{"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. \n", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 725, "end": 735}, "arguments": [{"role": "Theme", "text": "Id2", "start": 713, "end": 716}]}, {"trigger": {"text": "expression", "start": 725, "end": 735}, "arguments": [{"role": "Theme", "text": "Id3", "start": 721, "end": 724}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 583, "end": 598}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 602, "end": 607}]}, {"trigger": {"text": "phosphorylation", "start": 583, "end": 598}, "arguments": [{"role": "Theme", "text": "5", "start": 608, "end": 609}]}, {"trigger": {"text": "phosphorylation", "start": 583, "end": 598}, "arguments": [{"role": "Theme", "text": "8", "start": 610, "end": 611}]}, {"trigger": {"text": "phosphorylation", "start": 631, "end": 646}, "arguments": [{"role": "Theme", "text": "Smad", "start": 626, "end": 630}]}, {"trigger": {"text": "phosphorylation", "start": 1329, "end": 1344}, "arguments": [{"role": "Theme", "text": "Smad1", "start": 1348, "end": 1353}]}, {"trigger": {"text": "phosphorylation", "start": 1329, "end": 1344}, "arguments": [{"role": "Theme", "text": "5", "start": 1354, "end": 1355}]}, {"trigger": {"text": "phosphorylation", "start": 1329, "end": 1344}, "arguments": [{"role": "Theme", "text": "8", "start": 1356, "end": 1357}]}], "positive regulation": [{"trigger": {"text": "upregulation", "start": 85, "end": 97}, "arguments": [{"role": "Theme", "text": "Id1", "start": 101, "end": 104}]}, {"trigger": {"text": "induced", "start": 575, "end": 582}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 561, "end": 566}, {"role": "Theme", "text": "phosphorylation", "start": 583, "end": 598}]}, {"trigger": {"text": "followed", "start": 651, "end": 659}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 631, "end": 646}, {"role": "Theme", "text": "upregulation", "start": 663, "end": 675}]}, {"trigger": {"text": "upregulation", "start": 663, "end": 675}, "arguments": [{"role": "Theme", "text": "Id1", "start": 679, "end": 682}]}, {"trigger": {"text": "upregulation", "start": 663, "end": 675}, "arguments": [{"role": "Theme", "text": "Id1", "start": 692, "end": 695}]}, {"trigger": {"text": "upregulate", "start": 1189, "end": 1199}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1200, "end": 1205}]}, {"trigger": {"text": "induced", "start": 1321, "end": 1328}, "arguments": [{"role": "Cause", "text": "BMP-6", "start": 1280, "end": 1285}, {"role": "Theme", "text": "phosphorylation", "start": 1329, "end": 1344}]}, {"trigger": {"text": "followed", "start": 1358, "end": 1366}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 1329, "end": 1344}, {"role": "Theme", "text": "upregulation", "start": 1373, "end": 1385}]}, {"trigger": {"text": "upregulation", "start": 1373, "end": 1385}, "arguments": [{"role": "Theme", "text": "Id1", "start": 1389, "end": 1392}]}], "regulation": [{"trigger": {"text": "affected", "start": 744, "end": 752}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 631, "end": 646}, {"role": "Theme", "text": "expression", "start": 725, "end": 735}]}]}}, "schema": []} {"input": "Background\nMembers 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. \n", "output": {"json_structures": {"binding": [{"trigger": {"text": "ligation", "start": 1549, "end": 1557}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1498, "end": 1503}]}, {"trigger": {"text": "heterodimerzation", "start": 1562, "end": 1579}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1498, "end": 1503}]}, {"trigger": {"text": "ligation", "start": 1784, "end": 1792}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1755, "end": 1760}, {"role": "Theme2", "text": "Act-RIA", "start": 1817, "end": 1824}]}, {"trigger": {"text": "ligation", "start": 1784, "end": 1792}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1755, "end": 1760}, {"role": "Theme2", "text": "BMP-RIA", "start": 1826, "end": 1833}]}, {"trigger": {"text": "ligation", "start": 1784, "end": 1792}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1755, "end": 1760}, {"role": "Theme2", "text": "BMP-RIB", "start": 1839, "end": 1846}]}, {"trigger": {"text": "ligation", "start": 1784, "end": 1792}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1755, "end": 1760}, {"role": "Theme2", "text": "BMP-RII", "start": 1873, "end": 1880}]}, {"trigger": {"text": "ligation", "start": 1784, "end": 1792}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1755, "end": 1760}, {"role": "Theme2", "text": "Act-RIIA", "start": 1882, "end": 1890}]}, {"trigger": {"text": "ligation", "start": 1784, "end": 1792}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1755, "end": 1760}, {"role": "Theme2", "text": "Act-RIIB", "start": 1895, "end": 1903}]}, {"trigger": {"text": "form complexes", "start": 2009, "end": 2023}, "arguments": [{"role": "Theme", "text": "Smad-1", "start": 1962, "end": 1968}, {"role": "Theme2", "text": "Smad4", "start": 2042, "end": 2047}]}, {"trigger": {"text": "form complexes", "start": 2009, "end": 2023}, "arguments": [{"role": "Theme", "text": "Smad-5", "start": 1970, "end": 1976}, {"role": "Theme2", "text": "Smad4", "start": 2042, "end": 2047}]}, {"trigger": {"text": "form complexes", "start": 2009, "end": 2023}, "arguments": [{"role": "Theme", "text": "Smad-8", "start": 1982, "end": 1988}, {"role": "Theme2", "text": "Smad4", "start": 2042, "end": 2047}]}], "gene expression": [{"trigger": {"text": "expression", "start": 2402, "end": 2412}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 2434, "end": 2439}]}, {"trigger": {"text": "producing", "start": 2623, "end": 2632}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 2633, "end": 2638}]}], "localization": [{"trigger": {"text": "translocated", "start": 2057, "end": 2069}, "arguments": [{"role": "Theme", "text": "Smad-1", "start": 1962, "end": 1968}, {"role": "ToLoc", "text": "nucleus", "start": 2079, "end": 2086}]}, {"trigger": {"text": "translocated", "start": 2057, "end": 2069}, "arguments": [{"role": "Theme", "text": "Smad-5", "start": 1970, "end": 1976}, {"role": "ToLoc", "text": "nucleus", "start": 2079, "end": 2086}]}, {"trigger": {"text": "translocated", "start": 2057, "end": 2069}, "arguments": [{"role": "Theme", "text": "Smad-8", "start": 1982, "end": 1988}, {"role": "ToLoc", "text": "nucleus", "start": 2079, "end": 2086}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1923, "end": 1938}, "arguments": [{"role": "Theme", "text": "Smad-1", "start": 1962, "end": 1968}]}, {"trigger": {"text": "phosphorylation", "start": 1923, "end": 1938}, "arguments": [{"role": "Theme", "text": "Smad-5", "start": 1970, "end": 1976}]}, {"trigger": {"text": "phosphorylation", "start": 1923, "end": 1938}, "arguments": [{"role": "Theme", "text": "Smad-8", "start": 1982, "end": 1988}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 1347, "end": 1356}, "arguments": [{"role": "Theme", "text": "BMP-6", "start": 1327, "end": 1332}]}, {"trigger": {"text": "lead", "start": 1911, "end": 1915}, "arguments": [{"role": "Cause", "text": "ligation", "start": 1784, "end": 1792}, {"role": "Theme", "text": "phosphorylation", "start": 1923, "end": 1938}]}, {"trigger": {"text": "induced", "start": 2440, "end": 2447}, "arguments": [{"role": "Cause", "text": "expression", "start": 2402, "end": 2412}, {"role": "Theme", "text": "regulation", "start": 2506, "end": 2516}]}], "regulation": [{"trigger": {"text": "regulation", "start": 2506, "end": 2516}, "arguments": [{"role": "Theme", "text": "Id1", "start": 2537, "end": 2540}]}, {"trigger": {"text": "regulation", "start": 2506, "end": 2516}, "arguments": [{"role": "Theme", "text": "Id4", "start": 2541, "end": 2544}]}]}}, "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 /= 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. \n", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "secretion", "start": 407, "end": 416}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 402, "end": 406}]}], "positive regulation": [{"trigger": {"text": "high", "start": 397, "end": 401}, "arguments": [{"role": "Theme", "text": "secretion", "start": 407, "end": 416}]}]}}, "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. \n", "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. \n", "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). \n", "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. \n", "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. \n", "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. \n", "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. \n", "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. \n", "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. \n", "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. \n", "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), \n", "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. \n", "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. \n", "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). \n", "output": {"json_structures": {}}, "schema": []} {"input": "Supporting Information\nPhenotype of In Vitro Differentiated T Cells\nAfter two round of differentiation cultures, T cells were stimulated by plate-immobilized anti-CD3/CD28 and 3H-thymidine incorporation as measurement of proliferation was analyzed after 3 d of culture (A). In parallel, T cells were analyzed for Treg relevant surface receptor expression as indicated on the x-axis (B). \n(1.0 MB AI). \nClick here for additional data file. \nIn Vivo Treatment of Mice with IL-4 Antibody-Cytokine Complexes\nB6 mice were given every other day ip injections of phosphate-buffered saline (PBS), recombinant mouse IL-4 (rmIL-4), anti-IL-4 mAb (anti-IL-4 mAb, 11B11, or MAB404), or a mixture of rmIL-4 plus anti-IL-4 mAbs (11B11 or MAB404). Mice were analyzed on day 7 by flow cytometry for CD3, CD4, and CD25 expression. Shown is CD25 versus CD4 expression in CD3+ CD4+ spleen cells (A-F). Numbers indicate percentages of CD4+ CD25high CD3+ cells. Total cell counts (G) of CD4+ CD25high cells in spleen from mice in (A-F) are shown as mean +/- SD. The data are representative of three independent experiments. \n(369 KB AI). \nClick here for additional data file. \nEffect of IL-4 on Already Existing Natural or Inducible Treg Cells\n(A) CD4+CD25high nTreg cells were FACS-sorted and activated with plate-bound anti-CD3/CD28 plus IL-2 during 3 d and in the presence or absence of IL-4 (100 ng/ml) and harvested for real-time PCR analysis. The results shown represent the mean +/- SD of three independent experiments. \n(B) iTreg cells were induced in vitro. FOXP3 espression was assessed by real-time PCR analysis in resting cells, in cells re-stimulated with plate-bound anti-CD3/CD28, with or without TGF-beta, plus IL-2 during 3 d and in the presence (black bar) or absence (white bar) of IL-4 (100 ng/ml). \n(C) Activation dramatically increases CD4+CD25+ Treg cells suppressive capacity of CD4+CD25+ nTreg cells. CD4+CD25+ nTreg cells were preactivated during 2 d in the presence or absence of an increasing IL-4 concentration. After vigorous washing, their suppressive capacity on responder CD4+CD25- was tested. IL-4 pretreatment did not affect the suppressive capacity of FACS-sorted CD4+CD25high cells. 1 x 104 CD4+CD25+ nTreg cells were added to 5 x 104 CD4+CD25- and 5 x 104 irradiated PBMCs. The results are representative of three independent experiments. \n(269 KB AI). \nClick here for additional data file. \nSchematic Structure of the FOXP3 Gene and Location of the GATA3 Sites\nThe scheme shows the location of the 11 exons spaced by a large intron (6000 bp) from the 5'untranslated region (UTR). Human, murine, and rat sequences are aligned and transcription start site (TSS) is indicated with an arrow. \n(529 KB AI). \nClick here for additional data file. \n", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 802, "end": 812}, "arguments": [{"role": "Theme", "text": "CD3", "start": 783, "end": 786}]}, {"trigger": {"text": "expression", "start": 802, "end": 812}, "arguments": [{"role": "Theme", "text": "CD4", "start": 788, "end": 791}]}, {"trigger": {"text": "expression", "start": 802, "end": 812}, "arguments": [{"role": "Theme", "text": "CD25", "start": 797, "end": 801}]}, {"trigger": {"text": "expression", "start": 839, "end": 849}, "arguments": [{"role": "Theme", "text": "CD25", "start": 823, "end": 827}]}, {"trigger": {"text": "expression", "start": 839, "end": 849}, "arguments": [{"role": "Theme", "text": "CD4", "start": 835, "end": 838}]}, {"trigger": {"text": "espression", "start": 1552, "end": 1562}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 1546, "end": 1551}]}], "positive regulation": [{"trigger": {"text": "increasing", "start": 1989, "end": 1999}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 2000, "end": 2004}]}]}}, "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. \n", "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. \n", "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. \n", "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. \n", "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": 404, "end": 412}, "arguments": [{"role": "Theme", "text": "GATA3", "start": 388, "end": 393}]}, {"trigger": {"text": "staining", "start": 404, "end": 412}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 398, "end": 403}]}], "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 /=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. \nIn 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). \nImmunohistochemistry.\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). \nIn 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. \nCloning 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). \nTransfections 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. \nRNA 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. \nRNA 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. \nReal-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. \nFlow 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. \nWestern 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). \nPull-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. \nPromoter 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). \nChIP.\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). \nQuantification 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). \nOnline 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. \n", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 4094, "end": 4101}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 4088, "end": 4093}]}, {"trigger": {"text": "binding", "start": 4194, "end": 4201}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 4188, "end": 4193}]}], "gene expression": [{"trigger": {"text": "expression", "start": 595, "end": 605}, "arguments": [{"role": "Theme", "text": "Foxp3-GFP", "start": 585, "end": 594}]}, {"trigger": {"text": "expressed", "start": 4894, "end": 4903}, "arguments": [{"role": "Theme", "text": "GFP", "start": 4890, "end": 4893}]}, {"trigger": {"text": "expression", "start": 6561, "end": 6571}, "arguments": [{"role": "Theme", "text": "RUNX", "start": 6556, "end": 6560}]}, {"trigger": {"text": "overexpression", "start": 8581, "end": 8595}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 8599, "end": 8604}]}, {"trigger": {"text": "overexpression", "start": 8581, "end": 8595}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 8608, "end": 8613}]}, {"trigger": {"text": "transfected", "start": 8813, "end": 8824}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 8864, "end": 8869}]}, {"trigger": {"text": "transfected", "start": 8813, "end": 8824}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 8873, "end": 8878}]}, {"trigger": {"text": "expression", "start": 8965, "end": 8975}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 8959, "end": 8964}]}, {"trigger": {"text": "expression", "start": 12089, "end": 12099}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 12083, "end": 12088}]}, {"trigger": {"text": "protein level", "start": 12909, "end": 12922}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 12877, "end": 12882}]}, {"trigger": {"text": "protein level", "start": 12909, "end": 12922}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 12887, "end": 12892}]}, {"trigger": {"text": "transfected", "start": 13866, "end": 13877}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 13883, "end": 13888}]}, {"trigger": {"text": "transfected", "start": 13866, "end": 13877}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 13892, "end": 13897}]}, {"trigger": {"text": "transfected", "start": 15087, "end": 15098}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 15104, "end": 15109}]}, {"trigger": {"text": "transfected", "start": 15087, "end": 15098}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 15113, "end": 15118}]}, {"trigger": {"text": "expression", "start": 19233, "end": 19243}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 19227, "end": 19232}]}, {"trigger": {"text": "overexpression", "start": 19627, "end": 19641}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 19645, "end": 19650}]}, {"trigger": {"text": "overexpression", "start": 19627, "end": 19641}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 19655, "end": 19660}]}, {"trigger": {"text": "expression", "start": 19753, "end": 19763}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 19747, "end": 19752}]}], "localization": [{"trigger": {"text": "secretion", "start": 18664, "end": 18673}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 18611, "end": 18615}]}, {"trigger": {"text": "secretion", "start": 18664, "end": 18673}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 18617, "end": 18621}]}, {"trigger": {"text": "secretion", "start": 18664, "end": 18673}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 18623, "end": 18627}]}, {"trigger": {"text": "secretion", "start": 18664, "end": 18673}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 18629, "end": 18634}]}, {"trigger": {"text": "secretion", "start": 18664, "end": 18673}, "arguments": [{"role": "Theme", "text": "IL-13", "start": 18636, "end": 18641}]}, {"trigger": {"text": "secretion", "start": 18664, "end": 18673}, "arguments": [{"role": "Theme", "text": "IL-17", "start": 18643, "end": 18648}]}, {"trigger": {"text": "secretion", "start": 18664, "end": 18673}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 18654, "end": 18663}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 388, "end": 397}, "arguments": [{"role": "Theme", "text": "Rag", "start": 384, "end": 387}]}, {"trigger": {"text": "depletion", "start": 2437, "end": 2446}, "arguments": [{"role": "Theme", "text": "CD45RO", "start": 2430, "end": 2436}]}, {"trigger": {"text": "siRNA", "start": 5822, "end": 5827}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 5806, "end": 5811}]}, {"trigger": {"text": "siRNA", "start": 5822, "end": 5827}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 5816, "end": 5821}]}, {"trigger": {"text": "siRNAs", "start": 9337, "end": 9343}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 9348, "end": 9353}]}, {"trigger": {"text": "siRNAs", "start": 9407, "end": 9413}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 9418, "end": 9423}]}, {"trigger": {"text": "knockdown", "start": 11794, "end": 11803}, "arguments": [{"role": "Theme", "text": "RUNX", "start": 11789, "end": 11793}]}, {"trigger": {"text": "decreased", "start": 19128, "end": 19137}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 19154, "end": 19158}, {"role": "Cause", "text": "knockdown", "start": 19203, "end": 19212}]}, {"trigger": {"text": "decreased", "start": 19128, "end": 19137}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 19154, "end": 19158}, {"role": "Cause", "text": "decreased", "start": 19217, "end": 19226}]}, {"trigger": {"text": "knockdown", "start": 19203, "end": 19212}, "arguments": [{"role": "Theme", "text": "expression", "start": 19233, "end": 19243}]}, {"trigger": {"text": "decreased", "start": 19217, "end": 19226}, "arguments": [{"role": "Theme", "text": "expression", "start": 19233, "end": 19243}, {"role": "Cause", "text": "knockdown", "start": 19288, "end": 19297}]}, {"trigger": {"text": "knockdown", "start": 19288, "end": 19297}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 19272, "end": 19277}]}, {"trigger": {"text": "knockdown", "start": 19288, "end": 19297}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 19282, "end": 19287}]}, {"trigger": {"text": "siRNA", "start": 19430, "end": 19435}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 19414, "end": 19419}]}, {"trigger": {"text": "siRNA", "start": 19430, "end": 19435}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 19424, "end": 19429}]}], "positive regulation": [{"trigger": {"text": "mediated", "start": 11780, "end": 11788}, "arguments": [{"role": "Theme", "text": "knockdown", "start": 11794, "end": 11803}]}, {"trigger": {"text": "induction", "start": 19000, "end": 19009}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 19037, "end": 19041}]}, {"trigger": {"text": "mediated", "start": 19194, "end": 19202}, "arguments": [{"role": "Theme", "text": "knockdown", "start": 19203, "end": 19212}]}, {"trigger": {"text": "induction", "start": 19594, "end": 19603}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 19607, "end": 19612}, {"role": "Cause", "text": "overexpression", "start": 19627, "end": 19641}]}], "regulation": [{"trigger": {"text": "effect", "start": 8571, "end": 8577}, "arguments": [{"role": "Cause", "text": "overexpression", "start": 8581, "end": 8595}, {"role": "Theme", "text": "FOXP3", "start": 8617, "end": 8622}]}, {"trigger": {"text": "effect", "start": 19740, "end": 19746}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 19714, "end": 19718}, {"role": "Theme", "text": "expression", "start": 19753, "end": 19763}]}, {"trigger": {"text": "effect", "start": 19740, "end": 19746}, "arguments": [{"role": "Cause", "text": "IFN-gamma", "start": 19723, "end": 19732}, {"role": "Theme", "text": "expression", "start": 19753, "end": 19763}]}], "transcription": [{"trigger": {"text": "mRNA", "start": 11179, "end": 11183}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 11163, "end": 11168}]}, {"trigger": {"text": "mRNA", "start": 11179, "end": 11183}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 11173, "end": 11178}]}, {"trigger": {"text": "mRNA", "start": 11766, "end": 11770}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 11760, "end": 11765}]}, {"trigger": {"text": "mRNA", "start": 19037, "end": 19041}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 19013, "end": 19018}]}, {"trigger": {"text": "mRNA", "start": 19037, "end": 19041}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 19020, "end": 19025}]}, {"trigger": {"text": "mRNA", "start": 19037, "end": 19041}, "arguments": [{"role": "Theme", "text": "FOXP3", "start": 19031, "end": 19036}]}, {"trigger": {"text": "mRNA", "start": 19154, "end": 19158}, "arguments": [{"role": "Theme", "text": "RUNX1", "start": 19138, "end": 19143}]}, {"trigger": {"text": "mRNA", "start": 19154, "end": 19158}, "arguments": [{"role": "Theme", "text": "RUNX3", "start": 19148, "end": 19153}]}]}}, "schema": []} {"input": "Supplementary Material\n[Supplemental Material]\n", "output": {"json_structures": {}}, "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. \n", "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. \n", "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 \n", "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. \n", "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. \n", "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. \n", "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": "[Supplemental Material]\n", "output": {"json_structures": {}}, "schema": []} {"input": "Involvement of adenylate cyclase and p70(S6)-kinase activation in IL-10 up-regulation in human monocytes by gp41 envelope protein of human immunodeficiency virus type 1. \nOur previous results show that recombinant gp41 (aa565-647), the extracellular domain of HIV-1 transmembrane glycoprotein, stimulates interleukin-10 (IL-10) production in human monocytes. The signal cascade transducing this effect is not yet clear. In this study, we examined whether gp41-induced IL-10 up-regulation is mediated by the previously described synergistic activation of cAMP and NF-kappaB pathways. gp41 induced cAMP accumulation in monocytes in a time- and concentration-dependent manner and the adenylate cyclase inhibitor SQ 22536 suppressed gp41-induced IL-10 production in monocytes. In contrast, gp41 failed to stimulate NF-kappaB binding activity in as much as no NF-kappaB bound to the main NF-kappaB-binding site 2 of the IL-10 promoter after addition of gp41. We also examined the involvement of other signal transduction pathways. Specific inhibitors of p70(S6)-kinase (rapamycin), and Gi protein (pertussis toxin), prevented induction of IL-10 production by gp41 in monocytes, while inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase) (wortmannin) and mitogen-activated protein kinase (MAPK) pathway (PD 98059) did not. Thus HIV-1 gp41-induced IL-10 up-regulation in monocytes may not involve NF-kappaB, MAPK, or PI 3-kinase activation, but rather may operate through activation of adenylate cyclase and pertussis-toxin-sensitive Gi/Go protein to effect p70(S6)-kinase activation. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "bound", "start": 865, "end": 870}, "arguments": [{"role": "Site", "text": "main NF-kappaB-binding site 2", "start": 878, "end": 907}, {"role": "Theme", "text": "IL-10", "start": 915, "end": 920}]}], "gene expression": [{"trigger": {"text": "production", "start": 328, "end": 338}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 321, "end": 326}]}, {"trigger": {"text": "production", "start": 748, "end": 758}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 742, "end": 747}]}, {"trigger": {"text": "production", "start": 1140, "end": 1150}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1134, "end": 1139}]}], "negative regulation": [{"trigger": {"text": "suppressed", "start": 718, "end": 728}, "arguments": [{"role": "Theme", "text": "induced", "start": 734, "end": 741}]}, {"trigger": {"text": "prevented", "start": 1111, "end": 1120}, "arguments": [{"role": "Theme", "text": "induction", "start": 1121, "end": 1130}]}], "positive regulation": [{"trigger": {"text": "activation", "start": 52, "end": 62}, "arguments": [{"role": "Theme", "text": "p70(S6)-kinase", "start": 37, "end": 51}]}, {"trigger": {"text": "up-regulation", "start": 72, "end": 85}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 66, "end": 71}, {"role": "Cause", "text": "gp41", "start": 108, "end": 112}]}, {"trigger": {"text": "stimulates", "start": 294, "end": 304}, "arguments": [{"role": "Theme", "text": "production", "start": 328, "end": 338}]}, {"trigger": {"text": "induced", "start": 460, "end": 467}, "arguments": [{"role": "Cause", "text": "gp41", "start": 455, "end": 459}, {"role": "Theme", "text": "up-regulation", "start": 474, "end": 487}]}, {"trigger": {"text": "up-regulation", "start": 474, "end": 487}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 468, "end": 473}]}, {"trigger": {"text": "mediated", "start": 491, "end": 499}, "arguments": [{"role": "Theme", "text": "induced", "start": 460, "end": 467}]}, {"trigger": {"text": "induced", "start": 734, "end": 741}, "arguments": [{"role": "Cause", "text": "gp41", "start": 729, "end": 733}, {"role": "Theme", "text": "production", "start": 748, "end": 758}]}, {"trigger": {"text": "induction", "start": 1121, "end": 1130}, "arguments": [{"role": "Theme", "text": "production", "start": 1140, "end": 1150}, {"role": "Cause", "text": "gp41", "start": 1154, "end": 1158}]}, {"trigger": {"text": "induced", "start": 1342, "end": 1349}, "arguments": [{"role": "Cause", "text": "gp41", "start": 1337, "end": 1341}, {"role": "Theme", "text": "up-regulation", "start": 1356, "end": 1369}]}, {"trigger": {"text": "up-regulation", "start": 1356, "end": 1369}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1350, "end": 1355}]}, {"trigger": {"text": "operate", "start": 1458, "end": 1465}, "arguments": [{"role": "Theme", "text": "induced", "start": 1342, "end": 1349}]}], "regulation": [{"trigger": {"text": "Involvement", "start": 0, "end": 11}, "arguments": [{"role": "Cause", "text": "activation", "start": 52, "end": 62}, {"role": "Theme", "text": "up-regulation", "start": 72, "end": 85}]}]}}, "schema": []} {"input": "Constitutive activation of NF-kappaB in primary adult T-cell leukemia cells. \nHuman T-cell leukemia virus type I (HTLV-I) is an etiologic agent of adult T-cell leukemia (ATL). The viral protein Tax induces the activation and nuclear translocalization of transcription factor NF-kappaB, which is proposed to play a crucial role in the transformation of T cells by HTLV-I. However, the HTLV-I genes including Tax are not expressed significantly in primary leukemic cells from ATL patients. In this study, we examined the basis for NF-kappaB activation in freshly isolated leukemic cells from ATL patients. We found that leukemic cells from ATL patients, like HTLV-I-infected T-cell lines, display constitutive NF-kappaB DNA binding activity and increased degradation of IkappaBalpha (an inhibitor of NF-kappaB). Whereas the NF-kappaB binding activity in Tax-expressing T-cell lines consisted mostly of p50/c-Rel, fresh ATL samples contained p50/p50 and p50/p65 heterodimers. One T-cell line derived from ATL leukemic cells, TL-Om1, displayed constitutive NF-kappaB activity, as well as enhanced degradation of IkappaBalpha, despite the lack of detectable Tax expression. Interestingly, the NF-kappaB in TL-Om1 consists of p50/p50 and p50/p65 like that in fresh primary leukemic cells. Our results suggest that activation of NF-kappaB occurs through a Tax-independent mechanism in leukemic cells of ATL patients, possibly due to differential NF-kappaB subunit activation. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressed", "start": 419, "end": 428}, "arguments": [{"role": "Theme", "text": "Tax", "start": 407, "end": 410}]}, {"trigger": {"text": "expression", "start": 1157, "end": 1167}, "arguments": [{"role": "Theme", "text": "Tax", "start": 1153, "end": 1156}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 743, "end": 752}, "arguments": [{"role": "Theme", "text": "degradation", "start": 753, "end": 764}]}, {"trigger": {"text": "enhanced", "start": 1084, "end": 1092}, "arguments": [{"role": "Theme", "text": "degradation", "start": 1093, "end": 1104}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 753, "end": 764}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 768, "end": 780}]}, {"trigger": {"text": "degradation", "start": 1093, "end": 1104}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1108, "end": 1120}]}]}}, "schema": []} {"input": "Apoptosis-resistant T cells have a deficiency in NF-kappaB-mediated induction of Fas ligand transcription. \nApoptosis induced through the TCR in CD4+ T cells is mostly mediated by the inducible expression of Fas ligand (FasL) as a primary event leading to the commitment to death. To gain a better understanding of the transcriptional events that regulate this expression, we took advantage of our previously described mutant Jurkat cells. These cells are deficient in FasL expression and apoptosis induced upon TCR triggering, although their cytokine (IL-2 and IFN-gamma) production is normal. Here we show that both a FasL- and a consensus NF-kappaB- reporter construct are inefficiently induced in these cells compared to wild-type cells. In addition, we demonstrate that the inducible transcriptional activity of the FasL reporter is abolished by specific inhibitors of NF-kappaB activation. Thus, we could trace the deficit of the mutant cells to an inefficient NF-kappaB activation, evidencing a relevant role for NF-kappaB in the regulation of FasL expression in activated T cells. Furthermore, our results suggest that the induction of FasL versus cytokine gene expression is differentially sensitive to NF-kappaB deprivation. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 194, "end": 204}, "arguments": [{"role": "Theme", "text": "FasL", "start": 220, "end": 224}]}, {"trigger": {"text": "expression", "start": 474, "end": 484}, "arguments": [{"role": "Theme", "text": "FasL", "start": 469, "end": 473}]}, {"trigger": {"text": "production", "start": 573, "end": 583}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 553, "end": 557}]}, {"trigger": {"text": "production", "start": 573, "end": 583}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 562, "end": 571}]}, {"trigger": {"text": "expression", "start": 1056, "end": 1066}, "arguments": [{"role": "Theme", "text": "FasL", "start": 1051, "end": 1055}]}, {"trigger": {"text": "expression", "start": 1170, "end": 1180}, "arguments": [{"role": "Theme", "text": "FasL", "start": 1144, "end": 1148}]}], "negative regulation": [{"trigger": {"text": "deficiency", "start": 35, "end": 45}, "arguments": [{"role": "Theme", "text": "induction", "start": 68, "end": 77}]}, {"trigger": {"text": "deficient", "start": 456, "end": 465}, "arguments": [{"role": "Theme", "text": "induced", "start": 499, "end": 506}]}, {"trigger": {"text": "normal", "start": 587, "end": 593}, "arguments": [{"role": "Theme", "text": "production", "start": 573, "end": 583}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 68, "end": 77}, "arguments": [{"role": "Theme", "text": "transcription", "start": 92, "end": 105}]}, {"trigger": {"text": "inducible", "start": 184, "end": 193}, "arguments": [{"role": "Theme", "text": "expression", "start": 194, "end": 204}]}, {"trigger": {"text": "induced", "start": 499, "end": 506}, "arguments": [{"role": "Theme", "text": "expression", "start": 474, "end": 484}]}, {"trigger": {"text": "induction", "start": 1131, "end": 1140}, "arguments": [{"role": "Theme", "text": "expression", "start": 1170, "end": 1180}]}], "regulation": [{"trigger": {"text": "role", "start": 1011, "end": 1015}, "arguments": [{"role": "Theme", "text": "regulation", "start": 1037, "end": 1047}]}, {"trigger": {"text": "regulation", "start": 1037, "end": 1047}, "arguments": [{"role": "Theme", "text": "expression", "start": 1056, "end": 1066}]}], "transcription": [{"trigger": {"text": "transcription", "start": 92, "end": 105}, "arguments": [{"role": "Theme", "text": "Fas ligand", "start": 81, "end": 91}]}]}}, "schema": []} {"input": "NF-kappaB regulates Fas/APO-1/CD95- and TCR- mediated apoptosis of T lymphocytes. \nThe maintenance of lymphocyte homeostasis by apoptosis is a critical regulatory mechanism in the normal immune system. The transcription factor NF-kappaB has been shown to play a role in protecting cells against death mediated by TNF We show here that NF-kappaB also has a role in regulating Fas/APO-1/CD95-mediated death, a major pathway of peripheral T cell death. Transfection of Jurkat cells with the NF-kappaB subunits p50 and p65 confers resistance against Fas-mediated apoptosis. Reciprocally, inhibition of NF-kappaB activation by a soluble peptide inhibitor or a dominant form of the NF-kappaB inhibitor, IkappaB, makes the cells more susceptible to Fas-mediated apoptosis. Furthermore, inhibition of NF-kappaB activation by a soluble peptide inhibitor rendered a T cell hybridoma more susceptible to TCR-mediated apoptosis. Correspondingly, transfection of p50 and p65 provided considerable protection from TCR-mediated apoptosis. These observations were corroborated by studies on Fas-mediated death in primary T cells. Concanavalin A-activated cycling T cell blasts from mice that are transgenic for the dominant IkappaB molecule have increased sensitivity to Fas-mediated apoptosis, associated with a down-regulation of NF-kappaB complexes in the nucleus. In addition, blocking TNF, itself a positive regulator of NF-kappaB, with neutralizing antibodies renders the cells more susceptible to anti-Fas-mediated apoptosis. In summary, our results provide compelling evidence that NF-kappaB protects against Fas-mediated death and is likely to be an important regulator of T cell homeostasis and tolerance. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Transfection", "start": 450, "end": 462}, "arguments": [{"role": "Theme", "text": "p50", "start": 507, "end": 510}]}, {"trigger": {"text": "Transfection", "start": 450, "end": 462}, "arguments": [{"role": "Theme", "text": "p65", "start": 515, "end": 518}]}, {"trigger": {"text": "transfection", "start": 934, "end": 946}, "arguments": [{"role": "Theme", "text": "p50", "start": 950, "end": 953}]}, {"trigger": {"text": "transfection", "start": 934, "end": 946}, "arguments": [{"role": "Theme", "text": "p65", "start": 958, "end": 961}]}], "positive regulation": [{"trigger": {"text": "Transfection", "start": 450, "end": 462}, "arguments": [{"role": "Theme", "text": "Transfection", "start": 450, "end": 462}]}, {"trigger": {"text": "transfection", "start": 934, "end": 946}, "arguments": [{"role": "Theme", "text": "transfection", "start": 934, "end": 946}]}]}}, "schema": []} {"input": "Evidence for distinct intracellular signaling pathways in CD34+ progenitor to dendritic cell differentiation from a human cell line model. \nIntracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC-->DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC-->DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 1286, "end": 1296}, "arguments": [{"role": "Theme", "text": "CD83", "start": 1257, "end": 1261}]}, {"trigger": {"text": "expression", "start": 1286, "end": 1296}, "arguments": [{"role": "Theme", "text": "CD86", "start": 1263, "end": 1267}]}, {"trigger": {"text": "expression", "start": 1286, "end": 1296}, "arguments": [{"role": "Theme", "text": "CD14", "start": 1269, "end": 1273}]}, {"trigger": {"text": "expression", "start": 1323, "end": 1333}, "arguments": [{"role": "Theme", "text": "RelB", "start": 1302, "end": 1306}]}, {"trigger": {"text": "expression", "start": 1323, "end": 1333}, "arguments": [{"role": "Theme", "text": "DC-CK1", "start": 1311, "end": 1317}]}, {"trigger": {"text": "expression", "start": 1534, "end": 1544}, "arguments": [{"role": "Theme", "text": "TNF receptors 1", "start": 1548, "end": 1563}]}, {"trigger": {"text": "expression", "start": 1534, "end": 1544}, "arguments": [{"role": "Theme", "text": "2", "start": 1568, "end": 1569}]}, {"trigger": {"text": "expression", "start": 1534, "end": 1544}, "arguments": [{"role": "Theme", "text": "PKC isoforms alpha", "start": 1571, "end": 1589}]}, {"trigger": {"text": "expression", "start": 1534, "end": 1544}, "arguments": [{"role": "Theme", "text": "beta I", "start": 1591, "end": 1597}]}, {"trigger": {"text": "expression", "start": 1534, "end": 1544}, "arguments": [{"role": "Theme", "text": "beta II", "start": 1599, "end": 1606}]}, {"trigger": {"text": "expression", "start": 1534, "end": 1544}, "arguments": [{"role": "Theme", "text": "mu", "start": 1612, "end": 1614}]}, {"trigger": {"text": "expression", "start": 1534, "end": 1544}, "arguments": [{"role": "Theme", "text": "RelB", "start": 1620, "end": 1624}]}], "positive regulation": [{"trigger": {"text": "important", "start": 1672, "end": 1681}, "arguments": [{"role": "Theme", "text": "expression", "start": 1534, "end": 1544}, {"role": "Cause", "text": "TNF receptors 1", "start": 1548, "end": 1563}]}, {"trigger": {"text": "important", "start": 1672, "end": 1681}, "arguments": [{"role": "Theme", "text": "expression", "start": 1534, "end": 1544}, {"role": "Cause", "text": "2", "start": 1568, "end": 1569}]}, {"trigger": {"text": "important", "start": 1672, "end": 1681}, "arguments": [{"role": "Theme", "text": "expression", "start": 1534, "end": 1544}, {"role": "Cause", "text": "PKC isoforms alpha", "start": 1571, "end": 1589}]}, {"trigger": {"text": "important", "start": 1672, "end": 1681}, "arguments": [{"role": "Theme", "text": "expression", "start": 1534, "end": 1544}, {"role": "Cause", "text": "beta I", "start": 1591, "end": 1597}]}, {"trigger": {"text": "important", "start": 1672, "end": 1681}, "arguments": [{"role": "Theme", "text": "expression", "start": 1534, "end": 1544}, {"role": "Cause", "text": "beta II", "start": 1599, "end": 1606}]}, {"trigger": {"text": "important", "start": 1672, "end": 1681}, "arguments": [{"role": "Theme", "text": "expression", "start": 1534, "end": 1544}, {"role": "Cause", "text": "mu", "start": 1612, "end": 1614}]}, {"trigger": {"text": "important", "start": 1672, "end": 1681}, "arguments": [{"role": "Theme", "text": "expression", "start": 1534, "end": 1544}, {"role": "Cause", "text": "RelB", "start": 1620, "end": 1624}]}]}}, "schema": []} {"input": "IL-2-mediated cell cycle progression and inhibition of apoptosis does not require NF-kappa B or activating protein-1 activation in primary human T cells. \nThe IL-2 growth hormone is the major growth factor of activated T lymphocytes during a developing immune response. IL-2 is required not only for cell cycle progression but also to protect Ag-activated T cells from programmed cell death. In several cell types, activation of NF-kappa B and/or activating protein-1 (AP-1) has been demonstrated to be extremely important in blocking apoptosis. To determine whether either or both of these transcription factors are involved in cell survival or cell cycle progression in response to IL-2, primary human T cells responsive to the growth factor were analyzed for NF-kappa B and AP-1 activation. The current study clearly demonstrates that IL-2 does not induce I kappa B alpha degradation or NF-kappa B activation in primary human T cells that respond to IL-2 by entering the cell cycle and avoiding apoptosis. Similarly, IL-2 neither activates JNK nor increases AP-1 binding activity to a consensus o-tetradecanoylphorbol 13-acetate (TPA) response element. On the other hand, the growth factor does induce the activation of STAT3 and STAT5 in these cells, as has been previously demonstrated. These data show that neither NF-kappa B nor AP-1 activation is required for IL-2-mediated survival or cell cycle progression in activated primary human T cells. ", "output": {"json_structures": {"positive regulation": [{"trigger": {"text": "induce", "start": 852, "end": 858}, "arguments": [{"role": "Cause", "text": "IL-2", "start": 838, "end": 842}, {"role": "Theme", "text": "degradation", "start": 875, "end": 886}]}, {"trigger": {"text": "induce", "start": 1198, "end": 1204}, "arguments": [{"role": "Cause", "text": "IL-2", "start": 1020, "end": 1024}, {"role": "Theme", "text": "activation", "start": 1209, "end": 1219}]}, {"trigger": {"text": "activation", "start": 1209, "end": 1219}, "arguments": [{"role": "Theme", "text": "STAT3", "start": 1223, "end": 1228}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 875, "end": 886}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 859, "end": 874}]}]}}, "schema": []} {"input": "Impaired fetal thymocyte development after efficient adenovirus-mediated inhibition of NF-kappa B activation. \nWe introduce a new experimental system combining adenovirus-mediated gene transfer and fetal thymic organ culture (FTOC). This system allowed us to efficiently express in developing thymocytes a mutant form of the NF-kappa B inhibitor I kappa B alpha (mut-I kappa B) and to study the maturation defects occurring when NF-kappa B activation is inhibited during fetal development. Fetal thymocytes infected with adenovirus containing mut-I kappa B were found to develop normally until the CD44-CD25+, CD4-CD8- double-negative stage, while production of more mature double-positive and single-positive populations was strongly decreased. Proliferation, as measured by the percentage of cells in cycle appeared normal, as did rearrangement and expression of the TCR beta-chain. However, apoptosis was much higher in FTOC infected with adenovirus containing mut-I kappa B than in FTOC infected with a control virus. Taken together, these results suggest that NF-kappa B plays a crucial role in ensuring the differentiation and survival of thymocytes in the early stages of their development. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "express", "start": 271, "end": 278}, "arguments": [{"role": "Theme", "text": "mut-I kappa B", "start": 363, "end": 376}]}, {"trigger": {"text": "expression", "start": 851, "end": 861}, "arguments": [{"role": "Theme", "text": "TCR beta-chain", "start": 869, "end": 883}]}], "regulation": [{"trigger": {"text": "appeared normal", "start": 809, "end": 824}, "arguments": [{"role": "Theme", "text": "expression", "start": 851, "end": 861}]}]}}, "schema": []} {"input": "Differential regulation of 4E-BP1 and 4E-BP2, two repressors of translation initiation, during human myeloid cell differentiation. \nHuman myeloid differentiation is accompanied by a decrease in cell proliferation. Because the translation rate is an important determinant of cell proliferation, we have investigated translation initiation during human myeloid cell differentiation using the HL-60 promyelocytic leukemia cell line and the U-937 monoblastic cell line. A decrease in the translation rate is observed when the cells are induced to differentiate along the monocytic/macrophage pathway or along the granulocytic pathway. The inhibition in protein synthesis correlates with specific regulation of two repressors of translation initiation, 4E-BP1 and 4E-BP2. Induction of HL-60 and U-937 cell differentiation into monocytes/macrophages by IFN-gamma or PMA results in a dephosphorylation and consequent activation of 4E-BP1. Dephosphorylation of 4E-BP1 was also observed when U-937 cells were induced to differentiate into monocytes/macrophages following treatment with retinoic acid or DMSO. In contrast, treatment of HL-60 cells with retinoic acid or DMSO, which results in a granulocytic differentiation of these cells, decreases 4E-BP1 amount without affecting its phosphorylation and strongly increases 4E-BP2 amount. Taken together, these data provide evidence for differential regulation of the translational machinery during human myeloid differentiation, specific to the monocytic/macrophage pathway or to the granulocytic pathway. ", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "decreases", "start": 1230, "end": 1239}, "arguments": [{"role": "Theme", "text": "4E-BP1", "start": 1240, "end": 1246}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1276, "end": 1291}, "arguments": [{"role": "Theme", "text": "4E-BP1", "start": 1240, "end": 1246}]}], "positive regulation": [{"trigger": {"text": "results", "start": 864, "end": 871}, "arguments": [{"role": "Theme", "text": "activation", "start": 910, "end": 920}]}, {"trigger": {"text": "activation", "start": 910, "end": 920}, "arguments": [{"role": "Theme", "text": "4E-BP1", "start": 924, "end": 930}]}, {"trigger": {"text": "increases", "start": 1305, "end": 1314}, "arguments": [{"role": "Theme", "text": "4E-BP2", "start": 1315, "end": 1321}]}], "regulation": [{"trigger": {"text": "regulation", "start": 13, "end": 23}, "arguments": [{"role": "Theme", "text": "4E-BP1", "start": 27, "end": 33}]}, {"trigger": {"text": "regulation", "start": 13, "end": 23}, "arguments": [{"role": "Theme", "text": "4E-BP2", "start": 38, "end": 44}]}, {"trigger": {"text": "regulation", "start": 692, "end": 702}, "arguments": [{"role": "Theme", "text": "4E-BP1", "start": 748, "end": 754}]}, {"trigger": {"text": "regulation", "start": 692, "end": 702}, "arguments": [{"role": "Theme", "text": "4E-BP2", "start": 759, "end": 765}]}, {"trigger": {"text": "affecting", "start": 1262, "end": 1271}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1276, "end": 1291}]}]}}, "schema": []} {"input": "Stimulation of CD40 on immunogenic human malignant melanomas augments their cytotoxic T lymphocyte-mediated lysis and induces apoptosis. \nHere, we report the functional expression of CD40 on human malignant melanomas (MMs). Comparison of tumor specimen from MM precursor lesions, primary tumors, and metastases revealed that CD40 surface expression is down-regulated during tumor progression. CD40 expression was confirmed in 7 human MM cell lines established from immunogenic primary tumors or metastases, whereas 11 cell lines established from advanced stages were CD40 negative. CD40 expression could be enhanced in CD40-positive MM by stimulation with IFN-gamma and tumor necrosis factor-alpha but not by interleukin (IL)-1beta or CD40 triggering. CD40 ligation on MM by CD40L-transfected murine L-cells or by a soluble CD40L fusion protein up-regulated their expression of intercellular adhesion molecule-1 and MHC class I and class II molecules and their secretion of IL-6, IL-8, tumor necrosis factor-a, and granulocyte macrophage colony-stimulating factor and also induced a rapid activation of the transcription factor nuclear factor kappaB. Furthermore, CD40 ligation of a HLA-A2+, MelanA/MART1+ MM cell line enhanced its susceptibility to specific lysis by a HLA-A2-restricted, MelanA/MART-1-specific CTL clone. Finally, CD40 ligation induced growth inhibition and apoptosis in MM. These results indicate that CD40-CD40L interactions may play an important role in augmenting antitumor immunity and inducing apoptosis in some CD40-positive immunogenic human MMs. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "ligation", "start": 757, "end": 765}, "arguments": [{"role": "Theme", "text": "CD40", "start": 752, "end": 756}, {"role": "Theme2", "text": "CD40L", "start": 824, "end": 829}]}, {"trigger": {"text": "ligation", "start": 1169, "end": 1177}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1164, "end": 1168}]}, {"trigger": {"text": "ligation", "start": 1337, "end": 1345}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1332, "end": 1336}]}, {"trigger": {"text": "interactions", "start": 1432, "end": 1444}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1421, "end": 1425}, {"role": "Theme2", "text": "CD40L", "start": 1426, "end": 1431}]}], "gene expression": [{"trigger": {"text": "expression", "start": 169, "end": 179}, "arguments": [{"role": "Theme", "text": "CD40", "start": 183, "end": 187}]}, {"trigger": {"text": "expression", "start": 338, "end": 348}, "arguments": [{"role": "Theme", "text": "CD40", "start": 325, "end": 329}]}, {"trigger": {"text": "expression", "start": 398, "end": 408}, "arguments": [{"role": "Theme", "text": "CD40", "start": 393, "end": 397}]}, {"trigger": {"text": "negative", "start": 572, "end": 580}, "arguments": [{"role": "Theme", "text": "CD40", "start": 567, "end": 571}]}, {"trigger": {"text": "expression", "start": 587, "end": 597}, "arguments": [{"role": "Theme", "text": "CD40", "start": 582, "end": 586}]}, {"trigger": {"text": "expression", "start": 864, "end": 874}, "arguments": [{"role": "Theme", "text": "intercellular adhesion molecule-1", "start": 878, "end": 911}]}], "localization": [{"trigger": {"text": "secretion", "start": 961, "end": 970}, "arguments": [{"role": "Theme", "text": "granulocyte macrophage colony-stimulating factor", "start": 1015, "end": 1063}]}, {"trigger": {"text": "secretion", "start": 961, "end": 970}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 974, "end": 978}]}, {"trigger": {"text": "secretion", "start": 961, "end": 970}, "arguments": [{"role": "Theme", "text": "IL-8", "start": 980, "end": 984}]}, {"trigger": {"text": "secretion", "start": 961, "end": 970}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor-a", "start": 986, "end": 1009}]}], "negative regulation": [{"trigger": {"text": "down-regulated", "start": 352, "end": 366}, "arguments": [{"role": "Theme", "text": "expression", "start": 338, "end": 348}]}], "positive regulation": [{"trigger": {"text": "Stimulation", "start": 0, "end": 11}, "arguments": [{"role": "Theme", "text": "CD40", "start": 15, "end": 19}]}, {"trigger": {"text": "enhanced", "start": 607, "end": 615}, "arguments": [{"role": "Theme", "text": "expression", "start": 587, "end": 597}, {"role": "Cause", "text": "IFN-gamma", "start": 656, "end": 665}]}, {"trigger": {"text": "enhanced", "start": 607, "end": 615}, "arguments": [{"role": "Theme", "text": "expression", "start": 587, "end": 597}, {"role": "Cause", "text": "tumor necrosis factor-alpha", "start": 670, "end": 697}]}, {"trigger": {"text": "enhanced", "start": 607, "end": 615}, "arguments": [{"role": "Theme", "text": "expression", "start": 587, "end": 597}, {"role": "Cause", "text": "interleukin (IL)-1beta", "start": 709, "end": 731}]}, {"trigger": {"text": "enhanced", "start": 607, "end": 615}, "arguments": [{"role": "Theme", "text": "expression", "start": 587, "end": 597}, {"role": "Cause", "text": "CD40", "start": 735, "end": 739}]}, {"trigger": {"text": "up-regulated", "start": 845, "end": 857}, "arguments": [{"role": "Cause", "text": "ligation", "start": 757, "end": 765}, {"role": "Theme", "text": "expression", "start": 864, "end": 874}]}, {"trigger": {"text": "up-regulated", "start": 845, "end": 857}, "arguments": [{"role": "Cause", "text": "ligation", "start": 757, "end": 765}, {"role": "Theme", "text": "secretion", "start": 961, "end": 970}]}]}}, "schema": []} {"input": "Essential role of alveolar macrophages in intrapulmonary activation of NF-kappaB. \nAcute inflammatory injury in rat lung induced by deposition of immunoglobulin G immune complexes requires expression of cytokines and chemokines as well as activation of the transcription factor nuclear factor (NF)-kappaB. There is little direct evidence regarding the role of alveolar macrophages in these activation events. In the present studies, rat lungs were depleted of alveolar macrophages by airway instillation of liposome-encapsulated dichloromethylene diphosphonate. These procedures, which greatly reduced the number of retrievable alveolar macrophages, suppressed activation of lung NF-kappaB in the inflammatory model. In addition, bronchoalveolar lavage levels of tumor necrosis factor-alpha (TNF-alpha) and the CXC chemokine, macrophage inflammatory protein-2, were substantially reduced. In parallel, upregulation of the lung vascular adhesion molecule, intercellular adhesion molecule-1, was greatly reduced by intrapulmonary instillation of phosphonate-containing liposomes. Neutrophil accumulation and development of lung injury were also substantially diminished. Lung instillation of TNF-alpha in alveolar macrophage-depleted rats restored the NF-kappaB activation response in whole lung. These data suggest that, in this inflammatory model, initial activation of NF-kappaB occurs in alveolar macrophages and the ensuing production of TNF-alpha may propagate NF-kappaB activation to other cell types in the lung. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 1427, "end": 1437}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1441, "end": 1450}]}], "negative regulation": [{"trigger": {"text": "reduced", "start": 880, "end": 887}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 792, "end": 801}]}, {"trigger": {"text": "reduced", "start": 880, "end": 887}, "arguments": [{"role": "Theme", "text": "macrophage inflammatory protein-2", "start": 826, "end": 859}]}, {"trigger": {"text": "reduced", "start": 1002, "end": 1009}, "arguments": [{"role": "Theme", "text": "upregulation", "start": 902, "end": 914}]}], "positive regulation": [{"trigger": {"text": "upregulation", "start": 902, "end": 914}, "arguments": [{"role": "Theme", "text": "intercellular adhesion molecule-1", "start": 955, "end": 988}]}]}}, "schema": []} {"input": "Role of cellular tumor necrosis factor receptor-associated factors in NF-kappaB activation and lymphocyte transformation by herpesvirus Saimiri STP. \nThe STP oncoproteins of the herpesvirus saimiri (HVS) subgroup A strain 11 and subgroup C strain 488 are now found to be stably associated with tumor necrosis factor receptor-associated factor (TRAF) 1, 2, or 3. Mutational analyses identified residues of PXQXT/S in STP-A11 as critical for TRAF association. In addition, a somewhat divergent region of STP-C488 is critical for TRAF association. Mutational analysis also revealed that STP-C488 induced NF-kappaB activation that was correlated with its ability to associate with TRAFs. The HVS STP-C488 P10-->R mutant was deficient in human T-lymphocyte transformation to interleukin-2-independent growth but showed wild-type phenotype for marmoset T-lymphocyte transformation in vitro and in vivo. The STP-C488 P10-->R mutant was also defective in Rat-1 fibroblast transformation, and fibroblast cell transformation was blocked by a TRAF2 dominant-negative mutant. These data implicate TRAFs in STP-C488-mediated transformation of human lymphocytes and rodent fibroblasts. Other factors are implicated in immortalization of common marmoset T lymphocytes and may also be critical in the transformation of human lymphocytes and rodent fibroblasts. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "associated", "start": 278, "end": 288}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor receptor-associated factor (TRAF) 1", "start": 294, "end": 351}]}, {"trigger": {"text": "associated", "start": 278, "end": 288}, "arguments": [{"role": "Theme", "text": "2", "start": 353, "end": 354}]}, {"trigger": {"text": "associated", "start": 278, "end": 288}, "arguments": [{"role": "Theme", "text": "3", "start": 359, "end": 360}]}, {"trigger": {"text": "association", "start": 445, "end": 456}, "arguments": [{"role": "Theme", "text": "STP-A11", "start": 416, "end": 423}]}, {"trigger": {"text": "association", "start": 532, "end": 543}, "arguments": [{"role": "Theme", "text": "STP-C488", "start": 502, "end": 510}]}, {"trigger": {"text": "associate", "start": 662, "end": 671}, "arguments": [{"role": "Theme", "text": "STP-C488", "start": 584, "end": 592}]}], "positive regulation": [{"trigger": {"text": "transformation", "start": 860, "end": 874}, "arguments": [{"role": "Theme", "text": "associated", "start": 278, "end": 288}, {"role": "Cause", "text": "STP-C488", "start": 692, "end": 700}]}]}}, "schema": []} {"input": "Regulation of Fas ligand expression and cell death by apoptosis-linked gene 4. \nProgrammed cell death is a process required for the normal development of an organism. One of the best understood apoptotic pathways occurs in T lymphocytes and is mediated by Fas/Fas ligand (FasL) interaction. During studies of apoptosis induced by T cell-receptor engagement, we identified ALG-4F, a truncated transcript that prevents T cell-receptor-induced FasL upregulation and cell death. Overexpression of full-length ALG-4 induced transcription of FasL and, consequently, apoptosis. These results indicate that ALG-4 is necessary and sufficient for FasL expression. Fas/FasL interaction initiates cell death in many other systems, and its dysregulation is a mechanism by which several pathologic conditions arise. Understanding the molecular mechanisms of FasL regulation could be very useful in elucidating how these diseases develop and in identifying potential therapeutic targets. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 278, "end": 289}, "arguments": [{"role": "Theme", "text": "Fas", "start": 256, "end": 259}, {"role": "Theme2", "text": "FasL", "start": 272, "end": 276}]}, {"trigger": {"text": "interaction", "start": 663, "end": 674}, "arguments": [{"role": "Theme", "text": "Fas", "start": 654, "end": 657}, {"role": "Theme2", "text": "FasL", "start": 658, "end": 662}]}], "gene expression": [{"trigger": {"text": "expression", "start": 25, "end": 35}, "arguments": [{"role": "Theme", "text": "Fas ligand", "start": 14, "end": 24}]}, {"trigger": {"text": "Overexpression", "start": 475, "end": 489}, "arguments": [{"role": "Theme", "text": "ALG-4", "start": 505, "end": 510}]}, {"trigger": {"text": "expression", "start": 642, "end": 652}, "arguments": [{"role": "Theme", "text": "FasL", "start": 637, "end": 641}]}], "negative regulation": [{"trigger": {"text": "prevents", "start": 408, "end": 416}, "arguments": [{"role": "Theme", "text": "induced", "start": 433, "end": 440}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 433, "end": 440}, "arguments": [{"role": "Theme", "text": "upregulation", "start": 446, "end": 458}]}, {"trigger": {"text": "upregulation", "start": 446, "end": 458}, "arguments": [{"role": "Theme", "text": "FasL", "start": 441, "end": 445}]}, {"trigger": {"text": "Overexpression", "start": 475, "end": 489}, "arguments": [{"role": "Theme", "text": "Overexpression", "start": 475, "end": 489}]}, {"trigger": {"text": "induced", "start": 511, "end": 518}, "arguments": [{"role": "Cause", "text": "Overexpression", "start": 475, "end": 489}, {"role": "Theme", "text": "transcription", "start": 519, "end": 532}]}, {"trigger": {"text": "necessary and sufficient", "start": 608, "end": 632}, "arguments": [{"role": "Cause", "text": "ALG-4", "start": 599, "end": 604}, {"role": "Theme", "text": "expression", "start": 642, "end": 652}]}], "regulation": [{"trigger": {"text": "Regulation", "start": 0, "end": 10}, "arguments": [{"role": "Theme", "text": "expression", "start": 25, "end": 35}, {"role": "Cause", "text": "apoptosis-linked gene 4", "start": 54, "end": 77}]}, {"trigger": {"text": "dysregulation", "start": 727, "end": 740}, "arguments": [{"role": "Theme", "text": "interaction", "start": 663, "end": 674}]}], "transcription": [{"trigger": {"text": "transcription", "start": 519, "end": 532}, "arguments": [{"role": "Theme", "text": "FasL", "start": 536, "end": 540}]}]}}, "schema": []} {"input": "High molecular weight dextran sulfate increases the activity of NF-kappaB-regulated promoter in monocyte-derived macrophages. \nIt is known that sulfated polysaccharides can mimic the action of common T-cell mitogens. To investigate the molecular basis of the mitogenic effect of high molecular weight dextran sulfate (HMDS), monocyte-derived macrophages were transfected with recombinant plasmid containing chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV-1 long terminal repeat (LTR) promoter, which is regulated by transcription factor NF-kappaB. We observed that HMDS, similar to bacterial lipopolysaccharide (LPS), increases the expression of CAT reporter gene suggesting increased activity of NF-kappaB. The activation of NF-kappaB correlated with the increased expression of B7.1 molecules. It was postulated that this NF-kappaB-regulated promoter might play a role in the activation of the accessory cells as well as the rate of replication of HIV-1 in monocyte-derived macrophages. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 802, "end": 812}, "arguments": [{"role": "Theme", "text": "B7.1", "start": 816, "end": 820}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 792, "end": 801}, "arguments": [{"role": "Theme", "text": "expression", "start": 802, "end": 812}]}]}}, "schema": []} {"input": "Inhibition of NF-kappa B activity in human T lymphocytes induces caspase-dependent apoptosis without detectable activation of caspase-1 and -3. \nNF-kappa B is involved in the transcriptional control of various genes that act as extrinsic and intrinsic survival factors for T cells. Our findings show that suppression of NF-kappa B activity with cell-permeable SN50 peptide, which masks the nuclear localization sequence of NF-kappa B1 dimers and prevents their nuclear localization, induces apoptosis in resting normal human PBL. Inhibition of NF-kappa B resulted in the externalization of phosphatidylserine, induction of DNA breaks, and morphological changes consistent with apoptosis. DNA fragmentation was efficiently blocked by the caspase inhibitor Z-VAD-fmk and partially blocked by Ac-DEVD-fmk, suggesting that SN50-mediated apoptosis is caspase-dependent. Interestingly, apoptosis induced by NF-kappa B suppression, in contrast to that induced by TPEN (N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine) or soluble Fas ligand (CD95), was observed in the absence of active death effector proteases caspase-1-like (IL-1 converting enzyme), caspase-3-like (CPP32/Yama/apopain), and caspase-6-like and without cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and DNA fragmentation factor-45. These findings suggest either low level of activation is required or that different caspases are involved. Preactivation of T cells resulting in NF-kappa B nuclear translocation protected cells from SN50-induced apoptosis. Our findings demonstrate an essential role of NF-kappa B in survival of naive PBL. ", "output": {"json_structures": {"positive regulation": [{"trigger": {"text": "activation", "start": 1354, "end": 1364}, "arguments": [{"role": "Theme", "text": "CPP32", "start": 1165, "end": 1170}]}, {"trigger": {"text": "activation", "start": 1354, "end": 1364}, "arguments": [{"role": "Theme", "text": "caspase-6", "start": 1190, "end": 1199}]}]}}, "schema": []} {"input": "Signaling events induced by lipopolysaccharide-activated toll-like receptor 2. \nHuman Toll-like receptor 2 (TLR2) is a signaling receptor that responds to LPS and activates NF-kappaB. Here, we investigate further the events triggered by TLR2 in response to LPS. We show that TLR2 associates with the high-affinity LPS binding protein membrane CD14 to serve as an LPS receptor complex, and that LPS treatment enhances the oligomerization of TLR2. Concomitant with receptor oligomerization, the IL-1R-associated kinase (IRAK) is recruited to the TLR2 complex. Intracellular deletion variants of TLR2 lacking C-terminal 13 or 141 aa fail to recruit IRAK, which is consistent with the inability of these mutants to transmit LPS cellular signaling. Moreover, both deletion mutants could still form complexes with wild-type TLR2 and act in a dominant-negative (DN) fashion to block TLR2-mediated signal transduction. DN constructs of myeloid differentiation protein, IRAK, TNF receptor-associated factor 6, and NF-kappaB-inducing kinase, when coexpressed with TLR2, abrogate TLR2-mediated NF-kappaB activation. These results reveal a conserved signaling pathway for TLR2 and IL-1Rs and suggest a molecular mechanism for the inhibition of TLR2 by DN variants. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "associates", "start": 280, "end": 290}, "arguments": [{"role": "Theme", "text": "TLR2", "start": 275, "end": 279}, {"role": "Theme2", "text": "CD14", "start": 343, "end": 347}]}, {"trigger": {"text": "recruit", "start": 638, "end": 645}, "arguments": [{"role": "Theme", "text": "TLR2", "start": 593, "end": 597}]}, {"trigger": {"text": "form complexes", "start": 788, "end": 802}, "arguments": [{"role": "Theme", "text": "TLR2", "start": 593, "end": 597}, {"role": "Theme2", "text": "TLR2", "start": 818, "end": 822}]}], "gene expression": [{"trigger": {"text": "coexpressed", "start": 1037, "end": 1048}, "arguments": [{"role": "Theme", "text": "NF-kappaB-inducing kinase", "start": 1005, "end": 1030}]}, {"trigger": {"text": "coexpressed", "start": 1037, "end": 1048}, "arguments": [{"role": "Theme", "text": "TLR2", "start": 1054, "end": 1058}]}, {"trigger": {"text": "coexpressed", "start": 1037, "end": 1048}, "arguments": [{"role": "Theme", "text": "TNF receptor-associated factor 6", "start": 967, "end": 999}]}], "negative regulation": [{"trigger": {"text": "inhibition", "start": 1218, "end": 1228}, "arguments": [{"role": "Theme", "text": "TLR2", "start": 1232, "end": 1236}]}], "positive regulation": [{"trigger": {"text": "activated", "start": 47, "end": 56}, "arguments": [{"role": "Theme", "text": "toll-like receptor 2", "start": 57, "end": 77}]}], "regulation": [{"trigger": {"text": "responds", "start": 143, "end": 151}, "arguments": [{"role": "Theme", "text": "TLR2", "start": 108, "end": 112}]}]}}, "schema": []} {"input": "Activation of the Janus kinase 3-STAT5a pathway after CD40 triggering of human monocytes but not of resting B cells. \nCD40/CD40 ligand interactions play a key role in the immune responses of B lymphocytes, monocytes, and dendritic cells. The signal transduction events triggered by cross-linking of the CD40 receptor have been widely studied in B cell lines, but little is known about signaling following CD40 stimulation of monocytes and resting tonsillar B cells. Therefore, we studied the CD40 pathway in highly purified human monocytes and resting B cells. After CD40 triggering, a similar activation of the NF-kappaB (but not of the AP-1) transcription factor complex occurred in both cell preparations. However, the components of the NF-kappaB complexes were different in monocytes and B cells, because p50 is part of the NF-kappaB complex induced by CD40 triggering in both monocytes and B cells, whereas p65 was only induced in B cells. In contrast, although the Janus kinase 3 tyrosine kinase was associated with CD40 molecules in both monocytes and resting B cells, Janus kinase 3 phosphorylation induction was observed only in CD40-activated monocytes, with subsequent induction of STAT5a DNA binding activity in the nucleus. These results suggest that the activation signals in human B cells and monocytes differ following CD40 stimulation. This observation is consistent with the detection of normal CD40-induced monocyte activation in patients with CD40 ligand+ hyper IgM syndrome in whom a defect in CD40-induced B cell activation has been reported. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interactions", "start": 135, "end": 147}, "arguments": [{"role": "Theme", "text": "CD40", "start": 118, "end": 122}, {"role": "Theme2", "text": "CD40 ligand", "start": 123, "end": 134}]}, {"trigger": {"text": "associated", "start": 1006, "end": 1016}, "arguments": [{"role": "Theme", "text": "Janus kinase 3", "start": 971, "end": 985}, {"role": "Theme2", "text": "CD40", "start": 1022, "end": 1026}]}, {"trigger": {"text": "binding", "start": 1204, "end": 1211}, "arguments": [{"role": "Theme", "text": "STAT5a", "start": 1193, "end": 1199}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1091, "end": 1106}, "arguments": [{"role": "Theme", "text": "Janus kinase 3", "start": 1076, "end": 1090}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 846, "end": 853}, "arguments": [{"role": "Theme", "text": "p50", "start": 809, "end": 812}]}, {"trigger": {"text": "induced", "start": 925, "end": 932}, "arguments": [{"role": "Theme", "text": "p65", "start": 912, "end": 915}]}, {"trigger": {"text": "induction", "start": 1107, "end": 1116}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1091, "end": 1106}]}, {"trigger": {"text": "induction", "start": 1180, "end": 1189}, "arguments": [{"role": "Theme", "text": "binding", "start": 1204, "end": 1211}]}]}}, "schema": []} {"input": "Suppression of NF-kappaB activation in normal T cells by supernatant fluid from human renal cell carcinomas. \nT lymphocytes from patients with renal cell carcinoma (RCC) show reduced immune function and impaired activation of the transcription factor, NF-kappaB. We determined the mechanism of NF-kappaB suppression in T cells of RCC patient and determined whether supernatant fluid from RCC explants (RCC-S) induced the same phenotype of NF-kappaB suppression in normal T cells that is observed in patient T cells. The pattern of kappaB-binding activity in T cells of RCC patient was altered as compared to that seen in T cells obtained from normal volunteers. In some patients, no activation of RelA/NFkappaB1-binding activity was detectable, while in others kappaB-binding activity was modestly induced but the duration was reduced. IkappaBalpha was degraded normally following stimulation in both normal controls and T cells from RCC patients. RCC-S did not alter the cytoplasmic levels of RelA and NF-kappaB1 but did suppress their nuclear localization and inhibited the activation of RelA/NF-kappaB1 binding complexes. These results show that RCC-S can induce in normal T cells the same phenotype of impaired NF-kappaB activation that is detected in T cells of RCC patient. It also appears that NF-kappaB suppression by RCC-S may contribute to the immunosuppression of host immunity. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 712, "end": 719}, "arguments": [{"role": "Theme", "text": "RelA", "start": 697, "end": 701}, {"role": "Theme2", "text": "NFkappaB1", "start": 702, "end": 711}]}], "localization": [{"trigger": {"text": "localization", "start": 1045, "end": 1057}, "arguments": [{"role": "Theme", "text": "NF-kappaB1", "start": 1003, "end": 1013}, {"role": "ToLoc", "text": "nuclear", "start": 1037, "end": 1044}]}, {"trigger": {"text": "localization", "start": 1045, "end": 1057}, "arguments": [{"role": "Theme", "text": "RelA", "start": 994, "end": 998}, {"role": "ToLoc", "text": "nuclear", "start": 1037, "end": 1044}]}], "negative regulation": [{"trigger": {"text": "suppress", "start": 1022, "end": 1030}, "arguments": [{"role": "Theme", "text": "localization", "start": 1045, "end": 1057}]}], "positive regulation": [{"trigger": {"text": "activation", "start": 683, "end": 693}, "arguments": [{"role": "Theme", "text": "binding", "start": 712, "end": 719}]}, {"trigger": {"text": "following", "start": 871, "end": 880}, "arguments": [{"role": "Theme", "text": "degraded", "start": 853, "end": 861}]}], "protein catabolism": [{"trigger": {"text": "degraded", "start": 853, "end": 861}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 836, "end": 848}]}], "regulation": [{"trigger": {"text": "alter", "start": 962, "end": 967}, "arguments": [{"role": "Theme", "text": "NF-kappaB1", "start": 1003, "end": 1013}]}, {"trigger": {"text": "alter", "start": 962, "end": 967}, "arguments": [{"role": "Theme", "text": "RelA", "start": 994, "end": 998}]}]}}, "schema": []} {"input": "Monoclonal anti-endothelial cell antibodies from a patient with Takayasu arteritis activate endothelial cells from large vessels. \nOBJECTIVE: To create monoclonal anti-endothelial cell antibodies (mAECA) from a patient with Takayasu arteritis to evaluate their ability to activate human umbilical vein endothelial cells (HUVEC), and to characterize the mechanism of EC activation. METHODS: A panel of mAECA was generated from peripheral blood lymphocytes of a patient with Takayasu arteritis, using Epstein-Barr virus transformation. Activity against macrovascular EC (HUVEC) and microvascular EC (human bone marrow EC immortalized by SV40) antigens was detected by enzyme-linked immunosorbent assay. Inhibition studies were used to select the monoclonal antibodies (mAECA) which share the same EC epitope binding specificity as the total IgG-AECA from the Takayasu arteritis patient. The binding of the mAECA to human aortic EC was studied by immunohistochemistry. The secretion levels of interleukin-6 (IL-6) and von Willebrand factor (vWF) were determined, to serve as markers for EC activation. The activated EC were examined for the adherence of a monocytic cell line (U937), as well as for expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and E-selectin. In addition, nuclear extracts of the mAECA-treated EC were analyzed for the induction of translocation of nuclear factor kappaB (NF-kappaB), using a specific NF-kappaB oligoprobe in an electrophoretic mobility shift assay. RESULTS: Six mAECA were selected, the mixture of which produced 100% inhibition of binding of the original IgG (from the patient with Takayasu arteritis) to HUVEC. All mAECA possessed high activity against macrovascular EC, but none had significant antimicrovascular EC activity. The mAECA, but not normal human IgG, had anti-human aortic EC activity. Four of the 6 mAECA activated EC, manifested by increased IL-6 and vWF secretion. The 4 mAECA induced EC expression of adhesion molecules and increased adhesion of U937 monocytic cells to EC. In addition, these mAECA stimulated the nuclear translocation of the NF-kappaB transcription factor. CONCLUSION: Our findings suggest that AECA may directly stimulate EC in Takayasu arteritis through elevation of adhesion molecule expression associated with NF-kappaB activation and adhesion of monocytes, and may therefore play a pathogenic role in the development of the vasculopathy in Takayasu arteritis. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 1196, "end": 1206}, "arguments": [{"role": "Theme", "text": "vWF", "start": 1038, "end": 1041}]}, {"trigger": {"text": "expression", "start": 1196, "end": 1206}, "arguments": [{"role": "Theme", "text": "intercellular adhesion molecule 1", "start": 1245, "end": 1278}]}, {"trigger": {"text": "expression", "start": 1196, "end": 1206}, "arguments": [{"role": "Theme", "text": "E-selectin", "start": 1284, "end": 1294}]}], "localization": [{"trigger": {"text": "secretion", "start": 970, "end": 979}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1005, "end": 1009}]}, {"trigger": {"text": "secretion", "start": 970, "end": 979}, "arguments": [{"role": "Theme", "text": "vWF", "start": 1038, "end": 1041}]}, {"trigger": {"text": "secretion", "start": 1942, "end": 1951}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1929, "end": 1933}]}, {"trigger": {"text": "secretion", "start": 1942, "end": 1951}, "arguments": [{"role": "Theme", "text": "vWF", "start": 1938, "end": 1941}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 1919, "end": 1928}, "arguments": [{"role": "Theme", "text": "secretion", "start": 1942, "end": 1951}]}]}}, "schema": []} {"input": "The Epstein-Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-kappaB activation. \nA site in the Epstein-Barr virus (EBV) transforming protein LMP1 that constitutively associates with the tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein TRADD to mediate NF-kappaB and c-Jun N-terminal kinase activation is critical for long-term lymphoblastoid cell proliferation. We now find that LMP1 signaling through TRADD differs from TNFR1 signaling through TRADD. LMP1 needs only 11 amino acids to activate NF-kappaB or synergize with TRADD in NF-kappaB activation, while TNFR1 requires approximately 70 residues. Further, LMP1 does not require TRADD residues 294 to 312 for NF-kappaB activation, while TNFR1 requires TRADD residues 296 to 302. LMP1 is partially blocked for NF-kappaB activation by a TRADD mutant consisting of residues 122 to 293. Unlike TNFR1, LMP1 can interact directly with receptor-interacting protein (RIP) and stably associates with RIP in EBV-transformed lymphoblastoid cell lines. Surprisingly, LMP1 does not require RIP for NF-kappaB activation. Despite constitutive association with TRADD or RIP, LMP1 does not induce apoptosis in EBV-negative Burkitt lymphoma or human embryonic kidney 293 cells. These results add a different perspective to the molecular interactions through which LMP1, TRADD, and RIP participate in B-lymphocyte activation and growth. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "engages", "start": 61, "end": 68}, "arguments": [{"role": "Theme", "text": "latent membrane protein 1", "start": 35, "end": 60}, {"role": "Theme2", "text": "TRADD", "start": 124, "end": 129}]}, {"trigger": {"text": "engages", "start": 61, "end": 68}, "arguments": [{"role": "Theme", "text": "latent membrane protein 1", "start": 35, "end": 60}]}, {"trigger": {"text": "associates", "start": 326, "end": 336}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 301, "end": 305}, {"role": "Theme2", "text": "TRADD", "start": 419, "end": 424}]}, {"trigger": {"text": "interact", "start": 1044, "end": 1052}, "arguments": [{"role": "Theme", "text": "TNFR1", "start": 1028, "end": 1033}, {"role": "Theme2", "text": "TRADD", "start": 1490, "end": 1495}]}, {"trigger": {"text": "interact", "start": 1044, "end": 1052}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1035, "end": 1039}]}, {"trigger": {"text": "associates", "start": 1113, "end": 1123}, "arguments": [{"role": "Theme", "text": "TNFR1", "start": 1028, "end": 1033}]}, {"trigger": {"text": "associates", "start": 1113, "end": 1123}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1035, "end": 1039}]}, {"trigger": {"text": "association", "start": 1266, "end": 1277}, "arguments": [{"role": "Theme", "text": "TRADD", "start": 1283, "end": 1288}, {"role": "Theme2", "text": "LMP1", "start": 1297, "end": 1301}]}, {"trigger": {"text": "association", "start": 1266, "end": 1277}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1297, "end": 1301}]}]}}, "schema": []} {"input": "Regulatory effects of interleukin-11 during acute lung inflammatory injury. \nThe role of interleukin-11 (IL-11) was evaluated in the IgG immune complex model of acute lung injury in rats. IL-11 mRNA and protein were both up-regulated during the course of this inflammatory response. Exogenously administered IL-11 substantially reduced, in a dose-dependent manner, the intrapulmonary accumulation of neutrophils and the lung vascular leak of albumin. These in vivo anti-inflammatory effects of IL-11 were associated with reduced NF-kappaB activation in lung, reduced levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage (BAL) fluids, and diminished up-regulation of lung vascular ICAM-1. It is interesting that IL-11 did not affect BAL fluid content of the CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-inducible neutrophil chemoattractant (CINC); the presence of IL-11 did not affect these chemokines. However, BAL content of C5a was reduced by IL-11. These data indicate that IL-11 is a regulatory cytokine in the lung and that, like other members of this family, its anti-inflammatory properties appear to be linked to its suppression of NF-kappaB activation, diminished production of TNF-alpha, and reduced up-regulation of lung vascular ICAM-1. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 1222, "end": 1232}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1236, "end": 1245}]}], "negative regulation": [{"trigger": {"text": "reduced levels", "start": 559, "end": 573}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 606, "end": 615}]}, {"trigger": {"text": "diminished", "start": 661, "end": 671}, "arguments": [{"role": "Theme", "text": "up-regulation", "start": 672, "end": 685}]}, {"trigger": {"text": "diminished", "start": 1211, "end": 1221}, "arguments": [{"role": "Theme", "text": "production", "start": 1222, "end": 1232}]}, {"trigger": {"text": "reduced", "start": 1251, "end": 1258}, "arguments": [{"role": "Theme", "text": "up-regulation", "start": 1259, "end": 1272}]}], "positive regulation": [{"trigger": {"text": "up-regulated", "start": 221, "end": 233}, "arguments": [{"role": "Theme", "text": "IL-11", "start": 188, "end": 193}]}, {"trigger": {"text": "up-regulation", "start": 672, "end": 685}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 703, "end": 709}]}, {"trigger": {"text": "up-regulation", "start": 1259, "end": 1272}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 1290, "end": 1296}]}], "regulation": [{"trigger": {"text": "affect", "start": 748, "end": 754}, "arguments": [{"role": "Cause", "text": "IL-11", "start": 734, "end": 739}, {"role": "Theme", "text": "MIP-2", "start": 831, "end": 836}]}, {"trigger": {"text": "affect", "start": 926, "end": 932}, "arguments": [{"role": "Theme", "text": "MIP-2", "start": 831, "end": 836}, {"role": "Cause", "text": "IL-11", "start": 912, "end": 917}]}]}}, "schema": []} {"input": "ETS transcription factors regulate an enhancer activity in the third intron of TNF-alpha. \nWe describe an enhancer site in the third intron of tumor necrosis factor alpha (TNF-alpha). A reporter construct containing the 5'-flanking region of the mouse TNF-alpha gene displayed weak activity when transfected into RAW264.7 macrophage-like cells. The addition of the third intron of TNF-alpha to this construct resulted in an enhancement of CAT protein. This enhancement was eliminated if a conserved 20-bp sequence was removed from the intron or if a dominant-negative ets-binding factor was co-transfected with the reporter gene. Mutations of this site that destroyed potential ets transcription factor binding sites had reduced transcriptional activity. The major transcription factor that bound to the oligonucleotide was confirmed to be GABP by supershift and competition analysis. In RAW264.7 cells, the binding was constitutive, however, in bone marrow-derived macrophages binding activity was shown to be interferon-gamma inducible. This may imply a role for ets transcription factors in the production of TNF-alpha. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "bound", "start": 791, "end": 796}, "arguments": [{"role": "Theme", "text": "GABP", "start": 840, "end": 844}]}], "gene expression": [{"trigger": {"text": "activity", "start": 282, "end": 290}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 252, "end": 261}]}, {"trigger": {"text": "production", "start": 1098, "end": 1108}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1112, "end": 1121}]}], "negative regulation": [{"trigger": {"text": "eliminated", "start": 473, "end": 483}, "arguments": [{"role": "Theme", "text": "resulted in an enhancement", "start": 409, "end": 435}]}], "positive regulation": [{"trigger": {"text": "when", "start": 291, "end": 295}, "arguments": [{"role": "Theme", "text": "activity", "start": 282, "end": 290}]}, {"trigger": {"text": "resulted in an enhancement", "start": 409, "end": 435}, "arguments": [{"role": "Theme", "text": "CAT", "start": 439, "end": 442}]}, {"trigger": {"text": "inducible", "start": 1028, "end": 1037}, "arguments": [{"role": "Theme", "text": "bound", "start": 791, "end": 796}, {"role": "Cause", "text": "interferon-gamma", "start": 1011, "end": 1027}]}], "regulation": [{"trigger": {"text": "regulate", "start": 26, "end": 34}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 79, "end": 88}]}, {"trigger": {"text": "role", "start": 1056, "end": 1060}, "arguments": [{"role": "Theme", "text": "production", "start": 1098, "end": 1108}]}]}}, "schema": []} {"input": "Abnormal NF-kappa B activity in T lymphocytes from patients with systemic lupus erythematosus is associated with decreased p65-RelA protein expression. \nNumerous cellular and biochemical abnormalities in immune regulation have been described in patients with systemic lupus erythematosus (SLE), including surface Ag receptor-initiated signaling events and lymphokine production. Because NF-kappa B contributes to the transcription of numerous inflammatory genes and has been shown to be a molecular target of antiinflammatory drugs, we sought to characterize the functional role of the NF-kappa B protein complex in lupus T cells. Freshly isolated T cells from lupus patients, rheumatoid arthritis (RA) patients, and normal individuals were activated physiologically via the TCR with anti-CD3 and anti-CD28 Abs to assess proximal membrane signaling, and with PMA and a calcium ionophore (A23187) to bypass membrane-mediated signaling events. We measured the NF-kappa B binding activity in nuclear extracts by gel shift analysis. When compared with normal cells, the activation of NF-kappa B activity in SLE patients was significantly decreased in SLE, but not in RA, patients. NF-kappa B binding activity was absent in several SLE patients who were not receiving any medication, including corticosteroids. Also, NF-kappa B activity remained absent in follow-up studies. In supershift experiments using specific Abs, we showed that, in the group of SLE patients who displayed undetectable NF-kappa B activity, p65 complexes were not formed. Finally, immunoblot analysis of nuclear extracts showed decreased or absent p65 protein levels. As p65 complexes are transcriptionally active in comparison to the p50 homodimer, this novel finding may provide insight on the origin of abnormal cytokine or other gene transcription in SLE patients. ", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "decreased or absent", "start": 1596, "end": 1615}, "arguments": [{"role": "Theme", "text": "p65", "start": 1616, "end": 1619}]}], "positive regulation": [{"trigger": {"text": "transcriptionally active", "start": 1657, "end": 1681}, "arguments": [{"role": "Theme", "text": "p65", "start": 1639, "end": 1642}]}, {"trigger": {"text": "transcriptionally active", "start": 1657, "end": 1681}, "arguments": [{"role": "Theme", "text": "p50", "start": 1703, "end": 1706}]}]}}, "schema": []} {"input": "Possible differences in the mechanism(s) of action of different glucocorticoid hormone compounds. \nDifferent glucocorticoid hormones (GCH) show differences in the intensity and in the kinetics of their immunomodulating activity. The mechanism(s) of action of GCH is under investigation, but is has been noted that they exert immune activity via the genomic pathway. We have studied the effects of prednisone (PDN), deflazacort (DFC), and dexamethasone (DXM) on the production of cytokines (IL-2, IL-6, TNF-alpha, IL-10) by peripheral T lymphocytes, and the effects on the inhibition of NF-kB DNA binding activity by activated Jurkat cell line. The data obtained show that the three GCH molecules exert an immunosuppression on cytokine production by T lymphocytes and a strong decrease in the nuclear translocation of NF-kB in Jurkat cells; moreover, (a) not all the cytokines investigated were affected, and not with the same intensity, by the three GCH and (b) DXM inhibited the binding activity of NF-kB less than that of DFC and PDN. These data are in agreement with the concept that different GCH compounds might differ in their binding and affinity properties, tissue-specific metabolism, and interaction with transcription factor. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 465, "end": 475}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 490, "end": 494}]}, {"trigger": {"text": "production", "start": 465, "end": 475}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 496, "end": 500}]}, {"trigger": {"text": "production", "start": 465, "end": 475}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 502, "end": 511}]}, {"trigger": {"text": "production", "start": 465, "end": 475}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 513, "end": 518}]}], "regulation": [{"trigger": {"text": "effects", "start": 386, "end": 393}, "arguments": [{"role": "Theme", "text": "production", "start": 465, "end": 475}]}]}}, "schema": []} {"input": "The p53 paradox in the pathogenesis of tumor progression. \nRecent evidence suggests that the p53 molecule appears in two different forms: the mutant p53 that stimulates tumor progression, and wild type p53 that inhibits tumor progression. In addition, it has been established that tumor necrosis factor-alpha (TNF-alpha) can activate the expression of wild type p53 in concert with the nuclear transcription factor, NF-kappa B. Both TNF-alpha and NF-kappa B are also involved in the stimulation of the pathway that leads to the expression of major histocompatibility complex (MHC) class I molecules and, hence, antigen presentation to the T cells. In this paper we shall advance the hypothesis that: (i) TNF-alpha indirectly controls immune surveillance; and (ii) TNF-alpha controls DNA repair and tumor suppression through the regulation of wild type p53. Thus, it is hypothesized that elevated TNF-alpha is primarily responsible for promoting tumor progression. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 338, "end": 348}, "arguments": [{"role": "Theme", "text": "p53", "start": 362, "end": 365}]}], "positive regulation": [{"trigger": {"text": "activate", "start": 325, "end": 333}, "arguments": [{"role": "Cause", "text": "TNF-alpha", "start": 310, "end": 319}, {"role": "Theme", "text": "expression", "start": 338, "end": 348}]}, {"trigger": {"text": "elevated", "start": 887, "end": 895}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 896, "end": 905}]}], "regulation": [{"trigger": {"text": "regulation", "start": 828, "end": 838}, "arguments": [{"role": "Cause", "text": "TNF-alpha", "start": 764, "end": 773}, {"role": "Theme", "text": "p53", "start": 852, "end": 855}]}]}}, "schema": []} {"input": "Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic IkappaB kinase signalsome activation and IkappaB proteolysis. \nThe inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor (TNF) are potent activators of NF-kappaB. This study compared the effect of these stimuli on endogenous IkappaB kinase (IKK) signalsome activation and IkappaB phosphorylation/proteolysis in human monocytic cells and investigated the role of the signalsome proteins IKK-alpha, IKK-beta, NF-kappaB-inducing kinase (NIK), IKK-gamma (NF-kappaB essential modulator), and IKK complex-associated protein. Kinase assays showed that TNF elicited a rapid but short-lived induction of IKK activity with a 3-fold greater effect on IKK-alpha than on IKK-beta, peaking at 5 min. In contrast, LPS predominantly stimulated IKK-beta activity, which slowly increased, peaking at 30 min. A second peak was observed at a later time point following LPS stimulation, which consisted of both IKK-alpha and -beta activity. The endogenous levels of the signalsome components were unaffected by stimulation. Furthermore, our studies showed association of the IKK-alpha/beta heterodimer with NIK, IkappaB-alpha and -epsilon in unstimulated cells. Exposure to LPS or TNF led to differential patterns of IkappaB-alpha and IkappaB-epsilon disappearance from and reassembly with the signalsome, whereas IKK-alpha, IKK-beta, and NIK remained complex-associated. NIK cannot phosphorylate IkappaB-alpha directly, but it appears to be a functionally important subunit, because mutated NIK inhibited stimulus-induced kappaB-dependent transcription more effectively than mutated IKK-alpha or -beta. Overexpression of IKK complex-associated protein inhibited stimulus-mediated transcription, whereas NF-kappaB essential modulator enhanced it. The understanding of LPS- and TNF-induced signaling may allow the development of specific strategies to treat sepsis-associated disease. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "association", "start": 1137, "end": 1148}, "arguments": [{"role": "Theme", "text": "NIK", "start": 1188, "end": 1191}]}, {"trigger": {"text": "association", "start": 1137, "end": 1148}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 1193, "end": 1206}]}, {"trigger": {"text": "association", "start": 1137, "end": 1148}, "arguments": [{"role": "Theme", "text": "-epsilon", "start": 1211, "end": 1219}]}, {"trigger": {"text": "disappearance", "start": 1332, "end": 1345}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 1298, "end": 1311}]}, {"trigger": {"text": "disappearance", "start": 1332, "end": 1345}, "arguments": [{"role": "Theme", "text": "IkappaB-epsilon", "start": 1316, "end": 1331}]}, {"trigger": {"text": "reassembly", "start": 1355, "end": 1365}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 1298, "end": 1311}]}, {"trigger": {"text": "reassembly", "start": 1355, "end": 1365}, "arguments": [{"role": "Theme", "text": "IkappaB-epsilon", "start": 1316, "end": 1331}]}, {"trigger": {"text": "associated", "start": 1441, "end": 1451}, "arguments": [{"role": "Theme", "text": "IKK-alpha", "start": 1395, "end": 1404}]}, {"trigger": {"text": "associated", "start": 1441, "end": 1451}, "arguments": [{"role": "Theme", "text": "IKK-beta", "start": 1406, "end": 1414}]}, {"trigger": {"text": "associated", "start": 1441, "end": 1451}, "arguments": [{"role": "Theme", "text": "NIK", "start": 1420, "end": 1423}]}], "phosphorylation": [{"trigger": {"text": "phosphorylate", "start": 1464, "end": 1477}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 1478, "end": 1491}]}], "positive regulation": [{"trigger": {"text": "stimulated", "start": 819, "end": 829}, "arguments": [{"role": "Theme", "text": "IKK-beta", "start": 830, "end": 838}]}, {"trigger": {"text": "increased", "start": 862, "end": 871}, "arguments": [{"role": "Theme", "text": "stimulated", "start": 819, "end": 829}]}, {"trigger": {"text": "stimulation", "start": 955, "end": 966}, "arguments": [{"role": "Theme", "text": "-beta", "start": 1006, "end": 1011}]}, {"trigger": {"text": "stimulation", "start": 955, "end": 966}, "arguments": [{"role": "Theme", "text": "IKK-alpha", "start": 992, "end": 1001}]}, {"trigger": {"text": "led", "start": 1266, "end": 1269}, "arguments": [{"role": "Theme", "text": "disappearance", "start": 1332, "end": 1345}]}, {"trigger": {"text": "led", "start": 1266, "end": 1269}, "arguments": [{"role": "Theme", "text": "reassembly", "start": 1355, "end": 1365}]}, {"trigger": {"text": "phosphorylate", "start": 1464, "end": 1477}, "arguments": [{"role": "Cause", "text": "NIK", "start": 1453, "end": 1456}, {"role": "Theme", "text": "phosphorylate", "start": 1464, "end": 1477}]}], "regulation": [{"trigger": {"text": "effect", "start": 732, "end": 738}, "arguments": [{"role": "Theme", "text": "IKK-alpha", "start": 742, "end": 751}]}, {"trigger": {"text": "effect", "start": 732, "end": 738}, "arguments": [{"role": "Theme", "text": "IKK-beta", "start": 760, "end": 768}]}, {"trigger": {"text": "remained", "start": 1424, "end": 1432}, "arguments": [{"role": "Theme", "text": "associated", "start": 1441, "end": 1451}]}]}}, "schema": []} {"input": "Activation of protein kinase C and elevation of cAMP interact synergistically to raise c-Fos and AP-1 activity in Jurkat cells. \nWe have earlier found that in Jurkat cells activation of protein kinase C (PKC) enhances the cyclic adenosine monophosphate (cAMP) accumulation induced by adenosine receptor stimulation or activation of Gs. Here we have therefore examined the effect of the phorbol ester PMA (phorbol 12-myristate 13-acetate) which stimulates PKC and a combination of the adenosine receptor agonist NECA (5'-(N-ethyl)-carboxamido adenosine) and forskolin to raise cAMP, on the levels of c-Fos and Jun and on the binding and transcriptional activity of the transcription factor, activator protein-1 (AP-1). PMA treatment caused a concentration- and time-dependent increase in both c-Fos and Jun immunoreactivity in contrast to cAMP elevation that had only a slight effect. Both PMA and the combination of NECA and forskolin acted together either to increase (c-Fos) or decrease (Jun) protein levels as well as increasing AP-1 binding, as judged by gel-shift assay, and AP-1 transcriptional activity. Furthermore there was a clear-cut synergy between the PKC stimulator and the cAMP elevating agents. The results demonstrate that the simultaneous activation of PKC and elevation of cAMP leads to an enhanced AP-1 transcriptional activity in a T-leukemia cell line, suggesting that the previously observed interaction between the parallel signal transduction pathways may have functional consequences at the level of gene transcription. ", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "decrease", "start": 980, "end": 988}, "arguments": [{"role": "Theme", "text": "Jun", "start": 990, "end": 993}]}], "positive regulation": [{"trigger": {"text": "interact synergistically to raise", "start": 53, "end": 86}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 87, "end": 92}]}, {"trigger": {"text": "stimulates", "start": 444, "end": 454}, "arguments": [{"role": "Theme", "text": "PKC", "start": 455, "end": 458}]}, {"trigger": {"text": "levels", "start": 589, "end": 595}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 599, "end": 604}]}, {"trigger": {"text": "levels", "start": 589, "end": 595}, "arguments": [{"role": "Theme", "text": "Jun", "start": 609, "end": 612}]}, {"trigger": {"text": "increase", "start": 775, "end": 783}, "arguments": [{"role": "Theme", "text": "immunoreactivity", "start": 806, "end": 822}]}, {"trigger": {"text": "had only a slight effect", "start": 858, "end": 882}, "arguments": [{"role": "Theme", "text": "immunoreactivity", "start": 806, "end": 822}]}, {"trigger": {"text": "increase", "start": 960, "end": 968}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 970, "end": 975}]}, {"trigger": {"text": "activation", "start": 1257, "end": 1267}, "arguments": [{"role": "Theme", "text": "PKC", "start": 1271, "end": 1274}]}], "regulation": [{"trigger": {"text": "effect", "start": 372, "end": 378}, "arguments": [{"role": "Theme", "text": "levels", "start": 589, "end": 595}]}, {"trigger": {"text": "immunoreactivity", "start": 806, "end": 822}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 792, "end": 797}]}, {"trigger": {"text": "immunoreactivity", "start": 806, "end": 822}, "arguments": [{"role": "Theme", "text": "Jun", "start": 802, "end": 805}]}]}}, "schema": []} {"input": "Functional interaction between the two zinc finger domains of the v-erb A oncoprotein. \nThe v-erb A oncogene of avian erythroblastosis virus is a mutated and virally transduced copy of a host cell gene encoding a thyroid hormone receptor. The protein expressed by the v-erb A oncogene binds to DNA and acts as a dominant negative inhibitor of both the thyroid hormone receptor and the closely related retinoic acid receptor. The v-erb A protein has sustained two amino acid alterations within its DNA-binding domain relative to that of c-erb A, one of which, at serine 61, is known to be important for v-erb A function in the neoplastic cell. We report here that the second alteration, at threonine 78, also plays an important, although more indirect, role: alteration of the sequence at threonine 78 such that it resembles that of c-erb A can act as an intragenic suppressor and can partially restore function to a v-erb A protein rendered defective due to a mutation at position 61. Threonine 78 lies within the D-box of the v-erb A protein, a region thought to mediate receptor-receptor dimerizations, and is not in physical proximity to the serine at position 61. It therefore appears that an indirect interaction occurs between these two sites and that this interaction is crucial for v-erb A function. ", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "acts as a dominant negative inhibitor", "start": 302, "end": 339}, "arguments": [{"role": "Theme", "text": "retinoic acid receptor", "start": 401, "end": 423}]}, {"trigger": {"text": "rendered defective", "start": 932, "end": 950}, "arguments": [{"role": "Theme", "text": "v-erb A", "start": 916, "end": 923}]}], "positive regulation": [{"trigger": {"text": "restore function", "start": 894, "end": 910}, "arguments": [{"role": "Theme", "text": "v-erb A", "start": 916, "end": 923}]}, {"trigger": {"text": "crucial", "start": 1278, "end": 1285}, "arguments": [{"role": "Theme", "text": "v-erb A", "start": 1290, "end": 1297}]}], "regulation": [{"trigger": {"text": "important", "start": 588, "end": 597}, "arguments": [{"role": "Theme", "text": "v-erb A", "start": 602, "end": 609}]}, {"trigger": {"text": "role", "start": 752, "end": 756}, "arguments": [{"role": "Theme", "text": "v-erb A", "start": 602, "end": 609}]}]}}, "schema": []} {"input": "Inhibition of transcription factors belonging to the rel/NF-kappa B family by a transdominant negative mutant. \nThe KBF1 factor, which binds to the enhancer A located in the promoter of the mouse MHC class I gene H-2Kb, is indistinguishable from the p50 DNA binding subunit of the transcription factor NF-kappa B, which regulates a series of genes involved in immune and inflammatory responses. The KBF1/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family, like the c-rel and v-rel (proto)oncogenes. The dimerization domain of KBF1/p50 is contained between amino acids 201 and 367. A mutant of KBF1/p50 (delta SP), unable to bind to DNA but able to form homo- or heterodimers, has been constructed. This protein reduces or abolishes in vitro the DNA binding activity of wild-type proteins of the same family (KBF1/p50, c- and v-rel). This mutant also functions in vivo as a trans-acting dominant negative regulator: the transcriptional inducibility of the HIV long terminal repeat (which contains two potential NF-kappa B binding sites) by phorbol ester (PMA) is inhibited when it is co-transfected into CD4+ T cells with the delta SP mutant. Similarly the basal as well as TNF or IL1-induced activity of the MHC class I H-2Kb promoter can be inhibited by this mutant in two different cell lines. These results constitute the first formal demonstration that these genes are regulated by members of the rel/NF-kappa B family. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 135, "end": 140}, "arguments": [{"role": "Theme", "text": "KBF1", "start": 116, "end": 120}, {"role": "Site2", "text": "enhancer A", "start": 148, "end": 158}, {"role": "Theme2", "text": "H-2Kb", "start": 213, "end": 218}]}, {"trigger": {"text": "binds as a homodimer", "start": 415, "end": 435}, "arguments": [{"role": "Theme", "text": "KBF1", "start": 399, "end": 403}]}, {"trigger": {"text": "binds as a homodimer", "start": 415, "end": 435}, "arguments": [{"role": "Theme", "text": "p50", "start": 404, "end": 407}]}, {"trigger": {"text": "form heterodimers", "start": 449, "end": 466}, "arguments": [{"role": "Theme", "text": "KBF1", "start": 399, "end": 403}, {"role": "Theme2", "text": "c-rel", "start": 531, "end": 536}]}, {"trigger": {"text": "form heterodimers", "start": 449, "end": 466}, "arguments": [{"role": "Theme", "text": "p50", "start": 404, "end": 407}, {"role": "Theme2", "text": "c-rel", "start": 531, "end": 536}]}, {"trigger": {"text": "form heterodimers", "start": 449, "end": 466}, "arguments": [{"role": "Theme", "text": "KBF1", "start": 399, "end": 403}, {"role": "Theme2", "text": "v-rel", "start": 541, "end": 546}]}, {"trigger": {"text": "form heterodimers", "start": 449, "end": 466}, "arguments": [{"role": "Theme", "text": "p50", "start": 404, "end": 407}, {"role": "Theme2", "text": "v-rel", "start": 541, "end": 546}]}, {"trigger": {"text": "bind", "start": 690, "end": 694}, "arguments": [{"role": "Theme", "text": "KBF1", "start": 659, "end": 663}]}, {"trigger": {"text": "bind", "start": 690, "end": 694}, "arguments": [{"role": "Theme", "text": "p50", "start": 664, "end": 667}]}, {"trigger": {"text": "form homo-", "start": 714, "end": 724}, "arguments": [{"role": "Theme", "text": "KBF1", "start": 659, "end": 663}]}, {"trigger": {"text": "form homo-", "start": 714, "end": 724}, "arguments": [{"role": "Theme", "text": "p50", "start": 664, "end": 667}]}, {"trigger": {"text": "heterodimers", "start": 728, "end": 740}, "arguments": [{"role": "Theme", "text": "c-rel", "start": 531, "end": 536}, {"role": "Theme2", "text": "KBF1", "start": 659, "end": 663}]}, {"trigger": {"text": "heterodimers", "start": 728, "end": 740}, "arguments": [{"role": "Theme", "text": "v-rel", "start": 541, "end": 546}, {"role": "Theme2", "text": "KBF1", "start": 659, "end": 663}]}, {"trigger": {"text": "heterodimers", "start": 728, "end": 740}, "arguments": [{"role": "Theme", "text": "c-rel", "start": 531, "end": 536}, {"role": "Theme2", "text": "p50", "start": 664, "end": 667}]}, {"trigger": {"text": "heterodimers", "start": 728, "end": 740}, "arguments": [{"role": "Theme", "text": "v-rel", "start": 541, "end": 546}, {"role": "Theme2", "text": "p50", "start": 664, "end": 667}]}, {"trigger": {"text": "binding", "start": 815, "end": 822}, "arguments": [{"role": "Theme", "text": "KBF1", "start": 874, "end": 878}]}, {"trigger": {"text": "binding", "start": 815, "end": 822}, "arguments": [{"role": "Theme", "text": "p50", "start": 879, "end": 882}]}, {"trigger": {"text": "binding", "start": 815, "end": 822}, "arguments": [{"role": "Theme", "text": "c-", "start": 884, "end": 886}]}, {"trigger": {"text": "binding", "start": 815, "end": 822}, "arguments": [{"role": "Theme", "text": "v-rel", "start": 891, "end": 896}]}], "negative regulation": [{"trigger": {"text": "reduces or abolishes", "start": 777, "end": 797}, "arguments": [{"role": "Cause", "text": "p50", "start": 664, "end": 667}, {"role": "Theme", "text": "binding", "start": 815, "end": 822}]}]}}, "schema": []} {"input": "NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I. \nThe effect of constitutive Tax expression on the interaction of NF-kappa B with its recognition sequence and on NF-kappa B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-kappa B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-kappa B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85, 75, and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressing", "start": 38, "end": 48}, "arguments": [{"role": "Theme", "text": "Tax", "start": 53, "end": 56}]}, {"trigger": {"text": "expression", "start": 140, "end": 150}, "arguments": [{"role": "Theme", "text": "Tax", "start": 136, "end": 139}]}, {"trigger": {"text": "expressing", "start": 369, "end": 379}, "arguments": [{"role": "Theme", "text": "Tax", "start": 365, "end": 368}]}, {"trigger": {"text": "expressing", "start": 739, "end": 749}, "arguments": [{"role": "Theme", "text": "Tax", "start": 735, "end": 738}]}, {"trigger": {"text": "Expression", "start": 837, "end": 847}, "arguments": [{"role": "Theme", "text": "Tax", "start": 851, "end": 854}]}, {"trigger": {"text": "expression", "start": 1190, "end": 1200}, "arguments": [{"role": "Theme", "text": "Tax", "start": 1186, "end": 1189}]}], "positive regulation": [{"trigger": {"text": "resulted", "start": 877, "end": 885}, "arguments": [{"role": "Cause", "text": "Expression", "start": 837, "end": 847}, {"role": "Theme", "text": "dependent", "start": 932, "end": 941}]}, {"trigger": {"text": "dependent", "start": 932, "end": 941}, "arguments": [{"role": "Theme", "text": "transcription", "start": 889, "end": 902}]}], "transcription": [{"trigger": {"text": "transcription", "start": 889, "end": 902}, "arguments": [{"role": "Theme", "text": "granulocyte-macrophage colony stimulating factor", "start": 942, "end": 990}]}]}}, "schema": []} {"input": "Anti-CD2 receptor antibodies activate the HIV long terminal repeat in T lymphocytes. \nThe CD2 T lymphocyte glycoprotein surface molecule mediates both cell to cell adhesion and T cell activation, two processes that are involved in the spread of HIV infection. Treatment of chronically HIV-infected PBMC with anti-CD2 mAb has been shown to induce the expression of infectious virus from these cultures. In this study we investigated the mechanisms whereby anti-CD2 antibodies stimulate viral production. We demonstrate that treatment of transiently transfected T lymphocytes with anti-CD2 antibodies results in activation of the HIV long terminal repeat. Furthermore, CAT assays using mutated HIV long terminal repeat-CAT constructs and gel shift assays demonstrate that this activation is dependent on the NF-kappa B enhancer. These studies suggest that interaction of CD2 with its natural ligand, LFA-3, may play a role in regulation of HIV expression. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 854, "end": 865}, "arguments": [{"role": "Theme", "text": "CD2", "start": 869, "end": 872}, {"role": "Theme2", "text": "LFA-3", "start": 898, "end": 903}]}]}}, "schema": []} {"input": "v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII. \nThe v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone (T3/T4) receptor c-erbA (type alpha). It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte-specific genes. Here, we show that v-erbA and c-erbA bind directly to sequences within the promoter of the erythrocyte-specific carbonic anhydrase II (CAII), a gene whose transcription is efficiently suppressed by v-erbA. This erbA-binding site confers thyroid hormone responsiveness to a heterologous promoter in transient expression experiments and is a target for efficient down-regulation of CAII transcription by the v-erbA oncoprotein. In stably transformed erythroblasts coexpressing the v-erbA oncoprotein and the c-erbA/T3 receptor at an approximately equimolar ratio, c-erbA activity is dominant over v-erbA. T3 efficiently induced erythroid differentiation in these cells, thus overcoming the v-erbA-mediated differentiation arrest. Likewise, T3 activated CAII transcription as well as transient expression of a T3-responsive reporter gene containing the CAII-specific erbA-binding site. The c-erbA-dependent activation of this CAII reporter construct could only be suppressed by very high amounts of v-erbA. Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 495, "end": 499}, "arguments": [{"role": "Theme", "text": "v-erbA", "start": 477, "end": 483}, {"role": "Theme2", "text": "CAII", "start": 593, "end": 597}]}, {"trigger": {"text": "bind", "start": 495, "end": 499}, "arguments": [{"role": "Theme", "text": "c-erbA", "start": 488, "end": 494}, {"role": "Theme2", "text": "CAII", "start": 593, "end": 597}]}], "gene expression": [{"trigger": {"text": "overexpression", "start": 7, "end": 21}, "arguments": [{"role": "Theme", "text": "v-erbA", "start": 0, "end": 6}]}, {"trigger": {"text": "coexpressing", "start": 920, "end": 932}, "arguments": [{"role": "Theme", "text": "v-erbA", "start": 937, "end": 943}]}, {"trigger": {"text": "coexpressing", "start": 920, "end": 932}, "arguments": [{"role": "Theme", "text": "c-erbA", "start": 964, "end": 970}]}, {"trigger": {"text": "overexpression", "start": 1487, "end": 1501}, "arguments": [{"role": "Theme", "text": "v-erbA", "start": 1505, "end": 1511}]}], "negative regulation": [{"trigger": {"text": "extinguish", "start": 37, "end": 47}, "arguments": [{"role": "Cause", "text": "overexpression", "start": 7, "end": 21}, {"role": "Theme", "text": "regulation", "start": 102, "end": 112}]}, {"trigger": {"text": "suppressed", "start": 642, "end": 652}, "arguments": [{"role": "Theme", "text": "transcription", "start": 613, "end": 626}, {"role": "Cause", "text": "v-erbA", "start": 656, "end": 662}]}, {"trigger": {"text": "down-regulation", "start": 819, "end": 834}, "arguments": [{"role": "Theme", "text": "transcription", "start": 843, "end": 856}]}], "positive regulation": [{"trigger": {"text": "activated", "start": 1199, "end": 1208}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1214, "end": 1227}]}, {"trigger": {"text": "overexpression", "start": 1487, "end": 1501}, "arguments": [{"role": "Theme", "text": "overexpression", "start": 1487, "end": 1501}]}, {"trigger": {"text": "required", "start": 1515, "end": 1523}, "arguments": [{"role": "Cause", "text": "overexpression", "start": 1487, "end": 1501}, {"role": "Theme", "text": "v-erbA", "start": 1505, "end": 1511}]}], "regulation": [{"trigger": {"text": "regulation", "start": 102, "end": 112}, "arguments": [{"role": "Cause", "text": "c-erbA", "start": 48, "end": 54}, {"role": "Theme", "text": "CAII", "start": 137, "end": 141}]}, {"trigger": {"text": "target", "start": 798, "end": 804}, "arguments": [{"role": "Theme", "text": "down-regulation", "start": 819, "end": 834}, {"role": "Cause", "text": "v-erbA", "start": 864, "end": 870}]}], "transcription": [{"trigger": {"text": "transcription", "start": 613, "end": 626}, "arguments": [{"role": "Theme", "text": "CAII", "start": 593, "end": 597}]}, {"trigger": {"text": "transcription", "start": 843, "end": 856}, "arguments": [{"role": "Theme", "text": "CAII", "start": 838, "end": 842}]}, {"trigger": {"text": "transcription", "start": 1214, "end": 1227}, "arguments": [{"role": "Theme", "text": "CAII", "start": 1209, "end": 1213}]}]}}, "schema": []} {"input": "NF-X2 that binds to the DRA X2-box is activator protein 1. Expression cloning of c-Jun. \nHuman class II MHC Ag are a family of cell surface glycoproteins. Their constitutive expression is limited to B lymphocytes and thymic epithelial cells. In many other cells their expression can be induced by IFN-gamma. Conserved upstream promoter sequences regulate this tissue-specific expression of class II genes. In the DRA promoter, one of these cis-acting regulatory motifs is the X2-box to which nuclear factor X2 (NF-X2) binds. Here, we present the isolation and characterization of the full-length cDNA clone encoding NF-X2. This cDNA clone was isolated by expression cDNA cloning, and encodes the human c-Jun protein, which together with c-Fos forms the heterodimeric activator protein-1 transcription complex. Whereas c-Fos/c-Jun heterodimers do not exist in B cells, they form and bind to the X2-box in class II nonexpressing cells. Thus, c-Fos/c-Jun heterodimers might contribute to the repression of DRA gene expression. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "complex", "start": 801, "end": 808}, "arguments": [{"role": "Theme", "text": "c-Jun", "start": 702, "end": 707}, {"role": "Theme2", "text": "c-Fos", "start": 737, "end": 742}]}, {"trigger": {"text": "form", "start": 873, "end": 877}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 818, "end": 823}, {"role": "Theme2", "text": "c-Jun", "start": 824, "end": 829}]}, {"trigger": {"text": "bind", "start": 882, "end": 886}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 818, "end": 823}]}, {"trigger": {"text": "bind", "start": 882, "end": 886}, "arguments": [{"role": "Theme", "text": "c-Jun", "start": 824, "end": 829}]}], "gene expression": [{"trigger": {"text": "exist", "start": 850, "end": 855}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 818, "end": 823}]}, {"trigger": {"text": "exist", "start": 850, "end": 855}, "arguments": [{"role": "Theme", "text": "c-Jun", "start": 824, "end": 829}]}, {"trigger": {"text": "expression", "start": 1012, "end": 1022}, "arguments": [{"role": "Theme", "text": "DRA", "start": 1003, "end": 1006}]}], "negative regulation": [{"trigger": {"text": "repression", "start": 989, "end": 999}, "arguments": [{"role": "Cause", "text": "c-Fos", "start": 940, "end": 945}, {"role": "Theme", "text": "expression", "start": 1012, "end": 1022}]}, {"trigger": {"text": "repression", "start": 989, "end": 999}, "arguments": [{"role": "Cause", "text": "c-Jun", "start": 946, "end": 951}, {"role": "Theme", "text": "expression", "start": 1012, "end": 1022}]}]}}, "schema": []} {"input": "Regulation of jun and fos gene expression in human monocytes by the macrophage colony-stimulating factor. \nThe macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. However, the signaling events responsible for these effects remain unclear. The present studies have examined the effects of M-CSF on potential signaling pathways involving expression of the jun and fos early response genes. Low levels of c-jun transcripts were detectable in resting human peripheral blood monocytes. Treatment of these cells with 10(3) units/ml human recombinant M-CSF was associated with rapid and transient increases in c-jun mRNA levels. Nuclear run-on assays and mRNA stability studies demonstrated that M-CSF regulates c-jun expression by both an increase in transcription rate and a prolongation in the half-life of c-jun transcripts. M-CSF treatment was also associated with a rapid induction of the jun-B gene, although expression of this gene was prolonged compared to that of c-jun. We further demonstrate that M-CSF increases c-fos mRNA levels in human monocytes through control at both the transcriptional and posttranscriptional levels. Maximal induction of the c-fos gene was followed by that for the fos-B gene. Moreover, M-CSF-induced expression of the fos-related gene, fra-1, was delayed compared to that for both c-fos and fos-B. Taken together, the results indicate that M-CSF treatment is associated with differential activation of multiple members of the jun/fos family and that expression of these genes could contribute to nuclear signaling mechanisms that regulate a specific program of monocyte differentiation. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "gene expression", "start": 26, "end": 41}, "arguments": [{"role": "Theme", "text": "jun", "start": 14, "end": 17}]}, {"trigger": {"text": "gene expression", "start": 26, "end": 41}, "arguments": [{"role": "Theme", "text": "fos", "start": 22, "end": 25}]}, {"trigger": {"text": "expression", "start": 403, "end": 413}, "arguments": [{"role": "Theme", "text": "jun", "start": 421, "end": 424}]}, {"trigger": {"text": "expression", "start": 403, "end": 413}, "arguments": [{"role": "Theme", "text": "fos", "start": 429, "end": 432}]}, {"trigger": {"text": "expression", "start": 778, "end": 788}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 772, "end": 777}]}, {"trigger": {"text": "expression", "start": 976, "end": 986}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 1034, "end": 1039}]}, {"trigger": {"text": "expression", "start": 976, "end": 986}, "arguments": [{"role": "Theme", "text": "jun-B", "start": 955, "end": 960}]}, {"trigger": {"text": "expression", "start": 1299, "end": 1309}, "arguments": [{"role": "Theme", "text": "fra-1", "start": 1335, "end": 1340}]}, {"trigger": {"text": "expression", "start": 1299, "end": 1309}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1380, "end": 1385}]}, {"trigger": {"text": "expression", "start": 1299, "end": 1309}, "arguments": [{"role": "Theme", "text": "fos-B", "start": 1390, "end": 1395}]}], "positive regulation": [{"trigger": {"text": "detectable", "start": 492, "end": 502}, "arguments": [{"role": "Theme", "text": "transcripts", "start": 475, "end": 486}]}, {"trigger": {"text": "increases", "start": 657, "end": 666}, "arguments": [{"role": "Theme", "text": "levels", "start": 681, "end": 687}]}, {"trigger": {"text": "increase", "start": 800, "end": 808}, "arguments": [{"role": "Theme", "text": "transcription", "start": 812, "end": 825}]}, {"trigger": {"text": "prolongation in the half-life", "start": 837, "end": 866}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 870, "end": 875}]}, {"trigger": {"text": "induction", "start": 938, "end": 947}, "arguments": [{"role": "Theme", "text": "jun-B", "start": 955, "end": 960}]}, {"trigger": {"text": "prolonged", "start": 1004, "end": 1013}, "arguments": [{"role": "Theme", "text": "expression", "start": 976, "end": 986}]}, {"trigger": {"text": "increases", "start": 1075, "end": 1084}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1085, "end": 1090}]}, {"trigger": {"text": "induction", "start": 1206, "end": 1215}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1223, "end": 1228}]}, {"trigger": {"text": "induction", "start": 1206, "end": 1215}, "arguments": [{"role": "Theme", "text": "fos-B", "start": 1263, "end": 1268}]}, {"trigger": {"text": "induced", "start": 1291, "end": 1298}, "arguments": [{"role": "Theme", "text": "expression", "start": 1299, "end": 1309}]}], "regulation": [{"trigger": {"text": "Regulation", "start": 0, "end": 10}, "arguments": [{"role": "Theme", "text": "gene expression", "start": 26, "end": 41}]}, {"trigger": {"text": "effects", "start": 344, "end": 351}, "arguments": [{"role": "Theme", "text": "involving", "start": 393, "end": 402}]}, {"trigger": {"text": "involving", "start": 393, "end": 402}, "arguments": [{"role": "Theme", "text": "expression", "start": 403, "end": 413}]}, {"trigger": {"text": "regulates", "start": 762, "end": 771}, "arguments": [{"role": "Theme", "text": "expression", "start": 778, "end": 788}]}], "transcription": [{"trigger": {"text": "transcripts", "start": 475, "end": 486}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 469, "end": 474}]}, {"trigger": {"text": "levels", "start": 681, "end": 687}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 670, "end": 675}]}, {"trigger": {"text": "transcription", "start": 812, "end": 825}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 870, "end": 875}]}]}}, "schema": []} {"input": "cAMP-dependent regulation of proenkephalin by JunD and JunB: positive and negative effects of AP-1 proteins. \nWe demonstrate that JunD, a component of the AP-1 transcription factor complex, activates transcription of the human proenkephalin gene in a fashion that is completely dependent upon the cAMP-dependent protein kinase, protein kinase A. Activation of proenkephalin transcription by JunD is dependent upon a previously characterized cAMP-, phorbol ester-, and Ca(2+)-inducible enhancer, and JunD is shown to bind the enhancer as a homodimer. Another component of the AP-1 transcription complex, JunB, is shown to inhibit activation mediated by JunD. As a homodimer JunB is unable to bind the enhancer; however in the presence of c-Fos, high-affinity binding is observed. Furthermore, JunD is shown to activate transcription of genes linked to both cAMP and phorbol ester response elements in a protein kinase A-dependent fashion, further blurring the distinction between these response elements. These results demonstrate that the transcriptional activity of an AP-1-related protein is regulated by the cAMP-dependent second-messenger pathway and suggest that JunD and other AP-1-related proteins may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 516, "end": 520}, "arguments": [{"role": "Theme", "text": "JunD", "start": 499, "end": 503}]}, {"trigger": {"text": "bind", "start": 691, "end": 695}, "arguments": [{"role": "Theme", "text": "JunB", "start": 673, "end": 677}]}, {"trigger": {"text": "binding", "start": 758, "end": 765}, "arguments": [{"role": "Theme", "text": "JunB", "start": 673, "end": 677}]}], "positive regulation": [{"trigger": {"text": "in the presence of", "start": 718, "end": 736}, "arguments": [{"role": "Cause", "text": "c-Fos", "start": 737, "end": 742}, {"role": "Theme", "text": "binding", "start": 758, "end": 765}]}]}}, "schema": []} {"input": "Induction of monocytic differentiation and NF-kappa B-like activities by human immunodeficiency virus 1 infection of myelomonoblastic cells. \nThe effects of human immunodeficiency virus 1 (HIV-1) infection on cellular differentiation and NF-kappa B DNA binding activity have been investigated in a new model of myeloid differentiation. PLB-985 cells represent a bipotential myelomonoblastic cell population capable of either granulocytic or monocytic differentiation after induction with appropriate inducers. By virtue of the presence of CD4 on the cell surface, PLB-985 cells were chronically infected with HIV-1 strain IIIB. PLB-IIIB cells clearly possessed a more monocytic phenotype than the parental myeloblasts, as determined by differential staining, increased expression of the myeloid-specific surface markers, and transcription of the c-fms proto-oncogene. NF-kappa B binding activity was inducible by tumor necrosis factor and phorbol myristate acetate in PLB-985. However, in PLB-IIIB cells, constitutive expression of a novel NF-kappa B complex was detected, composed of proteins ranging between 70 and 110 kD. These proteins interacted specifically with the symmetric NF-kappa B site from the interferon beta (IFN-beta) promoter. Mutations affecting the 5' guanine residues of the kappa B site were unable to compete for these NF-kappa B-related proteins. Inducibility of endogenous IFN-beta and IFN-alpha RNA was also increased in PLB-IIIB cells. These studies indicate that HIV-1 infection of myelomonoblastic cells may select for a more mature monocytic phenotype and that unique subunit associations of NF-kappa B DNA binding proteins may contribute to differential NF-kappa B-mediated gene expression. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interacted", "start": 1140, "end": 1150}, "arguments": [{"role": "Site", "text": "symmetric NF-kappa B site", "start": 1173, "end": 1198}, {"role": "Theme", "text": "interferon beta", "start": 1208, "end": 1223}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 759, "end": 768}, "arguments": [{"role": "Theme", "text": "transcription", "start": 825, "end": 838}]}, {"trigger": {"text": "increased", "start": 1434, "end": 1443}, "arguments": [{"role": "Theme", "text": "Inducibility", "start": 1371, "end": 1383}]}], "transcription": [{"trigger": {"text": "transcription", "start": 825, "end": 838}, "arguments": [{"role": "Theme", "text": "c-fms", "start": 846, "end": 851}]}, {"trigger": {"text": "Inducibility", "start": 1371, "end": 1383}, "arguments": [{"role": "Theme", "text": "IFN-beta", "start": 1398, "end": 1406}]}, {"trigger": {"text": "Inducibility", "start": 1371, "end": 1383}, "arguments": [{"role": "Theme", "text": "IFN-alpha", "start": 1411, "end": 1420}]}]}}, "schema": []} {"input": "The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter. \nStimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at -150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c-jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "induction", "start": 371, "end": 380}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 403, "end": 407}]}, {"trigger": {"text": "expression", "start": 1542, "end": 1552}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1526, "end": 1531}]}, {"trigger": {"text": "expression", "start": 1542, "end": 1552}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 1536, "end": 1541}]}], "negative regulation": [{"trigger": {"text": "abrogates", "start": 655, "end": 664}, "arguments": [{"role": "Theme", "text": "induction", "start": 665, "end": 674}]}, {"trigger": {"text": "abolishes", "start": 748, "end": 757}, "arguments": [{"role": "Theme", "text": "induction", "start": 758, "end": 767}]}, {"trigger": {"text": "abrogate", "start": 1356, "end": 1364}, "arguments": [{"role": "Theme", "text": "induction", "start": 1365, "end": 1374}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 758, "end": 767}, "arguments": [{"role": "Theme", "text": "chloramphenicol acetyltransferase", "start": 771, "end": 804}]}, {"trigger": {"text": "inducibility", "start": 1244, "end": 1256}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1264, "end": 1268}, {"role": "Site", "text": "promoter", "start": 1269, "end": 1277}]}, {"trigger": {"text": "induction", "start": 1365, "end": 1374}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1382, "end": 1386}, {"role": "Site", "text": "promoter", "start": 1387, "end": 1395}]}, {"trigger": {"text": "enhance", "start": 1571, "end": 1578}, "arguments": [{"role": "Cause", "text": "expression", "start": 1542, "end": 1552}, {"role": "Theme", "text": "induction", "start": 1579, "end": 1588}]}, {"trigger": {"text": "induction", "start": 1579, "end": 1588}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1596, "end": 1600}, {"role": "Site", "text": "promoter", "start": 1601, "end": 1609}]}], "regulation": [{"trigger": {"text": "role", "start": 356, "end": 360}, "arguments": [{"role": "Theme", "text": "induction", "start": 371, "end": 380}]}, {"trigger": {"text": "effect", "start": 1234, "end": 1240}, "arguments": [{"role": "Theme", "text": "inducibility", "start": 1244, "end": 1256}]}], "transcription": [{"trigger": {"text": "induction", "start": 665, "end": 674}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 678, "end": 682}]}]}}, "schema": []} {"input": "The role of jun and fos gene family members in 12-O-tetradecanoylphorbol-13-acetate induced hemopoietic differentiation. \nTerminal differentiation of the leukemic cell lines U-937 and HL-60 by 12-O-tetradecanoylphorbol-13-acetate is accompanied by marked changes in gene expression. In this study, we demonstrate that the expression of jun and fos gene family members is induced with variable kinetics during 12-O-tetradecanoylphorbol-13-acetate induced differentiation, with c-jun expression best paralleling differentiation. The generation of AP-1 complexes, as measured by DNA binding activity, closely parallels morphological differentiation. Furthermore, the ability of these complexes to regulate gene expression is demonstrated by increased transcription from an AP-1 driven reporter construct and marked increases in the expression of endogenous AP-1 regulated genes. Differentiation assays using water soluble phorbol esters reveal that differentiation becomes irreversible soon after AP-1 appears. This tight correlation between c-jun expression, the generation of AP-1 activity, and differentiation suggests a critical role for this gene and transcriptional complex during this process. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 482, "end": 492}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 476, "end": 481}]}, {"trigger": {"text": "expression", "start": 1045, "end": 1055}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 1039, "end": 1044}]}]}}, "schema": []} {"input": "Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein. \nDimerization among transcription factors has become a recurrent theme in the regulation of eukaryotic gene expression. Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) is a homeodomain-containing protein that functions as a dimer. A dimerization cofactor of HNF-1 alpha (DCoH) was identified that displayed a restricted tissue distribution and did not bind to DNA, but, rather, selectively stabilized HNF-1 alpha dimers. The formation of a stable tetrameric DCoH-HNF-1 alpha complex, which required the dimerization domain of HNF-1 alpha, did not change the DNA binding characteristics of HNF-1 alpha, but enhanced its transcriptional activity. However, DCoH did not confer transcriptional activation to the GAL4 DNA binding domain. These results indicate that DCoH regulates formation of transcriptionally active tetrameric complexes and may contribute to the developmental specificity of the complex. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 447, "end": 451}, "arguments": [{"role": "Theme", "text": "DCoH", "start": 366, "end": 370}]}, {"trigger": {"text": "complex", "start": 570, "end": 577}, "arguments": [{"role": "Theme", "text": "DCoH", "start": 553, "end": 557}, {"role": "Theme2", "text": "HNF-1 alpha", "start": 558, "end": 569}]}, {"trigger": {"text": "binding", "start": 657, "end": 664}, "arguments": [{"role": "Theme", "text": "HNF-1 alpha", "start": 684, "end": 695}]}], "localization": [{"trigger": {"text": "distribution", "start": 422, "end": 434}, "arguments": [{"role": "Theme", "text": "DCoH", "start": 366, "end": 370}]}], "positive regulation": [{"trigger": {"text": "stabilized", "start": 485, "end": 495}, "arguments": [{"role": "Cause", "text": "DCoH", "start": 366, "end": 370}, {"role": "Theme", "text": "HNF-1 alpha", "start": 496, "end": 507}]}, {"trigger": {"text": "required", "start": 585, "end": 593}, "arguments": [{"role": "Theme", "text": "complex", "start": 570, "end": 577}]}], "regulation": [{"trigger": {"text": "change", "start": 642, "end": 648}, "arguments": [{"role": "Cause", "text": "complex", "start": 570, "end": 577}, {"role": "Theme", "text": "binding", "start": 657, "end": 664}]}]}}, "schema": []} {"input": "Nuclear factor kappa B activates proenkephalin transcription in T lymphocytes. \nUpon activation, T lymphocytes accumulate high levels of the neuropeptide enkephalin which correlate with high levels of proenkephalin mRNA in the cells. Here we investigated the transcriptional basis for these changes. The proenkephalin promoter contains a sequence GGGGACGTCCCC, named B2, which is similar to the kappa B sequence GGGGACTTTCC, the binding site of the transcription factor nuclear factor (NF)-kappa B. Activation of T lymphocytes induces an NF-kappa B-like binding activity to the B2 site, concomitant with activation of the proenkephalin promoter. Mutations at the B2 site abolish this transcriptional activation. The purified homodimer (two p50s) of the DNA-binding subunit of NF-kappa B binds the B2 site of proenkephalin relatively better than does the heterotetramer (two p65s plus two p50s) form of the factor. Thus, it appears that the T-cell-specific activation of the proenkephalin promoter is mediated by NF-kappa B. However, as NF-kappa B is ubiquitous and the transcriptional activation through the B2 site is T cell specific, yet another T-cell-specific factor which synergizes with NF-kappa B should be considered. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "homodimer", "start": 725, "end": 734}, "arguments": [{"role": "Theme", "text": "p50", "start": 740, "end": 743}]}, {"trigger": {"text": "binds", "start": 787, "end": 792}, "arguments": [{"role": "Site", "text": "B2 site", "start": 797, "end": 804}, {"role": "Theme", "text": "proenkephalin", "start": 808, "end": 821}]}, {"trigger": {"text": "heterotetramer", "start": 854, "end": 868}, "arguments": [{"role": "Theme", "text": "p65", "start": 874, "end": 877}, {"role": "Theme2", "text": "p50", "start": 888, "end": 891}]}], "negative regulation": [{"trigger": {"text": "abolish", "start": 671, "end": 678}, "arguments": [{"role": "Theme", "text": "activation", "start": 604, "end": 614}]}], "positive regulation": [{"trigger": {"text": "activates", "start": 23, "end": 32}, "arguments": [{"role": "Theme", "text": "transcription", "start": 47, "end": 60}]}, {"trigger": {"text": "activation", "start": 604, "end": 614}, "arguments": [{"role": "Theme", "text": "proenkephalin", "start": 622, "end": 635}, {"role": "Site", "text": "promoter", "start": 636, "end": 644}]}, {"trigger": {"text": "activation", "start": 956, "end": 966}, "arguments": [{"role": "Theme", "text": "proenkephalin", "start": 974, "end": 987}, {"role": "Site", "text": "promoter", "start": 988, "end": 996}]}, {"trigger": {"text": "mediated", "start": 1000, "end": 1008}, "arguments": [{"role": "Theme", "text": "activation", "start": 956, "end": 966}]}], "transcription": [{"trigger": {"text": "transcription", "start": 47, "end": 60}, "arguments": [{"role": "Theme", "text": "proenkephalin", "start": 33, "end": 46}]}]}}, "schema": []} {"input": "ETS1 transactivates the human GM-CSF promoter in Jurkat T cells stimulated with PMA and ionomycin. \nActivation of T helper cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one such cytokine whose increased expression results partly from increases in transcription. Cis-acting elements with NF kappa B, AP-1 and ETS-like motifs have been identified in the promoter region of the GM-CSF gene, which are important for transcriptional activity following PMA and ionomycin stimulation. A number of the ETS family of transcription factors are expressed in T cells, including ETS1 and ELF1. Here we describe the ability of these factors to interact with a site (GM5), located within the CLE0 element, -47 to -40 upstream of the GM-CSF transcription initiation site. Exogenous ETS1, but not ELF1, can transactivate GM-CSF, through the GM5 site, in a PMA/ionomycin dependent manner. Other unidentified ETS-like factors present in Jurkat cells are also capable of binding GM5. Mutation of the core ETS binding site from -GGAA- to -GGAT- prevents the binding of ETS-like factors with the exception of ETS1. The GM-CSF promoter, modified in this way to be ETS1 specific, is fully responsive to PMA/ionomycin induction, in addition to ETS1 transactivation in the presence of PMA and ionomycin. Together these data suggest that ETS1 may be involved in mediating the increased GM-CSF production associated with T cell activation. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interact", "start": 797, "end": 805}, "arguments": [{"role": "Theme", "text": "ETS1", "start": 733, "end": 737}]}, {"trigger": {"text": "interact", "start": 797, "end": 805}, "arguments": [{"role": "Theme", "text": "ELF1", "start": 742, "end": 746}]}, {"trigger": {"text": "binding", "start": 1204, "end": 1211}, "arguments": [{"role": "Theme", "text": "ETS1", "start": 1254, "end": 1258}]}, {"trigger": {"text": "specific", "start": 1313, "end": 1321}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 1264, "end": 1270}, {"role": "Site", "text": "promoter", "start": 1271, "end": 1279}, {"role": "Theme2", "text": "ETS1", "start": 1308, "end": 1312}]}], "gene expression": [{"trigger": {"text": "expression", "start": 370, "end": 380}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 325, "end": 331}]}, {"trigger": {"text": "expressed", "start": 701, "end": 710}, "arguments": [{"role": "Theme", "text": "ETS1", "start": 733, "end": 737}]}, {"trigger": {"text": "expressed", "start": 701, "end": 710}, "arguments": [{"role": "Theme", "text": "ELF1", "start": 742, "end": 746}]}, {"trigger": {"text": "production", "start": 1533, "end": 1543}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 1526, "end": 1532}]}], "negative regulation": [{"trigger": {"text": "exception", "start": 1241, "end": 1250}, "arguments": [{"role": "Theme", "text": "binding", "start": 1204, "end": 1211}]}], "positive regulation": [{"trigger": {"text": "transactivates", "start": 5, "end": 19}, "arguments": [{"role": "Cause", "text": "ETS1", "start": 0, "end": 4}, {"role": "Theme", "text": "GM-CSF", "start": 30, "end": 36}, {"role": "Site", "text": "promoter", "start": 37, "end": 45}]}, {"trigger": {"text": "increased", "start": 360, "end": 369}, "arguments": [{"role": "Theme", "text": "expression", "start": 370, "end": 380}, {"role": "Cause", "text": "increases", "start": 401, "end": 410}]}, {"trigger": {"text": "increases", "start": 401, "end": 410}, "arguments": [{"role": "Theme", "text": "transcription", "start": 414, "end": 427}]}, {"trigger": {"text": "transactivate", "start": 957, "end": 970}, "arguments": [{"role": "Cause", "text": "ETS1", "start": 933, "end": 937}, {"role": "Theme", "text": "GM-CSF", "start": 971, "end": 977}, {"role": "Site", "text": "GM5", "start": 991, "end": 994}]}, {"trigger": {"text": "transactivate", "start": 957, "end": 970}, "arguments": [{"role": "Cause", "text": "ELF1", "start": 947, "end": 951}, {"role": "Theme", "text": "GM-CSF", "start": 971, "end": 977}, {"role": "Site", "text": "GM5", "start": 991, "end": 994}]}, {"trigger": {"text": "induction", "start": 1360, "end": 1369}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 1264, "end": 1270}, {"role": "Site", "text": "promoter", "start": 1271, "end": 1279}]}, {"trigger": {"text": "transactivation", "start": 1391, "end": 1406}, "arguments": [{"role": "Theme", "text": "ETS1", "start": 1386, "end": 1390}]}, {"trigger": {"text": "mediating", "start": 1502, "end": 1511}, "arguments": [{"role": "Theme", "text": "increased", "start": 1516, "end": 1525}]}, {"trigger": {"text": "increased", "start": 1516, "end": 1525}, "arguments": [{"role": "Theme", "text": "production", "start": 1533, "end": 1543}]}], "regulation": [{"trigger": {"text": "involved", "start": 1490, "end": 1498}, "arguments": [{"role": "Cause", "text": "ETS1", "start": 1478, "end": 1482}, {"role": "Theme", "text": "mediating", "start": 1502, "end": 1511}]}], "transcription": [{"trigger": {"text": "transcription", "start": 414, "end": 427}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 325, "end": 331}]}]}}, "schema": []} {"input": "Tissue-specific regulation of the rabbit 15-lipoxygenase gene in erythroid cells by a transcriptional silencer. \nThe 15-lipoxygenase (lox) gene is expressed in a tissue-specific manner, predominantly in erythroid cells but also in airway epithelial cells and eosinophils. We demonstrate in this report that the 5' flanking DNA of the 15-lox gene contains sequences which down-regulate its activity in a variety of non-erythroid cell lines but not in two erythroid cell lines. The element has characteristics of a transcriptional 'silencer' since it functions in both orientations. The main activity of the silencer has been mapped to the first 900 bp of 5' flanking DNA, which contains nine binding sites for a nuclear factor present in non-erythroid cells but not in erythroid cells. These binding sites have similar sequences and multiple copies of the binding sites confer tissue-specific down-regulation when attached to a minimal lox promoter fragment. The 5' flanking DNA also contains a cluster of three binding sites for the GATA family of transcription factors. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressed", "start": 147, "end": 156}, "arguments": [{"role": "Theme", "text": "lox", "start": 134, "end": 137}]}], "negative regulation": [{"trigger": {"text": "down-regulate", "start": 371, "end": 384}, "arguments": [{"role": "Theme", "text": "expressed", "start": 147, "end": 156}, {"role": "Cause", "text": "15-lox", "start": 334, "end": 340}]}], "regulation": [{"trigger": {"text": "regulation", "start": 16, "end": 26}, "arguments": [{"role": "Theme", "text": "15-lipoxygenase", "start": 41, "end": 56}]}]}}, "schema": []} {"input": "Coexpression of NF-kappa B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia. \nProductive infection of T cells with human immunodeficiency virus 1 (HIV-1) typically requires that the T cells be stimulated with antigens or mitogens. This requirement has been attributed to the activation of the transcription factor NF-kappa B, which synergizes with the constitutive transcription factor Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous replication of HIV-1 takes place in nonactivated memory T cells after syncytium formation with dendritic cells (DCs). These syncytia lack activated cells as determined by an absence of staining for Ki-67 cell cycle antigen. The expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in isolated T cells and DCs from humans and mice. We have used immunolabeling, Western blot analysis, and electrophoretic mobility shift and supershift assays. T cells lack active NF-kappa B but express Sp1 as expected. DCs express high levels of all known NF-kappa B and Rel proteins, with activity residing primarily within RelB, p50, and p65. However, DCs lack Sp1, which may explain the failure of HIV-1 to replicate in purified DCs. Coexpression of NF-kappa B and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1. Therefore, HIV-1-induced cell fusion brings together factors that upregulate virus transcription. Since DCs and memory T cells frequently traffic together in situ, these unusual heterologous syncytia could develop in infected individuals and lead to chronic HIV-1 replication without ostensible immune stimulation. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Coexpression", "start": 0, "end": 12}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 35, "end": 38}]}, {"trigger": {"text": "expression", "start": 744, "end": 754}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 786, "end": 789}]}, {"trigger": {"text": "express", "start": 1011, "end": 1018}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 1019, "end": 1022}]}, {"trigger": {"text": "lack", "start": 1175, "end": 1179}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 1180, "end": 1183}]}, {"trigger": {"text": "Coexpression", "start": 1254, "end": 1266}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 1285, "end": 1288}]}], "positive regulation": [{"trigger": {"text": "activity", "start": 759, "end": 767}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 786, "end": 789}]}]}}, "schema": []} {"input": "Costimulation requirement for AP-1 and NF-kappa B transcription factor activation in T cells. \nThe transcriptional activity of the IL-2 promoter requires T-cell costimulation delivered by the TCR and the auxiliary receptor CD28. Several transcription factors participate in IL-2 promoter activation, among which are AP-1-like factors and NF-kappa B. Protein phosphorylation has an important role in the regulation of these two factors: (1) it induces the transactivating capacity of the AP-1 protein c-Jun; and (2) it is involved in the release of the cytoplasmic inhibitor, I kappa B, from NF-kappa B, allowing translocation of the latter into the nucleus. We have recently shown that both phosphorylation processes require T-cell costimulation. Furthermore, in activated T cells, the kinetics of the two phosphorylation events are essentially similar. According to our results, however, the kinases responsible for the two processes are distinct entities. Whereas TPCK inhibits phosphorylation of I kappa B and, consequently, activation of NF-kappa B, it markedly enhances the activity of JNK, the MAP kinase-related kinase that phosphorylates the transactivation domain of c-Jun. We, therefore, propose the activation scheme presented in FIGURE 3 for T-cell costimulation. Costimulation results in the activation of a signaling pathway that leads to the simultaneous induction of the two transcription factors, AP-1 and NF-kappa B. Integration of the signals generated by TCR and CD28 engagement occurs along this pathway, which then bifurcates to induce I kappa B phosphorylation and NF-kappa B activation on the one hand, and JNK activation and c-Jun phosphorylation on the other. We are currently engaged in defining where the two signals integrate along the AP-1/NF-kappa B pathway. ", "output": {"json_structures": {"phosphorylation": [{"trigger": {"text": "phosphorylates", "start": 1131, "end": 1145}, "arguments": [{"role": "Theme", "text": "c-Jun", "start": 1176, "end": 1181}]}, {"trigger": {"text": "phosphorylation", "start": 1656, "end": 1671}, "arguments": [{"role": "Theme", "text": "c-Jun", "start": 1650, "end": 1655}]}], "positive regulation": [{"trigger": {"text": "requires", "start": 145, "end": 153}, "arguments": [{"role": "Theme", "text": "transcriptional activity", "start": 99, "end": 123}]}, {"trigger": {"text": "activation", "start": 288, "end": 298}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 274, "end": 278}, {"role": "Site", "text": "promoter", "start": 279, "end": 287}]}, {"trigger": {"text": "induce", "start": 1551, "end": 1557}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1656, "end": 1671}]}], "regulation": [{"trigger": {"text": "participate", "start": 259, "end": 270}, "arguments": [{"role": "Theme", "text": "activation", "start": 288, "end": 298}]}], "transcription": [{"trigger": {"text": "transcriptional activity", "start": 99, "end": 123}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 131, "end": 135}]}]}}, "schema": []} {"input": "Cupric ion blocks NF kappa B activation through inhibiting the signal-induced phosphorylation of I kappa B alpha. \nA transcription factor NF kappa B, which regulates expression of various cellular genes involved in immune responses and viral genes including HIV, is sequestered in the cytoplasm as a complex with an inhibitory protein I kappa B. Various extracellular signals induce phosphorylation and rapid degradation of I kappa B alpha to release NF kappa B. Cu2+ was found to inhibit the activation of NF kappa B induced by TNF-alpha, TPA, or H2O2. Deoxycholate treatment of the cytoplasmic extract prepared from cells stimulated by TNF-alpha in the presence of Cu2+ resulted in the release of NF kappa B from I kappa B alpha, indicating that Cu2+ interferes with the dissociation of the NF kappa B-I kappa B complex. Neither phosphorylation nor degradation of I kappa B alpha was observed upon TNF-alpha stimulation in the presence of Cu2+. These results indicate that Cu2+ inhibits the release of NF kappa B by blockade of a signal leading to the phosphorylation of I kappa B alpha. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "complex", "start": 300, "end": 307}, "arguments": [{"role": "Theme", "text": "inhibitory protein I kappa B", "start": 316, "end": 344}]}], "negative regulation": [{"trigger": {"text": "inhibiting", "start": 48, "end": 58}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 78, "end": 93}]}, {"trigger": {"text": "blockade", "start": 1018, "end": 1026}, "arguments": [{"role": "Theme", "text": "leading", "start": 1039, "end": 1046}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 78, "end": 93}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 97, "end": 112}]}, {"trigger": {"text": "phosphorylation", "start": 383, "end": 398}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 424, "end": 439}]}, {"trigger": {"text": "phosphorylation", "start": 831, "end": 846}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 866, "end": 881}]}, {"trigger": {"text": "phosphorylation", "start": 1054, "end": 1069}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 1073, "end": 1088}]}], "positive regulation": [{"trigger": {"text": "induce", "start": 376, "end": 382}, "arguments": [{"role": "Cause", "text": "induce", "start": 376, "end": 382}, {"role": "Theme", "text": "degradation", "start": 409, "end": 420}]}, {"trigger": {"text": "induce", "start": 376, "end": 382}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 383, "end": 398}]}, {"trigger": {"text": "in the presence of", "start": 922, "end": 940}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 831, "end": 846}]}, {"trigger": {"text": "in the presence of", "start": 922, "end": 940}, "arguments": [{"role": "Theme", "text": "degradation", "start": 851, "end": 862}]}, {"trigger": {"text": "leading", "start": 1039, "end": 1046}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1054, "end": 1069}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 409, "end": 420}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 424, "end": 439}]}, {"trigger": {"text": "degradation", "start": 851, "end": 862}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 866, "end": 881}]}]}}, "schema": []} {"input": "Salicylates inhibit lipopolysaccharide-induced transcriptional activation of the tissue factor gene in human monocytic cells. \nBinding of plasma Factor VII/VIIa to the tissue factor (TF) receptor initiates the coagulation protease cascades. TF expression by circulating monocytes is associated with thrombotic and inflammatory complications in a variety of diseases. Transcriptional activation of the human TF gene in monocytic cells exposed to bacterial lipopolysaccharide (LPS) is mediated by binding of c-Rel/p65 heterodimers to a kappa B site in the TF promoter. Here, we report that a family of anti-inflammatory agents, known as the salicylates, inhibited LPS induction of TF activity and TF gene transcription in human monocytes and monocytic THP-1 cells at clinically relevant doses. Furthermore, sodium salicylate blocked the LPS-induced proteolytic degradation of I kappa B alpha, which prevented the nuclear translocation of c-Rel/p65 heterodimers. In contrast, two other nonsteroidal anti-inflammatory drugs, ibuprofen and indomethacin, did not inhibit LPS induction of the TF gene. These results indicated that salicylates inhibited LPS induction of TF gene transcription in monocytic cells by preventing nuclear translocation of c-Rel/p65 heterodimers. The clinical benefits of salicylates in the treatment of several diseases, including atherosclerosis and rheumatoid arthritis, may be related to their ability to reduce monocyte gene expression. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "Binding", "start": 127, "end": 134}, "arguments": [{"role": "Theme", "text": "Factor VII/VIIa", "start": 145, "end": 160}, {"role": "Theme2", "text": "tissue factor (TF) receptor", "start": 168, "end": 195}]}, {"trigger": {"text": "binding", "start": 495, "end": 502}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 506, "end": 511}, {"role": "Theme2", "text": "TF", "start": 554, "end": 556}, {"role": "Site2", "text": "promoter", "start": 557, "end": 565}]}, {"trigger": {"text": "binding", "start": 495, "end": 502}, "arguments": [{"role": "Theme", "text": "p65", "start": 512, "end": 515}, {"role": "Theme2", "text": "TF", "start": 554, "end": 556}, {"role": "Site2", "text": "promoter", "start": 557, "end": 565}]}], "gene expression": [{"trigger": {"text": "expression", "start": 244, "end": 254}, "arguments": [{"role": "Theme", "text": "TF", "start": 241, "end": 243}]}], "localization": [{"trigger": {"text": "translocation", "start": 919, "end": 932}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 911, "end": 918}, {"role": "Theme", "text": "c-Rel", "start": 936, "end": 941}]}, {"trigger": {"text": "translocation", "start": 919, "end": 932}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 911, "end": 918}, {"role": "Theme", "text": "p65", "start": 942, "end": 945}]}, {"trigger": {"text": "translocation", "start": 1226, "end": 1239}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 1218, "end": 1225}, {"role": "Theme", "text": "c-Rel", "start": 1243, "end": 1248}]}, {"trigger": {"text": "translocation", "start": 1226, "end": 1239}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 1218, "end": 1225}, {"role": "Theme", "text": "p65", "start": 1249, "end": 1252}]}], "negative regulation": [{"trigger": {"text": "inhibited", "start": 652, "end": 661}, "arguments": [{"role": "Theme", "text": "induction", "start": 666, "end": 675}]}, {"trigger": {"text": "blocked", "start": 823, "end": 830}, "arguments": [{"role": "Theme", "text": "induced", "start": 839, "end": 846}]}, {"trigger": {"text": "prevented", "start": 897, "end": 906}, "arguments": [{"role": "Cause", "text": "blocked", "start": 823, "end": 830}, {"role": "Theme", "text": "translocation", "start": 919, "end": 932}]}, {"trigger": {"text": "inhibit", "start": 1057, "end": 1064}, "arguments": [{"role": "Theme", "text": "induction", "start": 1069, "end": 1078}]}, {"trigger": {"text": "inhibited", "start": 1136, "end": 1145}, "arguments": [{"role": "Theme", "text": "induction", "start": 1150, "end": 1159}, {"role": "Cause", "text": "preventing", "start": 1207, "end": 1217}]}, {"trigger": {"text": "preventing", "start": 1207, "end": 1217}, "arguments": [{"role": "Theme", "text": "translocation", "start": 1226, "end": 1239}]}], "positive regulation": [{"trigger": {"text": "Transcriptional activation", "start": 367, "end": 393}, "arguments": [{"role": "Theme", "text": "TF", "start": 407, "end": 409}]}, {"trigger": {"text": "mediated", "start": 483, "end": 491}, "arguments": [{"role": "Theme", "text": "Transcriptional activation", "start": 367, "end": 393}, {"role": "Cause", "text": "binding", "start": 495, "end": 502}]}, {"trigger": {"text": "induction", "start": 666, "end": 675}, "arguments": [{"role": "Theme", "text": "transcription", "start": 703, "end": 716}]}, {"trigger": {"text": "induced", "start": 839, "end": 846}, "arguments": [{"role": "Theme", "text": "proteolytic degradation", "start": 847, "end": 870}]}, {"trigger": {"text": "induction", "start": 1069, "end": 1078}, "arguments": [{"role": "Theme", "text": "TF", "start": 1086, "end": 1088}]}, {"trigger": {"text": "induction", "start": 1150, "end": 1159}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1171, "end": 1184}]}], "protein catabolism": [{"trigger": {"text": "proteolytic degradation", "start": 847, "end": 870}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 874, "end": 889}]}], "transcription": [{"trigger": {"text": "transcription", "start": 703, "end": 716}, "arguments": [{"role": "Theme", "text": "TF", "start": 695, "end": 697}]}, {"trigger": {"text": "transcription", "start": 1171, "end": 1184}, "arguments": [{"role": "Theme", "text": "TF", "start": 1163, "end": 1165}]}]}}, "schema": []} {"input": "CIITA activates the expression of MHC class II genes in mouse T cells. \nIt has long been a puzzle that MHC class II molecules are expressed in human T cells after activation but not in mouse T cells; this expression is believed to play a role in the cell mediated immune response. Recently the MHC class II transactivator (CIITA) has been reported to be a major regulatory factor for both the constitutive and IFN inducible expression of MHC class II genes. Here we show that human T cells expressing MHC class II have CIITA transcripts while MHC class II-negative human T cells and mouse T cells do not. The expression of MHC class II genes in mouse T cells can be reconstituted upon transfection with the human CIITA cDNA. These data indicate that the expression of CIITA explains the expression or lack of expression of MHC class II in human and mouse T cells respectively. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "transfection", "start": 685, "end": 697}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 713, "end": 718}]}, {"trigger": {"text": "expression", "start": 754, "end": 764}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 768, "end": 773}]}], "positive regulation": [{"trigger": {"text": "transfection", "start": 685, "end": 697}, "arguments": [{"role": "Theme", "text": "transfection", "start": 685, "end": 697}]}], "transcription": [{"trigger": {"text": "have", "start": 514, "end": 518}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 519, "end": 524}]}]}}, "schema": []} {"input": "Identification of an I kappa B alpha-associated protein kinase in a human monocytic cell line and determination of its phosphorylation sites on I kappa B alpha. \nNuclear factor kappa B (NF-kappa B) is stored in the cytoplasm as an inactive form through interaction with I kappa B. Stimulation of cells leads to a rapid phosphorylation of I kappa B alpha, which is presumed to be important for the subsequent degradation. We have recently reported the establishment of a lipopolysaccharide (LPS)-dependent cell-free activation system of NF-kappa B in association with the induction of I kappa B alpha phosphorylation. In this study, we have identified a kinase in cell extracts from the LPS-stimulated human monocytic cell line, THP-1, that specifically binds and phosphorylates I kappa B alpha. LPS stimulation transiently enhanced the I kappa B alpha-bound kinase activity in THP-1 cells. Mutational analyses of I kappa B alpha and competition experiments with the synthetic peptides identified major phosphorylation sites by the bound kinase as Ser and Thr residues in the C-terminal acidic domain of I kappa B alpha. Moreover, we show that the peptide, corresponding to the C-terminal acidic domain of I kappa B alpha, blocked the LPS-induced NF-kappa B activation as well as inducible phosphorylation of endogenous I kappa B alpha in a cell-free system using THP-1 cells. These results suggested that the bound kinase is involved in the signaling pathway of LPS by inducing the phosphorylation of the C-terminal region of I kappa B alpha and subsequent dissociation of the NF-kappa B.I kappa B alpha complex. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 753, "end": 758}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 778, "end": 793}]}, {"trigger": {"text": "bound", "start": 852, "end": 857}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 836, "end": 851}]}, {"trigger": {"text": "complex", "start": 1604, "end": 1611}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 1588, "end": 1603}]}], "negative regulation": [{"trigger": {"text": "blocked", "start": 1222, "end": 1229}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1289, "end": 1304}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 319, "end": 334}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 338, "end": 353}]}, {"trigger": {"text": "phosphorylation", "start": 600, "end": 615}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 584, "end": 599}]}, {"trigger": {"text": "phosphorylates", "start": 763, "end": 777}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 778, "end": 793}]}, {"trigger": {"text": "phosphorylation sites", "start": 1002, "end": 1023}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 1103, "end": 1118}]}, {"trigger": {"text": "phosphorylation", "start": 1289, "end": 1304}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 1319, "end": 1334}]}, {"trigger": {"text": "phosphorylation", "start": 1482, "end": 1497}, "arguments": [{"role": "Site", "text": "C-terminal region", "start": 1505, "end": 1522}, {"role": "Theme", "text": "I kappa B alpha", "start": 1526, "end": 1541}]}], "positive regulation": [{"trigger": {"text": "leads", "start": 302, "end": 307}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 319, "end": 334}]}, {"trigger": {"text": "be important", "start": 376, "end": 388}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 319, "end": 334}, {"role": "Theme", "text": "degradation", "start": 408, "end": 419}]}, {"trigger": {"text": "induction", "start": 571, "end": 580}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 600, "end": 615}]}, {"trigger": {"text": "enhanced", "start": 823, "end": 831}, "arguments": [{"role": "Theme", "text": "bound", "start": 852, "end": 857}]}, {"trigger": {"text": "by", "start": 1024, "end": 1026}, "arguments": [{"role": "Theme", "text": "phosphorylation sites", "start": 1002, "end": 1023}]}, {"trigger": {"text": "inducing", "start": 1469, "end": 1477}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1482, "end": 1497}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 408, "end": 419}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 338, "end": 353}]}]}}, "schema": []} {"input": "Triggering of the human interleukin-6 gene by interferon-gamma and tumor necrosis factor-alpha in monocytic cells involves cooperation between interferon regulatory factor-1, NF kappa B, and Sp1 transcription factors. \nWe investigated the molecular basis of the synergistic induction by interferon-gamma (IFN-gamma)/tumor necrosis factor-alpha (TNF-alpha) of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and compared it with the basis of this induction by lipopolysaccharide (LPS). Functional studies with IL-6 promoter demonstrated that three regions are the targets of the IFN-gamma and/or TNF-alpha action, whereas only one of these regions seemed to be implicated in LPS activation. The three regions concerned are: 1) a region between -73 and -36, which is the minimal element inducible by LPS or TNF-alpha; 2) an element located between -181 and -73, which appeared to regulate the response to IFN-gamma and TNF-alpha negatively; and 3) a distal element upstream of -224, which was inducible by IFN-gamma alone. LPS signaling was found to involve NF kappa B activation by the p50/p65 heterodimers. Synergistic induction of the IL-6 gene by IFN-gamma and TNF-alpha, in monocytic cells, involved cooperation between the IRF-1 and NF kappa B p65 homodimers with concomitant removal of the negative effect of the retinoblastoma control element present in the IL-6 promoter. This removal occurred by activation of the constitutive Sp1 factor, whose increased binding activity and phosphorylation were mediated by IFN-gamma. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding activity", "start": 1473, "end": 1489}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 1445, "end": 1448}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1494, "end": 1509}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 1445, "end": 1448}]}], "positive regulation": [{"trigger": {"text": "Triggering", "start": 0, "end": 10}, "arguments": [{"role": "Theme", "text": "interleukin-6", "start": 24, "end": 37}, {"role": "Cause", "text": "interferon-gamma", "start": 46, "end": 62}]}, {"trigger": {"text": "Triggering", "start": 0, "end": 10}, "arguments": [{"role": "Theme", "text": "interleukin-6", "start": 24, "end": 37}, {"role": "Cause", "text": "tumor necrosis factor-alpha", "start": 67, "end": 94}]}, {"trigger": {"text": "induction", "start": 274, "end": 283}, "arguments": [{"role": "Cause", "text": "IFN-gamma", "start": 305, "end": 314}, {"role": "Theme", "text": "IL-6", "start": 380, "end": 384}]}, {"trigger": {"text": "induction", "start": 274, "end": 283}, "arguments": [{"role": "Cause", "text": "TNF-alpha", "start": 345, "end": 354}, {"role": "Theme", "text": "IL-6", "start": 380, "end": 384}]}, {"trigger": {"text": "induction", "start": 456, "end": 465}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 380, "end": 384}]}, {"trigger": {"text": "activation", "start": 1077, "end": 1087}, "arguments": [{"role": "Theme", "text": "p50", "start": 1095, "end": 1098}]}, {"trigger": {"text": "activation", "start": 1077, "end": 1087}, "arguments": [{"role": "Theme", "text": "p65", "start": 1099, "end": 1102}]}, {"trigger": {"text": "induction", "start": 1129, "end": 1138}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1146, "end": 1150}, {"role": "Cause", "text": "IFN-gamma", "start": 1159, "end": 1168}]}, {"trigger": {"text": "induction", "start": 1129, "end": 1138}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1146, "end": 1150}, {"role": "Cause", "text": "TNF-alpha", "start": 1173, "end": 1182}]}, {"trigger": {"text": "involved", "start": 1204, "end": 1212}, "arguments": [{"role": "Theme", "text": "induction", "start": 1129, "end": 1138}, {"role": "Cause", "text": "IRF-1", "start": 1237, "end": 1242}]}, {"trigger": {"text": "involved", "start": 1204, "end": 1212}, "arguments": [{"role": "Theme", "text": "induction", "start": 1129, "end": 1138}, {"role": "Cause", "text": "p65", "start": 1258, "end": 1261}]}, {"trigger": {"text": "activation", "start": 1414, "end": 1424}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 1445, "end": 1448}]}, {"trigger": {"text": "increased", "start": 1463, "end": 1472}, "arguments": [{"role": "Theme", "text": "binding activity", "start": 1473, "end": 1489}]}, {"trigger": {"text": "mediated", "start": 1515, "end": 1523}, "arguments": [{"role": "Theme", "text": "increased", "start": 1463, "end": 1472}, {"role": "Cause", "text": "IFN-gamma", "start": 1527, "end": 1536}]}, {"trigger": {"text": "mediated", "start": 1515, "end": 1523}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1494, "end": 1509}, {"role": "Cause", "text": "IFN-gamma", "start": 1527, "end": 1536}]}], "regulation": [{"trigger": {"text": "involves cooperation", "start": 114, "end": 134}, "arguments": [{"role": "Theme", "text": "Triggering", "start": 0, "end": 10}, {"role": "Cause", "text": "interferon regulatory factor-1", "start": 143, "end": 173}]}, {"trigger": {"text": "involves cooperation", "start": 114, "end": 134}, "arguments": [{"role": "Theme", "text": "Triggering", "start": 0, "end": 10}, {"role": "Cause", "text": "Sp1", "start": 191, "end": 194}]}, {"trigger": {"text": "action", "start": 615, "end": 621}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 519, "end": 523}, {"role": "Site", "text": "promoter", "start": 524, "end": 532}, {"role": "Cause", "text": "IFN-gamma", "start": 588, "end": 597}]}, {"trigger": {"text": "action", "start": 615, "end": 621}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 519, "end": 523}, {"role": "Site", "text": "promoter", "start": 524, "end": 532}, {"role": "Cause", "text": "TNF-alpha", "start": 605, "end": 614}]}]}}, "schema": []} {"input": "The lymphotoxin promoter is stimulated by HTLV-I tax activation of NF-kappa B in human T-cell lines. \nThe HTLV-I transcriptional activator tax was used to gain insight into the mechanism of lymphotoxin (LT; TNF-beta) gene induction. Tax-expressing cell lines produce LT biologic activity. An LT promoter (LT-293) CAT construct that contained an NF-kappa B site was active in the LT-producing C81-66-45 cell line, which contains defective HTLV-I but expresses tax. The observation that a mutated LT-kappa B construct (M1-CAT) was inactive in C81-66-45, confirmed the importance of NF-kappa B in LT gene expression. Tax was transfected into HTLV-I-negative human T-cell lines. Jurkat T cells stably expressing tax contained elevated levels of NF-kappa B that directly bound to the LT-kappa B site. Tax co-transfected with reporter constructs into Jurkat cells maximally activated HTLV-I-LTR-CAT and kappa B-fos-CAT and also activated LT-293 to a lesser extent. In JM T cells, tax induced LT-293 activity by two- to four-fold, though there was no induction of M1-CAT. The increase in LT-293 CAT activity mirrored the increase in LT biologic activity seen under these conditions. These studies, the first to demonstrate induction of LT promoter activity over basal levels, indicate that HTLV-I tax causes low-level activation of both endogenous LT and the LT promoter, at least in part through activation of NF-kappa B. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "induction", "start": 222, "end": 231}, "arguments": [{"role": "Theme", "text": "TNF-beta", "start": 207, "end": 215}]}, {"trigger": {"text": "expresses", "start": 449, "end": 458}, "arguments": [{"role": "Theme", "text": "tax", "start": 459, "end": 462}]}, {"trigger": {"text": "expression", "start": 602, "end": 612}, "arguments": [{"role": "Theme", "text": "TNF-beta", "start": 207, "end": 215}]}, {"trigger": {"text": "expressing", "start": 697, "end": 707}, "arguments": [{"role": "Theme", "text": "tax", "start": 708, "end": 711}]}, {"trigger": {"text": "transfected", "start": 803, "end": 814}, "arguments": [{"role": "Theme", "text": "Tax", "start": 796, "end": 799}]}], "positive regulation": [{"trigger": {"text": "inactive", "start": 529, "end": 537}, "arguments": [{"role": "Theme", "text": "CAT", "start": 520, "end": 523}]}, {"trigger": {"text": "importance", "start": 566, "end": 576}, "arguments": [{"role": "Theme", "text": "expression", "start": 602, "end": 612}]}, {"trigger": {"text": "transfected", "start": 803, "end": 814}, "arguments": [{"role": "Theme", "text": "transfected", "start": 803, "end": 814}]}]}}, "schema": []} {"input": "Induction of tyrosine phosphorylation and T-cell activation by vanadate peroxide, an inhibitor of protein tyrosine phosphatases. \nRapid tyrosine phosphorylation of key cellular proteins is a crucial event in the transduction of activation signals to T-lymphocytes. The regulatory role of protein tyrosine phosphatases (PTPases) in this process was explored by studying the effects of a powerful PTPase inhibitor, vanadate peroxide (pervanadate), on the activation cascade of Jurkat human leukaemic T-cells. Pervanadate induced activation of the tyrosine kinases lck and fyn (4- and 3-fold respectively) and a dramatic increase in tyrosine phosphorylation of cellular proteins, notably phospholipase C gamma 1. After this event, we observed a rise in intracellular Ca2+ concentration, corresponding to an influx. This effect required surface expression of the CD45 PTPase and was not observed in CD45-deficient variants of Jurkat cells. In the CD45-negative variant, the effect of pervanadate on tyrosine phosphorylation was globally decreased and some phosphorylated substrates were specifically missing. Pervanadate also stimulated transcription of the c-fos gene and accumulation of its mRNA as well as several other hallmarks of T-lymphocyte activation such as surface expression of the CD69 antigen and the interleukin 2 receptor alpha-chain (CD25). Pervanadate synergized with signals delivered by T-cell antigen receptor engagement or by a phorbol ester to induce interleukin 2 production. Pervanadate activated NF-kappa B, as shown by an increase in DNA-binding activity of this transcription factor. We thus conclude that PTPases play a crucial role in the negative regulation of signal transduction culminating in T-lymphocyte activation. Moreover, induction of tyrosine phosphorylation appears sufficient per se to initiate a complete activation programme. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 841, "end": 851}, "arguments": [{"role": "Theme", "text": "CD45", "start": 859, "end": 863}]}, {"trigger": {"text": "expression", "start": 1272, "end": 1282}, "arguments": [{"role": "Theme", "text": "CD69", "start": 1290, "end": 1294}]}, {"trigger": {"text": "expression", "start": 1272, "end": 1282}, "arguments": [{"role": "Theme", "text": "CD25", "start": 1347, "end": 1351}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 639, "end": 654}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 630, "end": 638}, {"role": "Theme", "text": "phospholipase C gamma 1", "start": 685, "end": 708}]}], "positive regulation": [{"trigger": {"text": "activation", "start": 527, "end": 537}, "arguments": [{"role": "Theme", "text": "lck", "start": 562, "end": 565}]}, {"trigger": {"text": "activation", "start": 527, "end": 537}, "arguments": [{"role": "Theme", "text": "fyn", "start": 570, "end": 573}]}, {"trigger": {"text": "increase", "start": 618, "end": 626}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 639, "end": 654}]}, {"trigger": {"text": "stimulated", "start": 1122, "end": 1132}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1133, "end": 1146}]}, {"trigger": {"text": "stimulated", "start": 1122, "end": 1132}, "arguments": [{"role": "Theme", "text": "accumulation", "start": 1169, "end": 1181}]}, {"trigger": {"text": "stimulated", "start": 1122, "end": 1132}, "arguments": [{"role": "Theme", "text": "expression", "start": 1272, "end": 1282}]}, {"trigger": {"text": "accumulation", "start": 1169, "end": 1181}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1154, "end": 1159}]}], "transcription": [{"trigger": {"text": "transcription", "start": 1133, "end": 1146}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1154, "end": 1159}]}]}}, "schema": []} {"input": "The interleukin-8 AP-1 and kappa B-like sites are genetic end targets of FK506-sensitive pathway accompanied by calcium mobilization. \nFK506, an immunosuppressant, inhibits the production of several cytokines in T lymphocytes. We observed that FK506 suppressed the transcription of a chemotactic cytokine, interleukin-8 (IL-8) in a human T cell line, Jurkat cells, activated by phorbol 12-myristate 13-acetate (PMA) and calcium (Ca2+) ionophore (ionomycin). By deleted and mutated analysis of the IL-8 promoters, the AP-1 and kappa B-like sites were identified as the responsive elements for PMA and ionomycin. FK506 suppressed the transcriptions through the AP-1 or kappa B-like sites induced by PMA plus Ca(2+)-mobilizing agents, but not those induced by Ca(2+)-independent stimuli. In gel retardation analysis, FK506 had little effect on the binding to the AP-1 site of PMA/ionomycin-induced nuclear factors, which were recognized with anti-JunD or c-Fos antibody. In contrast, FK506 or EGTA (Ca2+ chelator) similarly affected the formation of kappa B-like site binding complexes, which were not recognized by any antibodies against the human Rel family proteins (c-Rel, p65, p50, and p49). Furthermore, we confirmed the previous report that FK506 suppressed the PMA/ionomycin-induced activation through authentic kappa B site of immunoglobulin (Ig) gene, to which NF-kappa B binding was also decreased by FK506, indicating that both IL-8 kappa B-like site and Ig kappa B site are FK506-sensitive in spite of the difference of binding factors. Our results indicate that not only the reported IL-2 NF-AT and NFIL-2A sites and Ig kappa B site, but also the IL-8 AP-1 and kappa B-like sites are terminals of FK506-sensitive pathway involving Ca2+ mobilization. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "recognized", "start": 923, "end": 933}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 952, "end": 957}]}], "negative regulation": [{"trigger": {"text": "suppressed", "start": 250, "end": 260}, "arguments": [{"role": "Theme", "text": "transcription", "start": 265, "end": 278}]}, {"trigger": {"text": "suppressed", "start": 617, "end": 627}, "arguments": [{"role": "Theme", "text": "induced", "start": 686, "end": 693}]}, {"trigger": {"text": "suppressed", "start": 617, "end": 627}, "arguments": [{"role": "Theme", "text": "induced", "start": 746, "end": 753}]}], "positive regulation": [{"trigger": {"text": "through", "start": 647, "end": 654}, "arguments": [{"role": "Theme", "text": "transcription", "start": 265, "end": 278}]}, {"trigger": {"text": "induced", "start": 686, "end": 693}, "arguments": [{"role": "Theme", "text": "through", "start": 647, "end": 654}]}, {"trigger": {"text": "induced", "start": 746, "end": 753}, "arguments": [{"role": "Theme", "text": "through", "start": 647, "end": 654}]}], "regulation": [{"trigger": {"text": "targets", "start": 62, "end": 69}, "arguments": [{"role": "Theme", "text": "interleukin-8", "start": 4, "end": 17}, {"role": "Site", "text": "AP-1", "start": 18, "end": 22}]}, {"trigger": {"text": "targets", "start": 62, "end": 69}, "arguments": [{"role": "Theme", "text": "interleukin-8", "start": 4, "end": 17}, {"role": "Site", "text": "kappa B-like", "start": 27, "end": 39}]}, {"trigger": {"text": "terminals", "start": 1695, "end": 1704}, "arguments": [{"role": "Theme", "text": "IL-8", "start": 1658, "end": 1662}, {"role": "Site", "text": "AP-1", "start": 1663, "end": 1667}]}, {"trigger": {"text": "terminals", "start": 1695, "end": 1704}, "arguments": [{"role": "Theme", "text": "IL-8", "start": 1658, "end": 1662}, {"role": "Site", "text": "kappa B-like", "start": 1672, "end": 1684}]}], "transcription": [{"trigger": {"text": "transcription", "start": 265, "end": 278}, "arguments": [{"role": "Theme", "text": "IL-8", "start": 321, "end": 325}]}]}}, "schema": []} {"input": "Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion. Signal integration and NF-kappa B translocation [see comments] \nAdhesion molecules that tether circulating leukocytes to endothelial cells may also transduce or modulate outside-in signals for cellular activation, providing an initial regulatory point in the inflammatory response. Adhesion of human monocytes to P-selectin, the most rapidly expressed endothelial tethering factor, increased the secretion of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) by the leukocytes when they were stimulated with platelet-activating factor. Increased cytokine secretion was specifically inhibited by G1, an anti-P-selectin mAb that prevents P-selectin from binding to its ligand (P-selectin glycoprotein ligand-1) on myeloid cells. Moreover, tethering by P-selectin specifically enhanced nuclear translocation of nuclear factor-kappa B (NF-kappa B), a transcription factor required for expression of MCP-1, TNF-alpha, and other immediate-early genes. These results demonstrate that P-selectin, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 802, "end": 809}, "arguments": [{"role": "Theme", "text": "P-selectin", "start": 786, "end": 796}, {"role": "Theme2", "text": "P-selectin glycoprotein ligand-1", "start": 825, "end": 857}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 459, "end": 468}, "arguments": [{"role": "Theme", "text": "P-selectin", "start": 430, "end": 440}]}, {"trigger": {"text": "expression", "start": 1031, "end": 1041}, "arguments": [{"role": "Theme", "text": "MCP-1", "start": 1045, "end": 1050}]}, {"trigger": {"text": "expression", "start": 1031, "end": 1041}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1052, "end": 1061}]}], "localization": [{"trigger": {"text": "secretion", "start": 106, "end": 115}, "arguments": [{"role": "Theme", "text": "monocyte chemotactic protein-1", "start": 43, "end": 73}]}, {"trigger": {"text": "secretion", "start": 106, "end": 115}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor-alpha", "start": 78, "end": 105}]}, {"trigger": {"text": "secretion", "start": 513, "end": 522}, "arguments": [{"role": "Theme", "text": "MCP-1", "start": 558, "end": 563}]}, {"trigger": {"text": "secretion", "start": 513, "end": 522}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 598, "end": 607}]}], "negative regulation": [{"trigger": {"text": "prevents", "start": 777, "end": 785}, "arguments": [{"role": "Theme", "text": "binding", "start": 802, "end": 809}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 499, "end": 508}, "arguments": [{"role": "Theme", "text": "secretion", "start": 513, "end": 522}]}, {"trigger": {"text": "when", "start": 627, "end": 631}, "arguments": [{"role": "Theme", "text": "secretion", "start": 513, "end": 522}]}, {"trigger": {"text": "required", "start": 1018, "end": 1026}, "arguments": [{"role": "Theme", "text": "expression", "start": 1031, "end": 1041}]}], "regulation": [{"trigger": {"text": "regulates", "start": 33, "end": 42}, "arguments": [{"role": "Theme", "text": "secretion", "start": 106, "end": 115}]}]}}, "schema": []} {"input": "HMG-I binds to GATA motifs: implications for an HPFH syndrome. \nWe have examined binding of the nuclear protein HMG-I to the human gamma-globin promoter. We find that HMG-I binds preferentially to the more 3' of a pair of GATA motifs in the gamma-globin promoter; this paired motif is bound by the erythroid factor GATA-1. A naturally occurring mutation (-175 T-C) in the area bound by HMG-I results in overexpression of gamma-globin in adult red blood cells (HPFH) and up-regulation of the gamma-globin promoter in in vitro expression assays; HMG-I does not bind to this mutant sequence. A survey of GATA motifs from other globin cis-elements demonstrates HMG-I binding to most of them. These findings implicate HMG-I in the HPFH phenotype; we speculate that it may participate in the formation of multiprotein complexes that regulate globin gene expression. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 6, "end": 11}, "arguments": [{"role": "Theme", "text": "HMG-I", "start": 0, "end": 5}]}, {"trigger": {"text": "binding", "start": 81, "end": 88}, "arguments": [{"role": "Theme", "text": "HMG-I", "start": 112, "end": 117}]}, {"trigger": {"text": "binds", "start": 173, "end": 178}, "arguments": [{"role": "Theme", "text": "HMG-I", "start": 167, "end": 172}]}, {"trigger": {"text": "bound", "start": 285, "end": 290}, "arguments": [{"role": "Theme", "text": "GATA-1", "start": 315, "end": 321}]}, {"trigger": {"text": "bound", "start": 377, "end": 382}, "arguments": [{"role": "Theme", "text": "HMG-I", "start": 386, "end": 391}]}, {"trigger": {"text": "bind", "start": 559, "end": 563}, "arguments": [{"role": "Theme", "text": "HMG-I", "start": 544, "end": 549}]}, {"trigger": {"text": "binding", "start": 663, "end": 670}, "arguments": [{"role": "Theme", "text": "HMG-I", "start": 657, "end": 662}]}]}}, "schema": []} {"input": "Positive and negative regulation of granulocyte-macrophage colony-stimulating factor promoter activity by AML1-related transcription factor, PEBP2. \nThe granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter contains a consensus sequence for the polyomavirus enhancer binding-protein 2 (PEBP2) transcription factor, which consists of alpha and beta subunits. There are at least two genes, alpha A and alpha B, encoding the alpha subunit. alpha B is the mouse homologue of human AML1 gene detected at the breakpoints of t(8;21) and t(3;21) myeloid leukemias. We examined alpha A1 (an alpha A-gene product) and alpha B1 and alpha B2 (two alpha B-encoded isomers) for their effects on the GM-CSF promoter. PEBP2 alpha A1, alpha B1, and alpha B2 proteins bound the PEBP2 site within the mouse GM-CSF promoter. PEBP2 alpha A1 and alpha B1 enhanced the expression of the GM-CSF promoter-driven reporter plasmid in unstimulated and 12-O-tetradecanoylphorbol 13-acetate/phytohemagglutinin-stimulated human Jurkat T cells. In contrast, the promoter activity was suppressed by alpha B2. Coexpression of alpha B1 and alpha B2 showed that the promoter activity could be determined by the alpha B1/alpha B2 ratio. Jurkat cell extract contained PEBP2 site-binding protein(s) that cross-reacted with antimouse alpha A1 antibodies. Northern blot analysis indicated the expression of human PEBP2 alpha A, alpha B (AML1), and beta genes in Jurkat cells. Although further studies are required to determine the precise role of PEBP2 in the GM-CSF promoter activity, the present findings suggested the importance of the relative ratio of different PEBP2 isoforms in regulating the levels of the promoter activity. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "bound", "start": 768, "end": 773}, "arguments": [{"role": "Theme", "text": "PEBP2 alpha A1", "start": 720, "end": 734}, {"role": "Site2", "text": "PEBP2 site", "start": 778, "end": 788}, {"role": "Theme2", "text": "GM-CSF", "start": 806, "end": 812}]}, {"trigger": {"text": "bound", "start": 768, "end": 773}, "arguments": [{"role": "Theme", "text": "alpha B1", "start": 736, "end": 744}, {"role": "Site2", "text": "PEBP2 site", "start": 778, "end": 788}, {"role": "Theme2", "text": "GM-CSF", "start": 806, "end": 812}]}, {"trigger": {"text": "bound", "start": 768, "end": 773}, "arguments": [{"role": "Theme", "text": "alpha B2", "start": 750, "end": 758}, {"role": "Site2", "text": "PEBP2 site", "start": 778, "end": 788}, {"role": "Theme2", "text": "GM-CSF", "start": 806, "end": 812}]}], "gene expression": [{"trigger": {"text": "Coexpression", "start": 1094, "end": 1106}, "arguments": [{"role": "Theme", "text": "alpha B1", "start": 1110, "end": 1118}]}, {"trigger": {"text": "Coexpression", "start": 1094, "end": 1106}, "arguments": [{"role": "Theme", "text": "alpha B2", "start": 1123, "end": 1131}]}, {"trigger": {"text": "expression", "start": 1370, "end": 1380}, "arguments": [{"role": "Theme", "text": "(AML1)", "start": 1413, "end": 1419}]}], "negative regulation": [{"trigger": {"text": "negative regulation", "start": 13, "end": 32}, "arguments": [{"role": "Theme", "text": "granulocyte-macrophage colony-stimulating factor", "start": 36, "end": 84}, {"role": "Site", "text": "promoter", "start": 85, "end": 93}]}, {"trigger": {"text": "suppressed", "start": 1070, "end": 1080}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 882, "end": 888}, {"role": "Site", "text": "promoter", "start": 889, "end": 897}, {"role": "Cause", "text": "alpha B2", "start": 1084, "end": 1092}]}], "positive regulation": [{"trigger": {"text": "regulation", "start": 22, "end": 32}, "arguments": [{"role": "Theme", "text": "granulocyte-macrophage colony-stimulating factor", "start": 36, "end": 84}, {"role": "Site", "text": "promoter", "start": 85, "end": 93}]}], "regulation": [{"trigger": {"text": "effects", "start": 688, "end": 695}, "arguments": [{"role": "Cause", "text": "alpha A1", "start": 587, "end": 595}, {"role": "Theme", "text": "GM-CSF", "start": 703, "end": 709}, {"role": "Site", "text": "promoter", "start": 710, "end": 718}]}, {"trigger": {"text": "effects", "start": 688, "end": 695}, "arguments": [{"role": "Cause", "text": "alpha B1", "start": 626, "end": 634}, {"role": "Theme", "text": "GM-CSF", "start": 703, "end": 709}, {"role": "Site", "text": "promoter", "start": 710, "end": 718}]}, {"trigger": {"text": "effects", "start": 688, "end": 695}, "arguments": [{"role": "Cause", "text": "alpha B2", "start": 639, "end": 647}, {"role": "Theme", "text": "GM-CSF", "start": 703, "end": 709}, {"role": "Site", "text": "promoter", "start": 710, "end": 718}]}, {"trigger": {"text": "determined", "start": 1175, "end": 1185}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 882, "end": 888}, {"role": "Site", "text": "promoter", "start": 889, "end": 897}, {"role": "Cause", "text": "alpha B1", "start": 1193, "end": 1201}]}, {"trigger": {"text": "determined", "start": 1175, "end": 1185}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 882, "end": 888}, {"role": "Site", "text": "promoter", "start": 889, "end": 897}, {"role": "Cause", "text": "alpha B2", "start": 1202, "end": 1210}]}, {"trigger": {"text": "role", "start": 1516, "end": 1520}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 1537, "end": 1543}, {"role": "Site", "text": "promoter", "start": 1544, "end": 1552}]}, {"trigger": {"text": "importance", "start": 1598, "end": 1608}, "arguments": [{"role": "Theme", "text": "regulating", "start": 1662, "end": 1672}]}, {"trigger": {"text": "regulating", "start": 1662, "end": 1672}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 1537, "end": 1543}, {"role": "Site", "text": "promoter", "start": 1544, "end": 1552}]}]}}, "schema": []} {"input": "Differential induction of the NF-AT complex during restimulation and the induction of T-cell anergy. \nStimulation of human CD4+ T-cell clones through the T-cell receptor (TcR) by high doses of specific peptide results in the induction of a long-lived state of nonresponsiveness that has been called anergy. During the induction of anergy, T cells are phenotypically similar to cells responding to an immunogenic stimulus. The amount of TcR at the cell surface is downmodulated, whereas the CD2 and CD25 receptors are increased. When restimulated, however, anergic T cells fail to up-regulate transcription of the IL-2 gene and in consequence do not produce IL-2. In this study, we have compared the ability of various transcription factors to bind to their appropriate site on DNA. Factors were isolated from the nuclei of T cells that were in the induction phase of anergy or were undergoing activation. The pattern of binding activity in restimulated T cells is consistent with the pattern that has previously been shown to regulate T-cell-specific expression of the IL-2 and the beta chain of the TcR genes. The measured binding to a TCF-1 site is the same in the nuclei of resting, activated, and anergized cells. The inducible factors NK-kappa B, beta E2, CD28RC, and AP-1 are not expressed in resting cells and are twofold lower in anergized as compared with activated cells. In contrast, anergic T cells express approximately eightfold lower amounts of NF-AT, a member of the class of inducible factors that regulates IL-2 gene transcription. (ABSTRACT TRUNCATED AT 250 WORDS) ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 1124, "end": 1131}, "arguments": [{"role": "Theme", "text": "TCF-1", "start": 1137, "end": 1142}]}], "gene expression": [{"trigger": {"text": "produce", "start": 649, "end": 656}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 657, "end": 661}]}, {"trigger": {"text": "expression", "start": 1051, "end": 1061}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1069, "end": 1073}]}, {"trigger": {"text": "expressed", "start": 1286, "end": 1295}, "arguments": [{"role": "Theme", "text": "CD28RC", "start": 1261, "end": 1267}]}], "positive regulation": [{"trigger": {"text": "up-regulate", "start": 580, "end": 591}, "arguments": [{"role": "Theme", "text": "transcription", "start": 592, "end": 605}]}, {"trigger": {"text": "in consequence", "start": 627, "end": 641}, "arguments": [{"role": "Cause", "text": "up-regulate", "start": 580, "end": 591}, {"role": "Theme", "text": "produce", "start": 649, "end": 656}]}], "regulation": [{"trigger": {"text": "regulate", "start": 1026, "end": 1034}, "arguments": [{"role": "Theme", "text": "expression", "start": 1051, "end": 1061}]}, {"trigger": {"text": "regulates", "start": 1515, "end": 1524}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1535, "end": 1548}]}], "transcription": [{"trigger": {"text": "transcription", "start": 592, "end": 605}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 613, "end": 617}]}, {"trigger": {"text": "transcription", "start": 1535, "end": 1548}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1525, "end": 1529}]}]}}, "schema": []} {"input": "The retinoblastoma gene product negatively regulates transcriptional activation mediated by the human cytomegalovirus IE2 protein. \nThe IE2 gene product of human cytomegalovirus (HCMV) is one of a few viral regulatory proteins expressed immediately upon infection of the host cell. It is a potent transcriptional activator of many viral and cellular promoters. We found that the retinoblastoma susceptibility gene product (Rb) dramatically suppressed this IE2 transactivation of various promoters. However, unlike another tumor suppressor protein, p53, Rb did not have any significant effect on basal levels of transcription, suggesting that Rb specifically interacts with IE2 rather than other cellular factors involved in the general transcription machinery. We found by protein-affinity chromatography that Rb in nuclear extracts or produced by in vitro translation directly bound to IE2. Our results suggest that Rb may regulate the life cycle of HCMV, which is endemic in the human population. Furthermore, these data may provide new insights into the slow rate of HCMV DNA replication in cells and the possible involvement of HCMV in tumorigenesis. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interacts", "start": 658, "end": 667}, "arguments": [{"role": "Theme", "text": "Rb", "start": 642, "end": 644}, {"role": "Theme2", "text": "IE2", "start": 673, "end": 676}]}, {"trigger": {"text": "bound", "start": 878, "end": 883}, "arguments": [{"role": "Theme", "text": "Rb", "start": 810, "end": 812}, {"role": "Theme2", "text": "IE2", "start": 887, "end": 890}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 227, "end": 236}, "arguments": [{"role": "Theme", "text": "IE2", "start": 136, "end": 139}]}], "positive regulation": [{"trigger": {"text": "upon", "start": 249, "end": 253}, "arguments": [{"role": "Theme", "text": "expressed", "start": 227, "end": 236}]}]}}, "schema": []} {"input": "Epstein-Barr virus replicative gene transcription during de novo infection of human thymocytes: simultaneous early expression of BZLF-1 and its repressor RAZ. \nEpstein-Barr virus (EBV) is known to infect B cells and epithelial cells. We and others have shown that EBV can also infect a subset of thymocytes. Infection of thymocytes was accompanied by the appearance of linear EBV genome within 8 hr of infection. Circularization of the EBV genome was not detected. This is in contrast to the infection in B cells where the genome can circularize within 24 hr of infection. The appearance of the BamHI ZLF-1 gene product, ZEBRA, by RT-PCR, was observed within 8 hr of infection. The appearance of a novel fusion transcript (RAZ), which comprised regions of the BZLF-1 locus and the adjacent BRLF-1 locus, was detected by RT-PCR. ZEBRA protein was also identified in infected thymocytes by immunoprecipitation. In addition, we demonstrated that the EBNA-1 gene in infected thymocytes was transcribed from the Fp promoter, rather than from the Cp/Wp promoter which is used in latently infected B cells. Transcripts encoding gp350/220, the major coat protein of EBV, were identified, but we did not find any evidence of transcription from the LMP-2A or EBER-1 loci in infected thymocytes. These observations suggest that de novo EBV infection of thymocytes differs from infection of B cells. The main difference is that with thymocytes, no evidence could be found that the virus ever circularizes. Rather, EBV remains in a linear configuration from which replicative genes are transcribed. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 115, "end": 125}, "arguments": [{"role": "Theme", "text": "BZLF-1", "start": 129, "end": 135}]}, {"trigger": {"text": "expression", "start": 115, "end": 125}, "arguments": [{"role": "Theme", "text": "RAZ", "start": 154, "end": 157}]}, {"trigger": {"text": "appearance", "start": 577, "end": 587}, "arguments": [{"role": "Theme", "text": "ZEBRA", "start": 621, "end": 626}]}, {"trigger": {"text": "appearance", "start": 682, "end": 692}, "arguments": [{"role": "Theme", "text": "RAZ", "start": 723, "end": 726}]}, {"trigger": {"text": "identified", "start": 851, "end": 861}, "arguments": [{"role": "Theme", "text": "ZEBRA", "start": 828, "end": 833}]}], "positive regulation": [{"trigger": {"text": "from", "start": 998, "end": 1002}, "arguments": [{"role": "Theme", "text": "transcribed", "start": 986, "end": 997}]}, {"trigger": {"text": "rather than", "start": 1020, "end": 1031}, "arguments": [{"role": "Theme", "text": "transcribed", "start": 986, "end": 997}]}], "transcription": [{"trigger": {"text": "transcribed", "start": 986, "end": 997}, "arguments": [{"role": "Theme", "text": "EBNA-1", "start": 947, "end": 953}]}, {"trigger": {"text": "Transcripts", "start": 1100, "end": 1111}, "arguments": [{"role": "Theme", "text": "gp350/220", "start": 1121, "end": 1130}]}, {"trigger": {"text": "transcription", "start": 1216, "end": 1229}, "arguments": [{"role": "Theme", "text": "LMP-2A", "start": 1239, "end": 1245}]}, {"trigger": {"text": "transcription", "start": 1216, "end": 1229}, "arguments": [{"role": "Theme", "text": "EBER-1", "start": 1249, "end": 1255}]}]}}, "schema": []} {"input": "Induction of Sp1 phosphorylation and NF-kappa B-independent HIV promoter domain activity in T lymphocytes stimulated by okadaic acid. \nIn contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor NF-kappa B, and the promoter domain of the LTR. The inducibility of HIV LTR-driven luciferase expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional Sp1 binding elements and the ability of the TATA box to bind the protein TBP. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed Sp1 protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or PHA and interleukin 2. Responsiveness of LTR constructs deleted of kappa B elements to HIV Tat expression was increased upon OKA but not TNF stimulation. Our results suggest that SP1 phosphorylation induced by OKA, a selective inhibitor of the serine-threonine phosphatase PP2A, facilitates the formation of a transcription complex involving general transcription factors, HIV Tat, and Sp1 proteins. The formation of this complex would increase, independently of an in synergy with NF-kappa B, the low basal activity of the HIV LTR observed in normal T lymphocytes. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "complex", "start": 1175, "end": 1182}, "arguments": [{"role": "Theme", "text": "Tat", "start": 1228, "end": 1231}, {"role": "Theme2", "text": "Sp1", "start": 1237, "end": 1240}]}], "gene expression": [{"trigger": {"text": "expression", "start": 946, "end": 956}, "arguments": [{"role": "Theme", "text": "Tat", "start": 942, "end": 945}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 17, "end": 32}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 13, "end": 16}]}, {"trigger": {"text": "phosphorylation", "start": 720, "end": 735}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 768, "end": 771}]}, {"trigger": {"text": "phosphorylation", "start": 1034, "end": 1049}, "arguments": [{"role": "Theme", "text": "SP1", "start": 1030, "end": 1033}]}], "positive regulation": [{"trigger": {"text": "Induction", "start": 0, "end": 9}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 17, "end": 32}]}, {"trigger": {"text": "induced", "start": 704, "end": 711}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 720, "end": 735}]}, {"trigger": {"text": "induced", "start": 1050, "end": 1057}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1034, "end": 1049}]}, {"trigger": {"text": "facilitates", "start": 1130, "end": 1141}, "arguments": [{"role": "Cause", "text": "induced", "start": 1050, "end": 1057}, {"role": "Theme", "text": "complex", "start": 1175, "end": 1182}]}]}}, "schema": []} {"input": "Human MHC class II gene transcription directed by the carboxyl terminus of CIITA, one of the defective genes in type II MHC combined immune deficiency. \nType II major histocompatibility complex combined immune deficiency (type II MHC CID or bare lymphocyte syndrome) is a congenital immunodeficiency disease characterized by absent MHC class II expression. Four distinct complementation groups have been identified. Recently, the defective gene in group II type II MHC CID has been isolated and termed CIITA. Here, we demonstrate that CIITA is an MHC class II gene-specific transcription activator. The transcription activation function is provided by the N-terminal acidic domain (amino acids 26-137), which is experimentally exchangeable with a heterologous viral transcription-activating domain. The specificity of CIITA for three major MHC class II genes, DR, DQ and DP, is mediated by its remaining C-terminal residues (amino acids 317-1130). The transactivation of multiple cis elements, especially S and X2, of the DR alpha proximal promoter in group II CID cells is CIITA dependent. Since CIITA overexpression in normal cells did not increase class II expression, we propose that initiation of CIITA expression serves as the on-off switch, while availability of downstream interactor(s) limits transcription. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "overexpression", "start": 1103, "end": 1117}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 1097, "end": 1102}]}, {"trigger": {"text": "expression", "start": 1208, "end": 1218}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 1202, "end": 1207}]}], "positive regulation": [{"trigger": {"text": "transactivation", "start": 952, "end": 967}, "arguments": [{"role": "Site", "text": "S", "start": 1005, "end": 1006}, {"role": "Theme", "text": "DR alpha", "start": 1022, "end": 1030}]}, {"trigger": {"text": "transactivation", "start": 952, "end": 967}, "arguments": [{"role": "Site", "text": "X2", "start": 1011, "end": 1013}, {"role": "Theme", "text": "DR alpha", "start": 1022, "end": 1030}]}, {"trigger": {"text": "overexpression", "start": 1103, "end": 1117}, "arguments": [{"role": "Theme", "text": "overexpression", "start": 1103, "end": 1117}]}, {"trigger": {"text": "initiation", "start": 1188, "end": 1198}, "arguments": [{"role": "Theme", "text": "expression", "start": 1208, "end": 1218}]}], "regulation": [{"trigger": {"text": "dependent", "start": 1080, "end": 1089}, "arguments": [{"role": "Theme", "text": "transactivation", "start": 952, "end": 967}, {"role": "Cause", "text": "CIITA", "start": 1074, "end": 1079}]}]}}, "schema": []} {"input": "Abnormal regulation of the IL-2 promoter in lpr CD4-CD8- T lymphocytes results in constitutive expression of a novel nuclear factor of activated T cells-binding factor. \nThe inert quality of MRL-Ipr/Ipr (Ipr) peripheral CD4-CD8- (CD4-8-) T cells manifests primarily as an inability to proliferate or produce IL-2 in response to TCR or mitogenic stimulation. Yet these same cells do initiate early TCR-mediated signaling events, such as generation of inositol phosphates and increased intracellular calcium. They also display constitutively high levels of p59fyn and CD3 zeta tyrosine phosphorylation. The generation of second messengers in T cells normally leads to downstream signaling that results in transcriptional activation of the IL-2 gene. We, therefore, compared the activation state of the IL-2 gene promoter region in freshly isolated and stimulated Ipr CD4-8- T cells with that of normal T lymphocytes. Levels of the octamer, NF-kappa B (p50-p65 heterodimer), and AP-1 transcriptional factors are constitutively elevated in freshly isolated Ipr CD4-8- T cells, consistent with the activated phenotype of these cells. Upon stimulation with mitogens, formation of the transactivating complex, nuclear factor of activated T cells (NF-AT), occurs with normal kinetics in Ipr CD4-8- T cells. Yet, the levels of the activating NF-AT complex never reach those observed in similarly stimulated normal T cells. Furthermore, nuclear extracts from Ipr CD4-8- T cells display high levels of a novel specific binding activity at the NF-AT site, which is present at much lower levels in freshly isolated normal T lymphocytes. Upon mitogenic stimulation, the binding activity of the novel NF-AT-binding factor is rapidly down-regulated in normal T cells, but persists at high levels in Ipr CD4-8- T cells. These two abnormalities at the NF-AT site provide a potential mechanism to account for the defect in IL-2 production from Ipr CD4-8- T cells. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "produce", "start": 300, "end": 307}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 308, "end": 312}]}, {"trigger": {"text": "production", "start": 1909, "end": 1919}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1904, "end": 1908}]}], "negative regulation": [{"trigger": {"text": "defect", "start": 1894, "end": 1900}, "arguments": [{"role": "Theme", "text": "production", "start": 1909, "end": 1919}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 584, "end": 599}, "arguments": [{"role": "Theme", "text": "p59fyn", "start": 555, "end": 561}, {"role": "Site", "text": "tyrosine", "start": 575, "end": 583}]}, {"trigger": {"text": "phosphorylation", "start": 584, "end": 599}, "arguments": [{"role": "Theme", "text": "CD3 zeta", "start": 566, "end": 574}, {"role": "Site", "text": "tyrosine", "start": 575, "end": 583}]}], "positive regulation": [{"trigger": {"text": "inability", "start": 272, "end": 281}, "arguments": [{"role": "Theme", "text": "produce", "start": 300, "end": 307}]}, {"trigger": {"text": "high levels", "start": 540, "end": 551}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 584, "end": 599}]}, {"trigger": {"text": "results", "start": 692, "end": 699}, "arguments": [{"role": "Theme", "text": "transcriptional activation", "start": 703, "end": 729}]}, {"trigger": {"text": "transcriptional activation", "start": 703, "end": 729}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 737, "end": 741}]}, {"trigger": {"text": "activation", "start": 776, "end": 786}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 800, "end": 804}]}, {"trigger": {"text": "account", "start": 1878, "end": 1885}, "arguments": [{"role": "Theme", "text": "production", "start": 1909, "end": 1919}]}], "regulation": [{"trigger": {"text": "Abnormal regulation", "start": 0, "end": 19}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 27, "end": 31}, {"role": "Site", "text": "promoter", "start": 32, "end": 40}]}]}}, "schema": []} {"input": "GM-CSF and IL-2 share common control mechanisms in response to costimulatory signals in T cells. \nAntigen complexed with major histocompatibility complex class I or II molecules on the surface of antigen presenting cells interacts with the T cell receptor (TCR) on the surface of T cells and initiates an activation cascade. So called costimulatory signals, mediated by other cell surface interactions or soluble cytokines produced by antigen presenting cells, are also required for complete T cell activation. High levels of cytokine gene expression in T cells also required both TCR and costimulatory signals. The granulocyte-macrophage colony-stimulating factor requires sequences in the promoter as well as a powerful enhancer located 3kb upstream to respond to TCR-like signals. These promoter and enhancer regions are mainly activated by the transcription factor nuclear factor of activated T cells (NFAT). The activation of NFAT by TCR signals has been well described for interleukin-2 (IL-2) and IL-4 gene transcription in T cells. Costimulatory signals, such as activation of the CD28 cell surface molecule on T cells, lead to activation through a distinct region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter. This region is termed the CK-1 or CD28RE and appears to bind specific members of the NF-kappa B family of transcription factors. Human T leukemia virus type 1 (HTLV-1) infects T cells and can lead to increase GM-CSF expression. We have found that the HTLV-1 transactivator protein, tax, acts as a costimulatory signal for GM-CSF and IL-2 gene transcription, in that it can cooperate with TCR signals to mediate high level gene expression. Tax activates the GM-CSF promoter through the CK-1/CD28RE region and also activates nuclear factor-kappa B binding to this region. However, other transcription factors or coactivators of NF-kappa B are required for tax activation but these remain to be identified. The CK-1/CD28RE of GM-CSF shows a high degree of similarity to the IL-2 CD28RE and the IL-3 gene also contains a related region. This observation, together with the fact that both GM-CSF and IL-2 respond to TCR signals via NFAT, implies a high degree of conservation in the regulation of cytokine gene expression in T cells. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 1464, "end": 1474}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 1457, "end": 1463}]}], "positive regulation": [{"trigger": {"text": "requires", "start": 665, "end": 673}, "arguments": [{"role": "Theme", "text": "respond", "start": 755, "end": 762}]}, {"trigger": {"text": "activation", "start": 1071, "end": 1081}, "arguments": [{"role": "Theme", "text": "CD28", "start": 1089, "end": 1093}]}, {"trigger": {"text": "activation", "start": 1136, "end": 1146}, "arguments": [{"role": "Cause", "text": "activation", "start": 1071, "end": 1081}, {"role": "Theme", "text": "granulocyte-macrophage colony-stimulating factor", "start": 1180, "end": 1228}, {"role": "Site", "text": "promoter", "start": 1238, "end": 1246}]}, {"trigger": {"text": "increase", "start": 1448, "end": 1456}, "arguments": [{"role": "Theme", "text": "expression", "start": 1464, "end": 1474}]}, {"trigger": {"text": "acts as a costimulatory signal", "start": 1535, "end": 1565}, "arguments": [{"role": "Cause", "text": "tax", "start": 1530, "end": 1533}, {"role": "Theme", "text": "gene transcription", "start": 1586, "end": 1604}]}, {"trigger": {"text": "activates", "start": 1691, "end": 1700}, "arguments": [{"role": "Cause", "text": "Tax", "start": 1687, "end": 1690}, {"role": "Theme", "text": "GM-CSF", "start": 1705, "end": 1711}, {"role": "Site", "text": "promoter", "start": 1712, "end": 1720}]}], "regulation": [{"trigger": {"text": "in response to", "start": 48, "end": 62}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 0, "end": 6}]}, {"trigger": {"text": "in response to", "start": 48, "end": 62}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 11, "end": 15}]}, {"trigger": {"text": "respond", "start": 755, "end": 762}, "arguments": [{"role": "Theme", "text": "granulocyte-macrophage colony-stimulating factor", "start": 616, "end": 664}]}, {"trigger": {"text": "respond", "start": 2148, "end": 2155}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 2132, "end": 2138}]}, {"trigger": {"text": "respond", "start": 2148, "end": 2155}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 2143, "end": 2147}]}], "transcription": [{"trigger": {"text": "gene transcription", "start": 1009, "end": 1027}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 1004, "end": 1008}]}, {"trigger": {"text": "gene transcription", "start": 1009, "end": 1027}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 994, "end": 998}]}, {"trigger": {"text": "gene transcription", "start": 1586, "end": 1604}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 1570, "end": 1576}]}, {"trigger": {"text": "gene transcription", "start": 1586, "end": 1604}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1581, "end": 1585}]}]}}, "schema": []} {"input": "MIP1 alpha nuclear protein (MNP), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human MIP-1 alpha gene. \nMurine macrophage inflammatory protein 1 alpha (MIP-1 alpha) and its human equivalent (GOS19, LD78, or AT464) are members of the -C-C family of low-molecular-weight chemokines. Secreted from activated T cells and macrophages, bone marrow-derived MIP-1 alpha/GOS19 inhibits primitive hematopoietic stem cells and appears to be involved in the homeostatic control of stem cell proliferation. It also induces chemotaxis and inflammatory responses in mature cell types. Therefore, it is important to understand the mechanisms which control the expression of MIP-1 alpha/GOS19. Previous work has shown that in Jurkat T cells, a set of widely expressed transcription factors (the ICK-1 family) affect the GOS19 promoter. One member, ICK-1A, behaves as a strong negative regulator. In this communication, we provide evidence that the pathway of induction in the macrophage cell line U937 is different from that in Jurkat cells. Furthermore, we show that the ICK-1 binding site does not confer negative regulation in U937 cells. We provide evidence for an additional binding site, the MIP-1 alpha nuclear protein (MNP) site, which overlaps the ICK-1 site. Interaction of nuclear extracts from various cell lines and tissue with the MNP site leads to the formation of fast-migrating protein-DNA complexes with similar but distinct electrophoretic mobilities. A mutation of the MNP site which does not abrogate ICK-1 binding inactivates the GOS19.1 promoter in U937 cells and reduces its activity by fourfold in Jurkat cells. We propose that the MNP protein(s) binding at the MNP site constitutes a novel transcription factor(s) expressed in hematopoietic cells. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 702, "end": 712}, "arguments": [{"role": "Theme", "text": "MIP-1 alpha", "start": 716, "end": 727}]}], "localization": [{"trigger": {"text": "Secreted", "start": 339, "end": 347}, "arguments": [{"role": "Theme", "text": "MIP-1 alpha", "start": 408, "end": 419}]}], "positive regulation": [{"trigger": {"text": "crucial", "start": 104, "end": 111}, "arguments": [{"role": "Theme", "text": "transcription", "start": 116, "end": 129}]}, {"trigger": {"text": "derived", "start": 400, "end": 407}, "arguments": [{"role": "Theme", "text": "MIP-1 alpha", "start": 408, "end": 419}]}, {"trigger": {"text": "induction", "start": 1000, "end": 1009}, "arguments": [{"role": "Theme", "text": "ICK-1A", "start": 889, "end": 895}]}], "regulation": [{"trigger": {"text": "control", "start": 690, "end": 697}, "arguments": [{"role": "Theme", "text": "expression", "start": 702, "end": 712}]}, {"trigger": {"text": "affect", "start": 850, "end": 856}, "arguments": [{"role": "Theme", "text": "GOS19", "start": 861, "end": 866}, {"role": "Site", "text": "promoter", "start": 867, "end": 875}]}], "transcription": [{"trigger": {"text": "transcription", "start": 116, "end": 129}, "arguments": [{"role": "Theme", "text": "MIP-1 alpha", "start": 143, "end": 154}]}]}}, "schema": []} {"input": "Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor. \nHigh-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. (ABSTRACT TRUNCATED AT 250 WORDS) ", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 1307, "end": 1311}, "arguments": [{"role": "Theme", "text": "GATA-1", "start": 1293, "end": 1299}, {"role": "Theme2", "text": "ER", "start": 1304, "end": 1306}]}, {"trigger": {"text": "associate", "start": 1438, "end": 1447}, "arguments": [{"role": "Theme", "text": "GATA-1", "start": 1424, "end": 1430}, {"role": "Theme2", "text": "ER", "start": 1435, "end": 1437}]}, {"trigger": {"text": "contacts", "start": 1548, "end": 1556}, "arguments": [{"role": "Theme", "text": "GATA-1", "start": 1512, "end": 1518}, {"role": "Theme2", "text": "ER", "start": 1527, "end": 1529}, {"role": "Site", "text": "finger region", "start": 1576, "end": 1589}]}, {"trigger": {"text": "contacts", "start": 1548, "end": 1556}, "arguments": [{"role": "Theme", "text": "GATA-1", "start": 1512, "end": 1518}, {"role": "Theme2", "text": "ER", "start": 1527, "end": 1529}, {"role": "Site", "text": "N-terminal activation domain", "start": 1598, "end": 1626}]}, {"trigger": {"text": "interaction", "start": 1752, "end": 1763}, "arguments": [{"role": "Theme", "text": "GATA-1", "start": 1712, "end": 1718}, {"role": "Theme2", "text": "ER", "start": 1773, "end": 1775}]}], "negative regulation": [{"trigger": {"text": "repression", "start": 17, "end": 27}, "arguments": [{"role": "Theme", "text": "GATA-1", "start": 66, "end": 72}, {"role": "Cause", "text": "estrogen receptor", "start": 80, "end": 97}]}, {"trigger": {"text": "down-regulation", "start": 312, "end": 327}, "arguments": [{"role": "Theme", "text": "alpha-", "start": 377, "end": 383}]}, {"trigger": {"text": "down-regulation", "start": 312, "end": 327}, "arguments": [{"role": "Theme", "text": "beta-globin", "start": 388, "end": 399}]}, {"trigger": {"text": "down-regulation", "start": 312, "end": 327}, "arguments": [{"role": "Theme", "text": "H5", "start": 459, "end": 461}]}], "regulation": [{"trigger": {"text": "effects", "start": 682, "end": 689}, "arguments": [{"role": "Theme", "text": "GATA-1", "start": 693, "end": 699}]}, {"trigger": {"text": "dependent", "start": 1460, "end": 1469}, "arguments": [{"role": "Theme", "text": "associate", "start": 1438, "end": 1447}]}, {"trigger": {"text": "modulating", "start": 1701, "end": 1711}, "arguments": [{"role": "Theme", "text": "GATA-1", "start": 1712, "end": 1718}]}]}}, "schema": []} {"input": "Activation of pp90rsk and early growth response-1 gene expression by pokeweed mitogen in human B cells. \nThe present studies have examined the effects of pokeweed mitogen (PWM) on the induction of early growth response-1 gene (EGR-1) in normal human B cells. PWM regulates EGR-1 gene expression by both transcriptional and post-transcriptional mechanisms. Transient transfection assays with EGR-1 promoter fragments linked to the chloramphenicol acetyltransferase (CAT) gene demonstrated that PWM induced EGR-1 transcription is conferred by the CArG motif (C C[AT]6GG) in the EGR-1 promoter. The results further demonstrated the activation of S6 kinase (pp90rsk), evidenced by phosphorylation of S6 and serum response factor (SRF) peptides, in PWM treated B cells. Taken together, these findings suggest that PWM is able to initiate an intracytoplasmic signalling cascade and EGR-1 induction in normal human B cells. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 55, "end": 65}, "arguments": [{"role": "Theme", "text": "early growth response-1", "start": 26, "end": 49}]}, {"trigger": {"text": "expression", "start": 284, "end": 294}, "arguments": [{"role": "Theme", "text": "EGR-1", "start": 273, "end": 278}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 677, "end": 692}, "arguments": [{"role": "Theme", "text": "SRF", "start": 726, "end": 729}]}], "positive regulation": [{"trigger": {"text": "Activation", "start": 0, "end": 10}, "arguments": [{"role": "Theme", "text": "expression", "start": 55, "end": 65}]}, {"trigger": {"text": "induction", "start": 184, "end": 193}, "arguments": [{"role": "Theme", "text": "EGR-1", "start": 227, "end": 232}]}, {"trigger": {"text": "induced", "start": 497, "end": 504}, "arguments": [{"role": "Theme", "text": "transcription", "start": 511, "end": 524}]}, {"trigger": {"text": "conferred", "start": 528, "end": 537}, "arguments": [{"role": "Theme", "text": "induced", "start": 497, "end": 504}, {"role": "CSite", "text": "CArG motif", "start": 545, "end": 555}, {"role": "Cause", "text": "EGR-1", "start": 576, "end": 581}]}, {"trigger": {"text": "induction", "start": 882, "end": 891}, "arguments": [{"role": "Theme", "text": "EGR-1", "start": 876, "end": 881}]}], "regulation": [{"trigger": {"text": "effects", "start": 143, "end": 150}, "arguments": [{"role": "Theme", "text": "induction", "start": 184, "end": 193}]}, {"trigger": {"text": "regulates", "start": 263, "end": 272}, "arguments": [{"role": "Theme", "text": "expression", "start": 284, "end": 294}]}], "transcription": [{"trigger": {"text": "transcription", "start": 511, "end": 524}, "arguments": [{"role": "Theme", "text": "EGR-1", "start": 505, "end": 510}]}]}}, "schema": []} {"input": "NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. \nThe eukaryotic transcription factor nuclear factor-kappa B (NF-kappa B) participates in many parts of the genetic program mediating T lymphocyte activation and growth. Nuclear expression of NF-kappa B occurs after its induced dissociation from its cytoplasmic inhibitor I kappa B alpha. Phorbol ester and tumor necrosis factor-alpha induction of nuclear NF-kappa B is associated with both the degradation of performed I kappa B alpha and the activation of I kappa B alpha gene expression. Transfection studies indicate that the I kappa B alpha gene is specifically induced by the 65-kilodalton transactivating subunit of NF-kappa B. Association of the newly synthesized I kappa B alpha with p65 restores intracellular inhibition of NF-kappa B DNA binding activity and prolongs the survival of this labile inhibitor. Together, these results show that NF-kappa B controls the expression of I kappa B alpha by means of an inducible autoregulatory pathway. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "Association", "start": 745, "end": 756}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 782, "end": 797}, {"role": "Theme2", "text": "p65", "start": 803, "end": 806}]}], "gene expression": [{"trigger": {"text": "expression", "start": 20, "end": 30}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 44, "end": 59}]}, {"trigger": {"text": "expression", "start": 589, "end": 599}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 568, "end": 583}]}, {"trigger": {"text": "expression", "start": 986, "end": 996}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 1000, "end": 1015}]}], "positive regulation": [{"trigger": {"text": "activation", "start": 554, "end": 564}, "arguments": [{"role": "Theme", "text": "expression", "start": 589, "end": 599}]}, {"trigger": {"text": "induced", "start": 677, "end": 684}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 640, "end": 655}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 505, "end": 516}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 530, "end": 545}]}], "regulation": [{"trigger": {"text": "controls", "start": 11, "end": 19}, "arguments": [{"role": "Theme", "text": "expression", "start": 20, "end": 30}]}, {"trigger": {"text": "controls", "start": 973, "end": 981}, "arguments": [{"role": "Theme", "text": "expression", "start": 986, "end": 996}]}]}}, "schema": []} {"input": "Activation of primary human T-lymphocytes through CD2 plus CD28 adhesion molecules induces long-term nuclear expression of NF-kappa B. \nStimulation of highly purified human T-cells via CD2 and CD28 adhesion molecules induces and maintains proliferation for more than 3 weeks. This potent interleukin 2 (IL-2)-dependent activation does not require monocytes or accessory cells. Long-lasting IL-2 receptivity is associated with high-level expression of the inducible IL-2 receptor alpha chain (IL-2R alpha) gene that is regulated at both transcriptional and posttranscriptional levels. Increase of IL-2R alpha gene transcription involves the enhanced binding of the transcription factor NF-kappa B to its consensus sequence in the 5'-regulatory region of the IL-2R alpha gene. To dissect the molecular basis for the unusually persistent transcription of the IL-2R alpha gene, we analyzed nuclear NF-kappa B binding to a radiolabeled IL-2R alpha kappa B-specific oligonucleotide probe during the time course of CD2 + CD28 activation. Resting T-cell nuclear extracts contained KBF1/p50 homodimer. After stimulation, two new kappa B-specific complexes were identified as NF-kappa B p50-p65 heterodimer and putative c-Rel homodimer or c-Rel-p65 heterodimer. Both inducible complexes persisted for at least 3 weeks. Their relative levels were very similar for the duration of proliferation. In parallel, CD2 + CD28 activation triggered a significant intracellular thiol decrease, suggesting that oxygen radicals are involved in the signaling pathway of adhesion molecules. Finally, micromolar amounts of pyrrolidine dithiocarbamate, an oxygen radical scavenger that efficiently blocked the nuclear appearance of NF-kappa B in T-lymphocytes, also inhibited IL-2 secretion, IL-2R alpha cell surface expression, and T-cell proliferation. Together, these results suggest that NF-kappa B plays an important role in long-term activation of human primary T-lymphocytes via CD2 + CD28. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 649, "end": 656}, "arguments": [{"role": "Theme", "text": "IL-2R alpha", "start": 757, "end": 768}]}, {"trigger": {"text": "heterodimer", "start": 1185, "end": 1196}, "arguments": [{"role": "Theme", "text": "p50", "start": 1177, "end": 1180}, {"role": "Theme2", "text": "p65", "start": 1181, "end": 1184}]}, {"trigger": {"text": "homodimer", "start": 1216, "end": 1225}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 1210, "end": 1215}]}, {"trigger": {"text": "heterodimer", "start": 1239, "end": 1250}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 1229, "end": 1234}, {"role": "Theme2", "text": "p65", "start": 1235, "end": 1238}]}], "gene expression": [{"trigger": {"text": "expression", "start": 437, "end": 447}, "arguments": [{"role": "Theme", "text": "IL-2R alpha", "start": 492, "end": 503}]}, {"trigger": {"text": "expression", "start": 1790, "end": 1800}, "arguments": [{"role": "Theme", "text": "IL-2R alpha", "start": 1765, "end": 1776}]}], "localization": [{"trigger": {"text": "secretion", "start": 1754, "end": 1763}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1749, "end": 1753}]}], "negative regulation": [{"trigger": {"text": "inhibited", "start": 1739, "end": 1748}, "arguments": [{"role": "Theme", "text": "secretion", "start": 1754, "end": 1763}]}, {"trigger": {"text": "inhibited", "start": 1739, "end": 1748}, "arguments": [{"role": "Theme", "text": "expression", "start": 1790, "end": 1800}]}], "positive regulation": [{"trigger": {"text": "Increase", "start": 584, "end": 592}, "arguments": [{"role": "Theme", "text": "transcription", "start": 613, "end": 626}]}, {"trigger": {"text": "enhanced", "start": 640, "end": 648}, "arguments": [{"role": "Theme", "text": "binding", "start": 649, "end": 656}]}, {"trigger": {"text": "activation", "start": 1019, "end": 1029}, "arguments": [{"role": "Theme", "text": "CD2", "start": 1008, "end": 1011}]}, {"trigger": {"text": "activation", "start": 1019, "end": 1029}, "arguments": [{"role": "Theme", "text": "CD28", "start": 1014, "end": 1018}]}, {"trigger": {"text": "identified", "start": 1152, "end": 1162}, "arguments": [{"role": "Theme", "text": "heterodimer", "start": 1185, "end": 1196}]}, {"trigger": {"text": "identified", "start": 1152, "end": 1162}, "arguments": [{"role": "Theme", "text": "homodimer", "start": 1216, "end": 1225}]}, {"trigger": {"text": "identified", "start": 1152, "end": 1162}, "arguments": [{"role": "Theme", "text": "heterodimer", "start": 1239, "end": 1250}]}, {"trigger": {"text": "activation", "start": 1408, "end": 1418}, "arguments": [{"role": "Theme", "text": "CD2", "start": 1397, "end": 1400}]}, {"trigger": {"text": "activation", "start": 1408, "end": 1418}, "arguments": [{"role": "Theme", "text": "CD28", "start": 1403, "end": 1407}]}], "regulation": [{"trigger": {"text": "regulated", "start": 518, "end": 527}, "arguments": [{"role": "Theme", "text": "expression", "start": 437, "end": 447}]}], "transcription": [{"trigger": {"text": "transcription", "start": 613, "end": 626}, "arguments": [{"role": "Theme", "text": "IL-2R alpha", "start": 596, "end": 607}]}, {"trigger": {"text": "transcription", "start": 835, "end": 848}, "arguments": [{"role": "Theme", "text": "IL-2R alpha", "start": 856, "end": 867}]}]}}, "schema": []} {"input": "Synergism between the CD3 antigen- and CD2 antigen-derived signals. Exploration at the level of induction of DNA-binding proteins and characterization of the inhibitory activity of cyclosporine. \nWe have demonstrated earlier that the crosslinkage of the CD3/TCR complex with the CD2 antigen results in the proliferation of normal human T cells. The effect of this synergism was perceptible at the level of induction of the IL-2 gene, a process critical for T cell growth. To further understand the molecular and nuclear basis for this synergism, we have explored the induction of DNA-binding proteins in highly purified normal human T cells signaled via the CD3 and/or CD2 proteins. The effect of transmembrane signaling of T cells with ionomycin, and/or sn-1,2 dioctanoyl glycerol, was also determined. The emergence of nuclear binding proteins was investigated using interleukin-2 sequence specific oligonucleotide probes in the electrophoretic mobility shift assay. Our studies demonstrate for the first time that CD3 antigen-derived signals and CD2 antigen-derived signals are synergistic in inducing the emergence of transcription factors that bind to the NF-AT1, AP-1, and NF-kB sites located in the promoter/enhancer region of the IL-2 gene. Moreover, cyclosporine, at concentrations readily accomplished in clinical practice, was found to inhibit the emergence of these DNA-binding proteins in normal human T cells signaled via cell surface proteins implicated in antigen-dependent T cell activation and in T cells stimulated by mobilization of cellular calcium and activation of protein kinase C. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "induction", "start": 406, "end": 415}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 423, "end": 427}]}]}}, "schema": []} {"input": "Antisense oligonucleotides to the p65 subunit of NF-kappa B block CD11b expression and alter adhesion properties of differentiated HL-60 granulocytes. \nNF-kappa B is a pleiotropic regulator of a variety of genes implicated in the cellular response to injury. This function has been attributed to the coordinated binding of subunits of NF-kappa B to distinct regions of the promoter elements of numerous genes, including cytokines, growth factor receptors, and adhesion molecules. Antisense phosphorothioate oligonucleotides to the p50 and p65 subunits of the NF-kappa B complex were used to define the physiologic role of this transcription factor in resting and stimulated granulocytes. A reduction in the expression of p65 was produced by treatment with the phosphorothioate antisense oligodeoxynucleotide. This reduction was accompanied by rapid changes in the cellular adhesion of dimethyl sulfoxide-differentiated HL-60 leukemia cells stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA). These effects were characterized by a marked reduction in CD11b integrin expression on the surface of treated cells. Furthermore, the p65 antisense oligomer effectively abolished an upregulation of CD11b that was produced by formyl-met-leu-phe and TPA. However, the p65 antisense phosphorothioate oligodeoxynucleotide had no significant effect on the production of reactive oxygen intermediates or on phagocytosis by these cells. These findings indicate that antisense oligomers to p65 can be used to define the role of NF-kappa B in the activation pathways of neutrophils. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 72, "end": 82}, "arguments": [{"role": "Theme", "text": "CD11b", "start": 66, "end": 71}]}, {"trigger": {"text": "expression", "start": 707, "end": 717}, "arguments": [{"role": "Theme", "text": "p65", "start": 721, "end": 724}]}, {"trigger": {"text": "expression", "start": 1071, "end": 1081}, "arguments": [{"role": "Theme", "text": "CD11b", "start": 1056, "end": 1061}]}], "negative regulation": [{"trigger": {"text": "block", "start": 60, "end": 65}, "arguments": [{"role": "Theme", "text": "expression", "start": 72, "end": 82}]}, {"trigger": {"text": "reduction", "start": 690, "end": 699}, "arguments": [{"role": "Theme", "text": "expression", "start": 707, "end": 717}]}, {"trigger": {"text": "reduction", "start": 1043, "end": 1052}, "arguments": [{"role": "Theme", "text": "expression", "start": 1071, "end": 1081}]}, {"trigger": {"text": "abolished", "start": 1167, "end": 1176}, "arguments": [{"role": "Theme", "text": "upregulation", "start": 1180, "end": 1192}]}], "positive regulation": [{"trigger": {"text": "upregulation", "start": 1180, "end": 1192}, "arguments": [{"role": "Theme", "text": "CD11b", "start": 1196, "end": 1201}]}]}}, "schema": []} {"input": "Human interferon regulatory factor 2 gene. Intron-exon organization and functional analysis of 5'-flanking region. \nInterferon regulatory factor 2 (IRF-2) is a transcriptional regulatory protein that terminates interferon beta expression initiated by interferon regulatory factor 1. In this study, we isolated the genomic DNA for human IRF-2 gene, determined the intron-exon structure of the human IRF-2 gene, mapped the major transcription initiation site, identified a number of potential regulatory elements in the 5'-flanking region, and localized the IRF-2 gene on human chromosome 4. The IRF-2 promoter region contains a CpG island, with several GC boxes, a putative NF-kappa B-binding site, and a CAAT box, but no TATA box. When the promoter region was linked with a heterologous reporter gene, we found that the promoter region is inducible by both interferons (interferon-alpha and -gamma) and interferon regulatory factor 1. The region which induced these inductions was identified as being confined to 40 nucleotides 5' to the major transcriptional initiation site by testing a series of clones with truncated promoter of IRF-2. This region contains elements which are shared with the transcriptional enhancers of other genes including interferon regulatory factor 1, interferon beta, and interferon-inducible genes. These data suggest that interferon regulatory factor 1 not only triggers the activation of the interferon signal transduction pathway, but also may play a role in limiting the duration of this response by activating the transcription of IRF-2. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 227, "end": 237}, "arguments": [{"role": "Theme", "text": "interferon beta", "start": 211, "end": 226}]}], "negative regulation": [{"trigger": {"text": "terminates", "start": 200, "end": 210}, "arguments": [{"role": "Cause", "text": "IRF-2", "start": 148, "end": 153}, {"role": "Theme", "text": "expression", "start": 227, "end": 237}]}], "positive regulation": [{"trigger": {"text": "initiated", "start": 238, "end": 247}, "arguments": [{"role": "Theme", "text": "expression", "start": 227, "end": 237}, {"role": "Cause", "text": "interferon regulatory factor 1", "start": 251, "end": 281}]}, {"trigger": {"text": "inducible", "start": 839, "end": 848}, "arguments": [{"role": "Theme", "text": "IRF-2", "start": 594, "end": 599}, {"role": "Site", "text": "promoter region", "start": 600, "end": 615}, {"role": "Cause", "text": "interferon-alpha", "start": 870, "end": 886}]}, {"trigger": {"text": "inducible", "start": 839, "end": 848}, "arguments": [{"role": "Theme", "text": "IRF-2", "start": 594, "end": 599}, {"role": "Site", "text": "promoter region", "start": 600, "end": 615}, {"role": "Cause", "text": "-gamma", "start": 891, "end": 897}]}, {"trigger": {"text": "inducible", "start": 839, "end": 848}, "arguments": [{"role": "Theme", "text": "IRF-2", "start": 594, "end": 599}, {"role": "Site", "text": "promoter region", "start": 600, "end": 615}, {"role": "Cause", "text": "interferon regulatory factor 1", "start": 903, "end": 933}]}, {"trigger": {"text": "activating", "start": 1533, "end": 1543}, "arguments": [{"role": "Cause", "text": "interferon regulatory factor 1", "start": 1352, "end": 1382}, {"role": "Theme", "text": "transcription", "start": 1548, "end": 1561}]}], "transcription": [{"trigger": {"text": "transcription", "start": 1548, "end": 1561}, "arguments": [{"role": "Theme", "text": "IRF-2", "start": 1565, "end": 1570}]}]}}, "schema": []} {"input": "Overproduction of NFKB2 (lyt-10) and c-Rel: a mechanism for HTLV-I Tax-mediated trans-activation via the NF-kappa B signalling pathway. \nMolecular, biochemical and epidemiological evidence implicate HTLV-I as an etiologic agent of adult T cell leukemia (ATL). The Tax protein of HTLV-I, a positive transcriptional activator of HTLV-I gene expression, is a viral oncogene that also increases transcription of cellular genes including GM-CSF, IL-2R alpha and IL-2. One of the cellular targets of the trans-activating effects of Tax is the NF-kappa B/Rel family of transcription factors, pleiotropic regulators of immunoregulatory, cytokine and viral gene expression. In this report, we demonstrate that NFKB2 (lyt-10) and c-Rel are overexpressed in HTLV-I infected and Tax-expressing cells and, together, account for the majority of the constitutive NF-kappa B binding activity in these cells before and after PMA stimulation. Most importantly, we show a Tax-dependent correlation between expression of NFKB2(p100) and processing to the DNA binding NFKB2(p52) form, induction of c-Rel, and trans-activation of NF-kappa B-mediated gene expression. Furthermore, the NFKB2 precursor is physically associated with c-Rel and with Tax in HTLV-I infected cells. We propose that NFKB2 synthesis and processing allows continuous nuclear expression of an otherwise cytoplasmic protein and, in conjunction with overexpression of c-Rel, NFKB2 alters the NF-kappa B signalling pathway and contributes to leukemic transformation of T cells by HTLV-I. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "associated", "start": 1192, "end": 1202}, "arguments": [{"role": "Theme", "text": "NFKB2 precursor", "start": 1162, "end": 1177}, {"role": "Theme2", "text": "c-Rel", "start": 1208, "end": 1213}]}, {"trigger": {"text": "associated", "start": 1192, "end": 1202}, "arguments": [{"role": "Theme", "text": "NFKB2 precursor", "start": 1162, "end": 1177}, {"role": "Theme2", "text": "Tax", "start": 1223, "end": 1226}]}], "gene expression": [{"trigger": {"text": "Overproduction", "start": 0, "end": 14}, "arguments": [{"role": "Theme", "text": "NFKB2", "start": 18, "end": 23}]}, {"trigger": {"text": "Overproduction", "start": 0, "end": 14}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 37, "end": 42}]}, {"trigger": {"text": "overexpressed", "start": 730, "end": 743}, "arguments": [{"role": "Theme", "text": "NFKB2", "start": 701, "end": 706}]}, {"trigger": {"text": "overexpressed", "start": 730, "end": 743}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 720, "end": 725}]}, {"trigger": {"text": "expression", "start": 987, "end": 997}, "arguments": [{"role": "Theme", "text": "p100", "start": 1007, "end": 1011}]}, {"trigger": {"text": "synthesis", "start": 1275, "end": 1284}, "arguments": [{"role": "Theme", "text": "NFKB2", "start": 1269, "end": 1274}]}, {"trigger": {"text": "overexpression", "start": 1398, "end": 1412}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 1416, "end": 1421}]}], "localization": [{"trigger": {"text": "expression", "start": 1326, "end": 1336}, "arguments": [{"role": "Theme", "text": "NFKB2", "start": 1269, "end": 1274}, {"role": "AtLoc", "text": "nuclear", "start": 1318, "end": 1325}]}], "positive regulation": [{"trigger": {"text": "Overproduction", "start": 0, "end": 14}, "arguments": [{"role": "Theme", "text": "Overproduction", "start": 0, "end": 14}]}, {"trigger": {"text": "increases", "start": 381, "end": 390}, "arguments": [{"role": "Cause", "text": "Tax", "start": 264, "end": 267}, {"role": "Theme", "text": "transcription", "start": 391, "end": 404}]}, {"trigger": {"text": "overexpressed", "start": 730, "end": 743}, "arguments": [{"role": "Theme", "text": "overexpressed", "start": 730, "end": 743}]}, {"trigger": {"text": "induction", "start": 1064, "end": 1073}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 1077, "end": 1082}]}, {"trigger": {"text": "allows", "start": 1300, "end": 1306}, "arguments": [{"role": "Cause", "text": "synthesis", "start": 1275, "end": 1284}, {"role": "Theme", "text": "expression", "start": 1326, "end": 1336}]}, {"trigger": {"text": "overexpression", "start": 1398, "end": 1412}, "arguments": [{"role": "Theme", "text": "overexpression", "start": 1398, "end": 1412}]}], "transcription": [{"trigger": {"text": "transcription", "start": 391, "end": 404}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 433, "end": 439}]}, {"trigger": {"text": "transcription", "start": 391, "end": 404}, "arguments": [{"role": "Theme", "text": "IL-2R alpha", "start": 441, "end": 452}]}, {"trigger": {"text": "transcription", "start": 391, "end": 404}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 457, "end": 461}]}]}}, "schema": []} {"input": "Autoregulation of the NF-kappa B transactivator RelA (p65) by multiple cytoplasmic inhibitors containing ankyrin motifs. \nRelA (p65) functions as the critical transactivating component of the heterodimeric p50-p65 NF-kappa B complex and contains a high-affinity binding site for its cytoplasmic inhibitor, I kappa B alpha. After cellular activation, I kappa B alpha is rapidly degraded in concert with the induced nuclear translocation of NF-kappa B. The present study demonstrates that tumor necrosis factor alpha-induced degradation of I kappa B alpha in human T cells is preceded by its rapid phosphorylation in vivo. However, these effects on I kappa B alpha result in nuclear mobilization of only a fraction of the entire cytoplasmic pool of RelA. Subsequent studies have revealed that (i) cytoplasmic RelA is stably associated not only with I kappa B alpha but also with other ankyrin motif-rich proteins including the products of the NF-kappa B2 (p100) and NF-kappa B1 (p105) genes; (ii) in contrast to RelA-I kappa B alpha, RelA-p100 cytoplasmic complexes are not dissociated following tumor necrosis factor alpha activation; (iii) p100 functions as a potent inhibitor of RelA-mediated transcription in vivo; (iv) the interaction of RelA and p100 involves the conserved Rel homology domain of both proteins but not the nuclear localization signal of RelA, which is required for I kappa B alpha binding; (v) p100 inhibition of RelA function requires the C-terminal ankyrin motif domain, which mediates cytoplasmic retention of RelA; and (vi) as observed with I kappa B alpha, nuclear RelA stimulates p100 mRNA and protein expression. These findings thus reveal the presence of a second inducible autoregulated inhibitory pathway that helps ensure the rapid but transient action of nuclear NF-kappa B. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "associated", "start": 822, "end": 832}, "arguments": [{"role": "Theme", "text": "RelA", "start": 807, "end": 811}, {"role": "Theme2", "text": "I kappa B alpha", "start": 847, "end": 862}]}, {"trigger": {"text": "associated", "start": 822, "end": 832}, "arguments": [{"role": "Theme", "text": "RelA", "start": 807, "end": 811}, {"role": "Theme2", "text": "NF-kappa B2", "start": 941, "end": 952}]}, {"trigger": {"text": "associated", "start": 822, "end": 832}, "arguments": [{"role": "Theme", "text": "RelA", "start": 807, "end": 811}, {"role": "Theme2", "text": "NF-kappa B1", "start": 964, "end": 975}]}, {"trigger": {"text": "interaction", "start": 1226, "end": 1237}, "arguments": [{"role": "Theme", "text": "RelA", "start": 1241, "end": 1245}, {"role": "Theme2", "text": "p100", "start": 1250, "end": 1254}]}, {"trigger": {"text": "binding", "start": 1402, "end": 1409}, "arguments": [{"role": "Theme", "text": "RelA", "start": 1358, "end": 1362}, {"role": "Theme2", "text": "I kappa B alpha", "start": 1386, "end": 1401}]}], "gene expression": [{"trigger": {"text": "expression", "start": 1629, "end": 1639}, "arguments": [{"role": "Theme", "text": "p100", "start": 1607, "end": 1611}]}], "localization": [{"trigger": {"text": "mobilization", "start": 681, "end": 693}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 673, "end": 680}, {"role": "Theme", "text": "RelA", "start": 747, "end": 751}]}, {"trigger": {"text": "retention", "start": 1521, "end": 1530}, "arguments": [{"role": "AtLoc", "text": "cytoplasmic", "start": 1509, "end": 1520}, {"role": "Theme", "text": "RelA", "start": 1534, "end": 1538}]}], "negative regulation": [{"trigger": {"text": "inhibition", "start": 1420, "end": 1430}, "arguments": [{"role": "Cause", "text": "p100", "start": 1415, "end": 1419}, {"role": "Theme", "text": "RelA", "start": 1434, "end": 1438}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 596, "end": 611}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 538, "end": 553}]}], "positive regulation": [{"trigger": {"text": "After", "start": 323, "end": 328}, "arguments": [{"role": "Theme", "text": "degraded", "start": 377, "end": 385}]}, {"trigger": {"text": "induced", "start": 515, "end": 522}, "arguments": [{"role": "Cause", "text": "tumor necrosis factor alpha", "start": 487, "end": 514}, {"role": "Theme", "text": "degradation", "start": 523, "end": 534}]}, {"trigger": {"text": "result in", "start": 663, "end": 672}, "arguments": [{"role": "Cause", "text": "induced", "start": 515, "end": 522}, {"role": "Theme", "text": "mobilization", "start": 681, "end": 693}]}, {"trigger": {"text": "required", "start": 1373, "end": 1381}, "arguments": [{"role": "CSite", "text": "nuclear localization signal", "start": 1327, "end": 1354}, {"role": "Cause", "text": "RelA", "start": 1358, "end": 1362}, {"role": "Theme", "text": "binding", "start": 1402, "end": 1409}]}, {"trigger": {"text": "requires", "start": 1448, "end": 1456}, "arguments": [{"role": "Theme", "text": "inhibition", "start": 1420, "end": 1430}]}, {"trigger": {"text": "mediates", "start": 1500, "end": 1508}, "arguments": [{"role": "Theme", "text": "retention", "start": 1521, "end": 1530}]}, {"trigger": {"text": "stimulates", "start": 1596, "end": 1606}, "arguments": [{"role": "Cause", "text": "RelA", "start": 1591, "end": 1595}, {"role": "Theme", "text": "expression", "start": 1629, "end": 1639}]}], "protein catabolism": [{"trigger": {"text": "degraded", "start": 377, "end": 385}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 350, "end": 365}]}, {"trigger": {"text": "degradation", "start": 523, "end": 534}, "arguments": [{"role": "Theme", "text": "I kappa B alpha", "start": 538, "end": 553}]}], "regulation": [{"trigger": {"text": "Autoregulation", "start": 0, "end": 14}, "arguments": [{"role": "Theme", "text": "RelA", "start": 48, "end": 52}]}], "transcription": [{"trigger": {"text": "expression", "start": 1629, "end": 1639}, "arguments": [{"role": "Theme", "text": "p100", "start": 1607, "end": 1611}]}]}}, "schema": []} {"input": "Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B. \nThe interleukin-2 (IL-2) promoter consists of several independent T cell receptor (TcR) responsive elements. The induction of promoters dependent on these elements is inhibitable by the immunosuppressants cyclosporin A (CsA) and tacrolimus (FK-506). Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the FK-506- and CsA-sensitive enzyme required for TcR mediated activation of the IL-2 promoter. We report that a constitutively active form of calcineurin partially substitutes for the Ca2+ co-stimulus required to activate the IL-2 promoter elements IL-2A (which binds the factors OAP and Oct-1) and IL-2E (which binds NF-AT), and completely substitutes for the Ca2+ co-stimulus required to stimulate an NF-kappa B-dependent element. Calcineurin stimulates the NF-kappa B element by enhancing inactivation of I kappa B/MAD3, an inhibitor of NF-kappa B, thereby increasing the amount of nuclear NF-kappa B DNA binding activity. These data provide the first demonstration in vivo that activation of a protein phosphatase can inactivate I kappa B, and suggest one possible explanation for mechanism-based toxicities associated with FK-506 and CsA by demonstrating that these drugs can inhibit the calcineurin-dependent activation of a virtually ubiquitous transcription factor. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 674, "end": 679}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 638, "end": 642}, {"role": "Site", "text": "IL-2A", "start": 661, "end": 666}, {"role": "Theme2", "text": "OAP", "start": 692, "end": 695}]}, {"trigger": {"text": "binds", "start": 674, "end": 679}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 638, "end": 642}, {"role": "Site", "text": "IL-2A", "start": 661, "end": 666}, {"role": "Theme2", "text": "Oct-1", "start": 700, "end": 705}]}], "negative regulation": [{"trigger": {"text": "inactivate", "start": 40, "end": 50}, "arguments": [{"role": "Theme", "text": "MAD3", "start": 61, "end": 65}]}, {"trigger": {"text": "inhibitable", "start": 263, "end": 274}, "arguments": [{"role": "Theme", "text": "induction", "start": 209, "end": 218}]}, {"trigger": {"text": "inactivation", "start": 904, "end": 916}, "arguments": [{"role": "Theme", "text": "MAD3", "start": 930, "end": 934}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 209, "end": 218}, "arguments": [{"role": "Theme", "text": "interleukin-2", "start": 100, "end": 113}, {"role": "Site", "text": "promoter", "start": 121, "end": 129}]}, {"trigger": {"text": "required", "start": 448, "end": 456}, "arguments": [{"role": "Theme", "text": "activation", "start": 474, "end": 484}]}, {"trigger": {"text": "activation", "start": 474, "end": 484}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 492, "end": 496}, {"role": "Site", "text": "promoter", "start": 497, "end": 505}]}, {"trigger": {"text": "activate", "start": 625, "end": 633}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 638, "end": 642}, {"role": "Site", "text": "IL-2A", "start": 661, "end": 666}]}, {"trigger": {"text": "enhancing", "start": 894, "end": 903}, "arguments": [{"role": "Theme", "text": "inactivation", "start": 904, "end": 916}]}]}}, "schema": []} {"input": "Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factors NF-IL6 and NF-kappa B [published erratum appears in Proc Natl Acad Sci U S A 1995 Apr 11;92(8):3632] \nThe host response to Mycobacterium tuberculosis includes granuloma formation at sites of infection and systemic symptoms. Cytokines have been identified by immunohistochemistry in granulomas in animal models of bacillus Calmette-Guerin (BCG) infection and are released by mononuclear phagocytes upon stimulation by mycobacterial proteins. In this regard, the cytokine interleukin 6 (IL-6) may play a role in the clinical manifestations and pathological events of tuberculosis infection. We have demonstrated that lipoarabinomannan (LAM) from the mycobacterial cell wall, which was virtually devoid of lipopolysaccharide (LPS), stimulated mononuclear phagocytes to release IL-6 in a dose-response manner. LAM and LPS were potent inducers of IL-6 gene expression in peripheral blood monocytes. Both LAM- and LPS-inducible IL-6 promoter activity was localized to a DNA fragment, positions -158 to -49, by deletion analysis and chloramphenicol acetyltransferase assay. Two nuclear factor NF-IL6 (positions -153 to -145 and -83 to -75) and one nuclear factor NF-kappa B (positions -72 to -63) motifs are present within this fragment. Site-directed mutagenesis of one or more of these motifs within the IL-6 promoter demonstrated that each has positive regulatory activity and that they could act in a function- and orientation-independent manner. Deletion of all three elements abolished inducibility of IL-6 promoter activity by both LAM and LPS. We conclude that the NF-IL6 and NF-kappa B sites mediate IL-6 induction in response to both LPS and LAM, acting as bacterial or mycobacterial response elements. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 972, "end": 982}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 962, "end": 966}]}], "localization": [{"trigger": {"text": "release", "start": 886, "end": 893}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 894, "end": 898}]}], "negative regulation": [{"trigger": {"text": "abolished", "start": 1595, "end": 1604}, "arguments": [{"role": "Theme", "text": "inducibility", "start": 1605, "end": 1617}]}], "positive regulation": [{"trigger": {"text": "Activation", "start": 0, "end": 10}, "arguments": [{"role": "Theme", "text": "interleukin 6", "start": 18, "end": 31}]}, {"trigger": {"text": "mediated", "start": 92, "end": 100}, "arguments": [{"role": "Theme", "text": "Activation", "start": 0, "end": 10}, {"role": "Cause", "text": "NF-IL6", "start": 120, "end": 126}]}, {"trigger": {"text": "to", "start": 883, "end": 885}, "arguments": [{"role": "Theme", "text": "release", "start": 886, "end": 893}]}, {"trigger": {"text": "inducers", "start": 950, "end": 958}, "arguments": [{"role": "Theme", "text": "expression", "start": 972, "end": 982}]}, {"trigger": {"text": "inducible", "start": 1032, "end": 1041}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1042, "end": 1046}, {"role": "Site", "text": "promoter", "start": 1047, "end": 1055}]}, {"trigger": {"text": "inducibility", "start": 1605, "end": 1617}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1621, "end": 1625}, {"role": "Site", "text": "promoter", "start": 1626, "end": 1634}]}, {"trigger": {"text": "mediate", "start": 1714, "end": 1721}, "arguments": [{"role": "Theme", "text": "induction", "start": 1727, "end": 1736}]}, {"trigger": {"text": "induction", "start": 1727, "end": 1736}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1722, "end": 1726}]}]}}, "schema": []} {"input": "Calcineurin activates transcription from the GM-CSF promoter in synergy with either protein kinase C or NF-kappa B/AP-1 in T cells. \nTwo cis-acting elements GM-kappa B/GC-box and CLE0, of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene are required for maximal induction in Jurkat T cells by costimulation with phorbol-12-myristate acetate (PMA) and Ca2+ ionophore (A23187). The GM-kappa B sequence is recognized by NF-kappa B, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced AP-1 and a PMA/A23187-induced NF-AT. We examined whether signal transducing components in T cells can activate transcription of the GM-CSF gene. Cotransfection of NF-kappa B (p50/p65)- or AP-1 (c-Jun/c-Fos)- expression vectors into Jurkat cells with a luciferase reporter containing the GM-CSF promoter did not stimulate transcription from the GM-CSF promoter. In contrast, cotransfection with a combination of NF-kappa B and AP-1 significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (AP-1/NF-AT). Expression of a constitutively active calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by NF-kappa B/AP-1. Both constitutively active forms of CN and protein kinase C (PKC) synergistically activated transcription from the GM-CSF promoter. These results suggest that cooperation among NF-kappa B-, AP-1- and NF-AT-binding sequences is required for induction of the GM-CSF gene through PKC- and Ca2+- signaling pathways downstream of T cell activation. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Cotransfection", "start": 691, "end": 705}, "arguments": [{"role": "Theme", "text": "p50", "start": 721, "end": 724}]}, {"trigger": {"text": "Cotransfection", "start": 691, "end": 705}, "arguments": [{"role": "Theme", "text": "p65", "start": 725, "end": 728}]}, {"trigger": {"text": "Cotransfection", "start": 691, "end": 705}, "arguments": [{"role": "Theme", "text": "c-Jun", "start": 740, "end": 745}]}, {"trigger": {"text": "Cotransfection", "start": 691, "end": 705}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 746, "end": 751}]}], "positive regulation": [{"trigger": {"text": "activates", "start": 12, "end": 21}, "arguments": [{"role": "Theme", "text": "transcription", "start": 22, "end": 35}]}, {"trigger": {"text": "induction", "start": 280, "end": 289}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 242, "end": 248}]}, {"trigger": {"text": "activate", "start": 648, "end": 656}, "arguments": [{"role": "Theme", "text": "transcription", "start": 657, "end": 670}]}, {"trigger": {"text": "Cotransfection", "start": 691, "end": 705}, "arguments": [{"role": "Theme", "text": "Cotransfection", "start": 691, "end": 705}]}, {"trigger": {"text": "required", "start": 1507, "end": 1515}, "arguments": [{"role": "Theme", "text": "induction", "start": 1520, "end": 1529}]}, {"trigger": {"text": "induction", "start": 1520, "end": 1529}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 1537, "end": 1543}]}], "regulation": [{"trigger": {"text": "required", "start": 259, "end": 267}, "arguments": [{"role": "CSite", "text": "GM-kappa B/GC-box", "start": 157, "end": 174}, {"role": "Cause", "text": "GM-CSF", "start": 242, "end": 248}, {"role": "Theme", "text": "induction", "start": 280, "end": 289}]}, {"trigger": {"text": "required", "start": 259, "end": 267}, "arguments": [{"role": "CSite", "text": "CLE0", "start": 179, "end": 183}, {"role": "Cause", "text": "GM-CSF", "start": 242, "end": 248}, {"role": "Theme", "text": "induction", "start": 280, "end": 289}]}], "transcription": [{"trigger": {"text": "transcription", "start": 22, "end": 35}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 45, "end": 51}]}, {"trigger": {"text": "transcription", "start": 657, "end": 670}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 678, "end": 684}]}]}}, "schema": []} {"input": "Cell-specific bifunctional role of Jun oncogene family members on glucocorticoid receptor-dependent transcription. \nInteraction between protein kinase C (PKC)- and glucocorticoid receptor (GR)-mediated signaling is suggested by the ability of the PKC activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to inhibit GR-dependent transcription of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Here we report that this interference is cell specific, as TPA augmented dexamethasone-induced transcriptional activation of the MMTV LTR in several T cell lines but was inhibitory in NIH-3T3 fibroblasts. TPA-GR synergism was determined to have occurred at the GR-responsive element (GRE) level by functional analysis of deletion mutants or synthetic GRE oligonucleotides driving chloramphenicol acetyl-transferase expression. Synergism required an intact GR DNA-binding domain, whereas amino- or carboxyl-terminal domains were dispensable. The effect was abrogated by the PKC inhibitor staurosporine, suggesting a role for PKC. Increased c-jun, jun-B, and jun-D expression above basal levels and increased transcriptional activity of AP-1/TPA responsive elements fused to chloramphenicol acetyl-transferase vectors were observed in T cells treated with TPA alone or in combination with dexamethasone. The ability of Jun proteins to cooperate with GR in T cells has been investigated after transfection of c-jun, jun-B, or jun-D expression vectors, which augmented GR-dependent transcription from either MMTV LTR or GRE. Conversely, c-jun and jun-B transfection blunted GR-dependent transcription in HeLa cells. The presence of c-fos had a negative influence on GR function and correlated with the cell-specific synergistic or antagonistic activity of Jun with respect to GR; high basal expression of c-fos as well as AP-1 DNA binding and transcriptional activity were observed in HeLa cells, but not in T cells. Furthermore overexpression of exogenous c-fos has an inhibitory effect on GR-dependent transcription from GRE in T cells. We propose that Jun plays a bifunctional role on GR-dependent transcriptional activation of GRE, selecting either synergistic or antagonistic activity depending on the cell-specific microenvironment. In this regard, intracellular levels of c-fos appear to be influential. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 1088, "end": 1098}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 1064, "end": 1069}]}, {"trigger": {"text": "expression", "start": 1088, "end": 1098}, "arguments": [{"role": "Theme", "text": "jun-B", "start": 1071, "end": 1076}]}, {"trigger": {"text": "expression", "start": 1088, "end": 1098}, "arguments": [{"role": "Theme", "text": "jun-D", "start": 1082, "end": 1087}]}, {"trigger": {"text": "expression", "start": 1812, "end": 1822}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1826, "end": 1831}]}, {"trigger": {"text": "overexpression", "start": 1950, "end": 1964}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1978, "end": 1983}]}], "negative regulation": [{"trigger": {"text": "had a negative influence", "start": 1659, "end": 1683}, "arguments": [{"role": "Cause", "text": "c-fos", "start": 1653, "end": 1658}, {"role": "Theme", "text": "GR", "start": 1687, "end": 1689}]}, {"trigger": {"text": "antagonistic activity", "start": 1752, "end": 1773}, "arguments": [{"role": "Theme", "text": "GR", "start": 1797, "end": 1799}]}], "positive regulation": [{"trigger": {"text": "Increased", "start": 1054, "end": 1063}, "arguments": [{"role": "Theme", "text": "expression", "start": 1088, "end": 1098}]}, {"trigger": {"text": "activity", "start": 1765, "end": 1773}, "arguments": [{"role": "Theme", "text": "GR", "start": 1797, "end": 1799}]}, {"trigger": {"text": "high", "start": 1801, "end": 1805}, "arguments": [{"role": "Theme", "text": "expression", "start": 1812, "end": 1822}]}, {"trigger": {"text": "overexpression", "start": 1950, "end": 1964}, "arguments": [{"role": "Theme", "text": "overexpression", "start": 1950, "end": 1964}]}]}}, "schema": []} {"input": "Defective translocation of protein kinase C in multidrug-resistant HL-60 cells confers a reversible loss of phorbol ester-induced monocytic differentiation. \nPrevious studies have demonstrated that human HL-60 myeloid leukemia cells differentiate in response to phorbol esters. This event is associated with induction of the c-jun early response gene and appearance of a monocytic phenotype. The present studies have examined the effects of vincristine-selected, multidrug resistance on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL-60 cell differentiation. The results demonstrate that multidrug-resistant HL-60 cells, designated HL-60/vinc, fail to respond to TPA with an increase in c-jun transcripts or other phenotypic characteristics of monocytic differentiation. By contrast, treatment of HL-60/vinc cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases, induces c-jun transcription, growth arrest, and expression of the c-fms gene. Studies were also performed with an HL-60/vinc revertant (HL-60/vinc/R) line that has regained partial sensitivity to vincristine. The finding that HL-60/vinc/R cells respond to TPA with induction of a monocytic phenotype, but not c-jun expression, suggests that c-jun induction is not obligatory for monocytic differentiation. Other studies further demonstrate that the jun-B and fra-1 genes are induced by TPA in both HL-60/vinc and HL-60/vinc/R cells, whereas c-fos expression is attenuated in the HL-60/vinc line. Since TPA activates protein kinase C (PKC), we examined translocation of PKC from the cytosol to the membrane fraction. Although HL-60 and HL-60/vinc/R cells demonstrated translocation of PKC activity, this subcellular redistribution was undetectable in HL-60/vinc cells. Activity of the mitogen-activated protein kinase family with associated phosphorylation of c-Jun Y-peptide was markedly diminished in TPA-treated HL-60/vinc cells, but not in response to okadaic acid. Taken together, these findings suggest that vincristine resistance confers insensitivity to TPA-induced differentiation and can include defects in PKC-mediated signaling events and induction of jun/fos early response gene expression. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 943, "end": 953}, "arguments": [{"role": "Theme", "text": "c-fms", "start": 961, "end": 966}]}, {"trigger": {"text": "expression", "start": 1210, "end": 1220}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 1204, "end": 1209}]}, {"trigger": {"text": "expression", "start": 1442, "end": 1452}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1436, "end": 1441}]}], "negative regulation": [{"trigger": {"text": "attenuated", "start": 1456, "end": 1466}, "arguments": [{"role": "Theme", "text": "expression", "start": 1442, "end": 1452}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 308, "end": 317}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 325, "end": 330}]}, {"trigger": {"text": "increase", "start": 682, "end": 690}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 694, "end": 699}]}, {"trigger": {"text": "induces", "start": 895, "end": 902}, "arguments": [{"role": "Theme", "text": "transcription", "start": 909, "end": 922}]}, {"trigger": {"text": "induces", "start": 895, "end": 902}, "arguments": [{"role": "Theme", "text": "expression", "start": 943, "end": 953}]}, {"trigger": {"text": "induction", "start": 1160, "end": 1169}, "arguments": [{"role": "Theme", "text": "expression", "start": 1210, "end": 1220}]}, {"trigger": {"text": "induction", "start": 1242, "end": 1251}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 1236, "end": 1241}]}, {"trigger": {"text": "induced", "start": 1370, "end": 1377}, "arguments": [{"role": "Theme", "text": "jun-B", "start": 1344, "end": 1349}]}, {"trigger": {"text": "induced", "start": 1370, "end": 1377}, "arguments": [{"role": "Theme", "text": "fra-1", "start": 1354, "end": 1359}]}], "transcription": [{"trigger": {"text": "transcription", "start": 909, "end": 922}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 903, "end": 908}]}]}}, "schema": []} {"input": "Initiation binding repressor, a factor that binds to the transcription initiation site of the histone h5 gene, is a glycosylated member of a family of cell growth regulators [corrected] [published erratum appears in Mol Cell Biol 1996 Feb;16(2):735] \nInitiation binding repressor [corrected] (IBR) is a chicken erythrocyte factor (apparent molecular mass, 70 to 73 kDa) that binds to the sequences spanning the transcription initiation site of the histone h5 gene, repressing its transcription. A variety of other cells, including transformed erythroid precursors, do not have IBR but a factor referred to as IBF (68 to 70 kDa) that recognizes the same IBR sites. We have cloned the IBR cDNA and studied the relationship of IBR and IBF. IBR is a 503-amino-acid-long acidic protein which is 99.0% identical to the recently reported human NRF-1/alpha-Pal factor and highly related to the invertebrate transcription factors P3A2 and erected wing gene product (EWG). We present evidence that IBR and IBF are most likely identical proteins, differing in their degree of glycosylation. We have analyzed several molecular aspects of IBR/F and shown that the factor associates as stable homodimers and that the dimer is the relevant DNA-binding species. The evolutionarily conserved N-terminal half of IBR/F harbors the DNA-binding/dimerization domain (outer limits, 127 to 283), one or several casein kinase II sites (37 to 67), and a bipartite nuclear localization signal (89 to 106) which appears to be necessary for nuclear targeting. Binding site selection revealed that the alternating RCGCRYGCGY consensus constitutes high-affinity IBR/F binding sites and that the direct-repeat palindrome TGCGCATGCGCA is the optimal site. A survey of genes potentially regulated by this family of factors primarily revealed genes involved in growth-related metabolism. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 44, "end": 49}, "arguments": [{"role": "Theme", "text": "Initiation binding repressor", "start": 0, "end": 28}, {"role": "Site2", "text": "transcription initiation site", "start": 57, "end": 86}, {"role": "Theme2", "text": "histone h5", "start": 94, "end": 104}]}, {"trigger": {"text": "binds", "start": 375, "end": 380}, "arguments": [{"role": "Theme", "text": "IBR", "start": 293, "end": 296}, {"role": "Site2", "text": "transcription initiation site", "start": 411, "end": 440}, {"role": "Theme2", "text": "histone h5", "start": 448, "end": 458}]}, {"trigger": {"text": "recognizes", "start": 633, "end": 643}, "arguments": [{"role": "Theme", "text": "IBF", "start": 609, "end": 612}]}, {"trigger": {"text": "associates as stable homodimers", "start": 1158, "end": 1189}, "arguments": [{"role": "Theme", "text": "IBR", "start": 1126, "end": 1129}]}, {"trigger": {"text": "associates as stable homodimers", "start": 1158, "end": 1189}, "arguments": [{"role": "Theme", "text": "F", "start": 1130, "end": 1131}]}, {"trigger": {"text": "binding", "start": 1229, "end": 1236}, "arguments": [{"role": "Theme", "text": "IBR", "start": 1126, "end": 1129}]}, {"trigger": {"text": "binding", "start": 1229, "end": 1236}, "arguments": [{"role": "Theme", "text": "F", "start": 1130, "end": 1131}]}], "gene expression": [{"trigger": {"text": "have", "start": 572, "end": 576}, "arguments": [{"role": "Theme", "text": "IBR", "start": 577, "end": 580}]}], "localization": [{"trigger": {"text": "targeting", "start": 1520, "end": 1529}, "arguments": [{"role": "Theme", "text": "IBR", "start": 1294, "end": 1297}, {"role": "AtLoc", "text": "nuclear", "start": 1512, "end": 1519}]}, {"trigger": {"text": "targeting", "start": 1520, "end": 1529}, "arguments": [{"role": "Theme", "text": "F", "start": 1298, "end": 1299}, {"role": "AtLoc", "text": "nuclear", "start": 1512, "end": 1519}]}], "negative regulation": [{"trigger": {"text": "repressing", "start": 465, "end": 475}, "arguments": [{"role": "Cause", "text": "binds", "start": 375, "end": 380}, {"role": "Theme", "text": "transcription", "start": 480, "end": 493}]}], "positive regulation": [{"trigger": {"text": "necessary", "start": 1498, "end": 1507}, "arguments": [{"role": "Theme", "text": "targeting", "start": 1520, "end": 1529}]}], "transcription": [{"trigger": {"text": "transcription", "start": 480, "end": 493}, "arguments": [{"role": "Theme", "text": "histone h5", "start": 448, "end": 458}]}]}}, "schema": []} {"input": "Inhibition of NF-AT signal transduction events by a dominant-negative form of calcineurin. \nAn inhibitory, \"dominant-negative,\" form of the calcineurin catalytic (A) subunit was prepared, which lacks the calmodulin-binding domain, autoinhibitory domain and most of its catalytic core but possesses the regulatory (B) subunit binding domain. When tested for its ability to block calcineurin-dependent signaling in Jurkat cells, expression of this \"B-subunit knock-out\" (BKO) construct suppressed reporter gene activity driven by NF-AT, the pivotal promoter element for interleukin (IL)-2 gene induction. Immunoprecipitation of epitope-labeled BKO demonstrated for the formation of a tight complex with endogenous B subunit in Jurkat cells, consistent with an inhibitory mechanism that involves the sequestration of the B subunit. Furthermore, the sharply reduced NF-AT activity produced by co-transfecting BKO could be \"rescued\" by overexpression of transfected B subunit, suggesting that depletion of this subunit was responsible for the inhibition. These data suggest the potential utility of agents that disrupt calcineurin-mediated signal transduction pathways by blocking formation of the catalytically active dimer of calcineurin A and B subunits. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "formation of a tight complex", "start": 667, "end": 695}, "arguments": [{"role": "Theme", "text": "endogenous B subunit", "start": 701, "end": 721}]}, {"trigger": {"text": "formation of the catalytically active dimer", "start": 1176, "end": 1219}, "arguments": [{"role": "Theme", "text": "calcineurin A", "start": 1223, "end": 1236}, {"role": "Theme2", "text": "B subunits", "start": 1241, "end": 1251}]}], "gene expression": [{"trigger": {"text": "induction", "start": 592, "end": 601}, "arguments": [{"role": "Theme", "text": "interleukin (IL)-2", "start": 568, "end": 586}]}, {"trigger": {"text": "overexpression", "start": 931, "end": 945}, "arguments": [{"role": "Theme", "text": "B subunit", "start": 961, "end": 970}]}], "localization": [{"trigger": {"text": "sequestration", "start": 797, "end": 810}, "arguments": [{"role": "Theme", "text": "B subunit", "start": 818, "end": 827}]}], "negative regulation": [{"trigger": {"text": "inhibitory mechanism", "start": 758, "end": 778}, "arguments": [{"role": "Theme", "text": "B subunit", "start": 818, "end": 827}]}, {"trigger": {"text": "depletion", "start": 988, "end": 997}, "arguments": [{"role": "Theme", "text": "B subunit", "start": 961, "end": 970}]}, {"trigger": {"text": "blocking", "start": 1167, "end": 1175}, "arguments": [{"role": "Theme", "text": "formation of the catalytically active dimer", "start": 1176, "end": 1219}]}], "positive regulation": [{"trigger": {"text": "involves", "start": 784, "end": 792}, "arguments": [{"role": "Theme", "text": "inhibitory mechanism", "start": 758, "end": 778}, {"role": "Cause", "text": "sequestration", "start": 797, "end": 810}]}, {"trigger": {"text": "overexpression", "start": 931, "end": 945}, "arguments": [{"role": "Theme", "text": "overexpression", "start": 931, "end": 945}]}], "regulation": [{"trigger": {"text": "promoter element", "start": 547, "end": 563}, "arguments": [{"role": "Theme", "text": "induction", "start": 592, "end": 601}]}]}}, "schema": []} {"input": "Modulation of endogenous IL-1 beta and IL-1 receptor antagonist results in opposing effects on HIV expression in chronically infected monocytic cells. \nA proportion of HIV-infected individuals experience episodes of localized or systemic bacterial infections caused by Gram-negative bacteria. Many of the clinical side effects of these infections are associated with the production of proinflammatory cytokines, which are induced primarily by LPS, a constituent of the bacterial cell wall of Gram-negative bacteria. The present study examines the mechanisms involved in LPS-mediated induction of HIV expression in U1 cells, a promonocytic cell line chronically infected with HIV. Stimulation of U1 cells by LPS alone induced minimal levels of HIV expression, which was significantly enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF). Costimulation of U1 cells with LPS plus GM-CSF resulted in the accumulation of steady-state levels of HIV RNA; however, only a weak induction of HIV long terminal repeat-driven transcription, which was not associated with the activation of the cellular transcription factor nuclear factor-kappa B, was noted. Costimulation of cells with LPS plus GM-CSF induced the production of proinflammatory cytokines, IL-8, IL-1 beta and IL-6, but not TNF-alpha. IL-1 receptor antagonist (ra) inhibited LPS enhancement of HIV expression in GM-CSF-stimulated cells, suggesting that endogenous IL-1 was involved in LPS-mediated viral production. In this regard, anti-inflammatory cytokines inhibited LPS plus GM-CSF-stimulated HIV expression, and this effect closely correlated with inhibition of IL-1 beta release and, in particular, with up-regulation of endogenous IL-1ra production. Thus, the balance between an endogenously produced viral inducer (IL-1 beta ) and an inhibitor (IL-1ra) may represent an important pathway leading to modulation of HIV expression from monocytic cells. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 1219, "end": 1229}, "arguments": [{"role": "Theme", "text": "IL-8", "start": 1260, "end": 1264}]}, {"trigger": {"text": "production", "start": 1219, "end": 1229}, "arguments": [{"role": "Theme", "text": "IL-1 beta", "start": 1266, "end": 1275}]}, {"trigger": {"text": "production", "start": 1219, "end": 1229}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1280, "end": 1284}]}, {"trigger": {"text": "production", "start": 1219, "end": 1229}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1294, "end": 1303}]}, {"trigger": {"text": "production", "start": 1715, "end": 1725}, "arguments": [{"role": "Theme", "text": "IL-1ra", "start": 1708, "end": 1714}]}, {"trigger": {"text": "produced", "start": 1769, "end": 1777}, "arguments": [{"role": "Theme", "text": "IL-1 beta", "start": 1793, "end": 1802}]}, {"trigger": {"text": "produced", "start": 1769, "end": 1777}, "arguments": [{"role": "Theme", "text": "IL-1ra", "start": 1823, "end": 1829}]}], "localization": [{"trigger": {"text": "release", "start": 1647, "end": 1654}, "arguments": [{"role": "Theme", "text": "IL-1 beta", "start": 1637, "end": 1646}]}], "negative regulation": [{"trigger": {"text": "inhibition", "start": 1623, "end": 1633}, "arguments": [{"role": "Theme", "text": "release", "start": 1647, "end": 1654}]}, {"trigger": {"text": "balance", "start": 1737, "end": 1744}, "arguments": [{"role": "Cause", "text": "IL-1 beta", "start": 1793, "end": 1802}, {"role": "Theme", "text": "IL-1ra", "start": 1823, "end": 1829}]}, {"trigger": {"text": "balance", "start": 1737, "end": 1744}, "arguments": [{"role": "Theme", "text": "IL-1 beta", "start": 1793, "end": 1802}, {"role": "Cause", "text": "IL-1ra", "start": 1823, "end": 1829}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 1207, "end": 1214}, "arguments": [{"role": "Theme", "text": "production", "start": 1219, "end": 1229}]}, {"trigger": {"text": "up-regulation", "start": 1680, "end": 1693}, "arguments": [{"role": "Theme", "text": "production", "start": 1715, "end": 1725}]}]}}, "schema": []} {"input": "Transcriptional regulation of the interleukin-2 gene in normal human peripheral blood T cells. Convergence of costimulatory signals and differences from transformed T cells. \nTo study transcriptional regulation in normal human T cells, we have optimized conditions for transient transfection. Interleukin-2 (IL-2) promoter-reporter gene behavior closely parallels the endogenous gene in response to T cell receptor and costimulatory signals. As assessed with mutagenized promoters, the most important IL-2 cis-regulatory elements in normal T cells are the proximal AP-1 site and the NF- kappaB site. Both primary activation, with phytohemagglutinin or antibodies to CD3, and costimulation, provided by pairs of CD2 antibodies or B7-positive (B cells) or B7-negative (endothelial) accessory cells, are mediated through the same cis-elements. Interestingly, the nuclear factor of activated T cell sites are much less important in normal T cells than in Jurkat T cells. We conclude that IL-2 transcriptional regulation differs in tumor cell lines compared with normal T cells and that different costimulatory signals converge on the same cis-elements in the IL-2 promoter. ", "output": {"json_structures": {"regulation": [{"trigger": {"text": "Transcriptional regulation", "start": 0, "end": 26}, "arguments": [{"role": "Theme", "text": "Transcriptional regulation", "start": 0, "end": 26}]}, {"trigger": {"text": "transcriptional regulation", "start": 989, "end": 1015}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 984, "end": 988}]}], "transcription": [{"trigger": {"text": "Transcriptional regulation", "start": 0, "end": 26}, "arguments": [{"role": "Theme", "text": "interleukin-2", "start": 34, "end": 47}]}]}}, "schema": []} {"input": "A hydrophobic domain of Ca2+-modulating cyclophilin ligand modulates calcium influx signaling in T lymphocytes. \nCa2+-modulating cyclophilin ligand (CAML) was originally described as a cyclophilin B-binding protein whose overexpression in T cells causes a rise in intracellular calcium, thus activating transcription factors responsible for the early immune response. As reported here, structure-function analysis of the CAML gene in Jurkat T cells indicates that two of CAML's putative membrane-spanning domains are necessary and sufficient for the modulation of intracellular calcium. We propose that the hydrophobic C-terminal tail of CAML forms its effector domain, thus implicating the N-terminal hydrophilic domain in a regulatory role. These findings define a novel protein motif that functions in intracellular calcium signaling. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "overexpression", "start": 221, "end": 235}, "arguments": [{"role": "Theme", "text": "CAML", "start": 149, "end": 153}]}], "positive regulation": [{"trigger": {"text": "overexpression", "start": 221, "end": 235}, "arguments": [{"role": "Theme", "text": "overexpression", "start": 221, "end": 235}]}]}}, "schema": []} {"input": "Interactions of a transcriptional activator in the env gene of the mouse mammary tumor virus with activation-dependent, T cell-specific transacting factors. \nThe mouse mammary tumor virus env gene contains a transcriptional activator (META) that can control transcription of the adjacent long terminal repeat region. Transcriptional control by META parallels that of several lymphokine genes, being specific to T cells, dependent on their activation, and inhibited by the immunosuppressive drug cyclosporine (CsA). DNase I footprinting indicated that nuclear factors from activated T lymphocytes bound a promoter-proximal site, META(P), and a promoter-distal site, META(D+), within the 400-base pair META region. Nuclear factors from unstimulated, but not from activated cells, bound a site, META(D-), adjacent to META(D+). META(D+) directed transcription of a linked luciferase gene, and gel shift analysis revealed binding of inducible, CsA-sensitive T cell factors, in parallel with transfection results. Authentic NFAT and NF-kappaB targets did not compete for the META(D+) binding factor(s). The SV40 core sequence competed for META(D+) binding factors, but META(D+) failed to compete for the complexes obtained with the SV40 probe. Our results, taken together, indicate that META(D+) is a novel transcriptional enhancer element that is similar in its cell-type specificity, activation dependence, and CsA sensitivity to the NFAT element. It may be relevant to the role of MMTV in expression of Mls antigens or the induction of T cell lymphomas. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "Interactions", "start": 0, "end": 12}, "arguments": [{"role": "Theme", "text": "env", "start": 51, "end": 54}]}]}}, "schema": []} {"input": "Monocytic cell type-specific transcriptional induction of collagenase. \nInterstitial collagenase (MMP-1), a metalloproteinase produced by resident and inflammatory cells during connective tissue turnover, cleaves type I collagen fibrils. This catalytic event is rate limiting in remodeling of tissues rich in fibrillar collagen such as the skin and lungs. The regulation of collagenase expression is cell-type specific; bacterial LPS and zymosan, a yeast cell wall derivative, are potent inducers of collagenase expression in macrophages, but do not alter fibroblast collagenase expression. Since promoter elements controlling collagenase transcription in monocytic cells have not been previously defined, we sought to delineate responsive cis-acting elements of the collagenase promoter in transiently transfected human (U937) and murine (J774) monocytic cell lines. Deletion constructs containing as little as 72 bp of 5' -flanking sequence of the collagenase promoter were sufficient for LPS- or zymosan-mediated transcriptional induction, whereas phorbol inducibility exhibited an absolute requirement for upstream elements including the polyoma enhancer A-binding protein-3 site (-83 to -91) and TTCA sequence (-102 to -105) in both monocytic cells and fibroblasts. Mutagenesis of the activator protein-1 [AP-1] site at -72 abolished basal promoter activity and LPS/zymosan inducibility, while mutagenesis of an NF-kappaB-like site at -20 to -10 had no effect. Nuclear extracts from LPS- and zymosan-treated cells showed strong AP-1 activity by gel-shift analysis, and supershift analysis showed the AP-1 complexes contained specific members of both the jun and fos gene families. These data indicate that, in contrast to most LPS effects, AP-1, but not nuclear factor-kappaB, mediates LPS induction of collagenase transcription in macrophagelike cells. Furthermore, as compared to regulation by phorbol ester, collagenase induction in monocytic cells by cell wall derivatives of bacteria or yeast is largely independent of upstream promoter sequences. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "produced", "start": 126, "end": 134}, "arguments": [{"role": "Theme", "text": "MMP-1", "start": 98, "end": 103}]}]}}, "schema": []} {"input": "Inhibitory effect of E3330, a novel quinone derivative able to suppress tumor necrosis factor-alpha generation, on activation of nuclear factor-kappa B. \n(2E)-3-[5-(2,3-Dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2- propenoic acid (E3330), is a novel agent with hepatoprotective activity. We report the effect of E3330 on transcriptional activation of tumor necrosis factor (TNF)-alpha gene and on nuclear factor (NF)-kappa B activation. Nuclear run-on experiments showed that E3330 decreases transcriptional activation of TNF-alpha gene induced by lipopolysaccharide (LPS) stimulation in human peripheral monocytes. To investigate the inhibitory mechanisms, we constructed a secreted-type placental alkaline phosphatase (PLAP) reporter gene whose transcription is controlled by a 1.4-kb human TNF-alpha promoter. A stable transformant of the PLAP reporter gene derived from human monocytic cell line showed very little activity on the promoter before stimulation, whereas LPS stimulation led to a dramatic increase in PLAP activity. E3330 inhibited this induced promoter activity in a dose-dependent manner. There are four putative NF-kappa B binding sites (kappa B-1, kappa B-2, kappa B-3, kappa B-4) in human TNF-alpha promoter. By using mutated promoter-PLAP plasmids, we established that these NF-kappa B sites were necessary for induction of TNF-alpha transcription on stimulation with LPS. A gel retardation experiment with synthetic double-stranded oligonucleotides showed that activated NF-kappa B consisting of p50/p65 heterodimer bound to all four putative NF-kappa B DNA probes, suggesting that all four putative NF-kappa B recognition sites play an important role in inducible TNF-alpha expression. E3330 decreased activated NF-kappa B in nuclei, suggesting that E3330 inhibits NF-kappa B activation and/or translocation of the nuclei. Western blotting analysis with anti-I kappa B-alpha antibody indicated that E3330 inhibited degradation of I kappa B-alpha, which is an inhibitory protein of NF-kappa B, in LPS-stimulated monocytes. E3330 may suppress the production of active oxygen species serving as common messengers to activate NF-kappa B. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "bound", "start": 1543, "end": 1548}, "arguments": [{"role": "Theme", "text": "p50", "start": 1523, "end": 1526}]}, {"trigger": {"text": "bound", "start": 1543, "end": 1548}, "arguments": [{"role": "Theme", "text": "p65", "start": 1527, "end": 1530}]}], "gene expression": [{"trigger": {"text": "generation", "start": 100, "end": 110}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor-alpha", "start": 72, "end": 99}]}, {"trigger": {"text": "expression", "start": 1702, "end": 1712}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1692, "end": 1701}]}], "negative regulation": [{"trigger": {"text": "suppress", "start": 63, "end": 71}, "arguments": [{"role": "Theme", "text": "generation", "start": 100, "end": 110}]}, {"trigger": {"text": "inhibited", "start": 1933, "end": 1942}, "arguments": [{"role": "Theme", "text": "degradation", "start": 1943, "end": 1954}]}], "positive regulation": [{"trigger": {"text": "necessary", "start": 1323, "end": 1332}, "arguments": [{"role": "Theme", "text": "induction", "start": 1337, "end": 1346}]}, {"trigger": {"text": "induction", "start": 1337, "end": 1346}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1360, "end": 1373}]}, {"trigger": {"text": "play an important role", "start": 1656, "end": 1678}, "arguments": [{"role": "Theme", "text": "expression", "start": 1702, "end": 1712}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 1943, "end": 1954}, "arguments": [{"role": "Theme", "text": "I kappa B-alpha", "start": 1958, "end": 1973}]}], "regulation": [{"trigger": {"text": "controlled", "start": 767, "end": 777}, "arguments": [{"role": "Theme", "text": "transcription", "start": 750, "end": 763}, {"role": "Cause", "text": "TNF-alpha", "start": 796, "end": 805}, {"role": "CSite", "text": "promoter", "start": 806, "end": 814}]}], "transcription": [{"trigger": {"text": "transcription", "start": 750, "end": 763}, "arguments": [{"role": "Theme", "text": "placental alkaline phosphatase", "start": 692, "end": 722}]}, {"trigger": {"text": "transcription", "start": 1360, "end": 1373}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1350, "end": 1359}]}]}}, "schema": []} {"input": "The role of p16 in the E2F-dependent thymidine kinase regulation. \nThe role of alterations of the MTS1 tumor suppressor gene on chromosome 9p21, which encodes p16, the inhibitor of cyclin-dependent-kinase-4 and 6, in tumorigenesis is not yet clear. Phosphorylation of the retinoblastoma protein by cyclin-dependent kinases 4 and 6 prevents its interaction with the transcription factor E2F, which subsequently promotes the expression of S phase regulated genes, such as thymidine kinase. Although a role of p16 in this regulation has been presumed, there is no proof so far that loss of this tumor suppressor gene really affects E2F-mediated regulations. We investigated the regulation of thymidine kinase in phytohemagglutinin-stimulated normal human lymphocytes and in the p16-negative human acute lymphoblastic leukemia cell lines, MOLT-4 and CEM. Compared to normal lymphocytes, MOLT-4 and CEM cells exhibited an altered cell cycle regulation of thymidine kinase, a much higher intracellular activity of this enzyme, and higher thymidine kinase mRNA expression. Transient expression of p16 in normal human lymphocytes caused arrest in G1, but was without effect on the cell growth of MOLT-4 and CEM cells, although all of them express functional retinoblastoma protein. Nevertheless, in the two leukemia cell lines transient overexpression of p16 reestablished the normal regulation of thymidine kinase, paralleled by an increase of the underphosphorylated form of retinoblastoma protein and decrease of free E2F bound to its motif in the thymidine kinase promoter. We demonstrate that loss of p16 causes upregulation of this DNA precursor pathway enzyme via activation of E2F by a mechanism involving retinoblastoma protein. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 344, "end": 355}, "arguments": [{"role": "Theme", "text": "retinoblastoma protein", "start": 272, "end": 294}]}, {"trigger": {"text": "bound", "start": 1517, "end": 1522}, "arguments": [{"role": "Site", "text": "its motif", "start": 1526, "end": 1535}, {"role": "Theme", "text": "thymidine kinase", "start": 1543, "end": 1559}]}], "gene expression": [{"trigger": {"text": "expression", "start": 423, "end": 433}, "arguments": [{"role": "Theme", "text": "thymidine kinase", "start": 470, "end": 486}]}, {"trigger": {"text": "negative", "start": 779, "end": 787}, "arguments": [{"role": "Theme", "text": "p16", "start": 775, "end": 778}]}, {"trigger": {"text": "expression", "start": 1076, "end": 1086}, "arguments": [{"role": "Theme", "text": "p16", "start": 1090, "end": 1093}]}, {"trigger": {"text": "express", "start": 1231, "end": 1238}, "arguments": [{"role": "Theme", "text": "retinoblastoma protein", "start": 1250, "end": 1272}]}, {"trigger": {"text": "overexpression", "start": 1329, "end": 1343}, "arguments": [{"role": "Theme", "text": "p16", "start": 1347, "end": 1350}]}], "negative regulation": [{"trigger": {"text": "inhibitor", "start": 168, "end": 177}, "arguments": [{"role": "Cause", "text": "p16", "start": 159, "end": 162}, {"role": "Theme", "text": "cyclin-dependent-kinase-4", "start": 181, "end": 206}]}, {"trigger": {"text": "inhibitor", "start": 168, "end": 177}, "arguments": [{"role": "Cause", "text": "p16", "start": 159, "end": 162}, {"role": "Theme", "text": "6", "start": 211, "end": 212}]}, {"trigger": {"text": "prevents", "start": 331, "end": 339}, "arguments": [{"role": "Cause", "text": "Phosphorylation", "start": 249, "end": 264}, {"role": "Theme", "text": "interaction", "start": 344, "end": 355}]}, {"trigger": {"text": "loss", "start": 579, "end": 583}, "arguments": [{"role": "Theme", "text": "p16", "start": 507, "end": 510}]}, {"trigger": {"text": "underphosphorylated form", "start": 1441, "end": 1465}, "arguments": [{"role": "Theme", "text": "retinoblastoma protein", "start": 1469, "end": 1491}]}, {"trigger": {"text": "decrease", "start": 1496, "end": 1504}, "arguments": [{"role": "Theme", "text": "bound", "start": 1517, "end": 1522}]}], "phosphorylation": [{"trigger": {"text": "Phosphorylation", "start": 249, "end": 264}, "arguments": [{"role": "Theme", "text": "retinoblastoma protein", "start": 272, "end": 294}]}, {"trigger": {"text": "underphosphorylated form", "start": 1441, "end": 1465}, "arguments": [{"role": "Theme", "text": "retinoblastoma protein", "start": 1469, "end": 1491}]}], "positive regulation": [{"trigger": {"text": "Phosphorylation", "start": 249, "end": 264}, "arguments": [{"role": "Theme", "text": "Phosphorylation", "start": 249, "end": 264}, {"role": "Cause", "text": "cyclin-dependent kinases 4", "start": 298, "end": 324}]}, {"trigger": {"text": "Phosphorylation", "start": 249, "end": 264}, "arguments": [{"role": "Theme", "text": "Phosphorylation", "start": 249, "end": 264}, {"role": "Cause", "text": "6", "start": 329, "end": 330}]}, {"trigger": {"text": "promotes", "start": 410, "end": 418}, "arguments": [{"role": "Cause", "text": "prevents", "start": 331, "end": 339}, {"role": "Theme", "text": "expression", "start": 423, "end": 433}]}, {"trigger": {"text": "regulation", "start": 936, "end": 946}, "arguments": [{"role": "Theme", "text": "thymidine kinase", "start": 950, "end": 966}]}, {"trigger": {"text": "overexpression", "start": 1329, "end": 1343}, "arguments": [{"role": "Theme", "text": "overexpression", "start": 1329, "end": 1343}]}, {"trigger": {"text": "reestablished", "start": 1351, "end": 1364}, "arguments": [{"role": "Cause", "text": "overexpression", "start": 1329, "end": 1343}, {"role": "Theme", "text": "regulation", "start": 1376, "end": 1386}]}, {"trigger": {"text": "increase", "start": 1425, "end": 1433}, "arguments": [{"role": "Theme", "text": "underphosphorylated form", "start": 1441, "end": 1465}]}, {"trigger": {"text": "causes upregulation", "start": 1602, "end": 1621}, "arguments": [{"role": "Theme", "text": "thymidine kinase", "start": 1390, "end": 1406}]}], "regulation": [{"trigger": {"text": "role", "start": 4, "end": 8}, "arguments": [{"role": "Cause", "text": "p16", "start": 12, "end": 15}, {"role": "Theme", "text": "regulation", "start": 54, "end": 64}]}, {"trigger": {"text": "regulation", "start": 54, "end": 64}, "arguments": [{"role": "Theme", "text": "thymidine kinase", "start": 37, "end": 53}]}, {"trigger": {"text": "alterations", "start": 79, "end": 90}, "arguments": [{"role": "Theme", "text": "MTS1", "start": 98, "end": 102}]}, {"trigger": {"text": "role", "start": 499, "end": 503}, "arguments": [{"role": "Theme", "text": "prevents", "start": 331, "end": 339}, {"role": "Cause", "text": "p16", "start": 507, "end": 510}]}, {"trigger": {"text": "regulation", "start": 1376, "end": 1386}, "arguments": [{"role": "Theme", "text": "thymidine kinase", "start": 1390, "end": 1406}]}, {"trigger": {"text": "causes", "start": 1602, "end": 1608}, "arguments": [{"role": "Cause", "text": "p16", "start": 1598, "end": 1601}, {"role": "Theme", "text": "retinoblastoma protein", "start": 1706, "end": 1728}]}], "transcription": [{"trigger": {"text": "expression", "start": 1054, "end": 1064}, "arguments": [{"role": "Theme", "text": "thymidine kinase", "start": 1032, "end": 1048}]}]}}, "schema": []} {"input": "Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor. \nThe c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 41, "end": 51}, "arguments": [{"role": "Theme", "text": "interleukin 3", "start": 55, "end": 68}]}, {"trigger": {"text": "production", "start": 41, "end": 51}, "arguments": [{"role": "Theme", "text": "granulocyte-macrophage colony-stimulating factor", "start": 73, "end": 121}]}, {"trigger": {"text": "expression", "start": 604, "end": 614}, "arguments": [{"role": "Theme", "text": "CD25", "start": 703, "end": 707}]}, {"trigger": {"text": "expression", "start": 604, "end": 614}, "arguments": [{"role": "Theme", "text": "CD69", "start": 710, "end": 714}]}, {"trigger": {"text": "expression", "start": 604, "end": 614}, "arguments": [{"role": "Theme", "text": "CD62", "start": 731, "end": 735}]}, {"trigger": {"text": "production", "start": 1391, "end": 1401}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 1405, "end": 1409}]}, {"trigger": {"text": "production", "start": 1391, "end": 1401}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1411, "end": 1420}]}, {"trigger": {"text": "production", "start": 1391, "end": 1401}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 1426, "end": 1435}]}, {"trigger": {"text": "expression", "start": 1461, "end": 1471}, "arguments": [{"role": "Theme", "text": "IL-3", "start": 1445, "end": 1449}]}, {"trigger": {"text": "expression", "start": 1461, "end": 1471}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 1454, "end": 1460}]}, {"trigger": {"text": "production", "start": 1779, "end": 1789}, "arguments": [{"role": "Theme", "text": "IL-3", "start": 1793, "end": 1797}]}, {"trigger": {"text": "production", "start": 1779, "end": 1789}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 1802, "end": 1808}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 4, "end": 13}, "arguments": [{"role": "Theme", "text": "Rel", "start": 0, "end": 3}]}, {"trigger": {"text": "defects", "start": 30, "end": 37}, "arguments": [{"role": "Theme", "text": "production", "start": 41, "end": 51}]}, {"trigger": {"text": "lacking", "start": 227, "end": 234}, "arguments": [{"role": "Theme", "text": "Rel", "start": 235, "end": 238}]}, {"trigger": {"text": "loss", "start": 458, "end": 462}, "arguments": [{"role": "Theme", "text": "Rel", "start": 466, "end": 469}]}, {"trigger": {"text": "lower", "start": 1101, "end": 1106}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1033, "end": 1042}]}, {"trigger": {"text": "lower", "start": 1101, "end": 1106}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 1067, "end": 1076}]}, {"trigger": {"text": "lower", "start": 1101, "end": 1106}, "arguments": [{"role": "Theme", "text": "IL-3", "start": 932, "end": 936}]}, {"trigger": {"text": "lower", "start": 1101, "end": 1106}, "arguments": [{"role": "Theme", "text": "IL-5", "start": 938, "end": 942}]}, {"trigger": {"text": "lower", "start": 1101, "end": 1106}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 995, "end": 1001}]}], "positive regulation": [{"trigger": {"text": "restores", "start": 1382, "end": 1390}, "arguments": [{"role": "Cause", "text": "IL-2", "start": 1277, "end": 1281}, {"role": "Theme", "text": "production", "start": 1391, "end": 1401}]}, {"trigger": {"text": "restores", "start": 1382, "end": 1390}, "arguments": [{"role": "Cause", "text": "IL-2", "start": 1277, "end": 1281}, {"role": "Theme", "text": "expression", "start": 1461, "end": 1471}]}, {"trigger": {"text": "produce higher", "start": 1602, "end": 1616}, "arguments": [{"role": "Theme", "text": "GM-CSF", "start": 1639, "end": 1645}]}, {"trigger": {"text": "required", "start": 1749, "end": 1757}, "arguments": [{"role": "Cause", "text": "Rel", "start": 1677, "end": 1680}, {"role": "Theme", "text": "production", "start": 1779, "end": 1789}]}]}}, "schema": []} {"input": "Human T lymphotropic virus-I infection of human T lymphocytes induces expression of the beta-galactoside-binding lectin, galectin-3. \nAnimal lectins play important roles in a variety of biological processes via their recognition of glycoconjugates. Galectin-3 is a beta-galactoside-binding lectin previously designated as epsilon BP (IgE-binding protein), CBP35, Mac-2, L-29, and L-34, and its expression has been associated with various physiological and pathological processes, including cell growth, tumor transformation, and metastasis. Galectin-3 is widely distributed in various tissues and cell types and is expressed in many leukocytes, with the notable exception of B and T lymphocytes. We now report that galectin-3 is abundantly expressed in a number of human T lymphotropic virus (HTLV)-I-infected human T cell lines, including F6T, HUT 102, K3T, MT-2, and SLB-I, but is not expressed in non-HTLV-I-infected T cell lines such as Jurkat, CEM, and MOLT-4. In addition, the galectin-3 level was markedly increased in human thymocytes after infection with HTLV-I as compared with uninfected thymocytes. The up-regulation of galectin-3 expression appeared to correlate well with HTLV-I gene expression, as undetectable or very low levels of galectin-3 were found in the S1T and ATL-1K cell lines, which are nonproductively infected with HTLV-I. In co-transfection experiments, the galectin-3 promoter was significantly up-regulated by expression vectors encoding the 40-kd Tax protein, a potent transactivator in HTLV-I. Analysis of various Tax mutants suggested that galectin-3 promoter induction is dependent on activation of the cyclic-AMP-responsive element binding protein/activation transcription factor family of transcription factors and, to a lesser extent, nuclear factor-kappa B/Rel induction. Transfection of human promonocytic U-937 cells with an HTLV-I Tax expression vector induced galectin-3 expression in this cell line. Functionally, galectin-3 was shown to activate interleukin-2 production in Jurkat T cells. Together, these findings raise the possibility that HTLV-I Tax production induces the transcription and subsequent synthesis and secretion of galectin-3, which in turn may further activate these T cells and contribute to the altered properties of cell growth found in adult T cell leukemia induced by HTLV-I. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 70, "end": 80}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 121, "end": 131}]}, {"trigger": {"text": "expression", "start": 394, "end": 404}, "arguments": [{"role": "Theme", "text": "Galectin-3", "start": 249, "end": 259}]}, {"trigger": {"text": "distributed", "start": 562, "end": 573}, "arguments": [{"role": "Theme", "text": "Galectin-3", "start": 541, "end": 551}]}, {"trigger": {"text": "expressed", "start": 615, "end": 624}, "arguments": [{"role": "Theme", "text": "Galectin-3", "start": 541, "end": 551}]}, {"trigger": {"text": "expressed", "start": 740, "end": 749}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 715, "end": 725}]}, {"trigger": {"text": "expressed", "start": 887, "end": 896}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 715, "end": 725}]}, {"trigger": {"text": "expression", "start": 1143, "end": 1153}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 1132, "end": 1142}]}, {"trigger": {"text": "undetectable", "start": 1213, "end": 1225}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 1248, "end": 1258}]}, {"trigger": {"text": "low levels", "start": 1234, "end": 1244}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 1248, "end": 1258}]}, {"trigger": {"text": "Transfection", "start": 1812, "end": 1824}, "arguments": [{"role": "Theme", "text": "Tax", "start": 1874, "end": 1877}]}, {"trigger": {"text": "expression", "start": 1915, "end": 1925}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 1904, "end": 1914}]}, {"trigger": {"text": "production", "start": 2006, "end": 2016}, "arguments": [{"role": "Theme", "text": "interleukin-2", "start": 1992, "end": 2005}]}, {"trigger": {"text": "production", "start": 2099, "end": 2109}, "arguments": [{"role": "Theme", "text": "Tax", "start": 2095, "end": 2098}]}], "localization": [{"trigger": {"text": "secretion", "start": 2165, "end": 2174}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 2178, "end": 2188}]}], "positive regulation": [{"trigger": {"text": "induces", "start": 62, "end": 69}, "arguments": [{"role": "Theme", "text": "expression", "start": 70, "end": 80}]}, {"trigger": {"text": "increased", "start": 1013, "end": 1022}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 983, "end": 993}]}, {"trigger": {"text": "up-regulation", "start": 1115, "end": 1128}, "arguments": [{"role": "Theme", "text": "expression", "start": 1143, "end": 1153}]}, {"trigger": {"text": "up-regulated", "start": 1426, "end": 1438}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 1388, "end": 1398}, {"role": "Site", "text": "promoter", "start": 1399, "end": 1407}, {"role": "Cause", "text": "Tax", "start": 1480, "end": 1483}]}, {"trigger": {"text": "induction", "start": 1595, "end": 1604}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 1575, "end": 1585}, {"role": "Site", "text": "promoter", "start": 1586, "end": 1594}]}, {"trigger": {"text": "dependent", "start": 1608, "end": 1617}, "arguments": [{"role": "Theme", "text": "induction", "start": 1595, "end": 1604}]}, {"trigger": {"text": "Transfection", "start": 1812, "end": 1824}, "arguments": [{"role": "Theme", "text": "Transfection", "start": 1812, "end": 1824}]}, {"trigger": {"text": "induced", "start": 1896, "end": 1903}, "arguments": [{"role": "Cause", "text": "Transfection", "start": 1812, "end": 1824}, {"role": "Theme", "text": "expression", "start": 1915, "end": 1925}]}, {"trigger": {"text": "activate", "start": 1983, "end": 1991}, "arguments": [{"role": "Cause", "text": "galectin-3", "start": 1959, "end": 1969}, {"role": "Theme", "text": "production", "start": 2006, "end": 2016}]}, {"trigger": {"text": "induces", "start": 2110, "end": 2117}, "arguments": [{"role": "Cause", "text": "production", "start": 2099, "end": 2109}, {"role": "Theme", "text": "transcription", "start": 2122, "end": 2135}]}, {"trigger": {"text": "induces", "start": 2110, "end": 2117}, "arguments": [{"role": "Cause", "text": "production", "start": 2099, "end": 2109}, {"role": "Theme", "text": "secretion", "start": 2165, "end": 2174}]}], "transcription": [{"trigger": {"text": "transcription", "start": 2122, "end": 2135}, "arguments": [{"role": "Theme", "text": "galectin-3", "start": 2178, "end": 2188}]}]}}, "schema": []} {"input": "Nuclear appearance of a factor that binds the CD28 response element within the interleukin-2 enhancer correlates with interleukin-2 production. \nActivation of T lymphocytes requires the combined signaling of the T cell receptor and costimulatory molecules such as CD28. The ability of T cells to produce interleukin-2 (IL-2) is a critical control point in T lymphocyte activation. The IL-2 enhancer contains a functional motif named CD28 response element (CD28RE) that serves a role as a target for mitogenic T cell activation signals. The CD28RE sequence reveals similarity to the consensus kappaB binding motif. Here we demonstrate that CD28RE binds an inducible protein with a molecular mass of approximately 35 kDa called nuclear factor of mitogenic-activated T cells (NF-MATp35) that is clearly different from the known NF- kappaB/Rel family members. Induction of NF-MATp35 was shown to depend on de novo protein synthesis and was restricted to T cells that received a mitogenic combination of T cell stimuli, not necessarily including CD28 signaling. Nonmitogenic T cell stimulation did not result in appearance of NF-MATp35. These results indicate that mitogenic combinations of T cell activation signals are integrated at the level of NF-MATp35 induction. Similar to its effect on IL-2 production, cyclosporin A inhibited the induction of NF-MATp35. Taken together, these data demonstrate that the nuclear appearance of NF-MATp35 shows excellent correlation with IL-2 production, which is a unique characteristic among nuclear factors implicated in the control of IL-2 gene expression. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 132, "end": 142}, "arguments": [{"role": "Theme", "text": "interleukin-2", "start": 118, "end": 131}]}, {"trigger": {"text": "produce", "start": 296, "end": 303}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 319, "end": 323}]}, {"trigger": {"text": "production", "start": 1294, "end": 1304}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1289, "end": 1293}]}, {"trigger": {"text": "production", "start": 1476, "end": 1486}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1471, "end": 1475}]}, {"trigger": {"text": "expression", "start": 1582, "end": 1592}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1572, "end": 1576}]}], "negative regulation": [{"trigger": {"text": "inhibited", "start": 1320, "end": 1329}, "arguments": [{"role": "Theme", "text": "production", "start": 1294, "end": 1304}]}], "regulation": [{"trigger": {"text": "control", "start": 1561, "end": 1568}, "arguments": [{"role": "Theme", "text": "expression", "start": 1582, "end": 1592}]}]}}, "schema": []} {"input": "Preassociation of STAT1 with STAT2 and STAT3 in separate signalling complexes prior to cytokine stimulation. \nA variety of cytokines and growth factors act through an induction of gene expression mediated by a family of latent transcription factors called STAT (signal transducers and activators of transcription) proteins. Ligand-induced tyrosine phosphorylation of the STATs promotes their homodimer and heterodimer formation and subsequent nuclear translocation. We demonstrate here that STAT protein heterocomplexes exist prior to cytokine treatment. When unstimulated HeLa cells are ruptured in hypotonic buffer without salt or detergent, immunoadsorption of either STAT1 or STAT2 from the resulting cytosol yields coimmunoadsorption of the other STAT protein. Similarly, STAT1-STAT3 heterocomplexes are coimmunoadsorbed from hypotonic cytosol. STAT1 and STAT2 or STAT1 and STAT3 translated in reticulocyte lysate spontaneously form heterocomplexes when the translation lysates are mixed at 0 degrees C. Our data suggest that interferon-alpha /beta-induced tyrosine phosphorylation increases the stability of a preexisting, latent, STAT1-STAT2 signaling complex. Newly translated STAT1 binds in equilibrium fashion to STAT2 and STAT3, but we show that STAT2 and STAT3 exist in separate heterocomplexes with STAT1, consistent with a model in which STAT1 contains a common binding site for other STAT proteins. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "yields coimmunoadsorption", "start": 713, "end": 738}, "arguments": [{"role": "Theme", "text": "STAT1", "start": 671, "end": 676}]}, {"trigger": {"text": "yields coimmunoadsorption", "start": 713, "end": 738}, "arguments": [{"role": "Theme", "text": "STAT2", "start": 680, "end": 685}]}, {"trigger": {"text": "heterocomplexes are coimmunoadsorbed", "start": 789, "end": 825}, "arguments": [{"role": "Theme", "text": "STAT1", "start": 777, "end": 782}, {"role": "Theme2", "text": "STAT3", "start": 783, "end": 788}]}, {"trigger": {"text": "form heterocomplexes", "start": 933, "end": 953}, "arguments": [{"role": "Theme", "text": "STAT1", "start": 850, "end": 855}, {"role": "Theme2", "text": "STAT2", "start": 860, "end": 865}]}, {"trigger": {"text": "form heterocomplexes", "start": 933, "end": 953}, "arguments": [{"role": "Theme", "text": "STAT1", "start": 869, "end": 874}, {"role": "Theme2", "text": "STAT3", "start": 879, "end": 884}]}, {"trigger": {"text": "binds", "start": 1191, "end": 1196}, "arguments": [{"role": "Theme", "text": "STAT1", "start": 1185, "end": 1190}, {"role": "Theme2", "text": "STAT2", "start": 1223, "end": 1228}]}, {"trigger": {"text": "binds", "start": 1191, "end": 1196}, "arguments": [{"role": "Theme", "text": "STAT1", "start": 1185, "end": 1190}, {"role": "Theme2", "text": "STAT3", "start": 1233, "end": 1238}]}, {"trigger": {"text": "exist in separate heterocomplexes", "start": 1273, "end": 1306}, "arguments": [{"role": "Theme", "text": "STAT2", "start": 1257, "end": 1262}, {"role": "Theme2", "text": "STAT1", "start": 1312, "end": 1317}]}, {"trigger": {"text": "exist in separate heterocomplexes", "start": 1273, "end": 1306}, "arguments": [{"role": "Theme", "text": "STAT3", "start": 1267, "end": 1272}, {"role": "Theme2", "text": "STAT1", "start": 1312, "end": 1317}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1071, "end": 1086}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 1062, "end": 1070}, {"role": "Theme", "text": "STAT1", "start": 1137, "end": 1142}]}, {"trigger": {"text": "phosphorylation", "start": 1071, "end": 1086}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 1062, "end": 1070}, {"role": "Theme", "text": "STAT2", "start": 1143, "end": 1148}]}], "positive regulation": [{"trigger": {"text": "when", "start": 954, "end": 958}, "arguments": [{"role": "Theme", "text": "form heterocomplexes", "start": 933, "end": 953}]}, {"trigger": {"text": "induced", "start": 1054, "end": 1061}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1071, "end": 1086}]}, {"trigger": {"text": "increases the stability", "start": 1087, "end": 1110}, "arguments": [{"role": "Cause", "text": "induced", "start": 1054, "end": 1061}, {"role": "Theme", "text": "STAT1", "start": 1137, "end": 1142}]}, {"trigger": {"text": "increases the stability", "start": 1087, "end": 1110}, "arguments": [{"role": "Cause", "text": "induced", "start": 1054, "end": 1061}, {"role": "Theme", "text": "STAT2", "start": 1143, "end": 1148}]}], "regulation": [{"trigger": {"text": "separate", "start": 48, "end": 56}, "arguments": [{"role": "Theme", "text": "STAT2", "start": 29, "end": 34}]}, {"trigger": {"text": "separate", "start": 48, "end": 56}, "arguments": [{"role": "Theme", "text": "STAT3", "start": 39, "end": 44}]}]}}, "schema": []} {"input": "Inhibition of p105 processing by NF-kappaB proteins in transiently transfected cells. \nRegulation of the transcription factor NF-kappaB involves proteasome-mediated processing of the NF-kappaB1 p105 precursor protein, which generates the p50 subunit of NF-kappaB. The processing of p105 occurs constitutively in vivo but can be markedly enhanced by various cellular activation agents, although the underlying regulatory mechanism is not yet clear. In the present study, we demonstrate that signal-mediated induction of p105 processing in human T cells is associated with de novo synthesis of this precursor protein. Transient transfection studies performed in COS7 cells revealed that the newly synthesized p105 protein appears to be more rapidly processed compared to its accumulated form that is already associated with the processed product p50. Interestingly, the processing rate of p105 is markedly inhibited in cells co-transfected with p50 or other NF-kappaB subunits, including RelA and c-Rel, that physically interact with p105. These findings suggest that the processing of p105 is subject to negative regulation by the various NF-kappaB subunits. We further demonstrate that p105 undergoes degradation in lipopolysaccharide-stimulated human monocytic cells. However, the inducible degradation of p105 is not coupled with the generation of p50. Together, these studies demonstrate that the processing and inducible degradation of p105 are differentially regulated. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "associated", "start": 806, "end": 816}, "arguments": [{"role": "Theme", "text": "p50", "start": 844, "end": 847}]}, {"trigger": {"text": "interact", "start": 1018, "end": 1026}, "arguments": [{"role": "Theme", "text": "p50", "start": 943, "end": 946}, {"role": "Theme2", "text": "p105", "start": 1032, "end": 1036}]}, {"trigger": {"text": "interact", "start": 1018, "end": 1026}, "arguments": [{"role": "Theme", "text": "RelA", "start": 986, "end": 990}, {"role": "Theme2", "text": "p105", "start": 1032, "end": 1036}]}, {"trigger": {"text": "interact", "start": 1018, "end": 1026}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 995, "end": 1000}, {"role": "Theme2", "text": "p105", "start": 1032, "end": 1036}]}], "gene expression": [{"trigger": {"text": "generation", "start": 1336, "end": 1346}, "arguments": [{"role": "Theme", "text": "p50", "start": 1350, "end": 1353}]}], "positive regulation": [{"trigger": {"text": "generates", "start": 224, "end": 233}, "arguments": [{"role": "Theme", "text": "p50", "start": 238, "end": 241}]}, {"trigger": {"text": "undergoes", "start": 1191, "end": 1200}, "arguments": [{"role": "Theme", "text": "degradation", "start": 1201, "end": 1212}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 1201, "end": 1212}, "arguments": [{"role": "Theme", "text": "p105", "start": 1186, "end": 1190}]}, {"trigger": {"text": "degradation", "start": 1292, "end": 1303}, "arguments": [{"role": "Theme", "text": "p105", "start": 1307, "end": 1311}]}, {"trigger": {"text": "degradation", "start": 1425, "end": 1436}, "arguments": [{"role": "Theme", "text": "p105", "start": 1440, "end": 1444}]}], "regulation": [{"trigger": {"text": "differentially regulated", "start": 1449, "end": 1473}, "arguments": [{"role": "Theme", "text": "degradation", "start": 1425, "end": 1436}]}]}}, "schema": []} {"input": "Globin gene switching. In vivo protein-DNA interactions of the human beta-globin locus in erythroid cells expressing the fetal or the adult globin gene program. \nTo characterize the protein-DNA interactions important for the developmental control of the human beta-globin locus, we analyzed by in vivo dimethyl sulfate footprinting erythroid cells expressing either the fetal or the adult globin developmental program. In the locus control region (LCR) of the beta-globin locus, in vivo footprints on NF-E2 (or AP-1) and GATA-1 motifs remained the same regardless of whether the fetal or the adult globin genes are expressed. In contrast, in vivo footprints on GT (CACCC) motifs differed between the cells expressing the fetal or the adult globin program. In promoter regions, the actively transcribed genes demonstrated extensive and consistent footprints over the canonical elements, such as CACCC and CCAAT motifs. The adult globin expressing cells displayed more extensive footprints than the fetal globin expressing cells in the 3' regulatory sequences of both the Agamma- and the beta-globin genes, suggesting a role of these 3' elements in beta-globin gene expression. Our results suggest that the bulk of protein-DNA interactions that underlies the developmental control of globin genes takes place in the gamma- and beta-globin gene promoters, and that GT motifs of the beta-globin locus LCR may play a role in the developmental regulation of human beta-globin gene expression, perhaps by increasing the probability of interaction of the LCR holocomplex with the fetal or the adult globin gene. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interactions", "start": 43, "end": 55}, "arguments": [{"role": "Theme", "text": "beta-globin", "start": 69, "end": 80}]}, {"trigger": {"text": "footprints", "start": 977, "end": 987}, "arguments": [{"role": "Site", "text": "3' regulatory sequences", "start": 1034, "end": 1057}, {"role": "Theme", "text": "Agamma-", "start": 1070, "end": 1077}]}, {"trigger": {"text": "footprints", "start": 977, "end": 987}, "arguments": [{"role": "Site", "text": "3' regulatory sequences", "start": 1034, "end": 1057}, {"role": "Theme", "text": "beta-globin", "start": 1086, "end": 1097}]}, {"trigger": {"text": "interaction", "start": 1528, "end": 1539}, "arguments": [{"role": "Theme", "text": "fetal", "start": 1572, "end": 1577}]}, {"trigger": {"text": "interaction", "start": 1528, "end": 1539}, "arguments": [{"role": "Theme", "text": "adult globin", "start": 1585, "end": 1597}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 615, "end": 624}, "arguments": [{"role": "Theme", "text": "fetal", "start": 579, "end": 584}]}, {"trigger": {"text": "expressed", "start": 615, "end": 624}, "arguments": [{"role": "Theme", "text": "adult globin genes", "start": 592, "end": 610}]}, {"trigger": {"text": "expressing", "start": 706, "end": 716}, "arguments": [{"role": "Theme", "text": "fetal", "start": 721, "end": 726}]}, {"trigger": {"text": "expressing", "start": 706, "end": 716}, "arguments": [{"role": "Theme", "text": "adult globin", "start": 734, "end": 746}]}, {"trigger": {"text": "expression", "start": 1164, "end": 1174}, "arguments": [{"role": "Theme", "text": "beta-globin", "start": 1147, "end": 1158}]}, {"trigger": {"text": "expression", "start": 1475, "end": 1485}, "arguments": [{"role": "Theme", "text": "beta-globin", "start": 1458, "end": 1469}]}], "positive regulation": [{"trigger": {"text": "important", "start": 207, "end": 216}, "arguments": [{"role": "Theme", "text": "control", "start": 239, "end": 246}]}, {"trigger": {"text": "expressing", "start": 348, "end": 358}, "arguments": [{"role": "Theme", "text": "fetal", "start": 370, "end": 375}]}, {"trigger": {"text": "expressing", "start": 348, "end": 358}, "arguments": [{"role": "Theme", "text": "adult globin", "start": 383, "end": 395}]}, {"trigger": {"text": "more extensive", "start": 962, "end": 976}, "arguments": [{"role": "Theme", "text": "footprints", "start": 977, "end": 987}]}, {"trigger": {"text": "play a role", "start": 1405, "end": 1416}, "arguments": [{"role": "Theme", "text": "regulation", "start": 1438, "end": 1448}, {"role": "Cause", "text": "increasing", "start": 1498, "end": 1508}]}, {"trigger": {"text": "increasing", "start": 1498, "end": 1508}, "arguments": [{"role": "Theme", "text": "interaction", "start": 1528, "end": 1539}]}], "regulation": [{"trigger": {"text": "control", "start": 239, "end": 246}, "arguments": [{"role": "Theme", "text": "beta-globin", "start": 260, "end": 271}]}, {"trigger": {"text": "role", "start": 1118, "end": 1122}, "arguments": [{"role": "Theme", "text": "expression", "start": 1164, "end": 1174}]}, {"trigger": {"text": "regulation", "start": 1438, "end": 1448}, "arguments": [{"role": "Theme", "text": "expression", "start": 1475, "end": 1485}]}]}}, "schema": []} {"input": "Interleukin 10 induced c-fos expression in human B cells by activation of divergent protein kinases. \nIL-10 is a potent mediator of human B cell growth and plasma cell formation. However, signal transduction of IL-10 in B cells is poorly understood. In this study the effect of IL-10 on the expression of the protooncogene c-fos was investigated, because Fos plays a potential role in the regulation of B cell proliferation and differentiation. B cells were purified from buffy coat preparations of healthy blood donors by positive selection using an anti CD20 monoclonal antibody and a MiniMACS separation unit. B cells were prestimulated with SAC for 48 hrs. Then, cells were incubated with medium or IL-10 (100 ng/ml) for 10 to 120 min. RNA was extracted by phenol/chloroform and c-fos expression was analyzed by PCR assisted mRNA assay. A significant 2-4 fold increase of c-fos expression was observed within 30 min of stimulation with IL-10 (p < 0.01). After 2 hrs c-fos expression declined to basal levels. The effect of IL-10 was dose-dependent with a maximum stimulation using 100 ng/ml of IL-10. The IL-10 effect on c-fos expression was not blocked by polymyxin B. Using the tyrosine kinase inhibitor genistein (10 microM) a complete inhibition of IL-10 induced c-fos expression was observed. In addition, H-7 (10 microM), a specific inhibitor of serine/threonine kinases, significantly blocked IL-10 mediated c-fos expression (p < 0.05). In conclusion, these data show that IL-10 induces c-fos expression in human B-cells by activation of tyrosine and serine/threonine kinases. Since this is the first report on IL-10 induced signal transduction, these data may help to identify the intracellular mechanisms by which IL-10 stimulates human B-cells. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 29, "end": 39}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 23, "end": 28}]}, {"trigger": {"text": "expression", "start": 291, "end": 301}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 323, "end": 328}]}, {"trigger": {"text": "expression", "start": 789, "end": 799}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 783, "end": 788}]}, {"trigger": {"text": "expression", "start": 882, "end": 892}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 876, "end": 881}]}, {"trigger": {"text": "expression", "start": 976, "end": 986}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 970, "end": 975}]}, {"trigger": {"text": "expression", "start": 1131, "end": 1141}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1125, "end": 1130}]}, {"trigger": {"text": "expression", "start": 1277, "end": 1287}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1271, "end": 1276}]}, {"trigger": {"text": "expression", "start": 1425, "end": 1435}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1419, "end": 1424}]}, {"trigger": {"text": "expression", "start": 1504, "end": 1514}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 1498, "end": 1503}]}], "negative regulation": [{"trigger": {"text": "declined", "start": 987, "end": 995}, "arguments": [{"role": "Theme", "text": "expression", "start": 976, "end": 986}]}, {"trigger": {"text": "blocked", "start": 1150, "end": 1157}, "arguments": [{"role": "Theme", "text": "effect", "start": 1115, "end": 1121}]}, {"trigger": {"text": "inhibition", "start": 1243, "end": 1253}, "arguments": [{"role": "Theme", "text": "induced", "start": 1263, "end": 1270}]}, {"trigger": {"text": "blocked", "start": 1396, "end": 1403}, "arguments": [{"role": "Theme", "text": "mediated", "start": 1410, "end": 1418}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 15, "end": 22}, "arguments": [{"role": "Theme", "text": "expression", "start": 29, "end": 39}]}, {"trigger": {"text": "increase", "start": 864, "end": 872}, "arguments": [{"role": "Theme", "text": "expression", "start": 882, "end": 892}]}, {"trigger": {"text": "induced", "start": 1263, "end": 1270}, "arguments": [{"role": "Cause", "text": "IL-10", "start": 1257, "end": 1262}, {"role": "Theme", "text": "expression", "start": 1277, "end": 1287}]}, {"trigger": {"text": "mediated", "start": 1410, "end": 1418}, "arguments": [{"role": "Cause", "text": "IL-10", "start": 1404, "end": 1409}, {"role": "Theme", "text": "expression", "start": 1425, "end": 1435}]}, {"trigger": {"text": "induces", "start": 1490, "end": 1497}, "arguments": [{"role": "Theme", "text": "expression", "start": 1504, "end": 1514}]}], "regulation": [{"trigger": {"text": "effect", "start": 268, "end": 274}, "arguments": [{"role": "Cause", "text": "IL-10", "start": 278, "end": 283}, {"role": "Theme", "text": "expression", "start": 291, "end": 301}]}, {"trigger": {"text": "effect", "start": 1115, "end": 1121}, "arguments": [{"role": "Cause", "text": "IL-10", "start": 1109, "end": 1114}, {"role": "Theme", "text": "expression", "start": 1131, "end": 1141}]}]}}, "schema": []} {"input": "IL-10 inhibits nuclear factor-kappa B/Rel nuclear activity in CD3-stimulated human peripheral T lymphocytes. \nIL-10 markedly reduces nuclear factor (NF)-kappa B/Rel nuclear activity induced in PBMC by stimulation with the anti-CD3 mAb OKT3. The inhibition is exerted specifically on the NF-kappa B/Rel activation induced by mAb OKT3, and not that produced by PMA. As judged by supershifting the DNA-protein complexes with Abs recognizing specific components of the NF-kappa B/Rel protein family, the p50/p65 (Rel A) heterodimeric form of NF-kappa B is primarily affected. The maximal effect is observed at the IL-10 concentration of 20 U/ml. IL-10 inhibitory activity is exerted on T lymphocytes and is mediated by monocytes. Indeed, monocytes pretreated with IL-10 are able so inhibit NF-kappa B nuclear activity in purified T lymphocytes stimulated with OKT3. Soluble factors do not appear to be involved in the mechanism of inhibition. On the other hand, the up-regulation of CD80 Ag, found on monocytes obtained from PBMC incubated with OKT3, is not detected after addition of IL-10, and the anti-CD28 mAb CLB-CD28/1 restores the NF-kappa B/Rel nuclear activity in IL-10-inhibited lymphocytes. Therefore, the NF-kappa B/Rel inhibition might be ascribed to a lack of cooperation between accessory cells and T lymphocytes, resulting from down-regulation of a costimulatory molecule, such as CD80, produced by IL-10 on activated monocytes. Our results demonstrate that IL-10 can inhibit the induction of NF-kappa B/Rel nuclear activity in CD3-stimulated T lymphocytes. Since inappropriate activation of kappa B-driven genes has a physiopathologic role in a number of diseases, such as HIV infection, our findings support the possibility of using this cytokine to suppress an undesirable activation of these transcription factors. ", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "affected", "start": 562, "end": 570}, "arguments": [{"role": "Theme", "text": "p50", "start": 500, "end": 503}]}, {"trigger": {"text": "affected", "start": 562, "end": 570}, "arguments": [{"role": "Theme", "text": "p65", "start": 504, "end": 507}]}, {"trigger": {"text": "down-regulation", "start": 1340, "end": 1355}, "arguments": [{"role": "Theme", "text": "CD80", "start": 1393, "end": 1397}, {"role": "Cause", "text": "IL-10", "start": 1411, "end": 1416}]}], "positive regulation": [{"trigger": {"text": "up-regulation", "start": 962, "end": 975}, "arguments": [{"role": "Theme", "text": "CD80 Ag", "start": 979, "end": 986}]}, {"trigger": {"text": "detected", "start": 1054, "end": 1062}, "arguments": [{"role": "Theme", "text": "up-regulation", "start": 962, "end": 975}, {"role": "Cause", "text": "IL-10", "start": 1081, "end": 1086}]}]}}, "schema": []} {"input": "Vitamin D3- and retinoic acid-induced monocytic differentiation: interactions between the endogenous vitamin D3 receptor, retinoic acid receptors, and retinoid X receptors in U-937 cells. \nRetinoic acid (RA) and 1,25 alpha-dihydroxycholecalciferol (VitD3) are potent regulators of hematopoletic differentiation. Yet, little is known as to how the RA and VitD3 receptor network operates in hematopoietic cells, and whether receptor interactions can explain the interplay between the RA- and VitD3-signaling pathways during differentiation. Therefore, we analyzed the expression, DNA binding, and transcriptional activity of the endogenous RA and VitD3 receptors [retinoic acid receptors (RARs), retinoid X receptors (RXRs), and VitD3 receptor (VDR)] in the U-937 cell line, in which RA and VitD3 induce distinct monocytic differentiation pathways. VitD3 induction resulted in the formation of VDR/RXR DNA-binding complexes on both VitD3 response elements and RA response elements (RAREs). However, transcriptional activation was only observed from a VitD3 response element-driven reporter construct. Several DNA-binding complexes were detected on RAREs in undifferentiated cells. Stimulation by RA resulted in increased RAR beta/RXR DNA binding, activated RARE-dependent transcription, and increased expression of RAR-beta. Concomitant stimulation by VitD3 inhibited the RA-stimulated formation of RAR beta/RXR heterodimers, favoring VDR/RXR binding to the RARE. Also, VitD3 inhibited the expression of CD23 and CD49f, characteristic markers of retinoid-induced U-937 cell differentiation. In contrast, neither the RA-stimulated, RARE-mediated transcription nor the induced RAR-beta expression was suppressed by VitD3, suggesting that VitD3 selectively inhibited the retinoid-induced differentiation program but not the RARE-mediated signal. These results demonstrate a complex role for VitD3 in modifying the retinoid differentiation pathway and may have implications for differentiation-inducing therapy of hematopoietic tumors. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 582, "end": 589}, "arguments": [{"role": "Theme", "text": "VDR", "start": 743, "end": 746}]}, {"trigger": {"text": "formation", "start": 879, "end": 888}, "arguments": [{"role": "Theme", "text": "VDR", "start": 892, "end": 895}]}, {"trigger": {"text": "binding", "start": 1236, "end": 1243}, "arguments": [{"role": "Theme", "text": "RAR beta", "start": 1219, "end": 1227}]}, {"trigger": {"text": "heterodimers", "start": 1410, "end": 1422}, "arguments": [{"role": "Theme", "text": "RAR beta", "start": 1397, "end": 1405}]}, {"trigger": {"text": "binding", "start": 1441, "end": 1448}, "arguments": [{"role": "Theme", "text": "VDR", "start": 1433, "end": 1436}]}], "gene expression": [{"trigger": {"text": "expression", "start": 566, "end": 576}, "arguments": [{"role": "Theme", "text": "VDR", "start": 743, "end": 746}]}, {"trigger": {"text": "expression", "start": 1299, "end": 1309}, "arguments": [{"role": "Theme", "text": "RAR-beta", "start": 1313, "end": 1321}]}, {"trigger": {"text": "expression", "start": 1488, "end": 1498}, "arguments": [{"role": "Theme", "text": "CD23", "start": 1502, "end": 1506}]}, {"trigger": {"text": "expression", "start": 1488, "end": 1498}, "arguments": [{"role": "Theme", "text": "CD49f", "start": 1511, "end": 1516}]}, {"trigger": {"text": "expression", "start": 1682, "end": 1692}, "arguments": [{"role": "Theme", "text": "RAR-beta", "start": 1673, "end": 1681}]}], "negative regulation": [{"trigger": {"text": "inhibited", "start": 1356, "end": 1365}, "arguments": [{"role": "Theme", "text": "stimulated", "start": 1373, "end": 1383}]}, {"trigger": {"text": "inhibited", "start": 1474, "end": 1483}, "arguments": [{"role": "Theme", "text": "expression", "start": 1488, "end": 1498}]}, {"trigger": {"text": "suppressed", "start": 1697, "end": 1707}, "arguments": [{"role": "Theme", "text": "expression", "start": 1682, "end": 1692}]}], "positive regulation": [{"trigger": {"text": "resulted", "start": 863, "end": 871}, "arguments": [{"role": "Theme", "text": "formation", "start": 879, "end": 888}]}, {"trigger": {"text": "resulted in increased", "start": 1197, "end": 1218}, "arguments": [{"role": "Theme", "text": "binding", "start": 1236, "end": 1243}]}, {"trigger": {"text": "increased", "start": 1289, "end": 1298}, "arguments": [{"role": "Theme", "text": "expression", "start": 1299, "end": 1309}]}, {"trigger": {"text": "stimulated", "start": 1373, "end": 1383}, "arguments": [{"role": "Theme", "text": "heterodimers", "start": 1410, "end": 1422}]}, {"trigger": {"text": "favoring", "start": 1424, "end": 1432}, "arguments": [{"role": "Cause", "text": "expression", "start": 1299, "end": 1309}, {"role": "Theme", "text": "binding", "start": 1441, "end": 1448}]}, {"trigger": {"text": "induced", "start": 1665, "end": 1672}, "arguments": [{"role": "Theme", "text": "expression", "start": 1682, "end": 1692}]}]}}, "schema": []} {"input": "Calcium-dependent immediate-early gene induction in lymphocytes is negatively regulated by p21Ha-ras. \nThe induction of immediate-early (IE) response genes, such as egr-1, c-fos, and c-jun, occurs rapidly after the activation of T lymphocytes. The process of activation involves calcium mobilization, activation of protein kinase C (PKC), and phosphorylation of tyrosine kinases. p21(ras), a guanine nucleotide binding factor, mediates T-cell signal transduction through PKC-dependent and PKC-independent pathways. The involvement of p21(ras) in the regulation of calcium-dependent signals has been suggested through analysis of its role in the activation of NF-AT. We have investigated the inductions of the IE genes in response to calcium signals in Jurkat cells (in the presence of activated p21(ras)) and their correlated consequences. The expression of activated p21(ras) negatively regulated the induction of IE genes by calcium ionophore. This inhibition of calcium-activated IE gene induction was reversed by treatment with cyclosporin A, suggesting the involvement of calcineurin in this regulation. A later result of inhibition of this activation pathway by p21(ras) was down-regulation of the activity of the transcription factor AP-1 and subsequent coordinate reductions in IL-2 gene expression and protein production. These results suggest that p2l(ras) is an essential mediator in generating not only positive but also negative modulatory mechanisms controlling the competence of T cells in response to inductive stimulations. ", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "reductions", "start": 1272, "end": 1282}, "arguments": [{"role": "Theme", "text": "gene expression", "start": 1291, "end": 1306}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 107, "end": 116}, "arguments": [{"role": "Theme", "text": "egr-1", "start": 165, "end": 170}]}, {"trigger": {"text": "induction", "start": 107, "end": 116}, "arguments": [{"role": "Theme", "text": "c-fos", "start": 172, "end": 177}]}, {"trigger": {"text": "induction", "start": 107, "end": 116}, "arguments": [{"role": "Theme", "text": "c-jun", "start": 183, "end": 188}]}, {"trigger": {"text": "occurs", "start": 190, "end": 196}, "arguments": [{"role": "Theme", "text": "induction", "start": 107, "end": 116}]}, {"trigger": {"text": "activated", "start": 785, "end": 794}, "arguments": [{"role": "Theme", "text": "p21(ras)", "start": 795, "end": 803}]}, {"trigger": {"text": "expression of activated", "start": 844, "end": 867}, "arguments": [{"role": "Theme", "text": "p21(ras)", "start": 868, "end": 876}]}], "transcription": [{"trigger": {"text": "gene expression", "start": 1291, "end": 1306}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1286, "end": 1290}]}]}}, "schema": []} {"input": "Apoptosis signaling pathways in normal T cells: differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity. \nFas-mediated apoptosis plays an important role in regulating the immune response in peripheral T cells. Restimulation of T cell blasts up-regulates Fas and Fas ligand expression, with subsequent interaction leading to cell death. Overexpression of Bcl-2 in tumor cells blocks apoptosis induced by many stimuli, but inhibition of Fas-mediated killing has not been consistently observed. To examine the behavior of Bcl-2 in normal cells, T cell blasts were transiently transfected with Bcl-2 and related gene products to determine the effect on apoptotic signaling. Transient overexpression of Bcl-2 in mouse and human T cell blasts did not block Fas-mediated apoptosis, whereas etoposide- and glucocorticoid-induced cytotoxicity was potently inhibited. Expression of Bcl-xL and adenovirus E1B 19K did not interfere with anti-Fas killing. In contrast, interleukin-1beta-converting enzyme family protease inhibitors Ac-DEVD-CHO and CrmA blocked Fas-mediated apoptosis. These results suggest that peripheral T cells use distinct apoptosis signaling pathways with differential sensitivity to Bcl-2 and interleukin-1beta-converting enzyme family protease inhibitors. Since T cells normally express Bcl-2 and Bcl-xL following activation, their inability to block Fas-mediated apoptosis may allow for the elimination of self-reactive cells and the appropriate regulation of immune responses. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 383, "end": 394}, "arguments": [{"role": "Theme", "text": "Fas", "start": 336, "end": 339}, {"role": "Theme2", "text": "Fas ligand", "start": 344, "end": 354}]}], "gene expression": [{"trigger": {"text": "expression", "start": 355, "end": 365}, "arguments": [{"role": "Theme", "text": "Fas", "start": 336, "end": 339}]}, {"trigger": {"text": "expression", "start": 355, "end": 365}, "arguments": [{"role": "Theme", "text": "Fas ligand", "start": 344, "end": 354}]}, {"trigger": {"text": "Overexpression", "start": 418, "end": 432}, "arguments": [{"role": "Theme", "text": "Bcl-2", "start": 436, "end": 441}]}, {"trigger": {"text": "transfected", "start": 655, "end": 666}, "arguments": [{"role": "Theme", "text": "Bcl-2", "start": 672, "end": 677}]}, {"trigger": {"text": "overexpression", "start": 762, "end": 776}, "arguments": [{"role": "Theme", "text": "Bcl-2", "start": 780, "end": 785}]}, {"trigger": {"text": "Expression", "start": 940, "end": 950}, "arguments": [{"role": "Theme", "text": "Bcl-xL", "start": 954, "end": 960}]}, {"trigger": {"text": "Expression", "start": 940, "end": 950}, "arguments": [{"role": "Theme", "text": "E1B 19K", "start": 976, "end": 983}]}, {"trigger": {"text": "express", "start": 1372, "end": 1379}, "arguments": [{"role": "Theme", "text": "Bcl-2", "start": 1380, "end": 1385}]}, {"trigger": {"text": "express", "start": 1372, "end": 1379}, "arguments": [{"role": "Theme", "text": "Bcl-xL", "start": 1390, "end": 1396}]}], "positive regulation": [{"trigger": {"text": "up-regulates", "start": 323, "end": 335}, "arguments": [{"role": "Theme", "text": "expression", "start": 355, "end": 365}]}, {"trigger": {"text": "Overexpression", "start": 418, "end": 432}, "arguments": [{"role": "Theme", "text": "Overexpression", "start": 418, "end": 432}]}, {"trigger": {"text": "transfected", "start": 655, "end": 666}, "arguments": [{"role": "Theme", "text": "transfected", "start": 655, "end": 666}]}, {"trigger": {"text": "overexpression", "start": 762, "end": 776}, "arguments": [{"role": "Theme", "text": "overexpression", "start": 762, "end": 776}]}, {"trigger": {"text": "following", "start": 1397, "end": 1406}, "arguments": [{"role": "Theme", "text": "express", "start": 1372, "end": 1379}]}]}}, "schema": []} {"input": "Interferons induce normal and aberrant retinoic-acid receptors type alpha in acute promyelocytic leukemia cells: potentiation of the induction of retinoid-dependent differentiation markers. \nTreatment of the acute promyelocytic (APL) cell line NB4 with interferon alpha (IFN(alpha)), as well as IFN(beta) and gamma, results in an increased expression of the transcripts coding for retinoic-acid receptor type alpha (RAR(alpha)) and the leukemia-specific retinoic acid receptor PML-RAR. Transcriptional induction of the RAR(alpha) and PML-RAR mRNAs is rapid and it is parallelled by an increase in the corresponding proteins. Up-regulation of RAR(alpha) and PML-RAR gene expression by IFN(alpha) is accompanied by a strong potentiation in the induction of 2 retinoid-dependent granulocytic markers, i.e., granulocyte-colony-stimulating factor receptor mRNA and leukocyte alkaline phosphatase. However, IFN(alpha) does not have any effects on the retinoid-dependent regulation of the myeloid surface markers CD11b and CD33. The IFN-dependent increase in RAR(alpha) levels and the enhancing effect of the cytokine on retinoid-dependent granulocytic markers expression may be a characteristic of PML-RAR positive cells, since the phenomena are not observed in HL-60 promyelocytes. Interferons as well as retinoids inhibit the growth of NB4 cells, although the 2 classes of compounds do not significantly interact in terms of anti-proliferative activity. These results suggest the possible use of combinations between IFNs and retinoic acid in the cyto-differentiating treatment of APL patients. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 670, "end": 680}, "arguments": [{"role": "Theme", "text": "RAR(alpha)", "start": 642, "end": 652}]}, {"trigger": {"text": "expression", "start": 670, "end": 680}, "arguments": [{"role": "Theme", "text": "PML-RAR", "start": 657, "end": 664}]}, {"trigger": {"text": "levels", "start": 1063, "end": 1069}, "arguments": [{"role": "Theme", "text": "RAR(alpha)", "start": 1052, "end": 1062}]}], "positive regulation": [{"trigger": {"text": "induce", "start": 12, "end": 18}, "arguments": [{"role": "Theme", "text": "retinoic-acid receptors type alpha", "start": 39, "end": 73}]}, {"trigger": {"text": "results in an increased", "start": 316, "end": 339}, "arguments": [{"role": "Theme", "text": "expression", "start": 340, "end": 350}]}, {"trigger": {"text": "Transcriptional induction", "start": 486, "end": 511}, "arguments": [{"role": "Theme", "text": "RAR(alpha)", "start": 519, "end": 529}]}, {"trigger": {"text": "Transcriptional induction", "start": 486, "end": 511}, "arguments": [{"role": "Theme", "text": "PML-RAR", "start": 534, "end": 541}]}, {"trigger": {"text": "parallelled", "start": 567, "end": 578}, "arguments": [{"role": "Cause", "text": "Transcriptional induction", "start": 486, "end": 511}, {"role": "Theme", "text": "increase", "start": 585, "end": 593}]}, {"trigger": {"text": "increase", "start": 585, "end": 593}, "arguments": [{"role": "Theme", "text": "RAR(alpha)", "start": 519, "end": 529}]}, {"trigger": {"text": "increase", "start": 585, "end": 593}, "arguments": [{"role": "Theme", "text": "PML-RAR", "start": 534, "end": 541}]}, {"trigger": {"text": "Up-regulation", "start": 625, "end": 638}, "arguments": [{"role": "Theme", "text": "expression", "start": 670, "end": 680}]}, {"trigger": {"text": "induction", "start": 742, "end": 751}, "arguments": [{"role": "Theme", "text": "granulocyte-colony-stimulating factor receptor", "start": 804, "end": 850}]}, {"trigger": {"text": "induction", "start": 742, "end": 751}, "arguments": [{"role": "Theme", "text": "leukocyte alkaline phosphatase", "start": 860, "end": 890}]}, {"trigger": {"text": "dependent", "start": 766, "end": 775}, "arguments": [{"role": "Theme", "text": "granulocyte-colony-stimulating factor receptor", "start": 804, "end": 850}]}, {"trigger": {"text": "dependent", "start": 766, "end": 775}, "arguments": [{"role": "Theme", "text": "leukocyte alkaline phosphatase", "start": 860, "end": 890}]}, {"trigger": {"text": "increase", "start": 1040, "end": 1048}, "arguments": [{"role": "Theme", "text": "levels", "start": 1063, "end": 1069}]}, {"trigger": {"text": "observed", "start": 1244, "end": 1252}, "arguments": [{"role": "Theme", "text": "RAR(alpha)", "start": 1052, "end": 1062}]}], "regulation": [{"trigger": {"text": "effects", "start": 930, "end": 937}, "arguments": [{"role": "Theme", "text": "regulation", "start": 964, "end": 974}]}, {"trigger": {"text": "regulation", "start": 964, "end": 974}, "arguments": [{"role": "Theme", "text": "CD11b", "start": 1006, "end": 1011}]}, {"trigger": {"text": "regulation", "start": 964, "end": 974}, "arguments": [{"role": "Theme", "text": "CD33", "start": 1016, "end": 1020}]}], "transcription": [{"trigger": {"text": "expression", "start": 340, "end": 350}, "arguments": [{"role": "Theme", "text": "RAR(alpha)", "start": 416, "end": 426}]}, {"trigger": {"text": "expression", "start": 340, "end": 350}, "arguments": [{"role": "Theme", "text": "PML-RAR", "start": 477, "end": 484}]}]}}, "schema": []} {"input": "An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein. \nExpression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 101, "end": 106}, "arguments": [{"role": "Theme", "text": "Stat5", "start": 107, "end": 112}]}, {"trigger": {"text": "binds", "start": 101, "end": 106}, "arguments": [{"role": "Theme", "text": "Elf-1", "start": 114, "end": 119}]}, {"trigger": {"text": "binds", "start": 101, "end": 106}, "arguments": [{"role": "Theme", "text": "HMG-I(Y)", "start": 121, "end": 129}]}, {"trigger": {"text": "bind", "start": 838, "end": 842}, "arguments": [{"role": "Theme", "text": "Stat5", "start": 790, "end": 795}]}, {"trigger": {"text": "bind", "start": 838, "end": 842}, "arguments": [{"role": "Theme", "text": "Elf-1", "start": 797, "end": 802}]}, {"trigger": {"text": "bind", "start": 838, "end": 842}, "arguments": [{"role": "Theme", "text": "HMG-I(Y)", "start": 804, "end": 812}]}], "gene expression": [{"trigger": {"text": "Expression", "start": 158, "end": 168}, "arguments": [{"role": "Theme", "text": "interleukin-2 (IL-2) receptor alpha chain", "start": 182, "end": 223}]}], "negative regulation": [{"trigger": {"text": "negatively regulate", "start": 1063, "end": 1082}, "arguments": [{"role": "Theme", "text": "IL-2R alpha", "start": 1093, "end": 1104}, {"role": "Site", "text": "promoter", "start": 1105, "end": 1113}]}], "positive regulation": [{"trigger": {"text": "upregulated", "start": 241, "end": 252}, "arguments": [{"role": "Theme", "text": "Expression", "start": 158, "end": 168}, {"role": "Cause", "text": "IL-2", "start": 272, "end": 276}]}, {"trigger": {"text": "induced", "start": 1126, "end": 1133}, "arguments": [{"role": "Cause", "text": "IL-2", "start": 1121, "end": 1125}, {"role": "Theme", "text": "IL-2R alpha", "start": 1134, "end": 1145}, {"role": "Site", "text": "promoter", "start": 1146, "end": 1154}]}, {"trigger": {"text": "requires", "start": 1164, "end": 1172}, "arguments": [{"role": "Theme", "text": "induced", "start": 1126, "end": 1133}]}]}}, "schema": []} {"input": "Active suppression of the class II transactivator-encoding AIR-1 locus is responsible for the lack of major histocompatibility complex class II gene expression observed during differentiation from B cells to plasma cells. \nIn this study the genetic control of major histocompatibility complex (MHC) class II gene expression during the transition from B cell to plasma cell has been analyzed. Class II molecules are not expressed in plasma cells because of an active suppression resulting in the abrogation of class II gene transcription. We show here that the plasma cell-specific repressor function, designated SIR (suppressor of immune response genes), does not act directly on the transcription of class II genes, but instead on the transcription of the AIR-1 gene, whose product, the class II transactivator (CIITA), is fundamental for the regulation of the constitutive and inducible expression of MHC class II genes. This was unambiguously demonstrated by the fact that plasmacytoma x B cell hybrids carrying an AIR-1 locus derived from CIITA-expressing cells do not express CIITA-specific transcripts. Transfection of a cDNA containing the human CIITA coding sequence under the control of an heterologous promoter restores expression of human MHC class II genes in the hybrids and is responsible for de novo expression of mouse MHC class II genes in both the mouse plasmacytoma cell line and the hybrids. These results confirm and extend the notion of the functional conservation of the AIR-1 gene product across species barriers. Interestingly, in CIITA-transfected cell hybrids, cell surface expression of the human HLA-DQ heterodimer was not observed. This result was not attributable to lack of HLA-DQ alpha or -DQ beta transcription, because both transcripts were present in the CIITA-transfected hybrids, although at reduced levels. These findings further support our previous observations on the distinct regulation of expression of the human HLA-DQ class II subset, which may be thus controlled at the posttranscriptional level by a CIITA-independent mechanism. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Transfection", "start": 1109, "end": 1121}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 1153, "end": 1158}]}, {"trigger": {"text": "transfected", "start": 1562, "end": 1573}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 1556, "end": 1561}]}, {"trigger": {"text": "present", "start": 1776, "end": 1783}, "arguments": [{"role": "Theme", "text": "HLA-DQ alpha", "start": 1706, "end": 1718}]}, {"trigger": {"text": "present", "start": 1776, "end": 1783}, "arguments": [{"role": "Theme", "text": "-DQ beta", "start": 1722, "end": 1730}]}, {"trigger": {"text": "transfected", "start": 1797, "end": 1808}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 1791, "end": 1796}]}], "negative regulation": [{"trigger": {"text": "suppression", "start": 7, "end": 18}, "arguments": [{"role": "Theme", "text": "class II transactivator", "start": 26, "end": 49}]}, {"trigger": {"text": "act", "start": 664, "end": 667}, "arguments": [{"role": "Theme", "text": "transcription", "start": 736, "end": 749}]}, {"trigger": {"text": "lack", "start": 1698, "end": 1702}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1731, "end": 1744}]}, {"trigger": {"text": "reduced levels", "start": 1830, "end": 1844}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1731, "end": 1744}, {"role": "Cause", "text": "transfected", "start": 1797, "end": 1808}]}], "positive regulation": [{"trigger": {"text": "Active", "start": 0, "end": 6}, "arguments": [{"role": "Theme", "text": "suppression", "start": 7, "end": 18}]}, {"trigger": {"text": "express", "start": 1073, "end": 1080}, "arguments": [{"role": "Theme", "text": "transcripts", "start": 1096, "end": 1107}]}, {"trigger": {"text": "Transfection", "start": 1109, "end": 1121}, "arguments": [{"role": "Theme", "text": "under the control", "start": 1175, "end": 1192}]}, {"trigger": {"text": "transfected", "start": 1562, "end": 1573}, "arguments": [{"role": "Theme", "text": "transfected", "start": 1562, "end": 1573}]}, {"trigger": {"text": "transfected", "start": 1797, "end": 1808}, "arguments": [{"role": "Theme", "text": "transfected", "start": 1797, "end": 1808}]}], "regulation": [{"trigger": {"text": "under the control", "start": 1175, "end": 1192}, "arguments": [{"role": "Theme", "text": "Transfection", "start": 1109, "end": 1121}]}, {"trigger": {"text": "attributable", "start": 1682, "end": 1694}, "arguments": [{"role": "Theme", "text": "lack", "start": 1698, "end": 1702}]}], "transcription": [{"trigger": {"text": "transcription", "start": 736, "end": 749}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 813, "end": 818}]}, {"trigger": {"text": "transcripts", "start": 1096, "end": 1107}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 1081, "end": 1086}]}, {"trigger": {"text": "transcription", "start": 1731, "end": 1744}, "arguments": [{"role": "Theme", "text": "HLA-DQ alpha", "start": 1706, "end": 1718}]}, {"trigger": {"text": "transcription", "start": 1731, "end": 1744}, "arguments": [{"role": "Theme", "text": "-DQ beta", "start": 1722, "end": 1730}]}]}}, "schema": []} {"input": "Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF. \nGlobin genes are subject to tissue-specific and developmental stage-specific regulation. A switch from human fetal (gamma)-to adult (beta)-globin expression occurs within erythroid precursor cells of the adult lineage. Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Kruppel-like factor (EKLF), is required for expression of the beta-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter. To examine the role of EKLF in the developmental regulation of the human gamma-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human beta-globin yeast artificial chromosome transgene. We find that in the absence of EKLF, while human beta-globin expression is dramatically reduced, gamma-globin transcripts are elevated approximately 5-fold. Impaired silencing of gamma-globin expression identifies EKLF as the first transcription factor participating quantitatively in the gamma-globin to beta-globin switch. Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the beta-globin gene promoter and the locus control region that excludes the gamma-globin gene. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 548, "end": 559}, "arguments": [{"role": "Theme", "text": "EKLF", "start": 462, "end": 466}]}, {"trigger": {"text": "interaction", "start": 1266, "end": 1277}, "arguments": [{"role": "Theme", "text": "beta-globin", "start": 1290, "end": 1301}, {"role": "Site", "text": "promoter", "start": 1307, "end": 1315}]}], "gene expression": [{"trigger": {"text": "expression", "start": 32, "end": 42}, "arguments": [{"role": "Theme", "text": "fetal globin", "start": 19, "end": 31}]}, {"trigger": {"text": "expression", "start": 271, "end": 281}, "arguments": [{"role": "Theme", "text": "human fetal (gamma)-", "start": 228, "end": 248}]}, {"trigger": {"text": "expression", "start": 271, "end": 281}, "arguments": [{"role": "Theme", "text": "adult (beta)-globin", "start": 251, "end": 270}]}, {"trigger": {"text": "expression", "start": 485, "end": 495}, "arguments": [{"role": "Theme", "text": "beta-globin", "start": 503, "end": 514}]}, {"trigger": {"text": "expression", "start": 889, "end": 899}, "arguments": [{"role": "Theme", "text": "beta-globin", "start": 877, "end": 888}]}, {"trigger": {"text": "expression", "start": 1020, "end": 1030}, "arguments": [{"role": "Theme", "text": "gamma-globin", "start": 1007, "end": 1019}]}], "negative regulation": [{"trigger": {"text": "Silencing", "start": 0, "end": 9}, "arguments": [{"role": "Theme", "text": "expression", "start": 32, "end": 42}]}, {"trigger": {"text": "impaired", "start": 46, "end": 54}, "arguments": [{"role": "Theme", "text": "Silencing", "start": 0, "end": 9}, {"role": "Cause", "text": "absence", "start": 62, "end": 69}]}, {"trigger": {"text": "absence", "start": 62, "end": 69}, "arguments": [{"role": "Theme", "text": "EKLF", "start": 118, "end": 122}]}, {"trigger": {"text": "switch", "start": 216, "end": 222}, "arguments": [{"role": "Theme", "text": "expression", "start": 271, "end": 281}]}, {"trigger": {"text": "reduced", "start": 916, "end": 923}, "arguments": [{"role": "Theme", "text": "expression", "start": 889, "end": 899}]}, {"trigger": {"text": "Impaired", "start": 985, "end": 993}, "arguments": [{"role": "Theme", "text": "silencing", "start": 994, "end": 1003}]}, {"trigger": {"text": "silencing", "start": 994, "end": 1003}, "arguments": [{"role": "Theme", "text": "expression", "start": 1020, "end": 1030}]}, {"trigger": {"text": "switch", "start": 1145, "end": 1151}, "arguments": [{"role": "Theme", "text": "gamma-globin", "start": 1117, "end": 1129}]}, {"trigger": {"text": "excludes", "start": 1350, "end": 1358}, "arguments": [{"role": "Cause", "text": "mediates", "start": 1233, "end": 1241}, {"role": "Theme", "text": "gamma-globin", "start": 1363, "end": 1375}]}], "positive regulation": [{"trigger": {"text": "switch", "start": 216, "end": 222}, "arguments": [{"role": "Theme", "text": "expression", "start": 271, "end": 281}]}, {"trigger": {"text": "required", "start": 472, "end": 480}, "arguments": [{"role": "Cause", "text": "EKLF", "start": 462, "end": 466}, {"role": "Theme", "text": "expression", "start": 485, "end": 495}]}, {"trigger": {"text": "through", "start": 540, "end": 547}, "arguments": [{"role": "Theme", "text": "required", "start": 472, "end": 480}, {"role": "Cause", "text": "interaction", "start": 548, "end": 559}]}, {"trigger": {"text": "role", "start": 635, "end": 639}, "arguments": [{"role": "Cause", "text": "EKLF", "start": 643, "end": 647}, {"role": "Theme", "text": "regulation", "start": 669, "end": 679}]}, {"trigger": {"text": "elevated", "start": 954, "end": 962}, "arguments": [{"role": "Theme", "text": "gamma-globin", "start": 925, "end": 937}]}, {"trigger": {"text": "participating", "start": 1081, "end": 1094}, "arguments": [{"role": "Cause", "text": "EKLF", "start": 1042, "end": 1046}, {"role": "Theme", "text": "switch", "start": 1145, "end": 1151}]}, {"trigger": {"text": "switch", "start": 1145, "end": 1151}, "arguments": [{"role": "Theme", "text": "beta-globin", "start": 1133, "end": 1144}]}, {"trigger": {"text": "mediates", "start": 1233, "end": 1241}, "arguments": [{"role": "Cause", "text": "EKLF", "start": 1228, "end": 1232}, {"role": "Theme", "text": "interaction", "start": 1266, "end": 1277}]}], "regulation": [{"trigger": {"text": "regulation", "start": 669, "end": 679}, "arguments": [{"role": "Theme", "text": "gamma-globin", "start": 693, "end": 705}]}]}}, "schema": []} {"input": "Nasal NK- and T-cell lymphomas share the same type of Epstein-Barr virus latency as nasopharyngeal carcinoma and Hodgkin's disease. \nNasal T/NK-cell lymphomas can be further separated into those of natural killer (NK) cell lineage or of T-cell lineage, with differences in cellular phenotype, T-cell receptor (TcR) gene rearrangement and TcR transcript expression. Both NK- and T-cell subtypes are closely associated with Epstein-Barr virus (EBV). In this study, EBV gene expression was determined in 23 cases of nasal lymphoma (NL) by in situ hybridisation (ISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IH). Of the 23 cases, 19 were classified as NK-cell and 4 as T-cell tumours. ISH for EBV-encoded small non-polyadenylated RNAs showed that all cases, whether NK or T, harboured EBV in virtually all tumour cells. RT-PCR demonstrated that NL of both subtypes expressed EBNAI of the QUK splice pattern, the latent membrane proteins, LMP1 and 2 and the BamHI A rightward transcripts in the absence of EBNA2 mRNAs, compatible with the latency type II pattern. In addition, analysis of EBV protein expression by IH revealed a heterogeneous pattern of EBV gene expression at the single-cell level consisting of both LMP1+ and LMP1- tumour cells, suggesting a mixture of latency I and II. Although 2 early lytic transcripts, BZLF1 and BHRF1, were also detected in 13 and 10 cases, respectively, the lack of ZEBRA staining in any case indicates that these lytic transcripts are most likely expressed by rare cells in the biopsies entering lytic cycle. The viral transcriptional pattern similar to that of nasopharyngeal carcinoma and Hodgkin's disease suggests that EBV can exploit common regulatory mechanisms for gene transcription in diverse host cell types. Down-regulation of immunogenic proteins (EBNA2-EBNA6) in nasal lymphoma may enable tumour cells to evade host cytotoxic T-cell surveillance. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressed", "start": 905, "end": 914}, "arguments": [{"role": "Theme", "text": "EBNAI", "start": 915, "end": 920}]}, {"trigger": {"text": "expressed", "start": 905, "end": 914}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 978, "end": 982}]}, {"trigger": {"text": "expressed", "start": 905, "end": 914}, "arguments": [{"role": "Theme", "text": "2", "start": 987, "end": 988}]}], "negative regulation": [{"trigger": {"text": "Down-regulation", "start": 1801, "end": 1816}, "arguments": [{"role": "Theme", "text": "EBNA2", "start": 1842, "end": 1847}]}, {"trigger": {"text": "Down-regulation", "start": 1801, "end": 1816}, "arguments": [{"role": "Theme", "text": "EBNA6", "start": 1848, "end": 1853}]}], "transcription": [{"trigger": {"text": "absence", "start": 1034, "end": 1041}, "arguments": [{"role": "Theme", "text": "EBNA2", "start": 1045, "end": 1050}]}, {"trigger": {"text": "detected", "start": 1392, "end": 1400}, "arguments": [{"role": "Theme", "text": "BZLF1", "start": 1365, "end": 1370}]}, {"trigger": {"text": "detected", "start": 1392, "end": 1400}, "arguments": [{"role": "Theme", "text": "BHRF1", "start": 1375, "end": 1380}]}, {"trigger": {"text": "lack", "start": 1439, "end": 1443}, "arguments": [{"role": "Theme", "text": "ZEBRA", "start": 1447, "end": 1452}]}, {"trigger": {"text": "expressed", "start": 1529, "end": 1538}, "arguments": [{"role": "Theme", "text": "BZLF1", "start": 1365, "end": 1370}]}, {"trigger": {"text": "expressed", "start": 1529, "end": 1538}, "arguments": [{"role": "Theme", "text": "BHRF1", "start": 1375, "end": 1380}]}]}}, "schema": []} {"input": "Identification and characterization of a leukocyte-specific component of the nuclear body. \nThe nuclear body (NB) is a cellular organelle that is involved in the pathogenesis of acute promyelocytic leukemia and viral infection. The NB is also a target of antibodies in the serum of patients with the autoimmune disease primary biliary cirrhosis. In this study, serum from a patient with primary biliary cirrhosis was used to identify a cDNA encoding a novel component of the NB, a 140-kDa protein designated Sp140. The predicted amino acid sequence of the amino-terminal portion of Sp140 was similar to Sp100, a previously identified NB protein. The carboxyl portion of Sp140 contained a zinc-finger domain and a bromodomain, motifs that are present in proteins regulating gene transcription. High levels of Sp140 mRNA were detected in human spleen and peripheral blood leukocytes, but not other human tissues. The level of SP140 mRNA in myeloid precursor cell lines HL60 and NB4 markedly increased in response to chemically induced cellular differentiation. Immunohistochemical techniques were used to demonstrate that SP140 localized to the NB in differentiated HL60 and NB4 cells. The location of Sp140 in the NB, and expression of this gene in cells involved in host defense, suggest that Sp140 may be involved in the pathogenesis of acute promyelocytic leukemia and viral infection. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 1221, "end": 1231}, "arguments": [{"role": "Theme", "text": "Sp140", "start": 1200, "end": 1205}]}], "localization": [{"trigger": {"text": "localized", "start": 1126, "end": 1135}, "arguments": [{"role": "Theme", "text": "SP140", "start": 1120, "end": 1125}, {"role": "ToLoc", "text": "NB", "start": 1143, "end": 1145}]}, {"trigger": {"text": "location", "start": 1188, "end": 1196}, "arguments": [{"role": "Theme", "text": "Sp140", "start": 1200, "end": 1205}, {"role": "AtLoc", "text": "NB", "start": 1213, "end": 1215}]}], "positive regulation": [{"trigger": {"text": "High levels", "start": 793, "end": 804}, "arguments": [{"role": "Theme", "text": "detected", "start": 824, "end": 832}]}, {"trigger": {"text": "increased", "start": 989, "end": 998}, "arguments": [{"role": "Theme", "text": "level", "start": 915, "end": 920}]}], "transcription": [{"trigger": {"text": "detected", "start": 824, "end": 832}, "arguments": [{"role": "Theme", "text": "Sp140", "start": 808, "end": 813}]}, {"trigger": {"text": "level", "start": 915, "end": 920}, "arguments": [{"role": "Theme", "text": "SP140", "start": 924, "end": 929}]}]}}, "schema": []} {"input": "Potent gene regulatory and antiproliferative activities of 20-methyl analogues of 1,25 dihydroxyvitamin D3. \nThe biological active form of vitamin D3, 1,25-dihydroxyvitamin D3 (VD), regulates cellular growth and differentiation. This provides the hormone with an interesting therapeutic potential. However, hypercalcemia is a side effect, which is caused by VD's classical action, the regulation of calcium homeostasis. This made the need for VD analogues with selectively increased cell regulatory properties. Studies with 20-epi analogues pointed out the importance of the carbon-20 position and led to the development of 20-methyl derivatives of VD. In this report the biological properties of the compounds ZK161422 and ZK157202, which are 20-methyl- and 20-methyl-23-eneanalogues, respectively, have been analyzed in comparison with VD. Both compounds show about 2-fold lower affinity to the VD receptor (VDR) than VD. However, compared to VD, their antiproliferative effect is up to 30-fold higher on human peripheral blood mononuclear cells and even up to 300-fold higher on human breast cancer MCF-7 cells. Whereas the hypercalcemic effect for ZK157202 is also increased 10-fold, ZK161422 has the same calcium-mobilizing potency as VD. Moreover, ZK161422, but not ZK157202, showed preference for gene activation from a promoter carrying a VD response element with a palindromic arrangement of two hexameric receptor binding sites spaced by 9 nucleotides (IP9) rather than for activation from a response element formed by a direct repeat spaced by 3 nucleotides (DR3). This observation supports a model, in which promoter selectivity reflects the selectively increased antiproliferative effect of VD analogues. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "lower affinity", "start": 875, "end": 889}, "arguments": [{"role": "Theme", "text": "VDR", "start": 910, "end": 913}]}]}}, "schema": []} {"input": "Second messenger up-regulation of androgen receptor gene transcription is absent in androgen insensitive human prostatic carcinoma cell lines, PC-3 and DU-145. \nA theoretical pathway of transcriptional regulation of the androgen receptor (AR) gene is via a cAMP response element (CRE) present in its promoter region (-508 to -501). After 20 h of stimulation with 8-bromo-cAMP, AR mRNA was upregulated in LNCaP but not in either PC-3 or DU-145 cell lines. We have demonstrated that the level of CRE binding protein (CREB) was the same in all cell lines and that the putative AR-CRE forms specific and compatible protein interactions with CREB. The ability to regulate AR gene transcription via the second messenger pathway is lost in the PC-3 and DU-145 cell lines. This may be an important primary mechanism of androgen insensitivity in prostate cancer. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interactions", "start": 619, "end": 631}, "arguments": [{"role": "Theme", "text": "CREB", "start": 637, "end": 641}]}], "negative regulation": [{"trigger": {"text": "lost", "start": 725, "end": 729}, "arguments": [{"role": "Theme", "text": "regulate", "start": 658, "end": 666}]}], "positive regulation": [{"trigger": {"text": "via", "start": 251, "end": 254}, "arguments": [{"role": "Theme", "text": "transcriptional regulation", "start": 186, "end": 212}]}, {"trigger": {"text": "upregulated", "start": 389, "end": 400}, "arguments": [{"role": "Theme", "text": "AR", "start": 377, "end": 379}]}], "regulation": [{"trigger": {"text": "transcriptional regulation", "start": 186, "end": 212}, "arguments": [{"role": "Theme", "text": "AR", "start": 239, "end": 241}]}, {"trigger": {"text": "regulate", "start": 658, "end": 666}, "arguments": [{"role": "Theme", "text": "transcription", "start": 675, "end": 688}]}], "transcription": [{"trigger": {"text": "transcription", "start": 675, "end": 688}, "arguments": [{"role": "Theme", "text": "AR", "start": 667, "end": 669}]}]}}, "schema": []} {"input": "Activation of the NF-kappaB transcription factor in a T-lymphocytic cell line by hypochlorous acid. \nReactive oxygen species (ROS) such as hydrogen peroxide serve as second messengers in the induction of the transcription factor NF-kappaB, and hence in the activation and replication of human immunodeficiency virus type 1 (HIV-1) in human cells. During inflammatory reactions, many oxidative species are produced, one of which is hypochlorous acid (HOCl), which is responsible for the microbicidal effects of activated human polymorphonuclear leukocytes. Treatment of a T-lymphocytic cell line with micromolar concentrations of HOCl promoted the appearance of transcription factor NF-kappaB (the heterodimer p50/p65) in the nucleus of the cells, even in the absence of de novo protein synthesis. Western blot analysis of the NF-kappaB inhibitory subunits (IkappaB) demonstrated that both IkappaB-alpha proteolysis and p105 processing were induced by the treatment. NF-kappaB activation was very effective when cells were subjected to hyperthermia before being treated with HOCl. Various antioxidants, such as pyrrolidine dithiocarbamate, p-bromophenacyl-bromide and nordihydroguaiaretic acid could strongly reduce NF-kappaB translocation, demonstrating the importance of oxidative species in the transduction mechanism. Moreover, ACH-2 cells treated with HOCl or H2O2 released tumour necrosis factor-alpha (TNF-alpha) in the supernatants. The importance of TNF-alpha release in NF-kappaB induction by HOCl or H2O2 was demonstrated by the fact that: (1) the nuclear appearance of NF-kappaB was promoted in untreated cells; and (2) synergism between TNF-alpha and HOCl was detected. Collectively, these results suggest that HOCl should be considered as an oxidative species capable of inducing NF-kappaB in a T-lymphocytic cell line through a transduction mechanism involving ROS, and having a long-distance effect through subsequent TNF-alpha release. ", "output": {"json_structures": {"localization": [{"trigger": {"text": "appearance", "start": 647, "end": 657}, "arguments": [{"role": "Theme", "text": "p50", "start": 709, "end": 712}, {"role": "AtLoc", "text": "nucleus", "start": 725, "end": 732}]}, {"trigger": {"text": "appearance", "start": 647, "end": 657}, "arguments": [{"role": "Theme", "text": "p65", "start": 713, "end": 716}, {"role": "AtLoc", "text": "nucleus", "start": 725, "end": 732}]}, {"trigger": {"text": "released", "start": 1369, "end": 1377}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1408, "end": 1417}]}, {"trigger": {"text": "release", "start": 1468, "end": 1475}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1458, "end": 1467}]}, {"trigger": {"text": "release", "start": 1943, "end": 1950}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1933, "end": 1942}]}], "positive regulation": [{"trigger": {"text": "promoted", "start": 634, "end": 642}, "arguments": [{"role": "Theme", "text": "appearance", "start": 647, "end": 657}]}, {"trigger": {"text": "induced", "start": 940, "end": 947}, "arguments": [{"role": "Theme", "text": "proteolysis", "start": 903, "end": 914}]}, {"trigger": {"text": "treated with", "start": 1343, "end": 1355}, "arguments": [{"role": "Theme", "text": "released", "start": 1369, "end": 1377}]}, {"trigger": {"text": "subsequent", "start": 1922, "end": 1932}, "arguments": [{"role": "Theme", "text": "release", "start": 1943, "end": 1950}]}], "protein catabolism": [{"trigger": {"text": "proteolysis", "start": 903, "end": 914}, "arguments": [{"role": "Theme", "text": "IkappaB-alpha", "start": 889, "end": 902}]}]}}, "schema": []} {"input": "Thymocytes control the CD4 gene differently from mature T lymphocytes. \nWe analyzed the activity of the enhancer, the promoter and the silencer of the human CD4 gene during T cell development using transgenic mice. Immunofluorescence studies on thymic populations of mice carrying transgenes in various combinations of these regulatory DNA elements revealed that thymocytes control the CD4 gene in a different manner than mature peripheral T lymphocytes. The 5'-positive regulatory unit, consisting of the promoter and the 5' enhancer, is already active at the CD4-CD8-double-negative (DN) stage of development. However, its activity becomes lower in the double-positive and a fraction of the CD4+ CD8int/- cell population, indicating that an additional enhancer, located in either the first or the third intron of the CD4 gene, is required for CD4 gene expression in this population. The other studied regulatory element is the minimal CD4 silencer which inhibits CD4 gene expression in peripheral CD8 T lymphocytes. This silencer is inactive in the most immature DN thymocytes, which probably use a distinct silencer mechanism to down-regulate CD4 gene expression. Unexpectedly, the CD4 silencer is also active in CD4+ CD8int/- cells of the thymus, implying that an anti-silencer may be required to resume CD4 expression in this cell population. Altogether, the CD4 gene is regulated by several positive and negative regulatory mechanisms which come into play in a developmentally coordinated manner. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 854, "end": 864}, "arguments": [{"role": "Theme", "text": "CD4", "start": 845, "end": 848}]}, {"trigger": {"text": "expression", "start": 974, "end": 984}, "arguments": [{"role": "Theme", "text": "CD4", "start": 965, "end": 968}]}, {"trigger": {"text": "expression", "start": 1155, "end": 1165}, "arguments": [{"role": "Theme", "text": "CD4", "start": 1146, "end": 1149}]}, {"trigger": {"text": "expression", "start": 1312, "end": 1322}, "arguments": [{"role": "Theme", "text": "CD4", "start": 1308, "end": 1311}]}], "negative regulation": [{"trigger": {"text": "double-negative", "start": 569, "end": 584}, "arguments": [{"role": "Theme", "text": "CD4", "start": 561, "end": 564}]}, {"trigger": {"text": "inhibits", "start": 956, "end": 964}, "arguments": [{"role": "Theme", "text": "expression", "start": 974, "end": 984}]}, {"trigger": {"text": "down-regulate", "start": 1132, "end": 1145}, "arguments": [{"role": "Theme", "text": "expression", "start": 1155, "end": 1165}]}, {"trigger": {"text": "resume", "start": 1301, "end": 1307}, "arguments": [{"role": "Theme", "text": "expression", "start": 1312, "end": 1322}]}, {"trigger": {"text": "regulated", "start": 1376, "end": 1385}, "arguments": [{"role": "Theme", "text": "CD4", "start": 1364, "end": 1367}]}], "positive regulation": [{"trigger": {"text": "double-positive", "start": 655, "end": 670}, "arguments": [{"role": "Theme", "text": "CD4", "start": 561, "end": 564}]}, {"trigger": {"text": "required", "start": 832, "end": 840}, "arguments": [{"role": "Theme", "text": "expression", "start": 854, "end": 864}]}, {"trigger": {"text": "required", "start": 1289, "end": 1297}, "arguments": [{"role": "Theme", "text": "resume", "start": 1301, "end": 1307}]}, {"trigger": {"text": "regulated", "start": 1376, "end": 1385}, "arguments": [{"role": "Theme", "text": "CD4", "start": 1364, "end": 1367}]}], "regulation": [{"trigger": {"text": "control", "start": 11, "end": 18}, "arguments": [{"role": "Theme", "text": "CD4", "start": 23, "end": 26}]}, {"trigger": {"text": "control", "start": 374, "end": 381}, "arguments": [{"role": "Theme", "text": "CD4", "start": 386, "end": 389}]}]}}, "schema": []} {"input": "Triggering of HLA-DR antigens differentially modulates tumor necrosis factor alpha release by B cells at distinct stage of maturation. \nTriggering of HLA class II antigens by the anti-HLA-DR monoclonal antibody (mAb) L243 significantly (P < 0.05) and differentially enhanced the release of tumor necrosis factor alpha (TNF-alpha) by the non-Hodgkin's lymphoma cells Ri-I, Ci-I, and Sc-I, which are at a distinct stage of B-cell differentiation, and by the more mature Burkitt lymphoma cell Raji; in contrast, it did not induce TNF-alpha release by the pre-B leukemia cells Nalm-6 and BV173. TNF-alpha release peaked at 24 h and decreased thereafter, and it was dose dependent and preceded by an increase of TNF-alpha mRNA detectable after 3 h of stimulation with mAb L243. Secreted TNF-alpha mediated the enhancement of nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) binding activity; in fact, the triggering of HLA-DR antigens in the presence of antihuman TNF-alpha-neutralizing antibodies did not upregulate NF-kappa B and AP-1. In contrast, released TNF-alpha was not responsible for the homotypic aggregation of Ri-I, Ci-I, Sc-I, and Raji cells induced by mAb L243, and it did not affect the proliferation of B cells investigated. Altogether, our data demonstrate that: (a) the ability of B cells to release TNF-alpha after triggering of HLA-DR antigens depends on their stage of differentiation; (b) levels of released TNF-alpha seem to correlate with the stage of B-cell maturation but do not correlate with the amounts of cell surface HLA-DR antigens; (c) secreted TNF-alpha regulates the levels of expression of NF-kappa B and AP-1 by an autocrine loop; and (d) intracellular signals mediating TNF-alpha release by B cells are distinct from those regulating homotypic aggregation and proliferation. ", "output": {"json_structures": {"localization": [{"trigger": {"text": "release", "start": 83, "end": 90}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor alpha", "start": 55, "end": 82}]}, {"trigger": {"text": "release", "start": 279, "end": 286}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 319, "end": 328}]}, {"trigger": {"text": "release", "start": 537, "end": 544}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 527, "end": 536}]}, {"trigger": {"text": "release", "start": 601, "end": 608}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 591, "end": 600}]}, {"trigger": {"text": "Secreted", "start": 773, "end": 781}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 782, "end": 791}]}, {"trigger": {"text": "released", "start": 1064, "end": 1072}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1073, "end": 1082}]}, {"trigger": {"text": "release", "start": 1324, "end": 1331}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1332, "end": 1341}]}, {"trigger": {"text": "levels of released", "start": 1425, "end": 1443}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1444, "end": 1453}]}, {"trigger": {"text": "secreted", "start": 1583, "end": 1591}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1592, "end": 1601}]}, {"trigger": {"text": "release", "start": 1732, "end": 1739}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1722, "end": 1731}]}], "positive regulation": [{"trigger": {"text": "enhanced", "start": 266, "end": 274}, "arguments": [{"role": "Theme", "text": "release", "start": 279, "end": 286}]}, {"trigger": {"text": "induce", "start": 520, "end": 526}, "arguments": [{"role": "Theme", "text": "release", "start": 537, "end": 544}]}, {"trigger": {"text": "peaked", "start": 609, "end": 615}, "arguments": [{"role": "Theme", "text": "release", "start": 601, "end": 608}]}, {"trigger": {"text": "preceded", "start": 680, "end": 688}, "arguments": [{"role": "Theme", "text": "release", "start": 601, "end": 608}, {"role": "Cause", "text": "increase", "start": 695, "end": 703}]}, {"trigger": {"text": "increase", "start": 695, "end": 703}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 707, "end": 716}]}, {"trigger": {"text": "after", "start": 1342, "end": 1347}, "arguments": [{"role": "Theme", "text": "release", "start": 1324, "end": 1331}]}, {"trigger": {"text": "mediating", "start": 1712, "end": 1721}, "arguments": [{"role": "Theme", "text": "release", "start": 1732, "end": 1739}]}], "regulation": [{"trigger": {"text": "modulates", "start": 45, "end": 54}, "arguments": [{"role": "Theme", "text": "release", "start": 83, "end": 90}]}]}}, "schema": []} {"input": "Cytomegalovirus immediate early genes upregulate interleukin-6 gene expression. \nBACKGROUND: The immediate early genes (IE) of human cytomegalovirus (CMV) can be expressed in monocytic cells and are known to regulate viral and cellular genes. Interleukin-6 (IL-6) plays a central role in numerous inflammatory and immune processes. Interleukin-6 levels are increased in lung transplant patients clinically diagnosed with CMV pneumonitis. The regulation of IL-6 is dependent on various stimuli that include lipopolysaccharide (LPS), viruses, and other cytokines. These studies examined the ability of CMV IE gene products to modulate IL-6 production. METHODS: THP-1 cells, a monocytic cell line, were transfected with the CMV IE genes. Interleukin-6 protein and IL-6 mRNA were measured in control and CMV immediate early transfected cells. Cotransfection of CMV IE genes and IL-6 chloramphenicol acetyl transferase (CAT) or IL-6 luciferase constructs were used to study IL-6 promoter activity. RESULTS: Interleukin-6 protein and mRNA production were significantly increased in cells transfected with the CMV IE genes and stimulated with LPS compared to LPS-stimulated control cells. Cytomegalovirus IE gene products significantly enhanced LPS stimulation of IL-6 promoter activity in both IL-6 CAT and IL-6 luciferase assays. A deletion construct that contains a NF-kappa B site but is missing the multiple response region demonstrated a continued increase in IL-6 luciferase activity in LPS-stimulated CMV transfected cells. CONCLUSION: Cytomegalovirus immediate early gene products significantly enhanced expression of IL-6 in LPS-stimulated cells. The increase in IL-6 luciferase activity occurs in the absence of the multiple response region, the area of the IL-6 promoter responsive to IL-1, TNF alpha, cyclic amp, and phorbol 12-myristate 13-acetate. The ability of CMV IE gene products to enhance IL-6 production may play an important role in immune inflammatory states associated with CMV infection. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 68, "end": 78}, "arguments": [{"role": "Theme", "text": "interleukin-6", "start": 49, "end": 62}]}, {"trigger": {"text": "production", "start": 638, "end": 648}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 633, "end": 637}]}, {"trigger": {"text": "expression", "start": 1606, "end": 1616}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1620, "end": 1624}]}, {"trigger": {"text": "production", "start": 1908, "end": 1918}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1903, "end": 1907}]}], "positive regulation": [{"trigger": {"text": "upregulate", "start": 38, "end": 48}, "arguments": [{"role": "Theme", "text": "expression", "start": 68, "end": 78}]}, {"trigger": {"text": "increased", "start": 357, "end": 366}, "arguments": [{"role": "Theme", "text": "Interleukin-6", "start": 332, "end": 345}]}, {"trigger": {"text": "increased", "start": 1063, "end": 1072}, "arguments": [{"role": "Theme", "text": "production", "start": 1033, "end": 1043}]}, {"trigger": {"text": "enhanced", "start": 1229, "end": 1237}, "arguments": [{"role": "Theme", "text": "stimulation", "start": 1242, "end": 1253}]}, {"trigger": {"text": "stimulation", "start": 1242, "end": 1253}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1257, "end": 1261}, {"role": "Site", "text": "promoter", "start": 1262, "end": 1270}]}, {"trigger": {"text": "enhanced", "start": 1597, "end": 1605}, "arguments": [{"role": "Theme", "text": "expression", "start": 1606, "end": 1616}]}, {"trigger": {"text": "enhance", "start": 1895, "end": 1902}, "arguments": [{"role": "Theme", "text": "production", "start": 1908, "end": 1918}]}], "regulation": [{"trigger": {"text": "regulation", "start": 442, "end": 452}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 456, "end": 460}]}, {"trigger": {"text": "modulate", "start": 624, "end": 632}, "arguments": [{"role": "Theme", "text": "production", "start": 638, "end": 648}]}, {"trigger": {"text": "responsive", "start": 1776, "end": 1786}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1762, "end": 1766}, {"role": "Site", "text": "promoter", "start": 1767, "end": 1775}, {"role": "Cause", "text": "TNF alpha", "start": 1796, "end": 1805}]}, {"trigger": {"text": "responsive", "start": 1776, "end": 1786}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1762, "end": 1766}, {"role": "Site", "text": "promoter", "start": 1767, "end": 1775}]}], "transcription": [{"trigger": {"text": "production", "start": 1033, "end": 1043}, "arguments": [{"role": "Theme", "text": "Interleukin-6", "start": 1002, "end": 1015}]}]}}, "schema": []} {"input": "Homodimerization of the human interleukin 4 receptor alpha chain induces Cepsilon germline transcripts in B cells in the absence of the interleukin 2 receptor gamma chain. \nThe cytokines interleukin (IL)-4 and IL-13 play a critical role in inducing Cepsilon germline transcripts and IgE isotype switching in human B cells. The IL-4 receptor (IL-4R) in B cells is composed of two chains, the IL-4-binding IL-4Ralpha chain, which is shared with the IL-13R, and the IL-2Rgamma (gammac) chain, which is shared with IL-7R, IL-9R, and IL-15R. IL-4 induces Cepsilon germline transcripts and IgE isotype switching in B cells from patients with gammac chain deficiency. Induction of Cepsilon germline transcripts by IL-4 in B cells that lack the gammac chain may involve signaling via the IL-13R. Alternatively, the IL-4Ralpha chain may transduce intracellular signals that lead to Cepsilon gene transcription independently of its association with other chains. We show that ligand-induced homodimerization of chimeric surface receptors consisting of the extracellular and transmembrane domains of the erythropoietin receptor and of the intracellular domain of IL-4Ralpha induces Janus kinase 1 (Jak1) activation, STAT6 activation, and Cepsilon germline transcripts in human B cell line BJAB. Disruption of the Jak1-binding proline-rich Box1 region of IL-4Ralpha abolished signaling by this chimeric receptor. Furthermore, B cells transfected with a chimeric CD8alpha/IL-4Ralpha receptor, which is expressed on the cell surface as a homodimer, constitutively expressed Cepsilon germline transcripts. These results suggest that homodimerization of the IL-4Ralpha chain is sufficient to transduce Jak1-dependent intracellular signals that lead to IgE isotype switching. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "Homodimerization", "start": 0, "end": 16}, "arguments": [{"role": "Theme", "text": "interleukin 4 receptor alpha chain", "start": 30, "end": 64}]}, {"trigger": {"text": "binding", "start": 396, "end": 403}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 391, "end": 395}, {"role": "Theme2", "text": "IL-4Ralpha chain", "start": 404, "end": 420}]}, {"trigger": {"text": "as a homodimer", "start": 1519, "end": 1533}, "arguments": [{"role": "Theme", "text": "chimeric CD8alpha/IL-4Ralpha receptor", "start": 1441, "end": 1478}]}, {"trigger": {"text": "homodimerization", "start": 1618, "end": 1634}, "arguments": [{"role": "Theme", "text": "IL-4Ralpha chain", "start": 1642, "end": 1658}]}], "gene expression": [{"trigger": {"text": "lack", "start": 728, "end": 732}, "arguments": [{"role": "Theme", "text": "gammac chain", "start": 737, "end": 749}]}, {"trigger": {"text": "transfected", "start": 1422, "end": 1433}, "arguments": [{"role": "Theme", "text": "chimeric CD8alpha/IL-4Ralpha receptor", "start": 1441, "end": 1478}]}, {"trigger": {"text": "expressed", "start": 1489, "end": 1498}, "arguments": [{"role": "Theme", "text": "chimeric CD8alpha/IL-4Ralpha receptor", "start": 1441, "end": 1478}]}], "negative regulation": [{"trigger": {"text": "deficiency", "start": 649, "end": 659}, "arguments": [{"role": "Theme", "text": "gammac chain", "start": 636, "end": 648}]}], "positive regulation": [{"trigger": {"text": "induces", "start": 1163, "end": 1170}, "arguments": [{"role": "Theme", "text": "activation", "start": 1193, "end": 1203}]}, {"trigger": {"text": "activation", "start": 1193, "end": 1203}, "arguments": [{"role": "Theme", "text": "Jak1", "start": 1187, "end": 1191}]}, {"trigger": {"text": "transfected", "start": 1422, "end": 1433}, "arguments": [{"role": "Theme", "text": "transfected", "start": 1422, "end": 1433}]}]}}, "schema": []} {"input": "Characterization of peripheral blood T-lymphocytes transduced with HTLV-I Tax mutants with different trans-activating phenotypes. \nTax1, a transcriptional trans-activator of the Human T-cell leukemia virus type I (HTLV-I), induces the expression of many cellular genes through interaction with at least three distinct cellular transcription factors; CREB/ATF, NF-kappaB, and SRF. This Tax1-induced activation of cellular genes is considered to be a critical event in T-cell transformation by HTLV-I. To elucidate the role of each Tax1-inducible transcriptional pathway in T-cell transformation, we introduced Tax1 mutants with different trans-activating phenotypes into peripheral blood lymphocytes (PBL) by retroviral vectors. Analysis of these PBLs revealed that activation of the NF-kappaB pathway is sufficient to promote the growth response to IL-2. However, for the clonal expansion of CD4+ T-cells, which is a characteristic result of HTLV-I infection, activation of the CREB/ATF and SRF pathways is also required. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 277, "end": 288}, "arguments": [{"role": "Theme", "text": "Tax1", "start": 131, "end": 135}, {"role": "Theme2", "text": "SRF", "start": 375, "end": 378}]}, {"trigger": {"text": "interaction", "start": 277, "end": 288}, "arguments": [{"role": "Theme", "text": "Tax1", "start": 131, "end": 135}]}], "gene expression": [{"trigger": {"text": "transduced", "start": 51, "end": 61}, "arguments": [{"role": "Theme", "text": "Tax", "start": 74, "end": 77}]}, {"trigger": {"text": "introduced", "start": 598, "end": 608}, "arguments": [{"role": "Theme", "text": "Tax1", "start": 609, "end": 613}]}], "positive regulation": [{"trigger": {"text": "transduced", "start": 51, "end": 61}, "arguments": [{"role": "Theme", "text": "transduced", "start": 51, "end": 61}]}, {"trigger": {"text": "introduced", "start": 598, "end": 608}, "arguments": [{"role": "Theme", "text": "introduced", "start": 598, "end": 608}]}]}}, "schema": []} {"input": "Involvement of the N-terminal region of the human mineralocorticoid receptor hormone-binding domain in agonist and antagonist binding as revealed by a new monoclonal antibody. \nTo gain a better understanding of the mechanism of binding to the human mineralocorticoid receptor (hMR), we developed a new monoclonal antibody (mAb) raised against the hormone-binding domain (HBD). For this purpose, mice were immunized with a fusion protein including the sequence Thr729-Lys984 of hMR. After ELISA screening, mAb 18C7 was selected for its specificity towards the HBD. This antibody recognized both the denatured and native MR forms, as well as the hetero-oligomeric MR form and the transformed MR state. By using several HBD subfragments, the mAb 18C7 epitope was located in the N-terminal region of the HBD from Thr729 to Leu765. We then studied the effect of the antibody on aldosterone and progesterone binding to the hMR. When 18C7 was incubated with liganded MR, it was able to partly displace (20%) the hormone from its binding site. When 18C7 was incubated with MR before aldosterone or progesterone, the antibody inhibited 75-80% of the binding. The effect of 18C7 on the binding was similar with both hormones. A sucrose gradient analysis indicated the simultaneous presence of two kinds of receptor complexes: the steroid-MR complex and the antibody-MR complex. After its associated proteins, especially the heat-shock protein hsp90, had been cross-linked with the hMR by dimethylpimelimidate, 18C7 was still able to react with the receptor. Our results indicated that the epitope recognized by 18C7 was directly implicated in hormone binding. The lack of steroid binding of HBD mutants with the Thr729-Leu765 sequence deleted [Jalaguier, Mesnier, Leger and Auzou (1996) J.Steroid Biochem.Mol.Biol.57, 43-50] supports this hypothesis. Because of the similar behaviours of aldosterone and progesterone, we conclude that the N-terminal Thr729-Leu765 region of the HBD is similarly involved in the binding of both hormones. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 126, "end": 133}, "arguments": [{"role": "Theme", "text": "mineralocorticoid receptor", "start": 50, "end": 76}]}, {"trigger": {"text": "binding", "start": 228, "end": 235}, "arguments": [{"role": "Theme", "text": "MR", "start": 278, "end": 280}]}, {"trigger": {"text": "recognized", "start": 578, "end": 588}, "arguments": [{"role": "Theme", "text": "MR", "start": 619, "end": 621}]}, {"trigger": {"text": "recognized", "start": 578, "end": 588}, "arguments": [{"role": "Theme", "text": "MR", "start": 662, "end": 664}]}, {"trigger": {"text": "binding", "start": 902, "end": 909}, "arguments": [{"role": "Theme", "text": "MR", "start": 918, "end": 920}]}, {"trigger": {"text": "liganded", "start": 951, "end": 959}, "arguments": [{"role": "Theme", "text": "MR", "start": 960, "end": 962}]}, {"trigger": {"text": "binding", "start": 1141, "end": 1148}, "arguments": [{"role": "Theme", "text": "MR", "start": 1065, "end": 1067}]}, {"trigger": {"text": "binding", "start": 1176, "end": 1183}, "arguments": [{"role": "Theme", "text": "MR", "start": 1065, "end": 1067}]}, {"trigger": {"text": "complex", "start": 1331, "end": 1338}, "arguments": [{"role": "Theme", "text": "MR", "start": 1328, "end": 1330}]}, {"trigger": {"text": "complex", "start": 1359, "end": 1366}, "arguments": [{"role": "Theme", "text": "MR", "start": 1356, "end": 1358}]}, {"trigger": {"text": "cross-linked", "start": 1449, "end": 1461}, "arguments": [{"role": "Theme", "text": "hsp90", "start": 1433, "end": 1438}, {"role": "Theme2", "text": "hMR", "start": 1471, "end": 1474}]}, {"trigger": {"text": "binding", "start": 1641, "end": 1648}, "arguments": [{"role": "Theme", "text": "MR", "start": 1065, "end": 1067}]}, {"trigger": {"text": "binding", "start": 2001, "end": 2008}, "arguments": [{"role": "Theme", "text": "MR", "start": 1065, "end": 1067}]}], "negative regulation": [{"trigger": {"text": "inhibited", "start": 1117, "end": 1126}, "arguments": [{"role": "Theme", "text": "binding", "start": 1141, "end": 1148}]}, {"trigger": {"text": "still able to", "start": 1509, "end": 1522}, "arguments": [{"role": "Cause", "text": "cross-linked", "start": 1449, "end": 1461}, {"role": "Theme", "text": "react", "start": 1523, "end": 1528}]}], "regulation": [{"trigger": {"text": "effect", "start": 847, "end": 853}, "arguments": [{"role": "Theme", "text": "binding", "start": 902, "end": 909}]}, {"trigger": {"text": "effect", "start": 1154, "end": 1160}, "arguments": [{"role": "Theme", "text": "binding", "start": 1176, "end": 1183}]}, {"trigger": {"text": "react", "start": 1523, "end": 1528}, "arguments": [{"role": "Theme", "text": "hMR", "start": 1471, "end": 1474}]}]}}, "schema": []} {"input": "Abnormal T lymphocyte development induced by targeted overexpression of IkappaB alpha. \nA role in thymic maturation for factors of the NF-kappaB family has long been suspected, but not yet proven. Transgenic mice with a lymphocyte-specific defect in NF-kappaB activation were produced by targeted expression of human IkappaB alpha. The thymic cellularity of these mice was significantly decreased. The proportion of mature, TCRhigh thymocytes of the alphabeta lineage was reduced, and the remaining TCRhigh population contained an unusually high proportion of double-positive cells. This defect in maturation resulted in a transgene dose-dependent reduction in peripheral T lymphocytes, with the CD8 lineage being more severely affected. These data provide direct evidence for the involvement of NF-kappaB/Rel family proteins in late stages of T lymphocyte development, coincident with positive and negative selection. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 297, "end": 307}, "arguments": [{"role": "Theme", "text": "IkappaB alpha", "start": 317, "end": 330}]}], "positive regulation": [{"trigger": {"text": "overexpression", "start": 54, "end": 68}, "arguments": [{"role": "Theme", "text": "IkappaB alpha", "start": 72, "end": 85}]}]}}, "schema": []} {"input": "Sequential development of structural and functional alterations in T cells from tumor-bearing mice. \nThe TCR alpha beta or -gamma delta chains bind the peptide ligand, whereas the associated CD3 deltaepsilongamma and TCR zeta subunits couple the TCR to intracellular signal transduction components. Recently, several groups have described marked alterations in signal transduction elements in T cells from cancer patients or in mice bearing tumor for a few weeks (>26 days). The sequence in which these alterations develop is unknown. The aim of this study was to explore the kinetics of the development of alterations in signal transduction molecules (TCR zeta chain, NF kappaB family proteins, and tyrosine kinase p56(lck)) in mice bearing MC38 colon adenocarcinoma. The results demonstrate that alterations in NF kappaB family proteins, specifically the failure of p65 translocation to the nucleus, occur earlier and more frequently than the decrease in zeta-chain. These defects are paralleled by an impaired ability to produce Th1 cytokines (IL-2 and IFN-gamma). These initial changes are followed by the eventual loss of TCR zeta chain and p56(lck) and a marked decrease in cytotoxic function. An increased rate of lysosomal degradation is one of the mechanisms responsible for the loss of zeta-chain. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "produce", "start": 1024, "end": 1031}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1047, "end": 1051}]}, {"trigger": {"text": "produce", "start": 1024, "end": 1031}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 1056, "end": 1065}]}], "localization": [{"trigger": {"text": "translocation", "start": 872, "end": 885}, "arguments": [{"role": "Theme", "text": "p65", "start": 868, "end": 871}, {"role": "ToLoc", "text": "nucleus", "start": 893, "end": 900}]}], "negative regulation": [{"trigger": {"text": "failure", "start": 857, "end": 864}, "arguments": [{"role": "Theme", "text": "translocation", "start": 872, "end": 885}]}, {"trigger": {"text": "decrease", "start": 945, "end": 953}, "arguments": [{"role": "Theme", "text": "TCR zeta chain", "start": 653, "end": 667}]}, {"trigger": {"text": "impaired ability", "start": 1004, "end": 1020}, "arguments": [{"role": "Theme", "text": "produce", "start": 1024, "end": 1031}]}, {"trigger": {"text": "loss", "start": 1119, "end": 1123}, "arguments": [{"role": "Theme", "text": "TCR zeta chain", "start": 1127, "end": 1141}]}, {"trigger": {"text": "loss", "start": 1119, "end": 1123}, "arguments": [{"role": "Theme", "text": "p56(lck)", "start": 1146, "end": 1154}]}], "positive regulation": [{"trigger": {"text": "occur", "start": 902, "end": 907}, "arguments": [{"role": "Theme", "text": "failure", "start": 857, "end": 864}]}, {"trigger": {"text": "paralleled", "start": 987, "end": 997}, "arguments": [{"role": "Cause", "text": "failure", "start": 857, "end": 864}, {"role": "Theme", "text": "impaired ability", "start": 1004, "end": 1020}]}, {"trigger": {"text": "followed", "start": 1094, "end": 1102}, "arguments": [{"role": "Cause", "text": "impaired ability", "start": 1004, "end": 1020}, {"role": "Theme", "text": "loss", "start": 1119, "end": 1123}]}, {"trigger": {"text": "followed", "start": 1094, "end": 1102}, "arguments": [{"role": "Cause", "text": "failure", "start": 857, "end": 864}, {"role": "Theme", "text": "loss", "start": 1119, "end": 1123}]}]}}, "schema": []} {"input": "An isotype-specific activator of major histocompatibility complex (MHC) class II genes that is independent of class II transactivator. \nPatients with one type of major histocompatibility complex class II combined immunodeficiency have mutations in a gene termed class II transactivator (CIITA), which coordinately controls the transcription of the three major human class II genes, HLA-DR, -DQ, and -DP. However, the experimentally derived B-lymphoblastoid cell line, clone 13, expresses high levels of HLADQ in the absence of HLA-DR and HLA-DP, despite its mapping by complementation analysis to this group. It was possible that one of the clone 13 CIITA alleles bore a mutation that allowed HLA-DQ, but not HLA-DR or -DP transcription. Alternatively, another factor, distinct from CIITA, might control HLA-DQ expression. We report here that ectopic expression of CIITA cDNAs derived by reverse transcriptase polymerase chain reaction from clone 13 do not restore expression of HLA-DQ in another CIITA-deficient cell line, RJ2.2.5. In addition, no CIITA protein is detectable in clone 13 nuclear extracts. In contrast, somatic cell fusion between clone 13 and RJ2.2.5 restored expression of the HLA-DQ haplotype encoded by the RJ2.2.5 DQB gene. Taken together, these data demonstrate the existence of an HLA-DQ isotype-specific trans-acting factor, which functions independently of CIITA. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 851, "end": 861}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 865, "end": 870}]}, {"trigger": {"text": "detectable", "start": 1066, "end": 1076}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 1049, "end": 1054}]}]}}, "schema": []} {"input": "Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets. \nBACKGROUND: Activated platelets tether and activate myeloid leukocytes. To investigate the potential relevance of this mechanism in acute myocardial infarction (AMI), we examined cytokine induction by leukocyte-platelet adhesion and the occurrence of leukocyte-platelet conjugates in patients with AMI. METHODS AND RESULTS: We obtained peripheral venous blood samples in 20 patients with AMI before and daily for 5 days after direct percutaneous transluminal coronary angioplasty (PTCA) and in 20 patients undergoing elective PTCA. Throughout the study period, CD41 immunofluorescence of leukocytes (flow cytometry) revealed increased leukocyte-platelet adhesion in patients with AMI compared with control patients (mean +/- SE of fluorescence [channels] before PTCA: 77 +/- 16 versus 35 +/- 9; P = .003). In vitro, thrombin-stimulated fixed platelets bound to neutrophils and monocytes. Within 2 hours, this resulted in increased mRNA for interleukin (IL),1 beta, IL-8, and monocyte chemoattractant protein (MCP)-1 in unfractionated leukocytes. After 4 hours, IL-1 beta and IL-8 concentration of the cell-free supernatant had increased by 268 +/- 36% and 210 +/- 7%, respectively, and cellular MCP-1 content had increased by 170 +/- 8%. Addition of activated platelets to adherent monocytes had a similar effect and was associated with nuclear factor-kappa B activation. Inhibition of binding by anti-P selectin antibodies reduced the effect of activated platelets on cytokine production. CONCLUSIONS: In patients with AMI, leukocyte-platelet adhesion is increased. Binding of activated platelets induces IL-1 beta, IL-8, and MCP-1 in leukocytes. Our findings suggest that leukocyte-platelet adhesion contributes to the regulation of inflammatory responses in AMI. ", "output": {"json_structures": {"positive regulation": [{"trigger": {"text": "resulted in increased", "start": 1002, "end": 1023}, "arguments": [{"role": "Theme", "text": "interleukin (IL),1 beta", "start": 1033, "end": 1056}]}, {"trigger": {"text": "resulted in increased", "start": 1002, "end": 1023}, "arguments": [{"role": "Theme", "text": "IL-8", "start": 1058, "end": 1062}]}, {"trigger": {"text": "resulted in increased", "start": 1002, "end": 1023}, "arguments": [{"role": "Theme", "text": "monocyte chemoattractant protein (MCP)-1", "start": 1068, "end": 1108}]}, {"trigger": {"text": "increased", "start": 1220, "end": 1229}, "arguments": [{"role": "Theme", "text": "IL-1 beta", "start": 1154, "end": 1163}]}, {"trigger": {"text": "increased", "start": 1220, "end": 1229}, "arguments": [{"role": "Theme", "text": "IL-8", "start": 1168, "end": 1172}]}, {"trigger": {"text": "increased", "start": 1306, "end": 1315}, "arguments": [{"role": "Theme", "text": "MCP-1", "start": 1288, "end": 1293}]}, {"trigger": {"text": "induces", "start": 1691, "end": 1698}, "arguments": [{"role": "Theme", "text": "IL-1 beta", "start": 1699, "end": 1708}]}, {"trigger": {"text": "induces", "start": 1691, "end": 1698}, "arguments": [{"role": "Theme", "text": "IL-8", "start": 1710, "end": 1714}]}, {"trigger": {"text": "induces", "start": 1691, "end": 1698}, "arguments": [{"role": "Theme", "text": "MCP-1", "start": 1720, "end": 1725}]}]}}, "schema": []} {"input": "The class II trans-activator CIITA interacts with the TBP-associated factor TAFII32. \nThe class II trans- activator (CIITA) is the main transcriptional co-activator for the expression of MHC class II proteins. Its N-terminal 125 amino acids function as an independent transcriptional activation domain. Analyses of the primary amino acid sequence of the activation domain predict the presence of three alpha-helices, each with a high proportion of acidic residues. Using site-directed mutagenesis, we found that two of these predicted alpha-helices are required for full transcriptional activation by CIITA. Moreover, a CIITA protein in which both functional alpha-helices have been deleted displays a dominant negative phenotype. This activation domain of CIITA interacts with the 32 kDa subunit of the general transcription complex TFIID, TAFII32. Decreased transcriptional activation by N-terminal deletions of CIITA is correlated directly with their reduced binding to TAFII32. We conclude that interactions between TAFII32 and CIITA are responsible for activation of class II genes. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interacts", "start": 35, "end": 44}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 29, "end": 34}, {"role": "Theme2", "text": "TAFII32", "start": 76, "end": 83}]}, {"trigger": {"text": "interacts", "start": 763, "end": 772}, "arguments": [{"role": "Site", "text": "activation domain", "start": 736, "end": 753}, {"role": "Theme", "text": "CIITA", "start": 757, "end": 762}, {"role": "Theme2", "text": "TAFII32", "start": 841, "end": 848}]}, {"trigger": {"text": "binding", "start": 962, "end": 969}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 914, "end": 919}, {"role": "Theme2", "text": "TAFII32", "start": 973, "end": 980}]}, {"trigger": {"text": "interactions", "start": 999, "end": 1011}, "arguments": [{"role": "Theme", "text": "TAFII32", "start": 1020, "end": 1027}, {"role": "Theme2", "text": "CIITA", "start": 1032, "end": 1037}]}], "negative regulation": [{"trigger": {"text": "displays a dominant negative phenotype", "start": 691, "end": 729}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 620, "end": 625}]}, {"trigger": {"text": "reduced", "start": 954, "end": 961}, "arguments": [{"role": "Theme", "text": "binding", "start": 962, "end": 969}]}]}}, "schema": []} {"input": "Lipopolysaccharide induction of the tumor necrosis factor-alpha promoter in human monocytic cells. Regulation by Egr-1, c-Jun, and NF-kappaB transcription factors. \nBiosynthesis of tumor necrosis factor-alpha (TNF-alpha) is predominantly by cells of the monocytic lineage. This study examined the role of various cis-acting regulatory elements in the lipopolysaccharide (LPS) induction of the human TNF-alpha promoter in cells of monocytic lineage. Functional analysis of monocytic THP-1 cells transfected with plasmids containing various lengths of TNF-alpha promoter localized enhancer elements in a region (-182 to -37 base pairs (bp)) that were required for optimal transcription of the TNF-alpha gene in response to LPS. Two regions were identified: region I (-182 to -162 bp) contained an overlapping Sp1/Egr-1 site, and region II (-119 to -88) contained CRE and NF-kappaB (designated kappaB3) sites. In unstimulated THP-1, CRE-binding protein and, to a lesser extent, c-Jun complexes were found to bind to the CRE site. LPS stimulation increased the binding of c-Jun-containing complexes. In addition, LPS stimulation induced the binding of cognate nuclear factors to the Egr-1 and kappaB3 sites, which were identified as Egr-1 and p50/p65, respectively. The CRE and kappaB3 sites in region II together conferred strong LPS responsiveness to a heterologous promoter, whereas individually they failed to provide transcriptional activation. Furthermore, increasing the spacing between the CRE and the kappaB3 sites completely abolished LPS induction, suggesting a cooperative interaction between c-Jun complexes and p50/p65. These studies indicate that maximal LPS induction of the TNF-alpha promoter is mediated by concerted participation of at least two separate cis-acting regulatory elements. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 1005, "end": 1009}, "arguments": [{"role": "Theme", "text": "c-Jun", "start": 975, "end": 980}]}, {"trigger": {"text": "binding", "start": 1057, "end": 1064}, "arguments": [{"role": "Theme", "text": "c-Jun", "start": 1068, "end": 1073}]}, {"trigger": {"text": "binding", "start": 1137, "end": 1144}, "arguments": [{"role": "Theme", "text": "Egr-1", "start": 1229, "end": 1234}]}, {"trigger": {"text": "binding", "start": 1137, "end": 1144}, "arguments": [{"role": "Theme", "text": "p50", "start": 1239, "end": 1242}]}, {"trigger": {"text": "binding", "start": 1137, "end": 1144}, "arguments": [{"role": "Theme", "text": "p65", "start": 1243, "end": 1246}]}], "gene expression": [{"trigger": {"text": "Biosynthesis", "start": 165, "end": 177}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 210, "end": 219}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 19, "end": 28}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor-alpha", "start": 36, "end": 63}, {"role": "Site", "text": "promoter", "start": 64, "end": 72}]}, {"trigger": {"text": "induction", "start": 376, "end": 385}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 399, "end": 408}, {"role": "Site", "text": "promoter", "start": 409, "end": 417}]}, {"trigger": {"text": "required", "start": 649, "end": 657}, "arguments": [{"role": "Theme", "text": "in response to", "start": 706, "end": 720}]}, {"trigger": {"text": "in response to", "start": 706, "end": 720}, "arguments": [{"role": "Theme", "text": "transcription", "start": 670, "end": 683}]}, {"trigger": {"text": "increased", "start": 1043, "end": 1052}, "arguments": [{"role": "Theme", "text": "binding", "start": 1057, "end": 1064}]}, {"trigger": {"text": "induction", "start": 1670, "end": 1679}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1687, "end": 1696}, {"role": "Site", "text": "promoter", "start": 1697, "end": 1705}]}], "regulation": [{"trigger": {"text": "Regulation", "start": 99, "end": 109}, "arguments": [{"role": "Theme", "text": "induction", "start": 19, "end": 28}, {"role": "Cause", "text": "Egr-1", "start": 113, "end": 118}]}, {"trigger": {"text": "Regulation", "start": 99, "end": 109}, "arguments": [{"role": "Theme", "text": "induction", "start": 19, "end": 28}, {"role": "Cause", "text": "c-Jun", "start": 120, "end": 125}]}, {"trigger": {"text": "role", "start": 297, "end": 301}, "arguments": [{"role": "Theme", "text": "induction", "start": 376, "end": 385}]}, {"trigger": {"text": "mediated", "start": 1709, "end": 1717}, "arguments": [{"role": "Theme", "text": "induction", "start": 1670, "end": 1679}]}], "transcription": [{"trigger": {"text": "transcription", "start": 670, "end": 683}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 691, "end": 700}]}]}}, "schema": []} {"input": "Involvement of Rel, Fos, and Jun proteins in binding activity to the IL-2 promoter CD28 response element/AP-1 sequence in human T cells. \nCD28 is an important costimulatory molecule in the activation of human T cells. Costimulation of T cells through both the Ag receptor and CD28 leads to high level IL-2 production, which is vital to the development of an immune response in vivo. Previous reports have suggested the CD28 stimulation contributes to the activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including AP-1 and nuclear factor-kappaB (NF-kappaB)/Rel family members as well as an uncharacterized transcription factor called CD28 response complex. While several lines of investigation have suggested that NF-kappaB/Rel family members make up the CD28 response complex transcription factor, other work has not supported this conclusion. Recent studies suggest that the CD28 response element (CD28RE) does not function independently but works instead in conjunction with the adjacent promoter proximal AP-1-binding site and this hypothesis is confirmed here. Also in the current study, binding activity to the CD28RE/AP-1 sequence of the IL-2 promoter is evaluated. Although four specific complexes can be detected binding to this sequence, only one of these complexes is specific for both the CD28RE and the adjacent AP-1 site. Of the NF-kappaB/Rel family members tested, this CD28RE/AP-1-specific complex contains predominantly c-Rel, despite the fact that both p50 and RelA can efficiently bind to the CD28RE. c-Fos and c-Jun are also found in this CD28RE/AP-1-specific complex. These data indicate that functional complexes encompassing both the CD28RE and the AP-1-binding sites influence IL-2 promoter activity in CD28-costimulated T cells. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding activity", "start": 1140, "end": 1156}, "arguments": [{"role": "Site", "text": "CD28RE/AP-1 sequence", "start": 1164, "end": 1184}, {"role": "Theme", "text": "IL-2", "start": 1192, "end": 1196}]}, {"trigger": {"text": "binding", "start": 1269, "end": 1276}, "arguments": [{"role": "Site", "text": "CD28RE/AP-1 sequence", "start": 1164, "end": 1184}, {"role": "Theme", "text": "IL-2", "start": 1192, "end": 1196}]}, {"trigger": {"text": "bind", "start": 1547, "end": 1551}, "arguments": [{"role": "Theme", "text": "p50", "start": 1518, "end": 1521}]}, {"trigger": {"text": "bind", "start": 1547, "end": 1551}, "arguments": [{"role": "Theme", "text": "RelA", "start": 1526, "end": 1530}]}], "gene expression": [{"trigger": {"text": "production", "start": 306, "end": 316}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 301, "end": 305}]}], "positive regulation": [{"trigger": {"text": "leads", "start": 281, "end": 286}, "arguments": [{"role": "Theme", "text": "high level", "start": 290, "end": 300}]}, {"trigger": {"text": "high level", "start": 290, "end": 300}, "arguments": [{"role": "Theme", "text": "production", "start": 306, "end": 316}]}, {"trigger": {"text": "contributes", "start": 436, "end": 447}, "arguments": [{"role": "Theme", "text": "activation", "start": 455, "end": 465}]}, {"trigger": {"text": "activation", "start": 455, "end": 465}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 473, "end": 477}, {"role": "Site", "text": "promoter", "start": 478, "end": 486}]}], "regulation": [{"trigger": {"text": "influence", "start": 1738, "end": 1747}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1748, "end": 1752}, {"role": "Site", "text": "promoter", "start": 1753, "end": 1761}]}]}}, "schema": []} {"input": "c-Myb and Ets proteins synergize to overcome transcriptional repression by ZEB. \nThe Zfh family of zinc finger/homeodomain proteins was first identified in Drosophila where it is required for differentiation of tissues such as the central nervous system and muscle. ZEB, a vertebrate homolog of Zfh-1, binds a subset of E boxes and blocks myogenesis through transcriptional repression of muscle genes. We present evidence here that ZEB also has an important role in controlling hematopoietic gene transcription. Two families of transcription factors that are required for normal hematopoiesis are c-Myb and Ets. These factors act synergistically to activate transcription, and this synergy is required for transcription of at least several important hematopoietic genes. ZEB blocks the activity of c-Myb and Ets individually, but together the factors synergize to resist this repression. Such repression imposes a requirement for both c-Myb and Ets for transcriptional activity, providing one explanation for why synergy between these factors is important. The balance between repression by ZEB and transcriptional activation by c-Myb/Ets provides a flexible regulatory mechanism for controlling gene expression in hematopoietic cells. We demonstrate that one target of this positive/negative regulation in vivo is the alpha4 integrin, which play a key role in normal hematopoiesis and function of mature leukocytes. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 302, "end": 307}, "arguments": [{"role": "Theme", "text": "ZEB", "start": 266, "end": 269}]}], "negative regulation": [{"trigger": {"text": "blocks", "start": 775, "end": 781}, "arguments": [{"role": "Cause", "text": "ZEB", "start": 771, "end": 774}, {"role": "Theme", "text": "c-Myb", "start": 798, "end": 803}]}, {"trigger": {"text": "synergize to resist", "start": 851, "end": 870}, "arguments": [{"role": "Theme", "text": "blocks", "start": 775, "end": 781}, {"role": "Cause", "text": "c-Myb", "start": 798, "end": 803}]}], "regulation": [{"trigger": {"text": "positive/negative regulation", "start": 1275, "end": 1303}, "arguments": [{"role": "Theme", "text": "alpha4 integrin", "start": 1319, "end": 1334}]}]}}, "schema": []} {"input": "GABP factors bind to a distal interleukin 2 (IL-2) enhancer and contribute to c-Raf-mediated increase in IL-2 induction. \nTriggering of the T-cell receptor-CD3 complex activates two major signal cascades in T lymphocytes, (i) Ca2+-dependent signal cascades and (ii) protein kinase cascades. Both signal cascades contribute to the induction of the interleukin 2 (IL-2) gene during T-cell activation. Prominent protein kinase cascades are those that activate mitogen-activated protein (MAP) kinases. We show here that c-Raf, which is at the helm of the classic MAP-Erk cascade, contributes to IL-2 induction through a distal enhancer element spanning the nucleotides from positions -502 to -413 in front of the transcriptional start site of the IL-2 gene. Induction of this distal IL-2 enhancer differs from induction of the proximal IL-2 promoter-enhancer, since it is induced by phorbol esters alone and independent from Ca2+ signals. In DNA-protein binding studies, we detected the binding of transcription factors GABP alpha and -beta to a dyad symmetry element (DSE) of the distal enhancer, which is formed by palindromic binding sites of Ets-like factors. Introduction of point mutations suppressing GABP binding to the DSE interfered with the induction of the distal enhancer and the entire IL-2 promoter-enhancer, while overexpression of both GABP factors enhanced the IL-2 promoter-enhancer induction. Overexpression of BXB, a constitutive active version of c-Raf, and of further members of the Ras-Raf-Erk signal cascade exerted an increase of GABP-mediated promoter-enhancer induction. In conjunction with previously published data on c-Raf-induced phosphorylation of GABP factors (E.Flory, A. Hoffmeyer, U.Smola, U.R.Rapp, and J.T.Bruder, J.Virol.70:2260- 2268, 1996), these results indicate a contribution of GABP factors to the Raf-mediated enhancement of IL-2 induction during T-cell activation. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 13, "end": 17}, "arguments": [{"role": "Theme", "text": "interleukin 2", "start": 30, "end": 43}, {"role": "Site", "text": "enhancer", "start": 51, "end": 59}]}, {"trigger": {"text": "binding", "start": 983, "end": 990}, "arguments": [{"role": "Theme", "text": "GABP alpha", "start": 1016, "end": 1026}]}, {"trigger": {"text": "binding", "start": 983, "end": 990}, "arguments": [{"role": "Theme", "text": "-beta", "start": 1031, "end": 1036}]}], "gene expression": [{"trigger": {"text": "overexpression", "start": 1326, "end": 1340}, "arguments": [{"role": "Theme", "text": "GABP alpha", "start": 1016, "end": 1026}]}, {"trigger": {"text": "overexpression", "start": 1326, "end": 1340}, "arguments": [{"role": "Theme", "text": "-beta", "start": 1031, "end": 1036}]}, {"trigger": {"text": "Overexpression", "start": 1409, "end": 1423}, "arguments": [{"role": "Theme", "text": "BXB", "start": 1427, "end": 1430}]}], "negative regulation": [{"trigger": {"text": "interfered", "start": 1228, "end": 1238}, "arguments": [{"role": "Theme", "text": "induction", "start": 1248, "end": 1257}]}], "positive regulation": [{"trigger": {"text": "increase", "start": 93, "end": 101}, "arguments": [{"role": "Cause", "text": "c-Raf", "start": 78, "end": 83}, {"role": "Theme", "text": "induction", "start": 110, "end": 119}]}, {"trigger": {"text": "induction", "start": 110, "end": 119}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 105, "end": 109}]}, {"trigger": {"text": "induction", "start": 330, "end": 339}, "arguments": [{"role": "Theme", "text": "(IL-2", "start": 361, "end": 366}]}, {"trigger": {"text": "induction", "start": 596, "end": 605}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 591, "end": 595}]}, {"trigger": {"text": "induction", "start": 806, "end": 815}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 832, "end": 836}, {"role": "Site", "text": "promoter-enhancer", "start": 837, "end": 854}]}, {"trigger": {"text": "induced", "start": 868, "end": 875}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 832, "end": 836}, {"role": "Site", "text": "promoter-enhancer", "start": 837, "end": 854}]}, {"trigger": {"text": "independent", "start": 904, "end": 915}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 832, "end": 836}, {"role": "Site", "text": "promoter-enhancer", "start": 837, "end": 854}]}, {"trigger": {"text": "induction", "start": 1248, "end": 1257}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1296, "end": 1300}, {"role": "Site", "text": "promoter-enhancer", "start": 1301, "end": 1318}]}, {"trigger": {"text": "overexpression", "start": 1326, "end": 1340}, "arguments": [{"role": "Theme", "text": "overexpression", "start": 1326, "end": 1340}]}, {"trigger": {"text": "enhanced", "start": 1362, "end": 1370}, "arguments": [{"role": "Cause", "text": "overexpression", "start": 1326, "end": 1340}, {"role": "Theme", "text": "induction", "start": 1398, "end": 1407}]}, {"trigger": {"text": "induction", "start": 1398, "end": 1407}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1375, "end": 1379}, {"role": "Site", "text": "promoter-enhancer", "start": 1380, "end": 1397}]}, {"trigger": {"text": "Overexpression", "start": 1409, "end": 1423}, "arguments": [{"role": "Theme", "text": "Overexpression", "start": 1409, "end": 1423}]}, {"trigger": {"text": "enhancement", "start": 1853, "end": 1864}, "arguments": [{"role": "Theme", "text": "induction", "start": 1873, "end": 1882}]}, {"trigger": {"text": "induction", "start": 1873, "end": 1882}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1868, "end": 1872}]}], "regulation": [{"trigger": {"text": "contribute", "start": 64, "end": 74}, "arguments": [{"role": "Theme", "text": "increase", "start": 93, "end": 101}]}, {"trigger": {"text": "contribute", "start": 312, "end": 322}, "arguments": [{"role": "Theme", "text": "induction", "start": 330, "end": 339}]}, {"trigger": {"text": "contribution", "start": 1804, "end": 1816}, "arguments": [{"role": "Theme", "text": "enhancement", "start": 1853, "end": 1864}]}]}}, "schema": []} {"input": "Genomic organization, sequence, and transcriptional regulation of the human eotaxin gene. \nEotaxin is an eosinophil specific beta-chemokine assumed to be involved in eosinophilic inflammatory diseases such as atopic dermatitis, allergic rhinitis, asthma and parasitic infections. Its expression is stimulus- and cell-specific. We here describe the genomic organisation (3 exons of 132, 112 and 542 bp and 2 introns of 1211 and 378 bp) and sequence including 3 kb of DNA from the immediate 5' upstream region of the human eotaxin gene. Among the regulatory promoter elements potentially regulating eotaxin gene expression and/or mediating the effects of anti-inflammatory drugs we identified consensus sequences known to interact with nuclear factors like NF-IL6, AP-1, a NF-kappa-B like consensus sequence and gamma-interferon- as well as glucocorticoid response elements. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interact", "start": 720, "end": 728}, "arguments": [{"role": "Theme", "text": "NF-IL6", "start": 755, "end": 761}]}], "gene expression": [{"trigger": {"text": "expression", "start": 284, "end": 294}, "arguments": [{"role": "Theme", "text": "Eotaxin", "start": 91, "end": 98}]}, {"trigger": {"text": "expression", "start": 610, "end": 620}, "arguments": [{"role": "Theme", "text": "eotaxin", "start": 597, "end": 604}]}], "regulation": [{"trigger": {"text": "Genomic organization", "start": 0, "end": 20}, "arguments": [{"role": "Theme", "text": "eotaxin", "start": 76, "end": 83}]}, {"trigger": {"text": "transcriptional regulation", "start": 36, "end": 62}, "arguments": [{"role": "Theme", "text": "eotaxin", "start": 76, "end": 83}]}, {"trigger": {"text": "regulating", "start": 586, "end": 596}, "arguments": [{"role": "Theme", "text": "expression", "start": 610, "end": 620}]}]}}, "schema": []} {"input": "Dysregulation of monocytic nuclear factor-kappa B by oxidized low-density lipoprotein. \nNuclear factor-kappa B (NF-kappa B)/Rel transcription factors may be involved in atherosclerosis, as is suggested by the presence of activated NF-kappa B in human atherosclerotic lesions. The aim of the present study was to investigate the effects of oxidized LDL (oxLDL) on the NF-kappa B system in human THP-1 monocytic cells as well as adherent monocytes. Our results demonstrate that short-term incubation of these cells with oxLDL activated p50/p65 containing NF-kappa B dimers and induced the expression of the target gene IL-8. This activation of NF-kappa B was inhibited by the antioxidant and H2O2 scavenger pyrrolidine dithiocarbamate and the proteasome inhibitor PSI. The oxLDL-induced NF-kappa B activation was accompanied by an initial depletion of I kappa B-alpha followed by a slight transient increase in the level of this inhibitor protein. In contrast, long-term treatment with oxLDL prevented the lipopolysaccharide-induced depletion of I kappa B-alpha, accompanied by an inhibition of both NF-kappa B activation and the expression of tumor necrosis factor-alpha and interleukin-1 beta genes. These observations provide additional evidence that oxLDL is a potent modulator of gene expression and suggest that (dys)regulation of NF-kappa B/Rel is likely to play an important role in atherogenesis. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 1128, "end": 1138}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor-alpha", "start": 1142, "end": 1169}]}, {"trigger": {"text": "expression", "start": 1128, "end": 1138}, "arguments": [{"role": "Theme", "text": "interleukin-1 beta", "start": 1174, "end": 1192}]}], "negative regulation": [{"trigger": {"text": "depletion", "start": 837, "end": 846}, "arguments": [{"role": "Theme", "text": "I kappa B-alpha", "start": 850, "end": 865}]}, {"trigger": {"text": "prevented", "start": 990, "end": 999}, "arguments": [{"role": "Theme", "text": "depletion", "start": 1031, "end": 1040}]}, {"trigger": {"text": "depletion", "start": 1031, "end": 1040}, "arguments": [{"role": "Theme", "text": "I kappa B-alpha", "start": 1044, "end": 1059}]}, {"trigger": {"text": "inhibition", "start": 1079, "end": 1089}, "arguments": [{"role": "Theme", "text": "expression", "start": 1128, "end": 1138}]}], "positive regulation": [{"trigger": {"text": "increase", "start": 897, "end": 905}, "arguments": [{"role": "Theme", "text": "I kappa B-alpha", "start": 850, "end": 865}]}]}}, "schema": []} {"input": "Suppression of MHC class II expression by human class II trans-activator constructs lacking the N-terminal domain. \nThe class II trans-activator (CIITA) is a bi- or multi-functional domain protein which plays a critical role in the expression of MHC class II genes. We report that removal of the N-terminal 151 amino acids, encompassing all of the acidic domain but leaving intact the proline/serine/threonine-rich domain, results in a mutant protein with potent suppressive properties for MHC class II expression. HeLa cells stably or transiently transfected with mutant CIITA constructs showed up to 99% suppression of MHC class II antigen induction by IFN-gamma and marked suppression of HLA-DRA mRNA expression. Transient transfection of a B lymphoma line resulted in up to 89% reduction of constitutive MHC class II expression within 5 days and suppression of HLA-DRA mRNA synthesis. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "transfected", "start": 548, "end": 559}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 572, "end": 577}]}, {"trigger": {"text": "transfection", "start": 726, "end": 738}, "arguments": [{"role": "Theme", "text": "CIITA", "start": 572, "end": 577}]}], "positive regulation": [{"trigger": {"text": "transfected", "start": 548, "end": 559}, "arguments": [{"role": "Theme", "text": "transfected", "start": 548, "end": 559}]}, {"trigger": {"text": "transfection", "start": 726, "end": 738}, "arguments": [{"role": "Theme", "text": "transfection", "start": 726, "end": 738}]}]}}, "schema": []} {"input": "CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival. \nCD30 is a cell-surface receptor that can augment lymphocyte activation and survival through its ability to induce the transcription factor NF-kappaB. CD30, however, has also been implicated in the induction of apoptotic cell death of lymphocytes. Here we show that one of the effects of CD30 signal transduction is to render cells sensitive to apoptosis induced by the type 1 tumor necrosis factor receptor (TNFR1). This sensitization is dependent on the TRAF-binding sites within the CD30 cytoplasmic domain. One of the proteins that binds to these sites is TRAF2, a signal transduction molecule that is also utilized by TNFR1 to mediate the activation of several downstream kinases and transcription factors. During CD30 signal transduction, we found that binding of TRAF2 to the cytoplasmic domain of CD30 results in the rapid depletion of TRAF2 and the associated protein TRAF1 by proteolysis. These data suggest a model in which CD30 limits its own ability to transduce cell survival signals through signal-coupled depletion of TRAF2. Depletion of intracellular TRAF2 and its coassociated proteins also increased the sensitivity of the cell to undergoing apoptosis during activation of death-inducing receptors such as TNFR1. Consistent with this hypothesis, expression of a dominant-negative form of TRAF2 was found to potentiate TNFR1-mediated death. These studies provide a potential mechanism through which CD30, as well as other TRAF-binding members of the TNFR superfamily, can negatively regulate cell survival. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binds", "start": 662, "end": 667}, "arguments": [{"role": "Site", "text": "TRAF-binding sites", "start": 582, "end": 600}, {"role": "Theme", "text": "CD30", "start": 612, "end": 616}, {"role": "Theme2", "text": "TRAF2", "start": 686, "end": 691}]}, {"trigger": {"text": "binding", "start": 885, "end": 892}, "arguments": [{"role": "Theme", "text": "TRAF2", "start": 896, "end": 901}, {"role": "Theme2", "text": "CD30", "start": 931, "end": 935}]}], "positive regulation": [{"trigger": {"text": "results", "start": 936, "end": 943}, "arguments": [{"role": "Cause", "text": "binding", "start": 885, "end": 892}, {"role": "Theme", "text": "proteolysis", "start": 1012, "end": 1023}]}, {"trigger": {"text": "activation", "start": 1304, "end": 1314}, "arguments": [{"role": "Theme", "text": "TNFR1", "start": 1351, "end": 1356}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 15, "end": 26}, "arguments": [{"role": "Theme", "text": "TRAF2", "start": 30, "end": 35}]}, {"trigger": {"text": "proteolysis", "start": 1012, "end": 1023}, "arguments": [{"role": "Theme", "text": "TRAF2", "start": 970, "end": 975}]}], "regulation": [{"trigger": {"text": "dependent", "start": 5, "end": 14}, "arguments": [{"role": "Cause", "text": "CD30", "start": 0, "end": 4}, {"role": "Theme", "text": "degradation", "start": 15, "end": 26}]}]}}, "schema": []} {"input": "TNFalpha cooperates with the protein kinase A pathway to synergistically increase HIV-1 LTR transcription via downstream TRE-like cAMP response elements. \nActivating protein-1 (AP-1) binding TPA responsive elements (TRE) are located downstream of the transcription initiation site in the U5 region of the HIV-1 long terminal repeat (LTR). These downstream sequence elements, termed DSE, can bind both AP-1 and CREB/ATF transcription factors. Recently, we demonstrated that the DSE are also cAMP-responsive elements (CRE), since they mediated activation signals elicited by cholera toxin (Ctx), a potent activator of the cAMP-dependent protein kinase A (PKA) signal transduction pathway. In the present study, we demonstrate that the HIV-1 DSE can mediate the transcriptional synergy elicited by the combination of Ctx and TNFalpha. Ctx combined with TNFalpha or IL-1beta to produce a synergistic increase in p24 antigen production in U1 promonocytic cells. Transfection studies of LTR reporter constructs indicated that mutation of the DSE sites abrogated the LTR-mediated synergy induced by Ctx and TNFalpha, whereas the synergy induced by Ctx and IL-1beta was unaffected, suggesting TNFalpha and IL-1beta cooperate differently with the cAMP/PKA activation pathway to induce HIV-1 expression in U1 cells. Because the DSE are also TRE sites, we assessed the effect of the agonist combinations on AP-1-dependent transcription. TNFalpha as well as IL-1beta cooperated with Ctx to produce a synergistic activation of AP-1-mediated transcription. These data indicate that the TRE-like cAMP-responsive DSE sites within the 5'-untranslated leader can mediate the transcriptional cooperativity between TNFalpha and the cAMP/PKA pathway. Since the DSE and TRE sites cannot bind CREB/ATF homodimers, we propose a mechanism in which the HIV-1 DSE bind heterodimers composed of both AP-1 and CREB/ATF proteins. Copyright 1997 Academic Press. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 920, "end": 930}, "arguments": [{"role": "Theme", "text": "p24 antigen", "start": 908, "end": 919}]}], "positive regulation": [{"trigger": {"text": "to produce a synergistic increase", "start": 871, "end": 904}, "arguments": [{"role": "Cause", "text": "TNFalpha", "start": 850, "end": 858}, {"role": "Theme", "text": "production", "start": 920, "end": 930}]}, {"trigger": {"text": "to produce a synergistic increase", "start": 871, "end": 904}, "arguments": [{"role": "Cause", "text": "IL-1beta", "start": 862, "end": 870}, {"role": "Theme", "text": "production", "start": 920, "end": 930}]}]}}, "schema": []} {"input": "The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB. \nThe Epstein-Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxyl-terminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacted with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucine-mutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-kappaB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-kappaB activation also appears to be a critical component of long-term outgrowth. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "engages", "start": 66, "end": 73}, "arguments": [{"role": "Theme", "text": "latent membrane protein 1", "start": 40, "end": 65}]}, {"trigger": {"text": "interacting", "start": 1236, "end": 1247}, "arguments": [{"role": "Theme", "text": "TRADD", "start": 1336, "end": 1341}]}, {"trigger": {"text": "interacted", "start": 1376, "end": 1386}, "arguments": [{"role": "Theme", "text": "TRADD", "start": 1344, "end": 1349}]}, {"trigger": {"text": "associated", "start": 1451, "end": 1461}, "arguments": [{"role": "Theme", "text": "TRADD", "start": 1445, "end": 1450}, {"role": "Theme2", "text": "LMP1", "start": 1477, "end": 1481}]}, {"trigger": {"text": "associated", "start": 1451, "end": 1461}, "arguments": [{"role": "Theme", "text": "TRADD", "start": 1445, "end": 1450}, {"role": "Theme2", "text": "LMP1", "start": 1514, "end": 1518}]}, {"trigger": {"text": "associated", "start": 1564, "end": 1574}, "arguments": [{"role": "Theme", "text": "TRADD", "start": 1543, "end": 1548}, {"role": "Theme2", "text": "LMP1", "start": 1580, "end": 1584}]}], "positive regulation": [{"trigger": {"text": "activated", "start": 425, "end": 434}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 400, "end": 404}]}]}}, "schema": []} {"input": "Transcription factor NF-kappaB regulates inducible Oct-2 gene expression in precursor B lymphocytes. \nThe POU transcription factors Oct-1 and Oct-2 regulate the activity of octamer-dependent promoters, including those that direct transcription from rearranged immunoglobulin genes. Unlike Oct-1, which is constitutively expressed in many cell types, Oct-2 expression is restricted primarily to B lymphocytes and can be induced in precursor B cells by stimulation with bacterial lipopolysaccharide (LPS). However, the precise factors that mediate this induction mechanism remain unknown. In the present study, we monitored Oct-2 expression in cells arrested for the activation of NF-kappaB, an LPS-responsive member of the Rel transcription factor family. Despite stimulation with LPS, disruption of the NF-kappaB signaling pathway in precursor B cells led to the loss of inducible Oct-2 DNA binding activity in vitro and the suppression of Oct-2-directed transcription in vivo. This biochemical defect correlated with a specific block to Oct-2 gene expression at the level of transcription, whereas the expression of Oct-1 was unaffected. The finding that Oct-2 is under NF-kappaB control highlights an important cross-talk mechanism involving two distinct transcription factor families that regulate B lymphocyte function. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 891, "end": 898}, "arguments": [{"role": "Theme", "text": "Oct-2", "start": 881, "end": 886}]}], "gene expression": [{"trigger": {"text": "gene expression", "start": 57, "end": 72}, "arguments": [{"role": "Theme", "text": "Oct-2", "start": 51, "end": 56}]}, {"trigger": {"text": "expressed", "start": 320, "end": 329}, "arguments": [{"role": "Theme", "text": "Oct-1", "start": 289, "end": 294}]}, {"trigger": {"text": "expression", "start": 356, "end": 366}, "arguments": [{"role": "Theme", "text": "Oct-2", "start": 350, "end": 355}]}, {"trigger": {"text": "expression", "start": 628, "end": 638}, "arguments": [{"role": "Theme", "text": "Oct-2", "start": 622, "end": 627}]}, {"trigger": {"text": "expression", "start": 1103, "end": 1113}, "arguments": [{"role": "Theme", "text": "Oct-1", "start": 1117, "end": 1122}]}], "negative regulation": [{"trigger": {"text": "Despite", "start": 755, "end": 762}, "arguments": [{"role": "Theme", "text": "loss", "start": 863, "end": 867}]}, {"trigger": {"text": "loss", "start": 863, "end": 867}, "arguments": [{"role": "Theme", "text": "binding", "start": 891, "end": 898}]}, {"trigger": {"text": "block", "start": 1029, "end": 1034}, "arguments": [{"role": "Theme", "text": "expression at the level of transcription", "start": 1049, "end": 1089}]}, {"trigger": {"text": "unaffected", "start": 1127, "end": 1137}, "arguments": [{"role": "Theme", "text": "expression", "start": 1103, "end": 1113}]}], "positive regulation": [{"trigger": {"text": "inducible", "start": 41, "end": 50}, "arguments": [{"role": "Theme", "text": "gene expression", "start": 57, "end": 72}]}, {"trigger": {"text": "restricted", "start": 370, "end": 380}, "arguments": [{"role": "Theme", "text": "expressed", "start": 320, "end": 329}]}, {"trigger": {"text": "restricted", "start": 370, "end": 380}, "arguments": [{"role": "Theme", "text": "expression", "start": 356, "end": 366}]}, {"trigger": {"text": "induced", "start": 419, "end": 426}, "arguments": [{"role": "Theme", "text": "expression", "start": 356, "end": 366}]}, {"trigger": {"text": "mediate", "start": 538, "end": 545}, "arguments": [{"role": "Theme", "text": "induced", "start": 419, "end": 426}]}, {"trigger": {"text": "inducible", "start": 871, "end": 880}, "arguments": [{"role": "Theme", "text": "binding", "start": 891, "end": 898}]}], "regulation": [{"trigger": {"text": "regulates", "start": 31, "end": 40}, "arguments": [{"role": "Theme", "text": "gene expression", "start": 57, "end": 72}]}, {"trigger": {"text": "control", "start": 1181, "end": 1188}, "arguments": [{"role": "Theme", "text": "Oct-2", "start": 1156, "end": 1161}]}], "transcription": [{"trigger": {"text": "expression at the level of transcription", "start": 1049, "end": 1089}, "arguments": [{"role": "Theme", "text": "Oct-2", "start": 1038, "end": 1043}]}]}}, "schema": []} {"input": "Paternal expression of WT1 in human fibroblasts and lymphocytes. \nThe Wilms' tumor suppressor gene ( WT1 ) was previously identified as being imprinted, with frequent maternal expression in human placentae and fetal brains. We examined the allele-specific expression of WT1 in cultured human fibroblasts from 15 individuals. Seven of 15 fibroblast lines were heterozygous for polymorphic alleles, and the expression patterns were variable, i.e., equal, unequal or monoallelic paternal expression in three, two and two cases, respectively. Exclusive paternal expression of WT1 was also shown in non-cultured peripheral lymphocytes from the latter two individuals. The allele-specific expression profiles of other imprinted genes, IGF2 and H19, on human chromosome 11 were constant and consistent with those in other tissues. Our unexpected observations of paternal or biallelic expression of WT1 in fibroblasts and lymphocytes, together with the previous findings of maternal or biallelic expression in placentae and brains, suggest that the allele-specific regulatory system of WT1 is unique and may be controlled by a putative tissue- and individual-specific modifier. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Paternal expression", "start": 0, "end": 19}, "arguments": [{"role": "Theme", "text": "WT1", "start": 23, "end": 26}]}, {"trigger": {"text": "maternal expression", "start": 167, "end": 186}, "arguments": [{"role": "Theme", "text": "WT1", "start": 101, "end": 104}]}, {"trigger": {"text": "allele-specific expression", "start": 240, "end": 266}, "arguments": [{"role": "Theme", "text": "WT1", "start": 270, "end": 273}]}, {"trigger": {"text": "expression", "start": 405, "end": 415}, "arguments": [{"role": "Theme", "text": "WT1", "start": 270, "end": 273}]}, {"trigger": {"text": "paternal expression", "start": 549, "end": 568}, "arguments": [{"role": "Theme", "text": "WT1", "start": 572, "end": 575}]}, {"trigger": {"text": "allele-specific expression", "start": 667, "end": 693}, "arguments": [{"role": "Theme", "text": "IGF2", "start": 729, "end": 733}]}, {"trigger": {"text": "allele-specific expression", "start": 667, "end": 693}, "arguments": [{"role": "Theme", "text": "H19", "start": 738, "end": 741}]}, {"trigger": {"text": "biallelic expression", "start": 867, "end": 887}, "arguments": [{"role": "Theme", "text": "WT1", "start": 891, "end": 894}]}, {"trigger": {"text": "expression", "start": 877, "end": 887}, "arguments": [{"role": "Theme", "text": "WT1", "start": 891, "end": 894}]}, {"trigger": {"text": "biallelic expression", "start": 978, "end": 998}, "arguments": [{"role": "Theme", "text": "WT1", "start": 891, "end": 894}]}, {"trigger": {"text": "expression", "start": 988, "end": 998}, "arguments": [{"role": "Theme", "text": "WT1", "start": 891, "end": 894}]}], "regulation": [{"trigger": {"text": "allele-specific regulatory", "start": 1041, "end": 1067}, "arguments": [{"role": "Theme", "text": "WT1", "start": 1078, "end": 1081}]}, {"trigger": {"text": "controlled", "start": 1103, "end": 1113}, "arguments": [{"role": "Theme", "text": "allele-specific regulatory", "start": 1041, "end": 1067}]}]}}, "schema": []} {"input": "Extinction of immunoglobulin gene expression in B cells upon fusion with HeLa cells is preceded by rapid nuclear depletion of essential transcription factors and is accompanied by widespread inactivation of genes expressed in a B cell-specific manner. \nWhen immunoglobulin (Ig) expressing B cells are fused with non-B cells, Ig expression is rapidly suppressed at the level of transcription, a phenomenon termed extinction. Here we demonstrate that fusion of HeLa cells with either diploid or tetraploid B cells (Daudi) results in widespread extinction of several other B cell-encoded genes that are expressed in a B cell-specific manner. In contrast, expression of B cell-expressed genes that are not dependent on cell-specific controls is unaffected. We show that the molecular mechanism(s) underlying Ig gene extinction can be explained, at least in part, by a lack of transcription factors that are essential for Ig gene transcription. These transcription factors are either not produced due to block of transcription of their respective genes (Oct-2, OBF-1, PU.1), or are rendered inactive posttranslationally (NF-kappa B, E47). By isolating Daudi x HeLa heterokaryons a few hours after fusion, we have studied the initial fate of two B cell-specific transcription factors involved in Ig gene transcription, Oct-2 and NF-kappa B. This report provides the first demonstration that upon fusion with HeLa cells, the nuclear contents of B cell-expressed transcription factors are depleted within a few hours with kinetics that are as fast or faster than that of Ig gene extinction. Thus, the extinguishing mechanism is effective very early after fusion. We suggest that extinction of Ig genes is part of a global mechanism that suppresses the differentiation program foreign to the HeLa phenotype. ", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "block", "start": 999, "end": 1004}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1008, "end": 1021}]}, {"trigger": {"text": "inactive", "start": 1086, "end": 1094}, "arguments": [{"role": "Theme", "text": "E47", "start": 1128, "end": 1131}]}], "transcription": [{"trigger": {"text": "transcription", "start": 1008, "end": 1021}, "arguments": [{"role": "Theme", "text": "Oct-2", "start": 1049, "end": 1054}]}, {"trigger": {"text": "transcription", "start": 1008, "end": 1021}, "arguments": [{"role": "Theme", "text": "OBF-1", "start": 1056, "end": 1061}]}, {"trigger": {"text": "transcription", "start": 1008, "end": 1021}, "arguments": [{"role": "Theme", "text": "PU.1", "start": 1063, "end": 1067}]}]}}, "schema": []} {"input": "Regulation of Id3 cell cycle function by Cdk-2-dependent phosphorylation. \nThe functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins. These bHLH antagonists are induced during a mitogenic signalling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle, and this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro (E.Hara, M.Hall, and G.Peters, EMBO J.16:332-342, 1997). We now show that an analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or heterodimer complex formation in vitro and E-box-dependent reporter gene function in vivo. Furthermore, compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein. Cdk2-dependent phosphorylation therefore provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression and establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation. ", "output": {"json_structures": {"phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 57, "end": 72}, "arguments": [{"role": "Theme", "text": "Id3", "start": 14, "end": 17}]}, {"trigger": {"text": "phosphorylation", "start": 581, "end": 596}, "arguments": [{"role": "Site", "text": "Ser5", "start": 635, "end": 639}, {"role": "Theme", "text": "Id2", "start": 644, "end": 647}]}, {"trigger": {"text": "phosphorylation", "start": 960, "end": 975}, "arguments": [{"role": "Theme", "text": "Id3", "start": 979, "end": 982}]}], "positive regulation": [{"trigger": {"text": "dependent", "start": 47, "end": 56}, "arguments": [{"role": "Cause", "text": "Cdk-2", "start": 41, "end": 46}, {"role": "Theme", "text": "phosphorylation", "start": 57, "end": 72}]}, {"trigger": {"text": "dependent", "start": 571, "end": 580}, "arguments": [{"role": "Cause", "text": "cyclin E", "start": 538, "end": 546}, {"role": "Theme", "text": "phosphorylation", "start": 581, "end": 596}]}, {"trigger": {"text": "dependent", "start": 571, "end": 580}, "arguments": [{"role": "Cause", "text": "Cdk2", "start": 547, "end": 551}, {"role": "Theme", "text": "phosphorylation", "start": 581, "end": 596}]}, {"trigger": {"text": "dependent", "start": 571, "end": 580}, "arguments": [{"role": "Cause", "text": "cyclin A", "start": 557, "end": 565}, {"role": "Theme", "text": "phosphorylation", "start": 581, "end": 596}]}, {"trigger": {"text": "dependent", "start": 571, "end": 580}, "arguments": [{"role": "Cause", "text": "Cdk2", "start": 566, "end": 570}, {"role": "Theme", "text": "phosphorylation", "start": 581, "end": 596}]}], "regulation": [{"trigger": {"text": "Regulation", "start": 0, "end": 10}, "arguments": [{"role": "Theme", "text": "Id3", "start": 14, "end": 17}, {"role": "Cause", "text": "phosphorylation", "start": 57, "end": 72}]}, {"trigger": {"text": "regulated", "start": 950, "end": 959}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 960, "end": 975}]}]}}, "schema": []} {"input": "Cytokine rescue from glucocorticoid induced apoptosis in T cells is mediated through inhibition of IkappaBalpha. \nWe previously reported that dexamethasone (DEX), a synthetic glucocorticoid, causes apoptosis in mature Th cell lines, and that this induction of cell death is prevented by specific cytokines, namely, by IL-2 in Th1 cells and by IL-4 in Th2 cells. We now show that this differential rescue by specific cytokines in Th cells correlates with the level of IkappaBalpha that is regulated by DEX and cytokines. In both cell types the cellular levels of IkappaBalpha mRNA and protein were evaluated by DEX treatment. Interestingly, the DEX-mediated IkappaBalpha induction was completely inhibited by IL-2, but not IL-4, in Th1 cells, while the reverse profile was seen in Th2 cells. In both cell types, the cytokine that inhibits the induction of IkappaBalpha by DEX, also rescues these cells from DEX-induced apoptosis, although the rescue cytokine is different in Th1 and Th2 cells. Our results imply that T cells need to maintain a certain level of NF-kappaB transcriptional activity in order to survive; up- or down-regulation of nuclear NF kappaB through modulation of IkappaBalpha expression by cytokines or DEX may lead to cell survival or cell death, respectively. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "level", "start": 458, "end": 463}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 467, "end": 479}]}, {"trigger": {"text": "levels", "start": 552, "end": 558}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 562, "end": 574}]}, {"trigger": {"text": "expression", "start": 1195, "end": 1205}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1182, "end": 1194}]}], "negative regulation": [{"trigger": {"text": "inhibition", "start": 85, "end": 95}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 99, "end": 111}]}, {"trigger": {"text": "inhibited", "start": 695, "end": 704}, "arguments": [{"role": "Theme", "text": "induction", "start": 670, "end": 679}, {"role": "Cause", "text": "IL-2", "start": 708, "end": 712}]}, {"trigger": {"text": "inhibited", "start": 695, "end": 704}, "arguments": [{"role": "Theme", "text": "induction", "start": 670, "end": 679}, {"role": "Cause", "text": "IL-4", "start": 722, "end": 726}]}, {"trigger": {"text": "inhibits", "start": 829, "end": 837}, "arguments": [{"role": "Theme", "text": "induction", "start": 842, "end": 851}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 670, "end": 679}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 657, "end": 669}]}, {"trigger": {"text": "induction", "start": 842, "end": 851}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 855, "end": 867}]}], "regulation": [{"trigger": {"text": "regulated", "start": 488, "end": 497}, "arguments": [{"role": "Theme", "text": "level", "start": 458, "end": 463}]}, {"trigger": {"text": "modulation", "start": 1168, "end": 1178}, "arguments": [{"role": "Theme", "text": "expression", "start": 1195, "end": 1205}]}], "transcription": [{"trigger": {"text": "levels", "start": 552, "end": 558}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 562, "end": 574}]}]}}, "schema": []} {"input": "Xenogeneic human serum promotes leukocyte adhesion to porcine endothelium under flow conditions, possibly through the activation of the transcription factor NF-kappa B. \nEndothelial cell activation and leukocyte infiltration are a consistent feature of discordant xenograft rejection. Here we evaluated whether xenogeneic serum, as a source of xenoreactive natural antibodies and complement, induced endothelial cell activation with consequent leukocyte adhesion under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 1 hr 30 min or 5 hr with 10% homologous porcine serum (control) or 10% xenogeneic human serum and then perfused with total human leukocytes in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2). Adherent cells were counted by digital image analysis. Xenogeneic human serum significantly (P < 0.01) increased the number of adherent leukocytes as compared with porcine serum. A similar adhesive response was elicited by TNF alpha (100 U/ml), one of the most potent inducers of endothelial cell adhesive properties, here used as positive control. In order to elucidate possible mechanisms underlying endothelial cell activation by xenogeneic serum, we focussed on transcription factor NF-kappa B, a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. By confocal fluorescence microscopy, we observed a positive staining for NF-kappa B (p65 subunit) in the nuclei of PAEC exposed for 1 hr 30 min to human serum, which indicated NF-kappa B activation in this setting. At variance, in PAEC incubated with the homologous serum, NF-kappa B was strictly localized in the cell cytoplasm. Treatment of PAEC exposed to xenogeneic serum with the NF-kappa B inhibitors pyrrolidinedithiocarbamate (PDTC, 25 microM) and tosyl-phechloromethylketone (TPCK, 25 microM) significantly (P < 0.01) reduced leukocyte adhesion in respect to PAEC treated with human serum alone. Findings that xenogeneic serum promotes leukocyte-endothelium interaction possibly through NF-kappa B activation might be relevant for designing future therapeutic strategies aimed at prolonging xenograft survival. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "positive staining", "start": 1413, "end": 1430}, "arguments": [{"role": "Theme", "text": "p65", "start": 1447, "end": 1450}]}]}}, "schema": []} {"input": "IL-2-induced growth of CD8+ T cell prolymphocytic leukemia cells mediated by NF-kappaB induction and IL-2 receptor alpha expression. \nThe binding of interleukin-2 (IL-2) to its receptor on normal T cells induces nuclear expression of nuclear factor kappaB (NF-kappaB), activation of the IL-2 receptor (IL-2R) alpha chain gene, and cell proliferation. In the present study, the role of IL-2R signaling in the growth of CD8+ T cell prolymphocytic leukemia (T-PLL) cells has been investigated. Flow cytometry revealed that primary leukemia cells from a patient with CD8+ T-PLL expressed IL-2Ralpha and beta chains, and the cells showed a proliferative response and an increase in IL-2Ralpha expression on culture with exogeneous IL-2. Northern blot analysis failed to detect IL-2 mRNA, suggesting that IL-2 may act in a paracrine manner in vivo. Electrophoretic mobility-shift assays revealed that recombinant IL-2 increased NF-kappaB binding activity in nuclear extracts of the leukemia cells, and Northern blot analysis showed that IL-2 increased the abundance of mRNAs encoding the NF-kappaB components c-Rel and KBF1 in these cells. IL-2 binding analysis demonstrated that IL-2 markedly increased the number of low affinity IL-2Rs on the leukemia cells, without an effect on the number of high-affinity IL-2Rs. These results show that IL-2 is capable of inducing the nuclear expression of NF-kappaB in primary CD8+ T-PLL cells, and that this effect is mediated, at least in part, at a pretranslational level. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 138, "end": 145}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 164, "end": 168}]}, {"trigger": {"text": "binding", "start": 1139, "end": 1146}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 1134, "end": 1138}]}], "gene expression": [{"trigger": {"text": "expression", "start": 121, "end": 131}, "arguments": [{"role": "Theme", "text": "IL-2 receptor alpha", "start": 101, "end": 120}]}, {"trigger": {"text": "expressed", "start": 574, "end": 583}, "arguments": [{"role": "Theme", "text": "IL-2Ralpha", "start": 584, "end": 594}]}, {"trigger": {"text": "expressed", "start": 574, "end": 583}, "arguments": [{"role": "Theme", "text": "beta", "start": 599, "end": 603}]}, {"trigger": {"text": "expression", "start": 688, "end": 698}, "arguments": [{"role": "Theme", "text": "IL-2Ralpha", "start": 677, "end": 687}]}], "positive regulation": [{"trigger": {"text": "induces", "start": 204, "end": 211}, "arguments": [{"role": "Cause", "text": "binding", "start": 138, "end": 145}, {"role": "Theme", "text": "activation", "start": 269, "end": 279}]}, {"trigger": {"text": "activation", "start": 269, "end": 279}, "arguments": [{"role": "Theme", "text": "IL-2 receptor (IL-2R) alpha chain", "start": 287, "end": 320}]}, {"trigger": {"text": "increase", "start": 665, "end": 673}, "arguments": [{"role": "Theme", "text": "expression", "start": 688, "end": 698}, {"role": "Cause", "text": "IL-2", "start": 726, "end": 730}]}, {"trigger": {"text": "increased", "start": 1036, "end": 1045}, "arguments": [{"role": "Cause", "text": "IL-2", "start": 1031, "end": 1035}, {"role": "Theme", "text": "abundance of mRNAs", "start": 1050, "end": 1068}]}], "transcription": [{"trigger": {"text": "detect", "start": 765, "end": 771}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 772, "end": 776}]}, {"trigger": {"text": "abundance of mRNAs", "start": 1050, "end": 1068}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 1103, "end": 1108}]}, {"trigger": {"text": "abundance of mRNAs", "start": 1050, "end": 1068}, "arguments": [{"role": "Theme", "text": "KBF1", "start": 1113, "end": 1117}]}]}}, "schema": []} {"input": "Molecular and cellular analysis of human immunodeficiency virus-induced apoptosis in lymphoblastoid T-cell-line-expressing wild-type and mutated CD4 receptors. \nWe have previously shown that the presence of the CD4 cytoplasmic tail is critical for human immunodeficiency virus (HIV)-induced apoptosis (J.Corbeil, M.Tremblay, and D.D.Richman, J.Exp.Med.183:39-48, 1996). We have pursued our investigation of the role of the CD4 transduction pathway in HIV-induced apoptosis. To do this, wild-type and mutant forms of the CD4 cytoplasmic tail were stably expressed in the lymphoblastoid T-cell line A2.01. Apoptosis was prevented when CD4 truncated at residue 402 was expressed; however, cells expressing mutated receptors that do not associate with p56(lck) (mutated at the dicysteine motif and truncated at residue 418) but which conserved proximal domains of the cytoplasmic tail underwent apoptosis like wild- type CD4. The differences between wild-type and mutated receptors in the induction of apoptosis were not related to levels of p56(lck) or NF- kappaB activation. Initial signaling through the CD4 receptor played a major role in the sensitization of HIV-infected T cells to undergo apoptosis. Incubation of HIV-infected cells with monoclonal antibody (MAb) 13B8-2, which binds to CD4 in a region critical for dimerization of the receptor, prevented apoptosis without inhibiting HIV replication. Moreover, the apoptotic process was not related to Fas-Fas ligand interaction; however, an antagonistic anti-Fas MAb (ZB-4) enhanced apoptosis in HIV-infected cells without inducing apoptosis in uninfected cells. These observations demonstrate that CD4 signaling mediates HIV-induced apoptosis by a mechanism independent of Fas-Fas ligand interaction, does not require p56(lck) signaling, and may involve a critical region for CD4 dimerization. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "associate", "start": 733, "end": 742}, "arguments": [{"role": "Theme", "text": "p56(lck)", "start": 748, "end": 756}]}, {"trigger": {"text": "binds", "start": 1281, "end": 1286}, "arguments": [{"role": "Theme", "text": "CD4", "start": 1290, "end": 1293}]}, {"trigger": {"text": "interaction", "start": 1471, "end": 1482}, "arguments": [{"role": "Theme", "text": "Fas", "start": 1456, "end": 1459}, {"role": "Theme2", "text": "Fas ligand", "start": 1460, "end": 1470}]}, {"trigger": {"text": "interaction", "start": 1744, "end": 1755}, "arguments": [{"role": "Theme", "text": "Fas", "start": 1729, "end": 1732}, {"role": "Theme2", "text": "Fas ligand", "start": 1733, "end": 1743}]}, {"trigger": {"text": "dimerization", "start": 1836, "end": 1848}, "arguments": [{"role": "Theme", "text": "CD4", "start": 1832, "end": 1835}]}], "gene expression": [{"trigger": {"text": "expressing", "start": 112, "end": 122}, "arguments": [{"role": "Theme", "text": "CD4 receptor", "start": 145, "end": 157}]}, {"trigger": {"text": "expressed", "start": 553, "end": 562}, "arguments": [{"role": "Theme", "text": "CD4", "start": 520, "end": 523}]}, {"trigger": {"text": "expressed", "start": 666, "end": 675}, "arguments": [{"role": "Theme", "text": "CD4", "start": 633, "end": 636}]}, {"trigger": {"text": "expressing", "start": 692, "end": 702}, "arguments": [{"role": "Theme", "text": "CD4", "start": 917, "end": 920}]}]}}, "schema": []} {"input": "NF-kappaB only partially mediates Epstein-Barr virus latent membrane protein 1 activation of B cells. \nThe latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is required for EBV-induced immortalization of human B cells and causes tumorigenic transformation of cell lines. LMP1 expression induces phenotypic changes resembling B cell activation, such as cell size increase and up-regulation of cell surface activation markers. LMP1 contains two domains that activate the transcription factor NF-kappaB, one through interactions with TRAF proteins and the other with the TRADD protein. The purpose of the present study was to investigate the importance of NF-kappaB induction in the up-regulation of the B cell activation markers ICAM-1 and CD71 by LMP1. This study shows that expression of LMP1 activates transcription from p50/p65- and c-Rel- responsive promoters, and that this activity can be completely inhibited by expression of a dominant inhibitory IkappaB mutant. ICAM-1 and CD71 are nevertheless up-regulated by LMP1 in primary B cells and cell lines expressing the dominant IkappaB. Furthermore, LMP1-induced cell size increase of primary B cells was unaffected by IkappaB expression. It was concluded that even when LMP1 is unable to activate NF-kappaB, it is still capable of inducing certain characteristics of activated B cells, strongly suggesting that LMP1 can also activate cells independently of NF-kappaB. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 287, "end": 297}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 282, "end": 286}]}, {"trigger": {"text": "expression", "start": 785, "end": 795}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 799, "end": 803}]}], "positive regulation": [{"trigger": {"text": "up-regulation", "start": 691, "end": 704}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 738, "end": 744}, {"role": "Cause", "text": "LMP1", "start": 757, "end": 761}]}, {"trigger": {"text": "up-regulation", "start": 691, "end": 704}, "arguments": [{"role": "Theme", "text": "CD71", "start": 749, "end": 753}, {"role": "Cause", "text": "LMP1", "start": 757, "end": 761}]}, {"trigger": {"text": "up-regulated", "start": 1014, "end": 1026}, "arguments": [{"role": "Theme", "text": "ICAM-1", "start": 981, "end": 987}, {"role": "Cause", "text": "LMP1", "start": 1030, "end": 1034}]}, {"trigger": {"text": "up-regulated", "start": 1014, "end": 1026}, "arguments": [{"role": "Theme", "text": "CD71", "start": 992, "end": 996}, {"role": "Cause", "text": "LMP1", "start": 1030, "end": 1034}]}], "regulation": [{"trigger": {"text": "importance", "start": 650, "end": 660}, "arguments": [{"role": "Theme", "text": "up-regulation", "start": 691, "end": 704}]}]}}, "schema": []} {"input": "Relationship between IkappaBalpha constitutive expression, TNFalpha synthesis, and apoptosis in EBV-infected lymphoblastoid cells. \nIn order to understand the role of NF-kappaB in EBV transformation we have established stably transfected IkappaBalpha into lymphoblastoid cells. Two clones were obtained in which the loss of NF-kappaB binding activity correlated with the constitutive expression of the transgenic IkappaBalpha. Protein latency expression was determined by immunocytochemistry. Expression of surface markers, intracytoplasmic content of cytokines cell cycle analysis after BrdU incorporation and DNA staining with propidium iodide were studied by flow cytometry. Percentage of apoptotic cells was determined by in-situ labelling of DNA strand breaks. No significative changes in EBV latency nor in cell surface marker expression was found. In contrast, intracytoplasmic TNFalpha levels were strongly reduced in transfected clones. Furthermore, 30% of IkappaBalpha transfected cells were apoptotic after 8 h of TNFalpha treatment. This correlated with a strong reduction of BrdU incorporation after 24 h of TNFalpha treatment. No effect was seen with non transfected cells or with cells transfected with a control plasmid. Our results suggest that the TNFalpha gene could be one of the targets of NF-kappaB in EBV infected cells and that NF-kappaB protects EBV-infected cells from apoptosis induced by TNFalpha, which may favour the proliferative effect of this cytokine. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 47, "end": 57}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 21, "end": 33}]}, {"trigger": {"text": "synthesis", "start": 68, "end": 77}, "arguments": [{"role": "Theme", "text": "TNFalpha", "start": 59, "end": 67}]}, {"trigger": {"text": "stably transfected", "start": 219, "end": 237}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 238, "end": 250}]}, {"trigger": {"text": "constitutive expression", "start": 371, "end": 394}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 413, "end": 425}]}, {"trigger": {"text": "transfected", "start": 979, "end": 990}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 966, "end": 978}]}], "negative regulation": [{"trigger": {"text": "reduced", "start": 915, "end": 922}, "arguments": [{"role": "Theme", "text": "TNFalpha", "start": 885, "end": 893}]}], "positive regulation": [{"trigger": {"text": "stably transfected", "start": 219, "end": 237}, "arguments": [{"role": "Theme", "text": "stably transfected", "start": 219, "end": 237}]}, {"trigger": {"text": "transfected", "start": 979, "end": 990}, "arguments": [{"role": "Theme", "text": "transfected", "start": 979, "end": 990}]}], "regulation": [{"trigger": {"text": "targets", "start": 1300, "end": 1307}, "arguments": [{"role": "Theme", "text": "TNFalpha", "start": 1266, "end": 1274}]}]}}, "schema": []} {"input": "Uncoupling activation-dependent HS1 phosphorylation from nuclear factor of activated T cells transcriptional activation in Jurkat T cells: differential signaling through CD3 and the costimulatory receptors CD2 and CD28. \nCD3, CD2, and CD28 are functionally distinct receptors on T lymphocytes. Engagement of any of these receptors induces the rapid tyrosine phosphorylation of a shared group of intracellular signaling proteins, including Vav, Cbl, p85 phosphoinositide 3-kinase, and the Src family kinases Lck and Fyn. Ligation of CD3 also induces the tyrosine phosphorylation of HS1, a 75-kDa hematopoietic cell-specific intracellular signaling protein of unknown function. We have examined changes in HS1 phosphorylation after differential stimulation of CD3, CD2, and CD28 to elucidate its role in T cells and to further delineate the signaling pathways recruited by these receptors. Unlike ligation of CD3, stimulation with anti-CD28 mAb or CHO cells expressing the CD28 ligands CD80 or CD86 did not lead to tyrosine phosphorylation of HS1 in Jurkat T cells. Additionally, no tyrosine phosphorylation of HS1 was induced by mitogenic pairs of anti-CD2 mAbs capable of activating the transcription factor NFAT (nuclear factor of activated T cells). Costimulation through CD28 and/or CD2 did not modulate the CD3-dependent phosphorylation of HS1. In vivo studies indicated that CD3-induced HSI phosphorylation was dependent upon both the Src family tyrosine kinase Lck and the tyrosine phosphatase CD45, did not require MEK1 kinase activity, and was regulated by protein kinase C activation. Thus, although CD3, CD28, and CD2 activate many of the same signaling molecules, they differed in their capacity to induce the tyrosine phosphorylation of HSI. Furthermore, activation-dependent tyrosine phosphorylation of HS1 was not required for NFAT transcriptional activation. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "Engagement", "start": 294, "end": 304}, "arguments": [{"role": "Theme", "text": "CD2", "start": 226, "end": 229}]}, {"trigger": {"text": "Engagement", "start": 294, "end": 304}, "arguments": [{"role": "Theme", "text": "CD28", "start": 235, "end": 239}]}, {"trigger": {"text": "ligands", "start": 976, "end": 983}, "arguments": [{"role": "Theme", "text": "CD28", "start": 971, "end": 975}, {"role": "Theme2", "text": "CD80", "start": 984, "end": 988}]}, {"trigger": {"text": "ligands", "start": 976, "end": 983}, "arguments": [{"role": "Theme", "text": "CD28", "start": 971, "end": 975}, {"role": "Theme2", "text": "CD86", "start": 992, "end": 996}]}], "gene expression": [{"trigger": {"text": "expressing", "start": 956, "end": 966}, "arguments": [{"role": "Theme", "text": "CD80", "start": 984, "end": 988}]}, {"trigger": {"text": "expressing", "start": 956, "end": 966}, "arguments": [{"role": "Theme", "text": "CD86", "start": 992, "end": 996}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 36, "end": 51}, "arguments": [{"role": "Theme", "text": "HS1", "start": 32, "end": 35}]}, {"trigger": {"text": "phosphorylation", "start": 358, "end": 373}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 349, "end": 357}, {"role": "Theme", "text": "Vav", "start": 439, "end": 442}]}, {"trigger": {"text": "phosphorylation", "start": 358, "end": 373}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 349, "end": 357}, {"role": "Theme", "text": "Cbl", "start": 444, "end": 447}]}, {"trigger": {"text": "phosphorylation", "start": 358, "end": 373}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 349, "end": 357}, {"role": "Theme", "text": "Lck", "start": 507, "end": 510}]}, {"trigger": {"text": "phosphorylation", "start": 358, "end": 373}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 349, "end": 357}, {"role": "Theme", "text": "Fyn", "start": 515, "end": 518}]}, {"trigger": {"text": "phosphorylation", "start": 562, "end": 577}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 553, "end": 561}, {"role": "Theme", "text": "HS1", "start": 581, "end": 584}]}, {"trigger": {"text": "phosphorylation", "start": 708, "end": 723}, "arguments": [{"role": "Theme", "text": "HS1", "start": 704, "end": 707}]}, {"trigger": {"text": "phosphorylation", "start": 1022, "end": 1037}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 1013, "end": 1021}, {"role": "Theme", "text": "HS1", "start": 1041, "end": 1044}]}, {"trigger": {"text": "phosphorylation", "start": 1090, "end": 1105}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 1081, "end": 1089}, {"role": "Theme", "text": "HS1", "start": 1109, "end": 1112}]}, {"trigger": {"text": "phosphorylation", "start": 1325, "end": 1340}, "arguments": [{"role": "Theme", "text": "HS1", "start": 1344, "end": 1347}]}, {"trigger": {"text": "phosphorylation", "start": 1396, "end": 1411}, "arguments": [{"role": "Theme", "text": "HSI", "start": 1392, "end": 1395}]}, {"trigger": {"text": "phosphorylation", "start": 1730, "end": 1745}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 1721, "end": 1729}, {"role": "Theme", "text": "HSI", "start": 1749, "end": 1752}]}, {"trigger": {"text": "phosphorylation", "start": 1797, "end": 1812}, "arguments": [{"role": "Site", "text": "tyrosine", "start": 1788, "end": 1796}, {"role": "Theme", "text": "HS1", "start": 1816, "end": 1819}]}], "positive regulation": [{"trigger": {"text": "dependent", "start": 22, "end": 31}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 36, "end": 51}]}, {"trigger": {"text": "induces", "start": 331, "end": 338}, "arguments": [{"role": "Cause", "text": "Engagement", "start": 294, "end": 304}, {"role": "Theme", "text": "phosphorylation", "start": 358, "end": 373}]}, {"trigger": {"text": "induces", "start": 541, "end": 548}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 562, "end": 577}]}, {"trigger": {"text": "stimulation", "start": 743, "end": 754}, "arguments": [{"role": "Theme", "text": "CD2", "start": 763, "end": 766}]}, {"trigger": {"text": "stimulation", "start": 743, "end": 754}, "arguments": [{"role": "Theme", "text": "CD28", "start": 772, "end": 776}]}, {"trigger": {"text": "lead", "start": 1005, "end": 1009}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1022, "end": 1037}]}, {"trigger": {"text": "induced", "start": 1117, "end": 1124}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1090, "end": 1105}]}, {"trigger": {"text": "dependent", "start": 1315, "end": 1324}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1325, "end": 1340}]}, {"trigger": {"text": "induced", "start": 1384, "end": 1391}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1396, "end": 1411}]}, {"trigger": {"text": "dependent", "start": 1416, "end": 1425}, "arguments": [{"role": "Theme", "text": "induced", "start": 1384, "end": 1391}, {"role": "Cause", "text": "Lck", "start": 1467, "end": 1470}]}, {"trigger": {"text": "dependent", "start": 1416, "end": 1425}, "arguments": [{"role": "Theme", "text": "induced", "start": 1384, "end": 1391}, {"role": "Cause", "text": "CD45", "start": 1500, "end": 1504}]}, {"trigger": {"text": "require", "start": 1514, "end": 1521}, "arguments": [{"role": "Theme", "text": "induced", "start": 1384, "end": 1391}, {"role": "Cause", "text": "MEK1", "start": 1522, "end": 1526}]}, {"trigger": {"text": "induce", "start": 1710, "end": 1716}, "arguments": [{"role": "Cause", "text": "CD28", "start": 1614, "end": 1618}, {"role": "Theme", "text": "phosphorylation", "start": 1730, "end": 1745}]}, {"trigger": {"text": "induce", "start": 1710, "end": 1716}, "arguments": [{"role": "Cause", "text": "CD2", "start": 1624, "end": 1627}, {"role": "Theme", "text": "phosphorylation", "start": 1730, "end": 1745}]}, {"trigger": {"text": "dependent", "start": 1778, "end": 1787}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1797, "end": 1812}]}], "regulation": [{"trigger": {"text": "changes", "start": 693, "end": 700}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 708, "end": 723}, {"role": "Cause", "text": "stimulation", "start": 743, "end": 754}]}, {"trigger": {"text": "modulate", "start": 1298, "end": 1306}, "arguments": [{"role": "Theme", "text": "dependent", "start": 1315, "end": 1324}]}, {"trigger": {"text": "regulated", "start": 1552, "end": 1561}, "arguments": [{"role": "Theme", "text": "induced", "start": 1384, "end": 1391}]}]}}, "schema": []} {"input": "IL-7 reconstitutes multiple aspects of v-Abl-mediated signaling. \nThe mechanism by which early lymphoid cells are selectively transformed by v-Abl is currently unknown. Previous studies have shown constitutive activation of IL-4 and IL-7 signaling pathways, as measured by activation of Janus protein kinase (JAK)1, JAK3, STAT5, and STAT6, in pre-B cells transformed by v-Abl. To determine whether activation of these cytokine signaling pathways by v-Abl is important in the cellular events induced by the Abelson murine leukemia virus, the effects of IL-4 and IL-7 on pre-B cells transformed with a temperature-sensitive v-Abl mutant were examined. Whereas IL-4 had little or no effect, IL-7 delayed both the apoptosis and cell cycle arrest that occur upon v-Abl kinase inactivation. IL-7 also delayed the decreases in the levels of c-Myc, Bcl-2, and Bcl-xL that occur upon loss of v-Abl kinase activity. IL-7 did not maintain v-Abl-mediated differentiation arrest of the pre-B cells, as activation of NF-kappaB and RAG gene transcription was unaffected by IL-7. These results identify a potential role for IL-7 signaling pathways in transformation by v-Abl while demonstrating that a combination of IL-4 and IL-7 signaling cannot substitute for an active v-Abl kinase in transformed pre-B cells. ", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "delayed", "start": 795, "end": 802}, "arguments": [{"role": "Cause", "text": "IL-7", "start": 785, "end": 789}, {"role": "Theme", "text": "decreases", "start": 807, "end": 816}]}, {"trigger": {"text": "decreases", "start": 807, "end": 816}, "arguments": [{"role": "Theme", "text": "c-Myc", "start": 834, "end": 839}]}, {"trigger": {"text": "decreases", "start": 807, "end": 816}, "arguments": [{"role": "Theme", "text": "Bcl-2", "start": 841, "end": 846}]}, {"trigger": {"text": "decreases", "start": 807, "end": 816}, "arguments": [{"role": "Theme", "text": "Bcl-xL", "start": 852, "end": 858}]}], "positive regulation": [{"trigger": {"text": "reconstitutes", "start": 5, "end": 18}, "arguments": [{"role": "Cause", "text": "IL-7", "start": 0, "end": 4}, {"role": "Theme", "text": "signaling", "start": 54, "end": 63}]}, {"trigger": {"text": "signaling", "start": 54, "end": 63}, "arguments": [{"role": "Theme", "text": "v-Abl", "start": 39, "end": 44}]}, {"trigger": {"text": "activation", "start": 273, "end": 283}, "arguments": [{"role": "Theme", "text": "Janus protein kinase (JAK)1", "start": 287, "end": 314}]}, {"trigger": {"text": "activation", "start": 273, "end": 283}, "arguments": [{"role": "Theme", "text": "JAK3", "start": 316, "end": 320}]}, {"trigger": {"text": "activation", "start": 273, "end": 283}, "arguments": [{"role": "Theme", "text": "STAT6", "start": 333, "end": 338}]}]}}, "schema": []} {"input": "p21ras initiates Rac-1 but not phosphatidyl inositol 3 kinase/PKB, mediated signaling pathways in T lymphocytes. \np21ras is activated by the T cell antigen receptor (TCR) and then co-ordinates important signaling pathways for T lymphocyte activation. Effector pathways for this guanine nucleotide binding protein in T cells are mediated by the serine/threonine kinase Raf-1 and the Ras-related GTPase Rac-1. In fibroblasts, an important effector for the Ras oncogene is Phosphatidylinositol 3-kinase (PtdIns 3-kinase). Activation of this lipid kinase is able to induce critical Rac-1 signaling pathways and can couple p21ras to cell survival mechanisms via the serine/threonine kinase Akt/PKB. The role of PtdIns 3-kinase in Ras signaling in T cells has not been explored. In the present study, we examined the ability of PtdIns 3-kinase to initiate the Rac-1 signaling pathways important for T cell activation. We also examined the possibility that Akt/PKB is regulated by Ras signaling pathways in T lymphocytes. The results show that Ras can initiate a Rac-1 mediated pathway that regulates the transcriptional function of AP-1 complexes. PtdIns 3-kinase signals cannot mimic p21ras and induce the Rac mediated responses of AP-1 transcriptional activation. Moreover, neither TCR or Ras activation of AP-1 is dependent on PtdIns 3-kinase. PKB is activated in response to triggering of the T cell antigen receptor; PtdIns 3-kinase activity is both required and sufficient for this TCR response. In contrast, p21ras signals are unable to induce Akt/PKB activity in T cell nor is Ras function required for Akt/PKB activation in response to the TCR. The present data thus highlight that PtdIns 3-kinase and Akt/PKB are not universal Ras effector molecules. Ras can initiate Rac-1 regulated signaling pathways in the context of T cell antigen receptor function independently of PtdIns 3-kinase activity. ", "output": {"json_structures": {"positive regulation": [{"trigger": {"text": "activated", "start": 124, "end": 133}, "arguments": [{"role": "Theme", "text": "p21ras", "start": 114, "end": 120}]}, {"trigger": {"text": "mediated", "start": 328, "end": 336}, "arguments": [{"role": "Theme", "text": "Effector pathways", "start": 251, "end": 268}, {"role": "Cause", "text": "Raf-1", "start": 368, "end": 373}]}, {"trigger": {"text": "mediated", "start": 328, "end": 336}, "arguments": [{"role": "Theme", "text": "Effector pathways", "start": 251, "end": 268}, {"role": "Cause", "text": "Rac-1", "start": 401, "end": 406}]}, {"trigger": {"text": "couple", "start": 611, "end": 617}, "arguments": [{"role": "Theme", "text": "Akt", "start": 685, "end": 688}]}, {"trigger": {"text": "activated", "start": 1348, "end": 1357}, "arguments": [{"role": "Theme", "text": "PKB", "start": 1341, "end": 1344}]}, {"trigger": {"text": "required and sufficient", "start": 1449, "end": 1472}, "arguments": [{"role": "Theme", "text": "activated", "start": 1348, "end": 1357}]}, {"trigger": {"text": "induce", "start": 1538, "end": 1544}, "arguments": [{"role": "Cause", "text": "p21ras", "start": 1509, "end": 1515}, {"role": "Theme", "text": "Akt", "start": 1545, "end": 1548}]}, {"trigger": {"text": "required", "start": 1592, "end": 1600}, "arguments": [{"role": "Cause", "text": "p21ras", "start": 1509, "end": 1515}, {"role": "Theme", "text": "activation", "start": 1613, "end": 1623}]}, {"trigger": {"text": "activation", "start": 1613, "end": 1623}, "arguments": [{"role": "Theme", "text": "Akt", "start": 1605, "end": 1608}]}], "regulation": [{"trigger": {"text": "Effector pathways", "start": 251, "end": 268}, "arguments": [{"role": "Theme", "text": "p21ras", "start": 114, "end": 120}]}, {"trigger": {"text": "regulated", "start": 961, "end": 970}, "arguments": [{"role": "Theme", "text": "Akt", "start": 950, "end": 953}]}]}}, "schema": []} {"input": "Signal transduction abnormalities in T lymphocytes from patients with advanced renal carcinoma: clinical relevance and effects of cytokine therapy. \nStudies have demonstrated abnormalities of the CD3/T-cell antigen receptor (TCR) and pathways of signal transduction in T lymphocytes from animals and patients with advanced malignancy. Diminished expression of TCRzeta and p56(lck) that are associated with the TCR and reduced nuclear localization of RelA containing nuclear factor kappaB (NFkappaB) complexes have been noted. These defects have been described in T cells from patients with malignant melanoma, renal cell carcinoma (RCC), ovarian cancer, and colorectal cancer. Preliminary observations also indicate possible correlation with clinical variables such as stage in selected instances. To further characterize altered expression of TCRzeta, p56(lck), and impaired activation of NFkappaB, T lymphocytes were obtained from 65 patients with RCC, the majority of whom were receiving combination cytokine therapy [interleukin (IL)-2, IFN alpha-containing regimens] and 37 control individuals. In 29 of these patients, levels of TCRzeta and p56(lck) were determined by Western blots of T-cell lysates and semiquantitated using densitometry. Relative levels were then correlated with a series of clinical variables including response to therapy, performance status, survival, disease sites, age, and others. In another group of 28 patients (three individuals from the first group), the frequency of abnormal NFkappaB activation was studied using electrophoretic mobility shift assays after activation of T cells with phorbol myristate acetate/ionomycin or anti-CD3 monoclonal antibody. Changes in these signaling molecules during cytokine treatment were also investigated. TCRzeta and p56(lck) were detected in the peripheral blood T cells in 27 of 29 patients, and overall, reduced levels were noted visually in 12 of 29 (41%) and 13 of 29 (45%) individuals, respectively. When levels were semiquantitated using densitometry, significant decreases of TCRzeta (P = 0.029) and p56(lck) (P = 0.029) but not CD3epsilon (P = 0.131), compared with control levels, were found. In patients treated with IL-2/IFN alpha-based therapy, relative levels of TCRzeta increased significantly (P = 0.002) on day 15 of cycle one compared with the baseline. Correlations of TCRzeta or p56(lck) levels with response or disease variables, except for lower TCRzeta levels (P < 0.001) in the presence of bone metastases, were not found. Abnormal NFkappaB activation after stimulation with phorbol myristate acetate/ionomycin and/or anti-CD3 monoclonal antibody was found in 59% of patients (17 of 28) and was not accounted for by the advanced age of the study cohort. Activation of NFkappaB in peripheral blood T cells was inducible during cytokine therapy in four of six individuals who displayed impaired NFkappaB activity prior to therapy. Moreover, impaired activation of NFkappaB does not appear linked to a reduction of TCRzeta expression, because in five patients, normal TCRzeta levels were present although kappaB binding was not inducible. In the majority of patients with advanced RCC, peripheral blood T cells express TCRzeta and p56(lck), and in a subset, reduced levels of these TCRzeta associated molecules are seen that may increase during cytokine-based therapy. Abnormal activation of NFkappaB is also present in >50% of patients and may also revert to normal during IL-2/IFN alpha-based treatment. This alteration in NFkappaB activation occurred in the presence of normal expression of TCRzeta-associated signaling elements. The clinical significance of these findings remains unclear. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "associated", "start": 390, "end": 400}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 360, "end": 367}]}, {"trigger": {"text": "associated", "start": 390, "end": 400}, "arguments": [{"role": "Theme", "text": "p56(lck)", "start": 372, "end": 380}]}], "gene expression": [{"trigger": {"text": "expression", "start": 346, "end": 356}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 360, "end": 367}]}, {"trigger": {"text": "expression", "start": 346, "end": 356}, "arguments": [{"role": "Theme", "text": "p56(lck)", "start": 372, "end": 380}]}, {"trigger": {"text": "expression", "start": 830, "end": 840}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 844, "end": 851}]}, {"trigger": {"text": "expression", "start": 830, "end": 840}, "arguments": [{"role": "Theme", "text": "p56(lck)", "start": 853, "end": 861}]}, {"trigger": {"text": "levels", "start": 1125, "end": 1131}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 1135, "end": 1142}]}, {"trigger": {"text": "levels", "start": 1125, "end": 1131}, "arguments": [{"role": "Theme", "text": "p56(lck)", "start": 1147, "end": 1155}]}, {"trigger": {"text": "detected", "start": 1804, "end": 1812}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 1778, "end": 1785}]}, {"trigger": {"text": "detected", "start": 1804, "end": 1812}, "arguments": [{"role": "Theme", "text": "p56(lck)", "start": 1790, "end": 1798}]}, {"trigger": {"text": "expression", "start": 3017, "end": 3027}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 3009, "end": 3016}]}, {"trigger": {"text": "express", "start": 3205, "end": 3212}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 3213, "end": 3220}]}, {"trigger": {"text": "express", "start": 3205, "end": 3212}, "arguments": [{"role": "Theme", "text": "p56(lck)", "start": 3225, "end": 3233}]}], "localization": [{"trigger": {"text": "localization", "start": 434, "end": 446}, "arguments": [{"role": "AtLoc", "text": "nuclear", "start": 426, "end": 433}, {"role": "Theme", "text": "RelA", "start": 450, "end": 454}]}], "negative regulation": [{"trigger": {"text": "Diminished", "start": 335, "end": 345}, "arguments": [{"role": "Theme", "text": "expression", "start": 346, "end": 356}]}, {"trigger": {"text": "reduced", "start": 418, "end": 425}, "arguments": [{"role": "Theme", "text": "localization", "start": 434, "end": 446}]}, {"trigger": {"text": "defects", "start": 532, "end": 539}, "arguments": [{"role": "Theme", "text": "Diminished", "start": 335, "end": 345}]}, {"trigger": {"text": "defects", "start": 532, "end": 539}, "arguments": [{"role": "Theme", "text": "reduced", "start": 418, "end": 425}]}, {"trigger": {"text": "altered", "start": 822, "end": 829}, "arguments": [{"role": "Theme", "text": "expression", "start": 830, "end": 840}]}, {"trigger": {"text": "reduced", "start": 1880, "end": 1887}, "arguments": [{"role": "Theme", "text": "detected", "start": 1804, "end": 1812}]}, {"trigger": {"text": "decreases", "start": 2044, "end": 2053}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 2057, "end": 2064}]}, {"trigger": {"text": "decreases", "start": 2044, "end": 2053}, "arguments": [{"role": "Theme", "text": "p56(lck)", "start": 2081, "end": 2089}]}, {"trigger": {"text": "decreases", "start": 2044, "end": 2053}, "arguments": [{"role": "Theme", "text": "CD3epsilon", "start": 2110, "end": 2120}]}, {"trigger": {"text": "lower", "start": 2435, "end": 2440}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 2441, "end": 2448}]}, {"trigger": {"text": "reduction", "start": 2996, "end": 3005}, "arguments": [{"role": "Theme", "text": "expression", "start": 3017, "end": 3027}]}, {"trigger": {"text": "reduced levels", "start": 3252, "end": 3266}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 3213, "end": 3220}]}, {"trigger": {"text": "reduced levels", "start": 3252, "end": 3266}, "arguments": [{"role": "Theme", "text": "p56(lck)", "start": 3225, "end": 3233}]}], "positive regulation": [{"trigger": {"text": "Changes", "start": 1691, "end": 1698}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 1135, "end": 1142}]}, {"trigger": {"text": "Changes", "start": 1691, "end": 1698}, "arguments": [{"role": "Theme", "text": "p56(lck)", "start": 1147, "end": 1155}]}, {"trigger": {"text": "increased", "start": 2258, "end": 2267}, "arguments": [{"role": "Theme", "text": "TCRzeta", "start": 2250, "end": 2257}]}, {"trigger": {"text": "increase", "start": 3323, "end": 3331}, "arguments": [{"role": "Theme", "text": "reduced levels", "start": 3252, "end": 3266}]}]}}, "schema": []} {"input": "Transcriptional regulation of lysosomal acid lipase in differentiating monocytes is mediated by transcription factors Sp1 and AP-2. \nHuman lysosomal acid lipase (LAL) is a hydrolase required for the cleavage of cholesteryl esters and triglycerides derived from plasma lipoproteins. It is shown here that during monocyte to macrophage differentiation, the expression of LAL-mRNA is induced. This induction is dependent on protein kinase C activity and protein synthesis. The cell type-specific increase in LAL expression is further investigated in the THP-1 cell line with respect to transcriptional regulation. The human monocytic leukemia cell line THP-1 differentiates into macrophage-like cells when treated with phorbol esters. In order to determine the cis-acting elements necessary for both basal and phorbol 12-myristate-13 acetate (PMA)-enhanced promoter activity, we performed deletion analysis and reporter gene assays. A PMA responsive element has been identified between -182 bp and -107 bp upstream of the major transcription start site. Gel mobility shift assays demonstrated that binding of Sp1 and AP-2 to the LAL promoter is increased by PMA in THP-1 cells. Co-transfections with expression plasmids for Sp1 and AP-2 further emphasized the important role of these transcription factors in both basal and PMA-enhanced LAL expression. Our data suggest that differentiation dependent increase of lysosomal acid lipase (LAL) expression in THP-1 cells is mediated by a concerted action of Sp1 and AP-2. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 1095, "end": 1102}, "arguments": [{"role": "Theme", "text": "Sp1", "start": 1106, "end": 1109}, {"role": "Theme2", "text": "LAL", "start": 1126, "end": 1129}, {"role": "Site2", "text": "promoter", "start": 1130, "end": 1138}]}, {"trigger": {"text": "binding", "start": 1095, "end": 1102}, "arguments": [{"role": "Theme", "text": "LAL", "start": 1126, "end": 1129}, {"role": "Site", "text": "promoter", "start": 1130, "end": 1138}]}], "gene expression": [{"trigger": {"text": "expression", "start": 509, "end": 519}, "arguments": [{"role": "Theme", "text": "LAL", "start": 505, "end": 508}]}, {"trigger": {"text": "expression", "start": 1338, "end": 1348}, "arguments": [{"role": "Theme", "text": "LAL", "start": 1334, "end": 1337}]}, {"trigger": {"text": "expression", "start": 1438, "end": 1448}, "arguments": [{"role": "Theme", "text": "LAL", "start": 1433, "end": 1436}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 381, "end": 388}, "arguments": [{"role": "Theme", "text": "expression", "start": 355, "end": 365}]}, {"trigger": {"text": "dependent", "start": 408, "end": 417}, "arguments": [{"role": "Theme", "text": "induced", "start": 381, "end": 388}]}, {"trigger": {"text": "increase", "start": 493, "end": 501}, "arguments": [{"role": "Theme", "text": "expression", "start": 509, "end": 519}]}, {"trigger": {"text": "increased", "start": 1142, "end": 1151}, "arguments": [{"role": "Theme", "text": "binding", "start": 1095, "end": 1102}]}, {"trigger": {"text": "enhanced", "start": 1325, "end": 1333}, "arguments": [{"role": "Theme", "text": "expression", "start": 1338, "end": 1348}]}, {"trigger": {"text": "increase", "start": 1398, "end": 1406}, "arguments": [{"role": "Theme", "text": "expression", "start": 1438, "end": 1448}]}], "regulation": [{"trigger": {"text": "Transcriptional regulation", "start": 0, "end": 26}, "arguments": [{"role": "Theme", "text": "lysosomal acid lipase", "start": 30, "end": 51}]}, {"trigger": {"text": "mediated", "start": 84, "end": 92}, "arguments": [{"role": "Theme", "text": "Transcriptional regulation", "start": 0, "end": 26}, {"role": "Cause", "text": "Sp1", "start": 118, "end": 121}]}, {"trigger": {"text": "transcriptional regulation", "start": 583, "end": 609}, "arguments": [{"role": "Theme", "text": "LAL", "start": 505, "end": 508}]}, {"trigger": {"text": "important role", "start": 1257, "end": 1271}, "arguments": [{"role": "Cause", "text": "Sp1", "start": 1221, "end": 1224}, {"role": "Theme", "text": "enhanced", "start": 1325, "end": 1333}]}, {"trigger": {"text": "important role", "start": 1257, "end": 1271}, "arguments": [{"role": "Cause", "text": "Sp1", "start": 1221, "end": 1224}, {"role": "Theme", "text": "expression", "start": 1338, "end": 1348}]}, {"trigger": {"text": "dependent", "start": 1388, "end": 1397}, "arguments": [{"role": "Theme", "text": "increase", "start": 1398, "end": 1406}]}, {"trigger": {"text": "mediated", "start": 1467, "end": 1475}, "arguments": [{"role": "Theme", "text": "dependent", "start": 1388, "end": 1397}]}], "transcription": [{"trigger": {"text": "expression", "start": 355, "end": 365}, "arguments": [{"role": "Theme", "text": "LAL", "start": 369, "end": 372}]}]}}, "schema": []} {"input": "A nongenomic mechanism for progesterone-mediated immunosuppression: inhibition of K+ channels, Ca2+ signaling, and gene expression in T lymphocytes. \nThe mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin-resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl- channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression. ", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "reducing", "start": 1166, "end": 1174}, "arguments": [{"role": "Theme", "text": "Kv1.3", "start": 1180, "end": 1185}]}]}}, "schema": []} {"input": "Role of IKK1 and IKK2 in lipopolysaccharide signaling in human monocytic cells. \nMononuclear phagocytes play a major role in immune and inflammatory responses. Bacterial lipopolysaccharide (LPS) induces monocytes to express a variety of genes by activating the NF-kappaB/Rel transcription factor family. Recently, we have reported that the tumor necrosis factor and interleukin 1 signaling pathways activate two kinases, IKK1 and IKK2. Phosphorylation of the IkappaB cytoplasmic inhibitors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, by these kinases triggers proteolytic degradation and the release of NF-kappaB/Rel proteins into the nucleus. At present, the role of the IKKs in LPS signaling has not been investigated. Here, we report that LPS induces IKK activity in human monocytes and THP-1 monocytic cells. The kinetics of activation of kinase activity in monocytic cells are relatively slow with maximal activity observed at 60 min, which coincides with the degradation of IkappaBs and the nuclear translocation of NF-kappaB. In transfection experiments, overexpression of wild type IKK1, a dominant negative mutant IKK1 (K44M), or wild type IKK2 did not affect LPS-induced kappaB-dependent transcription in monocytic cells. In contrast, a dominant negative mutant of IKK2 inhibited LPS induction of kappaB-dependent transcription in a dose-dependent manner. These results indicate that LPS induction of kappaB-dependent gene expression in human monocytic cells requires activation of IKK2. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "overexpression", "start": 1066, "end": 1080}, "arguments": [{"role": "Theme", "text": "IKK1", "start": 1094, "end": 1098}]}, {"trigger": {"text": "overexpression", "start": 1066, "end": 1080}, "arguments": [{"role": "Theme", "text": "IKK1", "start": 1127, "end": 1131}]}], "phosphorylation": [{"trigger": {"text": "Phosphorylation", "start": 436, "end": 451}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 491, "end": 503}]}, {"trigger": {"text": "Phosphorylation", "start": 436, "end": 451}, "arguments": [{"role": "Theme", "text": "IkappaBbeta", "start": 505, "end": 516}]}, {"trigger": {"text": "Phosphorylation", "start": 436, "end": 451}, "arguments": [{"role": "Theme", "text": "IkappaBepsilon", "start": 522, "end": 536}]}], "positive regulation": [{"trigger": {"text": "activate", "start": 399, "end": 407}, "arguments": [{"role": "Theme", "text": "IKK1", "start": 421, "end": 425}]}, {"trigger": {"text": "activate", "start": 399, "end": 407}, "arguments": [{"role": "Theme", "text": "IKK2", "start": 430, "end": 434}]}, {"trigger": {"text": "by", "start": 538, "end": 540}, "arguments": [{"role": "Cause", "text": "IKK1", "start": 421, "end": 425}, {"role": "Theme", "text": "Phosphorylation", "start": 436, "end": 451}]}, {"trigger": {"text": "by", "start": 538, "end": 540}, "arguments": [{"role": "Cause", "text": "IKK2", "start": 430, "end": 434}, {"role": "Theme", "text": "Phosphorylation", "start": 436, "end": 451}]}, {"trigger": {"text": "triggers", "start": 555, "end": 563}, "arguments": [{"role": "Cause", "text": "by", "start": 538, "end": 540}, {"role": "Theme", "text": "proteolytic degradation", "start": 564, "end": 587}]}, {"trigger": {"text": "overexpression", "start": 1066, "end": 1080}, "arguments": [{"role": "Theme", "text": "overexpression", "start": 1066, "end": 1080}]}, {"trigger": {"text": "activation", "start": 1482, "end": 1492}, "arguments": [{"role": "Theme", "text": "IKK2", "start": 1496, "end": 1500}]}], "protein catabolism": [{"trigger": {"text": "proteolytic degradation", "start": 564, "end": 587}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 491, "end": 503}]}, {"trigger": {"text": "proteolytic degradation", "start": 564, "end": 587}, "arguments": [{"role": "Theme", "text": "IkappaBbeta", "start": 505, "end": 516}]}, {"trigger": {"text": "proteolytic degradation", "start": 564, "end": 587}, "arguments": [{"role": "Theme", "text": "IkappaBepsilon", "start": 522, "end": 536}]}]}}, "schema": []} {"input": "Effects of overexpression of IL-1 receptor-associated kinase on NFkappaB activation, IL-2 production and stress-activated protein kinases in the murine T cell line EL4. \nThe association and activation of the IL-1 receptor-associated protein kinase (IRAK) to the IL-1 receptor complex is one of the earliest events detectable in IL-1 signal transduction. We generated permanent clones of the murine T cell line EL4 6.1 overexpressing human (h)IRAK to evaluate the role of this kinase in IL-1 signaling. Overexpression of hIRAK enhanced IL-1-stimulated activation of the transcription factor NFkappaB, whereas a truncated form (N-IRAK) specifically inhibited IL-1-dependent NFkappaB activity. In clones stably overexpressing hIRAK a weak constitutive activation of NFkappaB correlated with a low basal IL-2 production which was enhanced in an IL-1-dependent manner. Compared to the parental cell line the dose-response curve of IL-1-induced IL-2 production was shifted in both potency and efficacy. These results demonstrate that IRAK directly triggers NFkappaB-mediated gene expression in EL4 cells. Qualitatively different effects were observed for the IL-1-induced activation of stress-activated protein (SAP) kinases: permanent overexpression of IRAK did not affect the dose dependence but prolonged the kinetics of IL-1-induced activation of SAP kinases, suggesting that this signaling branch may be regulated by distinct mechanisms. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 90, "end": 100}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 85, "end": 89}]}, {"trigger": {"text": "production", "start": 805, "end": 815}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 800, "end": 804}]}, {"trigger": {"text": "production", "start": 944, "end": 954}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 939, "end": 943}]}], "positive regulation": [{"trigger": {"text": "enhanced", "start": 826, "end": 834}, "arguments": [{"role": "Theme", "text": "production", "start": 805, "end": 815}]}, {"trigger": {"text": "induced", "start": 931, "end": 938}, "arguments": [{"role": "Theme", "text": "production", "start": 944, "end": 954}]}], "regulation": [{"trigger": {"text": "Effects", "start": 0, "end": 7}, "arguments": [{"role": "Theme", "text": "production", "start": 90, "end": 100}]}]}}, "schema": []} {"input": "Decreased IL-12 production and Th1 cell development by acetyl salicylic acid-mediated inhibition of NF-kappaB. \nIL-12 is a 75-kDa heterodimeric cytokine composed of two covalently linked p35 and p40 chains. This pro-inflammatory cytokine plays a prominent role in the development of Th1 cell-mediated immune responses. Th1 cell-mediated immune responses have been implicated in the pathogenesis of chronic inflammatory autoimmune diseases. Thus, IL-12 appears to be a critical factor in the generation and maintenance of chronic inflammatory conditions. In this study, we investigated the effects of a commonly prescribed anti-inflammatory drug, acetyl salicylic acid (ASA), on IL-12 production and Th1 cell development. ASA was found to inhibit secretion of the IL-12 heterodimer as well as p40 monomer by human monocytic cells. This was associated with the down-regulation of IL-12p40 mRNA expression. Analysis of the regulation of the p40 gene promoter revealed that ASA inhibited NF-kappaB activation and binding to the p40-kappaB site in the p40 promoter, leading to transcriptional repression of the p40 gene. Addition of ASA to an in vitro T helper cell differentiation system, at concentrations compatible with plasma levels reached during anti-inflammatory therapy, resulted in reduced development of Th1 cells. These results suggest that the inhibition of NF-kappaB activation by ASA leads to down-regulation of IL-12 production and inhibition of Th1 cell development. ", "output": {"json_structures": {"localization": [{"trigger": {"text": "secretion", "start": 746, "end": 755}, "arguments": [{"role": "Theme", "text": "p40", "start": 792, "end": 795}]}], "negative regulation": [{"trigger": {"text": "inhibit", "start": 738, "end": 745}, "arguments": [{"role": "Theme", "text": "secretion", "start": 746, "end": 755}]}, {"trigger": {"text": "down-regulation", "start": 859, "end": 874}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 887, "end": 902}]}], "positive regulation": [{"trigger": {"text": "leading to transcriptional repression", "start": 1061, "end": 1098}, "arguments": [{"role": "Theme", "text": "p40", "start": 1106, "end": 1109}]}], "regulation": [{"trigger": {"text": "regulation", "start": 920, "end": 930}, "arguments": [{"role": "Theme", "text": "p40", "start": 938, "end": 941}, {"role": "Site", "text": "promoter", "start": 947, "end": 955}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 887, "end": 902}, "arguments": [{"role": "Theme", "text": "IL-12p40", "start": 878, "end": 886}]}]}}, "schema": []} {"input": "Membrane-associated lymphotoxin on natural killer cells activates endothelial cells via an NF-kappaB-dependent pathway. \nBACKGROUND: Inhibition of complement in small animal models of xenotransplantation has demonstrated graft infiltration with natural killer (NK) cells and monocytes associated with endothelial cell (EC) activation. We have previously demonstrated that human NK cells activate porcine EC in vitro, which results in adhesion molecule expression and cytokine secretion. In this study, we used the NK cell line NK92 to define the molecular and cellular basis of NK cell-mediated EC activation. METHODS: EC were transfected with either reporter constructs containing the luciferase gene driven either by E-selectin or interleukin (IL)-8 promoters or a synthetic NF-kappaB-dependent promoter. In addition, a dominant-negative mutant tumor necrosis factor receptor I (TNFRI) expression vector was co-transfected in inhibition studies. Forty-eight hours after transfection, EC were stimulated with NK cells or NK cell membrane extracts for 7 hr and activation was measured by a luciferase assay. RESULTS: Co-culture of NK cells with transfected EC enhanced E-selectin, IL-8, and NF-kappaB-dependent promoter activity. NK cell membrane extracts retained the capacity to activate EC and induced nuclear translocation of NF-kappaB (p50 and p65). Western blotting of NK cell and membrane extracts detected the presence of Lymphotoxin-alpha (LTalpha) but not tumor necrosis factor-alpha. Furthermore, LTalpha was secreted in NK:EC co-cultures. Co-transfection with dominant-negative mutant TNFRI inhibited EC activation by NK cell membrane extracts and by NK cells by 80% and 47%, respectively. The same pattern of inhibition was observed using anti-human LT sera. CONCLUSIONS: Human NK cell membrane-bound LT signals across species via TNFRI, leading to NF-kappaB nuclear translocation and transcription of E-selectin and IL-8, which results in EC activation. The discrepancy in the degree of inhibition by membrane extracts and NK cells with mutant TNFRI suggests that additional pathways are utilized by NK cells to activate EC. ", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "co-transfected", "start": 910, "end": 924}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor receptor I", "start": 847, "end": 879}]}, {"trigger": {"text": "presence", "start": 1418, "end": 1426}, "arguments": [{"role": "Theme", "text": "LTalpha", "start": 1449, "end": 1456}]}, {"trigger": {"text": "presence", "start": 1418, "end": 1426}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor-alpha", "start": 1466, "end": 1493}]}, {"trigger": {"text": "Co-transfection", "start": 1551, "end": 1566}, "arguments": [{"role": "Theme", "text": "TNFRI", "start": 1597, "end": 1602}]}], "localization": [{"trigger": {"text": "translocation", "start": 1313, "end": 1326}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 1305, "end": 1312}, {"role": "Theme", "text": "p50", "start": 1341, "end": 1344}]}, {"trigger": {"text": "translocation", "start": 1313, "end": 1326}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 1305, "end": 1312}, {"role": "Theme", "text": "p65", "start": 1349, "end": 1352}]}, {"trigger": {"text": "secreted", "start": 1520, "end": 1528}, "arguments": [{"role": "Theme", "text": "LTalpha", "start": 1508, "end": 1515}]}], "positive regulation": [{"trigger": {"text": "co-transfected", "start": 910, "end": 924}, "arguments": [{"role": "Theme", "text": "co-transfected", "start": 910, "end": 924}]}, {"trigger": {"text": "induced", "start": 1297, "end": 1304}, "arguments": [{"role": "Theme", "text": "translocation", "start": 1313, "end": 1326}]}, {"trigger": {"text": "Co-transfection", "start": 1551, "end": 1566}, "arguments": [{"role": "Theme", "text": "Co-transfection", "start": 1551, "end": 1566}]}, {"trigger": {"text": "leading", "start": 1851, "end": 1858}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1898, "end": 1911}]}], "transcription": [{"trigger": {"text": "transcription", "start": 1898, "end": 1911}, "arguments": [{"role": "Theme", "text": "E-selectin", "start": 1915, "end": 1925}]}, {"trigger": {"text": "transcription", "start": 1898, "end": 1911}, "arguments": [{"role": "Theme", "text": "IL-8", "start": 1930, "end": 1934}]}]}}, "schema": []} {"input": "The involvement of multiple tumor necrosis factor receptor (TNFR)-associated factors in the signaling mechanisms of receptor activator of NF-kappaB, a member of the TNFR superfamily. \nReceptor activator of NF-kappaB (RANK) is a recently identified member of the tumor necrosis factor receptor superfamily and is expressed on activated T cells and dendritic cells. Its cognate ligand (RANKL) plays significant roles in the activation of dendritic cell function and osteoclast differentiation. We demonstrate here the interaction of RANK with tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 both in vitro and in cells. Mapping of the structural requirements for TRAF/RANK interaction revealed multiple TRAF binding sites clustered in two distinct domains in the RANK cytoplasmic tail. These TRAF binding domains were shown to be functionally important for the RANK-dependent induction of NF-kappaB and c-Jun NH2-terminal kinase activities. Site-directed mutagenesis demonstrated that these TRAF binding sites exhibited selective binding for different TRAF proteins. In particular, TRAF6 interacted with membrane-proximal determinants distinct from those binding TRAFs 1, 2, 3, and 5. When this membrane-proximal TRAF6 interaction domain was deleted, RANK-mediated NF-kappaB signaling was completely inhibited while c-Jun NH2-terminal kinase activation was partially inhibited. An NH2-terminal truncation mutant of TRAF6 inhibited RANKL-mediated NF-kappaB activation, but failed to affect constitutive signaling induced by receptor overexpression, revealing a selective role for TRAF6 in ligand-induced activation events. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 516, "end": 527}, "arguments": [{"role": "Theme", "text": "RANK", "start": 531, "end": 535}, {"role": "Theme2", "text": "tumor necrosis factor receptor-associated factors (TRAFs) 1", "start": 541, "end": 600}]}, {"trigger": {"text": "interaction", "start": 516, "end": 527}, "arguments": [{"role": "Theme", "text": "RANK", "start": 531, "end": 535}, {"role": "Theme2", "text": "2", "start": 602, "end": 603}]}, {"trigger": {"text": "interaction", "start": 516, "end": 527}, "arguments": [{"role": "Theme", "text": "RANK", "start": 531, "end": 535}, {"role": "Theme2", "text": "3", "start": 605, "end": 606}]}, {"trigger": {"text": "interaction", "start": 516, "end": 527}, "arguments": [{"role": "Theme", "text": "RANK", "start": 531, "end": 535}, {"role": "Theme2", "text": "5", "start": 608, "end": 609}]}, {"trigger": {"text": "interaction", "start": 516, "end": 527}, "arguments": [{"role": "Theme", "text": "RANK", "start": 531, "end": 535}, {"role": "Theme2", "text": "6", "start": 615, "end": 616}]}, {"trigger": {"text": "interaction", "start": 698, "end": 709}, "arguments": [{"role": "Theme", "text": "RANK", "start": 693, "end": 697}]}, {"trigger": {"text": "interacted", "start": 1113, "end": 1123}, "arguments": [{"role": "Theme", "text": "TRAF6", "start": 1107, "end": 1112}]}, {"trigger": {"text": "binding", "start": 1180, "end": 1187}, "arguments": [{"role": "Theme", "text": "TRAFs 1", "start": 1188, "end": 1195}]}, {"trigger": {"text": "binding", "start": 1180, "end": 1187}, "arguments": [{"role": "Theme", "text": "2", "start": 1197, "end": 1198}]}, {"trigger": {"text": "binding", "start": 1180, "end": 1187}, "arguments": [{"role": "Theme", "text": "3", "start": 1200, "end": 1201}]}, {"trigger": {"text": "binding", "start": 1180, "end": 1187}, "arguments": [{"role": "Theme", "text": "5", "start": 1207, "end": 1208}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 312, "end": 321}, "arguments": [{"role": "Theme", "text": "RANK", "start": 217, "end": 221}]}]}}, "schema": []} {"input": "Regulation of interleukin-1beta transcription by Epstein-Barr virus involves a number of latent proteins via their interaction with RBP. \nEpstein-Barr virus (EBV) infects B cells, resulting in the outgrowth of immortalised lymphoblastoid cell lines (LCLs). Here, we demonstrate through the use of intracellular staining that interleukin-1beta (IL-1beta) is expressed in LCLs and investigate the influence of the individual latent proteins on the expression of IL-1beta. Using RT-PCR, IL-1beta was shown to be up-regulated in EBV-transformed LCLs as well as in group III Burkitt's lymphoma (BL) cell lines, compared with group I BL cell lines. The up-regulation of IL-1beta message could be mediated by the latent membrane protein-1, EBV nuclear proteins 2, 3, 4, and 6 genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that the -300 region of the IL-1beta promoter, which contains a nuclear factor-kappaB (NF-kappaB) binding site, contained a functional RBP binding site. Binding of RBP to this site could be inhibited by addition of EBV nuclear proteins 3 and 6, suggesting that these proteins displace RBP from its recognition sequence, removing transcriptional repression and allowing gene transcription to occur. In group I BL cells, containing low levels of NF-kappaB, only RBP binding was observed in EMSAs, whereas NF-kappaB binding could be demonstrated in EBV-transformed B cell lines containing high levels of activated NF-kappaB. In addition, the expression of latent membrane protein-1 led to activation of NF-kappaB that was capable of binding the IL-1beta promoter. The study demonstrates that EBV can up-regulate IL-1beta expression, possibly by using RBP, NF-kappaB, or both. Copyright 1998 Academic Press. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 1565, "end": 1572}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1577, "end": 1585}, {"role": "Site", "text": "promoter", "start": 1586, "end": 1594}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 357, "end": 366}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 344, "end": 352}]}, {"trigger": {"text": "expression", "start": 446, "end": 456}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 460, "end": 468}]}, {"trigger": {"text": "expression", "start": 1474, "end": 1484}, "arguments": [{"role": "Theme", "text": "latent membrane protein-1", "start": 1488, "end": 1513}]}, {"trigger": {"text": "expression", "start": 1653, "end": 1663}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 1644, "end": 1652}]}], "positive regulation": [{"trigger": {"text": "up-regulated", "start": 509, "end": 521}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 484, "end": 492}]}, {"trigger": {"text": "up-regulation", "start": 647, "end": 660}, "arguments": [{"role": "Theme", "text": "IL-1beta", "start": 664, "end": 672}]}, {"trigger": {"text": "mediated", "start": 690, "end": 698}, "arguments": [{"role": "Theme", "text": "up-regulation", "start": 647, "end": 660}, {"role": "Cause", "text": "latent membrane protein-1", "start": 706, "end": 731}]}, {"trigger": {"text": "mediated", "start": 690, "end": 698}, "arguments": [{"role": "Theme", "text": "up-regulation", "start": 647, "end": 660}, {"role": "Cause", "text": "EBV nuclear proteins 2", "start": 733, "end": 755}]}, {"trigger": {"text": "mediated", "start": 690, "end": 698}, "arguments": [{"role": "Theme", "text": "up-regulation", "start": 647, "end": 660}, {"role": "Cause", "text": "3", "start": 757, "end": 758}]}, {"trigger": {"text": "mediated", "start": 690, "end": 698}, "arguments": [{"role": "Theme", "text": "up-regulation", "start": 647, "end": 660}, {"role": "Cause", "text": "4", "start": 760, "end": 761}]}, {"trigger": {"text": "mediated", "start": 690, "end": 698}, "arguments": [{"role": "Theme", "text": "up-regulation", "start": 647, "end": 660}, {"role": "Cause", "text": "6 genes", "start": 767, "end": 774}]}, {"trigger": {"text": "led", "start": 1514, "end": 1517}, "arguments": [{"role": "Cause", "text": "expression", "start": 1474, "end": 1484}, {"role": "Theme", "text": "activation", "start": 1521, "end": 1531}]}, {"trigger": {"text": "activation", "start": 1521, "end": 1531}, "arguments": [{"role": "Theme", "text": "binding", "start": 1565, "end": 1572}]}, {"trigger": {"text": "up-regulate", "start": 1632, "end": 1643}, "arguments": [{"role": "Theme", "text": "expression", "start": 1653, "end": 1663}]}], "regulation": [{"trigger": {"text": "Regulation", "start": 0, "end": 10}, "arguments": [{"role": "Theme", "text": "transcription", "start": 32, "end": 45}]}, {"trigger": {"text": "involves", "start": 68, "end": 76}, "arguments": [{"role": "Theme", "text": "Regulation", "start": 0, "end": 10}]}, {"trigger": {"text": "influence", "start": 395, "end": 404}, "arguments": [{"role": "Theme", "text": "expression", "start": 446, "end": 456}]}], "transcription": [{"trigger": {"text": "transcription", "start": 32, "end": 45}, "arguments": [{"role": "Theme", "text": "interleukin-1beta", "start": 14, "end": 31}]}]}}, "schema": []} {"input": "Characterization of the human elk-1 promoter. Potential role of a downstream intronic sequence for elk-1 gene expression in monocytes. \nTo characterize the human elk-1 promoter, we mapped the transcriptional start site and isolated elk-1-specific genomic phage clones that contained extensive upstream and downstream sequences. A TATA-like motif was identified immediately upstream of the transcriptional start site. Functional analyses of DNA fragments containing the TATA element and the identification of a DNase I-hypersensitive chromatin site (HS 1) in close proximity to the TATA box suggest that the identified TATA motif is important for elk-1 transcription in vivo. Sequences upstream and downstream from the TATA box were found to contribute to elk-1 promoter activity. A second hypersensitive site (HS 2) was identified within the first intron in pre-monocytic cells, which express Elk-1 only when differentiating to monocytes. In a variety of other cell types, which display a constitutive Elk-1 expression, HS 2 did not exist, suggesting that inducibility of elk-1 expression is associated with the presence of HS 2. Egr-1 and the serum response factor were found to interact specifically with the intronic sequence at +265 and +448, respectively. Because Egr-1 mRNA and protein levels were observed to increase significantly before induction of elk-1 expression, we propose that Egr-1 is important for the regulation of elk-1 transcription in differentiating monocytes. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "interact", "start": 1180, "end": 1188}, "arguments": [{"role": "Theme", "text": "Egr-1", "start": 1130, "end": 1135}]}, {"trigger": {"text": "interact", "start": 1180, "end": 1188}, "arguments": [{"role": "Theme", "text": "serum response factor", "start": 1144, "end": 1165}]}], "gene expression": [{"trigger": {"text": "gene expression", "start": 105, "end": 120}, "arguments": [{"role": "Theme", "text": "elk-1", "start": 99, "end": 104}]}, {"trigger": {"text": "express", "start": 885, "end": 892}, "arguments": [{"role": "Theme", "text": "Elk-1", "start": 893, "end": 898}]}, {"trigger": {"text": "expression", "start": 1008, "end": 1018}, "arguments": [{"role": "Theme", "text": "Elk-1", "start": 1002, "end": 1007}]}, {"trigger": {"text": "expression", "start": 1078, "end": 1088}, "arguments": [{"role": "Theme", "text": "elk-1", "start": 1072, "end": 1077}]}, {"trigger": {"text": "expression", "start": 1365, "end": 1375}, "arguments": [{"role": "Theme", "text": "elk-1", "start": 1359, "end": 1364}]}], "positive regulation": [{"trigger": {"text": "contribute", "start": 741, "end": 751}, "arguments": [{"role": "Theme", "text": "elk-1", "start": 755, "end": 760}, {"role": "Site", "text": "promoter", "start": 761, "end": 769}]}, {"trigger": {"text": "inducibility", "start": 1056, "end": 1068}, "arguments": [{"role": "Theme", "text": "expression", "start": 1078, "end": 1088}]}, {"trigger": {"text": "increase", "start": 1316, "end": 1324}, "arguments": [{"role": "Theme", "text": "Egr-1", "start": 1269, "end": 1274}]}, {"trigger": {"text": "induction", "start": 1346, "end": 1355}, "arguments": [{"role": "Theme", "text": "expression", "start": 1365, "end": 1375}]}], "regulation": [{"trigger": {"text": "role", "start": 56, "end": 60}, "arguments": [{"role": "Theme", "text": "gene expression", "start": 105, "end": 120}]}, {"trigger": {"text": "important", "start": 632, "end": 641}, "arguments": [{"role": "Theme", "text": "transcription", "start": 652, "end": 665}]}], "transcription": [{"trigger": {"text": "transcription", "start": 652, "end": 665}, "arguments": [{"role": "Theme", "text": "elk-1", "start": 646, "end": 651}]}, {"trigger": {"text": "transcription", "start": 1440, "end": 1453}, "arguments": [{"role": "Theme", "text": "elk-1", "start": 1434, "end": 1439}]}]}}, "schema": []} {"input": "Functional association of Nmi with Stat5 and Stat1 in IL-2- and IFNgamma-mediated signaling. \nUsing the coiled-coil region of Stat5b as the bait in a yeast two-hybrid screen, we identified the association of Nmi, a protein of unknown function previously reported as an N-Myc interactor. We further show that Nmi interacts with all STATs except Stat2. We evaluated two cytokine systems, IL-2 and IFNgamma, and demonstrate that Nmi augments STAT-mediated transcription in response to these cytokines. Interestingly, Nmi lacks an intrinsic transcriptional activation domain; instead, Nmi enhances the association of CBP/p300 coactivator proteins with Stat1 and Stat5, and together with CBP/p300 can augment IL-2- and IFNgamma-dependent transcription. Therefore, our data not only reveal that Nmi can potentiate STAT-dependent transcription, but also suggest that it can augment coactivator protein recruitment to at least some members of a group of sequence-specific transcription factors. ", "output": {"json_structures": {"binding": [{"trigger": {"text": "association", "start": 193, "end": 204}, "arguments": [{"role": "Theme", "text": "Stat5b", "start": 126, "end": 132}, {"role": "Theme2", "text": "Nmi", "start": 208, "end": 211}]}, {"trigger": {"text": "interacts", "start": 312, "end": 321}, "arguments": [{"role": "Theme", "text": "Nmi", "start": 308, "end": 311}, {"role": "Theme2", "text": "Stat2", "start": 344, "end": 349}]}, {"trigger": {"text": "association", "start": 598, "end": 609}, "arguments": [{"role": "Theme", "text": "Stat1", "start": 648, "end": 653}]}], "positive regulation": [{"trigger": {"text": "enhances", "start": 585, "end": 593}, "arguments": [{"role": "Cause", "text": "Nmi", "start": 581, "end": 584}, {"role": "Theme", "text": "association", "start": 598, "end": 609}]}]}}, "schema": []}