", "text": "Rel homology domain", "start": 258, "end": 277}, {"role": "Theme", "text": "binding", "start": 325, "end": 332}]}, {"trigger": {"text": "for", "start": 284, "end": 287}, "arguments": [{"role": "Cause", "text": "p52", "start": 209, "end": 212}, {"role": "CSite", "text": "Rel homology domain", "start": 258, "end": 277}, {"role": "Theme", "text": "dimerization", "start": 304, "end": 316}]}, {"trigger": {"text": "for", "start": 284, "end": 287}, "arguments": [{"role": "Cause", "text": "p52", "start": 209, "end": 212}, {"role": "CSite", "text": "Rel homology domain", "start": 258, "end": 277}, {"role": "Theme", "text": "binding", "start": 325, "end": 332}]}, {"trigger": {"text": "by", "start": 1091, "end": 1093}, "arguments": [{"role": "Theme", "text": "degraded", "start": 1082, "end": 1090}]}, {"trigger": {"text": "by", "start": 1189, "end": 1191}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1156, "end": 1171}, {"role": "Cause", "text": "MSK1", "start": 1200, "end": 1204}]}, {"trigger": {"text": "by", "start": 1219, "end": 1221}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1156, "end": 1171}, {"role": "Cause", "text": "PKCzeta", "start": 1222, "end": 1229}]}, {"trigger": {"text": "by", "start": 1245, "end": 1247}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1156, "end": 1171}, {"role": "Cause", "text": "IKKalpha", "start": 1248, "end": 1256}]}, {"trigger": {"text": "increases", "start": 1306, "end": 1315}, "arguments": [{"role": "Cause", "text": "Acetylation", "start": 1266, "end": 1277}, {"role": "Theme", "text": "binding", "start": 1324, "end": 1331}]}, {"trigger": {"text": "induces", "start": 2583, "end": 2590}, "arguments": [{"role": "Cause", "text": "binding", "start": 2540, "end": 2547}, {"role": "Theme", "text": "activation", "start": 2617, "end": 2627}]}, {"trigger": {"text": "activation", "start": 2617, "end": 2627}, "arguments": [{"role": "Theme", "text": "procaspase 8", "start": 2644, "end": 2656}]}, {"trigger": {"text": "activate", "start": 3040, "end": 3048}, "arguments": [{"role": "Cause", "text": "binds", "start": 2817, "end": 2822}, {"role": "Theme", "text": "transcription", "start": 3054, "end": 3067}]}, {"trigger": {"text": "induced", "start": 3691, "end": 3698}, "arguments": [{"role": "Cause", "text": "Tat", "start": 3687, "end": 3690}, {"role": "Theme", "text": "degradation", "start": 3732, "end": 3743}]}, {"trigger": {"text": "mediated", "start": 3839, "end": 3847}, "arguments": [{"role": "Cause", "text": "SIRT-1", "start": 3832, "end": 3838}, {"role": "Theme", "text": "deacetylation", "start": 3848, "end": 3861}]}], "protein catabolism": [{"trigger": {"text": "degraded", "start": 1082, "end": 1090}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 916, "end": 929}]}, {"trigger": {"text": "degradation", "start": 3732, "end": 3743}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 3747, "end": 3760}]}], "transcription": [{"trigger": {"text": "transcription", "start": 3054, "end": 3067}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor beta", "start": 2965, "end": 2991}]}, {"trigger": {"text": "transcription", "start": 3054, "end": 3067}, "arguments": [{"role": "Theme", "text": "interleukin-6", "start": 3011, "end": 3024}]}], "ubiquitination": [{"trigger": {"text": "ubiquitylated", "start": 1043, "end": 1056}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 916, "end": 929}, {"role": "Site", "text": "Lys21", "start": 1060, "end": 1065}]}, {"trigger": {"text": "ubiquitylated", "start": 1043, "end": 1056}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 916, "end": 929}, {"role": "Site", "text": "Lys22", "start": 1070, "end": 1075}]}]}}, "schema": []}
{"input": "Plasmids\nThe plasmids pcDNA-3xHA-IkappaB-alpha, p3xFLAG-CMV-Tat, p3xFLAG-CMV-Tat C(22,25,27)A, p3xFLAG-CMV-Tat R(49,52,53,55,56,57)A, pGEX-2T-Tat, pGEX-2T-Tat C(22,25,27)A and pGEX-2T-Tat R(49,52,53,55,56,57)A were previously described (50). The plasmids pNL4-3.Luc.R-E- and pHXB2-env were obtained from the AIDS Research & Reference Reagent Program, Division of AIDS, NIAID, NIH, USA; pkappaBluc and pSV-beta-Gal were purchased from Promega (Madison, WI, USA). The plasmids pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65deltaC(1-318), pRc/CMV-3xHA-p65deltaN(122-551), p3xFLAG-CMV-Tat T,N(23,24)A, p3xFLAG-CMV-Tat K(50,51)A, pGEX-2T-Tat T,N(23,24)A, pGEX-2T-Tat K(50,51)A and pNL4-3.FLAG-Tat.R-E- were generated as described in Supplementary Data.", "output": {"json_structures": {}}, "schema": []}
{"input": "Cells, transfection, treatments and luciferase assay\nHeLa, p50-/-p65-/- mouse embryonic fibroblasts (MEFs) (52) and 293T cells were cultured in Dulbecco's modified Eagle's medium; Jurkat, U937 cells and human peripheral blood mononuclear cells (PBMCs) were cultured in RPMI 1640. PBMCs were isolated as previously described (53). Media were supplemented with 10% heat-inactivated fetal calf serum and 2 mM l-glutamine (Lonza Cologne AG, Germany). HeLa, p50-/-p65-/- MEFs and 293T were transfected with DNA by using FuGENE HD (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer's protocol; total DNA amounts were equalized by transfection of pRc/CMV empty vector (Invitrogen, Carlsbad, CA, USA). For pulse-stimulation, HeLa cells were treated with phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St Louis, MO, USA) (20 ng/ml) for 5 min, or tumor necrosis factor-alpha (TNF-alpha; Sigma-Aldrich) (20 ng/ml) for 30 min, washed twice in complete culture medium and then returned to culture. For luciferase assays, pSV-beta-Gal was co-transfected with pkappaBluc to monitor the transfection efficiency. Forty-eight-hour post-transfection, cells were lysed in lysis buffer of Dual Light Luciferase System (Tropix, Bedford, MA, USA) and the luciferase and beta-galactosidase activities were evaluated by using Dual Light Luciferase System (Tropix) in a bioluminometer (Turner Biosystem, Sunnyvale, CA, USA). The ratio of firefly luciferase activity to beta-galactosidase activity was expressed as relative light units.", "output": {"json_structures": {}}, "schema": []}
{"input": "RNA interference\nJurkat or U937 cells were transfected by electroporation using a Bio-Rad apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, aliquots (5 x 106 cells) were suspended in 0.3 ml of RPMI 1640 supplemented with 20% fetal calf serum and subjected to a double electrical pulse (0.22 V, 960 microF) in the presence of annealed siRNA (200 pmol); electroporated cells were washed and cultured in complete medium. RNA interference was performed with: siRNA Tat sense, CUGCUUGUACCAAUUGCUAUU and siRNA Tat antisense, UAGCAAUUGGUACAAGCAGUU; siRNA control sense, CUGCUUGUCACA AUUGCUAUU and siRNA control antisense, UAGCAAUUGUGACAAGCAGUU. RNA interference of p65 and IkappaB-alpha was performed with SMART pool siRNA p65 and IkappaB-alpha (Dharmacon, Chicago, IL, USA).", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "interference", "start": 654, "end": 666}, "arguments": [{"role": "Theme", "text": "p65", "start": 670, "end": 673}]}, {"trigger": {"text": "interference", "start": 654, "end": 666}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 678, "end": 691}]}]}}, "schema": []}
{"input": "Pseudotyped virions and single round infection\n293T cells (1 x 107) were transfected with pNL4-3.Luc.R-E- or pNL4-3.FLAG-Tat.R-E- (10 microg) together with pHXB2 Env (10 microg), and 48-h post-transfection cell supernatant was collected. Enzyme-linked immunosorbent assay (ELISA) using anti-p24 antibody measured virion concentration. PBMCs, Jurkat or U937 cells (5 x 107) were infected with HXB2 Env-pseudotyped virions (500 ng of p24) by spinoculation, as previously described (50).", "output": {"json_structures": {}}, "schema": []}
{"input": "Cell extracts, western blotting, IKK activity and NF-kappaB DNA binding\nTotal, nuclear and cytosolic extracts were performed as previously described (54); details are reported in Supplementary Data. Western blotting analysis was performed by resuspending protein aliquots in loading buffer (125 mM Tris-HCl, pH 6.8, 5% SDS, 1% bromophenol blue, 10% beta-mercaptoethanol, 25% glycerol), resolved on 12% SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) and incubated with primary antibodies (1:1000) followed by incubation with horseradish-peroxidase-linked mouse or rabbit IgG (1:2000) (GE Healthcare Amersham, Little Chalfont, Buckinghamshire, UK) in PBS containing 5% non-fat dry milk (Bio-Rad Laboratories). Proteins were detected by chemiluminescence using the ECL System (GE Healthcare Amersham). Primary antibodies were purchased from: Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-HA F7, anti-IkappaB-alpha C15, anti-Histone H1, anti-Hexokinase-II); Sigma-Aldrich (anti-FLAG M2, anti-gamma-Tubulin); Upstate, Lake Placid, NY, USA (anti-p65). Densitometry of single bands was analysed by ImageJ software package (NIH, USA). IKK activity was evaluated in cytosolic extracts using the HTScan IKK kinase assay kit (Cell Signaling Technology, Danvers, MA, USA). Binding of p65, p50 and FLAG-Tat to the double-stranded NF-kappaB oligonucleotide was measured using NF-kappaB Combo Transcription Factor Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). Electrophoretic Mobility Shift Assay (EMSA) was performed as previously described (55); details are described in Supplementary Data.", "output": {"json_structures": {"binding": [{"trigger": {"text": "Binding", "start": 1310, "end": 1317}, "arguments": [{"role": "Theme", "text": "p65", "start": 1321, "end": 1324}]}, {"trigger": {"text": "Binding", "start": 1310, "end": 1317}, "arguments": [{"role": "Theme", "text": "p50", "start": 1326, "end": 1329}]}, {"trigger": {"text": "Binding", "start": 1310, "end": 1317}, "arguments": [{"role": "Theme", "text": "FLAG-Tat", "start": 1334, "end": 1342}]}]}}, "schema": []}
{"input": "In vitro translation\nHA-IkappaB-alpha, p65 and Tat were expressed under the T7 promoter and in vitro translated using the TnT quick coupled transcription/translation system (Promega), as previously reported (50). Details are described in Supplementary Data.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressed", "start": 56, "end": 65}, "arguments": [{"role": "Theme", "text": "HA-IkappaB-alpha", "start": 21, "end": 37}]}, {"trigger": {"text": "expressed", "start": 56, "end": 65}, "arguments": [{"role": "Theme", "text": "p65", "start": 39, "end": 42}]}, {"trigger": {"text": "expressed", "start": 56, "end": 65}, "arguments": [{"role": "Theme", "text": "Tat", "start": 47, "end": 50}]}], "positive regulation": [{"trigger": {"text": "under", "start": 66, "end": 71}, "arguments": [{"role": "Theme", "text": "expressed", "start": 56, "end": 65}]}]}}, "schema": []}
{"input": "Immunoprecipitation assay and GST-pull down\nImmunoprecipitation, GST-pull down, and production of GST proteins in Escherichia coli strain BL21 were performed as previously reported (50). Details are described in Supplementary Data.", "output": {"json_structures": {}}, "schema": []}
{"input": "Real-time PCR\nTotal RNA was extracted from cells by using the TRIzol reagent (Invitrogen); RNA aliquots (200 ng) were reverse transcribed using Random Examers (Roche) and Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol. Real-time PCR was performed with the iQ Green Super mix (Bio-Rad Laboratories) and carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) under the following conditions: 95degreesC, 1 min; (94degreesC, 10 s; 60degreesC, 30 s) x40. Primers for Tat and MIP-1alpha are listed in Supplementary Data. Real-time PCR of CSF3, LTA, NFKBIA, TLR2, GAPDH and ACTB was performed using the RT2 profiler PCR Array-Human NF-kappaB signaling pathway (QIAGEN Sciences, MD, USA). Reactions were carried out in triplicate, and gene expression levels were calculated relative to GAPDH mRNA levels as endogenous control. Relative expression was calculated as 2(Ct gene under investigation - Ct GAPDH).", "output": {"json_structures": {"transcription": [{"trigger": {"text": "levels", "start": 862, "end": 868}, "arguments": [{"role": "Theme", "text": "GAPDH", "start": 851, "end": 856}]}]}}, "schema": []}
{"input": "Chromatin immunoprecipitation assay\nCells were fixed by adding formaldehyde (Sigma-Aldrich) at the final concentration of 1%. After 10 min, ice-cold PBS plus 0.125 M glycine was added, and plates were transferred on ice, washed extensively with PBS, and scraped. After centrifugation, cells were 10 min lysed in lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40) supplemented with 1x Complete Protease Inhibitor (Roche Diagnostic GmbH). Nuclei were pelletted (1000 x g, 5 min), and resuspended in sonication buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA). Chromatin was sonicated using Bandelin Sonoplus GM70 (Bandelin Electronic, Berlin, Germany), centrifuged (14 000 x g, 15 min), and supernatant was 10-fold diluted in dilution buffer (0.01% SDS, 16.7 mM Tris-HCl pH 8.0, 1.1% Triton X-100, 167 mM NaCl, 1.2 mM EDTA). Samples were pre-cleared by 3-h incubation with 20 microl of protein G agarose beads followed by incubation with antibodies against the analysed proteins. Primary antibodies were: anti-p65 (sc-372), anti-IkappaBalpha (sc-203) and rabbit IgG (sc-2027) from Santa Cruz Biotechnology; anti-FLAG M2 from Sigma-Aldrich. Immunoprecipitations were carried out at 4degreesC overnight and immune complexes were collected with protein G agarose beads, washed five times with low salt buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), four times with high salt buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), once with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA), and extracted in TE buffer containing 2% SDS. Protein-DNA cross-links were reverted by heating at 65degreesC overnight. DNA was further purified by QIAquick PCR purification kit (QIAGEN) and eluted in 50 microl sterile distilled water. Specific enrichment in NF-kappaB enhancer sequences was measured by real-time PCR of chromatin immunoprecipitation (ChIP) eluates using SYBR GreenER Master Mix (Invitrogen). Reactions were carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) using the following conditions: 95degreesC, 1 min; (94degreesC, 10 s; 60degreesC, 30 s) x40. Primers used for MIP-1alpha, GAPDH and ACTB are listed in Supplementary Data. Real-time PCR of CSF3, LTA, NFKBIA and TLR2 were performed using the Custom ChIP array (QIAGEN). For each sample, values were normalized to input DNA and reported as % of input over the rabbit IgG control.", "output": {"json_structures": {}}, "schema": []}
{"input": "Statistical analysis\nStatistical analysis was performed by two-tail unpaired Student's t-test. Data were reported as means +/- SE. Differences between the means were considered as statistically significant at the 95% level (P < 0.05).", "output": {"json_structures": {}}, "schema": []}
{"input": "Tat enhances the NF-kappaB activity by hijacking IkappaB-alpha and inhibiting the post-activation turn off of NF-kappaB\nWe analysed the kinetic of NF-kappaB activation in single round HIV-1 infection by modulating the expression of Tat with RNA interference. Jurkat cells were transfected with siRNA Tat, siRNA control, or left untransfected, and 24 h later cells were infected with HXB2 Env-pseudotyped NL4-3.Luc.R-E- virions. By cytofluorimetric analysis, ~40% of cells was siRNA-transfected and HIV-1-infected at 12-h post-infection (Supplementary Figure S1). The expression of Tat was detected at 1-h post-infection and progressively increased up to 12 h in untransfected and siRNA control-transfected cells, while it was barely detected in siRNA Tat-transfected cells (Figure 1A). The LTR-dependent luciferase expression was also progressively induced at 1- to 12-h post-infection in untransfected and siRNA control-transfected cells, while it was barely observed in siRNA Tat-transfected cells, as expected for the Tat-dependent transactivation of the HIV-1 LTR (Figure 1A).\nThe progressive increase in NF-kappaB activity, as measured by p65-p50 binding to an NF-kappaB consensus oligonucleotide (Figure 1B) and nuclear p65 (Figure 1C, nucleus), was observed at 1- to 3-h post-infection in both Tat-positive (no siRNA or siRNA control) and Tat-negative (siRNA Tat) cells; however, at 12-h post-infection, the p65 DNA binding and nuclear p65 persisted elevated in presence of Tat (no siRNA and siRNA control), while they dropped in absence of Tat (siRNA Tat) (Figure 1B and C). IkappaB-alpha was degraded in the cytosol within 3-h post-infection and de novo synthesized at 6 h with full replenishment at 12-h post-infection in both Tat-positive and Tat-negative cells (Figure 1C, cytosol). Consistently, the IKK activity was induced at 1-h post-infection, and decreased at 3-h post-infection independently of the presence of Tat (Figure 1D). Similar kinetics of NF-kappaB activity, IKK activation and IkappaB-alpha degradation/re-synthesis were observed in single-round HIV-1 infection of PBMCs using a cocktail of Azidothymidine (AZT) and Lamivudine (3TC) as reverse transcriptase inhibitors (Supplementary Figure S2). These results indicated that in single-round HIV-1 infection the NF-kappaB activation initially correlated with the IKK activation and IkappaB-alpha degradation independently of the presence of Tat, and it was kept elevated in presence of Tat following the decay of the IKK activity and new synthesis of IkappaB-alpha.\nNext, we tested the single action of Tat on the kinetic of NF-kappaB activity induced by PMA. To this end, HeLa cells were transfected with p3x-FLAG-Tat, or empty vector, stimulated with PMA for 5 min, and extensively washed to avoid the oscillatory kinetic of NF-kappaB activation (17,18). In un-stimulated cells, the p65 DNA binding (Figure 2A) and nuclear p65 (Figure 2B, nucleus) were slightly enhanced by Tat, while the IkappaB-alpha content was not significantly affected (Figure 2B, cytosol). Transient stimulation with PMA increased the NF-kappaB DNA binding activity and nuclear p65 at 5 min peaking at 60-120 min independently of the presence of Tat (Figure 2A and B); however, while the p65 DNA binding and nuclear p65 persisted elevated at 240-min post-treatment in Tat-positive cells, they dropped in Tat-negative cells (Figure 2A and B). In addition, Tat bound to the NF-kappaB oligonucleotide in un-stimulated cells and its binding increased following PMA stimulation (Figure 2A), suggesting that the viral protein was a component of the NF-kappaB complex bound to DNA. Degradation of IkappaB-alpha was progressively induced at 5-30 min, followed by de novo synthesis at 60 min with full replenishment at 240 min in both Tat-positive and negative cells (Figure 2B, cytosol). Consistently, the IKK activation started at 5 min, and turned off at 30 min independently of the presence of Tat (Figure 2C). These results indicated that Tat enhanced the NF-kappaB activity in un-stimulated and PMA-stimulated cells without affecting the IKK activity and IkappaB-alpha content.\nAs we previously found that Tat binds to the sixth ankyrin of IkappaB-alpha (50,51), we tested whether Tat counteracted the generation of the IkappaB-alpha/NF-kappaB complex. To this end, the association of IkappaB-alpha with p65 was analysed in vivo at 0 and 240 min after short-pulse PMA, which corresponded to the time of un-stimulated condition and post-activation de novo synthesis of IkappaB-alpha, respectively. Coimmunoprecipitation of IkappaB-alpha with p65 was detected at 0- and 240-min post-treatment in Tat-negative cells (Figure 2D, lanes 1 and 3), and was halved in Tat-positive cells, where Tat coimmunoprecipitated with IkappaB-alpha (Figure 2D, lanes 2 and 4), indicating that Tat competed the IkappaB-alpha binding to p65.", "output": {"json_structures": {"binding": [{"trigger": {"text": "hijacking", "start": 39, "end": 48}, "arguments": [{"role": "Theme", "text": "Tat", "start": 0, "end": 3}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 49, "end": 62}]}, {"trigger": {"text": "binding", "start": 1152, "end": 1159}, "arguments": [{"role": "Theme", "text": "p65", "start": 1144, "end": 1147}]}, {"trigger": {"text": "binding", "start": 1152, "end": 1159}, "arguments": [{"role": "Theme", "text": "p50", "start": 1148, "end": 1151}]}, {"trigger": {"text": "binding", "start": 1423, "end": 1430}, "arguments": [{"role": "Theme", "text": "p65", "start": 1415, "end": 1418}]}, {"trigger": {"text": "binding", "start": 2871, "end": 2878}, "arguments": [{"role": "Theme", "text": "p65", "start": 2863, "end": 2866}]}, {"trigger": {"text": "binding", "start": 3250, "end": 3257}, "arguments": [{"role": "Theme", "text": "p65", "start": 3242, "end": 3245}]}, {"trigger": {"text": "bound", "start": 3413, "end": 3418}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3409, "end": 3412}]}, {"trigger": {"text": "binding", "start": 3483, "end": 3490}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3409, "end": 3412}]}, {"trigger": {"text": "binds", "start": 4161, "end": 4166}, "arguments": [{"role": "Theme", "text": "Tat", "start": 4157, "end": 4160}, {"role": "Site2", "text": "sixth ankyrin", "start": 4174, "end": 4187}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 4191, "end": 4204}]}, {"trigger": {"text": "generation", "start": 4253, "end": 4263}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 4271, "end": 4284}]}, {"trigger": {"text": "association", "start": 4321, "end": 4332}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 4336, "end": 4349}, {"role": "Theme2", "text": "p65", "start": 4355, "end": 4358}]}, {"trigger": {"text": "Coimmunoprecipitation", "start": 4548, "end": 4569}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 4573, "end": 4586}, {"role": "Theme2", "text": "p65", "start": 4592, "end": 4595}]}, {"trigger": {"text": "coimmunoprecipitated", "start": 4740, "end": 4760}, "arguments": [{"role": "Theme", "text": "Tat", "start": 4736, "end": 4739}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 4766, "end": 4779}]}, {"trigger": {"text": "binding", "start": 4855, "end": 4862}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 4841, "end": 4854}, {"role": "Theme2", "text": "p65", "start": 4866, "end": 4869}]}], "gene expression": [{"trigger": {"text": "expression", "start": 218, "end": 228}, "arguments": [{"role": "Theme", "text": "Tat", "start": 232, "end": 235}]}, {"trigger": {"text": "expression", "start": 567, "end": 577}, "arguments": [{"role": "Theme", "text": "Tat", "start": 581, "end": 584}]}, {"trigger": {"text": "expression", "start": 815, "end": 825}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 804, "end": 814}]}, {"trigger": {"text": "synthesized", "start": 1663, "end": 1674}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 1583, "end": 1596}]}, {"trigger": {"text": "re-synthesis", "start": 2032, "end": 2044}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 2006, "end": 2019}]}, {"trigger": {"text": "synthesis", "start": 2516, "end": 2525}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 2529, "end": 2542}]}, {"trigger": {"text": "synthesis", "start": 3717, "end": 3726}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 3644, "end": 3657}]}, {"trigger": {"text": "synthesis", "start": 4506, "end": 4515}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 4519, "end": 4532}]}], "negative regulation": [{"trigger": {"text": "barely detected", "start": 726, "end": 741}, "arguments": [{"role": "Theme", "text": "expression", "start": 567, "end": 577}, {"role": "Cause", "text": "siRNA Tat", "start": 745, "end": 754}]}, {"trigger": {"text": "dropped", "start": 1526, "end": 1533}, "arguments": [{"role": "Theme", "text": "binding", "start": 1423, "end": 1430}, {"role": "Cause", "text": "absence", "start": 1537, "end": 1544}]}, {"trigger": {"text": "dropped", "start": 1526, "end": 1533}, "arguments": [{"role": "Theme", "text": "p65", "start": 1443, "end": 1446}, {"role": "Cause", "text": "absence", "start": 1537, "end": 1544}]}, {"trigger": {"text": "absence", "start": 1537, "end": 1544}, "arguments": [{"role": "Theme", "text": "Tat", "start": 1548, "end": 1551}]}, {"trigger": {"text": "dropped", "start": 3347, "end": 3354}, "arguments": [{"role": "Theme", "text": "binding", "start": 3250, "end": 3257}, {"role": "Cause", "text": "negative", "start": 3362, "end": 3370}]}, {"trigger": {"text": "dropped", "start": 3347, "end": 3354}, "arguments": [{"role": "Theme", "text": "p65", "start": 3270, "end": 3273}, {"role": "Cause", "text": "negative", "start": 3362, "end": 3370}]}, {"trigger": {"text": "negative", "start": 3362, "end": 3370}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3358, "end": 3361}]}, {"trigger": {"text": "counteracted", "start": 4236, "end": 4248}, "arguments": [{"role": "Cause", "text": "Tat", "start": 4232, "end": 4235}, {"role": "Theme", "text": "generation", "start": 4253, "end": 4263}]}, {"trigger": {"text": "halved", "start": 4700, "end": 4706}, "arguments": [{"role": "Theme", "text": "Coimmunoprecipitation", "start": 4548, "end": 4569}, {"role": "Cause", "text": "Tat", "start": 4710, "end": 4713}]}, {"trigger": {"text": "competed", "start": 4828, "end": 4836}, "arguments": [{"role": "Cause", "text": "Tat", "start": 4824, "end": 4827}, {"role": "Theme", "text": "binding", "start": 4855, "end": 4862}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 638, "end": 647}, "arguments": [{"role": "Theme", "text": "expression", "start": 567, "end": 577}]}, {"trigger": {"text": "dependent", "start": 794, "end": 803}, "arguments": [{"role": "Theme", "text": "expression", "start": 815, "end": 825}]}, {"trigger": {"text": "induced", "start": 849, "end": 856}, "arguments": [{"role": "Theme", "text": "dependent", "start": 794, "end": 803}]}, {"trigger": {"text": "elevated", "start": 1457, "end": 1465}, "arguments": [{"role": "Theme", "text": "binding", "start": 1423, "end": 1430}, {"role": "Cause", "text": "Tat", "start": 1481, "end": 1484}]}, {"trigger": {"text": "elevated", "start": 1457, "end": 1465}, "arguments": [{"role": "Theme", "text": "p65", "start": 1443, "end": 1446}, {"role": "Cause", "text": "Tat", "start": 1481, "end": 1484}]}, {"trigger": {"text": "observed", "start": 2050, "end": 2058}, "arguments": [{"role": "Theme", "text": "degradation", "start": 2020, "end": 2031}]}, {"trigger": {"text": "observed", "start": 2050, "end": 2058}, "arguments": [{"role": "Theme", "text": "re-synthesis", "start": 2032, "end": 2044}]}, {"trigger": {"text": "enhanced", "start": 2942, "end": 2950}, "arguments": [{"role": "Theme", "text": "binding", "start": 2871, "end": 2878}, {"role": "Cause", "text": "Tat", "start": 2954, "end": 2957}]}, {"trigger": {"text": "enhanced", "start": 2942, "end": 2950}, "arguments": [{"role": "Theme", "text": "p65", "start": 2903, "end": 2906}, {"role": "Cause", "text": "Tat", "start": 2954, "end": 2957}]}, {"trigger": {"text": "increased", "start": 3075, "end": 3084}, "arguments": [{"role": "Theme", "text": "p65", "start": 3132, "end": 3135}]}, {"trigger": {"text": "elevated", "start": 3284, "end": 3292}, "arguments": [{"role": "Theme", "text": "binding", "start": 3250, "end": 3257}, {"role": "Cause", "text": "Tat", "start": 3322, "end": 3325}]}, {"trigger": {"text": "elevated", "start": 3284, "end": 3292}, "arguments": [{"role": "Theme", "text": "p65", "start": 3270, "end": 3273}, {"role": "Cause", "text": "Tat", "start": 3322, "end": 3325}]}, {"trigger": {"text": "increased", "start": 3491, "end": 3500}, "arguments": [{"role": "Theme", "text": "binding", "start": 3483, "end": 3490}]}, {"trigger": {"text": "induced", "start": 3676, "end": 3683}, "arguments": [{"role": "Theme", "text": "Degradation", "start": 3629, "end": 3640}]}, {"trigger": {"text": "followed", "start": 3697, "end": 3705}, "arguments": [{"role": "Cause", "text": "induced", "start": 3676, "end": 3683}, {"role": "Theme", "text": "synthesis", "start": 3717, "end": 3726}]}], "protein catabolism": [{"trigger": {"text": "degraded", "start": 1601, "end": 1609}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 1583, "end": 1596}]}, {"trigger": {"text": "degradation", "start": 2020, "end": 2031}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 2006, "end": 2019}]}, {"trigger": {"text": "degradation", "start": 2374, "end": 2385}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 2360, "end": 2373}]}, {"trigger": {"text": "Degradation", "start": 3629, "end": 3640}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 3644, "end": 3657}]}], "regulation": [{"trigger": {"text": "modulating", "start": 203, "end": 213}, "arguments": [{"role": "Theme", "text": "expression", "start": 218, "end": 228}]}, {"trigger": {"text": "affected", "start": 3013, "end": 3021}, "arguments": [{"role": "Cause", "text": "Tat", "start": 2954, "end": 2957}, {"role": "Theme", "text": "IkappaB-alpha", "start": 2969, "end": 2982}]}, {"trigger": {"text": "independently", "start": 3167, "end": 3180}, "arguments": [{"role": "Theme", "text": "increased", "start": 3075, "end": 3084}, {"role": "Cause", "text": "Tat", "start": 3200, "end": 3203}]}, {"trigger": {"text": "affecting", "start": 4075, "end": 4084}, "arguments": [{"role": "Cause", "text": "Tat", "start": 3989, "end": 3992}, {"role": "Theme", "text": "IkappaB-alpha", "start": 4106, "end": 4119}]}]}}, "schema": []}
{"input": "Tat counteracts the IkappaB-alpha inhibition of p65 by competing the repressor binding\nWe further investigated the physical interaction of Tat with IkappaB-alpha using in vitro translated proteins in coimmunoprecipitation assays. For mapping the interaction domains, we used the following Tat mutants: Tat T,N(23,24)A, Tat C(22,25,27)A, Tat K(50,51)A and Tat R(49-57)A (Figure 3A). IkappaB-alpha coimmunoprecipitated with all Tat proteins, except Tat R(49-57)A (Figure 3B). These results indicated that the arginine-rich domain of Tat was involved in the binding to IkappaB-alpha, which was consistent with previous observations (50,51). Moreover, Tat associating with IkappaB-alpha competed the binding of IkappaB-alpha to p65 in a dose-dependent manner (Figure 3C, lanes 3-5), while Tat R(49-57)A, which lacked the binding site for IkappaB-alpha, did not associate with IkappaB-alpha and did not compete the binding of IkappaB-alpha to p65 (Figure 3C, lanes 6-8).\nNext, we evaluated whether Tat counteracted the IkappaB-alpha-mediated inhibition of p65 binding to DNA by incubating in vitro translated p65 and IkappaB-alpha proteins with the NF-kappaB probe in presence or absence of Tat followed by EMSA. The p65 DNA binding activity was inhibited by IkappaB-alpha and restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49-57) (Figure 3D). Further, we analysed the Tat effect on the IkappaB-alpha repression of p65 transcriptional activity by transfecting p50-/-p65-/-MEFs with the NF-kappaB-Luc reporter together with expression vectors of p65 and IkappaB-alpha, in presence or absence of Tat. IkappaB-alpha inhibited the p65-dependent expression of the luciferase gene, which was restored in a dose-dependent manner by wild-type Tat, and not by Tat R(49-57)A (Figure 3E). Altogether these results indicated that Tat counteracts the IkappaB-alpha repression of the p65 DNA-binding and transcriptional activity by associating with IkappaB-alpha and competing the repressor binding to p65.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 79, "end": 86}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 20, "end": 33}]}, {"trigger": {"text": "interaction", "start": 124, "end": 135}, "arguments": [{"role": "Theme", "text": "Tat", "start": 139, "end": 142}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 148, "end": 161}]}, {"trigger": {"text": "coimmunoprecipitated", "start": 396, "end": 416}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 382, "end": 395}, {"role": "Theme2", "text": "Tat", "start": 426, "end": 429}]}, {"trigger": {"text": "coimmunoprecipitated", "start": 396, "end": 416}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 382, "end": 395}, {"role": "Theme2", "text": "Tat R(49-57)A", "start": 447, "end": 460}]}, {"trigger": {"text": "binding", "start": 555, "end": 562}, "arguments": [{"role": "Theme", "text": "Tat", "start": 531, "end": 534}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 566, "end": 579}]}, {"trigger": {"text": "associating", "start": 652, "end": 663}, "arguments": [{"role": "Theme", "text": "Tat", "start": 648, "end": 651}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 669, "end": 682}]}, {"trigger": {"text": "binding", "start": 696, "end": 703}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 707, "end": 720}, {"role": "Theme2", "text": "p65", "start": 724, "end": 727}]}, {"trigger": {"text": "binding", "start": 817, "end": 824}, "arguments": [{"role": "Theme", "text": "Tat", "start": 648, "end": 651}, {"role": "Site", "text": "site", "start": 825, "end": 829}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 834, "end": 847}]}, {"trigger": {"text": "associate", "start": 857, "end": 866}, "arguments": [{"role": "Theme", "text": "Tat R(49-57)A", "start": 785, "end": 798}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 872, "end": 885}]}, {"trigger": {"text": "binding", "start": 910, "end": 917}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 921, "end": 934}, {"role": "Theme2", "text": "p65", "start": 938, "end": 941}]}, {"trigger": {"text": "binding", "start": 1055, "end": 1062}, "arguments": [{"role": "Theme", "text": "p65", "start": 1051, "end": 1054}]}, {"trigger": {"text": "binding", "start": 1220, "end": 1227}, "arguments": [{"role": "Theme", "text": "p65", "start": 1212, "end": 1215}]}, {"trigger": {"text": "binding", "start": 1897, "end": 1904}, "arguments": [{"role": "Theme", "text": "p65", "start": 1889, "end": 1892}]}, {"trigger": {"text": "associating", "start": 1937, "end": 1948}, "arguments": [{"role": "Theme", "text": "Tat", "start": 1837, "end": 1840}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 1954, "end": 1967}]}, {"trigger": {"text": "binding", "start": 1996, "end": 2003}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 1954, "end": 1967}, {"role": "Theme2", "text": "p65", "start": 2007, "end": 2010}]}], "gene expression": [{"trigger": {"text": "expression", "start": 1660, "end": 1670}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1678, "end": 1688}]}], "negative regulation": [{"trigger": {"text": "counteracts", "start": 4, "end": 15}, "arguments": [{"role": "Theme", "text": "inhibition", "start": 34, "end": 44}, {"role": "Cause", "text": "competing", "start": 55, "end": 64}]}, {"trigger": {"text": "inhibition", "start": 34, "end": 44}, "arguments": [{"role": "Cause", "text": "IkappaB-alpha", "start": 20, "end": 33}, {"role": "Theme", "text": "p65", "start": 48, "end": 51}]}, {"trigger": {"text": "competing", "start": 55, "end": 64}, "arguments": [{"role": "Cause", "text": "Tat", "start": 0, "end": 3}, {"role": "Theme", "text": "binding", "start": 79, "end": 86}]}, {"trigger": {"text": "competed", "start": 683, "end": 691}, "arguments": [{"role": "Cause", "text": "associating", "start": 652, "end": 663}, {"role": "Theme", "text": "binding", "start": 696, "end": 703}]}, {"trigger": {"text": "lacked", "start": 806, "end": 812}, "arguments": [{"role": "Theme", "text": "Tat", "start": 648, "end": 651}, {"role": "Site", "text": "site", "start": 825, "end": 829}]}, {"trigger": {"text": "compete", "start": 898, "end": 905}, "arguments": [{"role": "Cause", "text": "Tat R(49-57)A", "start": 785, "end": 798}, {"role": "Theme", "text": "binding", "start": 910, "end": 917}]}, {"trigger": {"text": "counteracted", "start": 997, "end": 1009}, "arguments": [{"role": "Cause", "text": "Tat", "start": 993, "end": 996}, {"role": "Theme", "text": "inhibition", "start": 1037, "end": 1047}]}, {"trigger": {"text": "inhibition", "start": 1037, "end": 1047}, "arguments": [{"role": "Cause", "text": "IkappaB-alpha", "start": 1014, "end": 1027}, {"role": "Theme", "text": "binding", "start": 1055, "end": 1062}]}, {"trigger": {"text": "inhibited", "start": 1241, "end": 1250}, "arguments": [{"role": "Theme", "text": "binding", "start": 1220, "end": 1227}, {"role": "Cause", "text": "IkappaB-alpha", "start": 1254, "end": 1267}]}, {"trigger": {"text": "restored", "start": 1272, "end": 1280}, "arguments": [{"role": "Theme", "text": "inhibited", "start": 1241, "end": 1250}, {"role": "Cause", "text": "Tat", "start": 1321, "end": 1324}]}, {"trigger": {"text": "restored", "start": 1272, "end": 1280}, "arguments": [{"role": "Theme", "text": "inhibited", "start": 1241, "end": 1250}, {"role": "Cause", "text": "Tat R(49-57)", "start": 1337, "end": 1349}]}, {"trigger": {"text": "inhibited", "start": 1632, "end": 1641}, "arguments": [{"role": "Cause", "text": "IkappaB-alpha", "start": 1618, "end": 1631}, {"role": "Theme", "text": "dependent", "start": 1650, "end": 1659}]}, {"trigger": {"text": "restored", "start": 1705, "end": 1713}, "arguments": [{"role": "Theme", "text": "inhibited", "start": 1632, "end": 1641}, {"role": "Cause", "text": "Tat", "start": 1754, "end": 1757}]}, {"trigger": {"text": "restored", "start": 1705, "end": 1713}, "arguments": [{"role": "Theme", "text": "inhibited", "start": 1632, "end": 1641}, {"role": "Cause", "text": "Tat R(49-57)A", "start": 1770, "end": 1783}]}, {"trigger": {"text": "counteracts", "start": 1841, "end": 1852}, "arguments": [{"role": "Theme", "text": "repression", "start": 1871, "end": 1881}, {"role": "Cause", "text": "associating", "start": 1937, "end": 1948}]}, {"trigger": {"text": "counteracts", "start": 1841, "end": 1852}, "arguments": [{"role": "Theme", "text": "repression", "start": 1871, "end": 1881}, {"role": "Cause", "text": "competing", "start": 1972, "end": 1981}]}, {"trigger": {"text": "repression", "start": 1871, "end": 1881}, "arguments": [{"role": "Cause", "text": "IkappaB-alpha", "start": 1857, "end": 1870}, {"role": "Theme", "text": "binding", "start": 1897, "end": 1904}]}, {"trigger": {"text": "competing", "start": 1972, "end": 1981}, "arguments": [{"role": "Cause", "text": "Tat", "start": 1837, "end": 1840}, {"role": "Theme", "text": "binding", "start": 1996, "end": 2003}]}], "positive regulation": [{"trigger": {"text": "involved", "start": 539, "end": 547}, "arguments": [{"role": "CSite", "text": "arginine-rich domain", "start": 507, "end": 527}, {"role": "Cause", "text": "Tat", "start": 531, "end": 534}, {"role": "Theme", "text": "binding", "start": 555, "end": 562}]}, {"trigger": {"text": "dependent", "start": 1650, "end": 1659}, "arguments": [{"role": "Cause", "text": "p65", "start": 1646, "end": 1649}, {"role": "Theme", "text": "expression", "start": 1660, "end": 1670}]}]}}, "schema": []}
{"input": "Tat increases the p65 affinity binding to DNA through association with p65\nWe tested whether Tat physically interacted with p65. HeLa cells were transfected with the expression vectors of HA-p65 and FLAG-Tat, and 24 h later Tat was immunoprecipitated from cell extracts. The p65 protein was coimmunoprecipitated with wild-type Tat or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49-57)A at similar levels, while a significant decrease in coimmunoprecipitation was observed for Tat C(22,25,27)A (Figure 4A). Consistently, GST-pull down of in vitro translated proteins showed the direct binding of p65 to wild-type Tat, or the mutants Tat T,N(23,24)A, Tat K(50,51)A and Tat R(49-57)A, and lack of binding to Tat C(22,25,27)A (Figure 4B). These results indicated that Tat associated with p65 through the cysteine-rich domain and ruled-out the requirement of bridging proteins to mediate such interaction.\nTo identify the p65 domain required for binding to Tat, HeLa cells were transfected with FLAG-Tat together with HA-p65 mutants, which were deleted of the transactivation domain [p65deltaC (1-318)], or the RHD [p65deltaN (122-551)] (Figure 4C). Tat coimmunoprecipitated with p65deltaC (1-318), and not with p65deltaN (122-551) (Figure 4D), indicating that the RHD of p65 was involved in the physical interaction with Tat.\nTo address the effect of Tat on the p65 DNA-binding affinity, purified recombinant p65 protein was incubated with 32P-labeled-NF-kappaB double-stranded oligonucleotide in presence or absence of wild-type Tat or Tat C(22,25,27)A, and p65 DNA-binding was analysed by EMSA. Whereas wild-type Tat significantly increased the binding of p65 to DNA, the mutant Tat C(22,25,27)A, lacking the binding site for p65, was ineffective (Figure 4E). The p65 binding to DNA was evaluated by competition with increasing amounts of cold competitor NF-kappaB oligonucleotide, which showed that Tat significantly enhanced the p65 affinity binding to DNA (Figure 4E and F).", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 31, "end": 38}, "arguments": [{"role": "Theme", "text": "p65", "start": 18, "end": 21}]}, {"trigger": {"text": "association", "start": 54, "end": 65}, "arguments": [{"role": "Theme", "text": "Tat", "start": 0, "end": 3}, {"role": "Theme2", "text": "p65", "start": 71, "end": 74}]}, {"trigger": {"text": "interacted", "start": 108, "end": 118}, "arguments": [{"role": "Theme", "text": "Tat", "start": 93, "end": 96}, {"role": "Theme2", "text": "p65", "start": 124, "end": 127}]}, {"trigger": {"text": "coimmunoprecipitated", "start": 291, "end": 311}, "arguments": [{"role": "Theme", "text": "p65", "start": 275, "end": 278}, {"role": "Theme2", "text": "Tat", "start": 327, "end": 330}]}, {"trigger": {"text": "coimmunoprecipitation", "start": 446, "end": 467}, "arguments": [{"role": "Theme", "text": "p65", "start": 275, "end": 278}, {"role": "Theme2", "text": "Tat C(22,25,27)A", "start": 485, "end": 501}]}, {"trigger": {"text": "binding", "start": 593, "end": 600}, "arguments": [{"role": "Theme", "text": "p65", "start": 604, "end": 607}, {"role": "Theme2", "text": "Tat", "start": 621, "end": 624}]}, {"trigger": {"text": "binding", "start": 703, "end": 710}, "arguments": [{"role": "Theme", "text": "p65", "start": 604, "end": 607}, {"role": "Theme2", "text": "Tat C(22,25,27)A", "start": 714, "end": 730}]}, {"trigger": {"text": "associated", "start": 777, "end": 787}, "arguments": [{"role": "Theme", "text": "Tat", "start": 773, "end": 776}, {"role": "Theme2", "text": "p65", "start": 793, "end": 796}, {"role": "Site", "text": "cysteine-rich domain", "start": 809, "end": 829}]}, {"trigger": {"text": "binding", "start": 950, "end": 957}, "arguments": [{"role": "Theme", "text": "p65", "start": 926, "end": 929}, {"role": "Theme2", "text": "Tat", "start": 961, "end": 964}]}, {"trigger": {"text": "coimmunoprecipitated", "start": 1158, "end": 1178}, "arguments": [{"role": "Theme", "text": "Tat", "start": 1154, "end": 1157}, {"role": "Theme2", "text": "p65deltaC (1-318)", "start": 1184, "end": 1201}]}, {"trigger": {"text": "interaction", "start": 1309, "end": 1320}, "arguments": [{"role": "Site", "text": "RHD", "start": 1269, "end": 1272}, {"role": "Theme", "text": "p65", "start": 1276, "end": 1279}, {"role": "Theme2", "text": "Tat", "start": 1326, "end": 1329}]}, {"trigger": {"text": "binding", "start": 1375, "end": 1382}, "arguments": [{"role": "Theme", "text": "p65", "start": 1367, "end": 1370}]}, {"trigger": {"text": "binding", "start": 1572, "end": 1579}, "arguments": [{"role": "Theme", "text": "p65", "start": 1564, "end": 1567}]}, {"trigger": {"text": "binding", "start": 1652, "end": 1659}, "arguments": [{"role": "Theme", "text": "p65", "start": 1663, "end": 1666}]}, {"trigger": {"text": "binding", "start": 1716, "end": 1723}, "arguments": [{"role": "Theme", "text": "Tat", "start": 1620, "end": 1623}, {"role": "Site", "text": "site", "start": 1724, "end": 1728}, {"role": "Theme2", "text": "p65", "start": 1733, "end": 1736}]}, {"trigger": {"text": "binding", "start": 1775, "end": 1782}, "arguments": [{"role": "Theme", "text": "p65", "start": 1771, "end": 1774}]}, {"trigger": {"text": "binding", "start": 1951, "end": 1958}, "arguments": [{"role": "Theme", "text": "p65", "start": 1938, "end": 1941}]}], "gene expression": [{"trigger": {"text": "immunoprecipitated", "start": 232, "end": 250}, "arguments": [{"role": "Theme", "text": "Tat", "start": 224, "end": 227}]}], "negative regulation": [{"trigger": {"text": "decrease", "start": 434, "end": 442}, "arguments": [{"role": "Theme", "text": "coimmunoprecipitation", "start": 446, "end": 467}, {"role": "Cause", "text": "Tat C(22,25,27)A", "start": 485, "end": 501}]}, {"trigger": {"text": "deleted", "start": 1049, "end": 1056}, "arguments": [{"role": "Theme", "text": "p65", "start": 926, "end": 929}, {"role": "Site", "text": "transactivation domain", "start": 1064, "end": 1086}]}, {"trigger": {"text": "deleted", "start": 1049, "end": 1056}, "arguments": [{"role": "Theme", "text": "p65", "start": 926, "end": 929}, {"role": "Site", "text": "RHD", "start": 1115, "end": 1118}]}, {"trigger": {"text": "lacking", "start": 1704, "end": 1711}, "arguments": [{"role": "Theme", "text": "Tat", "start": 1620, "end": 1623}, {"role": "Site", "text": "site", "start": 1724, "end": 1728}]}], "positive regulation": [{"trigger": {"text": "increases", "start": 4, "end": 13}, "arguments": [{"role": "Theme", "text": "binding", "start": 31, "end": 38}, {"role": "Cause", "text": "association", "start": 54, "end": 65}]}, {"trigger": {"text": "mediate", "start": 884, "end": 891}, "arguments": [{"role": "Theme", "text": "associated", "start": 777, "end": 787}]}, {"trigger": {"text": "required", "start": 937, "end": 945}, "arguments": [{"role": "Cause", "text": "p65", "start": 926, "end": 929}, {"role": "CSite", "text": "domain", "start": 930, "end": 936}, {"role": "Theme", "text": "binding", "start": 950, "end": 957}]}, {"trigger": {"text": "increased", "start": 1638, "end": 1647}, "arguments": [{"role": "Cause", "text": "Tat", "start": 1620, "end": 1623}, {"role": "Theme", "text": "binding", "start": 1652, "end": 1659}]}, {"trigger": {"text": "ineffective", "start": 1742, "end": 1753}, "arguments": [{"role": "Theme", "text": "binding", "start": 1652, "end": 1659}, {"role": "Cause", "text": "Tat C(22,25,27)A", "start": 1686, "end": 1702}]}, {"trigger": {"text": "enhanced", "start": 1925, "end": 1933}, "arguments": [{"role": "Cause", "text": "Tat", "start": 1907, "end": 1910}, {"role": "Theme", "text": "binding", "start": 1951, "end": 1958}]}], "regulation": [{"trigger": {"text": "effect", "start": 1346, "end": 1352}, "arguments": [{"role": "Cause", "text": "Tat", "start": 1356, "end": 1359}, {"role": "Theme", "text": "binding", "start": 1375, "end": 1382}]}]}}, "schema": []}
{"input": "Tat activates the p65-dependent expression of NF-kappaB-responsive genes, occupies the NF-kappaB enhancers and promotes the p65 recruitment with IkappaB-alpha displacement\nWe analysed the action of Tat on the expression of NF-kappaB-responsive genes in vivo. To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49-57)A, and 48 h later the expression of a number of NF-kappaB-dependent genes was analysed by real-time PCR. Tat significantly increased the expression of MIP-1alpha, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49-57)A were ineffective (Figure 5A-E). The Tat-dependent transactivation of the NF-kappaB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A-E). Differently, the expression of the NF-kappaB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G). These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-kappaB activity, suggesting that the up-regulation of NF-kappaB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IkappaB-alpha.\nMIP-1alpha, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56). Consistently with our findings, MIP-1alpha expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1alpha production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62). The transcriptional regulation of MIP-1alpha has been poorly characterized. Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-kappaB enhancers in the proximal promoter region of the MIP-1alpha gene: NF-kappaB1, -1023/-1014 nucleotides; NF-kappaB2, -661/-652 nucleotides; NF-kappaB3, +370/+379 nucleotides (Supplementary Figure S3A). To validate the predicted NF-kappaB binding sites of the MIP-1alpha promoter, we stimulated HeLa cells with TNF-alpha, a well-known NF-kappaB inducer, and measured the expression levels of MIP-1alpha by real-time PCR, and the p65 occupancy of the NF-kappaB sites by ChIP. TNF-alpha activated the expression of MIP-1alpha and induced the p65 recruitment to the NF-kappaB1 site of MIP-1alpha without affecting the occupancy of the putative NF-kappaB2 and NF-kappaB3 sites (Supplementary Figure S3B and S3C). Tat also promoted the recruitment of p65 to the NF-kappaB1 site of MIP-1alpha, while it did not affect the putative NF-kappaB2 and NF-kappaB3 sites (Supplementary Figure S3D). Altogether these results indicated that the NF-kappaB1 site was the only effective NF-kappaB enhancer of the MIP-1alpha promoter in response to TNF-alpha and Tat.\nSimilarly to MIP-1alpha, Tat increased the recruitment of p65 to the NF-kappaB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H). By ChIP, Tat bound to the NF-kappaB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction. As additional findings, IkappaB-alpha was chromatin-immunoprecipitated at the NF-kappaB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IkappaB-alpha RNA interference (Figure 5J), indicating that Tat displaced IkappaB-alpha from promoters.", "output": {"json_structures": {"binding": [{"trigger": {"text": "occupies", "start": 74, "end": 82}, "arguments": [{"role": "Theme", "text": "Tat", "start": 0, "end": 3}]}, {"trigger": {"text": "recruitment", "start": 128, "end": 139}, "arguments": [{"role": "Theme", "text": "p65", "start": 124, "end": 127}]}, {"trigger": {"text": "displacement", "start": 159, "end": 171}, "arguments": [{"role": "Theme", "text": "p65", "start": 124, "end": 127}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 145, "end": 158}]}, {"trigger": {"text": "interaction", "start": 1230, "end": 1241}, "arguments": [{"role": "Theme", "text": "Tat", "start": 1197, "end": 1200}, {"role": "Theme2", "text": "p65", "start": 1268, "end": 1271}]}, {"trigger": {"text": "occupancy", "start": 2249, "end": 2258}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 2208, "end": 2218}, {"role": "Theme2", "text": "p65", "start": 2245, "end": 2248}, {"role": "Site", "text": "NF-kappaB sites", "start": 2266, "end": 2281}]}, {"trigger": {"text": "recruitment", "start": 2360, "end": 2371}, "arguments": [{"role": "Theme", "text": "p65", "start": 2356, "end": 2359}, {"role": "Site2", "text": "NF-kappaB1 site", "start": 2379, "end": 2394}, {"role": "Theme2", "text": "MIP-1alpha", "start": 2398, "end": 2408}]}, {"trigger": {"text": "occupancy", "start": 2431, "end": 2440}, "arguments": [{"role": "Theme", "text": "p65", "start": 2356, "end": 2359}]}, {"trigger": {"text": "recruitment", "start": 2547, "end": 2558}, "arguments": [{"role": "Theme", "text": "p65", "start": 2562, "end": 2565}, {"role": "Site2", "text": "NF-kappaB1 site", "start": 2573, "end": 2588}, {"role": "Theme2", "text": "MIP-1alpha", "start": 2592, "end": 2602}]}, {"trigger": {"text": "recruitment", "start": 2907, "end": 2918}, "arguments": [{"role": "Theme", "text": "p65", "start": 2922, "end": 2925}, {"role": "Site2", "text": "NF-kappaB enhancers", "start": 2933, "end": 2952}, {"role": "Theme2", "text": "CSF3", "start": 2956, "end": 2960}]}, {"trigger": {"text": "recruitment", "start": 2907, "end": 2918}, "arguments": [{"role": "Theme", "text": "p65", "start": 2922, "end": 2925}, {"role": "Site2", "text": "NF-kappaB enhancers", "start": 2933, "end": 2952}, {"role": "Theme2", "text": "LTA", "start": 2962, "end": 2965}]}, {"trigger": {"text": "recruitment", "start": 2907, "end": 2918}, "arguments": [{"role": "Theme", "text": "p65", "start": 2922, "end": 2925}, {"role": "Site2", "text": "NF-kappaB enhancers", "start": 2933, "end": 2952}, {"role": "Theme2", "text": "NFKBIA", "start": 2967, "end": 2973}]}, {"trigger": {"text": "recruitment", "start": 2907, "end": 2918}, "arguments": [{"role": "Theme", "text": "p65", "start": 2922, "end": 2925}, {"role": "Site2", "text": "NF-kappaB enhancers", "start": 2933, "end": 2952}, {"role": "Theme2", "text": "TLR2", "start": 2978, "end": 2982}]}, {"trigger": {"text": "bound", "start": 3070, "end": 3075}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3066, "end": 3069}, {"role": "Theme2", "text": "GAPDH", "start": 3130, "end": 3135}, {"role": "Site2", "text": "promoters", "start": 3145, "end": 3154}]}, {"trigger": {"text": "occupancy", "start": 3241, "end": 3250}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3237, "end": 3240}]}, {"trigger": {"text": "interaction", "start": 3268, "end": 3279}, "arguments": [{"role": "Theme", "text": "Tat", "start": 3237, "end": 3240}, {"role": "Theme2", "text": "p65", "start": 3264, "end": 3267}]}, {"trigger": {"text": "chromatin-immunoprecipitated", "start": 3323, "end": 3351}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 3305, "end": 3318}]}, {"trigger": {"text": "from", "start": 3552, "end": 3556}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 3538, "end": 3551}]}], "gene expression": [{"trigger": {"text": "expression", "start": 494, "end": 504}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 508, "end": 518}]}, {"trigger": {"text": "expression", "start": 494, "end": 504}, "arguments": [{"role": "Theme", "text": "CSF3", "start": 520, "end": 524}]}, {"trigger": {"text": "expression", "start": 494, "end": 504}, "arguments": [{"role": "Theme", "text": "LTA", "start": 526, "end": 529}]}, {"trigger": {"text": "expression", "start": 494, "end": 504}, "arguments": [{"role": "Theme", "text": "NFKBIA", "start": 531, "end": 537}]}, {"trigger": {"text": "expression", "start": 494, "end": 504}, "arguments": [{"role": "Theme", "text": "TLR2", "start": 542, "end": 546}]}, {"trigger": {"text": "expression", "start": 1476, "end": 1486}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 1465, "end": 1475}]}, {"trigger": {"text": "production", "start": 1579, "end": 1589}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 1568, "end": 1578}]}, {"trigger": {"text": "expression", "start": 2187, "end": 2197}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 2208, "end": 2218}]}, {"trigger": {"text": "expression", "start": 2315, "end": 2325}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 2329, "end": 2339}]}], "negative regulation": [{"trigger": {"text": "loss", "start": 3161, "end": 3165}, "arguments": [{"role": "Theme", "text": "bound", "start": 3070, "end": 3075}, {"role": "Cause", "text": "interference", "start": 3195, "end": 3207}]}, {"trigger": {"text": "interference", "start": 3195, "end": 3207}, "arguments": [{"role": "Theme", "text": "p65", "start": 3187, "end": 3190}]}, {"trigger": {"text": "removed", "start": 3423, "end": 3430}, "arguments": [{"role": "Theme", "text": "chromatin-immunoprecipitated", "start": 3323, "end": 3351}, {"role": "Cause", "text": "Tat", "start": 3446, "end": 3449}]}, {"trigger": {"text": "removed", "start": 3423, "end": 3430}, "arguments": [{"role": "Theme", "text": "chromatin-immunoprecipitated", "start": 3323, "end": 3351}, {"role": "Cause", "text": "interference", "start": 3482, "end": 3494}]}, {"trigger": {"text": "interference", "start": 3482, "end": 3494}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 3464, "end": 3477}]}, {"trigger": {"text": "displaced", "start": 3528, "end": 3537}, "arguments": [{"role": "Cause", "text": "Tat", "start": 3524, "end": 3527}, {"role": "Theme", "text": "from", "start": 3552, "end": 3556}]}], "positive regulation": [{"trigger": {"text": "promotes", "start": 111, "end": 119}, "arguments": [{"role": "Cause", "text": "Tat", "start": 0, "end": 3}, {"role": "Theme", "text": "recruitment", "start": 128, "end": 139}]}, {"trigger": {"text": "promotes", "start": 111, "end": 119}, "arguments": [{"role": "Cause", "text": "Tat", "start": 0, "end": 3}, {"role": "Theme", 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"end": 3250}, {"role": "Cause", "text": "interaction", "start": 3268, "end": 3279}]}], "regulation": [{"trigger": {"text": "regulation", "start": 1677, "end": 1687}, "arguments": [{"role": "Theme", "text": "transcriptional", "start": 1661, "end": 1676}]}], "transcription": [{"trigger": {"text": "transcriptional", "start": 1661, "end": 1676}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 1691, "end": 1701}]}]}}, "schema": []}
{"input": "In HIV-1-infected monocytes Tat sustains the NF-kappaB activity and promotes the transcriptional activation of MIP-1alpha by interacting with IkappaB-alpha and p65\nWe next analysed whether Tat affected the NF-kappaB activity and the expression of MIP-1alpha in the course of HIV-1 infection. To detect the Tat protein, we produced HXB2 Env-pseudotyped NL4-3.FLAG-Tat.R-E- virions, which were used in single-round infection of U937, a human monocytic cell line. Cells were transfected with siRNA Tat, siRNA control or left untransfected to modulate the Tat expression, and then analysed for the kinetic of NF-kappaB activation following viral infection. The expression of Tat and p24 was detected at 3- to 12-h post-infection, while it was barely detected in siRNA Tat-transfected cells (Figure 6A).\nThe NF-kappaB activity, as measured by p65 DNA binding, was induced at 3-h post-infection in both Tat-positive (no siRNA and siRNA control) and Tat-negative cells (siRNA Tat), and increased at 12 h only in Tat-positive cells (Figure 6B). Degradation of IkappaB-alpha was observed at 3-h post-infection, and was followed by de novo synthesis of IkappaB-alpha at 12 h independently of the Tat presence (Figure 6C). Consistently, the IKK activity was induced at 3-h post-infection and turned off at 12 h independently of the Tat presence (Figure 6D). These results agreed with the kinetic of NF-kappaB activation in single-round HIV-1 infection of Jurkat cells (Figure 1), and further supported the requirement of viral expression to counteract the post-activation turn off of NF-kappaB.\nIn HIV-1-infected U937 cells, the endogenous Tat was coimmunoprecipitated with IkappaB-alpha and p65 (Figure 6E), and activated the MIP-1alpha expression in a p65-dependent manner, as shown by the lack of effect following RNA interference of Tat or p65 (Figure 6F). Moreover, Tat and p65 were both recruited to the NF-kappaB enhancer of MIP-1alpha, while the p65 occupancy was abolished by siRNA Tat (Figure 6G and H). Altogether these results indicated that in single round HIV-1 infection Tat associated with IkappaB-alpha and p65, and induced the p65-dependent activation of MIP-1alpha expression through occupancy of the MIP-1alpha promoter and increased recruitment of p65.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interacting", "start": 125, "end": 136}, "arguments": [{"role": "Theme", "text": "Tat", "start": 28, "end": 31}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 142, "end": 155}]}, {"trigger": {"text": "binding", "start": 846, "end": 853}, "arguments": [{"role": "Theme", "text": "p65", "start": 838, "end": 841}]}, {"trigger": {"text": "coimmunoprecipitated", "start": 1637, "end": 1657}, "arguments": [{"role": "Theme", "text": "Tat", "start": 1629, "end": 1632}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 1663, "end": 1676}]}, {"trigger": {"text": "recruited", "start": 1882, "end": 1891}, "arguments": [{"role": "Theme", "text": "Tat", "start": 1860, 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"text": "recruitment", "start": 2243, "end": 2254}]}], "protein catabolism": [{"trigger": {"text": "Degradation", "start": 1037, "end": 1048}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 1052, "end": 1065}]}], "regulation": [{"trigger": {"text": "affected", "start": 193, "end": 201}, "arguments": [{"role": "Cause", "text": "Tat", "start": 189, "end": 192}, {"role": "Theme", "text": "expression", "start": 233, "end": 243}]}, {"trigger": {"text": "modulate", "start": 539, "end": 547}, "arguments": [{"role": "Theme", "text": "expression", "start": 556, "end": 566}]}, {"trigger": {"text": "independently", "start": 1165, "end": 1178}, "arguments": [{"role": "Theme", "text": "synthesis", "start": 1130, "end": 1139}, {"role": "Cause", "text": "Tat", "start": 1186, "end": 1189}]}], "transcription": [{"trigger": {"text": "transcriptional", "start": 81, "end": 96}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 111, "end": 121}]}]}}, "schema": []}
{"input": "This study reports a novel mechanism of NF-kappaB activation by the HIV-1 Tat transactivator. Based on the evidence that Tat enhanced the transcriptional activity of the p65 subunit of NF-kappaB (49,63,64), and physically interacted with the IkappaB-alpha repressor (50,51), we investigated the possibility that Tat could activate NF-kappaB via direct interaction with IkappaB-alpha and p65. To this end, the NF-kappaB activity was monitored in single round HIV-1 infection using RNA interference to silence the Tat expression. By this approach, we avoided the perpetuation of NF-kappaB activation signaling due to subsequent rounds of viral entry in cell culture propagation (30,31). Upon HIV-1 infection, the early NF-kappaB activation occurred concomitantly with IKK activation and IkappaB-alpha degradation in the absence of Tat. Soon after the shut off of IKK activity and new synthesis of IkappaB-alpha, the NF-kappaB activity was kept elevated in the presence of Tat, while it was down regulated upon silencing of the Tat gene. These findings indicate that in single round HIV-1 infection, Tat enhanced the NF-kappaB activity without affecting the IKK activity and the half-life of IkappaB-alpha. Similar kinetic of NF-kappaB activation occurred upon short-pulse of PMA in Tat-transfected HeLa cells, where Tat inhibited the post-activation turn off of NF-kappaB in presence of newly synthesized IkappaB-alpha.\nThe Tat-dependent activation of NF-kappaB correlated with the association of the viral protein with IkappaB-alpha, which interfered with the generation of the IkappaB-alpha/p65 complex. This evidence demonstrated that Tat competed for the binding of IkappaB-alpha to p65 and prevented the IkappaB-alpha repression of p65. As additional mechanism of NF-kappaB activation, Tat associated with p65 and increased the p65 binding affinity to the NF-kappaB enhancer; this evidence was obtained with recombinant proteins indicating that the stronger binding of p65 to DNA was a consequence of direct association with Tat, which likely caused a conformational change of p65. We also demonstrated that the Tat cysteine-rich sequence (C22,25,27) was involved in the binding to the RHD of p65 and NF-kappaB activation. Differently, the Tat arginine-rich sequence (R49,52,53,55,56,57) was required for the binding to the sixth ankyrin of IkappaB-alpha (50,51), and for releasing p65 from the IkappaB-alpha inhibition. Altogether these results demonstrated that Tat abolished the negative feedback regulation of NF-kappaB by hijacking the IkappaB-alpha repressor, shielding p65 from the IkappaB-alpha embrace, and enforcing the p65 binding to DNA.\nFurther evidence of Tat activation of NF-kappaB showed that wild-type Tat, and not the Tat mutants lacking the arginine- or cysteine-rich domains, up-regulated in vivo the expression of a number of NF-kappaB-responsive genes, including MIP-1alpha, CSF3, LTA, NFKBIA and TLR2. By ChIP analysis, we observed that Tat increased the recruitment of p65 to the NF-kappaB enhancers of the activated genes, and was recovered at the same sites. RNA interference of p65 abolished the Tat occupancy of the NF-kappaB enhancers indicating that the Tat binding to the NF-kappaB sites was mediated by p65. These results were consistent with the evidence that Tat displaced p65 from the binding to IkappaB-alpha (Figures 2D and 3C), and increased the p65 DNA binding affinity through association (Figure 4E and F). Thus, Tat likely increased the recruitment of p65 to the NF-kappaB-dependent promoters by associating with p65 and by interfering with the assembly of p65 with the repressor IkappaB-alpha.\nOf interest, IkappaB-alpha was found associated to the NF-kappaB enhancers in absence of Tat, where it was displaced in presence of Tat. These results suggest a negative regulatory role of IkappaB-alpha in gene regulation through occupancy of specific promoters. Indeed, IkappaB-alpha was found associated with hystone deacetylases at the hes1 promoter, from where it was removed following TNF-alpha stimulation causing histone acetylation and hes1 transcriptional activation (65). A similar mechanism of gene regulation could apply to the NF-kappaB-dependent genes analysed in this study, where Tat could activate the gene expression by removing IkappaB-alpha and promoting the p65 loading (Figure 7).\nThe physiological relevance of Tat cross talk with NF-kappaB was demonstrated in the HIV-1 infection of human monocytes, where HIV-1-encoded Tat protein counteracted post-activation turn off of NF-kappaB, as shown by: (i) sustained NF-kappaB activity by Tat in presence of newly synthesized IkappaB-alpha; (ii) coimmunoprecipitation of Tat with IkappaB-alpha and p65; (iii) induction of MIP-1alpha expression dependent on Tat and p65; (iv) Tat occupancy of the MIP-1alpha NF-kappaB enhancer associated with increased recruitment of p65.\nThis study supports the pro-inflammatory action of Tat through physical interaction with p65 and IkappaB-alpha. Several inflammatory cytokines under the transcriptional control of NF-kappaB are hyper-expressed in HIV-1 infected individuals, including IL-6 (66,67), TNF-alpha (68), and MIP-1alpha (57-62,69-71). In the case of TNF-alpha and IL-6, Tat activated the cytokine expression by binding to the TAR-like stem loop of the 5' transcript, thus likely promoting the transcriptional elongation (33,37,47). Here, we have shown that the Tat-dependent transcriptional activation of a number of pro-inflammatory genes required the assembly of Tat with p65 at the NF-kappaB enhancer together with the displacement of IkappaB-alpha (Figure 5). Thus, in the set of distinct NF-kappaB-responsive genes, Tat acted as a component of the transcriptional initiation complex, which is consistent with previous reports on Tat promoting the transcription via DNA motifs (72,73).\nOverproduction of pro-inflammatory cytokines is considered a major mechanism of immune deregulation (26) and neuron dysfunction in AIDS (74,75). In this scenario, inhibition of NF-kappaB activity could lead to a therapeutic strategy for counteracting the abnormal production of pro-inflammatory cytokines and chemokines in HIV-1 infection. NF-kappaB inhibitors, such as anti-oxidants, proteasome and IKK inhibitors, significantly reduced the HIV-1 replication and the associated inflammatory response (76,77); however, these NF-kappaB inhibitors acted indiscriminately in both HIV-1-infected and uninfected cells. The evidence that Tat activates NF-kappaB through direct interaction with IkappaB-alpha and p65 may lead to specific inhibitors to counteract the Tat pro-inflammatory action.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interacted", "start": 222, "end": 232}, "arguments": [{"role": "Theme", "text": "Tat", "start": 121, "end": 124}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 242, "end": 255}]}, {"trigger": {"text": "interaction", "start": 352, "end": 363}, "arguments": [{"role": "Theme", "text": "Tat", "start": 312, "end": 315}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 369, "end": 382}]}, {"trigger": {"text": "association", "start": 1480, "end": 1491}, "arguments": [{"role": "Theme", "text": "Tat", "start": 1422, "end": 1425}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 1518, "end": 1531}]}, {"trigger": {"text": "generation", "start": 1559, "end": 1569}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 1577, "end": 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2076}, "arguments": [{"role": "Theme", "text": "p65", "start": 2080, "end": 2083}]}, {"trigger": {"text": "cross talk", "start": 4379, "end": 4389}, "arguments": [{"role": "Cause", "text": "Tat", "start": 4375, "end": 4378}, {"role": "Theme", "text": "Tat", "start": 4375, "end": 4378}]}], "transcription": [{"trigger": {"text": "transcriptional elongation", "start": 5350, "end": 5376}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 5207, "end": 5216}]}, {"trigger": {"text": "transcriptional elongation", "start": 5350, "end": 5376}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 5221, "end": 5225}]}]}}, "schema": []}
{"input": "Tat counteracts the post-activation turn off of NF-kappaB in single round HIV-1-infection. Jurkat cells (5 x 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.Luc.R-E- virions (500 ng of p24) and harvested at the indicated time. (A) Tat expression was measured in total RNA by real-time PCR, while the luciferase activity was measured in whole cell extracts. (B) Nuclear extracts (10 microg) were analysed for the p65 and p50 binding to the NF-kappaB double-stranded oligonucleotide using the NF-kappaB Transcription Factor ELISA Assay kit (Cayman). (C) The expression of p65 and IkappaB-alpha was analysed by 12% SDS-PAGE and western blotting of nuclear or cytosolic extracts (20 microg) using anti-p65 and anti-IkappaB-alpha antibodies. Histone H1 and Hexokinase II were detected with specific antibodies as markers of nuclear and cytosolic extracts, respectively. Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic extracts (100 microg) by using HTScan IKK Kinase Assay (Cell Signaling Technology). Values (mean +/- SE, n = 3) are shown.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 523, "end": 530}, "arguments": [{"role": "Theme", "text": "p65", "start": 511, "end": 514}]}, {"trigger": {"text": "binding", "start": 523, "end": 530}, "arguments": [{"role": "Theme", "text": "p50", "start": 519, "end": 522}]}], "gene expression": [{"trigger": {"text": "expression", "start": 334, "end": 344}, "arguments": [{"role": "Theme", "text": "Tat", "start": 330, "end": 333}]}, {"trigger": {"text": "expression", "start": 655, "end": 665}, "arguments": [{"role": "Theme", "text": "p65", "start": 669, "end": 672}]}, {"trigger": {"text": "expression", "start": 655, "end": 665}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 677, "end": 690}]}]}}, "schema": []}
{"input": "Tat enhances the NF-kappaB activity following PMA stimulation by associating with IkappaB-alpha and counteracting the NF-kappaB repression. HeLa cells (5 x 106) were transfected with p3xFLAG-Tat or p3xFLAG empty vector (5 microg), and 48 h later were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM and harvested at the indicated times. Nuclear and cytosolic extracts were prepared for further analysis. (A) Nuclear extracts were analysed for the p65, p50 and FLAG-Tat binding to the NF-kappaB double-stranded oligonucleotide using the NF-kappaB Transcription Factor ELISA assay kit (Cayman). (B) Nuclear and cytosolic extracts (20 microg) were separated by 12% SDS-PAGE and analysed by western blotting using the anti-p65, anti-FLAG, anti-Histone H1, anti-IkappaB-alpha and anti-Hexokinase-II antibodies. Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (C) IKK activity was measured in cytosolic extracts (100 microg) by using HTScan IKK kinase assay (Cell Signaling Technology). (D) HeLa cells (5 x 106) were transfected with or without p3xFLAG-Tat (5 microg); 48 h later, cells were stimulated with PMA (5 min, 20 ng/ml) or left unstimulated, washed twice with DMEM, and harvested after 240 min. Whole cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-IkappaB-alpha antibody. Immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IkappaB-alpha antibodies. Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). Values (mean +/- SE, n = 3) are shown.", "output": {"json_structures": {"binding": [{"trigger": {"text": "associating", "start": 65, "end": 76}, "arguments": [{"role": "Theme", "text": "Tat", "start": 0, "end": 3}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 82, "end": 95}]}, {"trigger": {"text": "binding", "start": 504, "end": 511}, "arguments": [{"role": "Theme", "text": "p65", "start": 482, "end": 485}]}, {"trigger": {"text": "binding", "start": 504, "end": 511}, "arguments": [{"role": "Theme", "text": "p50", "start": 487, "end": 490}]}, {"trigger": {"text": "binding", "start": 504, "end": 511}, "arguments": [{"role": "Theme", "text": "FLAG-Tat", "start": 495, "end": 503}]}]}}, "schema": []}
{"input": "Tat relieves p65 from the IkappaB-alpha inhibition. (A) Schematic representation of wild-type and mutant Tat proteins. (B) HA-IkappaB-alpha (5 microl) was incubated in presence or absence of FLAG-Tat, FLAG-Tat T,N(23,24)A, FLAG-Tat K(50,51)A, FLAG-Tat R(49-57)A or FLAG-Tat C(22,25,27)A (10 microl) using in vitro translated proteins. Wild-type and mutant Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies. (C) 35S-methionine-labeled p65 (5 microl) was incubated with in vitro translated HA-IkappaB-alpha (5 microl) in presence or absence of FLAG-Tat or FLAG-Tat R(49-57)A (5, 10 or 20 microl). HA-IkappaB-alpha was immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-HA and anti-FLAG antibodies, or autoradiography (35S-Met-p65). Densitometry values (D) of the bands were expressed as fold increase above the control (lane 1). (D) The p65 DNA binding activity was analysed by EMSA using in vitro translated proteins. 32P-labeled-NF-kappaB double-stranded oligonucleotide was incubated with p65 (0.5 microl) in presence or absence of HA-IkappaB-alpha (1 microl), FLAG-Tat or FLAG-Tat R(49-57)A (5 and 10 microl); competition of DNA binding was performed with 10 - up to 100-fold molar excess of unlabeled NF-kappaB double-stranded oligonucleotide. DNA/protein complexes were run on 6% PAGE-TBE and analysed by autoradiography. (E) p50-/-p65-/-MEFs (3 x 105 cells) were transfected with pkappaBLuc (0.5 microg) and pSV-beta-Gal (0.1 microg) with or without pRc/CMV-p65 (0.5 microg), pCMV4-HA-IkappaB-alpha (0.5 microg), p3xFLAG-Tat or p3xFLAG-Tat R(49-57)A (0.5, 1 and 2 microg). The luciferase activity was measured in cell extracts 48-h post-transfection and normalized to beta-galactosidase activity. Fold activation was calculated relative to transfection of the pkappaBLuc plasmid alone. Values (mean +/- SE, n = 3) are shown. As control of protein expression, aliquots of cell extracts (20 microg) were analysed by western blotting with anti-p65, anti-HA, anti-FLAG and anti-gamma-tubulin antibodies.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 1054, "end": 1061}, "arguments": [{"role": "Theme", "text": "p65", "start": 1046, "end": 1049}]}, {"trigger": {"text": "binding", "start": 1342, "end": 1349}, "arguments": [{"role": "Theme", "text": "p65", "start": 1201, "end": 1204}]}], "negative regulation": [{"trigger": {"text": "relieves", "start": 4, "end": 12}, "arguments": [{"role": "Cause", "text": "Tat", "start": 0, "end": 3}, {"role": "Theme", "text": "inhibition", "start": 40, "end": 50}]}, {"trigger": {"text": "inhibition", "start": 40, "end": 50}, "arguments": [{"role": "Theme", "text": "p65", "start": 13, "end": 16}, {"role": "Cause", "text": "IkappaB-alpha", "start": 26, "end": 39}]}, {"trigger": {"text": "competition", "start": 1323, "end": 1334}, "arguments": [{"role": "Cause", "text": "HA-IkappaB-alpha", "start": 1244, "end": 1260}, {"role": "Theme", "text": "binding", "start": 1342, "end": 1349}]}]}}, "schema": []}
{"input": "Tat associates with p65 increasing its DNA-affinity binding. (A) HeLa cells were transfected with pRc/CMV-3xHA-p65 (5 microg) in presence or absence of p3xFLAG-Tat, p3xFLAG-Tat T,N(23,24)A, p3xFLAG-Tat K(50,51)A, p3xFLAG-Tat R(49-57)A, or p3xFLAG-Tat C(22,25,27)A (5 microg). FLAG-Tat proteins were immunoprecipitated with anti-FLAG antibody; immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (B) In vitro translated p65 (5 microl) was incubated with GST-Tat, GST-Tat T,N(23,24)A, GST-Tat K(50,51)A, GST-Tat R(49-57)A, GST-Tat C(22,25,27)A, or GST (5 microg) conjugated with Glutathione-Sepharose. Protein complexes were recovered by GST-pull down, separated by 12% SDS-PAGE, and analysed by western blotting with anti-p65 or anti-GST antibodies. (C) Schematic representation of wild-type and mutant p65 proteins. (D) HeLa cells were transfected with p3xFLAG-Tat (5 microg) in the presence or absence of pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65deltaC (1-318), or pRc/CMV-3xHA-p65deltaN (122-551) (5 microg). Wild-type and mutant HA-p65 proteins were immunoprecipitated with anti-HA antibody; immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-FLAG or anti-HA antibodies. (E) Recombinant p65 protein (100 ng; Active Motif Carlsbad, CA, USA) was 20 min incubated with 32P-labeled NF-kappaB double-stranded oligonucleotide in presence or absence of in vitro translated FLAG-Tat or FLAG-Tat C(22,25,27)A (5 microl); competitions were performed with 1.25- up to 40-fold molar excess of unlabeled oligonucleotide. DNA/protein complexes were separated by 6% PAGE in 0.5 x TBE buffer and analysed by autoradiography. (F) Densitometry of band-shifts shown in E.", "output": {"json_structures": {"binding": [{"trigger": {"text": "associates", "start": 4, "end": 14}, "arguments": [{"role": "Theme", "text": "Tat", "start": 0, "end": 3}, {"role": "Theme2", "text": "p65", "start": 20, "end": 23}]}, {"trigger": {"text": "binding", "start": 52, "end": 59}, "arguments": [{"role": "Theme", "text": "p65", "start": 20, "end": 23}]}], "positive regulation": [{"trigger": {"text": "increasing", "start": 24, "end": 34}, "arguments": [{"role": "Cause", "text": "associates", "start": 4, "end": 14}, {"role": "Theme", "text": "binding", "start": 52, "end": 59}]}]}}, "schema": []}
{"input": "Tat activates the p65-dependent expression of NF-kappaB-responsive genes by occupying the NF-kappaB enhancers and promoting the p65 recruitment with IkappaB-alpha displacement. HeLa cells (5 x 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49-57) or empty vector (10 microg); for p65 RNA interference, cells (5 x 106) were transfected with p3xFLAG-Tat (10 microg) and siRNA p65 or siRNA control (200 pmol). Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1alpha (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 x 106) were transfected with p3xFLAG-Tat, or empty vector (10 microg) and 48 h later ChIP was performed with anti-p65 antibody. Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 x 106) were transfected with empty vector, or p3xFLAG-Tat (10 microg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody. Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 x 106) were transfected with empty vector, p3xFLAG-Tat (10 microg), or empty vector plus siRNA IkappaB-alpha or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IkappaB-alpha antibody. Real-time PCR was performed with primers specific for the indicated promoters. Values (mean +/- SE, n = 3) are shown. The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P < 0.05).", "output": {"json_structures": {"binding": [{"trigger": {"text": "occupying", "start": 76, "end": 85}, "arguments": [{"role": "Theme", "text": "Tat", "start": 0, "end": 3}]}, {"trigger": {"text": "recruitment", "start": 132, "end": 143}, "arguments": [{"role": "Theme", "text": "p65", "start": 128, "end": 131}]}, {"trigger": {"text": "displacement", "start": 163, "end": 175}, "arguments": [{"role": "Theme", "text": "p65", "start": 128, "end": 131}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 149, "end": 162}]}], "gene expression": [{"trigger": {"text": "expression", "start": 545, "end": 555}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 559, "end": 569}]}, {"trigger": {"text": "expression", "start": 545, "end": 555}, "arguments": [{"role": "Theme", "text": "CSF3", "start": 575, "end": 579}]}, {"trigger": {"text": "expression", "start": 545, "end": 555}, "arguments": [{"role": "Theme", "text": "LTA", "start": 585, "end": 588}]}, {"trigger": {"text": "expression", "start": 545, "end": 555}, "arguments": [{"role": "Theme", "text": "NFKBIA", "start": 594, "end": 600}]}, {"trigger": {"text": "expression", "start": 545, "end": 555}, "arguments": [{"role": "Theme", "text": "TLR2", "start": 606, "end": 610}]}, {"trigger": {"text": "expression", "start": 545, "end": 555}, "arguments": [{"role": "Theme", "text": "GAPDH", "start": 616, "end": 621}]}, {"trigger": {"text": "expression", "start": 545, "end": 555}, "arguments": [{"role": "Theme", "text": "ACTB", "start": 627, "end": 631}]}], "negative regulation": [{"trigger": {"text": "interference", "start": 320, "end": 332}, "arguments": [{"role": "Theme", "text": "p65", "start": 312, "end": 315}]}], "positive regulation": [{"trigger": {"text": "promoting", "start": 114, "end": 123}, "arguments": [{"role": "Cause", "text": "Tat", "start": 0, "end": 3}, {"role": "Theme", "text": "recruitment", "start": 132, "end": 143}]}, {"trigger": {"text": "promoting", "start": 114, "end": 123}, "arguments": [{"role": "Cause", "text": "Tat", "start": 0, "end": 3}, {"role": "Theme", "text": "displacement", "start": 163, "end": 175}]}]}}, "schema": []}
{"input": "In HIV-1 infection Tat sustains the NF-kappaB activity and enhances the MIP-1alpha expression via interaction with IkappaB-alpha and p65. U937 cells (5 x 107) were transfected with siRNA Tat, siRNA control (2 nmol), or left untransfected; 24 h later cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R-E- virions (500 ng of p24) and harvested at the indicated time. (A) Real-time PCR of total RNA measured the Tat expression; ELISA measured the amount of p24 in whole cell extracts. (B) The binding of p65 to the NF-kappaB double-stranded oligonucleotide was measured in nuclear extracts (10 microg) using the NF-kappaB Transcription Factor ELISA assay kit (Cayman). (C) The IkappaB-alpha content was analysed by 12% SDS-PAGE and western blotting of cytosolic extracts (20 microg) using anti-IkappaB-alpha antibody. Densitometry values (D) of the bands were expressed as fold increase above the control (mock). (D) IKK activity was measured in cytosolic cell extracts (100 microg) using HTScan IKK Kinase Assay (Cell Signaling Technology). (E) U937 cells (5 x 107) were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R-E- virions, or left uninfected. Twenty-four-hour post-infection, cell extracts (1 mg) were immunoprecipitated with protein G-Sepharose-coupled anti-FLAG antibody. Immunocomplexes were separated by 12% SDS-PAGE and analysed by western blotting with anti-p65, anti-FLAG and anti-IkappaB-alpha antibodies. (F) U937 cells (5 x 107) were transfected with siRNA Tat, siRNA p65, siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R-E- virions, or left uninfected. Twenty-four-hour post-infection, total RNA was analysed for MIP-1alpha expression by real-time PCR. (G and H) U937 cells (5 x 107) were transfected with siRNA Tat or siRNA control (2 nmol), or left untransfected; 24 h later, cells were infected with HXB2-pseudotyped NL4-3.FLAG-Tat.R-E- virions, or left uninfected. Twenty-four-hour post-infection, ChIP was performed with anti-p65 (G) or anti-FLAG (H). Real-time PCR was performed with primers specific for MIP-1alpha and GAPDH promoters. Values (mean +/- SE, n = 3) are shown. The asterisks indicate statistically significant differences compared to the control (mock), according to the Student's t-test (P < 0.05).", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 98, "end": 109}, "arguments": [{"role": "Theme", "text": "Tat", "start": 19, "end": 22}, {"role": "Theme2", "text": "IkappaB-alpha", "start": 115, "end": 128}]}, {"trigger": {"text": "binding", "start": 498, "end": 505}, "arguments": [{"role": "Theme", "text": "p65", "start": 509, "end": 512}]}], "gene expression": [{"trigger": {"text": "expression", "start": 83, "end": 93}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 72, "end": 82}]}, {"trigger": {"text": "expression", "start": 421, "end": 431}, "arguments": [{"role": "Theme", "text": "Tat", "start": 417, "end": 420}]}, {"trigger": {"text": "expression", "start": 1718, "end": 1728}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 1707, "end": 1717}]}], "positive regulation": [{"trigger": {"text": "enhances", "start": 59, "end": 67}, "arguments": [{"role": "Theme", "text": "expression", "start": 83, "end": 93}, {"role": "Cause", "text": "interaction", "start": 98, "end": 109}]}]}}, "schema": []}
{"input": "Model of MIP-1alpha transcriptional activation by HIV-1 Tat. (A) In absence of Tat, IkappaB-alpha occupies the NF-kappaB enhancer of the MIP-1alpha promoter and likely interacts with transcriptional repressors, such as HDACs (65), to inhibit gene transcription. (B) Tat activates the MIP-1alpha expression by removing the IkappaB-alpha repressor from the NF-kappaB enhancer, and by increasing the binding of p65 NF-kappaB complex to the NF-kappaB enhancer.", "output": {"json_structures": {"binding": [{"trigger": {"text": "occupies", "start": 98, "end": 106}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 84, "end": 97}, {"role": "Site2", "text": "NF-kappaB enhancer", "start": 111, "end": 129}, {"role": "Theme2", "text": "MIP-1alpha", "start": 137, "end": 147}]}, {"trigger": {"text": "interacts", "start": 168, "end": 177}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 84, "end": 97}]}, {"trigger": {"text": "binding", "start": 397, "end": 404}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 284, "end": 294}, {"role": "Site", "text": "NF-kappaB enhancer", "start": 437, "end": 455}]}], "gene expression": [{"trigger": {"text": "expression", "start": 295, "end": 305}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 284, "end": 294}]}], "negative regulation": [{"trigger": {"text": "absence", "start": 68, "end": 75}, "arguments": [{"role": "Theme", "text": "Tat", "start": 79, "end": 82}]}, {"trigger": {"text": "removing", "start": 309, "end": 317}, "arguments": [{"role": "Cause", "text": "Tat", "start": 266, "end": 269}, {"role": "Theme", "text": "IkappaB-alpha", "start": 322, "end": 335}]}], "positive regulation": [{"trigger": {"text": "activation", "start": 36, "end": 46}, "arguments": [{"role": "Theme", "text": "transcriptional", "start": 20, "end": 35}, {"role": "Cause", "text": "Tat", "start": 56, "end": 59}]}, {"trigger": {"text": "activates", "start": 270, "end": 279}, "arguments": [{"role": "Cause", "text": "Tat", "start": 266, "end": 269}, {"role": "Theme", "text": "expression", "start": 295, "end": 305}]}, {"trigger": {"text": "increasing", "start": 382, "end": 392}, "arguments": [{"role": "Cause", "text": "Tat", "start": 266, "end": 269}, {"role": "Theme", "text": "binding", "start": 397, "end": 404}]}], "transcription": [{"trigger": {"text": "transcriptional", "start": 20, "end": 35}, "arguments": [{"role": "Theme", "text": "MIP-1alpha", "start": 9, "end": 19}]}]}}, "schema": []}
{"input": "Inflammasome-Mediated IL-1beta Production in Humans with Cystic Fibrosis\nBackground\nInflammation and infection are major determinants of disease severity and consequently, the quality of life and outcome for patients with cystic fibrosis (CF). Interleukin-1 beta (IL-1beta) is a key inflammatory mediator. Secretion of biologically active IL-1beta involves inflammasome-mediated processing. Little is known about the contribution of IL-1beta and the inflammasomes in CF inflammatory disease. This study examines inflammasome-mediated IL-1beta production in CF bronchial epithelial cell lines and human patients with CF.\nResults\nBronchial epithelial cell lines were found to produce negligible amounts of basal or stimulated IL-1beta compared to hematopoeitic cells and they did not significantly upregulate caspase-1 activity upon inflammasome stimulation. In contrast, peripheral blood mononuclear cells (PBMCs) from both CF and healthy control subjects produced large amounts of IL-1beta and strongly upregulated caspase-1 activity upon inflammasome stimulation. PBMCs from CF patients and controls displayed similar levels of caspase-1 activation and IL-1beta production when stimulated with inflammasome activators. This IL-1beta production was dependent on NF-kappaB activity and could be enhanced by priming with LPS. Finally, chemical inhibition of CFTR activity in control PBMCs and THP-1 cells did not significantly alter IL-1beta or IL-8 production in response to P. aeruginosa.\nConclusion\nHematopoeitic cells appear to be the predominant source of inflammasome-induced pro-inflammatory IL-1beta in CF. PBMCs derived from CF subjects display preserved inflammasome activation and IL-1beta secretion in response to the major CF pathogen Pseudomonas aeruginosa. However, our data do not support the hypothesis that increased IL-1beta production in CF subjects is due to an intrinsic increase in NF-kappaB activity through loss of CFTR function.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Production", "start": 31, "end": 41}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 22, "end": 30}]}, {"trigger": {"text": "production", "start": 543, "end": 553}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 534, "end": 542}]}, {"trigger": {"text": "produce", "start": 674, "end": 681}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 724, "end": 732}]}, {"trigger": {"text": "produced", "start": 955, "end": 963}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 981, "end": 989}]}, {"trigger": {"text": "production", "start": 1163, "end": 1173}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1154, "end": 1162}]}, {"trigger": {"text": "production", "start": 1448, "end": 1458}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1431, "end": 1439}]}, {"trigger": {"text": "production", "start": 1842, "end": 1852}, "arguments": [{"role": "Theme", "text": " IL-1beta", "start": 1832, "end": 1841}]}], "localization": [{"trigger": {"text": "Secretion", "start": 306, "end": 315}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 339, "end": 347}]}, {"trigger": {"text": "secretion", "start": 1699, "end": 1708}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1690, "end": 1698}]}], "positive regulation": [{"trigger": {"text": "Mediated", "start": 13, "end": 21}, "arguments": [{"role": "Theme", "text": "Production", "start": 31, "end": 41}]}, {"trigger": {"text": "mediated", "start": 525, "end": 533}, "arguments": [{"role": "Theme", "text": "production", "start": 543, "end": 553}]}, {"trigger": {"text": "stimulated", "start": 713, "end": 723}, "arguments": [{"role": "Theme", "text": "produce", "start": 674, "end": 681}]}, {"trigger": {"text": "upon", "start": 1034, "end": 1038}, "arguments": [{"role": "Theme", "text": "produced", "start": 955, "end": 963}]}, {"trigger": {"text": "stimulated", "start": 1179, "end": 1189}, "arguments": [{"role": "Theme", "text": "production", "start": 1163, "end": 1173}]}, {"trigger": {"text": "in response to", "start": 1459, "end": 1473}, "arguments": [{"role": "Theme", "text": "production", "start": 1448, "end": 1458}]}, {"trigger": {"text": "induced", "start": 1572, "end": 1579}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1597, "end": 1605}]}, {"trigger": {"text": "preserved", "start": 1652, "end": 1661}, "arguments": [{"role": "Theme", "text": "secretion", "start": 1699, "end": 1708}]}, {"trigger": {"text": "increased", "start": 1823, "end": 1832}, "arguments": [{"role": "Theme", "text": "production", "start": 1842, "end": 1852}]}, {"trigger": {"text": "due", "start": 1871, "end": 1874}, "arguments": [{"role": "Theme", "text": "increased", "start": 1823, "end": 1832}]}], "regulation": [{"trigger": {"text": "involves", "start": 348, "end": 356}, "arguments": [{"role": "Theme", "text": "Secretion", "start": 306, "end": 315}]}, {"trigger": {"text": "alter", "start": 1425, "end": 1430}, "arguments": [{"role": "Theme", "text": "in response to", "start": 1459, "end": 1473}]}]}}, "schema": []}
{"input": "Cystic fibrosis is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a chloride ion channel. CF remains one of the most common lethal genetic diseases in populations of European descent with the current average lifespan of CF patients approximately 40 years of age [1], [2]. Recurrent inflammatory pulmonary exacerbation is the primary cause of lung disease progression and ultimately, death in CF. Controlled inflammation is important for fighting infection, but in excess, it becomes destructive to host cells and to the architecture of the lungs [2], [3]. In CF, airway epithelial cells have been shown to produce an exaggerated pro-inflammatory cytokine response to stimulation [4], [5]. It is unclear whether this heightened inflammatory response is intrinsic to cells lacking CFTR or whether it is a result of chronic polymicrobial infection [6], [7]. Regardless of this controversy, identifying and targeting relevant inflammatory mediators is a critical step in developing more specific therapeutic approaches to control inflammation and improve health outcomes in CF [8].\nInterleukin-1 beta (IL-1beta) is a major inflammatory mediator. Its physiological effects are diverse and potentially important to the pathogenesis of lung exacerbations in CF, including the generation of fever, the recruitment of inflammatory effector cells, the induction of other pro-inflammatory cytokines such as IL-6 and IL-8, and the shaping of T cell responses [9], [10]. Following initiation of the NF-kappaB signaling cascade, IL-1beta is produced in the cytosol as a biologically inactive full-length pro-IL-1beta. Pro-IL-1beta is subsequently converted into its active form by cytosolic protein complexes termed \"inflammasomes.\" Inflammasomes assemble in response to certain cellular danger signals and mediate the auto-activation of caspase-1 [9], [11], which cleaves pro-IL-1beta and pro-IL-18 into their biologically active forms for secretion. Four distinct inflammasomes have been recognized. These are the NLRP1 [12], NLRP3 [13], [14], NLRC4 [15], [16], and AIM2 inflammasomes [17], [18], which respond to a variety of different microbial signatures and danger signals [11].\nP. aeruginosa, one of the most common and clinically relevant pathogens among CF patients, activates the NLRC4 inflammasome [19], [20]. Infection with P. aeruginosa triggers an increase in levels of IL-1beta, IL-6, and IL-8 in bronchoalveolar lavage fluid (BALF) from patients with CF [21]. Inflammasome responses depend on NF-kappaB signaling, where NF-kappaB is important in both the upregulation of specific inflammasome components [22], [23], as well as IL-1beta expression [24], [25].\nPrevious studies support a role for IL-1beta in the pathogenesis of CF inflammatory lung disease. Levels of IL-1beta are increased in BALF from CF patients with infection [21], [26], [27], [28] and this increase has been temporally associated with a clinical response to treatment [21]. Polymorphisms in the IL1B gene have also been associated with varying degrees of disease severity in CF patients [29]. Murine models of CFTR dysfunction have exhibited significant increases in IL-1beta expression or secretion in macrophages [30], [31], and support the hypothesis that the loss of CFTR increases NF-kappaB activation under basal and stimulatory conditions [4], [5], [32], [33]. Finally, replacement of chloride ions with glutamate or gluconate in cell culture media increases secretion of IL-1beta in response to NLRP3 stimulation by adenosine triphosphate (ATP) [34], implying an inhibitory role for extracellular chloride in NLRP3 activation. Taken together, these data implicate the involvement of IL-1beta and consequently, the inflammasomes, in CF inflammatory disease.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 2728, "end": 2738}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 2719, "end": 2727}]}, {"trigger": {"text": "expression", "start": 3240, "end": 3250}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 3231, "end": 3239}]}], "localization": [{"trigger": {"text": "secretion", "start": 3254, "end": 3263}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 3231, "end": 3239}]}, {"trigger": {"text": "secretion", "start": 3530, "end": 3539}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 3543, "end": 3551}]}], "positive regulation": [{"trigger": {"text": "triggers", "start": 2426, "end": 2434}, "arguments": [{"role": "Theme", "text": "increase", "start": 2438, "end": 2446}]}, {"trigger": {"text": "increase", "start": 2438, "end": 2446}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 2460, "end": 2468}]}, {"trigger": {"text": "increase", "start": 2438, "end": 2446}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 2470, "end": 2474}]}, {"trigger": {"text": "increased", "start": 2872, "end": 2881}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 2859, "end": 2867}]}, {"trigger": {"text": "increases", "start": 3218, "end": 3227}, "arguments": [{"role": "Theme", "text": "expression", "start": 3240, "end": 3250}]}, {"trigger": {"text": "increases", "start": 3218, "end": 3227}, "arguments": [{"role": "Theme", "text": "secretion", "start": 3254, "end": 3263}]}, {"trigger": {"text": "increases", "start": 3520, "end": 3529}, "arguments": [{"role": "Theme", "text": "secretion", "start": 3530, "end": 3539}]}], "regulation": [{"trigger": {"text": "important", "start": 2625, "end": 2634}, "arguments": [{"role": "Theme", "text": "expression", "start": 2728, "end": 2738}]}]}}, "schema": []}
{"input": "Airway epithelial cells do not produce significant amounts of IL-1beta in response to inflammasome stimulation\nThe inflammasomes and their respective activators examined in this study are listed in Table 1. Cells were stimulated in accordance with the schedule in Figure 1. In CF, airway epithelial cells have been shown to possess a hyper-inflammatory phenotype and produce an exaggerated pro-inflammatory cytokine response [4], [5]. To determine if airway epithelial cells contribute to the increased IL-1beta production in patients with CF, CF and control bronchial epithelial cell lines were stimulated with the inflammasome inducers P. aeruginosa strain PAO1 (PAO1) and LPS followed by ATP. IL-1beta levels in cell culture supernatants were not greatly increased in either the CF or control cell lines (Fig. 2a-d), although a small increase in IL-1beta production was detected in NuLi-1 and CuFi-1 cells, but not in S9 and IB3-1 cells, by 24 hours. In contrast, these airway cells were highly responsive to other inflammatory stimuli, such as recombinant IL-1beta, producing large quantities of IL-8 (Fig. 2a-d inserts).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "produce", "start": 31, "end": 38}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 62, "end": 70}]}, {"trigger": {"text": "production", "start": 512, "end": 522}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 503, "end": 511}]}, {"trigger": {"text": "production", "start": 858, "end": 868}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 849, "end": 857}]}], "positive regulation": [{"trigger": {"text": "in response to", "start": 71, "end": 85}, "arguments": [{"role": "Theme", "text": "produce", "start": 31, "end": 38}]}, {"trigger": {"text": "contribute", "start": 475, "end": 485}, "arguments": [{"role": "Theme", "text": "increased", "start": 493, "end": 502}]}, {"trigger": {"text": "increased", "start": 493, "end": 502}, "arguments": [{"role": "Theme", "text": "production", "start": 512, "end": 522}]}, {"trigger": {"text": "increased", "start": 758, "end": 767}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 696, "end": 704}]}, {"trigger": {"text": "increase", "start": 837, "end": 845}, "arguments": [{"role": "Theme", "text": "production", "start": 858, "end": 868}]}]}}, "schema": []}
{"input": "Airway epithelial cells do not significantly upregulate caspase-1 activity in response to inflammasome stimulation\nTo examine if inflammasome activation occurs in these airway cells, caspase-1 activity was quantified by flow cytometry. There was no significant increase upon stimulation with live PAO1 or LPS+ATP at the times examined (Fig. 3a-b). Because previous studies have indicated a role for caspase-1 in the activation of NF-kappaB through Toll-like receptor (TLR) signaling [35], we examined whether chemical inhibition of caspase-1 altered NF-kappaB-dependent IL-6 production in response to P. aeruginosa. However, treatment with the caspase-1 inhibitor z-YVAD-fmk (YVAD) did not decrease IL-6 secretion by airway epithelial cells (Fig. 3c).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 575, "end": 585}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 570, "end": 574}]}], "localization": [{"trigger": {"text": "secretion", "start": 704, "end": 713}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 699, "end": 703}]}], "negative regulation": [{"trigger": {"text": "decrease", "start": 690, "end": 698}, "arguments": [{"role": "Theme", "text": "secretion", "start": 704, "end": 713}]}], "positive regulation": [{"trigger": {"text": "in response to", "start": 586, "end": 600}, "arguments": [{"role": "Theme", "text": "dependent", "start": 560, "end": 569}]}], "regulation": [{"trigger": {"text": "altered", "start": 542, "end": 549}, "arguments": [{"role": "Theme", "text": "in response to", "start": 586, "end": 600}]}, {"trigger": {"text": "dependent", "start": 560, "end": 569}, "arguments": [{"role": "Theme", "text": "production", "start": 575, "end": 585}]}]}}, "schema": []}
{"input": "CD14 positive monocytes from CF patients and controls show similar increases in caspase-1 activity upon inflammasome stimulation\nMonocytes were identified in PBMC populations using CD14 as a phenotyping marker. CD14 positive monocytes from CF patients and healthy controls showed a significant increase in caspase-1 activation upon stimulation with LPS+ATP, PAO1, and LPS+Poly(dA:dT) (Fig. 4a) but this activation was not different between CF and control subjects (Fig. 4b).", "output": {"json_structures": {}}, "schema": []}
{"input": "PBMCs from CF patients do not produce increased amounts of IL-1beta upon inflammasome stimulation\nPrevious studies have shown that the loss of CFTR results in increased NF-kappaB activity and pro-inflammatory cytokine secretion [4], [5], [32], [36], [37]. To further examine this relationship, PBMCs from CF patients and healthy adult controls were stimulated with PAO1, LPS+ATP, and LPS+Poly(dA:dT), to activate the NLRC4, NLRP3, and AIM2 inflammasomes, respectively. By 24 hours of stimulation, CF PBMCs did not produce increased amounts of IL-1beta (Fig. 5a) or IL-8 (Fig. 5b) when compared to healthy controls. However, we did notice a transient decrease (P<0.001) in the amount of IL-1beta produced by CF cells in response to LPS+ATP at 6 hours (data not shown). Stimulation of PBMCs with P. aeruginosa that lacks exsA (PAO1deltaexsA), a key regulator of type III secretion, produced three-fold less IL-1beta compared to the parental PAO1 strain by 24 hours (Fig. 5a). Inflammasome stimulation without priming did not result in any IL-1beta production in either CF or control PBMCs. Contrary to our hypothesis, these results indicate that PBMCs from CF patients do not display increased production of IL-1beta or IL-8 with inflammasome activation nor do they suggest any increased basal or induced NF-kappaB activity. These results are consistent with our observation that caspase-1 activity is not different between CF and control PBMCs (Fig. 4).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "produce", "start": 30, "end": 37}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 59, "end": 67}]}, {"trigger": {"text": "produce", "start": 514, "end": 521}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 543, "end": 551}]}, {"trigger": {"text": "produced", "start": 695, "end": 703}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 686, "end": 694}]}, {"trigger": {"text": "produced", "start": 880, "end": 888}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 905, "end": 913}]}, {"trigger": {"text": "production", "start": 1046, "end": 1056}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1037, "end": 1045}]}, {"trigger": {"text": "production", "start": 1192, "end": 1202}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1206, "end": 1214}]}, {"trigger": {"text": "production", "start": 1192, "end": 1202}, "arguments": [{"role": "Theme", "text": "IL-8", "start": 1218, "end": 1222}]}], "negative regulation": [{"trigger": {"text": "decrease", "start": 650, "end": 658}, "arguments": [{"role": "Theme", "text": "produced", "start": 695, "end": 703}]}, {"trigger": {"text": "less", "start": 900, "end": 904}, "arguments": [{"role": "Theme", "text": "produced", "start": 880, "end": 888}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 38, "end": 47}, "arguments": [{"role": "Theme", "text": "produce", "start": 30, "end": 37}]}, {"trigger": {"text": "increased", "start": 522, "end": 531}, "arguments": [{"role": "Theme", "text": "produce", "start": 514, "end": 521}]}, {"trigger": {"text": "in response to", "start": 716, "end": 730}, "arguments": [{"role": "Theme", "text": "decrease", "start": 650, "end": 658}]}, {"trigger": {"text": "result", "start": 1023, "end": 1029}, "arguments": [{"role": "Theme", "text": "production", "start": 1046, "end": 1056}]}, {"trigger": {"text": "increased", "start": 1182, "end": 1191}, "arguments": [{"role": "Theme", "text": "production", "start": 1192, "end": 1202}]}]}}, "schema": []}
{"input": "NF-kappaB activation is required for IL-1beta and IL-8 responses to P. aeruginosa\nWe confirmed the dependence of PAO1-induced IL-1beta and IL-8 production on NF-kappaB activation using THP-1 cells expressing a reporter driven by NF-kappaB and AP-1 response elements. We found that stimulation of primed THP-1 reporter cells with heat-killed PAO1 produced the highest levels of NF-kappaB/AP-1 activation (Fig. 6a) and this correlated with IL-8 secretion (Fig. 6b) but negligible amounts of IL-1beta were secreted (Fig. 6c). Stimulation of primed THP-1 reporter cells with live PAO1 did not significantly increase NF-kappaB/AP-1 activity (Fig. 6a) or IL-8 secretion (Fig. 6b) over priming alone. However, IL-1beta production was greatly augmented over primed cells stimulated with heat-killed PAO1 or unprimed cells stimulated with live PAO1 (Fig. 6c). This confirmed that NF-kappaB activation alone is not sufficient for maximal IL-1beta secretion, but increased priming of NF-kappaB is capable of augmenting IL-1beta production and secretion upon inflammasome stimulation. Dependency of these responses on NF-kappaB was confirmed by pharmacologic inhibition of NF-kappaB using the Bay11-7082 inhibitor of IkappaBalpha phosphorylation, which significantly reduced NF-kappaB/AP-1 activation (P<0.001) (Fig. 6d) and the subsequent production of IL-8 (P<0.01) (Fig. 6e) and IL-1beta (P<0.001) (Fig. 6f) in response to both heat-killed and live PAO1. These results were also verified in CF and control PBMCs for each inflammasome examined (P<0.001) (Fig. 6g-h). Overall these results confirm that NF-kappaB is an important modulator of IL-1beta production and that increased activation of NF-kappaB augments inflammasome-mediated production of IL-1beta.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 144, "end": 154}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 126, "end": 134}]}, {"trigger": {"text": "production", "start": 712, "end": 722}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 703, "end": 711}]}, {"trigger": {"text": "production", "start": 1328, "end": 1338}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1370, "end": 1378}]}, {"trigger": {"text": "production", "start": 1640, "end": 1650}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1631, "end": 1639}]}, {"trigger": {"text": "production", "start": 1725, "end": 1735}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1739, "end": 1747}]}], "localization": [{"trigger": {"text": "secreted", "start": 503, "end": 511}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 489, "end": 497}]}, {"trigger": {"text": "secretion", "start": 937, "end": 946}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 928, "end": 936}]}], "negative regulation": [{"trigger": {"text": "inhibitor", "start": 1192, "end": 1201}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1218, "end": 1233}]}, {"trigger": {"text": "reduced", "start": 1255, "end": 1262}, "arguments": [{"role": "Cause", "text": "inhibitor", "start": 1192, "end": 1201}, {"role": "Theme", "text": "in response to", "start": 1399, "end": 1413}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1218, "end": 1233}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1205, "end": 1217}]}], "positive regulation": [{"trigger": {"text": "required", "start": 24, "end": 32}, "arguments": [{"role": "Theme", "text": "responses", "start": 55, "end": 64}]}, {"trigger": {"text": "induced", "start": 118, "end": 125}, "arguments": [{"role": "Theme", "text": "production", "start": 144, "end": 154}]}, {"trigger": {"text": "augmented", "start": 735, "end": 744}, "arguments": [{"role": "Theme", "text": "production", "start": 712, "end": 722}]}, {"trigger": {"text": "sufficient", "start": 905, "end": 915}, "arguments": [{"role": "Theme", "text": "secretion", "start": 937, "end": 946}]}, {"trigger": {"text": "in response to", "start": 1399, "end": 1413}, "arguments": [{"role": "Theme", "text": "production", "start": 1328, "end": 1338}]}, {"trigger": {"text": "augments", "start": 1694, "end": 1702}, "arguments": [{"role": "Theme", "text": "mediated", "start": 1716, "end": 1724}]}, {"trigger": {"text": "mediated", "start": 1716, "end": 1724}, "arguments": [{"role": "Theme", "text": "production", "start": 1725, "end": 1735}]}], "regulation": [{"trigger": {"text": "responses", "start": 55, "end": 64}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 37, "end": 45}]}, {"trigger": {"text": "dependence", "start": 99, "end": 109}, "arguments": [{"role": "Theme", "text": "induced", "start": 118, "end": 125}]}, {"trigger": {"text": "modulator", "start": 1618, "end": 1627}, "arguments": [{"role": "Theme", "text": "production", "start": 1640, "end": 1650}]}]}}, "schema": []}
{"input": "Bay11-7082 inhibits pro-IL-1beta production in response to P. aeruginosa\nIn addition to inhibition of NF-kappaB activity, Bay11-7082 can also directly inhibit the NLRP3 inflammasome [38]. To validate its use in this study as an NF-kappaB inhibitor, western blots for pro-IL-1beta were performed alongside inhibition of CFTR activity by CFTRinh172 in response to PAO1 at 4 hours after stimulation (Fig. 7a). Our results indicate that Bay11-7082 prevents production of pro-IL-1beta whereas CFTRinh172 does not seem to affect it. This was further corroborated by the ability Bay11-7082 to inhibit IkappaBalpha degradation at 0.5, 1, and 1.5 hours post PAO1 stimulation (Fig. 7b).", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "inhibit", "start": 586, "end": 593}, "arguments": [{"role": "Theme", "text": "degradation", "start": 607, "end": 618}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 607, "end": 618}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 594, "end": 606}]}]}}, "schema": []}
{"input": "Disruption of CFTR activity does not increase IL-1beta production in PBMCs and THP-1 cells\nA previous study has indicated a role for chloride ion concentration in suppression of NLRP3 inflammasome activation [34]. To determine whether CFTR dysfunction alters IL-1beta production, THP-1 cells and PBMCs from CF patients and healthy controls were treated with the CFTR inhibitor, CFTRinh172, prior to simulation with live P. aeruginosa. Treatment with CFTRinh172 did not alter IL-1beta or IL-8 production in control subjects or CF patients (Fig. 8a-b). Similarly, IL-1beta production was not different in monocyte-derived macrophages or THP-1 reporter cells treated with CFTRinh172 (Fig. 8c-d). IL-8 (Fig. 8e) and NF-kappaB activity (Fig. 8f) were also unchanged in CFTRinh172-treated THP-1 reporter cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 55, "end": 65}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 46, "end": 54}]}, {"trigger": {"text": "production", "start": 268, "end": 278}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 259, "end": 267}]}, {"trigger": {"text": "production", "start": 492, "end": 502}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 475, "end": 483}]}, {"trigger": {"text": "production", "start": 571, "end": 581}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 562, "end": 570}]}], "positive regulation": [{"trigger": {"text": "increase", "start": 37, "end": 45}, "arguments": [{"role": "Theme", "text": "production", "start": 55, "end": 65}]}], "regulation": [{"trigger": {"text": "alters", "start": 252, "end": 258}, "arguments": [{"role": "Theme", "text": "production", "start": 268, "end": 278}]}, {"trigger": {"text": "alter", "start": 469, "end": 474}, "arguments": [{"role": "Theme", "text": "production", "start": 492, "end": 502}]}]}}, "schema": []}
{"input": "Levels of IL-1beta are increased in the BALF of CF patients but the cellular source of this cytokine and its production in the context of targeted inflammasome activation are still unclear. We first studied airway epithelial cells due to their role in barrier function, proximity to infection, and ability to produce high levels of pro-inflammatory cytokines. However, we found that bronchial epithelial cells do not produce significant amounts of IL-1beta and do not show a significant increase in caspase-1 activation in response to PAO1 and LPS+ATP, in comparison to hematopoeitic mononuclear cells. Hematopoeitically-derived cells, such as monocytes and macrophages, appear to be a principal source of IL-1beta. CFTR is expressed in alveolar macrophages [32], [39] as well as in PBMCs at both the mRNA and protein level [40], [41], and its loss is frequently associated with an augmented inflammatory phenotype.\nDespite our findings indicating that bronchial epithelial cells when grown in vitro are unlikely to be significantly involved in the direct production of IL-1beta (Fig. 2a-d), others have shown their capacity to respond to alveolar macrophage-derived IL-1beta and to amplify the inflammatory response through the induction of chemokines and recruitment of inflammatory effector cells [42]. This interaction may constitute a critical component to effective host defense and diminishing the capacity of host cells to respond to IL-1beta may leave the host susceptible to infections by pathogens such as P. aeruginosa [43]. Conversely, overproduction of IL-1beta can also play a key role in chronic inflammatory responses and cause damage to the lung parenchyma [44], [45].\nAlthough we hypothesized that CF cells would secrete increased amounts of IL-1beta, we found that IL-1beta production in CF PBMCs was not increased upon inflammasome stimulation as compared to controls (Fig. 5a-b). This was in contrast to a previous study from our group, which showed increased IL-1beta production by CF PBMCs in response to LPS [46], although this difference may be accounted for by technical issues in stimulation time and dose. Moreover, IL-1beta production was not increased in CF PBMCs with inflammasome stimulation alone as would be anticipated if there were basal levels of NF-kappaB activation. Cells deficient in CFTR are thought to exhibit an increased basal level of NF-kappaB activity, which leads to increased pro-inflammatory cytokine production including an increased availability of pro-IL-1beta for cleavage and secretion. This amplification of IL-1beta secretion was shown by priming THP-1 monocytes and PBMCs with heat-killed P. aeruginosa or LPS prior to stimulation with live P. aeruginosa. This dramatically increased IL-1beta secretion over stimulation with live P. aeruginosa without priming (Fig. 6c). Similarly, if CF PBMCs expressed increased basal NF-kappaB activity, there would be an increase in IL-1beta secretion in the absence of LPS priming. However, no increase in IL-1beta was observed under basal or primed conditions. Studies investigating the production of IL-1beta have been somewhat inconsistent. A study by Reininger et al. [43] provided evidence that human bronchial epithelial cells possessing the deltaF508 CFTR mutation had a slightly reduced capacity to produce IL-1beta and lacked the ability to induce an early NF-kappaB activation in response to P. aeruginosa. Conversely, a study by Kotrange et al. [31] found that murine bone marrow-derived macrophages expressing deltaF508-CFTR produced increased amounts of IL-1beta when compared to macrophages expressing normal CFTR in response to Burkholderia cenocepacia K56-2. The differentiation of monocytes into macrophages may partly account for the differences observed with the study by Kotrange. Inflammasome-mediated IL-1beta production by monocytes and PBMCs does differ from macrophages [47], [48], [49] and macrophages are found to have higher expression of CFTR over monocytes [50]. However, as monocytes and other PBMCs express CFTR [50], [51] and produce large amounts of IL-1beta, they are adequate models to examine the effects of CFTR function on IL-1beta production. Other hematopoeitic cells may also contribute to IL-1beta production. For example, neutrophil counts can be significantly increased in the lungs of CF patients [52], [53], [54] and may produce mature IL-1beta through caspase-1 independent mechanisms [55].\nThe role of NF-kappaB activation in inflammasome activation and IL-1beta secretion is not straightforward. Studies have revealed an essential role for NF-kappaB activation in the production of pro-IL-1beta and inflammasome components such as NLRP3 [22], [23]. In contrast, deletion of IKKbeta, a kinase essential in NF-kappaB activation, increases IL-1beta secretion in murine macrophages [56], [57] and demonstrates a dual role for NF-kappaB in regulation of IL-1beta. To address this uncertainty in our experiments we also quantified IL-8, an important CF cytokine and marker of NF-kappaB activation [58], and found no differences between CF and control subjects. Similarly, levels of intracellular pro-IL-1beta in THP-1 cells were dependent on NF-kappaB activity and did not increase with CFTRinh172 treatment. Subsequent treatment of THP-1 cells and PBMCs with the NF-kappaB inhibitor Bay11-7082 significantly inhibited both IL-1beta and IL-8 secretion (Fig. 6d-h). Therefore, the IL-1beta and IL-8 responses observed were both dependent upon NF-kappaB activation. Priming with heat-killed P. aeruginosa, like LPS, is unable to induce a strong IL-1beta response as compared to live P. aeruginosa (Fig. 6c), but generated greater NF-kappaB/AP-1 activation (Fig. 6a) and IL-8 secretion (Fig. 6b) than live bacteria despite stimulation at equivalent MOIs. This may be indicative of the different degree and quality of the inflammatory response generated by live as opposed to dead bacteria [59].\nPotential shortcomings of these experiments include its translatability to lung disease and issues related to the hypermutability of P. aeruginosa during the evolution of chronic infection. Although the responses measured in peripheral blood cells may not completely reflect the responses occurring in the CF lung, PBMCs have a number of useful advantages: (i) PBMCs are not subject to alterations that may emerge from long-term cell culture, cloning and immortalization, and (ii) PBMCs express a large repertoire of innate immune receptors and secrete a broad array of cytokines and chemokines allowing comprehensive analysis of the modulation of inflammatory responses by CFTR. Consideration must also be given to the nature of P. aeruginosa infection and genotypic changes in P. aeruginosa as infection progresses. P. aeruginosa mediates inflammasome activation through its type III secretion system (T3SS) and the NLRC4 inflammasome [19], [20]. However, clones of P. aeruginosa established during chronic infection may accumulate mutations in virulence factors such as exsA [60]. By employing a deletion mutant in exsA, the key regulator in T3SS transcription, we confirmed that the T3SS is important for IL-1beta secretion (Fig. 5a-b), and that depending on the adaptation in type III secretion, the host IL-1beta response may be up or downregulated [19], [60], [61].\nIn conclusion, our data are consistent with a role for hematopoietic cells, not airway epithelial cells, as the major source of inflammasome-mediated IL-1beta production in the lungs in response to ATP and P. aeruginosa. Furthermore, we find little evidence to support an increased IL-1beta inflammatory response to NF-kappaB/Inflammasome stimulation in CF patients. Further studies are warranted to determine if adaptations of P. aeruginosa during the course of chronic lung infection alters inflammasome activation, and whether this can be correlated with disease severity in CF.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 109, "end": 119}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 10, "end": 18}]}, {"trigger": {"text": "produce", "start": 417, "end": 424}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 448, "end": 456}]}, {"trigger": {"text": "production", "start": 1056, "end": 1066}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1070, "end": 1078}]}, {"trigger": {"text": "overproduction", "start": 1549, "end": 1563}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1567, "end": 1575}]}, {"trigger": {"text": "production", "start": 1794, "end": 1804}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1785, "end": 1793}]}, 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{"input": "Ethics Statement\nBlood samples were obtained with informed written consent from control subjects and CF patients at the BC Children's Hospital. Consent was obtained for children by their parent or legal guardian. Subjects 7 years of age and older were required to provide informed assent as well. Protocols were approved by the Clinical Research Ethics Board (H09-01192).", "output": {"json_structures": {}}, "schema": []}
{"input": "Cell Culture\nCF (IB3-1 and CuFi-1) and control (S9 and NuLi-1) cells were obtained from the American Type Culture Collection. IB3-1 cells were derived from a patient expressing the deltaF508 and W1282X mutations and CuFi-1 were derived from a deltaF508 homozygous patient. S9 cells are IB3-1 cells that have been transfected with CFTR using an adeno-associated viral vector and NuLi-1 cells were derived from a patient possessing a wild-type CFTR genotype. THP1-XBlue cells stably express a secreted embryonic alkaline phosphatase (SEAP) reporter inducible by NF-kappaB and AP-1 (Invivogen). Cells were cultured as recommended by their respective suppliers using standard protocols. S9 and IB3-1 cells were cultured in basal LHC-8 (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, and 1 mM sodium pyruvate. NuLi-1/CuFi-1 cells were cultured in BEBM serum-free medium (Lonza) with supplement bullet kit (EGF, hydrocortisone, bovine pituitary extract, transferrin, bovine insulin, triiodothyronine, epinephrine, retinoic acid), 2 mM L-glutamine, and 1 mM sodium pyruvate. PBMCs from CF patients and controls were cultured in RPMI-1640 (Hyclone) supplemented with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate (complete RPMI). THP1-XBlue cells were cultured in complete RPMI with the addition of zeocin (100 microg/ml) to select for cells expressing the SEAP NF-kappaB/AP-1 reporter. Prior to stimulation, bronchial epithelial cell lines were plated in coated [62] 96-well plates (BD Biosciences) at 3x104 cells/well unless indicated, and allowed to adhere overnight. Plates for S9 and IB3-1 stimulations were coated in a mixture of bovine serum albumin (100 microg/ml), fibronectin (10 microg/ml), and bovine collage type I (30 microg/ml) (BD Biosciences). Plates for NuLi-1 and CuFi-1 were coated with collagen type IV (60 microg/ml) (Sigma Aldrich). PBMCs were plated in 96-well plates at a density of 1.5x105 cells/well in 200 microl (7.5x105 cells/mL). THP-1 reporter cells were differentiated into a macrophage-like phenotype using 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich) for 24 hours at a density of 1x105 cells/well in 200 microl (5x105 cells/ml). Cells were washed with PBS and allowed to rest a further 42 hours prior to stimulation.", "output": {"json_structures": {}}, "schema": []}
{"input": "CF and control subject PBMCs\nThe diagnosis of CF was established by typical clinical features, increased sweat chloride concentrations (>60 mmol/l), and detection of CF-inducing mutations. All patients with CF were clinically stable at the time of blood donation, and we excluded any subjects who were receiving systemic corticosteroids due to potential immunomodulatory activity. Control samples were provided by healthy adult volunteers. In previously published work we have demonstrated that TLR-mediated inflammatory responses are stable in humans from birth to 60 years old [63], therefore did not age-match the CF patients and control subjects. Peripheral blood was collected in sodium heparin tubes (BD Biosciences) and PBMCs were isolated using density gradient centrifugation on Ficoll-Paque(TM) Plus (GE Healthcare). The layer containing PBMCs was isolated, washed twice in PBS and resuspended in complete RPMI. Cells were enumerated by trypan blue exclusion using the Countess automated cell counter (Invitrogen). For derivation of macrophages from monocytes, monocytes were allowed to adhere to plastic for 2 hours in RPMI 1640 after which non-adherent cells were removed. Monocytes were allowed to differentiate in RPMI 1640 supplemented with 10% human AB serum for 10 days.", "output": {"json_structures": {}}, "schema": []}
{"input": "Cell stimulation and cytokine quantification\nBronchial epithelial cells were plated and allowed to adhere overnight prior to stimulation. Bronchial epithelial cells were rested or primed with LPS for 5 hours and stimulated with live P. aeruginosa PAO1 or ATP for the times indicated. PBMCs and THP-1 reporter cells were either rested or primed with LPS (Invivogen) or heat-killed PAO1 overnight (16 hours). The next day the cells were challenged with live PAO1, PAO1deltaexsA, ATP (Invivogen), or Poly(dA:dT) (Sigma Aldrich) for the times indicated (see Fig. 1). For stimulations with Poly(dA:dT), lipofectamine LTX was used at a 1:1 (w:v) ratio of microg of DNA to microl of lipofectamine and was mixed 30 minutes prior to stimulation. The NF-kappaB inhibitor Bay11-7082 (Invivogen) was added to cultures 1 hour prior to priming. If no priming was involved, inhibitor was added 1 hour prior to inflammasome stimulation. The CFTR inhibitor CFTRinh172 (Sigma Aldrich) was added to cultures 18 hours prior to inflammasome stimulation. The caspase-1 inhibitor z-YVAD-fmk (Biovision) was added to cultures 1 hour prior to inflammasome stimulation. Supernatants were collected and stored at -20degreesC. Cytokines released into supernatants from PBMCs stimulated with inflammasome activators were quantified using sandwich ELISA (eBioscience).", "output": {"json_structures": {}}, "schema": []}
{"input": "Immunoblotting\n1x106 cells were seeded in 12-well plates, stimulated as indicated, and lysed in RIPA buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). Protein concentrations were determined by Bradford assay (Thermo Scientific). Lysates were resolved by electrophoreses on 10% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore). Blots were blocked for 1 hour at room temperature and probed overnight at 4degreesC for pro-IL-1beta (Santa Cruz), IkappaBalpha (Cell Signaling), or beta-actin (Cell Signaling). Blots were subsequently probed with fluorescently-labeled secondary antibodies, IRDye(R) 680 or 800CW (LI-COR Biosciences) for 1 hour. Both blocking and probing steps were carried out in tris-buffered saline (G Biosciences) containing 5% bovine serum albumin and 0.1% TWEEN 20 (Calbiochem). Blots were imaged on a LI-COR Odyssey infrared imaging system (LI-COR Biosciences) and quantified using the included analysis software.", "output": {"json_structures": {}}, "schema": []}
{"input": "Quantification of caspase-1 activity\nBronchial epithelial cells were plated in 6-well plates at 5x105 cells/well overnight. Cells were primed with LPS for 5 hours and stimulated with ATP for 1 hour or stimulated with live PAO1 for 3 hours. PBMCs were stimulated the same day as blood donation. PBMCs were seeded in a 96-well plate at a density of 4.5x105 cells/well (2.5x106 cells/ml), primed with LPS for 5 hours and then stimulated with ATP for 1 hour or Poly(dA:dT) for 3 hours or stimulated with live PAO1 for 3 hours. Caspase-1 activity was measured using FLICA (Immunochemistry Technologies), a cell-permeable fluorescent probe (FAM-YVAD-fmk) that binds active caspase-1. Cells were incubated 1 hour with FLICA at 37degreesC and stained with PE-Cy7-conjugated anti-CD14 antibodies (eBioscience) to identify monocytes. The gating strategy consisted of including live cells that were CD14 positive which were subsequently analyzed for the frequency of FLICA positive cells.", "output": {"json_structures": {}}, "schema": []}
{"input": "NF-kappaB/AP-1 Activity Assay\nSupernatants from THP-1 reporter cells were incubated with Quanti-Blue substrate (Invivogen) at 37degreesC and allowed to develop for 16-18 hours. Quanti-Blue contains a substrate for alkaline phosphatase and changes in the amount of NF-kappaB/AP-1 activity were quantified by optical density (lambda = 655) measured using a SpectraMax 384 Plus plate reader and SoftMax Pro software (Molecular Devices).", "output": {"json_structures": {}}, "schema": []}
{"input": "Bacterial strains\nP. aeruginosa laboratory strains PAO1 and the PAO1deltaexsA mutant were obtained from Dr. Robert Hancock. P. aeruginosa strains PAO1 and PAO1deltaexsA were grown from overnight cultures in Luria Bertani (LB) broth and LB+streptomycin (150 microg/ml) until mid-logarithmic phase. Cells were washed once in PBS and resuspended in PBS to an optical density of 0.5 (lambda = 600 nm). To prepare heat-killed bacteria, live PAO1 was resuspended in PBS to an optical density of 0.5 and heated at 60degreesC for 1 hour. For stimulations, live PAO1 was resuspended to an optical density of 0.5 in PBS and further diluted in culture medium prior to stimulation to achieve the desired multiplicity of infection. Heat-killed PAO1 was added in a volume equivalent to that used to achieve an MOI of 1 for live PAO1.", "output": {"json_structures": {}}, "schema": []}
{"input": "Statistics\nAll graphs display the mean +/- SEM and were generated with Prism 5 (Graphpad). Statistical significance was determined by performing one or two-way ANOVA and the Bonferroni post-test where applicable.", "output": {"json_structures": {}}, "schema": []}
{"input": "Cell stimulation and inhibitor schedule.\nSchedule outlines the timing of inhibitor addition and priming in relation to inflammasome stimulation (t = 0) for THP-1 reporter and PBMC cytokine quantification experiments. Inhibitor treatments and stimulations were carried out as described in the Materials and Methods section.", "output": {"json_structures": {}}, "schema": []}
{"input": "Airway epithelial cells do not significantly contribute to IL-1beta production in response to inflammasome stimuli.\nControl cell lines ((A) S9, (C) NuLi-1) and their corresponding CF cell lines ((B) IB3-1, and (D) CuFi-1) cells were stimulated with P. aeruginosa (MOI = 10), ATP (5 mM), or IL-1beta (10 ng/ml), for the indicated times (n = 3 individual experiments). Cells were primed with LPS (100 ng/ml) for 4 hours where appropriate. Cell culture supernatants were assayed for IL-1beta and IL-8 production by ELISA. Insert shows IL-8 secretion in response to stimulation with IL-1beta (10 ng/ml).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 68, "end": 78}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 59, "end": 67}]}, {"trigger": {"text": "production", "start": 498, "end": 508}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 480, "end": 488}]}], "positive regulation": [{"trigger": {"text": "contribute", "start": 45, "end": 55}, "arguments": [{"role": "Theme", "text": "in response to", "start": 79, "end": 93}]}, {"trigger": {"text": "in response to", "start": 79, "end": 93}, "arguments": [{"role": "Theme", "text": "production", "start": 68, "end": 78}]}]}}, "schema": []}
{"input": "Airway cells do not strongly upregulate caspase-1 activity in response to inflammasome stimuli.\nS9 and IB3-1 cells were examined for caspase-1 activation following inflammasome stimulation with P. aeruginosa (MOI = 10) and ATP (5 mM). Cells were primed with LPS for 5 hours where appropriate. A representative histogram of % caspase-1-active cells is shown in (A) and the averaged values are shown in (B) (n = 3 separate experiments). (C) IB3-1 cells (5x104 cells/well) were pre-treated for 1 hour with increasing concentrations of z-YVAD-fmk (10-30 microM) prior to stimulation with P. aeruginosa (MOI = 50). Cell culture supernatants were collected after 6 hours and assayed for IL-6 by ELISA (n = 3).", "output": {"json_structures": {}}, "schema": []}
{"input": "PBMCs from CF patients and controls show similar increases in caspase-1 activity upon inflammasome activation.\nPBMCs from CF patients (n = 6) and healthy controls (n = 6) were primed with LPS (10 ng/ml) for 5 hours prior to stimulation with ATP (5 mM) for 1 hour or Poly(dA:dT) (1 microg/ml) for 3 hours. PBMCs were stimulated with P. aeruginosa strain PAO1 for 3 hours. A representative histogram of the % caspase-1-active cells is shown in (A) with the averaged values shown in (B).", "output": {"json_structures": {}}, "schema": []}
{"input": "PBMCs from CF patients do not produce increased amounts of IL-1beta.\nPBMCs from CF patients (n = 17-20) and healthy controls (n = 15-19) were primed with LPS (10 ng/ml) overnight and stimulated with P. aeruginosa PAO1 (MOI = 1), P. aeruginosa PAO1 lacking exsA (MOI = 1), ATP (5 mM), or Poly(dA:dT) (1 microg/ml) for 24 hours. P. aeruginosa lacking exsA was used as a type III secretion control in comparison with wild-type P. aeruginosa. Supernatants were assayed for (A) IL-1beta and (B) IL-8.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "produce", "start": 30, "end": 37}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 59, "end": 67}]}], "negative regulation": [{"trigger": {"text": "lacking", "start": 248, "end": 255}, "arguments": [{"role": "Theme", "text": "exsA", "start": 256, "end": 260}]}, {"trigger": {"text": "lacking", "start": 341, "end": 348}, "arguments": [{"role": "Theme", "text": "exsA", "start": 349, "end": 353}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 38, "end": 47}, "arguments": [{"role": "Theme", "text": "produce", "start": 30, "end": 37}]}]}}, "schema": []}
{"input": "NF-kappaB activation potentiates the degree of IL-1beta production and secretion upon inflammasome activation.\nTHP-1 reporter cells were primed overnight with heat-killed P. aeruginosa and stimulated the next day with live P. aeruginosa or additional heat-killed P. aeruginosa for the times indicated. Cell culture supernatants were assayed for (A) NF-kappaB/AP-1 activity, (B) IL-8, and (C) IL-1beta secretion (n = 3-6 experiments). Using the same stimulation method, THP-1 reporter cells were treated with Bay11-7082 (20 microM) for 1 hour prior to priming with heat-killed PAO1 or live PAO1. Supernatants were assayed at 24 hours for (D) NF-kappaB/AP-1 activity, (E) IL-8, and (F) IL-1beta secretion (n = 3-5). PBMCs from CF patients (n = 11-15) and controls (n = 10-13) were treated with z-YVAD-fmk (20 microM) or Bay11-7082 (10 microM) and stimulated with live PAO1 (MOI = 1), ATP (5 mM), or Poly(dA:dT) (1 microg/ml) according to the schedule in Figure 1. (G) IL-1beta and (H) IL-8 levels were measured at 24 hours. Statistical analysis was performed using two way ANOVA with Bonferroni correction for multiple comparisons. *, **, and *** signify P<0.05, 0.01, and 0.001.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 56, "end": 66}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 47, "end": 55}]}], "localization": [{"trigger": {"text": "secretion", "start": 71, "end": 80}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 47, "end": 55}]}, {"trigger": {"text": "secretion", "start": 401, "end": 410}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 392, "end": 400}]}, {"trigger": {"text": "secretion", "start": 693, "end": 702}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 684, "end": 692}]}], "positive regulation": [{"trigger": {"text": "potentiates", "start": 21, "end": 32}, "arguments": [{"role": "Theme", "text": "production", "start": 56, "end": 66}]}, {"trigger": {"text": "potentiates", "start": 21, "end": 32}, "arguments": [{"role": "Theme", "text": "secretion", "start": 71, "end": 80}]}, {"trigger": {"text": "upon", "start": 81, "end": 85}, "arguments": [{"role": "Theme", "text": "potentiates", "start": 21, "end": 32}]}]}}, "schema": []}
{"input": "Bay11-7082 inhibits pro-IL-1beta production in response to P. aeruginosa.\nPMA-differentiated THP-1 cells were treated with 10 microM CFTRinh172 or 20 microM Bay11-7082 and harvested after (A) 4 hours (n = 3) or (B) 0.5, 1, and 1.5 hours (n = 3) stimulation with PAO1. One representative blot is shown with a graph of the averaged fluorescence intensity values over 3 experiments.", "output": {"json_structures": {}}, "schema": []}
{"input": "Disruption of CFTR activity does not increase IL-1beta production in PBMCs or macrophages.\nPBMCs from CF patients (n = 15) and controls (n = 13) were treated with CFTRinh172 (10 microM) for 18 hours prior to stimulation with live PAO1 (MOI = 1). (A) IL-1beta and (B) IL-8 production was measured at 24 hours. (C) Monocytes from controls (n = 3) were differentiated into macrophages. Macrophages were treated with CFTRinh172, stimulated as per monocytes, and measured for IL-1beta production at 24 hours. THP-1 reporter cells were treated with CFTRinh172 24 hours prior to stimulation with PAO1 and measured for (D) IL-1beta secretion, (E) IL-8, and (F) NF-kappaB/AP-1 activity at 24 hours (n = 4).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 55, "end": 65}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 46, "end": 54}]}, {"trigger": {"text": "production", "start": 272, "end": 282}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 250, "end": 258}]}, {"trigger": {"text": "production", "start": 480, "end": 490}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 471, "end": 479}]}], "localization": [{"trigger": {"text": "secretion", "start": 624, "end": 633}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 615, "end": 623}]}], "positive regulation": [{"trigger": {"text": "increase", "start": 37, "end": 45}, "arguments": [{"role": "Theme", "text": "production", "start": 55, "end": 65}]}]}}, "schema": []}
{"input": "Inflammasome Activators.", "output": {"json_structures": {}}, "schema": []}