{"input": "Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region\nAlthough the bcr-abl translocation has been shown to be the causative genetic aberration in chronic myeloid leukemia (CML), there is mounting evidence that the deregulation of other genes, such as the transcription factor interferon regulatory factor 4 (IRF-4), is also implicated in the pathogenesis of CML. Promoter methylation of CpG target sites or direct deletions/insertions of genes are mechanisms of a reversible or permanent silencing of gene expression, respectively. Therefore, we investigated whether IRF-4 promoter methylation or mutation may be involved in the regulation of IRF-4 expression in leukemia cells. Whereas promoter mutations or structural rearrangements could be excluded as a cause of altered IRF-4 expression in hematopoietic cells, the IRF-4 promoter methylation status was found to significantly influence IRF-4 transcription. First, treatment of IRF-4-negative lymphoid, myeloid and monocytic cell lines with the methylation-inhibitor 5-aza-2-deoxycytidine resulted in a time- and concentration-dependent increase of IRF-4 mRNA and protein levels. Second, using a restriction-PCR-assay and bisulfite-sequencing we identified specifically methylated CpG sites in IRF-4-negative but not in IRF-4-positive cells. Third, we clearly determined promoter methylation as a mechanism for IRF-4 down-regulation via reporter gene assays, but did not detect an association of methylational status and mRNA expression of DNA methyltransferases or methyl-CpG-binding proteins. Together, these data suggest CpG site-specific IRF-4 promoter methylation as a putative mechanism of down-regulated IRF-4 expression in leukemia.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 55, "end": 65}, "arguments": [{"role": "Theme", "text": "interferon regulatory factor 4", "start": 19, "end": 49}]}, {"trigger": {"text": "expression", "start": 740, "end": 750}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 734, "end": 739}]}, {"trigger": {"text": "expression", "start": 872, "end": 882}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 866, "end": 871}]}, {"trigger": {"text": "negative", "start": 1029, "end": 1037}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1023, "end": 1028}]}, {"trigger": {"text": "negative", "start": 1345, "end": 1353}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1339, "end": 1344}]}, {"trigger": {"text": "positive", "start": 1371, "end": 1379}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1365, "end": 1370}]}, {"trigger": {"text": "expression", "start": 1762, "end": 1772}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1756, "end": 1761}]}], "negative regulation": [{"trigger": {"text": "Down-regulation", "start": 0, "end": 15}, "arguments": [{"role": "Theme", "text": "expression", "start": 55, "end": 65}]}, {"trigger": {"text": "down-regulation", "start": 1462, "end": 1477}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1456, "end": 1461}]}, {"trigger": {"text": "down-regulated", "start": 1741, "end": 1755}, "arguments": [{"role": "Theme", "text": "expression", "start": 1762, "end": 1772}]}], "positive regulation": [{"trigger": {"text": "resulted", "start": 1134, "end": 1142}, "arguments": [{"role": "Theme", "text": "increase", "start": 1182, "end": 1190}]}, {"trigger": {"text": "increase", "start": 1182, "end": 1190}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1194, "end": 1199}]}], "regulation": [{"trigger": {"text": "deregulation", "start": 305, "end": 317}, "arguments": [{"role": "Theme", "text": "interferon regulatory factor 4", "start": 367, "end": 397}]}, {"trigger": {"text": "regulation", "start": 720, "end": 730}, "arguments": [{"role": "Theme", "text": "expression", "start": 740, "end": 750}]}, {"trigger": {"text": "altered", "start": 858, "end": 865}, "arguments": [{"role": "Theme", "text": "expression", "start": 872, "end": 882}]}, {"trigger": {"text": "influence", "start": 972, "end": 981}, "arguments": [{"role": "Theme", "text": "transcription", "start": 988, "end": 1001}]}], "transcription": [{"trigger": {"text": "transcription", "start": 988, "end": 1001}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 982, "end": 987}]}]}}, "schema": []}
{"input": "Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder with a typical three phased course (chronic, accelerated and blastic phase) reflecting the loss of differentiation and malignant progress which inevitably leads to death after the blastic phase (1,2). The hallmark genetic aberration of CML is a reciprocal chromosomal translocation t(9;22) leading to expression of a bcr-abl fusion gene, an aberrant activated tyrosine kinase (2). Treatment with interferon alpha (IFN-alpha) prolongs survival of CML patients and is associated with a complete cytogenetic response in 5-33% of CML patients (1,2). Recently, we described an impaired expression of the interferon regulatory factor 4 (IRF-4) in CML, correlating with poor response to IFN-alpha treatment (3). The cause of the silencing of IRF-4 level remained unclear.\nInterferon regulatory factors (IRFs) are a family of transcriptional regulators defined by a characteristic homology in their DNA-binding domain. They play an important role in the regulation of various genes (such as IFNs, interleukins, MHC class I/II), apoptosis and differentiation/maturation (4-6). IRF-4 (ICSAT/Pip/MUM1/LSIRF) is one member with very restricted expression pattern: Predominately B- and activated T-lymphocytes are IRF-4 positive (7-11). In contrast to other IRFs, expression of IRF-4 cannot be induced by IFNs, but by antigen stimulation, crosslinking of T- or B-cell receptors or phorbol-myristate-acetate (10,11). Consistent with the restriction of expression to immunocompetent cells, mice with deletion of IRF-4 failed to develop mature and functionally active B- and T-lymphocytes (12), and the impaired expression of IRF-4 in CML was predominately found in T-cells (3). These data suggest a crucial role for IRF-4 in the function of immune cells.\nMethylation of dinucleotide cytosine-guanosine motifs (CpG), especially in CpG islands located in promoter regions, is one of the mechanisms of gene regulation in mammals and a common event of gene silencing in human neoplasias (13,14). As opposed to normal cells, hypermethylation of CpG islands is a frequently observed phenomenon in every cancer type. De novo DNA methylation of genes such as cell cycle, DNA repair, apoptosis and tumor suppressor genes is therefore thought to be involved in tumorigenesis (15-17). Examples for such aberrated genes are MGMT, DAPK, p14ARF, p15INK4b, p16INK4a, BRCA1, CDH13 and APAF-1 (17-19). In CML, methylation is known to regulate expression of the c-abl, the bcr gene and others (20-23), and the extent of methylation in the c-abl promoter has been shown to be associated with advanced disease (24). Hypermethylation due to overexpression of DNA methyltransferases (DNMTs) remains one possible explanation for de novo methylation in tumorigenesis. Recently, DNMTs have been shown to be up-regulated in hematopoietic malignancies (25). Methyl-CpG-binding proteins (MBPs) are thought to inhibit the binding of transcriptional factors to the promoter and are therefore discussed as one mechanism of transcription inhibition by hypermethylation (26).\nIn this work, we studied mechanisms of IRF-4 gene expression silencing in leukemic cells. We analyzed the IRF-4 promoter region for genetic aberrations and methylational status in IRF-4-positive and -negative hematopoietic cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 372, "end": 382}, "arguments": [{"role": "Theme", "text": "bcr-abl fusion gene", "start": 388, "end": 407}]}, {"trigger": {"text": "expression", "start": 652, "end": 662}, "arguments": [{"role": "Theme", "text": "interferon regulatory factor 4", "start": 670, "end": 700}]}, {"trigger": {"text": "expression", "start": 1203, "end": 1213}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1139, "end": 1144}]}, {"trigger": {"text": "positive", "start": 1278, "end": 1286}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1272, "end": 1277}]}, {"trigger": {"text": "expression", "start": 1322, "end": 1332}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1336, "end": 1341}]}, {"trigger": {"text": "expression", "start": 1667, "end": 1677}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1681, "end": 1686}]}, {"trigger": {"text": "expression", "start": 2482, "end": 2492}, "arguments": [{"role": "Theme", "text": "c-abl", "start": 2500, "end": 2505}]}, {"trigger": {"text": "expression", "start": 2482, "end": 2492}, "arguments": [{"role": "Theme", "text": "bcr", "start": 2511, "end": 2514}]}, {"trigger": {"text": "expression", "start": 3149, "end": 3159}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 3138, "end": 3143}]}, {"trigger": {"text": "positive", "start": 3285, "end": 3293}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 3279, "end": 3284}]}, {"trigger": {"text": "negative", "start": 3299, "end": 3307}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 3279, "end": 3284}]}], "negative regulation": [{"trigger": {"text": "impaired", "start": 643, "end": 651}, "arguments": [{"role": "Theme", "text": "expression", "start": 652, "end": 662}]}, {"trigger": {"text": "silencing", "start": 793, "end": 802}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 806, "end": 811}]}, {"trigger": {"text": "deletion", "start": 1556, "end": 1564}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1568, "end": 1573}]}, {"trigger": {"text": "silencing", "start": 3160, "end": 3169}, "arguments": [{"role": "Theme", "text": "expression", "start": 3149, "end": 3159}]}], "positive regulation": [{"trigger": {"text": "leading", "start": 361, "end": 368}, "arguments": [{"role": "Theme", "text": "expression", "start": 372, "end": 382}]}, {"trigger": {"text": "induced", "start": 1352, "end": 1359}, "arguments": [{"role": "Theme", "text": "expression", "start": 1322, "end": 1332}]}], "regulation": [{"trigger": {"text": "regulate", "start": 2473, "end": 2481}, "arguments": [{"role": "Theme", "text": "expression", "start": 2482, "end": 2492}]}]}}, "schema": []}
{"input": "Cell lines\nK-562, Jurkat and U-937 were obtained from the ATCC (American Type Culture Collection, Rockville, USA) and EM-2, LAMA-84, CML-T1, BV-173, SD-1 and RPMI-8226 from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). All cell lines, except BV-173, SD-1 and RPMI-8226, were IRF-4-negative.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "negative", "start": 330, "end": 338}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 324, "end": 329}]}]}}, "schema": []}
{"input": "Cell culture and stimulation\nAll cell lines were maintained at 5% CO2 in RPMI 1640 medium with 1% glutamine (Gibco/BRL Eggenstein, Germany) supplemented with 10% fetal calf serum (Gibco/BRL), 1% penicillin/streptomycin (Biochrom, Berlin, Germany). When indicated, cells were treated with 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) (Sigma, Taufkirchen, Germany) for different time periods. Owing to their chemical instability fresh substances were re-added every 24 h.", "output": {"json_structures": {}}, "schema": []}
{"input": "Sequencing of the IRF-4 promoter\nFor analysis of the IRF-4 promoter region for permanent aberrations such as insertions/deletions or mutation, we PCR-amplified two fragments from genomic DNA, which was extracted from depicted cell lines with a commercial kit (Qiagen, Hilde, Germany) as recommended. The primers were 1-forward: 5'-TTGAGATGGAGTCTTGCTCTGT-3', 1-reverse: 5'-CCAGGACCTCAGGAGGCCAGTCA-3'; 2-forward: 5'-AGCGGTGAAACTGAGAGTGCGAGGT-3', 2-reverse: 5'-GCCACATCGCTGCAGTTTAG-3'. The products were cloned with the 'TOPO TA cloning kit' (Invitrogen, Groningen, The Netherlands). After bacterial amplification of the cloned PCR fragments by standard procedures, at least three clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Bio-systems, Foster City, USA) as recommended by the manufacturer.", "output": {"json_structures": {}}, "schema": []}
{"input": "Expression analysis\nTo analyze the IRF-4 transcriptional level, RNA was extracted from cells using the commercial RNAzol-kit (Paesel, Frankfurt, Germany). An aliquot of 1 mug total RNA was used for cDNA synthesis as described previously (27). RNA expression analysis for IRF-4 and the reference gene beta-actin was carried out by semi-quantitative PCR as described previously (3,27). PCR products were verified by automated sequencing. PCR primers and conditions for expression analysis of DNMT or MBP (DNMT1 DNMT3A, DNMT3B, MeCP, MBD1, MBD2 and MBD4) were published elsewhere (28).\nFor analysis of IRF-4 protein expression, a standard immunoblotting assay was performed as described previously (29). Briefly, protein lysates were generated by incubating 1 x 106 cells in 100 microl RIPA buffer (1% NP-40, 0.5% sodiumdesoxycholate, 0.1% SDS, 100 microg/ml phenylmethylsulfonyl fluoride, 10 microl/ml protease-inhibitory-mix, 1 micromol/ml sodiumorthovanadate in phosphate-buffered saline) for 30 min on ice. After centrifugation, protein concentration of the supernatant was determined by BCA-method (Pierce, Rockford, IL) as recommended. Protein lysates (70-100 microg) were electrophoresed on polyacrylamide gels and transferred to a PVDF-membrane (Immobilon P, 0.45 microm; Millipore, Eschborn, Germany). Membranes were blocked with 2.5% blocking reagent (Boehringer Mannheim, Germany) in TBST buffer (4.44 g/l Tris-HCL, 2.65 g/l TrisOH, 8.07 g/l NaCl, 0.2 g/l KCl and 500 microl/l Tween-20 in H2O) and subsequently incubated with primary antibody as indicated and horseradish peroxidase-conjugated secondary antibody, anti-mouse or anti-goat IgG (DAKO, Hamburg, Germany), respectively. The membranes were then developed with an ECL detection kit (Amersham Pharmacia Biotech, Freiburg, Germany). The primary antibodies were goat anti-IRF-4/ICSAT (M-17) (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-beta-actin (AC-74) (Sigma).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 467, "end": 477}, "arguments": [{"role": "Theme", "text": "(DNMT1", "start": 502, "end": 508}]}, {"trigger": {"text": "expression", "start": 467, "end": 477}, "arguments": [{"role": "Theme", "text": "DNMT3A", "start": 509, "end": 515}]}, {"trigger": {"text": "expression", "start": 467, "end": 477}, "arguments": [{"role": "Theme", "text": "DNMT3B", "start": 517, "end": 523}]}, {"trigger": {"text": "expression", "start": 467, "end": 477}, "arguments": [{"role": "Theme", "text": "MeCP", "start": 525, "end": 529}]}, {"trigger": {"text": "expression", "start": 467, "end": 477}, "arguments": [{"role": "Theme", "text": "MBD1", "start": 531, "end": 535}]}, {"trigger": {"text": "expression", "start": 467, "end": 477}, "arguments": [{"role": "Theme", "text": "MBD2", "start": 537, "end": 541}]}, {"trigger": {"text": "expression", "start": 467, "end": 477}, "arguments": [{"role": "Theme", "text": "MBD4", "start": 546, "end": 550}]}, {"trigger": {"text": "expression", "start": 613, "end": 623}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 599, "end": 604}]}], "transcription": [{"trigger": {"text": "transcriptional", "start": 41, "end": 56}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 35, "end": 40}]}, {"trigger": {"text": "RNA expression", "start": 243, "end": 257}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 271, "end": 276}]}, {"trigger": {"text": "RNA expression", "start": 243, "end": 257}, "arguments": [{"role": "Theme", "text": "beta-actin", "start": 300, "end": 310}]}]}}, "schema": []}
{"input": "Methylation-specific restriction-PCR-assay\nDNA was extracted with a commercial kit (Qiagen) as recommended. Since the restriction ability of several endonucleases is inhibited by methylation of their target sequence, we used methylation-sensitive enzymes HpaII and HaeII-isochizomer Bsp143II and Bsh1236I (MBI Fermentas, St Leon-Rot, Germany) (20,24). As control the methylation-resistant enzyme MspI and an enzyme with no recognition site in the target promoter, EcoRI, were used. DNA (0.8 microg) was digested by 40 U the respective enzyme for 6 h and, to ensure complete cleavage, additional 20 U for 16 h. Thereafter 100 ng of digested DNA was used to a PCR amplification of two fragments (F1 and F2) spanning part of the IRF-4 promoter (30) (GenBank U52683; see Figure 3A). The sequences of the primers were F1-forward: 5'-TTGAGATGGAGTCTTGCTCTGT-3', F1-reverse: ATCACTTCCAGACTTCAGTTCACCT-3' (341 bp); F2-forward: 5'-AAGGTGAACTGAAGTCTGGAAGTGA-3', F2-reverse: 5'-CCAGGACCTCAGGAGGCCAGTCA-3' (474 bp). The PCR conditions were described elsewhere (3). PCR was performed with an annealing temperature of 62degreesC and 35 cycles. When DNA was methylated at specific sites, the sensitive enzymes were not able to digest the DNA and amplification took place; in case of no methylation, DNA was digested and no product was generated. The PCR products were electrophoresed on a 3% agarose gel, were stained with ethidium bromide and photographed. PCR products were verified by automated sequencing.", "output": {"json_structures": {}}, "schema": []}
{"input": "Bisulfite treatment\nDNA was extracted as described above. Bisulfite treatment of DNA, leading to conversion of unmethylated cytosine to uracil residues and no change of methylated cytosine residues, was performed as described as follows. Briefly, 1 microg of DNA and 2 microg of poly(dA-dT)(poly(dA-dT) copolymers (Amersham Pharmacia Biotech) were denaturated for 20 min at 42degreesC in 0.3 M NaOH in a volume of 50 microl. Fresh solutions of 30 microl of 10 mM hydrochinon (Sigma) and 530 microl of 3 M sodium bisulfite (pH 5.0; Sigma) were added, the solution was gently mixed, overlayed with mineral oil and incubated in the dark for 12-13 h at 50degreesC. The aqueous phase was recovered using the 'Wizard DNA clean-up system' (Promega, Mannheim, Germany). The purified DNA was subsequently mixed with 1 M NaOH to a final concentration of 0.3 M and incubated for 20 min at 37degreesC to ensure complete desulfonisation. DNA was ethanol precipitated in the presence of 1/10 vol of 3 M sodium acetate, washed with 70% ethanol and resuspended in 50 microl H2O. Subsequent PCR amplification of 4 microl bisulfite-treated DNA was used for cloning of two fragments of the IRF-4 promoter (BS-I and BS-II) into pCR2.1 vector with the 'TOPO TA cloning kit' (Invitrogen) (see Figure 3A). The primers used for PCR amplification of the BS-I and BS-II fragments contain the putative altered sequence of the sense strand due to bisulfite treatment (converted cytosine residues are written in bold letters): BS-I-forward 5'-TATTTGGATTTTTAGGGAGTTTTTTTT-3', BS-I-reverse 5'-ACCCAACTCCCTTAAACTATTAAACT-3' (187 bp); BS-II-forward 5'-AGTTTAATAGTTTAAGGGAGTTGGGT-3', BS-II-reverse 5'-CTCACCCTAAACTCAAAACTAAAAAC-3' (674 bp). After bacterial amplification of the cloned PCR fragments by standard procedures, eight clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Biosystems).", "output": {"json_structures": {}}, "schema": []}
{"input": "In vitro methylation and reporter gene assays\nThe IRF-4 promoter-reporter gene construct was generously provided by J. Hiscott (31). Constructs were methylated in vitro with CpG Methylase (M.Sss I) as recommended by the manufacturer (NE Biolabs) and complete methylation was checked via restriction analysis (Figure 5A). Reporter gene assays using the dual luciferase assay (Promega) were performed similar to previous reports (29). Briefly, 5 nM of the reporter construct and the transfection control construct expressing the renilla luciferase gene were transientlyco-expressed via electroporation. The control construct served as an internal reference for transfection efficiency. Forty-eight hours after transfection, luciferase activity was measured with a LB 96 P microlumat (EG&G Berthold, Bad Wildbad, Germany). IRF-4 promoter activation was quantified as a ratio of measured firefly light units (flu) relative to renilla (rlu). Each experiment was carried out at least three times.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressing", "start": 512, "end": 522}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 535, "end": 545}]}, {"trigger": {"text": "expressed", "start": 570, "end": 579}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 535, "end": 545}]}], "positive regulation": [{"trigger": {"text": "activation", "start": 835, "end": 845}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 820, "end": 825}, {"role": "Site", "text": "promoter", "start": 826, "end": 834}]}]}}, "schema": []}
{"input": "Absence of IRF-4 expression in leukemia cells is not due to promoter alterations\nWe have previously demonstrated a lack of IRF-4 expression in leukemia patients and specifically in CML T-cells (3). Here, we demonstrate the absence of IRF-4 expression in various hematopoietic cell lines, such as Jurkat, a T-cell leukemia, CML-T1, a bcr-abl-positive T-cell line, K-562, a bcr-abl-positve erythroleukemia, U-937, a monocytic leukemia, EM-2 and LAMA-84, bcr-abl-positve myeloid leukemia, but not in SD-1, a bcr-abl-positive acute lymphoblastic leukemia (pre B-ALL), RPMI-8226, a multiple myeloma and BV-173, a bcr-abl-positive B-cell line (Figures 1A and 5D). After sequencing of the IRF-4 promoter, it could be excluded that absence of IRF-4 expression in any of the above cell lines was due to genetic aberrations. However, 2 bp changes (nucleotide -1081, T to C and -1068, A to C) could be detected in both the IRF-4-positive BV-173 and the IRF-4-negative LAMA-84, EM-2 and K-562 (Figure 1B). At position -116 an A to C substitution was found in EM-2, K-562 and CML-T1, whereas Jurkat, BV-173 and SD-1 exhibited a mixed A/C sequence and U-937, LAMA-84 and RPMI-8226 no substitution at all (Figure 1B). Consequently, these alterations are unlikely to affect IRF-4 expression.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 17, "end": 27}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 11, "end": 16}]}, {"trigger": {"text": "expression", "start": 129, "end": 139}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 123, "end": 128}]}, {"trigger": {"text": "expression", "start": 240, "end": 250}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 234, "end": 239}]}, {"trigger": {"text": "positive", "start": 341, "end": 349}, "arguments": [{"role": "Theme", "text": "bcr-abl", "start": 333, "end": 340}]}, {"trigger": {"text": "positve", "start": 380, "end": 387}, "arguments": [{"role": "Theme", "text": "bcr-abl", "start": 372, "end": 379}]}, {"trigger": {"text": "positve", "start": 460, "end": 467}, "arguments": [{"role": "Theme", "text": "bcr-abl", "start": 452, "end": 459}]}, {"trigger": {"text": "positive", "start": 513, "end": 521}, "arguments": [{"role": "Theme", "text": "bcr-abl", "start": 505, "end": 512}]}, {"trigger": {"text": "positive", "start": 616, "end": 624}, "arguments": [{"role": "Theme", "text": "bcr-abl", "start": 608, "end": 615}]}, {"trigger": {"text": "expression", "start": 741, "end": 751}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 735, "end": 740}]}, {"trigger": {"text": "positive", "start": 918, "end": 926}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 912, "end": 917}]}, {"trigger": {"text": "negative", "start": 948, "end": 956}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 942, "end": 947}]}, {"trigger": {"text": "expression", "start": 1264, "end": 1274}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1258, "end": 1263}]}], "negative regulation": [{"trigger": {"text": "Absence", "start": 0, "end": 7}, "arguments": [{"role": "Theme", "text": "expression", "start": 17, "end": 27}]}, {"trigger": {"text": "lack", "start": 115, "end": 119}, "arguments": [{"role": "Theme", "text": "expression", "start": 129, "end": 139}]}, {"trigger": {"text": "absence", "start": 724, "end": 731}, "arguments": [{"role": "Theme", "text": "expression", "start": 741, "end": 751}]}], "positive regulation": [{"trigger": {"text": "due", "start": 53, "end": 56}, "arguments": [{"role": "Theme", "text": "Absence", "start": 0, "end": 7}]}], "regulation": [{"trigger": {"text": "affect", "start": 1251, "end": 1257}, "arguments": [{"role": "Theme", "text": "expression", "start": 1264, "end": 1274}]}]}}, "schema": []}
{"input": "Increase of IRF-4 expression in hematopoietic cells after demethylating treatment\nWe next analyzed whether promoter methylation could be responsible for down-regulation of IRF-4 expression. A region including exon1 in the IRF-4 promoter exhibited a large number of CpG-rich sequences (Figure 3A). Several chemical substances such as 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) inhibit de novo and maintenance methylation, and thus can be used to discern promoter methylation (32,33). We used AzadC to generate unmethylated DNA. A 72 h AzadC-treatment resulted in a concentration-dependent activation of IRF-4 transcription in Jurkat and CML-T1 T-cells as well as in U-937, K-562 and EM-2 cell lines (Figure 2A). IRF-4 transcription was induced in a time-dependent manner and was observed as early as 24 h after treatment with AzadC and increased over time until 72 h (Figure 2B). Time and strength of the appearance of IRF-4 transcripts varied among cell lines, i.e. CML-T1 responded strongest to AzadC-treatment (data not shown). In line with this, AzadC-treatment of CML-T1 and LAMA-84 cells also translated in an induction of IRF-4 protein expression (Figure 2C). Accordingly, treatment of the IRF-4-positive cell line BV-173, SD-1 and RPMI-8226 with AzadC had no effect on IRF-4 expression (Figure 2D). There was no difference in the effects of AzaC versus AzadC, as both increased the IRF-4 mRNA level in CML-T1 cells as well (data not shown). This implied that promoter methylation may control IRF-4 expression, but an alternative explanation may be activation of positive transcriptional regulators of IRF-4 by AzadC (or AzaC).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 18, "end": 28}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 12, "end": 17}]}, {"trigger": {"text": "expression", "start": 178, "end": 188}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 172, "end": 177}]}, {"trigger": {"text": "positive", "start": 1213, "end": 1221}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1207, "end": 1212}]}, {"trigger": {"text": "expression", "start": 1293, "end": 1303}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1287, "end": 1292}]}, {"trigger": {"text": "expression", "start": 1516, "end": 1526}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1510, "end": 1515}]}], "positive regulation": [{"trigger": {"text": "Increase", "start": 0, "end": 8}, "arguments": [{"role": "Theme", "text": "expression", "start": 18, "end": 28}]}, {"trigger": {"text": "activation", "start": 599, "end": 609}, "arguments": [{"role": "Theme", "text": "transcription", "start": 619, "end": 632}]}, {"trigger": {"text": "induced", "start": 746, "end": 753}, "arguments": [{"role": "Theme", "text": "transcription", "start": 728, "end": 741}]}, {"trigger": {"text": "increased", "start": 846, "end": 855}, "arguments": [{"role": "Theme", "text": "transcription", "start": 728, "end": 741}]}, {"trigger": {"text": "increased", "start": 1386, "end": 1395}, "arguments": [{"role": "Theme", "text": "mRNA level", "start": 1406, "end": 1416}]}], "regulation": [{"trigger": {"text": "effect", "start": 1277, "end": 1283}, "arguments": [{"role": "Theme", "text": "expression", "start": 1293, "end": 1303}]}, {"trigger": {"text": "control", "start": 1502, "end": 1509}, "arguments": [{"role": "Theme", "text": "expression", "start": 1516, "end": 1526}]}], "transcription": [{"trigger": {"text": "transcription", "start": 619, "end": 632}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 613, "end": 618}]}, {"trigger": {"text": "transcription", "start": 728, "end": 741}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 722, "end": 727}]}, {"trigger": {"text": "transcripts", "start": 935, "end": 946}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 929, "end": 934}]}, {"trigger": {"text": "mRNA level", "start": 1406, "end": 1416}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1400, "end": 1405}]}]}}, "schema": []}
{"input": "Methylation-sensitive enzymes do not cut specific sites in the IRF-4 promoter in hematopoietic cells\nTo further investigate promoter methylation as a regulatory mechanism of IRF-4 gene expression, restriction-PCR-assays were performed (20,24), where only methylated DNA would not be cut enabling subsequent PCR amplification and vice versa. Genomic DNA from leukemic cells Jurkat, CML-T1, U-937, K-562, EM-2 and BV-173 was digested with the methylation-sensitive enzymes HpaII, Bsh1236I and HaeII-isochizomer Bsp143II. EcoRI, which has no recognition site within the IRF-4 promoter, and the methylation-resistant enzyme MspI served as controls. Two separate amplification reactions were performed, generating two fragments, F1 and F2 (Figure 3A). After digestion with HpaII and Bsp143II a sufficient PCR amplification of F1 and F2 was detected in DNA from IRF-4-negative Jurkat, CML-T1, U-937, K-562 and EM-2 cells, suggesting a promoter methylation (and restriction protection) at the respective recognition sites (Figure 3B and C). Notably, in IRF-4-positive SD-1 cells digestion with the methylation-sensitive enzymes completely inhibited amplification of F1 and F2. In IRF-4-positive BV-173 cells a HpaII, but not a Bsh1236I digestion, significantly reduced the amplifiable DNA message of F2 (Figure 3C), whereas amplification of F1 was not affected (Figure 3B). This implied that IRF-4 transcription in SD-1 and BV-173 cells is associated with less promoter methylation (in BV-173 especially at HpaII sites) as compared with the tested IRF-4-negative cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 185, "end": 195}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 174, "end": 179}]}, {"trigger": {"text": "negative", "start": 862, "end": 870}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 856, "end": 861}]}, {"trigger": {"text": "positive", "start": 1052, "end": 1060}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1046, "end": 1051}]}, {"trigger": {"text": "positive", "start": 1179, "end": 1187}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1173, "end": 1178}]}, {"trigger": {"text": "negative", "start": 1547, "end": 1555}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1541, "end": 1546}]}], "transcription": [{"trigger": {"text": "transcription", "start": 1391, "end": 1404}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1385, "end": 1390}]}]}}, "schema": []}
{"input": "Specific CpG sites in the IRF-4 promoter are methylated in hematopoietic cells\nIn order to exactly map the methylation sites within the IRF-4 promoter, we treated DNA of Jurkat, CML-T1, U-937, K-562 and EM-2 cells as well as of SD-1, RPMI-8226 and BV-173 control cells with bisulfite, which chemically converts unmethylated cytosine to uracil, whereas it has no effect on methylated cytosine, i.e. in CpG (34). This technique is especially useful for detection of unknown methylation patterns. PCR amplification, cloning and sequencing of the bisulfite-treated DNA showed a specific methylation pattern of the analyzed 62 CpG sites in all cell lines (Figure 4 and Table 1). In general, the methylational status ranged from one cell line with a nearly non-methylated IRF-4 promoter (SD-1, IRF-4-positive) to a completely methylated IRF-4 promoter in CML-T1 (IRF-4-negative). Interestingly, the percentage of CpG methylation in the IRF-4 promoter from IRF-4-positive cells was very low (mean 24%) as compared with IRF-4-negative cells (mean 94%) (Figure 4A and Table 1). A 5'-region (R1) with 13 hypermethylated CpG sites (mean number of methylated clones 5.5 of 8 with 77% methylated CpGs) was found in most cells (except SD-1 and RPMI-8226) and a 3'-region (R3) of 6 hypomethylated CpG sites (mean number of methylated clones 1.7 of 8 with 33% methylated CpGs) was found in most cells (except CML-T1 and U-937) (Figure 4A and Table 1).\nIntriguingly, a stretch of 13 CpG sites (#10-22; R2) was detected in between these regions, which were highly methylated in IRF-4-negative (mean number of methylated clones 7.1 of 8 with 89% methylated CpGs) but totally non-methylated in IRF-4-positive cells (Figure 4A and B). Furthermore, three CpG sites at the 5' end (#54, 56, 58) and two CpG motifs at the 3' end (#1, 2) showed this direct correlation between high methylation status and absence of IRF-4 expression. In addition, two CpG sites located in a NFkappaB (#48) and a SP1 element (#45) are less methylated in IRF-4-positive than in IRF-4-negative cells (mean number of methylated clones: 1/8 versus 8/8). These results indicate the involvement of CpG methylation in the regulation of IRF-4 expression in leukemic cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "positive", "start": 794, "end": 802}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 788, "end": 793}]}, {"trigger": {"text": "negative", "start": 863, "end": 871}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 857, "end": 862}]}, {"trigger": {"text": "positive", "start": 956, "end": 964}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 950, "end": 955}]}, {"trigger": {"text": "negative", "start": 1018, "end": 1026}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1012, "end": 1017}]}, {"trigger": {"text": "negative", "start": 1566, "end": 1574}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1560, "end": 1565}]}, {"trigger": {"text": "positive", "start": 1680, "end": 1688}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1674, "end": 1679}]}, {"trigger": {"text": "expression", "start": 1896, "end": 1906}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 1890, "end": 1895}]}, {"trigger": {"text": "positive", "start": 2016, "end": 2024}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 2010, "end": 2015}]}, {"trigger": {"text": "negative", "start": 2039, "end": 2047}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 2033, "end": 2038}]}, {"trigger": {"text": "expression", "start": 2191, "end": 2201}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 2185, "end": 2190}]}], "negative regulation": [{"trigger": {"text": "absence", "start": 1879, "end": 1886}, "arguments": [{"role": "Theme", "text": "expression", "start": 1896, "end": 1906}]}], "regulation": [{"trigger": {"text": "regulation", "start": 2171, "end": 2181}, "arguments": [{"role": "Theme", "text": "expression", "start": 2191, "end": 2201}]}]}}, "schema": []}
{"input": "In vitro methylation of an IRF-4 promoter-reporter construct decreases its activity\nTo provide evidence for a direct effect of methylational status on IRF-4 promoter activity we performed reporter gene assays with IRF-4 promoter constructs before and after their in vitro methylation. A complete methylation of these constructs was checked via restriction assays with methylation-sensitive endonucleases (Figure 5A). Intriguingly, methylation of the IRF-4 promoter significantly decreased promoter activity in IRF-4-positive SD-1 cells by 85.0% (Figure 5B). The silencing effect of CpG methylation was not restricted to IRF-4-positive cells, since in vitro methylation led to a 92.9% abrogation of promoter activity in IRF-4-negative Jurkat cells (Figure 5C). In contrast, control methylation of a reporter construct with a different promoter (FasL) as well as an empty vector had no effect on the reporter activity (data not shown). These data proved a direct association between methylation and activity of the IRF-4 promoter.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "positive", "start": 516, "end": 524}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 510, "end": 515}]}, {"trigger": {"text": "positive", "start": 626, "end": 634}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 620, "end": 625}]}, {"trigger": {"text": "negative", "start": 725, "end": 733}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 719, "end": 724}]}], "negative regulation": [{"trigger": {"text": "decreased", "start": 479, "end": 488}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 450, "end": 455}, {"role": "Site", "text": "promoter", "start": 489, "end": 497}]}, {"trigger": {"text": "abrogation", "start": 684, "end": 694}, "arguments": [{"role": "Site", "text": "promoter", "start": 698, "end": 706}, {"role": "Theme", "text": "IRF-4", "start": 719, "end": 724}]}]}}, "schema": []}
{"input": "mRNA expression of DNA methyltransferases and methyl-CpG-binding proteins may not be associated with IRF-4 promoter methylation\nSince abundance of DNMT and MBP contribute to promoter regulation via methylation (25,26,28), we studied their mRNA expression to investigate a possible mechanism for the observed methylation differences in the IRF-4 promoter. To this end, we did not detect a significant difference in DNMT (DNMT1, DNMT3A and DNMT3B) or MBP (MBD1, MBD2, MBD4 and MeCP) mRNA expression between IRF-4-positive and -negative cells (Figure 5D). In fact, all analyzed cells had moderate to high mRNA levels of these tested DNMT/MBPs and differences in expression were not correlated with IRF-4 status. These results indicate a distinct cause of the methylation differences in IRF-4-positive and -negative cells rather than changes in the DNMT and MBP mRNA transcription.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "positive", "start": 511, "end": 519}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 505, "end": 510}]}, {"trigger": {"text": "negative", "start": 525, "end": 533}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 505, "end": 510}]}, {"trigger": {"text": "positive", "start": 789, "end": 797}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 783, "end": 788}]}, {"trigger": {"text": "negative", "start": 803, "end": 811}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 783, "end": 788}]}], "regulation": [{"trigger": {"text": "regulation", "start": 183, "end": 193}, "arguments": [{"role": "Site", "text": "promoter", "start": 174, "end": 182}, {"role": "Theme", "text": "IRF-4", "start": 339, "end": 344}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 481, "end": 496}, "arguments": [{"role": "Theme", "text": "DNMT1", "start": 420, "end": 425}]}, {"trigger": {"text": "mRNA expression", "start": 481, "end": 496}, "arguments": [{"role": "Theme", "text": "DNMT3A", "start": 427, "end": 433}]}, {"trigger": {"text": "mRNA expression", "start": 481, "end": 496}, "arguments": [{"role": "Theme", "text": "DNMT3B", "start": 438, "end": 444}]}, {"trigger": {"text": "mRNA expression", "start": 481, "end": 496}, "arguments": [{"role": "Theme", "text": "MBD1", "start": 454, "end": 458}]}, {"trigger": {"text": "mRNA expression", "start": 481, "end": 496}, "arguments": [{"role": "Theme", "text": "MBD2", "start": 460, "end": 464}]}, {"trigger": {"text": "mRNA expression", "start": 481, "end": 496}, "arguments": [{"role": "Theme", "text": "MBD4", "start": 466, "end": 470}]}, {"trigger": {"text": "mRNA expression", "start": 481, "end": 496}, "arguments": [{"role": "Theme", "text": "MeCP", "start": 475, "end": 479}]}, {"trigger": {"text": "mRNA levels", "start": 602, "end": 613}, "arguments": [{"role": "Theme", "text": "DNMT1", "start": 420, "end": 425}]}, {"trigger": {"text": "mRNA levels", "start": 602, "end": 613}, "arguments": [{"role": "Theme", "text": "DNMT3A", "start": 427, "end": 433}]}, {"trigger": {"text": "mRNA levels", "start": 602, "end": 613}, "arguments": [{"role": "Theme", "text": "DNMT3B", "start": 438, "end": 444}]}, {"trigger": {"text": "mRNA levels", "start": 602, "end": 613}, "arguments": [{"role": "Theme", "text": "MBD1", "start": 454, "end": 458}]}, {"trigger": {"text": "mRNA levels", "start": 602, "end": 613}, "arguments": [{"role": "Theme", "text": "MBD2", "start": 460, "end": 464}]}, {"trigger": {"text": "mRNA levels", "start": 602, "end": 613}, "arguments": [{"role": "Theme", "text": "MBD4", "start": 466, "end": 470}]}, {"trigger": {"text": "mRNA levels", "start": 602, "end": 613}, "arguments": [{"role": "Theme", "text": "MeCP", "start": 475, "end": 479}]}, {"trigger": {"text": "expression", "start": 659, "end": 669}, "arguments": [{"role": "Theme", "text": "DNMT1", "start": 420, "end": 425}]}, {"trigger": {"text": "expression", "start": 659, "end": 669}, "arguments": [{"role": "Theme", "text": "DNMT3A", "start": 427, "end": 433}]}, {"trigger": {"text": "expression", "start": 659, "end": 669}, "arguments": [{"role": "Theme", "text": "DNMT3B", "start": 438, "end": 444}]}, {"trigger": {"text": "expression", "start": 659, "end": 669}, "arguments": [{"role": "Theme", "text": "MBD1", "start": 454, "end": 458}]}, {"trigger": {"text": "expression", "start": 659, "end": 669}, "arguments": [{"role": "Theme", "text": "MBD2", "start": 460, "end": 464}]}, {"trigger": {"text": "expression", "start": 659, "end": 669}, "arguments": [{"role": "Theme", "text": "MBD4", "start": 466, "end": 470}]}, {"trigger": {"text": "expression", "start": 659, "end": 669}, "arguments": [{"role": "Theme", "text": "MeCP", "start": 475, "end": 479}]}, {"trigger": {"text": "transcription", "start": 863, "end": 876}, "arguments": [{"role": "Theme", "text": "DNMT1", "start": 420, "end": 425}]}, {"trigger": {"text": "transcription", "start": 863, "end": 876}, "arguments": [{"role": "Theme", "text": "DNMT3A", "start": 427, "end": 433}]}, {"trigger": {"text": "transcription", "start": 863, "end": 876}, "arguments": [{"role": "Theme", "text": "DNMT3B", "start": 438, "end": 444}]}, {"trigger": {"text": "transcription", "start": 863, "end": 876}, "arguments": [{"role": "Theme", "text": "MBD1", "start": 454, "end": 458}]}, {"trigger": {"text": "transcription", "start": 863, "end": 876}, "arguments": [{"role": "Theme", "text": "MBD2", "start": 460, "end": 464}]}, {"trigger": {"text": "transcription", "start": 863, "end": 876}, "arguments": [{"role": "Theme", "text": "MBD4", "start": 466, "end": 470}]}, {"trigger": {"text": "transcription", "start": 863, "end": 876}, "arguments": [{"role": "Theme", "text": "MeCP", "start": 475, "end": 479}]}]}}, "schema": []}
{"input": "Many genetic lesions are known to influence gene expression of tumor suppressor genes. Whereas mutations and deletions or insertions have permanent effects, reversible mechanisms are gene methylation, or expression and activation of transcription factors, respectively. We studied a putative cause for absent IRF-4 expression in leukemia cells and first focused on genetic aberrations of the promoter. We observed no genetic alterations in the IRF-4 promoter, which can account for the lack of IRF-4 expression: The detected base pair changes at position -1081 (T to C substitution), at position -1068 (A to C substitution) and at position -116 (A to C substitution) are unlikely responsible for absent IRF-4-expression since the first two mutations were found both in IRF-4-positive and -negative cells whereas the latter change was not detected consistently in all IRF-4-negative or -positive cells and may thus be a polymorphism. All three substitutions did not change any known putative transcription factor binding sites (30,31) and also do not affect any restriction sites or primer binding sites of the used assays. However, permanent genetic variations in the IRF-4 coding sequence, such as deletions or mutations resulting in stop-codons have not been excluded by sequence analysis. Since IRF-4 expression in cell lines and CML can be induced by demethylation and successful IFN-alpha therapy (3), respectively, the existence of such genetic aberrations seems unlikely.\nWe then investigated whether the previously described down-regulation of IRF-4 expression in human myeloid leukemias was due to a differential hypermethylation of the promoter, since the presented re-expression due to AzadC-treatment might also be a result of activation of positive transcriptional regulators of IRF-4. Methylation of CpG sites is a common mechanism of silencing genes in leukemia and has also been shown for another IRF, IRF-7 (35) and for PU.1 (36), an interacting partner of IRF-4. To elucidate the relevance of this mechanism for the regulation of IRF-4 expression, various leukemic cells were treated with demethylating agents and promoters were sequenced after bisulfite treatment. We found that IRF-4 expression could indeed be connected to the methylation status of distinct CpG motifs in the IRF-4 promoter. In Figure 4A, those CpG sites are shown (bottom line), whose hypermethylation may account for the absence of IRF-4 expression in the respective cells. One of them (#54) is adjacent to an identified regulatory element (NFkappaB-site), indicating a possible involvement of this site. At two further CpG sites (#48, 45) the methylation status in IRF-4-positive was lower than that of IRF-4-negative cells. These CpG sites are located in an NFkappaB and an SP1 element (31) and thus may also play a role in regulation of IRF-4 expression. It has been shown that NFkappaB elements play an important role in IRF-4 induction as IRF-4 expression depends on binding of the transactivator c-Rel to these elements in the IRF-4 promoter (31,37). Furthermore, methylation of the central CpG in the NFkappaB element inhibits binding of the NFkappaB protein complexes (38), promoting the significance of the observed methylation differences in IRF-4-positive and -negative cells.\nVia in vitro methylation and reporter gene assays we could clearly appoint the silencing of the IRF-4 promoter to a methylation effect, which may thus be the mechanism of IRF-4 deregulation in vivo. One possible cause for the aberrant methylation in tumorigenesis is an increased level of DNMTs during the pathogenetic process. In colon, lung and hematologic malignancies, overexpression of DNMT1, a maintenance DNMT, has been detected (39-41). Furthermore, it has been shown that CML cells in the acute phase exhibited elevated levels of the three known DNMTs, while CML cells in chronic phase expressed normal levels of DNMTs if compared with normal bone marrow cells (25). Interestingly, a positive correlation between DNMT1 expression levels and hypermethylation of p15INK4b has been detected in AML (25). In this work, we did not detect significant mRNA expression differences of selected DNMT or MBP, making it an unlikely cause for the observed methylation and thus IRF-4 expression differences in leukemia cells.\nThe finding that IRF-4 expression is silenced by promoter hypermethylation might represent a mechanism that accounts for the previously observed loss of IRF-4 expression in CML. Indeed, several clinical trials with leukemia patients and patients with myelodysplastic syndromes demonstrated the potential clinical benefit of a treatment with demethylating agents (42-45).\nThe expression of another IRF, IFN consensus sequence binding protein (ICSBP/IRF-8), is impaired in myeloid leukemias especially CML (27,46,47). But in contrast to IRF-4, the loss of this IRF could not be reverted in ICSBP-negative cell lines (EM-2, CML-T1, K-562 and LAMA-84) by treatment with AzadC (Figure 6) and AzadC has no effect on ICSBP levels in ICSBP-positive U-937 cells (Figure 6). These data suggest a distinct regulatory mechanism for these two IRFs.\nIRF-4, similar to many other classical tumor suppressor genes p15INK4b, p16INK4a or p53, may thus be a subject of alterations in the promoter methylation status leading to expression changes, which might contribute to the initiation and/or progression of cancer. Still, the obvious functional diversity of IRF-4 remains remarkable and cannot be fully explained by the IRF-4 promoter methylation status. For example, IRF-4 is primarily known for its oncogenic features. In multiple myeloma (MM) a translocation on chromosome 14q was reported to lead to a fusion gene of immunoglobulin heavy-chain (IgH) and IRF-4 resulting in a subsequent overexpression of IRF-4 (48,49). In addition, abundant IRF-4 expression was found to be a marker for various subsets of lymphomas, such as diffuse large B-cell lymphomas, primary effusion lymphoma, and marginal zone lymphoma, and adult T-cell leukemia (11,31,50-52). This draws a more complex picture of the role of IRF-4. Down-regulation of IRF-4 may promote leukemogenesis in myeloid cell context (3), which was recently confirmed in IRF-4-/- ICSBP-/- double knock-out mice (53), while IRF-4 up-regulation may induce a growth advantage in lymphomas or MM (48).\nTaken together, our data suggest that IRF-4 promoter methylation regulates IRF-4 expression, and that aberrant expression of IRF-4 in certain types of leukemia may be a consequence of IRF-4 promoter hypermethylation.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interacting", "start": 1951, "end": 1962}, "arguments": [{"role": "Theme", "text": "PU.1", "start": 1937, "end": 1941}, {"role": "Theme2", "text": "IRF-4", "start": 1974, "end": 1979}]}, {"trigger": {"text": "binding", "start": 2962, "end": 2969}, "arguments": [{"role": "Site2", "text": "NFkappaB elements", "start": 2871, "end": 2888}, {"role": "Theme", "text": "c-Rel", "start": 2992, "end": 2997}, {"role": "Theme2", "text": "IRF-4", "start": 3023, "end": 3028}]}, 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"end": 325}]}, {"trigger": {"text": "lack", "start": 486, "end": 490}, "arguments": [{"role": "Theme", "text": "expression", "start": 500, "end": 510}]}, {"trigger": {"text": "absent", "start": 696, "end": 702}, "arguments": [{"role": "Theme", "text": "expression", "start": 709, "end": 719}]}, {"trigger": {"text": "down-regulation", "start": 1533, "end": 1548}, "arguments": [{"role": "Theme", "text": "expression", "start": 1558, "end": 1568}]}, {"trigger": {"text": "absence", "start": 2411, "end": 2418}, "arguments": [{"role": "Theme", "text": "expression", "start": 2428, "end": 2438}]}, {"trigger": {"text": "inhibits", "start": 3115, "end": 3123}, "arguments": [{"role": "Cause", "text": "IRF-4", "start": 3023, "end": 3028}, {"role": "CSite", "text": "CpG", "start": 3087, "end": 3090}, {"role": "Theme", "text": "binding", "start": 3124, "end": 3131}]}, {"trigger": {"text": "silencing", "start": 3357, "end": 3366}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 3374, "end": 3379}, {"role": "Site", "text": "promoter", "start": 3380, "end": 3388}]}, {"trigger": {"text": "loss", "start": 4444, "end": 4448}, "arguments": [{"role": "Theme", "text": "expression", "start": 4458, "end": 4468}]}, {"trigger": {"text": "impaired", "start": 4758, "end": 4766}, "arguments": [{"role": "Theme", "text": "expression", "start": 4674, "end": 4684}]}, {"trigger": {"text": "loss", "start": 4845, "end": 4849}, "arguments": [{"role": "Theme", "text": "IFN consensus sequence binding protein", "start": 4701, "end": 4739}]}, {"trigger": {"text": "reverted", "start": 4875, "end": 4883}, "arguments": [{"role": "Theme", "text": "loss", "start": 4845, "end": 4849}]}, {"trigger": {"text": "Down-regulation", "start": 6096, "end": 6111}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 6115, "end": 6120}]}], "positive regulation": [{"trigger": {"text": "responsible", "start": 680, "end": 691}, "arguments": [{"role": "Theme", "text": "absent", "start": 696, "end": 702}]}, {"trigger": {"text": "induced", "start": 1344, "end": 1351}, "arguments": [{"role": "Theme", "text": "expression", "start": 1304, "end": 1314}, {"role": "Cause", "text": "IFN-alpha", "start": 1384, "end": 1393}]}, {"trigger": {"text": "due", "start": 1600, "end": 1603}, "arguments": [{"role": "Theme", "text": "down-regulation", "start": 1533, "end": 1548}]}, {"trigger": {"text": "result", "start": 1729, "end": 1735}, "arguments": [{"role": "Theme", "text": "re-expression", "start": 1676, "end": 1689}]}, {"trigger": {"text": "account", "start": 2395, "end": 2402}, "arguments": [{"role": "Theme", "text": "absence", "start": 2411, "end": 2418}]}, {"trigger": {"text": "induction", "start": 2921, "end": 2930}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 2915, "end": 2920}]}, {"trigger": {"text": "depends", "start": 2951, "end": 2958}, "arguments": [{"role": "Theme", "text": "expression", "start": 2940, "end": 2950}, {"role": "Cause", "text": "binding", "start": 2962, "end": 2969}]}, {"trigger": {"text": "overexpression", "start": 3651, "end": 3665}, "arguments": [{"role": "Theme", "text": "DNMT1", "start": 3669, "end": 3674}]}, {"trigger": {"text": "resulting", "start": 5747, "end": 5756}, "arguments": [{"role": "Theme", "text": "overexpression", "start": 5773, "end": 5787}]}, {"trigger": {"text": "overexpression", "start": 5773, "end": 5787}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 5791, "end": 5796}]}, {"trigger": {"text": "up-regulation", "start": 6267, "end": 6280}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 6261, "end": 6266}]}, {"trigger": {"text": "consequence", "start": 6505, "end": 6516}, "arguments": [{"role": "Theme", "text": "expression", "start": 6447, "end": 6457}]}], "regulation": [{"trigger": {"text": "change", "start": 965, "end": 971}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 703, "end": 708}, {"role": "Site", "text": "transcription factor binding sites", "start": 991, "end": 1025}]}, {"trigger": {"text": "affect", "start": 1050, "end": 1056}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 703, "end": 708}, {"role": "Site", "text": "restriction sites", "start": 1061, "end": 1078}]}, {"trigger": {"text": "affect", "start": 1050, "end": 1056}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 703, "end": 708}, {"role": "Site", "text": "primer binding sites", "start": 1082, "end": 1102}]}, {"trigger": {"text": "regulation", "start": 2034, "end": 2044}, "arguments": [{"role": "Theme", "text": "expression", "start": 2054, "end": 2064}]}, {"trigger": {"text": "role", "start": 2808, "end": 2812}, "arguments": [{"role": "Theme", "text": "regulation", "start": 2816, "end": 2826}]}, {"trigger": {"text": "regulation", "start": 2816, "end": 2826}, "arguments": [{"role": "Theme", "text": "expression", "start": 2836, "end": 2846}]}, {"trigger": {"text": "important role", "start": 2897, "end": 2911}, "arguments": [{"role": "CSite", "text": "NFkappaB elements", "start": 2871, "end": 2888}, {"role": "Theme", "text": "induction", "start": 2921, "end": 2930}, {"role": "Cause", "text": "IRF-4", "start": 3023, "end": 3028}]}, {"trigger": {"text": "deregulation", "start": 3455, "end": 3467}, "arguments": [{"role": "Theme", "text": "IRF-4", "start": 3449, "end": 3454}]}, {"trigger": {"text": "effect", "start": 4999, "end": 5005}, "arguments": [{"role": "Theme", "text": "levels", "start": 5015, "end": 5021}]}, {"trigger": {"text": "changes", "start": 5318, "end": 5325}, "arguments": [{"role": "Theme", "text": "expression", "start": 5307, "end": 5317}]}, {"trigger": {"text": "regulates", "start": 6401, "end": 6410}, "arguments": [{"role": "Theme", "text": "expression", "start": 6417, "end": 6427}]}]}}, "schema": []}
{"input": "Foxp3 Represses Retroviral Transcription by Targeting Both NF-kappaB and CREB Pathways\nForkhead box (Fox)/winged-helix transcription factors regulate multiple aspects of immune responsiveness and Foxp3 is recognized as an essential functional marker of regulatory T cells. Herein we describe downstream signaling pathways targeted by Foxp3 that may negatively impact retroviral pathogenesis. Overexpression of Foxp3 in HEK 293T and purified CD4+ T cells resulted in a dose-dependent and time-dependent decrease in basal levels of nuclear factor-kappaB (NF-kappaB) activation. Deletion of the carboxyl-terminal forkhead (FKH) domain, critical for nuclear localization and DNA-binding activity, abrogated the ability of Foxp3 to suppress NF-kappaB activity in HEK 293T cells, but not in Jurkat or primary human CD4+ T cells. We further demonstrate that Foxp3 suppressed the transcription of two human retroviral promoters (HIV-1 and human T cell lymphotropic virus type I [HTLV-I]) utilizing NF-kappaB-dependent and NF-kappaB-independent mechanisms. Examination of the latter identified the cAMP-responsive element binding protein (CREB) pathway as a target of Foxp3. Finally, comparison of the percent Foxp3+CD4+CD25+ T cells to the HTLV-I proviral load in HTLV-I-infected asymptomatic carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis suggested that high Foxp3 expression is associated with low proviral load and absence of disease. These results suggest an expanded role for Foxp3 in regulating NF-kappaB- and CREB-dependent cellular and viral gene expression.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 675, "end": 682}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 718, "end": 723}]}], "gene expression": [{"trigger": {"text": "expression", "start": 1396, "end": 1406}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1390, "end": 1395}]}], "localization": [{"trigger": {"text": "localization", "start": 654, "end": 666}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 646, "end": 653}, {"role": "Theme", "text": "Foxp3", "start": 718, "end": 723}]}], "negative regulation": [{"trigger": {"text": "Deletion", "start": 576, "end": 584}, "arguments": [{"role": "Site", "text": "carboxyl-terminal forkhead (FKH) domain", "start": 592, "end": 631}, {"role": "Theme", "text": "Foxp3", "start": 718, "end": 723}]}], "positive regulation": [{"trigger": {"text": "Overexpression", "start": 392, "end": 406}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 410, "end": 415}]}, {"trigger": {"text": "critical", "start": 633, "end": 641}, "arguments": [{"role": "CSite", "text": "carboxyl-terminal forkhead (FKH) domain", "start": 592, "end": 631}, {"role": "Theme", "text": "localization", "start": 654, "end": 666}, {"role": "Cause", "text": "Foxp3", "start": 718, "end": 723}]}, {"trigger": {"text": "critical", "start": 633, "end": 641}, "arguments": [{"role": "CSite", "text": "carboxyl-terminal forkhead (FKH) domain", "start": 592, "end": 631}, {"role": "Theme", "text": "binding", "start": 675, "end": 682}, {"role": "Cause", "text": "Foxp3", "start": 718, "end": 723}]}, {"trigger": {"text": "high", "start": 1385, "end": 1389}, "arguments": [{"role": "Theme", "text": "expression", "start": 1396, "end": 1406}]}]}}, "schema": []}
{"input": "Immunological tolerance to self-antigens is the result of the deletion of self-reactive T lymphocytes in the thymus (central tolerance) and suppression of the activation of potentially self-reactive T lymphocytes in the periphery (peripheral tolerance) [1]. Suppression of pathogenic T cell responses is mediated by naturally arising CD4+CD25+ T regulatory cells (Tregs) [2,3]. Deficiencies in Treg development and function have been linked to the severe autoimmune disorder known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) [4]. In addition, recent studies have provided strong evidence that dysregulation of Treg development and/or function may be a significant factor in the pathogenesis of several autoimmune disorders (e.g., multiple sclerosis [5], myasthenia gravis [6], and type 1 diabetes [7]) and virus-induced immunologic disorders (e.g., human T lymphotropic virus type I [HTLV-I]-associated myelopathy/tropical spastic paraparesis [HAM/TSP], and HIV-induced AIDS [8-10]).\nThe transcription factor Foxp3 is a 431-amino acid (48-kDa) protein expressed at very high levels in CD4+CD25hi T cells and has previously been shown to be absolutely critical for Treg development and function [11-14]. Foxp3 contains a proline-rich amino-terminal domain reported to function as a nuclear factor of activated T cells (NF-AT) and nuclear factor-kappaB (NF-kappaB) binding domain, a central region containing a zinc finger and leucine zipper potentially important for protein-protein interactions, and a carboxyl-terminal forkhead (FKH) domain required for nuclear localization and DNA-binding activity [14-16]. Functional inactivation of Foxp3 by genetic mutations affecting the Foxp3 coding region, as demonstrated in IPEX, or repression of Foxp3 expression by the HTLV-I-encoded transactivator protein Tax, as recently reported in patients with HAM/TSP, results in loss of regulatory activity in CD4+CD25hi T cells [4,8,17]. Although it is clear that Foxp3 regulates T cell proliferation and cytokine production, very little is known concerning the molecular mechanisms of Foxp3 function.\nThe first evidence to indicate how Foxp3 promotes the development and function of regulatory T cells came from a report by Ziegler and colleagues [16], which suggested that Foxp3 could inhibit transcriptional activation by physically interacting with forkhead binding sites located immediately adjacent to critical cis-acting NF-AT binding sites found in various cytokine promoters (e.g., IL-2 promoter). That study also demonstrated that Foxp3 could repress activation of a synthetic reporter vector containing an SV40 promoter and three tandem copies of a forkhead binding site. These results provided additional evidence suggesting that Foxp3 transcriptional repression was mediated by binding in a sequence-specific manner to promoters containing forkhead binding sites. A recent study by Bettelli and colleagues [15] further demonstrated that Foxp3 could inhibit NF-AT as well as NF-kappaB activation, although the mechanism of suppression was shown to involve direct protein-protein interactions between NF-AT or NF-kappaB and Foxp3 rather than binding of Foxp3 to promoter elements adjacent to cis-acting NF-AT or NF-kappaB sites. Collectively, these data suggested that Foxp3 may function as a transcriptional repressor, potentially through the formation of both DNA-protein and protein-protein interactions.\nIn the present study, we expanded upon these observations by defining additional requirements of Foxp3-mediated repression of NF-kappaB activation, and investigated whether Foxp3 could target additional signaling pathways by examining transcriptional activation of NF-kappaB-dependent and NF-kappaB-independent retroviral pathogens. The characterization of the molecular targets of Foxp3 and the mechanism(s) utilized by Foxp3 to support Treg development and function will aid in our understanding of the role Tregs play in the pathogenesis of human autoimmune disease.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interactions", "start": 1521, "end": 1533}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1242, "end": 1247}]}, {"trigger": {"text": "binding", "start": 1623, "end": 1630}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1242, "end": 1247}]}, {"trigger": {"text": "interacting", "start": 2363, "end": 2374}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2302, "end": 2307}]}, {"trigger": {"text": "binding", "start": 2818, "end": 2825}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2769, "end": 2774}]}, {"trigger": {"text": "interactions", "start": 3118, "end": 3130}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 3162, "end": 3167}]}, {"trigger": {"text": "binding", "start": 3180, "end": 3187}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 3191, "end": 3196}]}, {"trigger": {"text": "interactions", "start": 3432, "end": 3444}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 3307, "end": 3312}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 1091, "end": 1100}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1048, "end": 1053}]}, {"trigger": {"text": "expression", "start": 1786, "end": 1796}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1780, "end": 1785}]}], "localization": [{"trigger": {"text": "localization", "start": 1602, "end": 1614}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1242, "end": 1247}, {"role": "ToLoc", "text": "nuclear", "start": 1594, "end": 1601}]}], "negative regulation": [{"trigger": {"text": "inactivation", "start": 1660, "end": 1672}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1676, "end": 1681}, {"role": "Cause", "text": "affecting", "start": 1703, "end": 1712}]}, {"trigger": {"text": "repression", "start": 1766, "end": 1776}, "arguments": [{"role": "Theme", "text": "expression", "start": 1786, "end": 1796}, {"role": "Cause", "text": "Tax", "start": 1842, "end": 1845}]}, {"trigger": {"text": "inhibit", "start": 2314, "end": 2321}, "arguments": [{"role": "Theme", "text": "transcriptional activation", "start": 2322, "end": 2348}, {"role": "Cause", "text": "interacting", "start": 2363, "end": 2374}]}], "positive regulation": [{"trigger": {"text": "important", "start": 1491, "end": 1500}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 1242, "end": 1247}, {"role": "CSite", "text": "leucine zipper", "start": 1464, "end": 1478}, {"role": "Theme", "text": "interactions", "start": 1521, "end": 1533}]}, {"trigger": {"text": "required", "start": 1581, "end": 1589}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 1242, "end": 1247}, {"role": "CSite", "text": "carboxyl-terminal forkhead (FKH) domain", "start": 1541, "end": 1580}, {"role": "Theme", "text": "localization", "start": 1602, "end": 1614}]}, {"trigger": {"text": "required", "start": 1581, "end": 1589}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 1242, "end": 1247}, {"role": "CSite", "text": "carboxyl-terminal forkhead (FKH) domain", "start": 1541, "end": 1580}, {"role": "Theme", "text": "binding", "start": 1623, "end": 1630}]}, {"trigger": {"text": "transcriptional activation", "start": 2322, "end": 2348}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 2518, "end": 2522}, {"role": "Site", "text": "promoter", "start": 2523, "end": 2531}]}, {"trigger": {"text": "formation", "start": 3382, "end": 3391}, "arguments": [{"role": "Theme", "text": "interactions", "start": 3432, "end": 3444}]}], "regulation": [{"trigger": {"text": "affecting", "start": 1703, "end": 1712}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1717, "end": 1722}, {"role": "Site", "text": "coding region", "start": 1723, "end": 1736}]}]}}, "schema": []}
{"input": "Foxp3 Suppresses NF-kappaB Dependent Transcriptional Activation\nTo ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-kappaB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20]. We analyzed the effect of Foxp3 overexpression on NF-kappaB activation in HEK 293T cells in dose-response and time course analyses. Transfection of HEK 293T cells with an NF-kappaB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression. Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-kappaB activation decreases proportionally (Figure 1A). Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B). Since NF-kappaB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-kappaB activation by Foxp3 compared to the control vector at each concentration (Figure 1A). Fold inhibition of NF-kappaB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR. To determine the level of Foxp3-mediated suppression of NF-kappaB activation over time, HEK 293T cells were transfected with an NF-kappaB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d. As shown in Figure 1C, NF-kappaB activation was suppressed by overexpression of Foxp3 at all time points. Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-kappaB activation (Figure 1D). These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-kappaB-dependent transcription in human cell lines as well as in primary human CD4+ T cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 672, "end": 682}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 666, "end": 671}]}, {"trigger": {"text": "concentration", "start": 895, "end": 908}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 912, "end": 917}]}, {"trigger": {"text": "expression", "start": 1107, "end": 1117}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1101, "end": 1106}]}, {"trigger": {"text": "expression", "start": 1689, "end": 1699}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1716, "end": 1721}]}], "positive regulation": [{"trigger": {"text": "overexpression", "start": 429, "end": 443}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 423, "end": 428}]}, {"trigger": {"text": "increases", "start": 941, "end": 950}, "arguments": [{"role": "Theme", "text": "concentration", "start": 895, "end": 908}]}, {"trigger": {"text": "overexpression", "start": 1833, "end": 1847}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1851, "end": 1856}]}, {"trigger": {"text": "overexpression", "start": 1973, "end": 1987}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1991, "end": 1996}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 848, "end": 863}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 842, "end": 847}]}, {"trigger": {"text": "mRNA expression", "start": 1471, "end": 1486}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1465, "end": 1470}]}]}}, "schema": []}
{"input": "The Carboxyl-Terminal FKH Domain Is Not Required for Suppression of NF-kappaB Activation in T Cells\nTo define the requirements of Foxp3 with respect to inhibition of NF-kappaB-dependent transcription, we utilized a mutant of Foxp3 lacking the FKH domain (Figure 2A) [16], similar to the scurfy mutant Foxp3 of mice, and a mutant Foxp3 protein from a patient with IPEX [4,11,14,17]. Unlike full-length Foxp3, which localizes almost exclusively to the nucleus and can bind in a sequence-specific manner to forkhead binding sites, the deltaFKH mutant fails to localize to the nucleus and thus cannot interact with promoter elements or nuclear proteins [16]. Therefore, we utilized the deltaFKH mutant to determine whether nuclear localization (or other function associated with the FKH domain) of Foxp3 was a prerequisite for inhibition of NF-kappaB activation. Although Foxp3 interaction with NF-kappaB presumably takes place in the nucleus, it may also be possible for a cytoplasmic Foxp3 protein to bind to NF-kappaB in the cytoplasm and prevent localization to the nucleus following an activation stimulus. Overexpression of full-length Foxp3, but not of deltaFKH, was able to suppress activation of a cotransfected NF-kappaB reporter vector in HEK 293T cells (Figure 2B). Both Foxp3 and deltaFKH were expressed at very high levels following transfection as detected by real-time RT-PCR (unpublished data). These data appear to suggest that the carboxyl-terminal FKH domain is critically important for Foxp3 to down-regulate NF-kappaB-dependent transcription. However, NF-kappaB activation was blocked to a similar extent by both full-length Foxp3 and deltaFKH in Jurkat T cells (Figure 2C) and primary human CD4+ T cells (Figure 2D). Western blot analysis of NF-kappaB p65 expression demonstrated that Foxp3 and deltaFKH does not block NF-kappaB activation at the level of p65 protein expression (Figure 2E). These results are very interesting with respect to Foxp3 function, because they suggest that the carboxyl-terminal FKH domain, and possibly nuclear localization, are dispensable for Foxp3 function in T cell populations. Alternative interpretations may include the possibility that the localization of deltaFKH differ between epithelial cells and T cells. In either case, these results suggest a cell type-specific mechanism of action for this Foxp3 mutant.", "output": {"json_structures": {"binding": [{"trigger": {"text": "bind", "start": 466, "end": 470}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 401, "end": 406}]}, {"trigger": {"text": "interact", "start": 597, "end": 605}, "arguments": [{"role": "Theme", "text": "deltaFKH mutant", "start": 532, "end": 547}]}, {"trigger": {"text": "interaction", "start": 874, "end": 885}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 868, "end": 873}]}, {"trigger": {"text": "bind", "start": 999, "end": 1003}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 982, "end": 987}]}], "gene expression": [{"trigger": {"text": "expression", "start": 1775, "end": 1785}, "arguments": [{"role": "Theme", "text": "p65", "start": 1771, "end": 1774}]}, {"trigger": {"text": "expression", "start": 1887, "end": 1897}, "arguments": [{"role": "Theme", "text": "p65", "start": 1875, "end": 1878}]}], "localization": [{"trigger": {"text": "localizes", "start": 414, "end": 423}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 401, "end": 406}, {"role": "ToLoc", "text": "nucleus", "start": 450, "end": 457}]}, {"trigger": {"text": "localize", "start": 557, "end": 565}, "arguments": [{"role": "Theme", "text": "deltaFKH mutant", "start": 532, "end": 547}, {"role": "ToLoc", "text": "nucleus", "start": 573, "end": 580}]}, {"trigger": {"text": "localization", "start": 727, "end": 739}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 719, "end": 726}, {"role": "Theme", "text": "Foxp3", "start": 794, "end": 799}]}, {"trigger": {"text": "localization", "start": 1046, "end": 1058}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 982, "end": 987}, {"role": "ToLoc", "text": "nucleus", "start": 1066, "end": 1073}]}, {"trigger": {"text": "localization", "start": 2059, "end": 2071}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 2051, "end": 2058}, {"role": "Theme", "text": "Foxp3", "start": 2093, "end": 2098}]}, {"trigger": {"text": "localization", "start": 2196, "end": 2208}, "arguments": [{"role": "Theme", "text": "deltaFKH", "start": 2212, "end": 2220}]}], "negative regulation": [{"trigger": {"text": "lacking", "start": 231, "end": 238}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 225, "end": 230}, {"role": "Site", "text": "FKH domain", "start": 243, "end": 253}]}, {"trigger": {"text": "prevent", "start": 1038, "end": 1045}, "arguments": [{"role": "Cause", "text": "bind", "start": 999, "end": 1003}, {"role": "Theme", "text": "localization", "start": 1046, "end": 1058}]}, {"trigger": {"text": "block", "start": 1832, "end": 1837}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 1804, "end": 1809}, {"role": "Theme", "text": "expression", "start": 1887, "end": 1897}]}, {"trigger": {"text": "block", "start": 1832, "end": 1837}, "arguments": [{"role": "Cause", "text": "deltaFKH", "start": 1814, "end": 1822}, {"role": "Theme", "text": "expression", "start": 1887, "end": 1897}]}], "positive regulation": [{"trigger": {"text": "following", "start": 1074, "end": 1083}, "arguments": [{"role": "Theme", "text": "localization", "start": 1046, "end": 1058}]}, {"trigger": {"text": "Overexpression", "start": 1108, "end": 1122}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1138, "end": 1143}]}, {"trigger": {"text": "Overexpression", "start": 1108, "end": 1122}, "arguments": [{"role": "Theme", "text": "deltaFKH", "start": 1156, "end": 1164}]}, {"trigger": {"text": "expressed", "start": 1303, "end": 1312}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1279, "end": 1284}]}, {"trigger": {"text": "expressed", "start": 1303, "end": 1312}, "arguments": [{"role": "Theme", "text": "deltaFKH", "start": 1289, "end": 1297}]}, {"trigger": {"text": "following", "start": 1333, "end": 1342}, "arguments": [{"role": "Theme", "text": "expressed", "start": 1303, "end": 1312}]}]}}, "schema": []}
{"input": "Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-kappaB\nIf Foxp3 functions as a repressor of NF-kappaB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-kappaB. To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-kappaB binding sites located between positions -102 and -81 with respect to the transcription initiation site [21]. NF-kappaB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21]. Overexpression of full-length Foxp3, but not deltaFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-kappaB reporter vector (Figure 2B). Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and deltaFKH in Jurkat T cells (Figure 3B). To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-kappaB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-kappaB sites located between -102 and -81 (HIV-1 delta-kappaB LTR) (Figure 3C). This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data). However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E). These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-kappaB binding sites. Residual levels of inhibition of the HIV-1 delta-kappaB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-kappaB sites within the HIV-1 LTR [22,23].", "output": {"json_structures": {"binding": [{"trigger": {"text": "interactions", "start": 1104, "end": 1116}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1048, "end": 1053}]}], "positive regulation": [{"trigger": {"text": "overexpression", "start": 194, "end": 208}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 188, "end": 193}]}, {"trigger": {"text": "Overexpression", "start": 672, "end": 686}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 702, "end": 707}]}, {"trigger": {"text": "Overexpression", "start": 672, "end": 686}, "arguments": [{"role": "Theme", "text": "deltaFKH", "start": 717, "end": 725}]}, {"trigger": {"text": "overexpression", "start": 1539, "end": 1553}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1533, "end": 1538}]}]}}, "schema": []}
{"input": "The Transactivation Functions of HTLV-I Tax Are Suppressed by Foxp3\nPrevious studies by Bettelli and colleagues have demonstrated that Foxp3 can repress both the basal levels of NF-kappaB activation as well as tumor necrosis factor-alpha-stimulated NF-kappaB activation [15]. Our next step was to determine whether Foxp3 could also suppress the activation of NF-kappaB caused by a strong viral transactivator protein. HTLV-I encodes a multifunctional transactivator protein, Tax, capable of activating both the NF-kappaB and CREB pathways [24-29]. Since the HTLV-I Tax protein can function at multiple levels in both the cytoplasm and the nucleus to stimulate activation of NF-kappaB [28,29], we hypothesized that overexpression of Foxp3 may interfere with this process. However, since Tax-dependent HTLV-I gene expression is independent of NF-kappaB [18], we also hypothesized that Foxp3 would not affect Tax-dependent activation of the HTLV-I LTR. To test these hypotheses, we overexpressed HTLV-I Tax, full-length Foxp3, and/or deltaFKH in HEK 293T cells cotransfected with an HTLV-I LTR or NF-kappaB reporter vector. As shown in Figure 4A, HTLV-I Tax strongly up-regulated NF-kappaB-dependent transcriptional activation (~60-fold). Interestingly, overexpression of Foxp3, but not deltaFKH, suppressed Tax-mediated activation of NF-kappaB-dependent transcription. These observations further suggest that the carboxyl-terminal FKH domain is required for inhibiting activation of NF-kappaB in the presence of Tax in HEK 293T cells, strikingly similar to the requirements of Foxp3 inhibition of basal NF-kappaB activation shown in Figure 2B. Transactivation of the HTLV-I LTR was stimulated about 55-fold by overexpression of Tax (Figure 4B), while transfection of Foxp3 suppressed Tax-dependent HTLV-I LTR activation, although HTLV-I LTR activation in the presence or absence of Tax is independent of NF-kappaB or NF-AT (another transcriptional activator known to interact with Foxp3). Furthermore, overexpression of deltaFKH also led to suppression of HTLV-I transactivation by Tax to a similar extent as full-length Foxp3 (Figure 4B). The suppressive effects shown in Figure 4A and 4B were not the result of Foxp3 down-regulating the expression of the transfected Tax plasmid as determined by real-time RT-PCR (Figure 4C). These results strongly suggest that Foxp3 interacts with transcriptional regulators in addition to NF-kappaB and NF-AT, and that the carboxyl-terminal FKH domain, and therefore localization to the nucleus, are not required for inhibition of Tax-mediated HTLV-I LTR activation (even in HEK 293T cells).\nTo determine whether Foxp3 inhibited the transactivation functions of Tax by directly associating with this viral protein, we generated an expression vector in which HTLV-I Tax was fused in-frame to the carboxyl terminus of the Gal4 DNA-binding domain (Gal4-BD). This Gal4-BD-Tax fusion protein activated transcription of a synthetic promoter containing five Gal4 binding sites, while Gal4-BD was insufficient to stimulate transcription by itself (Figure 4D). Transactivation of the Gal4-resposive promoter by Gal4-BD-Tax remained relatively unaffected by overexpression of either EGFP (control), Foxp3, or deltaFKH, suggesting that Foxp3 does not repress Tax transactivation by directly interfacing with the HTLV-I Tax protein. To confirm that Foxp3 had a direct effect on HTLV-I replication, we transfected HEK 293T cells with a well-characterized HTLV-I infectious molecular clone (termed ACH) [30] in the presence of full-length Foxp3, deltaFKH, or control vector. ACH has been previously shown to direct the expression of viral antigens, produce infectious virus, and transform CD4+ T cells both in vitro and in vivo [31,32]. After 24 h, the amount of viral antigen expression, in this case Tax mRNA, was detected by a sensitive real-time RT-PCR assay. As illustrated in Figure 5, the level of Tax mRNA synthesized from ACH was down-regulated in the presence of Foxp3 compared to the level produced in the presence of the control vector. deltaFKH did not have a discernable affect on Tax expression. These data indicate that Foxp3 is capable of repressing the expression of Tax from an infectious HTLV-I molecular clone.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interact", "start": 1965, "end": 1973}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1979, "end": 1984}]}, {"trigger": {"text": "interacts", "start": 2368, "end": 2377}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2362, "end": 2367}]}, {"trigger": {"text": "associating", "start": 2714, "end": 2725}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2649, "end": 2654}, {"role": "Theme2", "text": "Tax", "start": 2698, "end": 2701}]}], "gene expression": [{"trigger": {"text": "overexpression", "start": 1251, "end": 1265}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1269, "end": 1274}]}, {"trigger": {"text": "overexpression", "start": 1251, "end": 1265}, "arguments": [{"role": "Theme", "text": "deltaFKH", "start": 1284, "end": 1292}]}, {"trigger": {"text": "transfection", "start": 1749, "end": 1761}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1765, "end": 1770}]}, {"trigger": {"text": "expression", "start": 2237, "end": 2247}, "arguments": [{"role": "Theme", "text": "Tax", "start": 2267, "end": 2270}]}, {"trigger": {"text": "expression", "start": 4121, "end": 4131}, "arguments": [{"role": "Theme", "text": "Tax", "start": 4117, "end": 4120}]}, {"trigger": {"text": "expression", "start": 4193, "end": 4203}, "arguments": [{"role": "Theme", "text": "Tax", "start": 4207, "end": 4210}]}], "localization": [{"trigger": {"text": "localization", "start": 2503, "end": 2515}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2362, "end": 2367}, {"role": "ToLoc", "text": "nucleus", "start": 2523, "end": 2530}]}], "negative regulation": [{"trigger": {"text": "down-regulating", "start": 2217, "end": 2232}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 2211, "end": 2216}, {"role": "Theme", "text": "expression", "start": 2237, "end": 2247}]}, {"trigger": {"text": "interfacing", "start": 3316, "end": 3327}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 3261, "end": 3266}, {"role": "Theme", "text": "Tax", "start": 3344, "end": 3347}]}, {"trigger": {"text": "down-regulated", "start": 3961, "end": 3975}, "arguments": [{"role": "Theme", "text": "synthesized", "start": 3936, "end": 3947}, {"role": "Cause", "text": "Foxp3", "start": 3995, "end": 4000}]}, {"trigger": {"text": "repressing", "start": 4178, "end": 4188}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 4158, "end": 4163}, {"role": "Theme", "text": "expression", "start": 4193, "end": 4203}]}], "positive regulation": [{"trigger": {"text": "overexpression", "start": 714, "end": 728}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 732, "end": 737}]}, {"trigger": {"text": "overexpressed", "start": 979, "end": 992}, "arguments": [{"role": "Theme", "text": "Tax", "start": 1000, "end": 1003}]}, {"trigger": {"text": "overexpressed", "start": 979, "end": 992}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1017, "end": 1022}]}, {"trigger": {"text": "overexpressed", "start": 979, "end": 992}, "arguments": [{"role": "Theme", "text": "deltaFKH", "start": 1031, "end": 1039}]}, {"trigger": {"text": "overexpression", "start": 1708, "end": 1722}, "arguments": [{"role": "Theme", "text": "Tax", "start": 1726, "end": 1729}]}, {"trigger": {"text": "overexpression", "start": 2000, "end": 2014}, "arguments": [{"role": "Theme", "text": "deltaFKH", "start": 2018, "end": 2026}]}, {"trigger": {"text": "overexpression", "start": 3184, "end": 3198}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 3225, "end": 3230}]}, {"trigger": {"text": "overexpression", "start": 3184, "end": 3198}, "arguments": [{"role": "Theme", "text": "deltaFKH", "start": 3235, "end": 3243}]}], "regulation": [{"trigger": {"text": "affect", "start": 4107, "end": 4113}, "arguments": [{"role": "Cause", "text": "deltaFKH", "start": 4071, "end": 4079}, {"role": "Theme", "text": "expression", "start": 4121, "end": 4131}]}], "transcription": [{"trigger": {"text": "expression", "start": 3799, "end": 3809}, "arguments": [{"role": "Theme", "text": "Tax", "start": 3824, "end": 3827}]}, {"trigger": {"text": "synthesized", "start": 3936, "end": 3947}, "arguments": [{"role": "Theme", "text": "Tax", "start": 3927, "end": 3930}]}, {"trigger": {"text": "produced", "start": 4023, "end": 4031}, "arguments": [{"role": "Theme", "text": "Tax", "start": 3927, "end": 3930}]}]}}, "schema": []}
{"input": "Increased Foxp3 Protein Expression Is Associated with Low HTLV-I Proviral Load\nSince Foxp3 is expressed almost exclusively within CD4+CD25+ T cells, a major viral reservoir for HTLV-I [33], it was important to determine whether there was an association between Foxp3 and HTLV-I replication in infected patients. We therefore quantitated the Foxp3 protein expression in CD4+CD25+ T cells by flow cytometry and the HTLV-I proviral load (a surrogate marker of viral replication) by real-time PCR from eight patients with HAM/TSP and eight asymptomatic carriers (ACs). The data from this analysis is summarized in Table 1. As expected, patients with HAM/TSP exhibited significantly higher proviral loads (indicated as HTLV-I proviral DNA copies/100 cells) (34.68 +/- 23.19) compared to ACs (4.75 +/- 5.47) (p = 0.0008). The percentage of Foxp3+ cells within the CD4+CD25+ T cell population was significantly greater in ACs (43.23 +/- 12.95) than in HAM/TSP patients (18.59 +/- 5.77) (p = 0.0033). These data suggest that high levels of Foxp3 protein expression are associated with reduced HTLV-I replication in vivo. They also support our recent reports that high proviral loads, which have been shown to correlate with high Tax mRNA in HTLV-I-infected patients, are associated with reduced Foxp3 expression [8,34].", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Expression", "start": 24, "end": 34}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 10, "end": 15}]}, {"trigger": {"text": "expressed", "start": 94, "end": 103}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 85, "end": 90}]}, {"trigger": {"text": "expression", "start": 355, "end": 365}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 341, "end": 346}]}, {"trigger": {"text": "expression", "start": 1046, "end": 1056}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1032, "end": 1037}]}, {"trigger": {"text": "expression", "start": 1293, "end": 1303}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1287, "end": 1292}]}], "negative regulation": [{"trigger": {"text": "reduced", "start": 1279, "end": 1286}, "arguments": [{"role": "Theme", "text": "expression", "start": 1293, "end": 1303}]}], "positive regulation": [{"trigger": {"text": "Increased", "start": 0, "end": 9}, "arguments": [{"role": "Theme", "text": "Expression", "start": 24, "end": 34}]}, {"trigger": {"text": "high", "start": 1017, "end": 1021}, "arguments": [{"role": "Theme", "text": "expression", "start": 1046, "end": 1056}]}, {"trigger": {"text": "high", "start": 1216, "end": 1220}, "arguments": [{"role": "Theme", "text": "Tax", "start": 1221, "end": 1224}]}]}}, "schema": []}
{"input": "CREB Is a Target for Transcriptional Repression by Foxp3\nAlthough Foxp3 could down-regulate Tax-dependent transactivation of the HTLV-I LTR (Figure 4B) and inhibit Tax expression from an infectious molecular clone (Figure 5), Foxp3 failed to modulate Tax function in the absence of the viral promoter (Figure 4D). These results led us to hypothesize that Foxp3 acts on HTLV-I gene expression by interacting with proteins important for driving HTLV-I LTR activity in vivo. Previous studies have demonstrated that the Tax-responsive elements within the HTLV-I LTR play a crucial role in driving Tax-mediated transactivation of the HTLV-I LTR [18]. The Tax-responsive elements have been shown to resemble CREB binding sites, bind CREB in vitro and in vivo, and facilitate HTLV-I LTR activation both in the presence and in the absence of Tax [35,36]. Ching and colleagues [18] demonstrated that addition of a dominant-negative CREB expression vector resulted in nearly complete inhibition of Tax-mediated activation of the HTLV-I LTR, while blocking NF-kappaB activation by addition of a dominant-negative IKKbeta expression vector had no effect on Tax transactivation of the HTLV-I LTR. Therefore, we hypothesized that Foxp3 may inhibit Tax transactivation of the HTLV-I LTR via disruption of the CREB signaling pathway. To test this possibility, HEK 293T cells were transfected with an HTLV-I LTR or synthetic CREB reporter vector along with a control expression vector (EGFP) or expression vectors encoding Foxp3 or deltaFKH. As shown in Figure 6A, Foxp3 down-regulated basal activation of the HTLV-I LTR and transcription of a synthetic CREB reporter vector, suggesting that Foxp3 down-regulates HTLV-I LTR activation by targeting the CREB pathway. Deletion of the FKH domain of Foxp3 dampened the suppressive effect of Foxp3, but did not completely abrogate suppression, as is seen with NF-kappaB-responsive promoters in HEK 293T cells. Like NF-kappaB activation, CREB transcriptional activation was also suppressed by expression of Foxp3, and to a similar extent deltaFKH, in healthy donor CD4+ T cells (Figure 6B). Similarly, Foxp3 and deltaFKH also repressed basal HTLV-I LTR activation in primary human CD4+ T cells (Figure 6C). To our knowledge, this is the first evidence implicating CREB as a molecular target of Foxp3. As observed with NF-kappaB activation, deltaFKH was a more potent inhibitor of CREB activation in CD4+ T cells than in HEK 293T cells, further indicating that a cell type-specific mechanism of action may govern the function of this Foxp3 mutant.\nTo determine whether Foxp3 functioned by directly signaling through CREB, we utilized expression vectors encoding CREB-1 or c-Jun (a member of the activator protein 1 family of transcription factors) fused in-frame to the Gal4-BD (Gal4-BD-CREB-1 and Gal4-BD-c-Jun). As shown in Figure 6D, activation of a Gal4-responsive reporter vector by Gal4-BD-CREB-1 was down-regulated by Foxp3 compared to control vector (EGFP), indicating that Foxp3 functions by directly or indirectly interacting with CREB-1. However, Foxp3 failed to markedly affect transcriptional activation of Gal4-BD-c-Jun (c-Jun has also been demonstrated to bind to the HTLV-I LTR) and Gal4-BD-Tax (see Figure 5). Importantly, the mechanism of Foxp3-mediated inhibition of CREB-dependent transcription was not due to a block in CREB-1 protein expression, as determined by Western blot analysis (Figure 6E). Although these results demonstrate that Foxp3 functions as a co-repressor of CREB activation (in addition to NF-kappaB and NF-AT), we were unable to detect a direct physical interaction between CREB-1 and Foxp3 by coimmunoprecipitation or mammalian two-hybrid analysis (unpublished data). Therefore, our data suggest that Foxp3 may interfere with CREB signaling at an upstream event, such as phosphorylation of CREB or recruitment/function of coactivator proteins CREB-binding protein (CBP)/p300.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interacting", "start": 395, "end": 406}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 355, "end": 360}]}, {"trigger": {"text": "interacting", "start": 3050, "end": 3061}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 3008, "end": 3013}, {"role": "Theme2", "text": "CREB-1", "start": 3067, "end": 3073}]}, {"trigger": {"text": "bind", "start": 3197, "end": 3201}, "arguments": [{"role": "Theme", "text": "c-Jun", "start": 3161, "end": 3166}]}, {"trigger": {"text": "interaction", "start": 3620, "end": 3631}, "arguments": [{"role": "Theme", "text": "CREB-1", "start": 3640, "end": 3646}, {"role": "Theme2", "text": "Foxp3", "start": 3651, "end": 3656}]}, {"trigger": {"text": "recruitment", "start": 3865, "end": 3876}, "arguments": [{"role": "Theme", "text": "CREB-binding protein", "start": 3910, "end": 3930}]}, {"trigger": {"text": "recruitment", "start": 3865, "end": 3876}, "arguments": [{"role": "Theme", "text": "p300", "start": 3937, "end": 3941}]}], "gene expression": [{"trigger": {"text": "expression", "start": 168, "end": 178}, "arguments": [{"role": "Theme", "text": "Tax", "start": 164, "end": 167}]}, {"trigger": {"text": "expression", "start": 1110, "end": 1120}, "arguments": [{"role": "Theme", "text": "IKKbeta", "start": 1102, "end": 1109}]}, {"trigger": {"text": "expression", "start": 2020, "end": 2030}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2034, "end": 2039}]}, {"trigger": {"text": "expression", "start": 2020, "end": 2030}, "arguments": [{"role": "Theme", "text": "deltaFKH", "start": 2065, "end": 2073}]}, {"trigger": {"text": "expression", "start": 2660, "end": 2670}, "arguments": [{"role": "Theme", "text": "Gal4-BD-CREB-1", "start": 2805, "end": 2819}]}, {"trigger": {"text": "expression", "start": 2660, "end": 2670}, "arguments": [{"role": "Theme", "text": "Gal4-BD-c-Jun", "start": 2824, "end": 2837}]}, {"trigger": {"text": "expression", "start": 3382, "end": 3392}, "arguments": [{"role": "Theme", "text": "CREB-1", "start": 3367, "end": 3373}]}], "negative regulation": [{"trigger": {"text": "inhibit", "start": 156, "end": 163}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 66, "end": 71}, {"role": "Theme", "text": "expression", "start": 168, "end": 178}]}, {"trigger": {"text": "Deletion", "start": 1749, "end": 1757}, "arguments": [{"role": "Site", "text": "FKH domain", "start": 1765, "end": 1775}, {"role": "Theme", "text": "Foxp3", "start": 1779, "end": 1784}]}, {"trigger": {"text": "inhibitor", "start": 2394, "end": 2403}, "arguments": [{"role": "Cause", "text": "deltaFKH", "start": 2367, "end": 2375}, {"role": "Theme", "text": "activation", "start": 2412, "end": 2422}]}, {"trigger": {"text": "block", "start": 3358, "end": 3363}, "arguments": [{"role": "Theme", "text": "expression", "start": 3382, "end": 3392}]}, {"trigger": {"text": "interfere", "start": 3778, "end": 3787}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 3768, "end": 3773}, {"role": "Theme", "text": "signaling", "start": 3798, "end": 3807}]}], "positive regulation": [{"trigger": {"text": "activation", "start": 2412, "end": 2422}, "arguments": [{"role": "Theme", "text": "CREB", "start": 2407, "end": 2411}]}, {"trigger": {"text": "signaling", "start": 3798, "end": 3807}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 3865, "end": 3876}]}], "regulation": [{"trigger": {"text": "modulate", "start": 242, "end": 250}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 226, "end": 231}, {"role": "Theme", "text": "Tax", "start": 251, "end": 254}]}]}}, "schema": []}
{"input": "Foxp3 Antagonizes CREB Transcriptional Activation by Disrupting Coactivator Recruitment\nStimulation of CREB-dependent transcription by reagents that activate adenylate cyclase and increase cAMP levels (e.g., forskolin) increase the transactivation potential of CREB through phosphorylation of serine 133 by protein kinase A, which permits binding and recruitment of coactivators CBP/p300 to the promoter [37,38]. Phosphorylation of serine 133 does not, however, affect the DNA-binding activity of CREB in most cases [39-41]. Addition of forskolin to HEK 293T cells stimulated activation of a CREB reporter vector about 65 fold (Figure 7A). Overexpression of Foxp3 was capable of down-regulating forskolin-induced CREB transcriptional activation. The functional interaction between Foxp3 and CREB did not affect the DNA-binding activity of CREB-1, but did show a modest decrease in activating transcription factor 2 (ATF-2) DNA-binding activity in the presence of forskolin as determined by transcription factor ELISA (Figure 7B).\nWhile Foxp3 has been shown to bind to and repress activation of both NF-kappaB and NF-AT, exactly how Foxp3 functions to bring about this affect has not been elucidated. To determine how Foxp3 blocks CREB-dependent transcription, we examined whether Foxp3 was capable of (1) disrupting the recruitment of coactivator proteins and/or (2) preventing phosphorylation of CREB at serine 133 (which is a prerequisite for coactivator recruitment). Since both of these events are required for CREB-dependent gene expression, we hypothesized that Foxp3 may affect CREB activation at both steps. To determine whether Foxp3 can disrupt the function/recruitment of the coactivator protein p300, we introduced a Gal4 reporter vector and a Gal4-BD-CREB-1 expression vector into HEK 293T cells in the absence or presence of Foxp3, p300, and/or control expression vectors (Figure 7C). As expected, p300 overexpression stimulated transcription of the Gal4-BD-CREB-1 fusion protein. Foxp3, again, repressed basal levels of Gal4-BD-CREB-1 activation by more than 2-fold, while effectively neutralizing Gal4-BD-CREB-1 activation in the presence of p300. We next analyzed the effect of Foxp3 on phosphorylation of CREB at serine 133 in forskolin-treated HEK 293T cells by Western blot analysis. Overexpression of Foxp3 failed to reduce the detectable levels of CREB phosphorylation using a phosphospecific antibody for CREB-1 (unpublished data). However, when we attempted to determine whether Foxp3 could physically interact with the coactivator protein p300, we found that p300 immunoprecipitated Foxp3 when both proteins were overexpressed in HEK 293T cells (Figure 7D). Collectively, these results suggest that Foxp3 antagonizes CREB-dependent gene expression by directly interacting with coactivator p300 and interfering with its function and/or recruitment to CREB-responsive promoter sequences.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 339, "end": 346}, "arguments": [{"role": "Theme", "text": "CBP", "start": 379, "end": 382}]}, {"trigger": {"text": "binding", "start": 339, "end": 346}, "arguments": [{"role": "Theme", "text": "p300", "start": 383, "end": 387}]}, {"trigger": {"text": "interaction", "start": 761, "end": 772}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 781, "end": 786}]}, {"trigger": {"text": "binding", "start": 819, "end": 826}, "arguments": [{"role": "Theme", "text": "CREB-1", "start": 839, "end": 845}]}, {"trigger": {"text": "binding", "start": 927, "end": 934}, "arguments": [{"role": "Theme", "text": "activating transcription factor 2", "start": 881, "end": 914}]}, {"trigger": {"text": "bind", "start": 1060, "end": 1064}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1036, "end": 1041}]}, {"trigger": {"text": "interact", "start": 2526, "end": 2534}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2503, "end": 2508}, {"role": "Theme2", "text": "p300", "start": 2564, "end": 2568}]}, {"trigger": {"text": "immunoprecipitated", "start": 2589, "end": 2607}, "arguments": [{"role": "Theme", "text": "p300", "start": 2584, "end": 2588}, {"role": "Theme2", "text": "Foxp3", "start": 2608, "end": 2613}]}, {"trigger": {"text": "interacting", "start": 2785, "end": 2796}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2724, "end": 2729}, {"role": "Theme2", "text": "p300", "start": 2814, "end": 2818}]}, {"trigger": {"text": "recruitment", "start": 2860, "end": 2871}, "arguments": [{"role": "Theme", "text": "p300", "start": 2814, "end": 2818}]}], "gene expression": [{"trigger": {"text": "expression", "start": 1771, "end": 1781}, "arguments": [{"role": "Theme", "text": "Gal4-BD-CREB-1", "start": 1756, "end": 1770}]}, {"trigger": {"text": "expression", "start": 1867, "end": 1877}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1839, "end": 1844}]}, {"trigger": {"text": "expression", "start": 1867, "end": 1877}, "arguments": [{"role": "Theme", "text": "p300", "start": 1846, "end": 1850}]}, {"trigger": {"text": "overexpressed", "start": 2638, "end": 2651}, "arguments": [{"role": "Theme", "text": "p300", "start": 2584, "end": 2588}]}, {"trigger": {"text": "overexpressed", "start": 2638, "end": 2651}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2608, "end": 2613}]}], "negative regulation": [{"trigger": {"text": "decrease", "start": 869, "end": 877}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 781, "end": 786}, {"role": "Theme", "text": "binding", "start": 927, "end": 934}]}, {"trigger": {"text": "repressed", "start": 2009, "end": 2018}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 1995, "end": 2000}, {"role": "Theme", "text": "activation", "start": 2050, "end": 2060}]}, {"trigger": {"text": "neutralizing", "start": 2100, "end": 2112}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 1995, "end": 2000}, {"role": "Theme", "text": "activation", "start": 2128, "end": 2138}]}, {"trigger": {"text": "interfering", "start": 2823, "end": 2834}, "arguments": [{"role": "Cause", "text": "interacting", "start": 2785, "end": 2796}, {"role": "Theme", "text": "recruitment", "start": 2860, "end": 2871}]}], "positive regulation": [{"trigger": {"text": "permits", "start": 331, "end": 338}, "arguments": [{"role": "Theme", "text": "binding", "start": 339, "end": 346}]}, {"trigger": {"text": "Overexpression", "start": 640, "end": 654}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 658, "end": 663}]}, {"trigger": {"text": "overexpression", "start": 1917, "end": 1931}, "arguments": [{"role": "Theme", "text": "p300", "start": 1912, "end": 1916}]}, {"trigger": {"text": "stimulated", "start": 1932, "end": 1942}, "arguments": [{"role": "Cause", "text": "overexpression", "start": 1917, "end": 1931}, {"role": "Theme", "text": "transcription", "start": 1943, "end": 1956}]}, {"trigger": {"text": "activation", "start": 2050, "end": 2060}, "arguments": [{"role": "Theme", "text": "Gal4-BD-CREB-1", "start": 2035, "end": 2049}]}, {"trigger": {"text": "activation", "start": 2128, "end": 2138}, "arguments": [{"role": "Theme", "text": "Gal4-BD-CREB-1", "start": 2113, "end": 2127}]}, {"trigger": {"text": "Overexpression", "start": 2304, "end": 2318}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 2322, "end": 2327}]}, {"trigger": {"text": "when", "start": 2614, "end": 2618}, "arguments": [{"role": "Theme", "text": "immunoprecipitated", "start": 2589, "end": 2607}, {"role": "Cause", "text": "overexpressed", "start": 2638, "end": 2651}]}], "regulation": [{"trigger": {"text": "affect", "start": 804, "end": 810}, "arguments": [{"role": "Cause", "text": "interaction", "start": 761, "end": 772}, {"role": "Theme", "text": "binding", "start": 819, "end": 826}]}], "transcription": [{"trigger": {"text": "transcription", "start": 1943, "end": 1956}, "arguments": [{"role": "Theme", "text": "Gal4-BD-CREB-1", "start": 1964, "end": 1978}]}]}}, "schema": []}
{"input": "In the present study, we show that Foxp3 functions as a potent repressor of NF-kappaB- and CREB-dependent transcriptional activation. Furthermore, the carboxyl-terminal FKH domain appears to be dispensable for mediating these effects, at least in T cell populations. This observation may become important in light of recent reports suggesting that Foxp3 expression in thymic epithelial cells was crucial for directing development of T cells in the thymus [42]. Interestingly, the majority of the genetic mutations associated with IPEX, a severe autoimmune disorder caused by functional inactivation of Foxp3, map to the carboxyl-terminal FKH domain or the leucine zipper domain in the central region of the protein. Only one mutation associated with IPEX to date has been mapped to the amino-terminal proline-rich region [43]. It is possible that the FKH domain has a complex tertiary structure that is particularly sensitive to misfolding caused by genetic mutations and that an intact FKH domain is absolutely critical for promoting Foxp3 function in the nucleus, whereas the structure of the amino-terminal proline-rich region may tolerate certain mutations as long as the NF-kappaB/NF-AT binding motif remains unaltered. This motif may also include the zinc finger domain. A logical region that may be targeted by the amino-terminal proline-rich region of Foxp3 is the Rel homology domain found in both NF-kappaB and NF-AT family proteins. A region that may also be important with respect to Foxp3 function is the leucine zipper domain, as demonstrated by the number of mutations associated with IPEX that have been mapped in this region of Foxp3. The role of this domain in Foxp3 function remains uncharacterized, but may play a role in dimer formation as it does in other Foxp family members [44].\nBecause the pathogenesis of a number of retroviral-induced immunologic disorders such as HIV-1/AIDS and HTLV-I/HAM/TSP have been associated with dysregulation of Foxp3 expression [8,45], we also examined the role of Foxp3 in retroviral gene expression. HIV-1 LTR activation in CD4+ T cells is critically dependent on two tandem NF-kappaB sites located between nucleotide positions -102 and -81 within the HIV-1 enhancer region, whereas HTLV-I LTR activation in the presence or absence of the HTLV-I-encoded transactivator protein Tax is independent of NF-kappaB [18]. To our knowledge for the first time, Foxp3 was shown to have a direct effect on HIV-1 LTR transcription. Deletion of the NF-kappaB sites within the HIV-1 enhancer region reduced the responsiveness of the HIV-1 LTR to Foxp3-mediated suppression. In addition, the FKH domain of Foxp3 was required for this inhibitory effect in HEK 293T cells, but not in Jurkat T cells, similar to Foxp3-mediated suppression of a synthetic NF-kappaB reporter. The direct effect of Foxp3 down-regulating HIV-1 gene expression correlates well with recently reported evidence indicating that higher regulatory activity of CD4+CD25+ T cells from HIV-1-infected patients was associated with lower HIV-1 viral loads in these patients [46].\nFoxp3 also affected two well-known functions of HTLV-I Tax: transactivation of the NF-kappaB pathway and, most surprisingly, transactivation of the HTLV-I LTR. Transactivation of the HTLV-I LTR by Tax involves the interaction of ATF/CREB factors with Tax in the nucleus. Binding of Tax enhances ATF/CREB dimerization and promotes assembly of Tax-ATF/CREB complexes onto specific sequences in the viral promoter known as Tax-responsive elements. This series of steps allows Tax to recruit coactivator proteins CBP/p300 to the viral promoter and facilitate a high level of viral gene expression [24-26]. Transactivation of the NF-kappaB pathway by Tax was inhibited by overexpression of full-length Foxp3, but not deltaFKH, as seen with basal activation of the HIV-1 LTR and a synthetic NF-kappaB reporter in HEK 293T cells. However, Tax-mediated transactivation of the HTLV-I LTR was inhibited by overexpression of both full-length Foxp3 as well as deltaFKH in both HEK 293T cells and CD4+ T cells. We demonstrated that Foxp3 did not directly affect the functioning of Tax, but rather Foxp3 targeted the transcription factors required for Tax transactivation (i.e., NF-kappaB and a then-unknown cellular factor, which we identified in this study as CREB). The negative effect of Foxp3 on HTLV-I gene expression was confirmed utilizing an HTLV-I infectious molecular clone.\nImportantly, we demonstrated that HTLV-I-infected individuals with the highest levels of Foxp3 protein expression within the CD4+CD25+ T cells population exhibited lower proviral loads than did individuals with the lowest levels of Foxp3 protein expression. Previous studies have demonstrated that the HTLV-I proviral load directly correlates with HTLV-I Tax mRNA load, the frequency of immunopathogenic virus-specific CD8+ T cells, and disease severity in patients with HAM/TSP [47]. These results have important implications on the utility of Foxp3 in controlling viral gene expression and thus pathogenesis of HAM/TSP. Therefore, Foxp3 becomes an attractive target for the development of novel therapeutic applications directed at modulating the expression of this important regulatory protein, especially in light of recent observations that the expression of Foxp3 can also be down-regulated by HTLV-I Tax [8].\nAs the activation of the HTLV-I LTR depends primarily on ATF/CREB proteins (whether in the presence or the absence of Tax), we investigated whether Foxp3 could interact with this additional cellular signaling pathway. While the DNA-binding activity of CREB is, in most cases, constitutive, the transactivation potential of CREB is regulated by the phosphorylation of CREB and recruitment of CBP/p300 [48]. Our data demonstrate that Foxp3 interferes with the latter of these two processes and that the recruitment of the coactivator protein p300, and resulting transcriptional activation are blocked by Foxp3. This may be the result of the physical interaction we detected between Foxp3 and p300. With respect to HTLV-I LTR activity, while full-length Foxp3 inhibited both basal and Tax-dependent transcription by ~50%, deltaFKH appeared less effective in suppressing basal activation (~25% inhibition) compared to Tax-dependent activation (~50% inhibition). The effect of deltaFKH on basal activation of the HTLV-I LTR in HEK 293T cells was very similar to that shown for a synthetic CREB reporter, suggesting that the FKH domain of Foxp3 is important at some level. As observed with NF-kappaB activation, the Foxp3 mutant lacking the FKH domain was a stronger inhibitor of CREB activation in CD4+ T cells than in HEK 293T epithelial cells. Therefore, it appears that in CD4+ T cells, the FKH domain is dispensable for the proper functioning of Foxp3 with respect to both NF-kappaB and CREB activation.\nIn summary, this is, to our knowledge, the first direct evidence implicating a role for the Treg-specific transcription factor Foxp3 in regulating retroviral gene expression. In addition, we identify the CREB pathway as a molecular target of Foxp3. Since CREB has been shown to regulate multiple genes involved in transcription (e.g., JunD, c-Fos, signal transducer of activated T cells 3 [STAT3]), cell cycle (e.g., p15INK4b, cyclin A, cyclin D1), and immune regulation (e.g., IL-2, IL-6, T-cell receptor alpha) (reviewed in [48]), the findings presented in this report broaden the potential range of signaling pathways under the control of the regulatory protein Foxp3. Our evidence stresses the importance of Foxp3 expression and Treg function in the development and maintenance of protective immunity against HIV-1 and HTLV-I. Based on recent findings, Foxp3 may limit HIV-1 and HTLV-I transcription by interfering with activation of NF-kappaB and CREB pathways. However, observing that this inhibitory effect is not absolute, a low level of viral gene expression may persist in CD4+ T cells (in particular regulatory T cells, which are known reservoirs of HIV-1 and HTLV-I) and result in the accumulation of viral proteins that either stimulate NF-kappaB and/or CREB activation or directly inhibit Foxp3 expression or function. The imbalance of NF-kappaB and CREB activation caused by these viral gene products may be a crucial step in the pathogenesis of virus-induced immunological disorders such as AIDS and HAM/TSP. Future studies will be directed at identifying and characterizing cellular proteins that interact with Foxp3 both in the nucleus and cytoplasm, in order to better address how Foxp3 functions to guide the development and function of regulatory T cells in health and disease.", "output": {"json_structures": {"binding": [{"trigger": {"text": "targeted", "start": 1306, "end": 1314}, "arguments": [{"role": "Site", "text": "amino-terminal proline-rich region", "start": 1322, "end": 1356}, {"role": "Theme", "text": "Foxp3", "start": 1360, "end": 1365}]}, {"trigger": {"text": "dimer formation", "start": 1742, "end": 1757}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1679, "end": 1684}]}, {"trigger": {"text": "interaction", "start": 3301, "end": 3312}, "arguments": [{"role": "Theme", "text": "Tax", "start": 3338, "end": 3341}]}, {"trigger": {"text": "Binding", "start": 3358, "end": 3365}, "arguments": [{"role": "Theme", "text": "Tax", "start": 3369, "end": 3372}]}, {"trigger": {"text": "assembly", "start": 3417, "end": 3425}, "arguments": [{"role": "Theme", "text": "Tax", "start": 3429, "end": 3432}]}, {"trigger": {"text": "recruit", "start": 3567, "end": 3574}, "arguments": [{"role": "Theme", "text": "Tax", "start": 3560, "end": 3563}, {"role": "Theme2", "text": "CBP", "start": 3596, "end": 3599}]}, {"trigger": {"text": "recruit", "start": 3567, "end": 3574}, "arguments": [{"role": "Theme", "text": "Tax", "start": 3560, "end": 3563}, {"role": "Theme2", "text": "p300", "start": 3600, "end": 3604}]}, {"trigger": {"text": "recruitment", "start": 5751, "end": 5762}, "arguments": [{"role": "Theme", "text": "CBP", "start": 5766, "end": 5769}]}, {"trigger": {"text": "recruitment", "start": 5751, "end": 5762}, "arguments": [{"role": "Theme", "text": "p300", "start": 5770, "end": 5774}]}, {"trigger": {"text": "recruitment", "start": 5876, "end": 5887}, "arguments": [{"role": "Theme", "text": "p300", "start": 5915, "end": 5919}]}, {"trigger": {"text": "interaction", "start": 6023, "end": 6034}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 6055, "end": 6060}, {"role": "Theme2", "text": "p300", "start": 6065, "end": 6069}]}, {"trigger": {"text": "interact", "start": 8492, "end": 8500}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 8506, "end": 8511}]}], "gene expression": [{"trigger": {"text": "expression", "start": 354, "end": 364}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 348, "end": 353}]}, {"trigger": {"text": "expression", "start": 1972, "end": 1982}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 1966, "end": 1971}]}, {"trigger": {"text": "overexpression", "start": 3983, "end": 3997}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 4018, "end": 4023}]}, {"trigger": {"text": "overexpression", "start": 3983, "end": 3997}, "arguments": [{"role": "Theme", "text": "deltaFKH", "start": 4035, "end": 4043}]}, {"trigger": {"text": "expression", "start": 4562, "end": 4572}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 4548, "end": 4553}]}, {"trigger": {"text": "expression", "start": 4705, "end": 4715}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 4691, "end": 4696}]}, {"trigger": {"text": "expression", "start": 5208, "end": 5218}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 5092, "end": 5097}]}, {"trigger": {"text": "expression", "start": 5309, "end": 5319}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 5323, "end": 5328}]}, {"trigger": {"text": "expression", "start": 7596, "end": 7606}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 7590, "end": 7595}]}, {"trigger": {"text": "expression", "start": 8187, "end": 8197}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 8181, "end": 8186}]}], "negative regulation": [{"trigger": {"text": "inactivation", "start": 586, "end": 598}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 602, "end": 607}]}, {"trigger": {"text": "lowest", "start": 4674, "end": 4680}, "arguments": [{"role": "Theme", "text": "expression", "start": 4705, "end": 4715}]}, {"trigger": {"text": "down-regulated", "start": 5341, "end": 5355}, "arguments": [{"role": "Theme", "text": "expression", "start": 5309, "end": 5319}, {"role": "Cause", "text": "Tax", "start": 5366, "end": 5369}]}, {"trigger": {"text": "interferes", "start": 5813, "end": 5823}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 5751, "end": 5762}, {"role": "Cause", "text": "Foxp3", "start": 5807, "end": 5812}]}, {"trigger": {"text": "blocked", "start": 5966, "end": 5973}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 5876, "end": 5887}, {"role": "Cause", "text": "Foxp3", "start": 5977, "end": 5982}]}, {"trigger": {"text": "lacking", "start": 6598, "end": 6605}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 6585, "end": 6590}, {"role": "Site", "text": "FKH domain", "start": 6610, "end": 6620}]}, {"trigger": {"text": "inhibit", "start": 8173, "end": 8180}, "arguments": [{"role": "Theme", "text": "expression", "start": 8187, "end": 8197}]}], "positive regulation": [{"trigger": {"text": "promotes", "start": 3408, "end": 3416}, "arguments": [{"role": "Cause", "text": "Binding", "start": 3358, "end": 3365}, {"role": "Theme", "text": "assembly", "start": 3417, "end": 3425}]}, {"trigger": {"text": "overexpression", "start": 3754, "end": 3768}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 3784, "end": 3789}]}, {"trigger": {"text": "overexpression", "start": 3754, "end": 3768}, "arguments": [{"role": "Theme", "text": "deltaFKH", "start": 3799, "end": 3807}]}, {"trigger": {"text": "highest", "start": 4530, "end": 4537}, "arguments": [{"role": "Theme", "text": "expression", "start": 4562, "end": 4572}]}, {"trigger": {"text": "result", "start": 6000, "end": 6006}, "arguments": [{"role": "Theme", "text": "interferes", "start": 5813, "end": 5823}, {"role": "Cause", "text": "interaction", "start": 6023, "end": 6034}]}, {"trigger": {"text": "result", "start": 6000, "end": 6006}, "arguments": [{"role": "Theme", "text": "blocked", "start": 5966, "end": 5973}, {"role": "Cause", "text": "interaction", "start": 6023, "end": 6034}]}], "regulation": [{"trigger": {"text": "play a role", "start": 1727, "end": 1738}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 1496, "end": 1501}, {"role": "CSite", "text": "leucine zipper domain", "start": 1518, "end": 1539}, {"role": "Theme", "text": "dimer formation", "start": 1742, "end": 1757}]}, {"trigger": {"text": "does", "start": 1764, "end": 1768}, "arguments": [{"role": "Cause", "text": "Foxp3", "start": 1496, "end": 1501}, {"role": "CSite", "text": "leucine zipper domain", "start": 1518, "end": 1539}, {"role": "Theme", "text": "dimer formation", "start": 1742, "end": 1757}]}, {"trigger": {"text": "dysregulation", "start": 1949, "end": 1962}, "arguments": [{"role": "Theme", "text": "expression", "start": 1972, "end": 1982}]}, {"trigger": {"text": "modulating", "start": 5193, "end": 5203}, "arguments": [{"role": "Theme", "text": "expression", "start": 5208, "end": 5218}]}, {"trigger": {"text": "regulate", "start": 7156, "end": 7164}, "arguments": [{"role": "Theme", "text": "JunD", "start": 7213, "end": 7217}]}, {"trigger": {"text": "regulate", "start": 7156, "end": 7164}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 7219, "end": 7224}]}, {"trigger": {"text": "regulate", "start": 7156, "end": 7164}, "arguments": [{"role": "Theme", "text": "STAT3", "start": 7268, "end": 7273}]}, {"trigger": {"text": "regulate", "start": 7156, "end": 7164}, "arguments": [{"role": "Theme", "text": "p15INK4b", "start": 7295, "end": 7303}]}, {"trigger": {"text": "regulate", "start": 7156, "end": 7164}, "arguments": [{"role": "Theme", "text": "cyclin A", "start": 7305, "end": 7313}]}, {"trigger": {"text": "regulate", "start": 7156, "end": 7164}, "arguments": [{"role": "Theme", "text": "cyclin D1", "start": 7315, "end": 7324}]}, {"trigger": {"text": "regulate", "start": 7156, "end": 7164}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 7356, "end": 7360}]}, {"trigger": {"text": "regulate", "start": 7156, "end": 7164}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 7362, "end": 7366}]}, {"trigger": {"text": "regulate", "start": 7156, "end": 7164}, "arguments": [{"role": "Theme", "text": "T-cell receptor alpha", "start": 7368, "end": 7389}]}]}}, "schema": []}
{"input": "Cell culture.\nHEK 293T cells were cultured in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, California, United States). Jurkat T cells and primary human CD4+ T cells were cultured in RPMI-1640 medium (Invitrogen). Media were supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mug/ml streptomycin (Cambrex, East Rutherford, New Jersey, United States), and 10% fetal bovine serum (Atlanta Biologicals, Norcross, Georgia, United States).", "output": {"json_structures": {}}, "schema": []}
{"input": "Patients and cell preparation.\nPBMCs were prepared by centrifugation over Ficoll-Hypaque gradients (BioWhittaker, Walkersville, Maryland, United States) from eight HAM/TSP patients and eight ACs, and the cells were viably cryopreserved in liquid nitrogen until tested. HAM/TSP was diagnosed according to WHO guidelines [49]. HTLV-I seropositivity was determined by ELISA (Abbott Laboratories, Abbott Park, Illinois, United States), with confirmation by Western blot analysis (Genelabs Technologies, Redwood City, California, United States). Blood samples were obtained after informed consent as part of a clinical protocol reviewed and approved by the NIH institutional review panel.", "output": {"json_structures": {}}, "schema": []}
{"input": "Plasmids.\nExpression vectors encoding human Foxp3 (pCMV-Foxp3-IRES-EGFP) and human Foxp3 lacking the forkhead (FKH) domain (pCMV-deltaFKH-IRES-EGFP) were generous gifts from S. Ziegler (Benaroya Research Institute). pEGFP-C2 was provided by I. Lipinski (NIDDK/NIH). pcDNA3 was provided by K. T. Jeang (NIAID/NIH). pCMV4-Tax was a generous gift from W. Greene (University of California San Francisco). pGL4-luc2 and pGL4-TKhRluc2 were purchased from Promega (Madison, Wisconsin, United States). pUC18 was purchased from Stratagene (La Jolla, California, United States). HIV-1 wt LTR and HIV-1 delta-kappaB LTR luciferase reporter vectors were constructed by cloning the XhoI/HindIII LTR fragments from pHIV-CAT and pdelta-kappaB-HIV-CAT (AIDS Research and Reference Reagent Program, NIAID/NIH) into the multiple cloning site of pGL4-luc2. NF-kappaB, HTLV-I LTR, and CREB luciferase reporter and pCMV-p300-HA expression vectors were generously provided by B. Wigdahl (Drexel University College of Medicine). HTLV-I pACH infectious molecular clone has been described previously [30]. pFR-luc, pFA-CMV, pFA2-CREB-1, and pFA2-c-Jun were purchased from Stratagene. pFA-Tax (encoding a fusion protein consisting of the Gal4 DNA-binding domain fused in-frame to HTLV-I Tax) was constructed by PCR amplification of HTLV-I Tax using pCMV4-Tax as a template and BamHI/BglII-tagged primers. The amplified insert was digested and ligated into the BamHI/BglII sites of pFA-CMV. Plasmid contents were confirmed by DNA sequencing.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Expression", "start": 10, "end": 20}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 44, "end": 49}]}, {"trigger": {"text": "Expression", "start": 10, "end": 20}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 83, "end": 88}]}, {"trigger": {"text": "expression", "start": 907, "end": 917}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 870, "end": 880}]}, {"trigger": {"text": "expression", "start": 907, "end": 917}, "arguments": [{"role": "Theme", "text": "p300", "start": 899, "end": 903}]}], "negative regulation": [{"trigger": {"text": "lacking", "start": 89, "end": 96}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 83, "end": 88}, {"role": "Site", "text": "forkhead (FKH) domain", "start": 101, "end": 122}]}]}}, "schema": []}
{"input": "Isolation of primary human CD4+ T cells.\nCD4+ T cells were isolated from cryopreserved healthy donor PBMCs by negative selection with the CD4+ T Cell Isolation Kit II (Miltenyi Biotech, Bergisch Gladbach, Germany) according to manufacturer's guidelines. Purity of negatively selected CD4+ T cells was consistently higher than 96% as determined by flow cytometry.", "output": {"json_structures": {}}, "schema": []}
{"input": "Transient expression and luciferase assays.\nHEK 293T cells were plated at a density of 5 x 105 cells/well in six-well culture plates (BD Biosciences, San Diego, California, United States) 1 d prior to transfection with the appropriate plasmid DNA (~2 mug total) using FuGene 6 transfection reagent (Roche, Basel, Switzerland). Jurkat T cells were plated at 1 x 106 cells/well in six-well culture plates the day of transfection with the appropriate plasmid DNA (~2 mug total) using FuGene 6 transfection reagent. Primary human CD4+ T cells (2 x 106) were nucleofected with the specified plasmid DNA (5 mug total) using the Human T Cell Nucleofection Kit (Amaxa, Gaithersburg, Maryland, United States). Forskolin (10 muM; Calbiochem, San Diego, California, United States) was added in some experiments 20 h posttransfection. Cells were harvested 24 h posttransfection and luciferase activity was analyzed using the Dual-luciferase Reporter Assay System (Promega) and a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, California, United States) according to manufacturer's guidelines. pGL4-TKhRluc2 was used as an internal control to normalize for transfection efficiency. Nucleofected CD4+ T cells were also monitored for transfection efficiency and cell viability 24 h posttransfection as follows. Transfection efficiency was routinely ~30% as determined by flow cytometric analysis of EGFP expression. Cell viability, determined by staining with 7-amino-actinomycin D (7-AAD; BD Biosciences), was routinely ~70%. Both transfection efficiency and cell viability in nucleofected CD4+ T cells was independent of the plasmids used.", "output": {"json_structures": {}}, "schema": []}
{"input": "Foxp3 and HTLV-I Tax expression analysis by real-time RT-PCR.\nReal-time RT-PCR analysis of Foxp3 and HTLV-I Tax expression was performed as previously described [8,47]. Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, California, United States) according to manufacturer's guidelines, and cDNA was synthesized by reverse transcription using TaqMan Gold RT-PCR Kit using random hexamer primers (Applied Biosystems, Foster City, California, United States). Foxp3 and HTLV-I Tax mRNA expression was quantified by real-time PCR using ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The normalized values in each sample were calculated as the relative quantity of Foxp3 or HTLV-I Tax mRNA expression divided by the relative quantity of HPRT mRNA expression. The values were calculated by the following formula: normalized Foxp3 or HTLV-I Tax expression = 2Ct value of HPRT - Ct value of Foxp3 or HTLV-I Tax.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 21, "end": 31}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 0, "end": 5}]}, {"trigger": {"text": "expression", "start": 21, "end": 31}, "arguments": [{"role": "Theme", "text": "Tax", "start": 17, "end": 20}]}, {"trigger": {"text": "expression", "start": 112, "end": 122}, "arguments": [{"role": "Theme", "text": "Tax", "start": 108, "end": 111}]}, {"trigger": {"text": "expression", "start": 112, "end": 122}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 91, "end": 96}]}, {"trigger": {"text": "expression", "start": 881, "end": 891}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 861, "end": 866}]}, {"trigger": {"text": "expression", "start": 881, "end": 891}, "arguments": [{"role": "Theme", "text": "Tax", "start": 877, "end": 880}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 505, "end": 520}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 484, "end": 489}]}, {"trigger": {"text": "mRNA expression", "start": 505, "end": 520}, "arguments": [{"role": "Theme", "text": "Tax", "start": 501, "end": 504}]}, {"trigger": {"text": "mRNA expression", "start": 723, "end": 738}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 703, "end": 708}]}, {"trigger": {"text": "mRNA expression", "start": 723, "end": 738}, "arguments": [{"role": "Theme", "text": "Tax", "start": 719, "end": 722}]}, {"trigger": {"text": "mRNA expression", "start": 780, "end": 795}, "arguments": [{"role": "Theme", "text": "HPRT", "start": 775, "end": 779}]}]}}, "schema": []}
{"input": "Real-time PCR.\nReal-time PCR analysis of HTLV-I (Tax) proviral load was performed as previously described [47,50]. DNA was extracted from 1 x 106 cells using Puregene DNA Isolation Kit (Gentra, Minneapolis, Minnesota, United States), and 100 ng of the sample DNA solution was analyzed by this system. The HTLV-I proviral DNA load was calculated by the following formula: copy number of HTLV-I (pX) per 100 cells = (copy number of pX)/(copy number of beta-actin/2) x 100.", "output": {"json_structures": {}}, "schema": []}
{"input": "Western blot analysis.\nHEK 293T cells were plated at a density of 5 x 105 cells/well in six-well culture plates (BD Biosciences) 1 d prior to transfection with the appropriate plasmid DNA (2 mug total) using FuGene 6 transfection reagent (Roche). Cells were harvested 24 h posttransfection for whole-cell lysates in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Igepal (NP-40), 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1x Complete Mini Protease Inhibitor [Roche]). Protein concentration was determined by Lowry assay (Bio-Rad, Hercules, California, United States) and colorimetric reactions were read using a VersaMax microplate reader (Molecular Devices, Sunnyvale, California, United States) at an absorbance of 750 nm. Size fractionation was performed on 20 mug of protein/sample by SDS-PAGE, and the protein was transferred to nitrocellulose or PVDF membranes and subjected to immunoblotting using the indicated antibodies. Foxp3 was detected using rabbit anti-human Foxp3 polyclonal antibody (ab4728 or ab10563; Abcam, Cambridge, United Kingdom) and anti-rabbit IgG-HRP secondary antibody (Cell Signaling Technology, Beverly, Massachusetts, United States). NF-kappaB p65 and CREB-1 were detected using rabbit anti-human polyclonal (p65) or monoclonal antibody (CREB-1; 48H2) (Cell Signaling Technology). Beta-actin was detected using a mouse monoclonal antibody (AC-15; Sigma, St. Louis, Missouri, United States). For coimmunoprecipitation analysis, cell lysates were precleared with 30 mul of protein A/G plus-agarose beads (Santa Cruz Biotechnology, Santa Cruz, California, United States) and then incubated with mouse monoclonal anti-HA antibody (6E2; 1:100; Cell Signaling Technology) and 30 mul of protein A/G plus-agarose beads overnight. The immunoprecipitates were washed four times with RIPA buffer, resuspended in SDS sample buffer, and heated at 95 degreesC for 5 min. Proteins were then treated as described for Western blot analysis.", "output": {"json_structures": {}}, "schema": []}
{"input": "Transcription factor DNA-binding analysis (TF-ELISA).\nCREB-1 and ATF-2 DNA-binding activity was analyzed with the TransFactor Profiling (Inflammation 1) Kit (BD Biosciences) according to the manufacturer's protocol. Nuclear extracts were prepared from HEK 293T cells transfected with a control vector (EGFP) or Foxp3 expression vector (1,000 ng) in the presence or absence of forskolin (10 muM for 4 h) using the TransFactor Extraction Kit (BD Biosciences). Protein concentration was determined using a Biophotometer (Eppendorf, Hamburg, Germany). Nuclear extracts (20 mug) were incubated in preblocked wells containing plate-bound double-stranded oligonucleotides corresponding to an ATF/CREB consensus sequence (...TGACATCA...). Wells were washed, incubated with the appropriate primary antibody, washed, incubated with secondary antibody (HRP-labeled), washed again, and finally developed with TMB substrate. Colorimetric reactions were read using a VersaMax microplate reader (Molecular Devices) at an absorbance of 655 nm.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 75, "end": 82}, "arguments": [{"role": "Theme", "text": "CREB-1", "start": 54, "end": 60}]}, {"trigger": {"text": "binding", "start": 75, "end": 82}, "arguments": [{"role": "Theme", "text": "ATF-2", "start": 65, "end": 70}]}], "gene expression": [{"trigger": {"text": "expression", "start": 317, "end": 327}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 311, "end": 316}]}]}}, "schema": []}
{"input": "Flow cytometric analysis of Foxp3 protein expression.\nCryopreserved PBMCs from HAM/TSP patients or HTLV-I-infected ACs were thawed and washed with FACS buffer (1x PBS, 0.1% NaN3, 5% FBS). Cells (1.5 x 106) were fixed by sequential formaldehyde/methanol fixation as follows. Cells were carefully resuspended in FACS buffer and fixed with 100 mul of reagent A (Fix & Perm kit; Caltag Laboratories, Burlingame, California, United States) at room temperature for 3 min followed by 2 ml of 70% methanol for 5 min at 4 degreesC. Cells were washed twice and permeabilized with 100 mul of reagent B (Fix & Perm kit) and stained for intracellular Foxp3 with mouse anti-human Foxp3 monoclonal antibody (0.5 mug of ab22510; Abcam) or the appropriate isotype control for 30 min. Cells were washed twice and stained with Cy5-conjugated goat anti-mouse immunoglobulin F(ab')2 secondary antibody (Caltag Laboratories) for an additional 30 min. Cells were washed twice and stained for surface CD4 expression with PE-labeled anti-CD4 (BD) and CD25 expression with FITC-labeled anti-CD25 (BD). Cells were washed twice and analyzed on a FACSCalibur (BD). Data analysis was performed using FlowJo (Tree Star, Ashland, Oregon, United States).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 42, "end": 52}, "arguments": [{"role": "Theme", "text": "Foxp3", "start": 28, "end": 33}]}, {"trigger": {"text": "expression", "start": 981, "end": 991}, "arguments": [{"role": "Theme", "text": "CD4", "start": 977, "end": 980}]}, {"trigger": {"text": "expression", "start": 1031, "end": 1041}, "arguments": [{"role": "Theme", "text": "CD25", "start": 1026, "end": 1030}]}]}}, "schema": []}
{"input": "Statistical analyses.\nThe Mann-Whitney U test was used to compare the data between patients with HAM/TSP and AC.", "output": {"json_structures": {}}, "schema": []}
{"input": "EBV Latent Membrane Protein 1 Activates Akt, NFkappaB, and Stat3 in B Cell Lymphomas\nLatent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFkappaB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFkappaB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "inhibitors", "start": 444, "end": 454}, "arguments": [{"role": "Theme", "text": "Akt", "start": 458, "end": 461}]}, {"trigger": {"text": "inhibitors", "start": 444, "end": 454}, "arguments": [{"role": "Theme", "text": "Stat3", "start": 477, "end": 482}]}], "positive regulation": [{"trigger": {"text": "Activates", "start": 30, "end": 39}, "arguments": [{"role": "Cause", "text": "Latent Membrane Protein 1", "start": 4, "end": 29}, {"role": "Theme", "text": "Akt", "start": 40, "end": 43}]}, {"trigger": {"text": "Activates", "start": 30, "end": 39}, "arguments": [{"role": "Cause", "text": "Latent Membrane Protein 1", "start": 4, "end": 29}, {"role": "Theme", "text": "Stat3", "start": 59, "end": 64}]}], "regulation": [{"trigger": {"text": "deregulated", "start": 737, "end": 748}, "arguments": [{"role": "Theme", "text": "IL4", "start": 606, "end": 609}]}, {"trigger": {"text": "deregulated", "start": 737, "end": 748}, "arguments": [{"role": "Theme", "text": "Stat6", "start": 610, "end": 615}]}, {"trigger": {"text": "deregulated", "start": 737, "end": 748}, "arguments": [{"role": "Theme", "text": "Stat3", "start": 692, "end": 697}]}]}}, "schema": []}
{"input": "Epstein-Barr virus (EBV) is a ubiquitous gamma-herpesvirus that infects humans predominantly at an early age with greater than 90% of the adult population infected with EBV [1]. EBV is linked to the development of both B lymphocyte and epithelial cell malignancies, including Burkitt lymphoma, Hodgkin disease (HD), and nasopharyngeal carcinoma (NPC), and cancers linked to immunosuppression, including post-transplant lymphoma and AIDS-associated lymphomas [2,3]. In vitro infection of B lymphocytes with EBV induces permanent growth transformation, and this ability to affect cell growth regulation likely contributes to the development of cancer.\nMany of the viral proteins expressed in transformed cells, including the EBV nuclear antigens and latent membrane proteins, have profound effects on cell growth regulation and are required for EBV latent infection and B cell transformation [1]. Latent membrane protein 1 (LMP1) is considered the major oncoprotein of EBV, as it transforms rodent fibroblasts to tumorigenicity in nude mice and is expressed in HD, NPC, and immunosuppression-associated tumors [4-8]. In B lymphocytes, LMP1 mimics CD40 signaling, and both LMP1 and CD40 are essential for EBV-mediated B cell transformation [9-11]. While CD40 interacts with CD40 ligand expressed on activated T cells to induce B cell activation and differentiation, LMP1 acts as a constitutive signal through ligand-independent oligomerization. LMP1 and CD40 interact with the same tumor necrosis factor receptor-associated factors (TRAFs) leading to activation of NFkappaB, c-Jun N terminal kinase (JNK), and p38 MAPK signaling pathways [12-16]. Activation of NFkappaB is required for EBV-induced B cell transformation and its inhibition rapidly results in cell death [17,18]. Recent studies indicate that LMP1 also activates phosphatidylinositol 3 kinase (PI3K)/Akt signaling and that this activation is required for LMP1-mediated transformation of rodent fibroblasts [5,19].\nIn vitro, primary B cells can be maintained by CD40 ligation in combination with IL4 treatment. In vivo, CD40 signaling is necessary for germinal center (GC) formation such that mice deficient for CD40 or CD40L are unable to form GCs in response to T cell-dependent antigens [20,21]. Both the membrane proximal and distal cytoplasmic regions of CD40 that bind TRAF6 and TRAFs2/3/5, respectively, are necessary for GC formation, but either region is sufficient to induce extrafollicular B cell differentiation and restore low affinity antibody production [22]. Functionally, LMP1 can rescue CD40-deficient mice and restore immunoglobulin (Ig) class switching, most likely because LMP1 recruits similar TRAF molecules, TRAFs 1/2/3/5 and TRAF6, through the C-terminal activation regions 1 and 2 domains, respectively. However, LMP1 is unable to restore affinity maturation and GC formation [23].\nSeveral EBV transforming proteins have been studied in transgenic mouse models, however, only LMP1 induces tumor development when expressed under the control of the Ig heavy chain promoter and enhancer [24-26]. The LMP1 transgenic mice (IgLMP1) express LMP1 in B lymphocytes, and in mice older than 12 mo, lymphoma develops with increased incidence (40%-50%) compared to wild-type control mice (11%), suggesting that LMP1 contributes to tumor development [26]. The LMP1 lymphomas have rearranged Ig genes and have activated Akt, JNK, p38, and NFkappaB, with specific activation of the NFkappaB family member cRel [27].\nIn this study, the LMP1 transgenic lymphocytes and lymphomas were further characterized and their growth properties in vitro were determined. To obtain pure populations of malignant lymphocytes and to enable more detailed biochemical analyses, examples of primary lymphomas were inoculated and passaged in SCID mice. Interestingly, lymphoma development was restricted to B-1a lymphocytes, a self-replenishing population of cells that are prone to malignancy [28,29]. LMP1 transgenic lymphocytes had increased viability in vitro and viability was increased by the addition of IL4. In contrast, both LMP1-positive and -negative lymphoma cells were independent of IL4 co-stimulation for survival and proliferation in vitro with a complete absence of activated Stat6, the IL4 target. The lymphomas were also distinguished by constitutive activation of Stat3 and deregulation of the Rb cell cycle pathway. Inhibition of the PI3K/Akt, NFkappaB, and Stat3 signaling pathways blocked the enhanced growth of both LMP1 transgenic and malignant lymphocytes, suggesting that these pathways are required for their growth and survival. These appear to be the same targets that are deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. This study reveals that LMP1 promotes malignancy in cells with the inherent ability to proliferate and that the Akt, NFkappaB, and Stat3 signaling pathways are required for its growth stimulatory effects.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interacts", "start": 1256, "end": 1265}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1251, "end": 1255}, {"role": "Theme2", "text": "CD40 ligand", "start": 1271, "end": 1282}]}, {"trigger": {"text": "oligomerization", "start": 1425, "end": 1440}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1363, "end": 1367}]}, {"trigger": {"text": "interact", "start": 1456, "end": 1464}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1442, "end": 1446}]}, {"trigger": {"text": "interact", "start": 1456, "end": 1464}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1451, "end": 1455}]}, {"trigger": {"text": "ligation", "start": 2027, "end": 2035}, "arguments": [{"role": "Theme", "text": "CD40", "start": 2022, "end": 2026}]}, {"trigger": {"text": "bind", "start": 2330, "end": 2334}, "arguments": [{"role": "Site", "text": "membrane proximal", "start": 2268, "end": 2285}, {"role": "Theme", "text": "CD40", "start": 2320, "end": 2324}, {"role": "Theme2", "text": "TRAF6", "start": 2335, "end": 2340}]}, {"trigger": {"text": "bind", "start": 2330, "end": 2334}, "arguments": [{"role": "Site", "text": "distal cytoplasmic regions", "start": 2290, "end": 2316}, {"role": "Theme", "text": "CD40", "start": 2320, "end": 2324}, {"role": "Theme2", "text": "TRAFs2", "start": 2345, "end": 2351}]}, {"trigger": {"text": "bind", "start": 2330, "end": 2334}, "arguments": [{"role": "Site", "text": "distal cytoplasmic regions", "start": 2290, "end": 2316}, {"role": "Theme", "text": "CD40", "start": 2320, "end": 2324}, {"role": "Theme2", "text": "3", "start": 2352, "end": 2353}]}, {"trigger": {"text": "bind", "start": 2330, "end": 2334}, "arguments": [{"role": "Site", "text": "distal cytoplasmic regions", "start": 2290, "end": 2316}, {"role": "Theme", "text": "CD40", "start": 2320, "end": 2324}, {"role": "Theme2", "text": "5", "start": 2354, "end": 2355}]}, {"trigger": {"text": "recruits", "start": 2659, "end": 2667}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 2654, "end": 2658}, {"role": "Theme2", "text": "TRAFs 1", "start": 2692, "end": 2699}, {"role": "Site", "text": "C-terminal activation regions 1", "start": 2729, "end": 2760}]}, {"trigger": {"text": "recruits", "start": 2659, "end": 2667}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 2654, "end": 2658}, {"role": "Theme2", "text": "2", "start": 2700, "end": 2701}, {"role": "Site", "text": "C-terminal activation regions 1", "start": 2729, "end": 2760}]}, {"trigger": {"text": "recruits", "start": 2659, "end": 2667}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 2654, "end": 2658}, {"role": "Theme2", "text": "3", "start": 2702, "end": 2703}, {"role": "Site", "text": "C-terminal activation regions 1", "start": 2729, "end": 2760}]}, {"trigger": {"text": "recruits", "start": 2659, "end": 2667}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 2654, "end": 2658}, {"role": "Theme2", "text": "5", "start": 2704, "end": 2705}, {"role": "Site", "text": "C-terminal activation regions 1", "start": 2729, "end": 2760}]}, {"trigger": {"text": "recruits", "start": 2659, "end": 2667}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 2654, "end": 2658}, {"role": "Theme2", "text": "TRAF6", "start": 2710, "end": 2715}, {"role": "Site", "text": "2 domains", "start": 2765, "end": 2774}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 1046, "end": 1055}, "arguments": [{"role": "Theme", "text": "Latent membrane protein 1", "start": 895, "end": 920}]}, {"trigger": {"text": "expressed", "start": 1283, "end": 1292}, "arguments": [{"role": "Theme", "text": "CD40 ligand", "start": 1271, "end": 1282}]}, {"trigger": {"text": "expressed", "start": 2998, "end": 3007}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 2962, "end": 2966}]}, {"trigger": {"text": "express", "start": 3113, "end": 3120}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 3121, "end": 3125}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 2158, "end": 2167}, "arguments": [{"role": "Theme", "text": "CD40", "start": 2172, "end": 2176}]}, {"trigger": {"text": "deficient", "start": 2158, "end": 2167}, "arguments": [{"role": "Theme", "text": "CD40L", "start": 2180, "end": 2185}]}, {"trigger": {"text": "rescue", "start": 2558, "end": 2564}, "arguments": [{"role": "Theme", "text": "deficient", "start": 2570, "end": 2579}]}, {"trigger": {"text": "deficient", "start": 2570, "end": 2579}, "arguments": [{"role": "Theme", "text": "CD40", "start": 2565, "end": 2569}]}, {"trigger": {"text": "absence", "start": 4223, "end": 4230}, "arguments": [{"role": "Theme", "text": "activated", "start": 4234, "end": 4243}]}], "positive regulation": [{"trigger": {"text": "under the control", "start": 3008, "end": 3025}, "arguments": [{"role": "Theme", "text": "expressed", "start": 2998, "end": 3007}]}, {"trigger": {"text": "activated", "start": 3382, "end": 3391}, "arguments": [{"role": "Theme", "text": "Akt", "start": 3392, "end": 3395}]}, {"trigger": {"text": "activation", "start": 3435, "end": 3445}, "arguments": [{"role": "Theme", "text": "cRel", "start": 3476, "end": 3480}]}, {"trigger": {"text": "activated", "start": 4234, "end": 4243}, "arguments": [{"role": "Theme", "text": "Stat6", "start": 4244, "end": 4249}]}, {"trigger": {"text": "activation", "start": 4321, "end": 4331}, "arguments": [{"role": "Theme", "text": "Stat3", "start": 4335, "end": 4340}]}], "regulation": [{"trigger": {"text": "target", "start": 4259, "end": 4265}, "arguments": [{"role": "Theme", "text": "Stat6", "start": 4244, "end": 4249}, {"role": "Cause", "text": "IL4", "start": 4255, "end": 4258}]}, {"trigger": {"text": "deregulated", "start": 4654, "end": 4665}, "arguments": [{"role": "Theme", "text": "Akt", "start": 4411, "end": 4414}]}, {"trigger": {"text": "deregulated", "start": 4654, "end": 4665}, "arguments": [{"role": "Theme", "text": "Stat3", "start": 4430, "end": 4435}]}]}}, "schema": []}
{"input": "High Levels of LMP1 Expression Correlates with the Development of Lymphoma\nLMP1 expression in IgLMP1 mice was directed to B cells under the control of the Ig heavy chain promoter and enhancer. It has previously been shown that in these transgenic mice, LMP1 expression was restricted to B220+ B cells with lymphoma detected in greatly enlarged spleens [23,26]. To investigate whether LMP1 expression contributes to lymphoma development, B cells were purified from splenocytes by positive selection using anti-CD19 MACS magnetic beads, and equivalent amounts of B cells were analyzed by immunoblotting. LMP1 was detectable in LMP1 transgenic B cells, but upon development of lymphoma, LMP1 expression was stronger in 5/7 lymphomas analyzed with concomitant appearance of degradation products (Figure 1A). To determine whether the higher level of LMP1 detected was due to an expansion of malignant lymphocytes, expression of LMP1 in the spleen was further evaluated by immunohistochemical staining. Immunohistochemistry analysis of spleen sections detected LMP1 in the plasma membrane of cells in both the follicular white pulp and circulating lymphocytes in the red pulp (Figure 1B). LMP1 expression was heterogeneous with strong LMP1 staining interspersed amongst a background of cells staining weakly for LMP1. Upon development to lymphoma, LMP1 expression was more abundantly detected with multiple foci of intense LMP1 staining. This demonstrates that the increased LMP1 detected by immunoblotting upon malignant progression reflects an increase in LMP1 expression and an accumulation of cells expressing high levels of LMP1. This correlation between high LMP1 expression and the development of lymphoma suggests that progression to lymphoma results from increased levels of LMP1.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Expression", "start": 20, "end": 30}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 15, "end": 19}]}, {"trigger": {"text": "expression", "start": 80, "end": 90}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 75, "end": 79}]}, {"trigger": {"text": "expression", "start": 258, "end": 268}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 253, "end": 257}]}, {"trigger": {"text": "expression", "start": 389, "end": 399}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 384, "end": 388}]}, {"trigger": {"text": "detectable", "start": 611, "end": 621}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 602, "end": 606}]}, {"trigger": {"text": "expression", "start": 689, "end": 699}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 684, "end": 688}]}, {"trigger": {"text": "expression", "start": 909, "end": 919}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 923, "end": 927}]}, {"trigger": {"text": "detected", "start": 1046, "end": 1054}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1055, "end": 1059}]}, {"trigger": {"text": "expression", "start": 1188, "end": 1198}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1183, "end": 1187}]}, {"trigger": {"text": "expression", "start": 1347, "end": 1357}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1342, "end": 1346}]}, {"trigger": {"text": "expression", "start": 1557, "end": 1567}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1552, "end": 1556}]}, {"trigger": {"text": "expressing", "start": 1597, "end": 1607}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1623, "end": 1627}]}, {"trigger": {"text": "expression", "start": 1664, "end": 1674}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1659, "end": 1663}]}, {"trigger": {"text": "levels", "start": 1768, "end": 1774}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1778, "end": 1782}]}], "positive regulation": [{"trigger": {"text": "High Levels", "start": 0, "end": 11}, "arguments": [{"role": "Theme", "text": "Expression", "start": 20, "end": 30}]}, {"trigger": {"text": "restricted", "start": 273, "end": 283}, "arguments": [{"role": "Theme", "text": "expression", "start": 258, "end": 268}]}, {"trigger": {"text": "stronger", "start": 704, "end": 712}, "arguments": [{"role": "Theme", "text": "expression", "start": 689, "end": 699}]}, {"trigger": {"text": "higher level", "start": 829, "end": 841}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 845, "end": 849}]}, {"trigger": {"text": "strong", "start": 1222, "end": 1228}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1229, "end": 1233}]}, {"trigger": {"text": "weakly", "start": 1295, "end": 1301}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1306, "end": 1310}]}, {"trigger": {"text": "abundantly", "start": 1367, "end": 1377}, "arguments": [{"role": "Theme", "text": "expression", "start": 1347, "end": 1357}]}, {"trigger": {"text": "intense", "start": 1409, "end": 1416}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1417, "end": 1421}]}, {"trigger": {"text": "increased", "start": 1459, "end": 1468}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 1469, "end": 1473}]}, {"trigger": {"text": "increase", "start": 1540, "end": 1548}, "arguments": [{"role": "Theme", "text": "expression", "start": 1557, "end": 1567}]}, {"trigger": {"text": "high levels", "start": 1608, "end": 1619}, "arguments": [{"role": "Theme", "text": "expressing", "start": 1597, "end": 1607}]}, {"trigger": {"text": "high", "start": 1654, "end": 1658}, "arguments": [{"role": "Theme", "text": "expression", "start": 1664, "end": 1674}]}, {"trigger": {"text": "increased", "start": 1758, "end": 1767}, "arguments": [{"role": "Theme", "text": "levels", "start": 1768, "end": 1774}]}], "regulation": [{"trigger": {"text": "under the control", "start": 130, "end": 147}, "arguments": [{"role": "Theme", "text": "expression", "start": 80, "end": 90}]}]}}, "schema": []}
{"input": "LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion\nTo determine if LMP1 signaling affects B cell differentiation and to immunophenotype the lymphomas that arise from LMP1 expression, surface Ig expression of heavy chains (IgM, IgG, IgD) and light chains (kappa, lambda) were analyzed by flow cytometry. Similar numbers of naive (IgM+IgD+IgG-) splenic B cells with a strong bias towards kappa light chain were detected from wild-type or LMP1 transgenic mice, indicating that LMP1 signaling does not affect B cell maturation (unpublished data). Flow cytometry analysis of the SCID-passaged wild-type and LMP1 transgenic lymphomas revealed an IgMhighIgDlow phenotype (Figure 2A), indicative of marginal zone, B-1, or memory B cells. B-1 cells are further separated into CD5+ (B-1a) and CD5- (B-1b) subsets. To differentiate between these cell types, lymphoma cells were further analyzed for the B-1a marker CD5. All (5/5) of the tested LMP1 transgenic lymphomas displayed an IgMhighIgDlowCD5+ phenotype (Figure 2A), an expression pattern that distinguishes B-1a cells. Interestingly, a spontaneous wild-type lymphoma also developed in B-1a cells (Figure 2A). These cells are an interesting population that is self replenishing with an increased likelihood to become malignant in aged mice [30]. Analysis of LMP1 transgenic mice before the development of lymphoma showed similar numbers of splenic B-1a (CD19+CD5+) and B-1b or B-2 populations (CD19+CD5-), indicating that LMP1 does not affect B cell differentiation (Figure 2B).\nDue to allelic exclusion, mature B cells that have been exposed to antigen will typically express only one heavy chain isotype (IgG, IgE, or IgA) and either a kappa or lambda light chain. Interestingly, 2/5 LMP1 transgenic lymphomas analyzed (lymphomas 2 and 4) were doubly positive for low levels of both kappa and lambda light chains (Figure 2A). Previous characterization of the LMP1 lymphomas had revealed that the lymphomas were clonal as determined by Ig heavy chain rearrangement [26], and analysis of kappa chain rearrangement (Figure S1) of the samples analyzed in this study confirmed clonality. To further assess light chain expression, the passaged samples were tested by immunoblotting for kappa and lambda light chains (Figure 2C). Interestingly, very low levels of expression of both light chains were detected by flow cytometry. The low levels of expression may reflect a limitation of the total number of light chains that can be expressed on the surface of a B cell. In agreement with the flow cytometry analysis, LMP1 transgenic lymphomas 2 and 4 were also positive for kappa and lambda light chains by immunoblot analysis (Figure 2C), confirming that these lymphomas express both light chains. A previous study of mice that developed leukemia due to an expansion of self-reactive B-1a cells determined that the B-1a leukemias were also doubly positive for kappa and lambda light chains [31]. These findings indicate that expression of LMP1 in B-1a cells promotes the development of malignancy and can result in the aberrant escape from allelic exclusion.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 183, "end": 193}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 178, "end": 182}]}, {"trigger": {"text": "expression", "start": 2978, "end": 2988}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 2992, "end": 2996}]}]}}, "schema": []}
{"input": "LMP1 Promotes B Cell Survival and Proliferation In Vitro\nPrimary B cell cultures can be maintained through CD40 ligation and supplementation with IL4 [32]. To investigate whether LMP1 affects primary B cell survival and proliferation, splenocytes were cultured in the presence or absence of IL4 and analyzed by MTS as a metabolic marker, by ethidium monoazide (EMA) exclusion for viability, and by 5-bromo-2'-deoxy-uridine (BrdU) incorporation for proliferation. In the MTS assay, as expected, splenocytes from wild-type mice did not survive even with the addition of IL4 due to a lack of CD40 ligation (Figure 3A). In contrast, LMP1 splenocytes had increased metabolism even in the absence of IL4, which was further enhanced upon addition of IL4. Wild-type and LMP1 transgenic lymphoma cells had high levels of MTS activity even in the absence of IL4 (Figure 3A). The LMP1 transgenic lymphoma cells had approximately 4-fold higher MTS activity than the normal transgenic lymphocytes and were at least 2-fold higher than the control lymphoma. As previously published, lymphoma usually develops in mice over 12 mo of age and all mice are sacrificed by 18-20 mo. The ages of the transgenic mice with or without lymphoma ranged between 6 and 20 mo old. There was no correlation between age and MTS activity.\nEMA exclusion of CD19+ gated B cells prepared from two wild-type and two LMP1 transgenic mice indicated a 2-fold increase in viability in the LMP1 transgenic lymphocytes compared to wild-type lymphocytes. Two examples of LMP1 transgenic lymphoma cells had greatly increased viability that was not increased by IL4 treatment, indicating that the lymphoma cells are independent of IL4 co-stimulation (Figure 3B). Enhancement in MTS activity was observed in LMP1 transgenic lymphoma cells by the addition of IL4; however, EMA exclusion did not reveal a similar increase. This could reflect a difference for IL4 requirement in the metabolic activity versus the viability of LMP1 transgenic lymphoma cells. Although expression of LMP1 could enhance survival of non-malignant primary lymphocytes, BrdU incorporation revealed that LMP1 expression alone was not sufficient to induce proliferation in culture (unpublished data). Only lymphoma cells had detectable levels of BrdU incorporation detected by flow cytometry (Figure 3C). Interestingly, LMP1 lymphoma cells had significantly higher levels of proliferation in comparison to the spontaneous lymphoma that developed in an LMP1-negative littermate (25% versus 4%). This higher level of proliferation was observed in lymphomas that express both high (Table 1, LMP1-L2 and LMP1-L3) and low (Table 1, LMP1-L5) levels of LMP1, suggesting that even small amounts of LMP1 is sufficient to induce dramatic effects in proliferation. The level of proliferation was not enhanced upon IL4 addition, confirming the IL4 independence observed in the viability studies (Figure 3C; Table1).", "output": {"json_structures": {"binding": [{"trigger": {"text": "ligation", "start": 112, "end": 120}, "arguments": [{"role": "Theme", "text": "CD40", "start": 107, "end": 111}]}, {"trigger": {"text": "ligation", "start": 594, "end": 602}, "arguments": [{"role": "Theme", "text": "CD40", "start": 589, "end": 593}]}], "gene expression": [{"trigger": {"text": "expression", "start": 2016, "end": 2026}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 2030, "end": 2034}]}, {"trigger": {"text": "expression", "start": 2134, "end": 2144}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 2129, "end": 2133}]}, {"trigger": {"text": "express", "start": 2584, "end": 2591}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 2670, "end": 2674}]}], "negative regulation": [{"trigger": {"text": "lack", "start": 581, "end": 585}, "arguments": [{"role": "Theme", "text": "ligation", "start": 594, "end": 602}]}, {"trigger": {"text": "absence", "start": 683, "end": 690}, "arguments": [{"role": "Theme", "text": "IL4", "start": 694, "end": 697}]}, {"trigger": {"text": "absence", "start": 837, "end": 844}, "arguments": [{"role": "Theme", "text": "IL4", "start": 848, "end": 851}]}, {"trigger": {"text": "negative", "start": 2481, "end": 2489}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 2476, "end": 2480}]}], "positive regulation": [{"trigger": {"text": "high", "start": 2597, "end": 2601}, "arguments": [{"role": "Theme", "text": "express", "start": 2584, "end": 2591}]}, {"trigger": {"text": "low", "start": 2637, "end": 2640}, "arguments": [{"role": "Theme", "text": "express", "start": 2584, "end": 2591}]}]}}, "schema": []}
{"input": "Wild-Type and LMP1 Transgenic Lymphoma Cells Do Not Require IL4 and Stat6 Signaling\nTo investigate whether IL4 independence was due to endogenous IL4 expression, IL4 transcription was assessed by an Rnase protection assay (RPA). IL4 transcription was detectable with control RNA and faintly in the mouse lymphoma cell line K46mu (Figure 4A). However, IL4 transcription was not detectable in CD19+ MACS-purified B cells from wild-type lymphocytes (unpublished data), LMP1 transgenic lymphocytes, or lymphoma cells, although the GAPDH and L32 controls were effectively protected (Figure 4A). Activated Stat6 (pStat6), a target of the IL4 receptor pathway, was detected in the wild-type and LMP1 transgenic lymphocytes (Figure 4B). In contrast, pStat6 was barely detected in either the wild-type or LMP1 transgenic lymphoma cells. However, the pathway was not disabled, as treatment of the lymphoma cells with IL4 induced Stat6 phosphorylation (Figure 4C).\nAlthough wild-type lymphocytes cannot be maintained in culture with IL4 supplementation alone (Figure 3A), slight enhancement in MTS activity could be detected if the cells were analyzed at an earlier time point, at 1 d (Figure 4D) versus 3 d (Figure 3A) post-harvest. The enhancement of MTS activity induced by IL4 in wild-type lymphocytes could be neutralized by the addition of IL4 antibody (Figure 4D). However, neutralizing antibodies to IL4 did not affect the MTS activity of LMP1 transgenic lymphoma cells (Figure 4E). In summary, the wild-type and LMP1 transgenic lymphoma cells grew independently of IL4 treatment and did not require Stat6 signaling.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 150, "end": 160}, "arguments": [{"role": "Theme", "text": "IL4", "start": 146, "end": 149}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 925, "end": 940}, "arguments": [{"role": "Theme", "text": "Stat6", "start": 919, "end": 924}]}], "positive regulation": [{"trigger": {"text": "Activated", "start": 590, "end": 599}, "arguments": [{"role": "Theme", "text": "Stat6", "start": 600, "end": 605}]}, {"trigger": {"text": "induced", "start": 911, "end": 918}, "arguments": [{"role": "Cause", "text": "IL4", "start": 907, "end": 910}, {"role": "Theme", "text": "phosphorylation", "start": 925, "end": 940}]}], "regulation": [{"trigger": {"text": "target", "start": 618, "end": 624}, "arguments": [{"role": "Theme", "text": "Stat6", "start": 600, "end": 605}]}], "transcription": [{"trigger": {"text": "transcription", "start": 166, "end": 179}, "arguments": [{"role": "Theme", "text": "IL4", "start": 162, "end": 165}]}, {"trigger": {"text": "transcription", "start": 233, "end": 246}, "arguments": [{"role": "Theme", "text": "IL4", "start": 229, "end": 232}]}, {"trigger": {"text": "transcription", "start": 355, "end": 368}, "arguments": [{"role": "Theme", "text": "IL4", "start": 351, "end": 354}]}]}}, "schema": []}
{"input": "LMP1 Upregulates IL10 and Constitutively Activates Stat3\nTo identify cytokines that may contribute to the increased survival and growth of lymphomas, the expression levels of a panel of cytokines were screened on CD19+ MACS-purified B cells, using an RPA probe set for IL4, IL5, IL10, IL13, IL15, IL9, IL2, IL6, and IFNgamma. Expression levels were quantified with a phosphorimager and normalized to the ribosomal housekeeping gene L32. None of the tested cytokines were detected in wild-type lymphocytes, therefore cytokine:L32 ratios were set to 1 in the mouse B cell lymphoma line 967. Transcription of IL10, IL15, and IFNgamma were reproducibly detected in LMP1 transgenic lymphocytes and lymphoma cells and was higher than in the B cell lymphoma cell lines 967 and K46mu (Figure 5A). There was no significant difference in the expression of IL15 and IFNgamma between LMP1 transgenic lymphocytes and lymphoma cells, suggesting that upregulation of IL15 and IFNgamma is induced by LMP1 expression in healthy lymphocytes but is not a unique property of malignant lymphocytes. Strikingly, IL10, a B lymphocyte stimulatory cytokine, was increased 1.5- to 5-fold in the wild-type and LMP1 transgenic lymphoma cells compared to LMP1 transgenic lymphocytes (Figure 5A). Production of IL15 and IFNgamma has been associated with induction of cytotoxic effector responses in cells latently infected with EBV [33,34]. However, transformation and growth properties induced by EBV are associated with the upregulation of IL10 [35-38]; hence, the effects of IL10 upregulation on the growth properties of the lymphoma cells were further examined. Immunoblot analysis indicated that LMP1 transgenic lymphocytes and wild-type and LMP1 transgenic lymphoma cells had corresponding increased levels of phosphorylated alpha and beta isoforms of activated Stat3, a target of the IL10 receptor (Figure 5B). However, when comparing the same lymphomas, there was no correlation between the levels of IL10 induction and the levels of Stat3 activation. This suggests that the activation of Stat3 is not solely induced by IL10 or that Stat3 activation may be constitutive. Additionally, there was no correlation between the levels of LMP1 expression and the levels of IL10 induction (Figures 1A and 5A). This indicates that the induction of IL10 is a general property associated with enhanced survival and may only be indirectly affected by LMP1. Neutralizing antibodies to IL10 did not affect the survival of lymphoma cells as determined by the MTS assay (unpublished data), suggesting constitutive activation of Stat3. This was confirmed by immunoblot analysis such that in the presence of anti-IL10 neutralizing antibodies, pStat3 levels remained activated in lymphoma cells isolated from wild-type and LMP1 transgenic lymphomas (Figure 5C). Exogenous addition of IL10 enhanced pStat3 activation above constitutive levels, indicating that lymphoma cells are responsive to IL10 treatment (Figure 5C). This means that although the lymphoma cells have constitutive Stat3 activation, it may be further enhanced by IL10 induction. The neutralizing effect of the anti-IL10 antibody was confirmed by pre-incubation of IL10 with anti-IL10 antibody compared to a rat IgG1 isotype control (Figure 5C).\nNuclear translocation of pStat3 is a consequence of activation, and nuclear pStat3 was not detected by immunohistochemistry staining of spleen sections from control mice. However, nuclear pStat3 was detectable in LMP1 transgenic mice and wild-type lymphomas and was detected more homogeneously in LMP1 transgenic lymphomas (Figure 5D). The constitutive activation of pStat3 and abundant nuclear Stat3 suggests that Stat3 signaling contributes to LMP1-mediated lymphoma development.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "detected", "start": 471, "end": 479}, "arguments": [{"role": "Theme", "text": "IL4", "start": 269, "end": 272}]}, {"trigger": {"text": "detected", "start": 471, "end": 479}, "arguments": [{"role": "Theme", "text": "IL5", "start": 274, "end": 277}]}, {"trigger": {"text": "detected", "start": 471, "end": 479}, "arguments": [{"role": "Theme", "text": "IL10", "start": 279, "end": 283}]}, {"trigger": {"text": "detected", "start": 471, "end": 479}, "arguments": [{"role": "Theme", "text": "IL13", "start": 285, "end": 289}]}, {"trigger": {"text": "detected", "start": 471, "end": 479}, "arguments": [{"role": "Theme", "text": "IL15", "start": 291, "end": 295}]}, {"trigger": {"text": "detected", "start": 471, "end": 479}, "arguments": [{"role": "Theme", "text": "IL9", "start": 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3545}, "arguments": [{"role": "ToLoc", "text": "nuclear", "start": 3451, "end": 3458}, {"role": "Theme", "text": "pStat3", "start": 3459, "end": 3465}]}], "negative regulation": [{"trigger": {"text": "Neutralizing", "start": 2423, "end": 2435}, "arguments": [{"role": "Theme", "text": "IL10", "start": 2450, "end": 2454}]}, {"trigger": {"text": "neutralizing effect", "start": 3109, "end": 3128}, "arguments": [{"role": "Theme", "text": "IL10", "start": 3141, "end": 3145}]}], "phosphorylation": [{"trigger": {"text": "phosphorylated", "start": 1786, "end": 1800}, "arguments": [{"role": "Theme", "text": "Stat3", "start": 1838, "end": 1843}]}], "positive regulation": [{"trigger": {"text": "Upregulates", "start": 5, "end": 16}, "arguments": [{"role": "Cause", "text": "LMP1", "start": 0, "end": 4}, {"role": "Theme", "text": "IL10", "start": 17, "end": 21}]}, {"trigger": {"text": "Activates", "start": 41, "end": 50}, "arguments": [{"role": "Cause", "text": "LMP1", "start": 0, "end": 4}, {"role": 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{"input": "LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway\nLMP1 transformation of rodent fibroblasts requires activation of PI3K and Akt [5]. Additionally, activated pAkt is frequently detected in NPC and the neoplastic Reed-Sternberg cells of classical HD [39,40]. To determine if Akt signaling is activated in LMP1 transgenic mice, pAkt and several of its targets were assessed by immunoblotting of splenic CD19+ MACS-purified B cells. LMP1 transgenic B cells had increased levels of pAkt compared to wild-type lymphocytes; however, progression to lymphoma in both LMP1-positive and -negative lymphoma cells did not further increase pAkt levels. The Akt target glycogen synthase kinase 3 (GSK3) is inactivated by phosphorylation; however, increased phosphorylated GSK3 was not detected in the transgenic lymphocytes and was almost absent in the lymphoma samples (Figure 6A). This finding indicates that GSK3 is not a target of activated Akt in the LMP1 transgenic lymphocytes and lymphoma cells. Similarly, activation of Akt without phosphorylation of GSK3 has been previously shown in EBV-positive HD [40]. In contrast, the wild-type lymphocytes lacked activated Akt but did have detectable phosphorylated GSK3. This further suggests that additional pathways are involved in the regulation of GSK3.\nTo identify other potential Akt targets, immunoblot analysis for p-mTOR was performed. Activated p-mTOR was not increased in LMP1 transgenic lymphocytes or lymphoma cells, indicating that this pathway is not affected by LMP1-induced Akt activation and does not contribute to lymphoma development (Figure 6B). Akt is also known to phosphorylate and induce the degradation of the pro-apoptotic Forkhead family of transcription factors, leading to cell cycle progression and survival in some human tumors [41,42]. Immunoblot analysis of splenic B cells did not consistently detect p-FoxO1 levels, a signal that targets FoxO1 for degradation. Hence, degradation of FoxO1 was assessed by detection of total FoxO1 levels. Immunoblot analysis indicated that total FoxO1 levels were greatly decreased in wild-type and LMP1 transgenic lymphomas (Figure 6B), suggesting that inhibition of the Forkhead signaling pathway is an important target of Akt in lymphoma development. However, considering that Akt activation did not induce FoxO1 degradation in LMP1 transgenic B cells, Akt may not be the sole regulator of FoxO1, and it may be that progression to lymphoma requires modulation of multiple pathways.\nThe Forkhead family of transcription factors is known to induce the expression of the Cdk inhibitor p27 [43,44]. LMP1-transformed rodent fibroblasts have decreased expression of p27, upregulation of Cdk2, and subsequent phosphorylation and inactivation of the tumor suppressor gene Rb [45]. To investigate whether LMP1 affected cell cycle regulation through the Rb pathway in B cells, immunoblot analyses for pRb, Cdk2, and p27 were performed on splenic CD19+ MACS-purified B cells. LMP1 transgenic B cells had enhanced levels of pRb with concomitant stabilization of total Rb levels and Cdk2 compared to wild-type B lymphocytes (Figure 6C). Progression to lymphoma in both wild-type and LMP1 transgenic lymphoma cells led to increased levels of Rb, correspondingly high levels of Cdk2, and decreased levels of p27 (Figure 6C). These data indicate that the Rb pathway is deregulated in LMP1 transgenic lymphocytes and that lymphoma cells are distinguished by loss of FoxO1 and decreased p27.", "output": {"json_structures": {"binding": [{"trigger": {"text": "target", "start": 668, "end": 674}, "arguments": [{"role": "Theme", "text": "Akt", "start": 664, "end": 667}, {"role": "Theme2", "text": "glycogen synthase kinase 3", "start": 675, "end": 701}]}, {"trigger": {"text": "target", "start": 931, "end": 937}, "arguments": [{"role": "Theme", "text": "GSK3", "start": 917, "end": 921}, {"role": "Theme2", "text": "Akt", "start": 951, "end": 954}]}, {"trigger": {"text": "targets", "start": 1346, "end": 1353}, "arguments": [{"role": "Theme", "text": "Akt", "start": 1342, "end": 1345}]}], "gene expression": [{"trigger": {"text": "expression", "start": 2578, "end": 2588}, "arguments": [{"role": "Theme", "text": "p27", "start": 2610, "end": 2613}]}], "negative regulation": [{"trigger": {"text": "inactivated", "start": 712, 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{"input": "LMP1 Promotes Tumor Growth and Survival through Activation of Akt, NFkappaB, and Stat3 Pathways\nTo explore which pathways were required for the enhanced growth and survival of LMP1-induced lymphomas, splenocytes from wild-type and LMP1 transgenic mice were cultured in the presence of inhibitors for Akt, NFkappaB, Stat3, mTOR, or MAPK and assayed for growth and survival by the MTS assay. As previously shown, wild-type lymphocytes were not viable in culture and could not be tested with the inhibitors. However, the enhanced viability of LMP1 transgenic lymphocytes was effectively blocked by treatment with triciribine, BAY11-7085, cucurbitacin I, and slightly with SB203580, but not by treatment with rapamycin, U0126, or AG490 (Figure 7). Triciribine inhibits the activation of Akt and at 20 muM has been shown to induce growth arrest in cancer cells with aberrant Akt activity [46]. The effects of triciribine on cell growth of the transgenic lymphocytes and lymphomas were apparent as low as 1 muM, suggesting that activation of Akt is required for the survival and growth of LMP1 transgenic lymphocytes and lymphoma cells (Figure 7). The effects of the inhibitors were assessed by identifying phosphorylated Akt, Stat3, and total levels of IkappaBalpha (Figure 8). Treatment with triciribine effectively blocked phosphorylation of Akt, and phosphorylated Akt was no longer detected past 5 muM. Phosphorylated Stat3 and IkappaBalpha were still present at 25 muM. These findings suggest that triciribine specifically targets Akt and that Akt activation is required for the enhanced viability of the transgenic lymphocytes and lymphoma cells.\nInhibition of NFkappaB signaling rapidly induces cell death of EBV-transformed lymphocytes [17,18]. BAY11-7085, an inhibitor of NFkappaB signaling, also greatly decreased the viability of the LMP1 transgenic lymphocytes and lymphoma cells at doses as low as 1 muM, and at 5 muM the cells were completely nonviable (Figure 7). This is well within the reported IC50 of 10 muM. Phosphorylated Akt, Stat3, and total IkappaBalpha were still present up to treatment with 15 muM and then were no longer detected (Figure 8). This finding suggests that inhibition of NFkappaB can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells without significant effects on activation of Akt or Stat3.\nCucurbitacin I inhibits activation of Stat3 by suppressing the activation of its kinase JAK2. It has been shown to selectively inhibit the growth of tumors with constitutively activated Stat3 [47]. Similarly, LMP1 transgenic lymphocytes and lymphoma cells were susceptible to cucurbitacin I treatment starting at 0.1 muM, a dose that corresponds closely to the reported IC50 of 500 nM (Figure 7) [47]. Phosphorylated Akt, Stat3, and total IkappaBalpha were not detectable past 1 muM and at higher doses all protein levels were greatly decreased, indicative of the total loss of viability (Figure 8). A second reported inhibitor of Stat3, AG490, had no effect on growth (Figure 7), but activation of Akt, Stat3, or levels of IkappaBalpha were also not affected (Figure 8). These findings suggest that inhibition of Stat3 can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells, but Stat3 inhibition also has considerable crossover effects on Akt and NFkappaB signaling.\nLMP1 has also been shown to activate JNK and p38 MAPK pathways [13,48], and LMP1 transgenic lymphocytes were mildly susceptible to growth inhibition by SB202190, an inhibitor of p38 MAPK, but not U0126, an inhibitor of MEK1/2 activity. However, effects of SB202190 were only apparent at high doses (>10 muM), much higher than the reported IC50 of 0.35 muM, suggesting that p38 MAPK does not significantly contribute to the enhanced viability in LMP1 transgenic lymphocytes or lymphoma cells (Figure 7). Interestingly, both wild-type and LMP1 transgenic lymphomas were similarly susceptible to triciribine, BAY11-7085, and cucurbitacin I treatments, but not SB202190, AG490, or U0126 treatment, suggesting that activation of Akt, NFkappaB, and Stat3 but not MAPK pathways are characteristics associated with malignant transformation (Figure 7). rapamycin, an inhibitor of mTOR, did not affect the viability of the transgenic lymphocytes or lymphoma cells, confirming that mTOR is not targeted by Akt activation in LMP1 transgenic lymphocytes or malignant lymphoma cells (Figure 7).", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "inhibitors", "start": 285, "end": 295}, "arguments": [{"role": "Theme", "text": "Akt", "start": 300, "end": 303}]}, {"trigger": {"text": "inhibitors", "start": 285, "end": 295}, "arguments": [{"role": "Theme", "text": "Stat3", "start": 315, "end": 320}]}, {"trigger": {"text": "inhibitors", "start": 285, "end": 295}, "arguments": [{"role": 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{"input": "This study defines the oncogenic properties of LMP1 in promoting B cell lymphomagenesis. LMP1 transgenic mice have a higher incidence of lymphoma [26] and the progression to lymphoma correlates with higher expression levels of LMP1 (Figure 1A and 1B), suggesting that LMP1 is directly involved in tumor development. Table 1 summarizes the biological and molecular properties that were identified in wild-type and LMP1 transgenic lymphomas. Although many of the molecular properties studied were similar between wild-type and LMP1 transgenic lymphomas, there were distinguishing biological properties, namely the ability of LMP1 transgenic lymphomas to induce higher levels of survival and proliferation. Interestingly, although LMP1 transgenic mice develop lymphomas in the same B-1a cell type as spontaneous wild-type lymphomas (Figure 2), some signaling effects induced by LMP1 may explain the enhanced promotion to lymphomagenesis. Since CD40-deficient mice have decreased numbers of IgMhighIgDlow cells, a phenotype associated with B-1, marginal zone, and memory B cells, the mimicry of CD40 signaling by LMP1 could possibly contribute to the expansion of B-1 cells [20]. It is noteworthy that expression of LMP1 in transgenic mice has been shown to inhibit the formation of GCs [23,49], preventing typical B-2 cells from antigen-driven selection and expansion. The lack of GC reactions may contribute to the bias of LMP1 transgenic mice towards B-1 cell lymphomas. Interestingly, LMP2 signaling also favors development of B-1 cells, but this occurs in the absence of transformation. These results suggest that the mimicry of B cell receptor signaling by LMP2 promotes B-1 cell differentiation but not transformation [24,50,51]. This promotion of B-1 differentiation may account for the ability of LMP2 to exacerbate autoimmunity and bypass anergy induction [52,53]. In contrast, the preponderance of tumors of B-1a origin does not reflect effects of LMP1 signaling on B cell differentiation, as splenic B cells from healthy LMP1 transgenic mice contain similar numbers of B-1 and B-2 cells as wild-type mice. In support of this lack of effect, the differentiation of B-1 versus B-2 cells is thought to be independent of CD40 signaling [54].\nB-1 cells constitute the predominant lymphocyte population in the peritoneal and pleuropericardial cavities, while B-2 cells are mainly found in the spleen, lymph node, and peripheral blood. B-1 cells produce the main source of IgM and IgA antibodies in serum, which are involved in T cell-independent responses to common microbial antigens. Importantly, B-1 cells have the unique capacity to self replenish and are also predisposed to transformation [28,29]. Clonal expansion of B-1 cells can be detected in aging mice above 18 mo of age, and B-1 cells are thought to be the murine progenitor of B cell chronic lymphocytic leukemia [30]. The data presented in this study indicate that although LMP1 is expressed in all B lymphocytes in the transgenic mice, malignancy develops in this specific subset of B cells. The elevated expression of LMP1 in B-1a cells and the activation of specific pathways apparently induce malignant growth. These same pathways can also become sporadically activated in aged mice and also result in lymphoma development. This is similar to EBV-associated cancers in vivo, where pathways that are activated by LMP1 are also activated in the less prevalent EBV-negative forms of the cancers [40,55-58]. Thus, the contribution of EBV and LMP1 to tumor development is apparently the continuous activation of pathways that can also be sporadically activated and contribute to tumor development.\nThe lymphomas were marked by the upregulation of IL10, constitutive activation of Stat3 signaling, and a requirement for activation of Akt, NFkappaB, and Stat3 pathways (Figures 5 and 7). Induction of IL10 is associated with the transformation of B-1 lymphomas in mice [59,60] and is frequently associated with EBV-positive B cell malignancies acting as a B cell growth factor [35-38]. In addition, LMP1 has been shown to stimulate IL10 expression in Burkitt lymphoma cell lines [61,62]. This suggests that although Stat3 is constitutively activated in the lymphoma cells, the induction of IL10 may further enhance Stat3 activation or may contribute to other IL10-responsive signaling pathways.\nLMP1 activates both the canonical and non-canonical pathways of NFkappaB signaling [14,63-65], and inhibition of NFkappaB blocked the survival of LMP1 transgenic lymphocytes and LMP1-positive and -negative lymphoma cells. NFkappaB and PI3K signaling are crucial for CD40-induced proliferation, and mice deficient for cRel or the p85 regulatory subunit of PI3K are unresponsive to mitogenic stimuli, including CD40 ligation [66-68]. We have previously shown that cRel is specifically activated in both wild-type and LMP1 transgenic lymphomas, suggesting that activation of cRel is associated with B cell transformation [27]. Our observations suggest that similar to CD40-induced proliferation, LMP1 induces proliferation through PI3K-mediated activation of Akt and activation of NFkappaB components such as cRel. CD40 also induces downregulation of the cell cycle inhibitor p27 through a PI3K-dependent manner, and the LMP1 lymphoma cells also had decreased levels of p27 with phosphorylation of Rb and increased Cdk2 (Figure 6C) [66]. Although LMP1 has been shown to deregulate the Rb pathway in epithelial cells [69], to our knowledge this is the first demonstration of this property in B lymphocytes.\nThe requirement for Akt activation was confirmed by the striking inhibition of lymphoma viability by triciribine, an Akt inhibitor. However, the activated pAkt did not lead to phosphorylation and inactivation of the downstream target GSK3 (Figure 6A). This effect has also been described in EBV-positive HD biopsies [40]. In contrast, rapamycin, U0126, and SB202190 did not affect the survival of LMP1 transgenic lymphocytes or the wild-type and LMP1 transgenic lymphoma cells (Figure 7A, 7C, and 7E). This lack of effect by rapamycin confirmed the absence of activated p-mTOR levels (Figure 6B). These findings suggest that other Akt targets contribute to malignant progression. One key target is likely the inhibition of the Fox01 transcription factors. Repression of the pro-apoptotic transcription factor FoxO1 in a PI3K-dependent manner can inhibit expression of bcl6, a transcription factor necessary for GC formation [49,70]. It has been shown that overstimulation of CD40 signaling with agonistic antibodies inhibits GC formation [71]. Similarly, due to mimicry of CD40 signaling, transgenic LMP1 mice are also defective in GC formation [23,49]. The constitutive signaling by LMP1 likely blocks GC formation through downregulation of bcl6. Interestingly, clinical studies indicate that expression of LMP1 and bcl6 are mutually exclusive in non-HD and classical HD [72,73]. Thus, the LMP1 transgenic lymphomas mirror aspects of EBV-induced HD. Although the activation of Akt and the lack of Fox01 in the lymphoma cells suggest that LMP1 affects bcl6 and GC formation through this pathway, regulation of other Forkhead targets involved in cell cycle progression, such as p27 and CyclinD2, likely contribute to malignant transformation. Indeed, loss of FoxO1 expression in lymphoma cells correlated with a loss of p27 (Figure 6B and 6C). CyclinD2 has also been shown to be upregulated by LMP1 through release of FoxO1-mediated repression [70].\nIn summary, in this transgenic model of lymphomagenesis, LMP1 promotes malignancy in B-1a cells, a population that is predisposed to clonal expansion with age. The malignant lymphocytes were distinguished by constitutively active Stat3 signaling, decreased p27, and activated Akt and NFkappaB pathways, properties that are associated with promoting the growth and survival of B lymphocytes. Importantly, Akt, NFkappaB, and Stat3 pathways were critically required for the growth and survival of malignant lymphocytes as well as healthy LMP1 transgenic lymphocytes. The growth of EBV-transformed lymphocytes also requires activation of NFkappaB, and these studies provide insight into how LMP1 contributes to EBV-associated transformation. 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"start": 4662, "end": 4671}, "arguments": [{"role": "Theme", "text": "p85", "start": 4688, "end": 4691}]}, {"trigger": {"text": "downregulation", "start": 5189, "end": 5203}, "arguments": [{"role": "Theme", "text": "p27", "start": 5232, "end": 5235}]}, {"trigger": {"text": "decreased", "start": 5306, "end": 5315}, "arguments": [{"role": "Theme", "text": "p27", "start": 5326, "end": 5329}]}, {"trigger": {"text": "inhibitor", "start": 5683, "end": 5692}, "arguments": [{"role": "Theme", "text": "Akt", "start": 5679, "end": 5682}]}, {"trigger": {"text": "inactivation", "start": 5758, "end": 5770}, "arguments": [{"role": "Theme", "text": "GSK3", "start": 5796, "end": 5800}]}, {"trigger": {"text": "absence", "start": 6111, "end": 6118}, "arguments": [{"role": "Theme", "text": "activated", "start": 6122, "end": 6131}]}, {"trigger": {"text": "inhibition", "start": 6271, "end": 6281}, "arguments": [{"role": "Theme", "text": "Fox01", "start": 6289, "end": 6294}]}, {"trigger": {"text": "Repression", "start": 6318, "end": 6328}, "arguments": [{"role": "Theme", "text": "FoxO1", "start": 6371, "end": 6376}]}, {"trigger": {"text": "inhibit", "start": 6408, "end": 6415}, "arguments": [{"role": "Cause", "text": "Repression", "start": 6318, "end": 6328}, {"role": "Theme", "text": "expression", "start": 6416, "end": 6426}]}, {"trigger": {"text": "downregulation", "start": 6786, "end": 6800}, "arguments": [{"role": "Theme", "text": "bcl6", "start": 6804, "end": 6808}]}, {"trigger": {"text": "exclusive", "start": 6897, "end": 6906}, "arguments": [{"role": "Cause", "text": "expression", "start": 6856, "end": 6866}, {"role": "Theme", "text": "expression", "start": 6856, "end": 6866}]}, {"trigger": {"text": "lack", "start": 7052, "end": 7056}, "arguments": [{"role": "Theme", "text": "Fox01", "start": 7060, "end": 7065}]}, {"trigger": {"text": "loss", "start": 7312, "end": 7316}, "arguments": [{"role": "Theme", "text": "expression", "start": 7326, "end": 7336}]}, {"trigger": {"text": "loss", "start": 7373, "end": 7377}, "arguments": [{"role": "Theme", "text": "p27", "start": 7381, "end": 7384}]}, {"trigger": {"text": "decreased", "start": 7758, "end": 7767}, "arguments": [{"role": "Theme", "text": "p27", "start": 7768, "end": 7771}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 5335, "end": 5350}, "arguments": [{"role": "Theme", "text": "Rb", "start": 5354, "end": 5356}]}, {"trigger": {"text": "phosphorylation", "start": 5738, "end": 5753}, "arguments": [{"role": "Theme", "text": "GSK3", "start": 5796, "end": 5800}]}], "positive regulation": [{"trigger": {"text": "higher", "start": 199, "end": 205}, "arguments": [{"role": "Theme", "text": "expression", "start": 206, "end": 216}]}, {"trigger": {"text": "elevated", "start": 3064, "end": 3072}, "arguments": [{"role": "Theme", "text": "expression", "start": 3073, "end": 3083}]}, {"trigger": {"text": "upregulation", "start": 3697, "end": 3709}, "arguments": [{"role": "Theme", "text": "IL10", "start": 3713, "end": 3717}]}, {"trigger": {"text": "Induction", "start": 3852, "end": 3861}, "arguments": [{"role": "Theme", "text": "IL10", "start": 3865, "end": 3869}]}, {"trigger": {"text": "stimulate", "start": 4086, "end": 4095}, "arguments": [{"role": "Cause", "text": "LMP1", "start": 4063, "end": 4067}, {"role": "Theme", "text": "expression", "start": 4101, "end": 4111}]}, {"trigger": {"text": "activated", "start": 4204, "end": 4213}, "arguments": [{"role": "Theme", "text": "Stat3", "start": 4180, "end": 4185}]}, {"trigger": {"text": "induction", "start": 4241, "end": 4250}, "arguments": [{"role": "Theme", "text": "IL10", "start": 4254, "end": 4258}]}, {"trigger": {"text": "enhance", "start": 4271, "end": 4278}, "arguments": [{"role": "Cause", "text": "induction", "start": 4241, "end": 4250}, {"role": "Theme", "text": "activation", "start": 4285, "end": 4295}]}, {"trigger": {"text": "activation", "start": 4285, "end": 4295}, "arguments": [{"role": "Theme", "text": "Stat3", "start": 4279, "end": 4284}]}, {"trigger": {"text": "activated", "start": 4842, "end": 4851}, "arguments": [{"role": "Theme", "text": "cRel", "start": 4821, "end": 4825}]}, {"trigger": {"text": "activation", "start": 4917, "end": 4927}, "arguments": [{"role": "Theme", "text": "cRel", "start": 4931, "end": 4935}]}, {"trigger": {"text": "induces", "start": 5057, "end": 5064}, "arguments": [{"role": "Cause", "text": "LMP1", "start": 5052, "end": 5056}, {"role": "Theme", "text": "activation", "start": 5101, "end": 5111}]}, {"trigger": {"text": "induces", "start": 5057, "end": 5064}, "arguments": [{"role": "Cause", "text": "LMP1", "start": 5052, "end": 5056}, {"role": "Theme", "text": "activation", "start": 5123, "end": 5133}]}, {"trigger": {"text": "activation", "start": 5101, "end": 5111}, "arguments": [{"role": "Theme", "text": "Akt", "start": 5115, "end": 5118}]}, {"trigger": {"text": "activation", "start": 5123, "end": 5133}, "arguments": [{"role": "Theme", "text": "cRel", "start": 5165, "end": 5169}]}, {"trigger": {"text": "induces", "start": 5181, "end": 5188}, "arguments": [{"role": "Cause", "text": "CD40", "start": 5171, "end": 5175}, {"role": "Theme", "text": "downregulation", "start": 5189, "end": 5203}]}, {"trigger": {"text": "increased", "start": 5361, "end": 5370}, "arguments": [{"role": "Theme", "text": "Cdk2", "start": 5371, "end": 5375}]}, {"trigger": {"text": "requirement", "start": 5566, "end": 5577}, "arguments": [{"role": "Theme", "text": "activation", "start": 5586, "end": 5596}]}, {"trigger": {"text": "activation", "start": 5586, "end": 5596}, "arguments": [{"role": "Theme", "text": "Akt", "start": 5582, "end": 5585}]}, {"trigger": {"text": "activated", "start": 5707, "end": 5716}, "arguments": [{"role": "Theme", "text": "pAkt", "start": 5717, "end": 5721}]}, {"trigger": {"text": "lead", "start": 5730, "end": 5734}, "arguments": [{"role": "Cause", "text": "activated", "start": 5707, "end": 5716}, {"role": "Theme", "text": "phosphorylation", "start": 5738, "end": 5753}]}, {"trigger": {"text": "lead", "start": 5730, "end": 5734}, "arguments": [{"role": "Cause", "text": "activated", "start": 5707, "end": 5716}, {"role": "Theme", "text": "inactivation", "start": 5758, "end": 5770}]}, {"trigger": {"text": "activated", "start": 6122, "end": 6131}, "arguments": [{"role": "Theme", "text": "p-mTOR", "start": 6132, "end": 6138}]}, {"trigger": {"text": "activation", "start": 7026, "end": 7036}, "arguments": [{"role": "Theme", "text": "Akt", "start": 7040, "end": 7043}]}, {"trigger": {"text": "correlated", "start": 7355, "end": 7365}, "arguments": [{"role": "Cause", "text": "loss", "start": 7312, "end": 7316}, {"role": "Theme", "text": "loss", "start": 7373, "end": 7377}]}, {"trigger": {"text": "upregulated", "start": 7440, "end": 7451}, "arguments": [{"role": "Theme", "text": "CyclinD2", "start": 7405, "end": 7413}]}], "regulation": [{"trigger": {"text": "target", "start": 6250, "end": 6256}, "arguments": [{"role": "Cause", "text": "Akt", "start": 6193, "end": 6196}, {"role": "Theme", "text": "inhibition", "start": 6271, "end": 6281}]}, {"trigger": {"text": "affects", "start": 7106, "end": 7113}, "arguments": [{"role": "Theme", "text": "bcl6", "start": 7114, "end": 7118}, {"role": "Cause", "text": "pathway", "start": 7149, "end": 7156}]}, {"trigger": {"text": "pathway", "start": 7149, "end": 7156}, "arguments": [{"role": "Theme", "text": "Fox01", "start": 7060, "end": 7065}, {"role": "Cause", "text": "LMP1", "start": 7101, "end": 7105}]}, {"trigger": {"text": "regulation", "start": 7158, "end": 7168}, "arguments": [{"role": "Theme", "text": "p27", "start": 7239, "end": 7242}]}, {"trigger": {"text": "regulation", "start": 7158, "end": 7168}, "arguments": [{"role": "Theme", "text": "CyclinD2", "start": 7247, "end": 7255}]}]}}, "schema": []}
{"input": "Transgenic mice.\nGeneration of LMP1 mice under the Ig heavy chain promoter and enhancer have been described previously and were maintained as heterozygotes on a Balbc background [26]. LMP1 mice were genotyped by Southern blot and PCR analysis of tail DNA as described previously [26]. Spleen and liver sections were fixed in 4% paraformaldehyde and embedded in paraffin, and 5-mum sections were stained with hematoxylin and eosin for histopathological analysis. Lymphomas were passaged by intraperitoneal injection of 1 x 108 splenocytes into SCID mice and sacrificed upon development of an extended abdomen. Animals were housed in the Association for Assessment and Accreditation for Animal Care-approved animal facility at the University of North Carolina at Chapel Hill. All protocols were approved by the Institutional Animal Care and Use Committee.", "output": {"json_structures": {}}, "schema": []}
{"input": "Isolation and growth of B cells.\nSplenocytes were prepared by homogenizing spleen tissue with two frosted slides and debris was filtered through a 100-mum cell strainer. Erythrocytes were lysed using 0.8% ammonium chloride solution (StemCell Technologies) for 10 min on ice and washed twice with PBS. B cells were isolated using CD19-MACS beads according to the manufacturer's instructions (Miltenyi Biotec) and grown in Iscove's medium supplemented with heat-inactivated 10% fetal bovine serum and antibiotic/antimycotic (GIBCO). Splenocytes isolated from SCID-passaged lymphomas consisted of 80%-90% B cells as determined by flow cytometry, and were hence not further purified with CD19-MACS beads. Splenocytes were seeded at 1.25 x 106 cells/ml and where applicable, recombinant mouse IL4 was added at 100 ng/ml, recombinant mouse IL10 at 10 ng/ml, and rat IgG1 anti-mouse IL10 and rat IgG1 isotype control at 10 mug/ml (R&D Systems). For BrdU incorporation assays, splenocytes were pulsed for 24 h with 10 muM BrdU 1 d post-harvest.", "output": {"json_structures": {}}, "schema": []}
{"input": "MTS assay.\nThe 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) cell cytotoxicity/proliferation assays were performed using the CellTiter 96 aqueous one-solution cell proliferation assay (Promega), according to manufacturer's instructions. For IL4 studies, splenocytes were cultured for 3 d in the presence or absence of 100 ng/ml IL4. Cells were seeded on day 3 in triplicate in a 96-well plate at 2.5 x 106 cells/ml at 100 mul per well. MTS reagent was added for 4 h and absorbance was read at 540 nm; values plotted were subtracted from blanks. For neutralization assays, splenocytes were seeded at 5 x 106 cells/ml at 100 mul per well on day of harvest and IL4, rat IgG1 anti-mouse IL4, or rat IgG1 isotype control (R&D Systems) were added at the concentrations indicated in the figures. Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding. For inhibitor studies, splenocytes were seeded at 1 x 107 cells/ml at 100 mul per well on day of harvest, and inhibitors were added at the concentrations indicated in the figures. The inhibitors BAY11-7085, rapamycin, triciribine, U0126, SB202190, and cucurbitacin I were purchased from EMD Biosciences. For non-malignant splenocyte cultures, B cell activation was induced with 10 mug/ml of goat F(ab') anti-mouse IgM (Jackson ImmunoResearch). Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding.", "output": {"json_structures": {}}, "schema": []}
{"input": "Immunohistochemistry.\nParaffin-embedded spleen sections were deparaffinized in Histoclear (National Diagnostics) and rehydrated in graded ethanol. Sections were antigen retrieved by microwaving in citrate buffer (pH 6.0) for 15 min (LMP1 staining) and 10 min (pStat3 staining). For LMP1 staining, sections were blocked with 1% BSA and 0.1% cold fish skin gelatin followed with streptavidin/biotin block (Vector Labs). Rat IgG anti-LMP1 (clones 8G3 and 1G6, Ascenion) were used at 1:10 dilution from tissue culture supernatants, followed with 8 mug/ml biotinylated mouse F(ab') anti-rat IgG (H+L) pre-adsorbed to mouse serum (Jackson ImmunoResearch) and 2 mug/ml streptavidin-alkaline phosphatase conjugate (Jackson ImmunoResearch). Stains were developed with BCIP/NBT and counterstained in Nuclear Fast Red (Dako). Phospho-Stat3 was detected with 4 mug/ml of pStat3 antibody (Tyr705, Cell Signaling) and detected with anti-rabbit Poly-HRP IHC detection kit (Chemicon).", "output": {"json_structures": {}}, "schema": []}
{"input": "Flow cytometry.\nOne million splenocytes were stained with the appropriate primary antibody unconjugated or conjugated to FITC, PE, or APC diluted in stain buffer (PBS with 3% FBS). For BrdU detection, the FITC-BrdU flow kit was used as instructed by the manufacturer (BD Bioscience). Briefly, cells were exposed to EMA (Molecular Probes) for exclusion of dead cells, stained for surface antigens, fixed in paraformaldehyde, and permeabilized with saponin. To expose BrdU epitopes, cells were treated with Dnase and stained with FITC-conjugated anti-BrdU antibody. Flow cytometry was performed on FACScalibur using the CellQuest program (Becton Dickinson). Further analysis was conducted on the Summit v4.2 program (Dako).", "output": {"json_structures": {}}, "schema": []}
{"input": "Rnase protection assay.\nTotal RNA was isolated from CD19+ MACS-purified B cells using the Rneasy midi purification kit with Dnase treatment, according to the manufacturer's instructions (Qiagen). The mCK-1 probe template set (BD Biosciences) was labeled using the In Vitro Transcription Kit according to the manufacturer's instructions (BD Biosciences). Briefly, 50 ng of mCK-1 probe set was labeled with [alpha-32P]UTP using T7 RNA polymerase and purified using Sephadex G-50 columns (NucAway Spin column, Ambion). The labeled probe was quantitated using a scintillation counter, and 6 x 105 cherenkov cpm was used to hybridize to 4 mug of total RNA using the RPA kit (BD Biosciences). Samples were denatured at 90 degreesC and hybridized at 56 degreesC overnight. Single-stranded RNA was digested with a mixture of Rnase A and T1, and precipitated using isopropanol. Protected probes were resolved on a denaturing 4.75% acrylamide gel, dried, and imaged using a phosphorImager (Molecular Dynamics). Densitometry was performed using the ImageQuant TL v2005 software (GE Healthcare).", "output": {"json_structures": {}}, "schema": []}
{"input": "PCR analysis of kappa chain rearrangement.\nDNA was isolated from splenocytes using the Dneasy Tissue Kit (Qiagen), with Rnase treatment. PCR reactions contained 100 ng of genomic DNA, 0.2 muM each primer, 0.2 mM dNTPs, and 2.5 U Taq DNA polymerase (NEB) performed in 1X ThermoPol buffer (NEB). Primers used were Vkappacon and Jkappa5-1degrees and have been described previously [74]. PCR conditions were 94 degreesC for 2 min, 40 cycles of 94 degreesC for 30 s, 63 degreesC for 90 s, and 72 degreesC for 1 min, followed by 1 cycle of 72 degreesC for 5 min.", "output": {"json_structures": {}}, "schema": []}
{"input": "Immunoblot analysis.\nWhole cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate) supplemented with 2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1:100 protease/phosphatase inhibitor cocktails (Sigma). Crude lysates were centrifuged at 13,000 rpm for 10 min at 4 degreesC and the supernatants were collected for further analysis. Protein concentrations were determined with the Bio-Rad DC protein assay system. Lysates were boiled in the presence of 2.5% beta-mecaptoethanol, separated by denaturing SDS-PAGE, and transferred to 0.45-mum Optitran membranes (Schleicher & Schuell) in a Bio-Rad transfer unit. Membranes were immunoblotted with the appropriate primary antibody followed by horseradish peroxidase-tagged secondary antibodies (Amersham Biosciences and Dako) and detected with the SuperSignal West Pico System (Pierce).", "output": {"json_structures": {}}, "schema": []}
{"input": "Antibodies.\nFITC-conjugated goat anti-mouse IgM, rat IgG2akappa anti-mouse IgD (clone 11-26), rat IgG1kappa anti-mouse kappa (clone 187.1), rat IgG2bkappa anti-mouse lambda (clone JC5-1); PE-conjugated goat anti-mouse IgG; un-conjugated goat anti-mouse kappa and anti-mouse lambda were purchased from Southern Biotech. APC-conjugated rat IgG2akappa anti-mouse CD19 (clone 6D5), PE-conjugated mouse IgG2akappa anti-LMP1 (clone S12), un-conjugated mouse IgG2a anti-Rb (clone 2), and anti-Cdk2 (clone 55) were purchased from BD Bioscience. PE-conjugated rat IgG2akappa anti-mouse CD5 (clone 53-7.3) was purchased from eBioscience. Rat anti-LMP1 (clones 8G3, 1G6, 7E10, and 7G8) was purchased from Ascenion. Rabbit anti-pAkt (Ser473), anti-pGSK3alpha/beta (Ser21/9), anti-pStat3 (Tyr705), anti-pStat6 (Tyr641), anti-pmTOR (Ser2448), anti-Stat6, anti-Akt, anti-FoxO1, and anti-p27 were purchased from Cell Signaling. Rabbit anti-Stat3 (H-190), anti-IkappaBalpha (C-21), and goat anti-beta actin (I-19) were purchased from Santa Cruz Biotechnology. Mouse IgG1kappa anti-GSK3 was purchased from Upstate Biotechnology. Rabbit anti-pRb (Thr373) was purchased from EMD Biosciences.", "output": {"json_structures": {}}, "schema": []}
{"input": "High LMP1 Expression Correlates with the Development of Lymphoma\nLMP1 expression is shown by (A) immunoblotting of purified B cells (CD19+) and (B) immunohistochemistry staining of spleen tissue from wild-type (WT) and LMP1 transgenic mice.\n(A) Lymphomas are identified with a number (1-7). Arrows indicate the LMP1-specific band and its degradation products as well as a non-specific band. Actin was used as a loading control.\n(B) White and red pulps are shown, but this architecture is lost upon development of lymphoma. Scale bar, 20 mum.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Expression", "start": 10, "end": 20}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 5, "end": 9}]}, {"trigger": {"text": "expression", "start": 70, "end": 80}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 65, "end": 69}]}], "positive regulation": [{"trigger": {"text": "High", "start": 0, "end": 4}, "arguments": [{"role": "Theme", "text": "Expression", "start": 10, "end": 20}]}]}}, "schema": []}
{"input": "LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion\n(A) Flow cytometry analysis of splenocytes from a WT or LMP1 transgenic lymphoma for the pan-B cell (CD19), B-1a cell (CD5), and Ig heavy chain (IgM and IgD) and light chain (kappa and lambda) markers. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 4. This analysis was repeated on four other LMP1 transgenic lymphomas (1, 2, 3, and 6) showing a similar B-1a phenotype, of which lymphomas 2 and 4 were also doubly positive for kappa and lambda light chains.\n(B) Flow cytometry analysis of WT or LMP1 transgenic splenocytes for B-1a (CD19+CD5+) and B-1b or B2 subsets (CD19+CD5-). Percentages of B-1a and B-1b or B2 subsets are shown in each quadrant. This analysis was repeated on three other WT and two other LMP1 transgenic mice with similar results.\n(C) Immunoblot analysis for kappa and lambda light chains of B cells (CD19+) purified from WT and LMP1 transgenic mice. Actin was used as a loading control.", "output": {"json_structures": {}}, "schema": []}
{"input": "LMP1 Promotes B Cell Survival and Proliferation In Vitro\n(A) MTS assay of splenocytes from WT and LMP1 transgenic mice. Splenocytes were cultured in the presence (grey bars) or absence (black bars) of IL4 for 3 d. The results are the mean +/- SEM of triplicate samples averaged from multiple mice where \"n\" the number of mice analyzed is as follows: n = 2 for WT lymphocytes and WT lymphomas, n = 11 for LMP1 transgenic lymphocytes, and n = 13 for LMP1 transgenic lymphomas.\n(B) EMA exclusion of CD19+ gated splenocytes from WT and LMP1 transgenic mice showing percentage of viable B cells cultured with (white bars) or without (black bars) IL4 for 2 d.\n(C) Flow cytometry analysis for incorporated BrdU in WT and LMP1 transgenic lymphoma cells cultured with or without IL4 for 2 d. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 2. Percentages of cells in each quadrant are shown.", "output": {"json_structures": {}}, "schema": []}
{"input": "Wild-Type and LMP1 Transgenic Lymphoma Cells Survive Independently of IL4/Stat6 Signaling in Culture\n(A) Rnase protection assay for IL4 mRNA from purified B cells (CD19+) from WT and LMP1 transgenic splenocytes. The L32 and GAPDH housekeeping genes were used as a loading control. Arrow indicates the position of the protected probe.\n(B and C) Immunoblot analysis of WT and LMP1 transgenic mice for activated pStat6 in (B) purified B cells (CD19+) at the time of harvest or in (C) whole splenocytes cultured with or without IL4. (B) Actin was used as a loading control, and the white line indicates that intervening lanes have been spliced out.\n(D and E) MTS assay of (D) WT lymphocytes and (E) LMP1 transgenic lymphoma cells cultured with IL4, a neutralizing antibody to IL4, or a rat IgG isotype control at the indicated concentrations. Shown are the results from LMP1 transgenic lymphoma 3. The results are the mean +/- SEM of triplicate samples from a single representative experiment that was repeated twice with similar results.", "output": {"json_structures": {"positive regulation": [{"trigger": {"text": "activated", "start": 399, "end": 408}, "arguments": [{"role": "Theme", "text": "pStat6", "start": 409, "end": 415}]}]}}, "schema": []}
{"input": "LMP1 Upregulates IL10 Expression and Constitutively Activates Stat3\n(A) Relative expression of IL10, IL15, and IFNgamma mRNA in WT and LMP1 transgenic B cells (CD19+), as detected with an Rnase protection assay. Mouse lymphoma cell lines 967 and K46mu were used as controls. Expression levels were quantified with a phosphorimager and values were normalized to the ribosomal housekeeping gene L32. The cytokine:L32 ratio was set to 1 in the mouse B cell lymphoma line 967.\n(B and C) Immunoblot analysis of activated pStat3 in purified B cells (CD19+) from WT and LMP1 transgenic mice (B) at the time of harvest, and (C) 4 h after culture with or without IL10, a neutralizing antibody to IL10, or a rat IgG1 isotype control. (C) Shown are the results for WT lymphoma 1 and LMP1 transgenic lymphoma 1. Arrows indicate the positions of the alpha and beta isoforms of Stat3. Actin was used as a loading control.\n(D) Immunohistochemistry detection of activated nuclear pStat3 in the spleens of WT and LMP1 transgenic mice. Scale bar, 20 mum.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Expression", "start": 22, "end": 32}, "arguments": [{"role": "Theme", "text": "IL10", "start": 17, "end": 21}]}], "positive regulation": [{"trigger": {"text": "Upregulates", "start": 5, "end": 16}, "arguments": [{"role": "Cause", "text": "LMP1", "start": 0, "end": 4}, {"role": "Theme", "text": "Expression", "start": 22, "end": 32}]}, {"trigger": {"text": "Activates", "start": 52, "end": 61}, "arguments": [{"role": "Cause", "text": "LMP1", "start": 0, "end": 4}, {"role": "Theme", "text": "Stat3", "start": 62, "end": 67}]}, {"trigger": {"text": "activated", "start": 506, "end": 515}, "arguments": [{"role": "Theme", "text": "pStat3", "start": 516, "end": 522}]}, {"trigger": {"text": "activated", "start": 946, "end": 955}, "arguments": [{"role": "Theme", "text": "pStat3", "start": 964, "end": 970}]}], "transcription": [{"trigger": {"text": "expression", "start": 81, "end": 91}, "arguments": [{"role": "Theme", "text": "IL15", "start": 101, "end": 105}]}, {"trigger": {"text": "expression", "start": 81, "end": 91}, "arguments": [{"role": "Theme", "text": "IFNgamma", "start": 111, "end": 119}]}, {"trigger": {"text": "expression", "start": 81, "end": 91}, "arguments": [{"role": "Theme", "text": "IL10", "start": 95, "end": 99}]}]}}, "schema": []}
{"input": "LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway\n(A and B) Immunoblot analysis of purified B cells (CD19+) from the spleens of WT and LMP1 transgenic mice for Akt signaling, probing for (A) activated pAkt and downstream targets, including inactivated pGSK3alpha/beta, and (B) activated p-mTOR, and total levels of FoxO1. Arrows indicate the positions of alpha and beta isoforms of GSK3. The white line indicates that intervening lanes have been spliced out.\n(C) Immunoblot analysis for cell cycle proteins regulating the Rb pathway, probing for activated pRb, and total levels of Cdk2 and the Cdk inhibitor p27. Actin was used as a loading control.", "output": {"json_structures": {"positive regulation": [{"trigger": {"text": "inactivated", "start": 261, "end": 272}, "arguments": [{"role": "Theme", "text": "pGSK3alpha", "start": 273, "end": 283}]}, {"trigger": {"text": "inactivated", "start": 261, "end": 272}, "arguments": [{"role": "Theme", "text": "beta", "start": 284, "end": 288}]}, {"trigger": {"text": "activated", "start": 298, "end": 307}, "arguments": [{"role": "Theme", "text": "p-mTOR", "start": 308, "end": 314}]}, {"trigger": {"text": "activated", "start": 567, "end": 576}, "arguments": [{"role": "Theme", "text": "pRb", "start": 577, "end": 580}]}], "regulation": [{"trigger": {"text": "targets", "start": 242, "end": 249}, "arguments": [{"role": "Theme", "text": "pGSK3alpha", "start": 273, "end": 283}]}, {"trigger": {"text": "targets", "start": 242, "end": 249}, "arguments": [{"role": "Theme", "text": "beta", "start": 284, "end": 288}]}]}}, "schema": []}
{"input": "Akt, NFkappaB, and Stat3 Signaling Are Required for the Growth and Survival of Lymphoma Cells\nMTS assay of splenocytes from (A and B) WT or (C and D) LMP1 transgenic lymphomas and (E and F) LMP1 transgenic lymphoctyes. Splenocytes were cultured with or without inhibitors of NFkappaB (BAY11), mTOR (rapamycin), Akt (triciribine), MEK1/2 (U0126), p38 (SB202190), or Stat3 (cucurbitacin I and AG490) at the indicated concentrations. The results are the mean +/- SEM of triplicate samples. Shown are the results for (A and B) WT lymphoma 1, (C and D) LMP1 transgenic lymphoma 2, and (E and F) one out of two LMP1 transgenic mice analyzed. This analysis was repeated with LMP1 transgenic lymphoma 4 yielding similar results.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "inhibitors", "start": 261, "end": 271}, "arguments": [{"role": "Theme", "text": "mTOR", "start": 293, "end": 297}]}, {"trigger": {"text": "inhibitors", "start": 261, "end": 271}, "arguments": [{"role": "Theme", "text": "Akt", "start": 311, "end": 314}]}, {"trigger": {"text": "inhibitors", "start": 261, "end": 271}, "arguments": [{"role": "Theme", "text": "MEK1", "start": 330, "end": 334}]}, {"trigger": {"text": "inhibitors", "start": 261, "end": 271}, "arguments": [{"role": "Theme", "text": "2", "start": 335, "end": 336}]}, {"trigger": {"text": "inhibitors", "start": 261, "end": 271}, "arguments": [{"role": "Theme", "text": "Stat3", "start": 365, "end": 370}]}]}}, "schema": []}
{"input": "Analysis of Akt, NFkappaB, and Stat3 Pathways in Contribution to the Growth and Survival of Lymphoma Cells\n(A-D) Immunoblot analysis of wild-type and LMP1 transgenic lymphomas for Akt, NFkappaB, and Stat3 signaling after treatment with (A) an Akt inhibitor, triciribine, (B) an NFkappaB inhibitor, BAY11-7085, and the Stat3 inhibitors (C) cucurbitacin I and (D) AG490, at the indicated concentrations. Arrows indicate the positions of alpha and beta isoforms of Stat3. Actin was used as a loading control.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "inhibitor", "start": 247, "end": 256}, "arguments": [{"role": "Theme", "text": "Akt", "start": 243, "end": 246}]}, {"trigger": {"text": "inhibitors", "start": 324, "end": 334}, "arguments": [{"role": "Theme", "text": "Stat3", "start": 318, "end": 323}]}]}}, "schema": []}
{"input": "Summary of Analysis Performed on Wild-Type and LMP1 Transgenic Lymphomas", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Transgenic", "start": 52, "end": 62}, "arguments": [{"role": "Theme", "text": "LMP1", "start": 47, "end": 51}]}]}}, "schema": []}
{"input": "Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs\nActivation of naive CD8+ T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 644, "end": 654}, "arguments": [{"role": "Theme", "text": "interferon (IFN) gamma", "start": 621, "end": 643}]}, {"trigger": {"text": "expression", "start": 795, "end": 805}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 789, "end": 794}]}, {"trigger": {"text": "expression", "start": 817, "end": 827}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 883, "end": 892}]}, {"trigger": {"text": "expression", "start": 817, "end": 827}, "arguments": [{"role": "Theme", "text": "perforin", "start": 894, "end": 902}]}, {"trigger": {"text": "expression", "start": 817, "end": 827}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 908, "end": 918}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 487, "end": 496}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 500, "end": 505}]}, {"trigger": {"text": "induction", "start": 515, "end": 524}, "arguments": [{"role": "Theme", "text": "Eomesodermin", "start": 528, "end": 540}]}], "regulation": [{"trigger": {"text": "regulate", "start": 583, "end": 591}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 500, "end": 505}, {"role": "Theme", "text": "expression", "start": 644, "end": 654}]}, {"trigger": {"text": "regulate", "start": 583, "end": 591}, "arguments": [{"role": "Cause", "text": "Eomesodermin", "start": 528, "end": 540}, {"role": "Theme", "text": "expression", "start": 644, "end": 654}]}, {"trigger": {"text": "regulates", "start": 779, "end": 788}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 773, "end": 778}, {"role": "Theme", "text": "expression", "start": 795, "end": 805}]}, {"trigger": {"text": "regulates", "start": 779, "end": 788}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 773, "end": 778}, {"role": "Theme", "text": "expression", "start": 817, "end": 827}]}]}}, "schema": []}
{"input": "Naive CD8+T cells differentiate into effector CTLs with the ability to lyse antigen-bearing target cells by exocytosis of lytic granules containing perforin and granzymes, and to produce inflammatory cytokines such as IFN-gamma and TNF upon restimulation through the TCR (1,2). In vivo experiments have elucidated many critical parameters governing the development and evolution of primary CTL responses (3,4). In this study, we have used in vitro systems such as those developed to study CD4+T cell differentiation to define the molecular basis of effector CTL differentiation (5,6).The T-box transcription factors Eomesodermin (Eomes) and T-bet are needed for important aspects of effector and memory CTL differentiation (7). In uninfected mice, compound deletion of theTbx21(encoding T-bet) andeomesodermingenes is associated with a selective loss of CD8+T cells with an IL-2Rbeta-high, memory phenotype (8). Mice deficient for both T-bet and Eomes in T cells have impaired expression of cytolytic mediators, manifest poor cytolytic activity, and fail to control acute lymphocytic choriomeningitis virus infection (9). Nevertheless, the specific roles of T-bet and Eomes in clonal expansion and CTL differentiation have not yet been resolved: in particular, it is not known whether these transcription factors function redundantly to control effector CD8+T cell differentiation, and whether they do so directly by targeting specific effector cytokine and cytolytic genes.Runx proteins, a family of three DNA-binding transcription factors, control thymocyte differentiation and the CD4/CD8 lineage decision (10-13). Runx3 and perforin mRNA are expressed by double-positive (DP) thymocytes and CD8+single-positive (SP) thymocytes but not in CD4+SP cells (14). Although Runx3 is not expressed in naive CD4+T cells, its expression is up-regulated during Th1 cell differentiation, and Runx3 influences Th1 cell differentiation and function through direct regulation of theIl4andIfngcytokine genes (15,16). In contrast, all three Runx proteins are expressed in mature CD8+T cells (10,12), and Runx3-deficient CD8+T cells show reduced cytolytic activity (12,13). We therefore tested whether Runx3 influenced cytolytic T cell differentiation.In this report, we show that Runx3 and T-box factors synergistically regulate CTL differentiation and function. T-bet is induced quickly upon TCR stimulation and is required for early programming of cytokine production (17), whereas Eomes is induced later during differentiation and sustains IFN-gamma expression. Runx3 is required for Eomes and perforin expression, and both Eomes and Runx3 bind at thePrf1locus; in contrast, perforin expression is unaffected in T-bet-deficient cells. T cells lacking Runx3 show decreased expression of IFN-gamma and granzyme B, and Runx3 also binds the promoter regions of theIfngandGzmbgenes. Collectively, these results provide evidence for a complex transcriptional network in which Runx3 is a primary regulator ofGzmbexpression but synergizes with T-bet and Eomes, respectively, to promote transcription of theIfngandPrf1genes.", "output": {"json_structures": {"binding": [{"trigger": {"text": "targeting", "start": 1417, "end": 1426}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 1158, "end": 1163}]}, {"trigger": {"text": "targeting", "start": 1417, "end": 1426}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 1168, "end": 1173}]}, {"trigger": {"text": "bind", "start": 2629, "end": 2633}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 2613, "end": 2618}, {"role": "Theme2", "text": "Prf1", "start": 2640, "end": 2644}]}, {"trigger": {"text": "bind", "start": 2629, "end": 2633}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 2623, "end": 2628}, {"role": "Theme2", "text": "Prf1", "start": 2640, "end": 2644}]}, {"trigger": {"text": "binds", "start": 2816, "end": 2821}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 2805, "end": 2810}, {"role": "Site2", "text": "promoter regions", "start": 2826, "end": 2842}, {"role": "Theme2", "text": "Ifng", "start": 2849, "end": 2853}]}, {"trigger": {"text": "binds", "start": 2816, "end": 2821}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 2805, "end": 2810}, {"role": "Site2", "text": "promoter regions", "start": 2826, "end": 2842}, {"role": "Theme2", "text": "Gzmb", "start": 2856, "end": 2860}]}], "gene expression": [{"trigger": {"text": "produce", "start": 179, "end": 186}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 218, "end": 227}]}, {"trigger": {"text": "produce", "start": 179, "end": 186}, "arguments": [{"role": "Theme", "text": "TNF", "start": 232, "end": 235}]}, {"trigger": {"text": "expressed", "start": 1783, "end": 1792}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 1770, "end": 1775}]}, {"trigger": {"text": "expression", "start": 1819, "end": 1829}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 1770, "end": 1775}]}, {"trigger": {"text": "expression", "start": 2539, "end": 2549}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 2529, "end": 2538}]}, {"trigger": {"text": "expression", "start": 2592, "end": 2602}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 2573, "end": 2578}]}, {"trigger": {"text": "expression", "start": 2592, "end": 2602}, "arguments": [{"role": "Theme", "text": "perforin", "start": 2583, "end": 2591}]}, {"trigger": {"text": "expression", "start": 2673, "end": 2683}, "arguments": [{"role": "Theme", "text": "perforin", "start": 2664, "end": 2672}]}, {"trigger": {"text": "expression", "start": 2761, "end": 2771}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 2775, "end": 2784}]}, {"trigger": {"text": "expression", "start": 2761, "end": 2771}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 2789, "end": 2799}]}, {"trigger": {"text": "expression", "start": 2994, "end": 3004}, "arguments": [{"role": "Theme", "text": "Gzmb", "start": 2990, "end": 2994}]}], "negative regulation": [{"trigger": {"text": "deletion", "start": 757, "end": 765}, "arguments": [{"role": "Theme", "text": "Tbx21", "start": 772, "end": 777}]}, {"trigger": {"text": "deletion", "start": 757, "end": 765}, "arguments": [{"role": "Theme", "text": "eomesodermin", "start": 797, "end": 809}]}, {"trigger": {"text": "deficient", "start": 917, "end": 926}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 936, "end": 941}]}, {"trigger": {"text": "deficient", "start": 917, "end": 926}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 946, "end": 951}]}, {"trigger": {"text": "deficient", "start": 2096, "end": 2105}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 2090, "end": 2095}]}, {"trigger": {"text": "deficient", "start": 2707, "end": 2716}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 2701, "end": 2706}]}, {"trigger": {"text": "lacking", "start": 2732, "end": 2739}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 2740, "end": 2745}]}, {"trigger": {"text": "decreased", "start": 2751, "end": 2760}, "arguments": [{"role": "Cause", "text": "lacking", "start": 2732, "end": 2739}, {"role": "Theme", "text": "expression", "start": 2761, "end": 2771}]}], "positive regulation": [{"trigger": {"text": "up-regulated", "start": 1833, "end": 1845}, "arguments": [{"role": "Theme", "text": "expression", "start": 1819, "end": 1829}]}, {"trigger": {"text": "induced", "start": 2358, "end": 2365}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 2349, "end": 2354}]}, {"trigger": {"text": "induced", "start": 2479, "end": 2486}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 2470, "end": 2475}]}, {"trigger": {"text": "sustains", "start": 2520, "end": 2528}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 2470, "end": 2475}, {"role": "Theme", "text": "expression", "start": 2539, "end": 2549}]}, {"trigger": {"text": "required", "start": 2560, "end": 2568}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 2551, "end": 2556}, {"role": "Theme", "text": "expression", "start": 2592, "end": 2602}]}, {"trigger": {"text": "promote", "start": 3059, "end": 3066}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 3025, "end": 3030}, {"role": "Theme", "text": "transcription", "start": 3067, "end": 3080}]}, {"trigger": {"text": "promote", "start": 3059, "end": 3066}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 3035, "end": 3040}, {"role": "Theme", "text": "transcription", "start": 3067, "end": 3080}]}], "regulation": [{"trigger": {"text": "regulation", "start": 1953, "end": 1963}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 1883, "end": 1888}, {"role": "Theme", "text": "Il4", "start": 1970, "end": 1973}]}, {"trigger": {"text": "regulation", "start": 1953, "end": 1963}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 1883, "end": 1888}, {"role": "Theme", "text": "Ifng", "start": 1976, "end": 1980}]}, {"trigger": {"text": "unaffected", "start": 2687, "end": 2697}, "arguments": [{"role": "Theme", "text": "expression", "start": 2673, "end": 2683}, {"role": "Cause", "text": "deficient", "start": 2707, "end": 2716}]}, {"trigger": {"text": "regulator", "start": 2978, "end": 2987}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 2959, "end": 2964}, {"role": "Theme", "text": "expression", "start": 2994, "end": 3004}]}], "transcription": [{"trigger": {"text": "expressed", "start": 1646, "end": 1655}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 1618, "end": 1623}]}, {"trigger": {"text": "expressed", "start": 1646, "end": 1655}, "arguments": [{"role": "Theme", "text": "perforin", "start": 1628, "end": 1636}]}, {"trigger": {"text": "transcription", "start": 3067, "end": 3080}, "arguments": [{"role": "Theme", "text": "Ifng", "start": 3087, "end": 3091}]}, {"trigger": {"text": "transcription", "start": 3067, "end": 3080}, "arguments": [{"role": "Theme", "text": "Prf1", "start": 3094, "end": 3098}]}]}}, "schema": []}
{"input": "A cell culture system to monitor effector CTL differentiation\nWe used a simple cell culture system to examine the kinetics of effector gene expression during CD8+ T cell differentiation. Naive CD8+ T cells from P14 TCR transgenic mice were activated for 2 d with anti-CD3 and anti-CD28 or with splenic APCs in the presence of Gp33 peptide, and were cultured in media containing 100 U/ml of recombinant human IL-2 (rhIL-2). We used TCR transgenic mice for these experiments because they provide a reliable source of CD8+ T cells that are truly naive; however, we chose not to stimulate cells with antigen in most experiments so as to avoid contamination with proteins and nucleic acids derived from APCs. There were only minor differences in gene expression during differentiation induced by antigen/APC versus anti-CD3/anti-CD28, and the major conclusions presented in this report are the same for both activating conditions.\nUnder our culture conditions, activated CD8+ T cells expanded exponentially and accumulated for >8 d. We limited our analysis to the first 6-8 d after activation, a period that coincides with clonal expansion of CD8+ T cells after activation in vivo.", "output": {"json_structures": {}}, "schema": []}
{"input": "Distinct expression kinetics of perforin and granzyme B during CTL development in culture\nOur experiments revealed clear differences in the kinetics of perforin, granzyme B, and cytokine expression during CD8+ T cell activation (Fig. 1). Naive T cells showed detectable expression of perforin mRNA as well as perforin protein (Fig. 1, A-D). Relative to its expression in naive T cells, perforin (Prf1) mRNA expression did not increase appreciably at day 2 but showed a reproducible decrease at day 4, followed by robust reexpression between days 4 and 8 (Fig. 1, A-D). In contrast, granzyme B (Gzmb) mRNA was low or undetectable in naive T cells but was strongly up-regulated by day 2 after stimulation and increased progressively until day 6 (Fig. 1, A and B); similarly, granzyme B protein was expressed by day 4 and remained high until day 6 (Fig. 1 E). As expected, a small fraction of naive T cells expressed the cytokines IFN-gamma and TNF in response to stimulation, and this capacity increased significantly in differentiated cells (Fig. 1 E; see also Fig. 2 A).\nWe evaluated antigen-dependent cytolytic function in a short-term assay in which target cell death was measured within 2 h (Fig. 1 F). By limiting the duration of TCR stimulation, this strategy minimizes cytolysis secondary to new gene expression during the period of the assay. Naive T cells did not display significant cytolytic function in this short-term assay (unpublished data), most likely because they express immature (unprocessed) forms of perforin and lack the capacity to degranulate (18, 19). Even after activation for 2 or 4 d, the cells showed poor cytolytic activity (Fig. 1 F), in striking contrast to their capacity for efficient cytokine production (Fig. 1 E). Only cells cultured until day 6 displayed robust cytotoxicity, as judged by their ability to induce apoptosis in a large number of target cells (Fig. 1 F).\nThese results show that after a strong priming stimulus through TCRs and co-stimulatory receptors in vitro, granzyme B expression and the ability to produce effector cytokines are programmed early, whereas perforin expression and cytolytic function are induced later, during the phase of clonal expansion in IL-2. Therefore, the two major effector functions of CTL, cytokine production and cytolytic activity, are not intrinsically coregulated.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 9, "end": 19}, "arguments": [{"role": "Theme", "text": "perforin", "start": 32, "end": 40}]}, {"trigger": {"text": "expression", "start": 9, "end": 19}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 45, "end": 55}]}, {"trigger": {"text": "expression", "start": 187, "end": 197}, "arguments": [{"role": "Theme", "text": "perforin", "start": 152, "end": 160}]}, {"trigger": {"text": "expression", "start": 187, "end": 197}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 162, "end": 172}]}, {"trigger": {"text": "expression", "start": 270, "end": 280}, "arguments": [{"role": "Theme", "text": "perforin", "start": 284, "end": 292}]}, {"trigger": {"text": "expression", "start": 270, "end": 280}, "arguments": [{"role": "Theme", "text": "perforin", "start": 309, "end": 317}]}, {"trigger": {"text": "expression", "start": 357, "end": 367}, "arguments": [{"role": "Theme", "text": "perforin", "start": 386, "end": 394}]}, {"trigger": {"text": "expressed", "start": 904, "end": 913}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 928, "end": 937}]}, {"trigger": {"text": "expressed", "start": 904, "end": 913}, "arguments": [{"role": "Theme", "text": "TNF", "start": 942, "end": 945}]}, {"trigger": {"text": "express", "start": 1481, "end": 1488}, "arguments": [{"role": "Theme", "text": "perforin", "start": 1521, "end": 1529}]}, {"trigger": {"text": "expression", "start": 2026, "end": 2036}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 2015, "end": 2025}]}, {"trigger": {"text": "expression", "start": 2122, "end": 2132}, "arguments": [{"role": "Theme", "text": "perforin", "start": 2113, "end": 2121}]}], "negative regulation": [{"trigger": {"text": "decrease", "start": 482, "end": 490}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 402, "end": 417}]}], "positive regulation": [{"trigger": {"text": "increase", "start": 426, "end": 434}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 402, "end": 417}]}, {"trigger": {"text": "reexpression", "start": 520, "end": 532}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 402, "end": 417}]}, {"trigger": {"text": "up-regulated", "start": 663, "end": 675}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 582, "end": 592}]}, {"trigger": {"text": "increased", "start": 707, "end": 716}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 582, "end": 592}]}, {"trigger": {"text": "remained high", "start": 819, "end": 832}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 773, "end": 783}]}, {"trigger": {"text": "increased", "start": 992, "end": 1001}, "arguments": [{"role": "Theme", "text": "expressed", "start": 904, "end": 913}]}, {"trigger": {"text": "induced", "start": 2160, "end": 2167}, "arguments": [{"role": "Theme", "text": "expression", "start": 2122, "end": 2132}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 402, "end": 417}, "arguments": [{"role": "Theme", "text": "perforin", "start": 386, "end": 394}]}, {"trigger": {"text": "low or undetectable", "start": 609, "end": 628}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 582, "end": 592}]}]}}, "schema": []}
{"input": "Distinct kinetics of T-bet and Eomes expression during CTL differentiation\nThe T-box transcription factors T-bet and Eomes have been linked to the regulation of genes encoding effector cytokines (e.g., Ifng) and genes important for cytolytic function (e.g., Prf1 and GzmB) (20). We investigated the kinetics of expression of these transcription factors in our in vitro cultures (Fig. 1, A-D). T-bet mRNA and protein were not detectable in naive CD8+ T cells, but were strongly induced upon TCR priming (day 2) and remained expressed through day 6 of differentiation (Fig. 1, A and C; quantified in Fig. 1, B and D). In contrast, Eomes expression was low or undetectable at both the mRNA and protein levels in naive CD8+ T cells, and TCR priming in culture had only a modest effect on its expression at day 2 (Fig. 1, A and C). Strong induction of Eomes mRNA and protein was only observed at day 4 and later (Fig. 1, A and C). T-bet mRNA expression slightly preceded the expression of GzmB mRNA; similarly, Eomes mRNA and protein were expressed ~1 d ahead of the reexpression of perforin mRNA and protein, respectively (Fig. 1, B and D).\nThis detailed kinetic analysis suggested that, under our culture conditions, T-bet and Eomes contribute to distinct aspects of gene transcription during CTL differentiation. T-bet is required early for IFN-gamma production, and our data suggested that Eomes might not function during this early period but rather might contribute later to the control of perforin expression. Our data seemed most consistent with a model in which TCR signals induce T-bet, which in turn induces IFN-gamma (17) and possibly granzyme B; subsequently, Eomes is induced during the period of clonal expansion in IL-2 and activates perforin expression.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 37, "end": 47}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 21, "end": 26}]}, {"trigger": {"text": "expression", "start": 37, "end": 47}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 31, "end": 36}]}, {"trigger": {"text": "expression", "start": 311, "end": 321}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 107, "end": 112}]}, {"trigger": {"text": "expression", "start": 311, "end": 321}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 117, "end": 122}]}, {"trigger": {"text": "detectable", "start": 425, "end": 435}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 393, "end": 398}]}, {"trigger": {"text": "expressed", "start": 523, "end": 532}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 393, "end": 398}]}, {"trigger": {"text": "expression", "start": 635, "end": 645}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 629, "end": 634}]}, {"trigger": {"text": "expression", "start": 788, "end": 798}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 629, "end": 634}]}, {"trigger": {"text": "expression", "start": 970, "end": 980}, "arguments": [{"role": "Theme", "text": "GzmB", "start": 984, "end": 988}]}, {"trigger": {"text": "expressed", "start": 1034, "end": 1043}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 1006, "end": 1011}]}, {"trigger": {"text": "reexpression", "start": 1062, "end": 1074}, "arguments": [{"role": "Theme", "text": "perforin", "start": 1078, "end": 1086}]}, {"trigger": {"text": "production", "start": 1349, "end": 1359}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 1339, "end": 1348}]}, {"trigger": {"text": "expression", "start": 1500, "end": 1510}, "arguments": [{"role": "Theme", "text": "perforin", "start": 1491, "end": 1499}]}, {"trigger": {"text": "expression", "start": 1754, "end": 1764}, "arguments": [{"role": "Theme", "text": "perforin", "start": 1745, "end": 1753}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 477, "end": 484}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 393, "end": 398}]}, {"trigger": {"text": "remained", "start": 514, "end": 522}, "arguments": [{"role": "Theme", "text": "expressed", "start": 523, "end": 532}]}, {"trigger": {"text": "low or undetectable", "start": 650, "end": 669}, "arguments": [{"role": "Theme", "text": "expression", "start": 635, "end": 645}]}, {"trigger": {"text": "induction", "start": 834, "end": 843}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 847, "end": 852}]}, {"trigger": {"text": "required", "start": 1320, "end": 1328}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 1311, "end": 1316}, {"role": "Theme", "text": "production", "start": 1349, "end": 1359}]}, {"trigger": {"text": "contribute", "start": 1456, "end": 1466}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 1389, "end": 1394}, {"role": "Theme", "text": "control", "start": 1480, "end": 1487}]}, {"trigger": {"text": "induce", "start": 1578, "end": 1584}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 1585, "end": 1590}]}, {"trigger": {"text": "induces", "start": 1606, "end": 1613}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 1585, "end": 1590}, {"role": "Theme", "text": "IFN-gamma", "start": 1614, "end": 1623}]}, {"trigger": {"text": "induces", "start": 1606, "end": 1613}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 1585, "end": 1590}, {"role": "Theme", "text": "granzyme B", "start": 1642, "end": 1652}]}, {"trigger": {"text": "induced", "start": 1677, "end": 1684}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 1668, "end": 1673}]}, {"trigger": {"text": "activates", "start": 1735, "end": 1744}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 1668, "end": 1673}, {"role": "Theme", "text": "expression", "start": 1754, "end": 1764}]}], "regulation": [{"trigger": {"text": "linked", "start": 133, "end": 139}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 107, "end": 112}, {"role": "Theme", "text": "regulation", "start": 147, "end": 157}]}, {"trigger": {"text": "linked", "start": 133, "end": 139}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 117, "end": 122}, {"role": "Theme", "text": "regulation", "start": 147, "end": 157}]}, {"trigger": {"text": "regulation", "start": 147, "end": 157}, "arguments": [{"role": "Theme", "text": "Ifng", "start": 202, "end": 206}]}, {"trigger": {"text": "regulation", "start": 147, "end": 157}, "arguments": [{"role": "Theme", "text": "Prf1", "start": 258, "end": 262}]}, {"trigger": {"text": "regulation", "start": 147, "end": 157}, "arguments": [{"role": "Theme", "text": "GzmB", "start": 267, "end": 271}]}, {"trigger": {"text": "effect", "start": 774, "end": 780}, "arguments": [{"role": "Theme", "text": "expression", "start": 788, "end": 798}]}, {"trigger": {"text": "control", "start": 1480, "end": 1487}, "arguments": [{"role": "Theme", "text": "expression", "start": 1500, "end": 1510}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 932, "end": 947}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 926, "end": 931}]}]}}, "schema": []}
{"input": "Perforin and granzyme B expression are not appreciably regulated by T-bet\nTo test the model outlined in the previous paragraph directly, we compared the expression of IFN-gamma, perforin, and granzyme B in CD8+ T cells from WT and Tbx21 (T-bet)-deficient mice. As expected (17, 21), naive Tbx21-/- CD8+ T cells produced IFN-gamma poorly upon activation (Fig. 2 A). Notably, this deleterious effect of T-bet deficiency was only observed in differentiating CD8+ T cells until day 4 of culture but was almost completely mitigated by day 6 (Fig. 2 A). This most likely reflected compensation by Eomes, which was strongly induced between days 4 and 6 (Fig. 1). In contrast, T-bet-deficient T cells cultured for 6 d showed no defect in perforin mRNA expression (Fig. 2 B, compare lanes 1 and 4). We consistently observed a modest reduction in GzmB mRNA in T-bet-deficient T cells (Fig. 2 B, compare lanes 1 and 4), which did not translate into a decrease in expression of granzyme B protein (Fig. 2 C).\nTo examine the role of Eomes, we transduced naive CD8+ T cells from WT and Tbx21-/- mice with retroviruses containing internal ribosome entry site (IRES)-GFP that were either empty or encoded a strongly transactivating version of Eomes (Eo-VP16) (8), and expanded them for 6 d under our culture conditions. Eo-VP16, but not the empty GFP retrovirus, increased perforin expression in both WT and T-bet-deficient CD8+ T cells (Fig. 2 B, lanes 2, 3, 5, and 6). As expected, Eo-VP16 also rescued the early defect in IFN-gamma production observed in T-bet-deficient CD8+ T cells (Fig. 2 D). However, Eo-VP16 did not induce GzmB mRNA expression in either WT or T-bet-deficient cells; thus, the partial T-bet dependence of GzmB mRNA expression cannot be compensated for by Eo-VP16.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 24, "end": 34}, "arguments": [{"role": "Theme", "text": "Perforin", "start": 0, "end": 8}]}, {"trigger": {"text": "expression", "start": 24, "end": 34}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 13, "end": 23}]}, {"trigger": {"text": "expression", "start": 153, "end": 163}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 167, "end": 176}]}, {"trigger": {"text": "expression", "start": 153, "end": 163}, "arguments": [{"role": "Theme", "text": "perforin", "start": 178, "end": 186}]}, {"trigger": {"text": "expression", "start": 153, "end": 163}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 192, "end": 202}]}, {"trigger": {"text": "produced", "start": 311, "end": 319}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 320, "end": 329}]}, {"trigger": {"text": "expression", "start": 952, "end": 962}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 966, "end": 976}]}, {"trigger": {"text": "expression", "start": 1366, "end": 1376}, "arguments": [{"role": "Theme", "text": "perforin", "start": 1357, "end": 1365}]}, {"trigger": {"text": "production", "start": 1519, "end": 1529}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 1509, "end": 1518}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 245, "end": 254}, "arguments": [{"role": "Theme", "text": "Tbx21", "start": 231, "end": 236}]}, {"trigger": {"text": "deficiency", "start": 407, "end": 417}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 401, "end": 406}]}, {"trigger": {"text": "deficient", "start": 675, "end": 684}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 669, "end": 674}]}, {"trigger": {"text": "defect", "start": 720, "end": 726}, "arguments": [{"role": "Cause", "text": "deficient", "start": 675, "end": 684}, {"role": "Theme", "text": "mRNA expression", "start": 739, "end": 754}]}, {"trigger": {"text": "reduction", "start": 824, "end": 833}, "arguments": [{"role": "Theme", "text": "GzmB", "start": 837, "end": 841}, {"role": "Cause", "text": "deficient", "start": 856, "end": 865}]}, {"trigger": {"text": "deficient", "start": 856, "end": 865}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 850, "end": 855}]}, {"trigger": {"text": "decrease", "start": 940, "end": 948}, "arguments": [{"role": "Theme", "text": "expression", "start": 952, "end": 962}]}, {"trigger": {"text": "deficient", "start": 1398, "end": 1407}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 1392, "end": 1397}]}, {"trigger": {"text": "rescued", "start": 1481, "end": 1488}, "arguments": [{"role": "Cause", "text": "Eo-VP16", "start": 1468, "end": 1475}, {"role": "Theme", "text": "defect", "start": 1499, "end": 1505}]}, {"trigger": {"text": "defect", "start": 1499, "end": 1505}, "arguments": [{"role": "Theme", "text": "production", "start": 1519, "end": 1529}, {"role": "Cause", "text": "deficient", "start": 1548, "end": 1557}]}, {"trigger": {"text": "deficient", "start": 1548, "end": 1557}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 1542, "end": 1547}]}, {"trigger": {"text": "deficient", "start": 1658, "end": 1667}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 1652, "end": 1657}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 617, "end": 624}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 591, "end": 596}]}, {"trigger": {"text": "increased", "start": 1347, "end": 1356}, "arguments": [{"role": "Cause", "text": "Eo-VP16", "start": 1304, "end": 1311}, {"role": "Theme", "text": "expression", "start": 1366, "end": 1376}]}, {"trigger": {"text": "induce", "start": 1608, "end": 1614}, "arguments": [{"role": "Cause", "text": "Eo-VP16", "start": 1592, "end": 1599}, {"role": "Theme", "text": "mRNA expression", "start": 1620, "end": 1635}]}, {"trigger": {"text": "dependence", "start": 1699, "end": 1709}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 1693, "end": 1698}, {"role": "Theme", "text": "mRNA expression", "start": 1718, "end": 1733}]}], "regulation": [{"trigger": {"text": "regulated", "start": 55, "end": 64}, "arguments": [{"role": "Theme", "text": "expression", "start": 24, "end": 34}, {"role": "Cause", "text": "T-bet", "start": 68, "end": 73}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 739, "end": 754}, "arguments": [{"role": "Theme", "text": "perforin", "start": 730, "end": 738}]}, {"trigger": {"text": "mRNA expression", "start": 1620, "end": 1635}, "arguments": [{"role": "Theme", "text": "GzmB", "start": 1615, "end": 1619}]}, {"trigger": {"text": "mRNA expression", "start": 1718, "end": 1733}, "arguments": [{"role": "Theme", "text": "GzmB", "start": 1713, "end": 1717}]}]}}, "schema": []}
{"input": "Runx3 controls multiple aspects of the CTL differentiation program, in part through induction of Eomes\nBecause Runx3 is highly expressed in peripheral CD8+ T cells, and because of the T-bet-Runx3 cooperation we observed earlier in CD4+ T cells (15), we examined the role of Runx3 in effector CTL differentiation. We isolated CD8+ T cells from Runx3-/- (KO) mice of the outbred ICR background and their WT Runx3+/+ littermates by positive selection with anti-CD8 magnetic beads (Figs. S1 and S2, available at http://www.jem.org/cgi/content/full/jem.20081242/DC1). Strikingly, Runx3-/- CD8+ T cells were strongly impaired in their ability to differentiate into effector CTLs, as judged by expression of perforin, granzyme B, and IFN-gamma (Fig. 3). Compared with WT T cells, perforin mRNA and protein expression were essentially undetectable in Runx3-/- T cells at day 6 of culture (Fig. 3, A and B). Runx3-/- T cells also had no detectable Eomes expression; in contrast, T-bet expression was unimpaired (Fig. 3 A). Furthermore, Runx3 was required for maximal production of IFN-gamma, but not TNF or IL-2, by CD8+ T cells restimulated at day 6 (Fig. 3 C).\nWe previously reported that Th1 cell differentiation was regulated through a feed-forward loop in which T-bet is up-regulated early and induces Runx3, after which T-bet and Runx3 cooperate to induce IFN-gamma and silence IL-4, thus promoting stable differentiation toward the Th1 lineage (15, 22). Because (a) Runx3 appeared necessary for Eomes induction (Fig. 3 A), (b) the kinetics of Eomes expression paralleled those of perforin expression (Fig. 2), and (c) overexpression of Eo-VP16 in either WT or T-bet-deficient T cells led to an increase in both perforin and IFN-gamma expression (Fig. 2, B and D), we asked whether CTL differentiation was also potentially regulated by a feed-forward loop involving these same two classes of Runx and T-box transcription factors. Specifically, we asked whether Runx3, which was necessary for Eomes induction, then cooperated with Eomes to regulate transcription of the effector CTL markers perforin, IFN-gamma, and granzyme B.\nTo test this hypothesis, we used chromatin immunoprecipitation (ChIP) assays to ask whether Eomes and Runx3 bound regulatory regions of the Prf1, Ifng, and Gzmb genes (Fig. 3 D). Both proteins associated with gene regulatory regions in differentiated CTLs. Runx3 bound to the Prf1 and Gzmb transcription start sites (TSS); to a known IL-2 responsive enhancer located near -1 kb of the Prf1 gene (23); to the distal CTL-specific DNase I hypersensitive site 9 in the Prf1 locus (24); to the Ifng promoter near the TSS, as previously reported for Th1 cells (10); and to several DNase I hypersensitive sites in the Ifng locus (Fig. 3 D and not depicted) (25). Eomes bound primarily to the Prf1 TSS and the -1 kb enhancer; this binding was substantially greater than that observed at the promoter of the Il2rb gene, a known direct target of Eomes (8), and comparable to that observed at the Ifng TSS, a known target of T-box proteins in both Th1 and CD8+ T cells (Fig. 3 D) (17).\nTo determine whether Runx3 controlled the expression of CTL effector genes through its induction of Eomes, we retrovirally expressed Runx3 and Eo-VP16 in CD8+ T cells from Runx3-/- mice. Because of the limited number of CD8+ T cells in these mice, and because we saw no difference between Runx3-/- CD8+CD4- SP and CD8+CD4+ DP cells in our previous experiments, we used total Runx3-/- CD8+ T cells without further fractionation as recipients for retroviral transduction. Reconstitution of Runx3-/- CD8+ T cells with Runx3 restored expression of Eomes as well as perforin, granzyme B, and IFN-gamma (Fig. 4, A and B). In addition, Runx3-/- T cells showed a compensatory up-regulation of Runx1, which was suppressed upon reconstitution with Runx3, indicating that Runx1 is a target of repression by Runx3. Notably, Eo-VP16 did not up-regulate perforin expression when expressed in Runx3-/- cells, even though it restored the capacity to induce IFN-gamma expression upon TCR restimulation (Fig. 4, A and B). This result suggests strongly that perforin expression requires Runx3 and Eomes.\nAs expected from their defect in perforin and granzyme B expression, Runx3-/- CD8+ T cells showed defective cytolytic activity in a mixed lymphocyte reaction (12). However, TCR-stimulated Runx3-/- CD8+ cells were as effective as WT cells in killing tumor cells in a redirected CTL assay (12). Furthermore, CD8+ cells from the peritoneal cavity of Runx3-/- mice immunized with certain tumor cells effectively killed these targets (13). Therefore, although activation of the perforin/granzyme B machinery is defective in Runx3-/- CD8+ cells, these cells are not entirely devoid of cytolytic activity and could still effectively kill targets, possibly by alternative mechanisms such as the Fas-Fas ligand pathway.", "output": {"json_structures": {"binding": [{"trigger": {"text": "bound", "start": 2232, "end": 2237}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 2216, "end": 2221}, {"role": "Site2", "text": "regulatory regions", "start": 2238, "end": 2256}, {"role": "Theme2", "text": "Prf1", "start": 2264, "end": 2268}]}, {"trigger": {"text": "bound", "start": 2232, "end": 2237}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 2226, "end": 2231}, {"role": "Site2", "text": "regulatory regions", "start": 2238, "end": 2256}, {"role": "Theme2", "text": "Prf1", "start": 2264, "end": 2268}]}, {"trigger": {"text": "bound", "start": 2232, "end": 2237}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 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{"input": "Runx3 and T-box factors control a complex program of transcriptional regulation during CTL differentiation\nCollectively, these data provide evidence that Runx3, together with T-box factors, orchestrates a complex program of transcriptional regulation in differentiating CTL (Fig. 4 C). Runx3 is present in naive CD8+ T cells before activation (12). It represses Runx1 and has a positive role in the induction of Eomes, granzyme B, perforin, and IFN-gamma. Runx3 binds to promoters and putative regulatory regions of the latter three genes, suggesting a direct effect on gene expression. Additional experiments are needed to determine whether Eomes and Runx1 are also direct target genes of Runx3.\nSurprisingly, Runx3 contributed to the optimal expression of TNF, IL-2, and IFN-gamma at day 4 (Fig. S2). For TNF and IL-2, the requirement for Runx3 subsides by day 6 (Fig. 3 C), possibly because of compensation by Runx1, which is derepressed in Runx3-/- cells (Fig. 4 A). Runx3 continues to be required for IFN-gamma expression even at day 6, perhaps because of its role in the induction of Eomes expression (Fig. 4 A).\nAn unexpected finding was that the two T-box transcription factors, T-bet and Eomes, are up-regulated with very different kinetics in CD8+ T cells under our culture conditions and have nonredundant roles in the subsequent expression of key effector proteins (Fig. 4 C). T-bet is needed early to confer on activated CD8+ T cells the competence to produce IFN-gamma upon restimulation, but its function is less important at later times. Eomes, which is induced late and functions downstream of Runx3, may substitute for T-bet in promoting the acute expression of IFN-gamma in restimulated CTLs (8). Indeed, T-bet and Eomes both contribute to perforin expression in NK cells (8, 26), and Eomes induces granzyme B as effectively as T-bet in developing Th2 cells (7); thus, the relative roles of these T-box transcription factors vary depending on cell type.\nSurprisingly, however, Eomes and T-bet appeared nonredundant in their ability to induce two other markers of CTL function, Prf1 and Gzmb (Fig. 4 C). Rather, T-bet and Eomes were involved in regulating granzyme B and perforin expression, respectively: up-regulation of T-bet and Eomes mRNA and protein closely preceded up-regulation of Gzmb and Prf1 mRNA and protein, respectively. T-bet had no role in perforin expression under our culture conditions, and Eo-VP16 did not affect granzyme B expression when expressed in T-bet-/- or Runx3-/- cells. Because conventional Eomes-deficient mice die before precursor cells can be isolated for bone marrow transfers (27) and because T cells conditionally deficient in Eomes have only recently been described (9), we were unable to introduce Runx3 into Eomes-deficient CD8+ T cells to test formally whether Eomes cooperated with Runx3 to induce perforin expression.\nCollectively, our data are consistent with a transcriptional network in which preexisting Runx3 cooperates with the induced T-box factors T-bet and Eomes and IL-2Rbeta signals (unpublished data) to orchestrate CTL differentiation (Fig. 4 C). Our data recall the \"feed-forward\" interaction between T-bet and Runx3 that we previously described in CD4+ (Th1) T cells (15) but are distinct in two respects: in differentiating Th1 cells, T-bet is induced by TCR signals and IFN-gamma, and in turn induces Runx3 (15), whereas in differentiating CD8+ T cells, preexisting Runx3 is required to induce the T-box transcription factor Eomes. 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"start": 372, "end": 391}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 286, "end": 291}, {"role": "Theme", "text": "induction", "start": 399, "end": 408}]}, {"trigger": {"text": "induction", "start": 399, "end": 408}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 412, "end": 417}]}, {"trigger": {"text": "induction", "start": 399, "end": 408}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 419, "end": 429}]}, {"trigger": {"text": "induction", "start": 399, "end": 408}, "arguments": [{"role": "Theme", "text": "perforin", "start": 431, "end": 439}]}, {"trigger": {"text": "induction", "start": 399, "end": 408}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 445, "end": 454}]}, {"trigger": {"text": "contributed", "start": 717, "end": 728}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 711, "end": 716}, {"role": "Theme", "text": "expression", "start": 744, "end": 754}]}, {"trigger": {"text": "requirement", "start": 825, "end": 836}, "arguments": [{"role": "Theme", "text": "TNF", "start": 807, "end": 810}, {"role": "Cause", "text": "Runx3", "start": 841, "end": 846}]}, {"trigger": {"text": "requirement", "start": 825, "end": 836}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 815, "end": 819}, {"role": "Cause", "text": "Runx3", "start": 841, "end": 846}]}, {"trigger": {"text": "required", "start": 993, "end": 1001}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 971, "end": 976}, {"role": "Theme", "text": "expression", "start": 1016, "end": 1026}]}, {"trigger": {"text": "induction", "start": 1077, "end": 1086}, "arguments": [{"role": "Theme", "text": "expression", "start": 1096, "end": 1106}]}, {"trigger": {"text": "up-regulated", "start": 1208, "end": 1220}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 1187, "end": 1192}]}, {"trigger": {"text": "up-regulated", "start": 1208, "end": 1220}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 1197, "end": 1202}]}, {"trigger": {"text": "needed", "start": 1398, "end": 1404}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 1389, "end": 1394}, {"role": "Theme", "text": "produce", "start": 1465, "end": 1472}]}, {"trigger": {"text": "induced", "start": 1570, "end": 1577}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 1554, "end": 1559}]}, {"trigger": {"text": "promoting", "start": 1646, "end": 1655}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 1554, "end": 1559}, {"role": "Theme", "text": "expression", "start": 1666, "end": 1676}]}, {"trigger": {"text": "promoting", "start": 1646, "end": 1655}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 1637, "end": 1642}, {"role": "Theme", "text": "expression", "start": 1666, "end": 1676}]}, {"trigger": {"text": "contribute", "start": 1745, "end": 1755}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 1724, "end": 1729}, {"role": "Theme", "text": "expression", "start": 1768, "end": 1778}]}, {"trigger": {"text": "contribute", "start": 1745, "end": 1755}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 1734, "end": 1739}, {"role": "Theme", "text": "expression", "start": 1768, "end": 1778}]}, {"trigger": {"text": "induces", "start": 1810, "end": 1817}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 1804, "end": 1809}, {"role": "Theme", "text": "granzyme B", "start": 1818, "end": 1828}]}, {"trigger": {"text": "induces", "start": 1810, "end": 1817}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 1818, "end": 1828}, {"role": "Cause", "text": "T-bet", "start": 1847, "end": 1852}]}, {"trigger": {"text": "induce", "start": 2054, "end": 2060}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 1996, "end": 2001}, {"role": "Theme", "text": "Prf1", "start": 2096, "end": 2100}]}, {"trigger": {"text": "induce", "start": 2054, "end": 2060}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 1996, "end": 2001}, {"role": "Theme", "text": "Gzmb", "start": 2105, "end": 2109}]}, {"trigger": {"text": "induce", "start": 2054, "end": 2060}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 2006, "end": 2011}, {"role": "Theme", "text": "Prf1", "start": 2096, "end": 2100}]}, {"trigger": {"text": "induce", "start": 2054, "end": 2060}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 2006, "end": 2011}, {"role": "Theme", "text": "Gzmb", "start": 2105, "end": 2109}]}, {"trigger": {"text": "up-regulation", "start": 2224, "end": 2237}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 2241, "end": 2246}]}, {"trigger": {"text": "up-regulation", "start": 2224, "end": 2237}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 2251, "end": 2256}]}, {"trigger": {"text": "preceded", "start": 2282, "end": 2290}, "arguments": [{"role": "Cause", "text": "up-regulation", "start": 2224, "end": 2237}, {"role": "Theme", "text": "up-regulation", "start": 2291, "end": 2304}]}, {"trigger": {"text": "up-regulation", "start": 2291, "end": 2304}, "arguments": [{"role": "Theme", "text": "Gzmb", "start": 2308, "end": 2312}]}, {"trigger": {"text": "up-regulation", "start": 2291, "end": 2304}, "arguments": [{"role": "Theme", "text": "Prf1", "start": 2317, "end": 2321}]}, {"trigger": {"text": "induce", "start": 2852, "end": 2858}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 2821, "end": 2826}, {"role": "Theme", "text": "expression", "start": 2868, "end": 2878}]}, {"trigger": {"text": "induce", "start": 2852, "end": 2858}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 2843, "end": 2848}, {"role": "Theme", "text": "expression", "start": 2868, "end": 2878}]}, {"trigger": {"text": "induced", "start": 2996, "end": 3003}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 3018, "end": 3023}]}, {"trigger": {"text": "induced", "start": 2996, "end": 3003}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 3028, "end": 3033}]}, {"trigger": {"text": "induced", "start": 3322, "end": 3329}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 3313, "end": 3318}, {"role": "Cause", "text": "IFN-gamma", "start": 3349, "end": 3358}]}, {"trigger": {"text": "induces", "start": 3372, "end": 3379}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 3313, "end": 3318}, {"role": "Theme", "text": "Runx3", "start": 3380, "end": 3385}]}, {"trigger": {"text": "required", "start": 3454, "end": 3462}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 3445, "end": 3450}, {"role": "Theme", "text": "induce", "start": 3466, "end": 3472}]}, {"trigger": {"text": "induce", "start": 3466, "end": 3472}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 3504, "end": 3509}]}], "regulation": [{"trigger": {"text": "effect", "start": 560, "end": 566}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 456, "end": 461}, {"role": "Theme", "text": "expression", "start": 575, "end": 585}]}, {"trigger": {"text": "role", "start": 1065, "end": 1069}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 971, "end": 976}, {"role": "Theme", "text": "induction", "start": 1077, "end": 1086}]}, {"trigger": {"text": "functions downstream", "start": 1587, "end": 1607}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 1554, "end": 1559}, {"role": "Cause", "text": "Runx3", "start": 1611, "end": 1616}]}, {"trigger": {"text": "regulating", "start": 2163, "end": 2173}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 2130, "end": 2135}, {"role": "Theme", "text": "expression", "start": 2198, "end": 2208}]}, {"trigger": {"text": "regulating", "start": 2163, "end": 2173}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 2140, "end": 2145}, {"role": "Theme", "text": "expression", "start": 2198, "end": 2208}]}, {"trigger": {"text": "had no role", "start": 2360, "end": 2371}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 2354, "end": 2359}, {"role": "Theme", "text": "expression", "start": 2384, "end": 2394}]}, {"trigger": {"text": "affect", "start": 2445, "end": 2451}, "arguments": [{"role": "Cause", "text": "Eo-VP16", "start": 2429, "end": 2436}, {"role": "Theme", "text": "expression", "start": 2463, "end": 2473}]}, {"trigger": {"text": "interaction", "start": 3157, "end": 3168}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 3177, "end": 3182}, {"role": "Theme", "text": "T-bet", "start": 3177, "end": 3182}]}, {"trigger": {"text": "interaction", "start": 3157, "end": 3168}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 3177, "end": 3182}, {"role": "Theme", "text": "Runx3", "start": 3187, "end": 3192}]}, {"trigger": {"text": "interaction", "start": 3157, "end": 3168}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 3177, "end": 3182}, {"role": "Cause", "text": "Runx3", "start": 3187, "end": 3192}]}, {"trigger": {"text": "interaction", "start": 3157, "end": 3168}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 3187, "end": 3192}, {"role": "Theme", "text": "Runx3", "start": 3187, "end": 3192}]}]}}, "schema": []}
{"input": "Antibodies and reagents.\nThe following antibodies used for intracellular or surface stains were obtained from eBioscience: anti-IL-2, anti-IFN-gamma, anti-TNF, anti-granzyme B, anti-CD8, anti-CD25, and anti-CD44. Anti-CD69 was purchased from BD. For ChIP experiments, the anti-Eomes antibody was obtained from Abcam and the anti-Runx3 antibody was produced by the Groner laboratory. The following antibodies were used for immunoblotting: antiperforin (Abcam), anti-Eomes (Abcam), and anti-Pol-II (Santa Cruz Biotechnology, Inc.). The T-bet antibody was provided by L. Glimcher (Harvard School of Public Health, Boston, MA).\nThe following reagents were used for the experiments presented in this report: Annexin V-FITC Apoptosis Detection Kit (BD), CD8 Negative Isolation Kit (Invitrogen), CD8 MicroBeads (Miltenyi Biotec), and SYBR Green PCR Core Reagents (Applied Biosystems). The Gp33 peptide (KAVYNFATC) was synthesized by the Tufts University Core Facility, and 10 mM of stock solutions was prepared in DMSO.", "output": {"json_structures": {}}, "schema": []}
{"input": "Isolation and culture of primary CD8+ T cells.\nCD8+ T cells from 4-8-wk-old Tcra-/- x P14 TCR transgenic (Taconic), C57BL/6J WT, or Tbx21-/- (The Jackson Laboratory) mice were purified (>95% purity) by negative selection (Invitrogen) from pooled spleen and lymph node cells. CD8+ T cells from Runx3-/- mice on the ICR background were purified by positive selection (Miltenyi Biotec). All mice were maintained in specific pathogen-free barrier facilities and used according to protocols approved by the Immune Disease Institute and the Harvard Medical School Animal Care and Use Committees. For stimulation, purified CD8+ T cells were cultured at 106 cells/ml (10 ml) in T25 flasks coated with 1 mug/ml each of anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) by pretreatment with 300 mug/ml goat anti-hamster IgG. After 48 h, cells were removed from the TCR stimulation and recultured at a concentration of 5 x 105 cells/ml in media supplemented with 100 U/ml rhIL-2. Every 24 h, viable cells were counted and readjusted to 5 x 105 cells/ml with fresh media containing the corresponding amount of rhIL-2.", "output": {"json_structures": {}}, "schema": []}
{"input": "Isolation of CD8+ T cells from Runx3-/- mice.\nRunx3-deficient T cells fail to silence CD4 expression normally (Fig. S1) (12, 13). We therefore further fractionated the positively selected CD8+ T cells from Runx3 KO mice into CD8+CD4- SP or CD8+CD4+ DP cells by separation using anti-CD4 magnetic beads. This yielded a Runx3 KO SP \"enriched\" population that contained 75% CD8+CD4- cells and a KO DP enriched population that contained 85% CD8+CD4+ cells (Fig. S1). The cells were stimulated with anti-CD3+ anti-CD28 for 2 d before removing them from the TCR stimulus and culturing them in media containing 100 U/ml IL-2. As previously reported, TCR-induced proliferation of Runx3-/- CD8+ T cells was severely impaired, irrespective of CD4 expression (Fig. S1) (12, 13). However, the Runx3-/- cells showed cell-surface expression patterns indicative of activated cells, including up-regulation of CD25 and CD69 (Fig. S1). As expected from their ability to up-regulate CD25, Runx3-/- CD8+ T cells responded to IL-2 supplementation after day 2 and efficiently expanded until day 6 of the culture period, albeit at slower rates compared with WT cells (Fig. S1). Although a fraction of the KO DP cells silenced CD4 expression after activation, the ratio of SP/DP cells in each enriched population remained constant thereafter, and we did not observe any major differences between these two populations throughout the culture period, indicating that in terms of effector CTL differentiation and under our culture conditions, Runx3-/- CD8+ T cells that also coexpress CD4 are indistinguishable from those that do not. The data presented in Fig. S2 are from Runx3 KO SP cells, whereas those shown in Figs. 3 and 4 are from total Runx3 KO CD8 cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 90, "end": 100}, "arguments": [{"role": "Theme", "text": "CD4", "start": 86, "end": 89}]}, {"trigger": {"text": "expression", "start": 737, "end": 747}, "arguments": [{"role": "Theme", "text": "CD4", "start": 733, "end": 736}]}, {"trigger": {"text": "expression", "start": 1208, "end": 1218}, "arguments": [{"role": "Theme", "text": "CD4", "start": 1204, "end": 1207}]}, {"trigger": {"text": "coexpress", "start": 1549, "end": 1558}, "arguments": [{"role": "Theme", "text": "CD4", "start": 1559, "end": 1562}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 52, "end": 61}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 46, "end": 51}]}, {"trigger": {"text": "silence", "start": 78, "end": 85}, "arguments": [{"role": "Cause", "text": "deficient", "start": 52, "end": 61}, {"role": "Theme", "text": "expression", "start": 90, "end": 100}]}, {"trigger": {"text": "silenced", "start": 1195, "end": 1203}, "arguments": [{"role": "Theme", "text": "expression", "start": 1208, "end": 1218}]}], "positive regulation": [{"trigger": {"text": "up-regulation", "start": 877, "end": 890}, "arguments": [{"role": "Theme", "text": "CD25", "start": 894, "end": 898}]}, {"trigger": {"text": "up-regulation", "start": 877, "end": 890}, "arguments": [{"role": "Theme", "text": "CD69", "start": 903, "end": 907}]}, {"trigger": {"text": "up-regulate", "start": 953, "end": 964}, "arguments": [{"role": "Theme", "text": "CD25", "start": 965, "end": 969}]}]}}, "schema": []}
{"input": "FACS-based cytotoxicity assay.\nTo measure cytotoxicity, EL4 thymoma target cells were loaded with 0 or 1 muM Gp33 peptide for 2 h before a 2-h coincubation with P14 CD8+ T cells at the effector-to-target ratios indicated in the figures in 96-well round-bottom plates. After the coincubation period, cells were stained with Annexin V-FITC and anti-CD8-allophycocyanin. Data analysis was performed with FlowJo software (Tree Star, Inc.); EL4 target cells (CD8-negative events) were gated, and the percentage of Annexin V+ target cells was determined.", "output": {"json_structures": {}}, "schema": []}
{"input": "Cytokine and surface marker staining.\nTo assess cytokine production, cells were restimulated with 10 nM PMA + 1 muM ionomycin for 6 h (unless indicated otherwise in the figures), and intracellular cytokine stains were performed as previously described (28). To detect expression of surface molecules, cells were washed in PBS, resuspended in FACS wash buffer (3% FBS, 0.1% sodium azide, 30 mM Hepes, 1x PBS) containing the antibodies indicated in the figures at previously optimized concentrations, incubated for 15 min at room temperature (RT), washed, and resuspended in 2% formaldehyde fixative solution before acquisition on a FACSCalibur (BD).", "output": {"json_structures": {}}, "schema": []}
{"input": "Retroviral transduction of primary CD8+ T cells.\nFor transduction experiments, viral supernatants were generated by calcium phosphate transfection of Phoenix cells and concentration by overnight centrifugation at 6,000 g. At ~42 h after the initial TCR activation of 106 CD8+ T cells per well in 12-well plates, the culture media was removed and replaced with complete media supplemented with 8 mug/ml polybrene containing fresh plus concentrated virus. The plates were centrifuged at 700 g for 1 h at RT before returning to 37degreesC for an additional 5 h. Retroviral constructs for Eomes-VP16 and the MIG control empty vector were a gift from S.L. Reiner (University of Pennsylvania, Philadelphia, PA) (8).", "output": {"json_structures": {}}, "schema": []}
{"input": "ChIP and real-time PCR analysis.\n20 x 106 CD8+ T cells per immunoprecipitation were fixed by adding a 1/10th volume of fixation solution (11.1% formaldehyde, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes) to 1 volume of culture media and were incubated for 10 or 30 min at RT. Fixation was stopped with 120 mM glycine on ice for 5 min. Fixed cells were washed 2x with cold PBS, 1x with cold solution I (10 mM Tris [pH 7.5], 10 mM EDTA, 0.5 mM EGTA, 1% Triton X-100), and 1x with cold solution II (10 mM Tris [pH 7.5], 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). After washes, cell pellets were resuspended at 40 x 106 cells/ml in ChIP lysis buffer (150 mM NaCl, 25 mM Tris [pH 7.5], 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate plus protease and phosphatase inhibitors), and chromatin was sheared with a sonicator to yield 0.5-1-kb DNA fragments. After preclearing the sheared chromatin with protein A-sepharose beads and removing 5% as input chromatin, immunoprecipitation was performed by adding optimized antibody amounts (per 20 x 106 cell equivalents: 2.5 mug anti-Eomes, 1:100 dilution anti-Runx3), followed by overnight incubation at 4degreesC; protein A-sepharose beads were added for the last 3 h of the incubation period. Beads were washed 2x with RIPA buffer (50 mM Tris [pH 8], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate), 1x with high salt buffer (50 mM Tris [pH 8], 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS), and 1x with TE buffer. After the last wash, DNA was eluted by resuspending the beads in elution buffer (1% SDS, 100 mM NaHCO3). Both input and ChIP chromatin were then treated with RNase A (5 mug total) for 1 h at 37degreesC, followed by the addition of proteinase K (100 mug total) and overnight incubation at 65degreesC to reverse cross-linking. DNA was then purified with QIAquick columns (Gel Extraction Kit; QIAGEN) according to the manufacturer's instructions and resuspended in a 50-mul volume. For real-time PCR detection of immunoprecipitated targets using the SYBR Green PCR Kit, a standard curve was obtained with serial dilutions of input DNA for each sample, and 1 mul ChIP DNA was used per PCR reaction (performed in duplicates). Melt curves and agarose gels were analyzed to ensure amplification of specific target sequences. Refer to Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081242/DC1) for a list of primer sets. The data are presented as the number of immunoprecipitated target sequences relative to input chromatin, assuming two copies of target sequence per cell equivalent used for the ChIP.", "output": {"json_structures": {}}, "schema": []}
{"input": "Northern and Western blot analyses.\nRNA isolation and Northern blot analysis was performed as previously described (29). In brief, 10 mug of total RNA was loaded per lane and transferred to positively charged nylon membranes (Hybond-N+; GE Healthcare), which was confirmed by ethidium bromide staining of ribosomal RNA species on the membrane. Membranes were hybridized with 1 ng/ml alpha-[32P]dCTP-labeled trichloroacetic acid precipitable probe in ExpressHyb hybridization buffer (Clontech Laboratories, Inc.). All cDNA probes were confirmed to have the appropriate single-copy specificity under these conditions using genomic Southern blot analysis. Band intensities were acquired by phosphorimaging analysis.\nFor Western analysis, whole-cell protein lysates were obtained from CD8+ T cells at the time points indicated in the figures during clonal expansion in 100 U/ml IL-2 with lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1% NP-40) by resuspending samples in 10 mul per 106 cells and incubating on ice for 30 min in the presence of protease inhibitors. Immunoblot analysis was performed with the antibodies indicated in the figures after SDS-PAGE (10-30 mug of total protein was loaded per well). Quantification of detected protein was performed with an Intelligent Dark Box unit (LAS-3000; Fujifilm) and normalized for loading with the amount of RNA Pol-II detected in each lane.", "output": {"json_structures": {}}, "schema": []}
{"input": "Online supplemental material.\nFig. S1 shows the characterization of peripheral CD8+ T cells from Runx3-/- mice. Fig. S2 shows effector protein expression by Runx3 WT and KO cells at day 4 of in vitro culture. Primer sequences used for ChIP experiments are shown in Table S1. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20081242/DC1.", "output": {"json_structures": {}}, "schema": []}
{"input": "Kinetics of gene expression during CD8+ T cell differentiation. (A) Kinetics of Prf1, Gzmb, Tbx21 (T-bet), and Eomes mRNA expression in differentiating P14 CD8+ T cells analyzed by Northern blotting. RNA from day 7 Th1 cells was used as a control. Sizes of mRNA transcripts are indicated. (B) Quantification of relative mRNA amounts by phosphorimager analysis. (C) Kinetics of protein expression in differentiating P14 CD8+ T cells analyzed by immunoblotting. Sizes of protein bands are indicated. (D) Relative protein amounts quantified from the Western blots. (E) Intracellular staining for granzyme B, IFN-gamma, and TNF. Granzyme B staining was specific relative to an isotype control (not depicted). Cells were restimulated with PMA and ionomycin for 4 h. (F) FACS-based assay to measure cytolytic activity of P14 CD8+ T cells against EL4 targets loaded with 0 (-) or 1 (+) muM Gp33 peptide (effector-to-target ratio = 5:1). Percentage of Annexin V+ (apoptotic) target cells in the CD8-negative EL4 target population (dot plots) was determined (histograms). Cytolytic activity was blocked by incubation with 2 mM EGTA (not depicted), confirming involvement of the granule exocytosis (perforin-granzyme B) pathway. Data are representative of at least five (A-E) or three (F) independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "staining", "start": 580, "end": 588}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 593, "end": 603}]}, {"trigger": {"text": "staining", "start": 580, "end": 588}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 605, "end": 614}]}, {"trigger": {"text": "staining", "start": 580, "end": 588}, "arguments": [{"role": "Theme", "text": "TNF", "start": 620, "end": 623}]}, {"trigger": {"text": "staining", "start": 636, "end": 644}, "arguments": [{"role": "Theme", "text": "Granzyme B", "start": 625, "end": 635}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 117, "end": 132}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 111, "end": 116}]}, {"trigger": {"text": "mRNA expression", "start": 117, "end": 132}, "arguments": [{"role": "Theme", "text": "Prf1", "start": 80, "end": 84}]}, {"trigger": {"text": "mRNA expression", "start": 117, "end": 132}, "arguments": [{"role": "Theme", "text": "Gzmb", "start": 86, "end": 90}]}, {"trigger": {"text": "mRNA expression", "start": 117, "end": 132}, "arguments": [{"role": "Theme", "text": "Tbx21", "start": 92, "end": 97}]}]}}, "schema": []}
{"input": "Regulation of perforin, granzyme B, and IFN-gamma expression by T-bet and Eomes in differentiating CTLs. (A) IFN-gamma expression by WT (Tbx21+/+) and T-bet-deficient (Tbx21-/-) T cells. Naive CD8+ T cells, or cells activated and cultured for 4 or 6 d, were restimulated with PMA and ionomycin for 6 h, and IFN-gamma expression was assessed by intracellular staining. Numbers show the percentage of IFN-gamma+ cells. (B) Northern blot analysis of Prf1 and GzmB mRNA expression in WT or T-bet-deficient CD8+ T cells activated and either left uninfected (uninf) or transduced with retroviruses expressing Eomes-VP16 (Eo-VP16) or an empty IRES-GFP cassette (GFP). Total cellular RNA was analyzed on day 6 of culture. The frequency of transduced cells in the cultures was equivalent for both constructs (~65-70% GFP+ cells; not depicted). (C) Granzyme B and IFN-gamma expression by Tbx21+/+ and Tbx21-/- T cells analyzed in restimulated cells that had been cultured for 5 d. (D) IFN-gamma production by cells transduced with Eo-VP16 or control (GFP) retroviruses (RV) measured on day 4 after 6 h of restimulation with PMA and ionomycin. Numbers show the percentage of GFP+ IFN-gamma+ cells. Results are representative of three (A and C) or two (B and D) independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 50, "end": 60}, "arguments": [{"role": "Theme", "text": "perforin", "start": 14, "end": 22}]}, {"trigger": {"text": "expression", "start": 50, "end": 60}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 24, "end": 34}]}, {"trigger": {"text": "expression", "start": 50, "end": 60}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 40, "end": 49}]}, {"trigger": {"text": "expression", "start": 119, "end": 129}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 109, "end": 118}]}, {"trigger": {"text": "expression", "start": 317, "end": 327}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 307, "end": 316}]}, {"trigger": {"text": "expressing", "start": 592, "end": 602}, "arguments": [{"role": "Theme", "text": "Eomes-VP16", "start": 603, "end": 613}]}, {"trigger": {"text": "expression", "start": 864, "end": 874}, "arguments": [{"role": "Theme", "text": "Granzyme B", "start": 839, "end": 849}]}, {"trigger": {"text": "expression", "start": 864, "end": 874}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 854, "end": 863}]}, {"trigger": {"text": "production", "start": 985, "end": 995}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 975, "end": 984}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 157, "end": 166}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 151, "end": 156}]}, {"trigger": {"text": "deficient", "start": 492, "end": 501}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 486, "end": 491}]}], "regulation": [{"trigger": {"text": "Regulation", "start": 0, "end": 10}, "arguments": [{"role": "Theme", "text": "expression", "start": 50, "end": 60}, {"role": "Cause", "text": "T-bet", "start": 64, "end": 69}]}, {"trigger": {"text": "Regulation", "start": 0, "end": 10}, "arguments": [{"role": "Theme", "text": "expression", "start": 50, "end": 60}, {"role": "Cause", "text": "Eomes", "start": 74, "end": 79}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 461, "end": 476}, "arguments": [{"role": "Theme", "text": "Prf1", "start": 447, "end": 451}]}, {"trigger": {"text": "mRNA expression", "start": 461, "end": 476}, "arguments": [{"role": "Theme", "text": "GzmB", "start": 456, "end": 460}]}]}}, "schema": []}
{"input": "Key role for Runx3 in effector CTL differentiation. (A) Western analysis of Runx3, Eomes, T-bet, and perforin expression in Runx3+/+ versus Runx3-/- CD8+ SP T cells differentiated for 6 d. beta-Actin was used as a loading control. (B) Northern blot analysis of Prf1 mRNA expression in Runx3+/+ versus Runx3-/- CD8+ T cells differentiated for 6 d. beta-Actin was used as a loading control. (C) Expression of granzyme B, IFN-gamma, TNF, and IL-2 by resting or restimulated (6 h) Runx3+/+ versus Runx3-/- CD8+ SP T cells differentiated for 6 d. The vertical gray line indicates the granzyme B MFI for WT GFP+ cells. Results in A-C are representative of two independent experiments. (D) ChIP analysis of binding of endogenous Runx3 and Eomes to the Prf1 locus. Enrichment of the indicated genomic regions was evaluated by real-time PCR of DNA from immunoprecipitated and input chromatin. The data are the means of duplicate measurements from two chromatin preparations from two independent CD8+ T cell differentiations. The efficiency of recovery of input for the -1-kb region of Prf1 was 0.97% for the Runx3 ChIP and 0.5% for the Eomes ChIP.", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 700, "end": 707}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 722, "end": 727}, {"role": "Theme2", "text": "Prf1", "start": 745, "end": 749}]}, {"trigger": {"text": "binding", "start": 700, "end": 707}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 732, "end": 737}, {"role": "Theme2", "text": "Prf1", "start": 745, "end": 749}]}], "gene expression": [{"trigger": {"text": "expression", "start": 110, "end": 120}, "arguments": [{"role": "Theme", "text": "perforin", "start": 101, "end": 109}]}, {"trigger": {"text": "expression", "start": 110, "end": 120}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 76, "end": 81}]}, {"trigger": {"text": "expression", "start": 110, "end": 120}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 83, "end": 88}]}, {"trigger": {"text": "expression", "start": 110, "end": 120}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 90, "end": 95}]}, {"trigger": {"text": "Expression", "start": 393, "end": 403}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 407, "end": 417}]}, {"trigger": {"text": "Expression", "start": 393, "end": 403}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 419, "end": 428}]}, {"trigger": {"text": "Expression", "start": 393, "end": 403}, "arguments": [{"role": "Theme", "text": "TNF", "start": 430, "end": 433}]}, {"trigger": {"text": "Expression", "start": 393, "end": 403}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 439, "end": 443}]}], "transcription": [{"trigger": {"text": "mRNA expression", "start": 266, "end": 281}, "arguments": [{"role": "Theme", "text": "Prf1", "start": 261, "end": 265}]}]}}, "schema": []}
{"input": "Runx3 controls Eomes, perforin, granzyme B, and IFN-gamma expression in effector CTLs. Runx3+/+ or Runx3-/- CD8+ T cells were activated and transduced with retroviruses bearing an empty IRES-GFP cassette (GFP) or also encoding Eomes-VP16 (Eo-VP16) or Myc-Runx3 (Runx3). The frequency of transduced cells in the cultures was equivalent for all constructs (~75-90% GFP+ cells; not depicted). (A) Protein expression in whole-cell extracts (day 6) was analyzed by immunoblotting. Overexpression of Eomes-VP16 cannot be detected with the Eomes antibody, as the C-terminal epitope is within the region that has been replaced with the VP16 transactivation domain. (B) Expression of granzyme B and IFN-gamma after culture for 6 d and restimulation for 4 h with PMA and ionomycin was determined by intracellular staining. The percentage of positively stained cells is shown above the gate; the mean fluorescence intensity (MFI) of granzyme B staining for the total population is shown below the gate. The vertical gray lines indicate the MFI for WT GFP+ cells. Results are representative of at least two independent experiments. (C) Schematic diagram of the transcriptional network involving Runx3 and T-box factors. T-bet is induced by TCR signals and is essential for early IFN-gamma expression. Runx3 is present in naive CD8+ T cells and represses Runx1 and induces Eomes, perforin, granzyme B, and IFN-gamma expression. Eomes may participate in sustaining late IFN-gamma expression, whereas Runx3 and Eomes (but not T-bet) may cooperate to activate perforin expression. The dotted line indicates the partial effect of T-bet deficiency on Gzmb mRNA but not granzyme B protein expression.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 58, "end": 68}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 15, "end": 20}]}, {"trigger": {"text": "expression", "start": 58, "end": 68}, "arguments": [{"role": "Theme", "text": "perforin", "start": 22, "end": 30}]}, {"trigger": {"text": "expression", "start": 58, "end": 68}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 32, "end": 42}]}, {"trigger": {"text": "expression", "start": 58, "end": 68}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 48, "end": 57}]}, {"trigger": {"text": "Expression", "start": 661, "end": 671}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 675, "end": 685}]}, {"trigger": {"text": "Expression", "start": 661, "end": 671}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 690, "end": 699}]}, {"trigger": {"text": "staining", "start": 933, "end": 941}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 922, "end": 932}]}, {"trigger": {"text": "expression", "start": 1277, "end": 1287}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 1267, "end": 1276}]}, {"trigger": {"text": "present", "start": 1298, "end": 1305}, "arguments": [{"role": "Theme", "text": "Runx3", "start": 1289, "end": 1294}]}, {"trigger": {"text": "expression", "start": 1403, "end": 1413}, "arguments": [{"role": "Theme", "text": "Eomes", "start": 1360, "end": 1365}]}, {"trigger": {"text": "expression", "start": 1403, "end": 1413}, "arguments": [{"role": "Theme", "text": "perforin", "start": 1367, "end": 1375}]}, {"trigger": {"text": "expression", "start": 1403, "end": 1413}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 1377, "end": 1387}]}, {"trigger": {"text": "expression", "start": 1403, "end": 1413}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 1393, "end": 1402}]}, {"trigger": {"text": "expression", "start": 1466, "end": 1476}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 1456, "end": 1465}]}, {"trigger": {"text": "expression", "start": 1553, "end": 1563}, "arguments": [{"role": "Theme", "text": "perforin", "start": 1544, "end": 1552}]}, {"trigger": {"text": "expression", "start": 1670, "end": 1680}, "arguments": [{"role": "Theme", "text": "Gzmb", "start": 1633, "end": 1637}]}, {"trigger": {"text": "expression", "start": 1670, "end": 1680}, "arguments": [{"role": "Theme", "text": "granzyme B", "start": 1651, "end": 1661}]}], "negative regulation": [{"trigger": {"text": "represses", "start": 1332, "end": 1341}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 1289, "end": 1294}, {"role": "Theme", "text": "Runx1", "start": 1342, "end": 1347}]}, {"trigger": {"text": "deficiency", "start": 1619, "end": 1629}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 1613, "end": 1618}]}], "positive regulation": [{"trigger": {"text": "Overexpression", "start": 476, "end": 490}, "arguments": [{"role": "Theme", "text": "Eomes-VP16", "start": 494, "end": 504}]}, {"trigger": {"text": "induced", "start": 1217, "end": 1224}, "arguments": [{"role": "Theme", "text": "T-bet", "start": 1208, "end": 1213}]}, {"trigger": {"text": "essential", "start": 1247, "end": 1256}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 1208, "end": 1213}, {"role": "Theme", "text": "expression", "start": 1277, "end": 1287}]}, {"trigger": {"text": "induces", "start": 1352, "end": 1359}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 1289, "end": 1294}, {"role": "Theme", "text": "expression", "start": 1403, "end": 1413}]}, {"trigger": {"text": "activate", "start": 1535, "end": 1543}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 1486, "end": 1491}, {"role": "Theme", "text": "expression", "start": 1553, "end": 1563}]}, {"trigger": {"text": "activate", "start": 1535, "end": 1543}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 1496, "end": 1501}, {"role": "Theme", "text": "expression", "start": 1553, "end": 1563}]}, {"trigger": {"text": "activate", "start": 1535, "end": 1543}, "arguments": [{"role": "Cause", "text": "T-bet", "start": 1511, "end": 1516}, {"role": "Theme", "text": "expression", "start": 1553, "end": 1563}]}], "regulation": [{"trigger": {"text": "controls", "start": 6, "end": 14}, "arguments": [{"role": "Cause", "text": "Runx3", "start": 0, "end": 5}, {"role": "Theme", "text": "expression", "start": 58, "end": 68}]}, {"trigger": {"text": "participate", "start": 1425, "end": 1436}, "arguments": [{"role": "Cause", "text": "Eomes", "start": 1415, "end": 1420}, {"role": "Theme", "text": "expression", "start": 1466, "end": 1476}]}, {"trigger": {"text": "effect", "start": 1603, "end": 1609}, "arguments": [{"role": "Cause", "text": "deficiency", "start": 1619, "end": 1629}, {"role": "Theme", "text": "expression", "start": 1670, "end": 1680}]}]}}, "schema": []}
{"input": "Insights into the Regulation of TNF-alpha Production in Human Mononuclear Cells: The Effects of Non-Specific Phosphodiesterase Inhibition\nOBJECTIVE\nThe objective of this study was to determine the effect of nonspecific phosphodiesterase inhibition on transcription factor activation and tumor necrosis factor-alpha (TNF-alpha) production in lipopolysaccharide (LPS)-stimulated human mononuclear cells.\nINTRODUCTION\nThe production of TNF-alpha following LPS stimulation is one of the key steps in bacterial sepsis and inflammation. The mechanism by which phosphodiesterase inhibition alters TNF-alpha production in the presence of LPS remains unclear.\nMETHODS\nHuman mononuclear cells were stimulated with LPS (1 mug/mL), in the presence and absence of Pentoxifylline (PTX; 20 mM), a nonspecific phosphodiesterase inhibitor. Western blotting of phosphorylated cytoplasmic I-kappaBalpha, nuclear factor-kappaB p65 (NF-kappaB), and nuclear cAMP-response element binding protein (CREB) was performed. DNA binding of NF-kappaB and CREB was verified by electrophoretic mobility shift assay. TNF-alpha levels were determined in the supernatant of stimulated cells in the presence and absence Protein kinase A inhibition by an enzyme-linked immunosorbent assay (ELISA).\nRESULTS\nPTX was demonstrated to significantly reduce cytoplasmic I-kappaBalpha phosphorylation, nuclear p65 phosphorylation, and the DNA binding activity of NF-kappaB. In contrast, PTX markedly enhanced the phosphorylation and DNA binding activity of CREB. Cells concomitantly treated with PTX and LPS secreted similar levels of TNF-alpha in the presence and absence Protein kinase A inhibition.\nDISCUSSION\nThe increased level of cAMP that results from phosphodiesterase inhibition affects cytoplasmic and nuclear events, resulting in the attenuation of NF-kappaB and the activation of CREB transcriptional DNA binding through pathways that are partially Protein kinase A-independent.\nCONCLUSION\nPTX-mediated phosphodiesterase inhibition occurs partially through a Protein kinase A-independent pathway and may serve as a useful tool in the attenuation of LPS-induced inflammation.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Production", "start": 42, "end": 52}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 32, "end": 41}]}, {"trigger": {"text": "production", "start": 327, "end": 337}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor-alpha", "start": 287, "end": 314}]}, {"trigger": {"text": "production", "start": 419, "end": 429}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 433, "end": 442}]}, {"trigger": {"text": "production", "start": 600, "end": 610}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 590, "end": 599}]}], "localization": [{"trigger": {"text": "secreted", "start": 1563, "end": 1571}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1590, "end": 1599}]}], "negative regulation": [{"trigger": {"text": "reduce", "start": 1307, "end": 1313}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1340, "end": 1355}]}, {"trigger": {"text": "reduce", "start": 1307, "end": 1313}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1369, "end": 1384}]}], "phosphorylation": [{"trigger": {"text": "phosphorylated", "start": 843, "end": 857}, "arguments": [{"role": "Theme", "text": "I-kappaBalpha", "start": 870, "end": 883}]}, {"trigger": {"text": "phosphorylation", "start": 1340, "end": 1355}, "arguments": [{"role": "Theme", "text": "I-kappaBalpha", "start": 1326, "end": 1339}]}, {"trigger": {"text": "phosphorylation", "start": 1369, "end": 1384}, "arguments": [{"role": "Theme", "text": "p65", "start": 1365, "end": 1368}]}], "positive regulation": [{"trigger": {"text": "following", "start": 443, "end": 452}, "arguments": [{"role": "Theme", "text": "production", "start": 419, "end": 429}]}, {"trigger": {"text": "in the presence of", "start": 611, "end": 629}, "arguments": [{"role": "Theme", "text": "alters", "start": 583, "end": 589}]}], "regulation": [{"trigger": {"text": "Regulation", "start": 18, "end": 28}, "arguments": [{"role": "Theme", "text": "Production", "start": 42, "end": 52}]}, {"trigger": {"text": "effect", "start": 197, "end": 203}, "arguments": [{"role": "Theme", "text": "production", "start": 327, "end": 337}]}, {"trigger": {"text": "alters", "start": 583, "end": 589}, "arguments": [{"role": "Theme", "text": "production", "start": 600, "end": 610}]}, {"trigger": {"text": "presence", "start": 1607, "end": 1615}, "arguments": [{"role": "Theme", "text": "secreted", "start": 1563, "end": 1571}]}, {"trigger": {"text": "absence", "start": 1620, "end": 1627}, "arguments": [{"role": "Theme", "text": "secreted", "start": 1563, "end": 1571}]}]}}, "schema": []}
{"input": "The bacterial membrane component lipopolysaccharide (LPS) is capable of initiating phosphorylation and activation of multiple host intracellular protein kinases and transcription factors. Transcription factor activation, which results in the modulation of gene transcription and protein synthesis, is a critical element in the defense mechanism of the host immune system.1,2\nLPS-induced transcription factor activation has been shown to be a key regulator of tumor necrosis factor-alpha (TNF-alpha) production.3 TNF-alpha is a potent pro-inflammatory cytokine involved in a wide spectrum of cellular responses. Furthermore, TNF-alpha synthesis can be attenuated in immune cells exposed to phosphodiesterase (PDE) inhibition after challenge by a variety of pro-inflammatory stimulants.4 The signaling mechanisms affected by PDE inhibition, which ultimately lead to the downregulation of TNF-alpha production, have not been well characterized in inflammatory cells.\nClassically, it has been demonstrated that PDE inhibition results in the intracellular accumulation of the second messenger cyclic adenosine-3,5-monophosphate (cAMP) and subsequent activation of Protein kinase A (PKA).5 PKA activation then leads to the phosphorylation of the transcription factor cAMP-response element binding protein (CREB), transmission of signals into the nucleus, and the subsequent modulation of gene transcription.6 This apparently simple linear cascade does not fully explain the mechanism by which an elevation in the intracellular cAMP level exerts wide-ranging effects on multiple cellular functions. There is a growing body of evidence suggesting that cAMP may function through both PKA-dependent and -independent mechanisms.6-8\nLPS-induced activation of the transcription factor nuclear factor-kappaB (NF-kappaB) has also been the focus of a great deal of research. It has been clearly demonstrated that agents that increase intracellular cAMP also inhibit NF-kappaB-dependent pro-inflammatory gene transcription, particularly of the TNF-alpha gene.9\nControversy exists regarding the exact mechanism(s) by which PDE inhibition down-regulates TNF-alpha production. Possibilities include, but are not limited to, inhibition of NF-kappaB DNA binding activity, downregulation of NF-kappaB transcriptional activity, increased CREB activation, and competition between NF-kappaB and CREB for common co-activators such as CREB binding protein (CBP). It is also not known whether these processes rely solely upon the activation of PKA.\nTherefore, the objective of the present study is to determine the effects of nonspecific PDE inhibition with 1-[5-oxohexyl]-3,7-dimethylxanthine (Pentoxifylline; PTX) on NF-kappaB and CREB activation in vitro in human mononuclear cells. With the use of specific inhibitors, we also investigated the role of PKA in LPS-induced TNF-alpha production.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 499, "end": 509}, "arguments": [{"role": "Theme", "text": "tumor necrosis factor-alpha", "start": 459, "end": 486}]}, {"trigger": {"text": "synthesis", "start": 634, "end": 643}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 624, "end": 633}]}, {"trigger": {"text": "production", "start": 896, "end": 906}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 886, "end": 895}]}, {"trigger": {"text": "production", "start": 2145, "end": 2155}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 2135, "end": 2144}]}, {"trigger": {"text": "production", "start": 2856, "end": 2866}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 2846, "end": 2855}]}], "negative regulation": [{"trigger": {"text": "attenuated", "start": 651, "end": 661}, "arguments": [{"role": "Theme", "text": "synthesis", "start": 634, "end": 643}]}, {"trigger": {"text": "downregulation", "start": 868, "end": 882}, "arguments": [{"role": "Theme", "text": "production", "start": 896, "end": 906}]}, {"trigger": {"text": "inhibit", "start": 1942, "end": 1949}, "arguments": [{"role": "Theme", "text": "dependent", "start": 1960, "end": 1969}]}, {"trigger": {"text": "down-regulates", "start": 2120, "end": 2134}, "arguments": [{"role": "Theme", "text": "production", "start": 2145, "end": 2155}]}], "positive regulation": [{"trigger": {"text": "lead", "start": 856, "end": 860}, "arguments": [{"role": "Theme", "text": "downregulation", "start": 868, "end": 882}]}, {"trigger": {"text": "induced", "start": 2838, "end": 2845}, "arguments": [{"role": "Theme", "text": "production", "start": 2856, "end": 2866}]}], "regulation": [{"trigger": {"text": "regulator", "start": 446, "end": 455}, "arguments": [{"role": "Theme", "text": "production", "start": 499, "end": 509}]}, {"trigger": {"text": "dependent", "start": 1960, "end": 1969}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1992, "end": 2005}]}, {"trigger": {"text": "role", "start": 2819, "end": 2823}, "arguments": [{"role": "Theme", "text": "induced", "start": 2838, "end": 2845}]}], "transcription": [{"trigger": {"text": "transcription", "start": 1992, "end": 2005}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 2027, "end": 2036}]}]}}, "schema": []}
{"input": "This study was approved by the Human Research Protections Program and the Institutional Review Board. Written consent to participate in the study was obtained from all volunteers prior to blood donation.Heparinized Vacutainers were purchased from Becton Dickinson (San Jose, CA). 1.5-mL Eppendorf centrifuge tubes were purchased from Fisher Scientific (Pittsburg, PA). Hank's balanced salt solution (HBSS) and RPMI 1640 were obtained from Irvine Scientific (Santa Ana, CA). LPS fromEscherichia coliserotype 0111:B4 and PTX were purchased from Sigma (St. Louis, MO). The PKA inhibitor, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoguinolinesulfonamide-2HCl] (H89), was purchased from Calbiochem (La Jolla, CA). Antibodies for phosphorylated CREB (serine 133), phosphorylated I-kappaBalpha (serines 32 and 36), phosphorylated NF-kappaB p65 subunit (serine 276), and secondary antibodies were purchased from Cell Signaling (Beverly, MA).Dextran T500 and Percoll were received from GE Healthcare/Amersham Biosciences (Piscataway, NJ). The enzyme-linked immunosorbent assay (ELISA) for TNF-alpha was obtained from Quantikine R&D Systems (Minneapolis, MN).Tris-glycine and DNA retardation gels, nitrocellulose membranes, and western blot running buffers were obtained from Invitrogen (Carlsbad, CA). NE-PER(R)nuclear and cytoplasmic extraction reagents, the BCA protein reagent kit, the Supersignal West Pico Chemiluminescent Kit, and the LightShift Chemiluminescent EMSA Kit were purchased from Pierce (Rockland, IL). CREB and NF-kappaB DNA oligonucleotide probes were synthesized by IDT (Coralville, IA). Nylon membranes were purchased from Roche Applied Sciences (Indianapolis, IN). Western blot band quantification was performed with the UN-SCAN-IT Gel Digitizing software (Silk Scientific, Orem, UT).", "output": {"json_structures": {"phosphorylation": [{"trigger": {"text": "phosphorylated", "start": 721, "end": 735}, "arguments": [{"role": "Theme", "text": "CREB", "start": 736, "end": 740}, {"role": "Site", "text": "serine 133", "start": 742, "end": 752}]}, {"trigger": {"text": "phosphorylated", "start": 755, "end": 769}, "arguments": [{"role": "Theme", "text": "I-kappaBalpha", "start": 770, "end": 783}, {"role": "Site", "text": "serines 32", "start": 785, "end": 795}]}, {"trigger": {"text": "phosphorylated", "start": 755, "end": 769}, "arguments": [{"role": "Theme", "text": "I-kappaBalpha", "start": 770, "end": 783}, {"role": "Site", "text": "36", "start": 800, "end": 802}]}, {"trigger": {"text": "phosphorylated", "start": 805, "end": 819}, "arguments": [{"role": "Theme", "text": "p65", "start": 830, "end": 833}, {"role": "Site", "text": "serine 276", "start": 843, "end": 853}]}]}}, "schema": []}
{"input": "Human Peripheral Blood Mononuclear Cell Isolation and Stimulation\nHuman mononuclear cells were isolated from the peripheral blood of four healthy human volunteers. The sample size was chosen from our previously published work, which demonstrated reliable results.10 Cell isolation and all subsequent experiments were conducted under sterile and pyrogen-free conditions. Blood collected in heparin tubes was incubated with Dextran T500 and the red cells were sedimented for 40 minutes at room temperature. The resultant serum was then washed with HBSS and separated by Percoll gradient centrifugation according to the manufacturer's instructions. Cell viability was assessed by trypan blue dye exclusion, with purity of greater than 95%. Isolated cells were resuspended in RPMI 1640 supplemented with 10% FBS and 5 mM HEPES at a concentration of 1 x 107cells/mL. Each subsequent experiment listed below was conducted on samples of 5 x 106 cells per treatment group. Isolated cells were stimulated with either HBSS as a negative control, LPS (1 mug/mL), PTX (20 mM), or concomitant LPS / PTX, at the above-mentioned concentrations, for 30 minutes at 37degreesC. Cells were placed on ice for 10 minutes to stop the reaction and the samples were then stored at -70degreesC for further analysis. The stimulation times and concentrations of LPS and PTX utilized in this study were determined in previous pilot studies done in our laboratory, which examined the effects of increasing concentrations of PTX on LPS-induced TNF-alpha production. LPS at a concentration of 1 mug/mL and PTX at a concentration of 20 mM were the minimum concentrations necessary to produce a reproducible and reliable up-regulation and down-regulation in TNF-alpha production, respectively, in quantitative assays (data not shown).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 1524, "end": 1534}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1514, "end": 1523}]}, {"trigger": {"text": "production", "start": 1735, "end": 1745}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 1725, "end": 1734}]}], "negative regulation": [{"trigger": {"text": "down-regulation", "start": 1706, "end": 1721}, "arguments": [{"role": "Theme", "text": "production", "start": 1735, "end": 1745}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 1506, "end": 1513}, "arguments": [{"role": "Theme", "text": "production", "start": 1524, "end": 1534}]}, {"trigger": {"text": "up-regulation", "start": 1688, "end": 1701}, "arguments": [{"role": "Theme", "text": "production", "start": 1735, "end": 1745}]}]}}, "schema": []}
{"input": "Effect of PTX on LPS-induced TNF-alpha production by mononuclear cells\nThe concentration of TNF-alpha was quantitatively determined by ELISA in the media of cells exposed to the treatments described above after stimulation for up to a maximum of 18 hours, at 3-hour intervals. The results are expressed in pg/mL.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 39, "end": 49}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 29, "end": 38}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 21, "end": 28}, "arguments": [{"role": "Theme", "text": "production", "start": 39, "end": 49}]}]}}, "schema": []}
{"input": "SDS-PAGE and Immunoblotting (I-kappaBalpha, NF-kappaB p65, and CREB)\nFollowing stimulation, PBMCs were washed with ice-cold PBS and centrifuged to collect a cell pellet. The pellet was resuspended in ice-cold SDS sample buffer supplemented with 100 mM dithiothreitol. Cytoplasmic and nuclear extracts were isolated with NE-PER nuclear and cytoplasmic extraction reagents with 1x Halt Protease Inhibitor Cocktail according to the manufacturer's instructions. Protein concentrations were determined with the BCA protein reagent kit for each sample according to a standardized curve for albumin. Cell lysates were collected by boiling the samples in a 100degreesC water bath for 5 minutes. Ten mug of protein per sample was separated by SDS-polyacrylamide gel electrophoresis through 8-16% tris-glycine polyacrylamide gradient gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in Tris-buffered saline/Tween 20 (Fischer Scientific, Pittsburgh, PA) for 1 hour. Cytoplasmic extracts were incubated with phosphorylated I-kappaBalpha antibody (1:200), while nuclear extracts were incubated with either phosphorylated NF-kappaB p65 antibody (1:500) or phosphorylated CREB antibody (1:500) overnight at 4degreesC in separate experiments. The membranes were washed with Tris-buffered saline/Tween 20 and incubated for 1 hour at room temperature with the secondary antibody, horseradish peroxidase-linked anti-rabbit IgG diluted 1:2000 in blocking solution. After repeated washing of the membrane, the Supersignal West Pico Chemiluminescent Kit was applied for antibody detection per the manufacturer's instructions.\nWestern blot data is presented as a percentage of LPS stimulation. The percentage of LPS stimulation was calculated by dividing the mean band pixel total for each treatment arm divided by the mean pixel total of samples stimulated with LPS and multiplying by 100. Thus, LPS stimulation is reported as 100% and each of the treatment arms is reported as a percent of LPS stimulation.", "output": {"json_structures": {"phosphorylation": [{"trigger": {"text": "phosphorylated", "start": 1037, "end": 1051}, "arguments": [{"role": "Theme", "text": "I-kappaBalpha", "start": 1052, "end": 1065}]}, {"trigger": {"text": "phosphorylated", "start": 1134, "end": 1148}, "arguments": [{"role": "Theme", "text": "p65", "start": 1159, "end": 1162}]}]}}, "schema": []}
{"input": "NF-kappaB and CREB Electrophoretic Mobility Shift Assay (EMSA)\nThe non-radioactive LightShift Chemiluminescent EMSA Kit was used to detect DNA-protein interactions. The NF-kappaB 3' biotin end-labeled DNA oligonucleotide used as a probe for the EMSA was a 42-bp double stranded construct (5'-TTGTTACAA-GGGGACTTTCCGCTGGGGACTTTCCAGGGAGGC - 3') containing two tandemly repeated NF-kappaB binding sites (underlined). Specificity was determined by a competition assay with the addition of 200 molar excess of unlabeled double stranded NF-kappaB oligonucleotide. The CREB 3' biotin end-labeled DNA oligonucleotide used as a probe for the EMSA was a 23-bp double stranded construct (5'-TTT TCG AGC TCTGACGTCAGA-GC - 3') containing the CRE consensus sequence (underlined). Specificity was determined by a competition assay with the addition of 200 molar excess of unlabeled double stranded CREB oligonucleotide.\nNuclear extracts (10 mug) were incubated with 5 nM NF-kappaB or CREB probe (NF-kB: 1x binding buffer, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.1% NP40, 10% glycerol, and 50 ng/mul poly dI-dC, CREB: 1x binding buffer, 20 mM Tris, pH 7.5, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.10% NP40, 6% glycerol, 0.1 mg/mL BSA and 50 ng/mul poly dI-dC) and were then electrophoresed through a 6% DNA retardation gel at 100V for 90 minutes. The gels were electrophoretically transferred at 380mA for 1 hour on ice to a positively charged nylon membrane and immediately cross-linked for 15 minutes with a UV transilluminator equipped with a 312 nm bulb. Streptavidin-horseradish peroxidase conjugate and the LightShift Chemiluminescent Substrate were used to detect the biotin end-labeled DNA. The nylon membranes were exposed to x-ray film for 1-3 minutes for detection.", "output": {"json_structures": {}}, "schema": []}
{"input": "Role of PKA on LPS-induced mononuclear cell TNF-alpha production\nTo evaluate the role of PKA on TNF-alpha production in mononuclear cells and its involvement in the attenuation of LPS-induced TNF-alpha production observed following PTX treatment, H89, a specific PKA inhibitor, was utilized. Isolated mononuclear cells were incubated according to the treatment groups described above in the presence and absence of pretreatment with H89 (10 muM) for 1 hour at 37degreesC. The dose of H89 was chosen based on previous work which demonstrated specific and complete inhibition of PKA at this concentration.10 Since the activity of this inhibitor is both specific and consistent at this specified dose, we did not include experiments with additional PKA inhibitors with this set of experiments. The TNF-alpha concentration in the supernatant was measured quantitatively by ELISA. The results are expressed in pg/mL.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 54, "end": 64}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 44, "end": 53}]}, {"trigger": {"text": "production", "start": 106, "end": 116}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 96, "end": 105}]}, {"trigger": {"text": "production", "start": 202, "end": 212}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 192, "end": 201}]}], "negative regulation": [{"trigger": {"text": "attenuation", "start": 165, "end": 176}, "arguments": [{"role": "Theme", "text": "induced", "start": 184, "end": 191}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 19, "end": 26}, "arguments": [{"role": "Theme", "text": "production", "start": 54, "end": 64}]}, {"trigger": {"text": "induced", "start": 184, "end": 191}, "arguments": [{"role": "Theme", "text": "production", "start": 202, "end": 212}]}], "regulation": [{"trigger": {"text": "Role", "start": 0, "end": 4}, "arguments": [{"role": "Theme", "text": "induced", "start": 19, "end": 26}]}, {"trigger": {"text": "role", "start": 81, "end": 85}, "arguments": [{"role": "Theme", "text": "production", "start": 106, "end": 116}]}, {"trigger": {"text": "involvement", "start": 146, "end": 157}, "arguments": [{"role": "Theme", "text": "attenuation", "start": 165, "end": 176}]}]}}, "schema": []}
{"input": "Statistical Analysis\nThe experimental results obtained in this study were derived from four separate experiments with healthy volunteer donors. Each assay was performed in duplicate or triplicate where appropriate. Data is presented as the mean +/- SEM. Statistical differences between groups were determined by one-way analysis of variance (ANOVA) with a Bonferroni correction. Statistical significance was defined as P < 0.05.", "output": {"json_structures": {}}, "schema": []}
{"input": "The effects of PTX on LPS-induced TNF-alpha production\nTo determine the duration of PDE inhibition on TNF-alpha production after LPS stimulation, time course studies were conducted. In accordance with prior work, PTX had a rapid and sustained effect on TNF-alpha production in vitro (Figure 1).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 44, "end": 54}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 34, "end": 43}]}, {"trigger": {"text": "production", "start": 112, "end": 122}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 102, "end": 111}]}, {"trigger": {"text": "production", "start": 263, "end": 273}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 253, "end": 262}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 26, "end": 33}, "arguments": [{"role": "Theme", "text": "production", "start": 44, "end": 54}]}, {"trigger": {"text": "stimulation", "start": 133, "end": 144}, "arguments": [{"role": "Theme", "text": "production", "start": 112, "end": 122}]}], "regulation": [{"trigger": {"text": "effects", "start": 4, "end": 11}, "arguments": [{"role": "Theme", "text": "induced", "start": 26, "end": 33}]}, {"trigger": {"text": "effect", "start": 243, "end": 249}, "arguments": [{"role": "Theme", "text": "production", "start": 263, "end": 273}]}]}}, "schema": []}
{"input": "PTX decreases LPS-induced NF-kappaB activation\nSince phosphorylation leads to ubiquitination and degradation of I-kappaBalpha and subsequent nuclear translocation of NF-kappaB, we first examined the effects of PTX on I-kappaBalpha. Cytoplasmic I-kappaBalpha phosphorylation was markedly increased following LPS stimulation when compared to control (100 +/- 0 vs. 20 +/- 18; P < 0.05). The addition of PTX significantly downregulated LPS-induced I-kappaBalpha phosphorylation (P = 0.02; Figure 2)\nIn a similar fashion, nuclear NF-kappaB p65 phosphorylation was increased following LPS stimulation (100 +/- 0 vs. 38 +/- 20; P < 0.01). PTX similarly decreased LPS-induced NF-kappaB p65 nuclear phosphorylation, a marker of NF-kappaB activation and nuclear translocation (100 +/- 0 vs. 40 +/- 6; P =0.03; Figure 3A).\nEMSAs were then performed to verify the PTX-induced alterations in nuclear phosphorylation resulted in similar effects on the DNA binding activity of NF-kappaB. The addition of PTX to LPS-stimulated mononuclear cells resulted in comparable downregulation of DNA binding activity, similar to the observed downregulation of NF-kappaB phosphorylation (Figure 3B).", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "downregulated", "start": 419, "end": 432}, "arguments": [{"role": "Theme", "text": "induced", "start": 437, "end": 444}]}, {"trigger": {"text": "decreased", "start": 647, "end": 656}, "arguments": [{"role": "Theme", "text": "induced", "start": 661, "end": 668}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 258, "end": 273}, "arguments": [{"role": "Theme", "text": "I-kappaBalpha", "start": 244, "end": 257}]}, {"trigger": {"text": "phosphorylation", "start": 459, "end": 474}, "arguments": [{"role": "Theme", "text": "I-kappaBalpha", "start": 445, "end": 458}]}, {"trigger": {"text": "phosphorylation", "start": 540, "end": 555}, "arguments": [{"role": "Theme", "text": "p65", "start": 536, "end": 539}]}, {"trigger": {"text": "phosphorylation", "start": 691, "end": 706}, "arguments": [{"role": "Theme", "text": "p65", "start": 679, "end": 682}]}], "positive regulation": [{"trigger": {"text": "leads", "start": 69, "end": 74}, "arguments": [{"role": "Theme", "text": "degradation", "start": 97, "end": 108}]}, {"trigger": {"text": "increased", "start": 287, "end": 296}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 258, "end": 273}]}, {"trigger": {"text": "following", "start": 297, "end": 306}, "arguments": [{"role": "Theme", "text": "increased", "start": 287, "end": 296}]}, {"trigger": {"text": "induced", "start": 437, "end": 444}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 459, "end": 474}]}, {"trigger": {"text": "increased", "start": 560, "end": 569}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 540, "end": 555}]}, {"trigger": {"text": "following", "start": 570, "end": 579}, "arguments": [{"role": "Theme", "text": "increased", "start": 560, "end": 569}]}, {"trigger": {"text": "induced", "start": 661, "end": 668}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 691, "end": 706}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 97, "end": 108}, "arguments": [{"role": "Theme", "text": "I-kappaBalpha", "start": 112, "end": 125}]}], "regulation": [{"trigger": {"text": "effects", "start": 199, "end": 206}, "arguments": [{"role": "Theme", "text": "I-kappaBalpha", "start": 217, "end": 230}]}]}}, "schema": []}
{"input": "PTX upregulates CREB phosphorylation and activation after LPS stimulation\nPhosphorylation of nuclear CREB was used as a marker for CREB activation. LPS stimulation caused a negligible increase in CREB phosphorylation when compared to control (Figure 4A). PTX alone caused a marked increase in CREB phosphorylation (P < 0.01 vs. HBSS). When LPS and PTX exposure occurred simultaneously, CREB phosphorylation was significantly higher than with LPS stimulation alone (543 +/- 92 vs. 100 +/- 0; P < 0.01). In addition, the amount of CREB phosphorylation seen with concomitant LPS and PTX treatment was less than that seen with PTX alone, although this difference was not statistically significant (P = 0.08).\nTo determine if CREB-DNA binding was affected by PTX in a manner similar to CREB phosphorylation, an EMSA was performed. Exposure of LPS-stimulated cells to PTX similarly increased CREB DNA-binding compared to LPS alone (Figure 4B).", "output": {"json_structures": {"binding": [{"trigger": {"text": "binding", "start": 895, "end": 902}, "arguments": [{"role": "Theme", "text": "CREB", "start": 886, "end": 890}]}], "positive regulation": [{"trigger": {"text": "increased", "start": 876, "end": 885}, "arguments": [{"role": "Theme", "text": "binding", "start": 895, "end": 902}]}]}}, "schema": []}
{"input": "Effect of PTX on TNF-alpha production in the presence of PKA inhibition\nThe role of PKA in PTX-induced downregulation of TNF-alpha production in LPS-stimulated mononuclear cells was assessed by treating mononuclear cells with H89, a PKA inhibitor, prior to LPS and PTX exposure. The addition PTX to LPS-stimulated cells resulted in a reduction in TNF-alpha, similar to that observed in our previous experiments (529 pg/mL +/- 112 vs. 37 pg/mL +/- 10; P < 0.01). Pre-incubation of cells with H89 prior to LPS and PTX exposure did not result in any significant modulation of TNF-alpha production when compared to that demonstrated with PTX alone (P = 0.2; Figure 5). These results suggest that downregulation of TNF-alpha production by PTX in LPS-stimulated mononuclear cells is, at least in part, PKA-independent.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "production", "start": 27, "end": 37}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 17, "end": 26}]}, {"trigger": {"text": "production", "start": 131, "end": 141}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 121, "end": 130}]}, {"trigger": {"text": "production", "start": 583, "end": 593}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 573, "end": 582}]}, {"trigger": {"text": "production", "start": 720, "end": 730}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 710, "end": 719}]}], "negative regulation": [{"trigger": {"text": "downregulation", "start": 103, "end": 117}, "arguments": [{"role": "Theme", "text": "production", "start": 131, "end": 141}]}, {"trigger": {"text": "reduction", "start": 334, "end": 343}, "arguments": [{"role": "Theme", "text": "TNF-alpha", "start": 347, "end": 356}]}, {"trigger": {"text": "downregulation", "start": 692, "end": 706}, "arguments": [{"role": "Theme", "text": "production", "start": 720, "end": 730}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 95, "end": 102}, "arguments": [{"role": "Theme", "text": "downregulation", "start": 103, "end": 117}]}, {"trigger": {"text": "resulted", "start": 320, "end": 328}, "arguments": [{"role": "Theme", "text": "reduction", "start": 334, "end": 343}]}], "regulation": [{"trigger": {"text": "Effect", "start": 0, "end": 6}, "arguments": [{"role": "Theme", "text": "production", "start": 27, "end": 37}]}, {"trigger": {"text": "in the presence of", "start": 38, "end": 56}, "arguments": [{"role": "Theme", "text": "Effect", "start": 0, "end": 6}]}, {"trigger": {"text": "role", "start": 76, "end": 80}, "arguments": [{"role": "Theme", "text": "induced", "start": 95, "end": 102}]}, {"trigger": {"text": "result", "start": 533, "end": 539}, "arguments": [{"role": "Theme", "text": "modulation", "start": 559, "end": 569}]}, {"trigger": {"text": "modulation", "start": 559, "end": 569}, "arguments": [{"role": "Theme", "text": "production", "start": 583, "end": 593}]}, {"trigger": {"text": "independent", "start": 800, "end": 811}, "arguments": [{"role": "Theme", "text": "downregulation", "start": 692, "end": 706}]}]}}, "schema": []}
{"input": "The exposure of inflammatory cells to a variety of extracellular stimuli results in the initiation of transcriptional and posttranscriptional events, which culminate in the production of pro-inflammatory mediators.11-14 TNF-alpha is released very early following shock and is considered an important mediator of the inflammatory cascade, since it regulates the synthesis of several other critical cytokines and chemokines.15 A clear relationship between increased TNF-alpha synthesis and the development of shock and multiple organ dysfunction has been observed in animal models of sepsis.15-17\nBecause of its importance in orchestrating multiple steps of the inflammatory response, strategies aimed at decreasing TNF-alpha levels or attenuating its production seem attractive. PDE inhibitors have been shown to downregulate TNF-alpha production in distinct human cell populations. We have previously shown that, in general, TNF-alpha levels continue to increase after LPS exposure and that concomitant infusion of phosphodiesterase inhibitors can markedly decrease TNF-alpha levels.18-19 In this in vitro study, we verified the sustained attenuation of LPS-induced TNF-alpha expression over time in an isolated human mononuclear cell population.\nThe mechanisms by which PTX, a non-specific phosphodiesterase inhibitor, significantly and consistently decreases TNF-alpha production have not been completely elucidated. It has been postulated that drugs that increase intracellular cAMP exert their anti-inflammatory effects through the activation of PKA. This classic pathway has been challenged as the sole mechanism involved in the modulation of inflammation by cAMP-enhancing drugs, and alternative mechanisms involving PKA-independent pathways have been proposed.6-8 In this series of experiments, we observed a significant attenuation of TNF-alpha production in LPS-stimulated human mononuclear cells following PTX administration, independent of PKA activation.\nWe used a nonspecific phosphodiesterase inhibitor, PTX, in the present study because it has been used clinically in the treatment of a variety of conditions in which inflammation is an important component of the pathophysiology of the disease process.19 Additionally, the use of PTX in sepsis as an adjunct to other treatments to maintain adequate organ function has been explored based upon its effects on TNF-alpha synthesis. However, the reported beneficial effects of PTX are not only related to the downregulation of TNF-alphabut also to PTX's hemorrheologic properties, its ability to reduce neutrophil activation, and its beneficial effects on microcirculation, cardiac performance, and organ injury.20-25 Herein, we demonstrated that the downregulatory effects of PTX in human mononuclear cells involves modulation of pathways that involve NF-kappaB and CREB, two major transcription factors involved in the inflammatory cascade.\nNF-kappaB/Rel constitutes a family of transcriptional factors involved in the regulation of numerous cytokine genes and immune responses in different cell populations. The most abundant form of NF-kappaB is the p50-p65 heterodimer.26 The inactive form of NF-kappaB exists in the cytoplasm bound to an inhibitory complex containing I-kappaBalpha. Following cellular stimulation with LPS and chemotactic factors, NF-kappaB inducible kinase phosphorylates and activates the I-kappaB kinase (IKK) complex consisting of IKK-1 and IKK-2, which in turn phosphorylates the I-kappaBalpha subunit. Phosphorylation, ubiquitination, and subsequent degradation of I-kappaBalpha results in NF-kappaB nuclear translocation. This allows the transcription factor to bind to promoter regions of specific pro-inflammatory genes and influence transcription.27,28 Because a number of potential kappaB sites are present in the nucleotide sequences of the TNF-alpha gene, NF-kappaB is considered to be a critical transcriptional factor involved in TNF-alpha gene expression and protein synthesis.29,30\nIn this set of experiments, we demonstrated that PTX downregulates cytoplasmic I-kappaBalpha phosphorylation, nuclear NF-kappaB p65 phosphorylation/translocation, as well as NF-kappaB DNA-binding after LPS stimulation, suggesting that PTX exerts its function proximal to or at the level of I-kappaBalpha phosphorylation.\nPrevious studies that have evaluated the effects of cAMP-elevating drugs on I-kappaBalpha and NF-kappaB have reported conflicting results. Haddad et al. reported results similar to ours with respect to the effects of PTX on I-kappaBalpha and NF-kappaB in pulmonary epithelial cells.31 Conversely, Neumann et al. proposed that increased intracellular cAMP stabilizes the interaction between I-kappaBalpha and NF-kappaB, therefore reducing NF-kappaB activation.32 In contrast, Takahashi et al. showed no difference in I-kappaBalpha degradation in Jurkat T-lymphocytes in the presence and absence of forskolin, an alternative cAMP inducing compound. This group also demonstrated that forskolin did not inhibit the DNA-binding activity of NF-kappaB and that CREB was not involved in forskolin-induced decrease in p65 transcriptional activity.33 Similar results were reported by Ollivier et al. using THP-1 cells and endothelial cells treated with forskolin or dibutyryl cAMP, two agents known to function through cAMP elevation and subsequent PKA activation.9 The differences between our results and those mentioned above may be explained by the use of different cell types and exogenous stimulants. It is also possible that PTX exerts its anti-inflammatory effects on the NF-kappaB pathway independent of PKA, as suggested by the continued attenuation of LPS-induced TNF-alpha production by PTX in the presence or absence of PKA inhibition observed in this study. This hypothesis is currently under investigation in our laboratory. Furthermore, we utilized a broad PKA inhibitor in this study in addition to H89, but did not examine the effect of specific inhibition of various PKA isoforms. This information may yield more insight into the mechanism by which PTX exerts its down-regulatory effects.\nPDE inhibitors may also downregulate NF-kappaB transcriptional activity by altering the competitive binding of CREB and NF-kappaB to the promoter regions of pro-inflammatory genes. Herein, we showed that the activation and DNA binding activity of CREB is upregulated in a dose-dependent manner by PTX. Of note, the increase in CREB activity was slightly alleviated by the addition of LPS, suggesting competition between PTX-induced CREB-associated gene transcription and those factors, such as NF-kappaB, which may be upregulated by LPS exposure. This competition has been postulated to involve the recruitment of the co-activator CBP and its homologue p30034, suggesting an alternative mechanism that may be involved in the anti-inflammatory actions of PTX. Future studies are planned to delineate the validity of this potential mechanism.\nThe effects of PTX on other transcription factors, such as AP-1, c-fos, and c-jun, cannot be ruled out by our experiments. Activation of AP-1 and its components (c-fos and c-jun) by agents that enhance mononuclear cell activity has been implicated in TNF-alpha expression. Moreover, AP-1 and CREB recognize a similar DNA binding sequence in the promoter region of the TNF-alpha gene35. Given these relationships, it is conceivable that PTX may exert some of its anti-inflammatory actions by affecting AP-1 activation and favoring CREB binding, thus inhibiting TNF-alpha gene transcription. Additional studies are necessary to elucidate the complex interactions between CREB, NF-kappaB, and AP-1 following LPS exposure and PDE inhibition. Furthermore, the effect of PTX on the cGMP pathway after LPS stimulation has not been explored, although we plan to do so in the future.\nIn summary, we have demonstrated that PDE inhibition of human mononuclear cells downregulates TNF-alpha production, at least in part, through a PKA-independent mechanism. In addition, PTX attenuates the activity of NF-B while upregulating CREB activation after LPS stimulation, which may result in modulation of pro-inflammatory mediator synthesis. 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{"input": "Interleukin-10 Production by Th1 Cells Requires Interleukin-12-Induced STAT4 Transcription Factor and ERK MAP Kinase Activation by High Antigen Dose\nSummary\nCD4+ T cells producing interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) are reported in chronic infections. However, the signals that direct the development of IL-10-producing T helper 1 (Th1) cells are undefined. We showed that development of IL-10-producing Th1 cells required high T cell receptor (TCR) ligation, sustained ERK1 and ERK2 MAP kinases phosphorylation, and IL-12-induced STAT4 transcription factor activation. Repeated TCR triggering led to enhanced IL-10 production by Th1 cells, and continued IL-12 action and high-dose TCR signaling were required for the development and maintenance of IL-10-producing Th1 cells. Although Th1, Th2, and Th17 cells require the activation of distinct STATs for their differentiation, activation of ERK1 and ERK2 was a common requirement for production of IL-10 by all Th cell subsets. IL-10 expression also correlated with c-maf expression. Despite having distinct functions in protection against pathogens, all Th cells share the important task of controlling overexuberant immune responses by means of IL-10 production.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Production", "start": 15, "end": 25}, "arguments": [{"role": "Theme", "text": "Interleukin-10", "start": 0, "end": 14}]}, {"trigger": {"text": "producing", "start": 170, "end": 179}, "arguments": [{"role": "Theme", "text": "interleukin-10", "start": 180, "end": 194}]}, {"trigger": {"text": "producing", "start": 170, "end": 179}, "arguments": [{"role": "Theme", "text": "interferon-gamma", "start": 207, "end": 223}]}, {"trigger": {"text": "producing", "start": 330, "end": 339}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 324, "end": 329}]}, {"trigger": {"text": "producing", "start": 414, "end": 423}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 408, "end": 413}]}, {"trigger": {"text": "production", "start": 636, "end": 646}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 630, "end": 635}]}, {"trigger": {"text": "producing", "start": 775, "end": 784}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 769, "end": 774}]}, {"trigger": {"text": "production", "start": 955, "end": 965}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 969, "end": 974}]}, {"trigger": {"text": "expression", "start": 1005, "end": 1015}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 999, "end": 1004}]}, {"trigger": {"text": "expression", "start": 1043, "end": 1053}, "arguments": [{"role": "Theme", "text": "c-maf", "start": 1037, "end": 1042}]}, {"trigger": {"text": "production", "start": 1224, "end": 1234}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1218, "end": 1223}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 516, "end": 531}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 490, "end": 494}]}, {"trigger": {"text": "phosphorylation", "start": 516, "end": 531}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 499, "end": 503}]}], "positive regulation": [{"trigger": {"text": "Requires", "start": 39, "end": 47}, "arguments": [{"role": "Theme", "text": "Production", "start": 15, "end": 25}, {"role": "Cause", "text": "Activation", "start": 117, "end": 127}]}, {"trigger": {"text": "Requires", "start": 39, "end": 47}, "arguments": [{"role": "Theme", "text": "Production", "start": 15, "end": 25}, {"role": "Cause", "text": "Induced", "start": 63, "end": 70}]}, {"trigger": {"text": "Induced", "start": 63, "end": 70}, "arguments": [{"role": "Cause", "text": "Interleukin-12", "start": 48, "end": 62}, {"role": "Theme", "text": "STAT4", "start": 71, "end": 76}]}, {"trigger": {"text": "Activation", "start": 117, "end": 127}, "arguments": [{"role": "Theme", "text": "ERK", "start": 102, "end": 105}]}, {"trigger": {"text": "induced", "start": 543, "end": 550}, "arguments": [{"role": "Cause", "text": "IL-12", "start": 537, "end": 542}, {"role": "Theme", "text": "activation", "start": 578, "end": 588}]}, {"trigger": {"text": "activation", "start": 578, "end": 588}, "arguments": [{"role": "Theme", "text": "STAT4", "start": 551, "end": 556}]}, {"trigger": {"text": "enhanced", "start": 621, "end": 629}, "arguments": [{"role": "Theme", "text": "production", "start": 636, "end": 646}]}, {"trigger": {"text": "required", "start": 721, "end": 729}, "arguments": [{"role": "Theme", "text": "producing", "start": 775, "end": 784}]}, {"trigger": {"text": "activation", "start": 898, "end": 908}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 912, "end": 916}]}, {"trigger": {"text": "activation", "start": 898, "end": 908}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 921, "end": 925}]}, {"trigger": {"text": "requirement", "start": 939, "end": 950}, "arguments": [{"role": "Cause", "text": "activation", "start": 898, "end": 908}, {"role": "Theme", "text": "production", "start": 955, "end": 965}]}]}}, "schema": []}
{"input": "Interleukin-10 (IL-10) is a cytokine with broad anti-inflammatory properties that inhibits macrophage and dendritic cell (DC) function (Moore et al., 2001). IL-10 limits the immune and inflammatory responses to pathogens and gut flora and prevents damage to the host (Moore et al., 2001; O'Garra and Vieira, 2004), but when dysregulated may result in chronic infection (Brooks et al., 2006; Ejrnaes et al., 2006; Moore et al., 2001). IL-10 is expressed by T helper 2 (Th2) cells, B cells, DCs, and macrophages (Moore et al., 2001), and also by Th1 cells (Anderson et al., 2007; Assenmacher et al., 1994; Del Prete et al., 1993; Gerosa et al., 1996; Jankovic et al., 2007; Pohl-Koppe et al., 1998) and (reviewed in O'Garra and Vieira, 2007; Trinchieri, 2007), certain regulatory (Treg) T cells (Moore et al., 2001; O'Garra and Vieira, 2004; Roncarolo et al., 2006), and Th17 cells (Awasthi et al., 2007; Fitzgerald et al., 2007; McGeachy et al., 2007; Stumhofer et al., 2007).\nIn vitro human CD4+ and CD8+ T cell clones, or mouse CD4+ T cells that produce both interferon-gamma (IFN-gamma) and IL-10, can be differentiated by T cell receptor (TCR)-stimulation in the presence of IL-12 (Chang et al., 2007; Gerosa et al., 1996; Jeannin et al., 1996; Meyaard et al., 1996; Windhagen et al., 1996). Furthermore, Th1 cell clones coproducing IFN-gamma and IL-10 have been isolated from bronchoalveolar lavage (BAL) of active pulmonary tuberculosis (TB) patients (Gerosa et al., 1999). IL-10 production by Th1 cells was also reported in animals infected with Toxoplasma gondii (Jankovic et al., 2002; Shaw et al., 2006) or with Leishmania major (Anderson et al., 2007) and shown to be required for regulation of the immune response in these infections (Anderson et al., 2007; Jankovic et al., 2007). The relative amounts of IL-10 and IFN-gamma produced by Th1 cells may influence the balance between clearance and persistent infection with certain pathogens (Moore et al., 2001; Trinchieri, 2007), thus determining whether chronic infection or immunopathology ensues.\nTh1, Th2, and Th17 cell responses differentiate along distinct signaling pathways (Glimcher and Murphy, 2000; Ivanov et al., 2007; Stockinger and Veldhoen, 2007). Th1 cell development requires signal transducer and activator of transcription (STAT)1 activation, induced by type I IFN or IFN-gamma, the transcription factor T-box 21 (T-bet), and IL-12-induced STAT4 signaling, which can couple with IL-18-induced IRAK and NF-kappaB transcription factors to drive the high amounts of IFN-gamma required to eradicate intracellular pathogens (Glimcher and Murphy, 2000). Th2 cell development, with expression of IL-4, IL-5, and IL-13, requires IL-4, STAT6, and the transcription factor GATA binding protein (GATA)-3 (Glimcher and Murphy, 2000). The development of Th17 cells requires IL-6, TGF-beta, and the STAT3-dependent expression of the transcription factor RORgammat (Ivanov et al., 2007; Stockinger and Veldhoen, 2007).\nTh1 and Th2 cell responses can also be induced by varying the dose of antigen presented to the naive T cell by the antigen-presenting cell (APC). Whereas high doses of antigen, with sustained TCR signaling and extracellular-signal regulated (ERK) mitogen-activated protein kinase (MAPK) phosphorylation, result in Th1 cells producing IFN-gamma via an IL-12-independent mechanism, low doses of antigen, with transient ERK1 and ERK2 activation, favor Th2 responses and IL-4 secretion (Constant et al., 1995; Hosken et al., 1995; Jorritsma et al., 2003; Yamane et al., 2005).\nBecause Th1 and Th2 cells cross regulate each other's development and function and can suppress Th17 cell responses, and all differentiate along distinct signaling pathways (Glimcher and Murphy, 2000; Stockinger and Veldhoen, 2007), IL-10 produced by all these Th cells may thus act as a feedback regulator to control the pathology associated with an overexuberant, albeit efficacious, inflammatory response. Whether IL-10 production by these different Th cell subsets is induced by independent and/or common mechanisms is unknown.\nHere, we showed that in vitro differentiation of IL-10-producing Th1 cells from naive CD4+ T cells required IL-12-induced STAT4 signaling, strong TCR activation (high antigen dose), and sustained ERK1 and ERK2 phosphorylation. Furthermore, we showed that activation of ERK1 and ERK2 is a requirement for production of IL-10 by Th1, Th2, and Th17 cell subsets. This common but highly regulated pathway for IL-10 induction and maintenance ensures its function as a feedback loop to control damage to the host and also allows a protective response to ensue as opposed to chronic infection.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressed", "start": 443, "end": 452}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 434, "end": 439}]}, {"trigger": {"text": "produce", "start": 1047, "end": 1054}, "arguments": [{"role": "Theme", "text": "interferon-gamma", "start": 1060, "end": 1076}]}, {"trigger": {"text": "produce", "start": 1047, "end": 1054}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1093, "end": 1098}]}, {"trigger": {"text": "coproducing", "start": 1324, "end": 1335}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 1336, "end": 1345}]}, {"trigger": {"text": "coproducing", "start": 1324, "end": 1335}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1350, "end": 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{"input": "IL-12 and High Doses of Antigen Induce the Development of Th1 Cells Producing IL-10\nTo study the differentiation of Th1 cells coproducing IFN-gamma and IL-10, we cultured purified TCR-transgenic DO11.10 naive CD4+ T cells with purified DCs as APCs and increasing doses of ovalbumin peptide 323-339 (OVA). Culture with high doses of antigen for 7 days gave rise to Th1 cells expressing IFN-gamma upon restimulation (Constant et al., 1995; Hosken et al., 1995), but not IL-10 (Figure S1A available online). Culture with low antigen doses under the same conditions led to the differentiation of Th2 cells, which expressed both IL-4 and IL-10 upon restimulation (Figure S1A). Culture of naive CD4+ T cells in an APC-free system by stimulation with anti-CD3 and anti-CD28 antibodies in the presence of IL-12 resulted in IFN-gamma-producing Th1 cells, a proportion of which coproduced IL-10, as did Th2 cells resulting from culture in IL-4 (Figure S1B).\nTo investigate whether the lack of IL-10 produced by Th1 cells resulted from inhibition of IL-10 production by DCs and high antigen dose or alternatively required IL-12, we cultured naive CD4+ T cells with increasing doses of antigen presented by DC in the presence of IL-12. At low doses of antigen, IL-12 abrogated the development of Th2 cells and induced IFN-gamma expression but only low levels of IL-10 expression, suggesting that IL-12 per se was not sufficient to induce significant IL-10 production in Th1 cells (Figure 1A). Strikingly, as the antigen dose was increased, Th1 populations driven with IL-12 now contained higher numbers of IL-10-producing cells (Figure 1A) and produced more IL-10 protein upon restimulation (Figures 1A and 1B). Thus, the development of Th1 cells producing IL-10 required both IL-12 and high doses of antigen. Th1 cells differentiated to produce large amounts of IL-10 and IFN-gamma and lost their capacity to produce IL-2 (Figure 1B). Because the presence of IL-12 reduced the proliferation of CD4+ T cells at both high and low antigen doses (Table S1), and only the former showed IL-10 production, the development of high IL-10-producing cells is most likely not related to limited IL-2. Naive CD4+ T cells from DO11.10/recombination-activating gene 1 (Rag1)-deficient animals, cultured with high doses of antigen in the presence of IL-12, also resulted in IL-10 expression by Th1 cells, showing that this expression was not dependent on the presence of effector or memory T cells or Treg cells (Figure S2). Although it has been suggested that TGF-beta can induce IL-10 in CD4+ T cells (Kitani et al., 2003; Schiott et al., 2000), we found that in developing Th1 and Th2 cells this was not the case (data not shown). In fact, neutralization of TGF-beta led to increased IL-10 production by both T cell subsets (Figure S3). Thus, the development of IL-10-producing Th1 cells only depended on the presence of IL-12 together with high antigen dose and not on other soluble factors such as IL-2 or TGF-beta or on the presence of other T cell types.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Producing", "start": 68, "end": 77}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 78, "end": 83}]}, {"trigger": {"text": "coproducing", "start": 126, "end": 137}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 138, "end": 147}]}, {"trigger": {"text": "coproducing", "start": 126, "end": 137}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 152, "end": 157}]}, {"trigger": {"text": "expressing", "start": 374, "end": 384}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 385, "end": 394}]}, {"trigger": {"text": "expressing", "start": 374, "end": 384}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 468, "end": 473}]}, {"trigger": {"text": 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{"input": "IL-10 Production by Th1 Cells Is Dependent on STAT4 but Not on STAT6, IFN-gamma, or IL-4 Signaling\nTo further elucidate the mechanisms required for the development of Th1 cells producing IL-10, we investigated the role of STAT4, one of the signaling pathways activated by IL-12 (Murphy et al., 2000). Naive CD4+ D011.10 T cells deficient in STAT4 (Ouyang et al., 1998) were cultured in the presence of IL-12 and OVA. Again, IL-10-producing Th1 cells were differentiated at the high antigen dose in the presence of IL-12 in DO11.10 T cells (Figure 2A). In contrast, in the absence of STAT4, the percentage of cells expressing IFN-gamma was dramatically diminished as expected and resulted in an increase in the percentage of cells expressing IL-4, but not IL-10 (Figure 2A), suggesting that STAT4 contributes to IL-10 expression by Th1 cells.\nBecause IL-10 expression is associated with an IL-4-induced Th2 cell phenotype, we investigated whether the differentiation of the IL-10-producing Th1 cells depended on signaling through the IL-4 receptor via STAT6 activation (Glimcher and Murphy, 2000; Murphy et al., 2000). The absence of STAT6 did not impair the differentiation of IL-10-producing Th1 cells in the presence of IL-12 and OVA (Figure 2A). In fact, a higher percentage of STAT6-deficient cells compared with WT cells produced both IL-10 and IFN-gamma (Figure 2A), which may be the result of the loss of Th2 cell control over a Th1 cell response. As expected, lack of STAT6 abrogated both IL-4 and IL-10 production by T cells developed with IL-4 or with low antigen dose (Figures 2B and 2C). However, in the absence of STAT4 signaling, IL-10 and IL-4 production by Th2 cells was if anything increased (Figures 2B and 2C). Thus, in contrast to what was observed under Th1 conditions, IL-10 expression by Th2 cells depended on STAT6, but not on STAT4, signaling (Figures 2B and 2C).\nTo investigate whether the inability of STAT4-deficient T cells to produce IL-10 might be due to the absence of IFN-gamma, as suggested before (Shaw et al., 2006), we differentiated DO11.10 or DO11.10 IFN-gamma-deficient naive CD4+ T cells in the presence of IL-12 and increasing doses of OVA. The secretion of IL-10 as induced by high antigen dose, and IL-12 was not affected by an absence of IFN-gamma (Figure 2D), showing that the expression of IL-10 by Th1 cells is independent of IFN-gamma. In the absence of IFN-gamma, we observed an increase in the secreted IL-4 as expected (data not shown).\nWe also tested for any potential role of IL-4 in the development of Th1 cells producing IL-10 by culturing DO11.10 or DO11.10 IL-4-deficient naive CD4+ T cells with IL-12 and increasing doses of antigen. As observed in the absence of STAT6 (Figure 2A), IL-4 deficiency had no effect on the development of Th1 cells producing IL-10 (Figure 2E), but compromised the development of Th2 cells producing IL-10 (Figure 2F). Thus, our data suggested that IL-10 production by Th1 or Th2 cells was dependent on the specific signaling pathways required for their differentiation, given that STAT4 is required for the induction of IL-10 production by Th1 cells and STAT6 for Th2 cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Production", "start": 6, "end": 16}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 0, "end": 5}]}, {"trigger": {"text": "producing", "start": 177, "end": 186}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 187, "end": 192}]}, {"trigger": {"text": "producing", "start": 430, "end": 439}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 424, "end": 429}]}, {"trigger": {"text": "expressing", "start": 614, "end": 624}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 625, "end": 634}]}, {"trigger": {"text": "expressing", "start": 730, "end": 740}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 741, "end": 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"start": 2454, "end": 2458}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 328, "end": 337}, "arguments": [{"role": "Theme", "text": "STAT4", "start": 341, "end": 346}]}, {"trigger": {"text": "absence", "start": 572, "end": 579}, "arguments": [{"role": "Theme", "text": "STAT4", "start": 583, "end": 588}]}, {"trigger": {"text": "diminished", "start": 652, "end": 662}, "arguments": [{"role": "Cause", "text": "absence", "start": 572, "end": 579}, {"role": "Theme", "text": "expressing", "start": 614, "end": 624}]}, {"trigger": {"text": "absence", "start": 1122, "end": 1129}, "arguments": [{"role": "Theme", "text": "STAT6", "start": 1133, "end": 1138}]}, {"trigger": {"text": "deficient", "start": 1287, "end": 1296}, "arguments": [{"role": "Theme", "text": "STAT6", "start": 1281, "end": 1286}]}, {"trigger": {"text": "lack", "start": 1468, "end": 1472}, "arguments": [{"role": "Theme", "text": "STAT6", "start": 1476, "end": 1481}]}, {"trigger": {"text": "abrogated", 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"absence", "start": 2392, "end": 2399}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 2403, "end": 2412}]}, {"trigger": {"text": "deficient", "start": 2620, "end": 2629}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 2615, "end": 2619}]}, {"trigger": {"text": "absence", "start": 2712, "end": 2719}, "arguments": [{"role": "Theme", "text": "STAT6", "start": 2723, "end": 2728}]}, {"trigger": {"text": "deficiency", "start": 2747, "end": 2757}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 2742, "end": 2746}]}], "positive regulation": [{"trigger": {"text": "Dependent", "start": 33, "end": 42}, "arguments": [{"role": "Theme", "text": "Production", "start": 6, "end": 16}]}, {"trigger": {"text": "activated", "start": 259, "end": 268}, "arguments": [{"role": "Theme", "text": "STAT4", "start": 222, "end": 227}, {"role": "Cause", "text": "IL-12", "start": 272, "end": 277}]}, {"trigger": {"text": "increase", "start": 694, "end": 702}, "arguments": [{"role": "Cause", "text": "absence", "start": 572, "end": 579}, {"role": "Theme", "text": "expressing", "start": 730, "end": 740}]}, {"trigger": {"text": "contributes", "start": 796, "end": 807}, "arguments": [{"role": "Cause", "text": "STAT4", "start": 790, "end": 795}, {"role": "Theme", "text": "expression", "start": 817, "end": 827}]}, {"trigger": {"text": "activation", "start": 1057, "end": 1067}, "arguments": [{"role": "Theme", "text": "STAT6", "start": 1051, "end": 1056}]}, {"trigger": {"text": "in the presence of", "start": 1203, "end": 1221}, "arguments": [{"role": "Theme", "text": "producing", "start": 1183, "end": 1192}, {"role": "Cause", "text": "IL-12", "start": 1222, "end": 1227}]}, {"trigger": {"text": "in the presence of", "start": 1203, "end": 1221}, "arguments": [{"role": "Theme", "text": "producing", "start": 1183, "end": 1192}, {"role": "Cause", "text": "OVA", "start": 1232, "end": 1235}]}, {"trigger": {"text": "increased", "start": 1699, "end": 1708}, "arguments": [{"role": 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{"text": "induction", "start": 3096, "end": 3105}, "arguments": [{"role": "Theme", "text": "production", "start": 3115, "end": 3125}, {"role": "Cause", "text": "STAT6", "start": 3143, "end": 3148}]}], "regulation": [{"trigger": {"text": "role", "start": 214, "end": 218}, "arguments": [{"role": "Theme", "text": "producing", "start": 177, "end": 186}, {"role": "Cause", "text": "STAT4", "start": 222, "end": 227}]}, {"trigger": {"text": "affected", "start": 2257, "end": 2265}, "arguments": [{"role": "Theme", "text": "IL-12", "start": 2243, "end": 2248}, {"role": "Cause", "text": "absence", "start": 2272, "end": 2279}]}, {"trigger": {"text": "independent", "start": 2359, "end": 2370}, "arguments": [{"role": "Theme", "text": "expression", "start": 2323, "end": 2333}, {"role": "Cause", "text": "IFN-gamma", "start": 2374, "end": 2383}]}, {"trigger": {"text": "role", "start": 2522, "end": 2526}, "arguments": [{"role": "Cause", "text": "IL-4", "start": 2530, "end": 2534}, {"role": "Theme", 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{"input": "High Antigen Doses and STAT4 Are Required for the In Vivo Generation of IL-10-Producing Th1 Cells\nTo address the mechanisms regulating IL-10 production by Th1 cells in vivo, we transferred DO11.10 cells into BALB/c recipient mice and immunized the recipients with very high doses of OVA-protein with or without added lipopolysacharide (LPS). T cells were recovered from the inguinal lymph nodes 3 days after priming and restimulated in vitro with OVA peptide for 48 hr. This in vivo immunization induced IL-10 and IFN-gamma production, and the amount of IL-10 production was enhanced by addition of LPS in the immunization (Figure 3A) and with higher doses of OVA (3 muM versus 1 muM, data not shown). To test the role of STAT4 and STAT6 signaling in the in vivo development of IL-10-producing Th1 cells, we transferred STAT4- or STAT6-deficient or WT DO11.10 cells into recipient BALB/c mice and immunized with OVA-protein plus LPS as before. In vivo expression of both IL-10 and IFN-gamma was markedly reduced but not completely abrogated in the absence of STAT4 signaling (Figures 3B and 3C), suggesting the existence of compensatory mechanisms that were absent in the in vitro system. Signaling through STAT6 had no effect on IL-10 production by Th1 cells as shown by intracellular cytokine staining (ICS) and by immunoassay in STAT6-deficient T cells (Figures 3B and 3C).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Producing", "start": 78, "end": 87}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 72, "end": 77}]}, {"trigger": {"text": "production", "start": 141, "end": 151}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 135, "end": 140}]}, {"trigger": {"text": "production", "start": 524, "end": 534}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 504, "end": 509}]}, {"trigger": {"text": "production", "start": 524, "end": 534}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 514, "end": 523}]}, {"trigger": {"text": "production", "start": 560, "end": 570}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 554, "end": 559}]}, {"trigger": {"text": "producing", "start": 784, "end": 793}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 778, "end": 783}]}, {"trigger": {"text": "expression", "start": 952, "end": 962}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 971, "end": 976}]}, {"trigger": {"text": "expression", "start": 952, "end": 962}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 981, "end": 990}]}, {"trigger": {"text": "production", "start": 1236, "end": 1246}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1230, "end": 1235}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 836, "end": 845}, "arguments": [{"role": "Theme", "text": "STAT4", "start": 820, "end": 825}]}, {"trigger": {"text": "deficient", "start": 836, "end": 845}, "arguments": [{"role": "Theme", "text": "STAT6", "start": 830, "end": 835}]}, {"trigger": {"text": "reduced", "start": 1004, "end": 1011}, "arguments": [{"role": "Theme", "text": "expression", "start": 952, "end": 962}]}, {"trigger": {"text": "completely abrogated", "start": 1020, "end": 1040}, "arguments": [{"role": "Theme", "text": "expression", "start": 952, "end": 962}]}, {"trigger": {"text": "deficient", "start": 1338, "end": 1347}, "arguments": [{"role": "Theme", "text": "STAT6", "start": 1332, "end": 1337}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 496, "end": 503}, "arguments": [{"role": "Theme", "text": "production", "start": 524, "end": 534}]}, {"trigger": {"text": "enhanced", "start": 575, "end": 583}, "arguments": [{"role": "Theme", "text": "production", "start": 560, "end": 570}, {"role": "Cause", "text": "OVA", "start": 660, "end": 663}]}], "regulation": [{"trigger": {"text": "regulating", "start": 124, "end": 134}, "arguments": [{"role": "Theme", "text": "production", "start": 141, "end": 151}]}, {"trigger": {"text": "effect", "start": 1220, "end": 1226}, "arguments": [{"role": "Theme", "text": "production", "start": 1236, "end": 1246}]}]}}, "schema": []}
{"input": "IL-10 Production Is Maintained by High TCR Signal Strength and IL-12\nWe next investigated whether repeated strong TCR activation is a compensatory signal for IL-12-induced STAT4 signaling in the induction of IL-10 in Th1 cells. For this, CD4+ T cells were differentiated for 2 consecutive weeks with high antigen doses in the presence or absence of IL-12 throughout (Figures 4A-4D). High antigen dose and IL-12 cooperated to induce maximal IL-10 production (Figures 4A and 4B), given that this combination resulted in the highest numbers of IL-10-producing Th1 cells. Repeated high antigen dose stimulation in the absence of exogenously added IL-12 resulted in the production of IL-10 by Th1 cells, suggesting that repeated strong TCR triggering may overcome the need for IL-12 for IL-10 induction (Figures 4C and 4D). However, IL-10 induction under these conditions was abrogated when IL-12p40-deficient DCs were used as APCs (Figure 4E). Thus, IL-12 is essential during both primary and secondary antigenic stimulation for production of IL-10 by Th1 cells.\nTo determine the requirements for stability of the IL-10-producing Th1 cells, we differentiated CD4+ T cells for 1 week with high antigen doses with or without IL-12 (Figures 4A and 4C), washed them, and then restimulated them for an additional week with a low antigen dose, in the absence or presence of IL-12 (Figure 4F). Th1 cells induced in the first week to produce IL-10 by culture with high antigen doses and IL-12 lost their ability to express IL-10 when recultured with low doses of OVA, which could be compensated for, to some extent, by addition of IL-12 to the secondary cultures (Figure 4F), again suggesting that antigen dose and IL-12 signals cooperate for the induction of IL-10. Finally, DO11.10 CD4+ cells that were exposed to low doses of antigen and IL-12 during the primary differentiation phase produced high amounts of IFN-gamma but little IL-10, but they could be induced to produce IL-10 when both high antigen dose and IL-12 were present during the recall phase (Figure S4). Thus, high antigen dose and IL-12 are required for sustaining the induction of IL-10 production by Th1 cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Production", "start": 6, "end": 16}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 0, "end": 5}]}, {"trigger": {"text": "production", "start": 446, "end": 456}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 440, "end": 445}]}, {"trigger": {"text": "producing", "start": 547, "end": 556}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 541, "end": 546}]}, {"trigger": {"text": "production", "start": 665, "end": 675}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 679, "end": 684}]}, {"trigger": {"text": "production", "start": 1025, "end": 1035}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1039, "end": 1044}]}, {"trigger": {"text": "producing", "start": 1116, "end": 1125}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1110, "end": 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"start": 63, "end": 68}]}, {"trigger": {"text": "induction", "start": 195, "end": 204}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 208, "end": 213}]}, {"trigger": {"text": "induce", "start": 425, "end": 431}, "arguments": [{"role": "Cause", "text": "IL-12", "start": 405, "end": 410}, {"role": "Theme", "text": "production", "start": 446, "end": 456}]}, {"trigger": {"text": "resulted", "start": 649, "end": 657}, "arguments": [{"role": "Theme", "text": "production", "start": 665, "end": 675}]}, {"trigger": {"text": "need", "start": 763, "end": 767}, "arguments": [{"role": "Cause", "text": "IL-12", "start": 772, "end": 777}, {"role": "Theme", "text": "induction", "start": 788, "end": 797}]}, {"trigger": {"text": "induction", "start": 788, "end": 797}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 782, "end": 787}]}, {"trigger": {"text": "induction", "start": 834, "end": 843}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 828, "end": 833}]}, {"trigger": {"text": "essential", "start": 955, "end": 964}, "arguments": [{"role": "Cause", "text": "IL-12", "start": 946, "end": 951}, {"role": "Theme", "text": "production", "start": 1025, "end": 1035}]}, {"trigger": {"text": "induced", "start": 1393, "end": 1400}, "arguments": [{"role": "Theme", "text": "produce", "start": 1422, "end": 1429}, {"role": "Cause", "text": "IL-12", "start": 1475, "end": 1480}]}, {"trigger": {"text": "addition", "start": 1607, "end": 1615}, "arguments": [{"role": "Theme", "text": "IL-12", "start": 1619, "end": 1624}]}, {"trigger": {"text": "cooperate", "start": 1717, "end": 1726}, "arguments": [{"role": "Theme", "text": "induction", "start": 1735, "end": 1744}]}, {"trigger": {"text": "induced", "start": 1947, "end": 1954}, "arguments": [{"role": "Theme", "text": "produce", "start": 1958, "end": 1965}, {"role": "Cause", "text": "IL-12", "start": 2004, "end": 2009}]}, {"trigger": {"text": "induction", "start": 2126, "end": 2135}, "arguments": [{"role": 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{"input": "IL-10 Production by Th1 Cells Requires ERK1 and ERK2 Activation\nOur data showed that the maintenance of IL-10 induction in Th1 cells required stimulation with high antigen dose, which to some extent could be compensated for by the addition of IL-12. Signaling through the TCR with high doses of antigen induced stronger ERK1 and ERK2 activation than that induced by low antigen dose, not only in naive CD4+ T cells (data not shown) as previously demonstrated (Jorritsma et al., 2003) but also in CD4+ T cells restimulated with the same high and low antigen doses (Figure 5A). Although the apparent peak and amount of ERK1 and ERK2 activation varied slightly between experiments, a consistent finding was that high antigen dose differentiated Th1 cells always showed enhanced and prolonged ERK1 and ERK2 activation in the presence of IL-12, regardless of whether they were restimulated with high or low antigen dose (Figure 5B).\nWe then investigated whether ERK1 and ERK2 activation was required for the induction of IL-10 in Th1 cells by using U0126 (Figure 5C), a compound that blocks downstream ERK activation. To ensure that only T cell signaling was being affected by U0126, we used an APC-free system in which the T cells were differentiated in the presence of increasing doses of anti-CD3 and a constant amount of IL-12. As in the APC-driven cultures, stronger TCR stimulation together with IL-12 led to higher percentages of cells producing both IL-10 and IFN-gamma after 1 week of culture (Figure 5C). Addition of U0126 to the cultures abrogated the production of IL-10 at all doses of anti-CD3 (Figure 5C). Because U0126 inhibits the MEK5-catalyzed activation of ERK5, as well as the MEK1- and MEK2-catalyzed activation of ERK1 and ERK2 (Bain et al., 2007; Mody et al., 2001), we also used the more specific, structurally unrelated MEK1 and MEK2 inhibitor PD184352 at concentrations in which it inhibits MEK1 and MEK2 but not MEK5 (Bain et al., 2007; Mody et al., 2001). PD184352 caused a similar inhibition of IL-10 production by Th1 cells in a dose-dependent fashion (Figure 5D and Figure S5A). Upon addition of inhibitors to other signaling pathways, including a p38 MAPK inhibitor, SB203580, or the GSK3beta inhibitor, CT99021 (Bain et al., 2007), no effect on IL-10 production was observed (Figure S5B). Our data thus suggested that IL-10 production by Th1 cells in response to high antigen dose and IL-12 requires ERK1 and ERK2 signaling, but not the activation of the p38 or the GSK3beta pathways.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Production", "start": 6, "end": 16}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 0, "end": 5}]}, {"trigger": {"text": "induction", "start": 110, "end": 119}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 104, "end": 109}]}, {"trigger": {"text": "producing", "start": 1438, "end": 1447}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1453, "end": 1458}]}, {"trigger": {"text": "producing", "start": 1438, "end": 1447}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 1463, "end": 1472}]}, {"trigger": {"text": "production", "start": 1558, "end": 1568}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1572, "end": 1577}]}, {"trigger": {"text": "production", "start": 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{"trigger": {"text": "inhibitor", "start": 1855, "end": 1864}, "arguments": [{"role": "Theme", "text": "MEK2", "start": 1850, "end": 1854}]}, {"trigger": {"text": "inhibits", "start": 1904, "end": 1912}, "arguments": [{"role": "Theme", "text": "MEK1", "start": 1913, "end": 1917}]}, {"trigger": {"text": "inhibits", "start": 1904, "end": 1912}, "arguments": [{"role": "Theme", "text": "MEK2", "start": 1922, "end": 1926}]}, {"trigger": {"text": "inhibits", "start": 1904, "end": 1912}, "arguments": [{"role": "Theme", "text": "MEK5", "start": 1935, "end": 1939}]}, {"trigger": {"text": "inhibition", "start": 2006, "end": 2016}, "arguments": [{"role": "Theme", "text": "production", "start": 2026, "end": 2036}]}, {"trigger": {"text": "inhibitor", "start": 2184, "end": 2193}, "arguments": [{"role": "Theme", "text": "p38 MAPK", "start": 2175, "end": 2183}]}, {"trigger": {"text": "inhibitor", "start": 2221, "end": 2230}, "arguments": [{"role": "Theme", "text": "GSK3beta", "start": 2212, "end": 2220}]}], "positive regulation": [{"trigger": {"text": "Requires", "start": 30, "end": 38}, "arguments": [{"role": "Cause", "text": "Activation", "start": 53, "end": 63}, {"role": "Theme", "text": "activation", "start": 2466, "end": 2476}]}, {"trigger": {"text": "Activation", "start": 53, "end": 63}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 39, "end": 43}]}, {"trigger": {"text": "Activation", "start": 53, "end": 63}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 48, "end": 52}]}, {"trigger": {"text": "maintenance", "start": 89, "end": 100}, "arguments": [{"role": "Theme", "text": "induction", "start": 110, "end": 119}]}, {"trigger": {"text": "required", "start": 133, "end": 141}, "arguments": [{"role": "Theme", "text": "maintenance", "start": 89, "end": 100}]}, {"trigger": {"text": "addition", "start": 231, "end": 239}, "arguments": [{"role": "Theme", "text": "IL-12", "start": 243, "end": 248}]}, {"trigger": {"text": "induced", "start": 303, "end": 310}, "arguments": [{"role": "Theme", "text": "activation", "start": 334, "end": 344}]}, {"trigger": {"text": "activation", "start": 334, "end": 344}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 320, "end": 324}]}, {"trigger": {"text": "activation", "start": 334, "end": 344}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 329, "end": 333}]}, {"trigger": {"text": "induced", "start": 355, "end": 362}, "arguments": [{"role": "Theme", "text": "activation", "start": 334, "end": 344}]}, {"trigger": {"text": "activation", "start": 631, "end": 641}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 617, "end": 621}]}, {"trigger": {"text": "activation", "start": 631, "end": 641}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 626, "end": 630}]}, {"trigger": {"text": "enhanced", "start": 766, "end": 774}, "arguments": [{"role": "Theme", "text": "activation", "start": 803, "end": 813}, {"role": "Cause", "text": "IL-12", "start": 833, "end": 838}]}, {"trigger": 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"text": "IL-10", "start": 1016, "end": 1021}]}, {"trigger": {"text": "catalyzed", "start": 1648, "end": 1657}, "arguments": [{"role": "Cause", "text": "MEK5", "start": 1643, "end": 1647}, {"role": "Theme", "text": "activation", "start": 1658, "end": 1668}]}, {"trigger": {"text": "activation", "start": 1658, "end": 1668}, "arguments": [{"role": "Theme", "text": "ERK5", "start": 1672, "end": 1676}]}, {"trigger": {"text": "catalyzed", "start": 1708, "end": 1717}, "arguments": [{"role": "Cause", "text": "MEK1", "start": 1693, "end": 1697}, {"role": "Theme", "text": "activation", "start": 1718, "end": 1728}]}, {"trigger": {"text": "catalyzed", "start": 1708, "end": 1717}, "arguments": [{"role": "Cause", "text": "MEK2", "start": 1703, "end": 1707}, {"role": "Theme", "text": "activation", "start": 1718, "end": 1728}]}, {"trigger": {"text": "activation", "start": 1718, "end": 1728}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 1732, "end": 1736}]}, {"trigger": {"text": "activation", "start": 1718, "end": 1728}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 1741, "end": 1745}]}, {"trigger": {"text": "in response to", "start": 2377, "end": 2391}, "arguments": [{"role": "Theme", "text": "production", "start": 2353, "end": 2363}, {"role": "Cause", "text": "IL-12", "start": 2414, "end": 2419}]}, {"trigger": {"text": "requires", "start": 2420, "end": 2428}, "arguments": [{"role": "Theme", "text": "in response to", "start": 2377, "end": 2391}, {"role": "Cause", "text": "activation", "start": 2466, "end": 2476}]}, {"trigger": {"text": "activation", "start": 2466, "end": 2476}, "arguments": [{"role": "Theme", "text": "p38", "start": 2484, "end": 2487}]}, {"trigger": {"text": "activation", "start": 2466, "end": 2476}, "arguments": [{"role": "Theme", "text": "GSK3beta", "start": 2495, "end": 2503}]}], "regulation": [{"trigger": {"text": "effect", "start": 2264, "end": 2270}, "arguments": [{"role": "Cause", "text": "inhibitor", "start": 2184, "end": 2193}, {"role": "Theme", "text": "production", "start": 2280, "end": 2290}]}, {"trigger": {"text": "effect", "start": 2264, "end": 2270}, "arguments": [{"role": "Cause", "text": "inhibitor", "start": 2221, "end": 2230}, {"role": "Theme", "text": "production", "start": 2280, "end": 2290}]}]}}, "schema": []}
{"input": "IL-10 Production by Th2 and Th17 Cells Also Requires ERK1 and ERK2 Activation\nTo address whether IL-10 production by Th2 and Th17 cells was also dependent on ERK1 and ERK2 activation, we differentiated these cells with anti-CD3 and anti-CD28 in the absence of APCs (Shoemaker et al., 2006; Veldhoen et al., 2009; Veldhoen et al., 2006), in the presence or absence of the MEK inhibitor (PD184352). We showed that ERK1 and ERK2 activation is a common pathway required for induction of IL-10 in different Th cell subsets because IL-10 production by both Th2 and Th17 cells was markedly inhibited in the presence of the MEK inhibitor (PD184352) (Figure 5D and Figure S5B). In contrast, inhibitors of p38 MAPK or of GSK-3beta activation did not affect the expression of IL-10 by these subsets (Figure S5B). Activation of the ERK1 and ERK2 signaling pathway is therefore a common requirement for the induction of IL-10 production by Th1, Th2, and Th17 cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Production", "start": 6, "end": 16}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 0, "end": 5}]}, {"trigger": {"text": "production", "start": 103, "end": 113}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 97, "end": 102}]}, {"trigger": {"text": "induction", "start": 470, "end": 479}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 483, "end": 488}]}, {"trigger": {"text": "production", "start": 532, "end": 542}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 526, "end": 531}]}, {"trigger": {"text": "expression", "start": 751, "end": 761}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 765, "end": 770}]}, {"trigger": {"text": "production", "start": 913, "end": 923}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 907, "end": 912}]}], "negative regulation": [{"trigger": {"text": "inhibitor", "start": 375, "end": 384}, "arguments": [{"role": "Theme", "text": "MEK", "start": 371, "end": 374}]}, {"trigger": {"text": "inhibited", "start": 583, "end": 592}, "arguments": [{"role": "Theme", "text": "production", "start": 532, "end": 542}, {"role": "Cause", "text": "inhibitor", "start": 620, "end": 629}]}, {"trigger": {"text": "inhibitor", "start": 620, "end": 629}, "arguments": [{"role": "Theme", "text": "MEK", "start": 616, "end": 619}]}, {"trigger": {"text": "inhibitors", "start": 682, "end": 692}, "arguments": [{"role": "Theme", "text": "activation", "start": 721, "end": 731}]}], "positive regulation": [{"trigger": {"text": "Requires", "start": 44, "end": 52}, "arguments": [{"role": "Theme", "text": "Production", "start": 6, "end": 16}, {"role": "Cause", "text": "Activation", "start": 67, "end": 77}]}, {"trigger": {"text": "Activation", "start": 67, "end": 77}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 53, "end": 57}]}, {"trigger": {"text": "Activation", "start": 67, "end": 77}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 62, "end": 66}]}, {"trigger": {"text": "dependent", "start": 145, "end": 154}, "arguments": [{"role": "Theme", "text": "production", "start": 103, "end": 113}, {"role": "Cause", "text": "activation", "start": 172, "end": 182}]}, {"trigger": {"text": "activation", "start": 172, "end": 182}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 158, "end": 162}]}, {"trigger": {"text": "activation", "start": 172, "end": 182}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 167, "end": 171}]}, {"trigger": {"text": "activation", "start": 426, "end": 436}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 412, "end": 416}]}, {"trigger": {"text": "activation", "start": 426, "end": 436}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 421, "end": 425}]}, {"trigger": {"text": "required", "start": 457, "end": 465}, "arguments": [{"role": "Cause", "text": "activation", "start": 426, "end": 436}, {"role": "Theme", "text": "induction", "start": 470, "end": 479}]}, {"trigger": {"text": "activation", "start": 721, "end": 731}, "arguments": [{"role": "Theme", "text": "p38 MAPK", "start": 696, "end": 704}]}, {"trigger": {"text": "activation", "start": 721, "end": 731}, "arguments": [{"role": "Theme", "text": "GSK-3beta", "start": 711, "end": 720}]}, {"trigger": {"text": "requirement", "start": 874, "end": 885}, "arguments": [{"role": "Theme", "text": "induction", "start": 894, "end": 903}]}, {"trigger": {"text": "induction", "start": 894, "end": 903}, "arguments": [{"role": "Theme", "text": "production", "start": 913, "end": 923}]}], "regulation": [{"trigger": {"text": "affect", "start": 740, "end": 746}, "arguments": [{"role": "Cause", "text": "inhibitors", "start": 682, "end": 692}, {"role": "Theme", "text": "expression", "start": 751, "end": 761}]}]}}, "schema": []}
{"input": "c-maf Expression Correlates with IL-10 Production in Th1, Th2, and Th17 Cells\nTo investigate further the downstream factors involved in regulating IL-10 production, we differentiated DO11.10 CD4+ T cells with increasing doses of OVA, in the presence or absence of IL-12, and quantified the expression of cytokines and transcription factors by real-time RT-PCR. Low-dose antigen resulted in transcription of Il4 and this was abrogated by both high antigen doses and IL-12 (Figure 6A). A low amount of transcription of Ifngamma was induced by IL-12 when cells were differentiated with low antigen dose, but this effect of IL-12 was markedly upregulated with increasing doses of antigen (Figure 6A). A low amount of Il10 transcription was observed at low doses of antigen accompanying Il4 expression (Th2 cell response), and this was abrogated by increased doses of antigen as was Il4 expression (Figure 6A). At low doses of antigen, IL-12 had little effect to increase IL-10 mRNA expression (Figure 6A) in keeping with the protein data (Figure 1A). However, IL-12 induced a high amount of Il10 transcription as well as Ifngamma expression with increased antigen doses (Figure 6A), again in keeping with the protein data (Figure 1A).\nCD4+ T cells differentiated with increasing doses of antigen did not express high amounts of Tbx-21 (T-bet) mRNA, unless they were cocultured with IL-12 (Figure 6B). In contrast, high amounts of GATA-3 mRNA expression were only observed under Th2 cell differentiation conditions (low-dose antigen) (Figure 6B), and this expression was markedly downregulated by both increasing antigen dose and coculture in IL-12 (Figure 6B). Differentiation of T cells under low antigen dose led to expression of c-maf, in keeping with the Th2 cell profile (Ho et al., 1996), which was almost completely abrogated by increasing doses of antigen (Figure 6B). Interestingly, IL-12 sustained the high expression of c-Maf mRNA even at the highest antigen dose (Figure 6B). Moreover, IL-12 maintenance of c-maf expression required STAT4 activation (data not shown).\nIn Th17 cells that expressed IL-17a as well as IL-10 mRNA (Figure 6C), T-bet and GATA-3 mRNA were undetectable (data not shown), whereas that of ROR-gammat was high (Figure 6C) (Ivanov et al., 2007). Th17 cells also expressed high amounts of c-maf (Figure 6C), confirming a recent report (Bauquet et al., 2009). c-Maf is therefore expressed in all IL-10-expressing T cell populations tested (Figures 6B and 6C) and may not be just a Th2 cell-specific transcription factor as originally thought (Ho et al., 1996). We showed also that like Il10 expression, c-maf expression was inhibited in Th1 and Th17 cells in the presence of the MEK1 and MEK2 inhibitor (PD184352), whereas T-bet and RORgammat expression was hardly affected (Figure 6D).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Expression", "start": 6, "end": 16}, "arguments": [{"role": "Theme", "text": "c-maf", "start": 0, "end": 5}]}, {"trigger": {"text": "Production", "start": 39, "end": 49}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 33, "end": 38}]}, {"trigger": {"text": "production", "start": 153, "end": 163}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 147, "end": 152}]}, {"trigger": {"text": "expression", "start": 786, "end": 796}, "arguments": [{"role": "Theme", "text": "Il4", "start": 782, "end": 785}]}, {"trigger": {"text": "expression", "start": 882, "end": 892}, "arguments": [{"role": "Theme", "text": "Il4", "start": 878, "end": 881}]}, {"trigger": {"text": "expression", 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1588}, "arguments": [{"role": "Theme", "text": "mRNA expression", "start": 1433, "end": 1448}, {"role": "Cause", "text": "IL-12", "start": 1638, "end": 1643}]}, {"trigger": {"text": "abrogated", "start": 1819, "end": 1828}, "arguments": [{"role": "Theme", "text": "expression", "start": 1714, "end": 1724}]}, {"trigger": {"text": "undetectable", "start": 2174, "end": 2186}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 2164, "end": 2168}]}, {"trigger": {"text": "inhibited", "start": 2652, "end": 2661}, "arguments": [{"role": "Theme", "text": "expression", "start": 2619, "end": 2629}, {"role": "Cause", "text": "inhibitor", "start": 2721, "end": 2730}]}, {"trigger": {"text": "inhibited", "start": 2652, "end": 2661}, "arguments": [{"role": "Theme", "text": "expression", "start": 2637, "end": 2647}, {"role": "Cause", "text": "inhibitor", "start": 2721, "end": 2730}]}, {"trigger": {"text": "inhibitor", "start": 2721, "end": 2730}, "arguments": [{"role": "Theme", "text": "MEK1", "start": 2707, "end": 2711}]}, {"trigger": {"text": "inhibitor", "start": 2721, "end": 2730}, "arguments": [{"role": "Theme", "text": "MEK2", "start": 2716, "end": 2720}]}], "positive regulation": [{"trigger": {"text": "resulted", "start": 378, "end": 386}, "arguments": [{"role": "Theme", "text": "transcription", "start": 390, "end": 403}]}, {"trigger": {"text": "induced", "start": 530, "end": 537}, "arguments": [{"role": "Theme", "text": "transcription", "start": 500, "end": 513}, {"role": "Cause", "text": "IL-12", "start": 541, "end": 546}]}, {"trigger": {"text": "this effect", "start": 605, "end": 616}, "arguments": [{"role": "Theme", "text": "transcription", "start": 500, "end": 513}, {"role": "Cause", "text": "IL-12", "start": 620, "end": 625}]}, {"trigger": {"text": "upregulated", "start": 639, "end": 650}, "arguments": [{"role": "Theme", "text": "this effect", "start": 605, "end": 616}]}, {"trigger": {"text": "increase", "start": 958, "end": 966}, "arguments": [{"role": "Theme", 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{"input": "IL-10 expression by cells of the innate and adaptive immune systems reflects the importance of this cytokine in the tight regulation of the immune response, to minimize pathology during infection. IL-10 expression by Th1 cells has been reported to regulate the immune response in leishmaniasis and toxoplasmosis. However, in many situations, IL-10 is not produced by Th1 cells in response to antigenic stimulation. Our goal was to address the molecular signals that determine whether Th1 cells develop to produce IL-10 (Trinchieri, 2007). Here, we showed that Th1 cells required high-antigen-dose-induced ERK1 and ERK2 phosphorylation and IL-12-induced STAT-4 activation to produce IL-10. Our findings that ERK1 and ERK2 activation was a common pathway required for the production of IL-10 by Th1, Th2, and Th17 cell subsets, which differentiate along distinct pathways, such that IL-10 provides a highly regulated feedback loop to avoid the extremes of excessive inflammation or chronic infections and also allow a protective response to diverse pathogens.\nIn certain viral or parasitic infections (Anderson et al., 2007; Brooks et al., 2006; Ejrnaes et al., 2006), high amounts of stimulation may lead to the chronic nonhealing infection shown to be regulated by IL-10. During the course of infection, after initial triggering with antigen, T cells migrate to the tissue encountering high doses of antigen and factors produced by the innate immune response. Under these conditions, we speculate that Th1 cells will be induced to express high amounts of IL-10, in keeping with reports that IL-10-producing Th1 cells were found in CD4+ clones isolated from BAL but not blood of TB patients (Gerosa et al., 1999). Similarly, the immune response to a clinical isolate of L. major, which produces heavily infected nonhealing lesions, was found to be regulated by IL-10 derived from Foxp3- Th1 cells that coproduce IL-10 and IFN-gamma (Anderson et al., 2007), and the immune response during T. gondii infection was found also to be regulated by Foxp3- Th1 cells (Jankovic et al., 2007). It is likely that IL-10 production by Th1 cells is evoked under conditions of high inflammation and antigenic stimulation, whereas regulatory CD4+ T cells producing IL-10 may operate to regulate the immune response under conditions in which the pathogen is clinically controlled, such as in infection with L. major (Friedlin strain) (Belkaid et al., 2002; Suffia et al., 2006). We now also reported that CD4+ T cells cultured with high antigen dose and IL-12 differentiate into canonical Th1 effector cells, which, in addition to expressing large amounts of IFN-gamma and IL-10, lose their IL-2 expression as described before in certain chronic infection models (Sallusto et al., 2004). Our demonstration that loss of IL-2 is accompanied by production of IL-10 offers potential additional mechanisms whereby effector T cell responses may be dampened during chronic disease.\nUsing an in vivo transfer model of DO11.10 TCR transgenic cells (Castro et al., 2000), we showed that IL-10-producing Th1 cells were differentiated in the presence of high doses of OVA protein and LPS. We showed here that this induction of IL-10 in Th1 cells in vivo was markedly, but not totally, reduced in STAT4-deficient T cells as observed during T. gondii infection (Jankovic et al., 2002). A high antigenic activation during T. gondii infection or high antigen doses delivered in the presence of LPS, as seen in our system, may compensate for an absolute requirement for IL-12 in the induction of IL-10 by Th1 cells.\nIn our in vitro system, repeated stimulation of Th1 cells with high antigen doses allowed the development of Th1 cells producing IL-10 in an IL-12-dependent manner. IL-10 production by Th1 cells induced by high antigen dose and IL-12 was independent of IFN-gamma, in keeping with previous findings (Jankovic et al., 2002). However, a role for IFN-gamma in mediating IL-10 reactivation by Th1 cells during secondary infection with T. gondii has been suggested (Shaw et al., 2006). We have found that CD4+ T cells exposed to a high dose of antigen do not express IL-10 upon restimulation, but can be induced to produce IL-10 upon re-exposure to a high dose of antigen in the recall phase in the absence of added IL-12. However, this is dependent on the induction of IL-12 by antigen-presenting DCs. The combination of both high antigen dose and IL-12 resulted in the highest levels of IL-10 production and correlated with the high levels of ERK1 and ERK2 activation. The increased expression of IFN-gamma observed during the secondary phase will induce increased IL-12 production by DCs and suggests that repeated high-level TCR activation feeds back to upregulate IL-12 production by DC. It is thus likely that in T. gondii infection in vivo (Shaw et al., 2006), the requirement for IFN-gamma to induce IL-10, was for feedback upregulation of IL-12 by DCs, which in turn induced IL-10 in the Th1 cells.\nAlthough IL-10 may be differentially regulated in Th1 and Th2 cells as has been reported (Chang et al., 2007; Wang et al., 2005), some studies suggest the existence of common pathways, but the molecular basis for these is as yet unclear. Costimulatory OX-40 signals have been shown to negatively regulate IL-10 production (Ito et al., 2005) both in Th1 and Th2 cells, whereas ICOS signaling has been suggested to induce IL-10 (Ito et al., 2007; Witsch et al., 2002) in both Th1 and Th2 cells. However, in some cases, ICOS signaling also regulates IL-4 production and Th2 responses (Greenwald et al., 2005). We now provide a common mechanism of ERK1 and ERK2 activation for the regulation of IL-10 production in Th1, Th2, and Th17 cells, although each subset differentiates along a distinct and subset-specific transcriptional pathway. This reinforces the fact that IL-10 is not a Th cell-subset-specific cytokine, but instead is produced in a tightly regulated fashion during each differentiation pathway. Of note, a role for ERK1 and ERK2 activation in the induction of IL-10 production has already been described for macrophages and DC (Agrawal et al., 2006; Hacker et al., 1999).\nDifferential transcriptional regulation of IL-10 in Th1 and Th2 cells has been suggested (Chang et al., 2007; Wang et al., 2005), and extensive histone acetylation of the IL-10 gene is detectable in fully polarized Th2 cells, but not Th1 cells (Chang et al., 2007). We provide evidence that IL-10 is produced in canonical Th1 cells and that its expression correlates with the expression of T-bet and the highest IFN-gamma production, in keeping with our observations that high-dose antigen stimulation and IL-12 signaling are required for IL-10 and IFN-gamma expression. It has also been shown that maintenance of IL-10 expression is conditional on IL-12 or IL-4 unless the IL-10 gene is imprinted by GATA-3 (Chang et al., 2007), which can remodel the IL-10 locus, thus explaining the highest amounts of IL-10 produced by Th2 cells (Chang et al., 2007; Shoemaker et al., 2006). We show here that high antigen dose and IL-12 drastically downregulate Gata-3 expression, suggesting that additional factors are in place to induce IL-10 expression in Th1 cells, albeit transiently. Expression of c-maf was greatly diminished by high antigen doses in T cells and yet was unexpectedly maintained by IL-12 and present in Th17 cells. That c-maf expression is common to IL-10-producing Th1, Th2, and Th17 cells and, like IL-10, is dependent on ERK activation in Th1 and Th17 cells for its expression is of interest because c-Maf has been shown to be an essential transcription factor for IL-10 expression in macrophages (Cao et al., 2005).\nIn summary, we show that although Th1, Th2, and Th17 CD4+ T cell subsets differentiate along distinct signaling and transcriptional pathways, they can all be induced to make IL-10. ERK1 and ERK2 activation is required for IL-10 production by all these Th cell subsets. With regard to the expression of IL-10 by Th1 cells, our data provide a mechanism for how IL-10 expression is induced and then amplified and regulated by the levels of antigen and IL-12 encountered in the environment. This provides a mechanism whereby a Th1 cell responds to extrinsic signals, reflecting increased inflammation in the tissue, to tightly regulate the production of IL-10 so as to allow a protective response to eradicate a pathogen with minimal damage to the host and also prevent chronic infection. Moreover, our findings have important implications for the regulation of IL-10 production during an inflammatory Th1 response in infection and may be of relevance for the design of vaccines and for strategies in immunotherapy in infectious diseases.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 6, "end": 16}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 0, "end": 5}]}, {"trigger": {"text": "expression", "start": 203, "end": 213}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 197, "end": 202}]}, {"trigger": {"text": "produced", "start": 355, "end": 363}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 342, "end": 347}]}, {"trigger": {"text": "produce", "start": 505, "end": 512}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 513, "end": 518}]}, {"trigger": {"text": "produce", "start": 674, "end": 681}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 682, "end": 687}]}, {"trigger": 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"induce", "start": 5396, "end": 5402}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 5403, "end": 5408}]}, {"trigger": {"text": "activation", "start": 5641, "end": 5651}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 5627, "end": 5631}]}, {"trigger": {"text": "activation", "start": 5641, "end": 5651}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 5636, "end": 5640}]}, {"trigger": {"text": "activation", "start": 6023, "end": 6033}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 6009, "end": 6013}]}, {"trigger": {"text": "activation", "start": 6023, "end": 6033}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 6018, "end": 6022}]}, {"trigger": {"text": "induction", "start": 6041, "end": 6050}, "arguments": [{"role": "Theme", "text": "production", "start": 6060, "end": 6070}]}, {"trigger": {"text": "required", "start": 6692, "end": 6700}, "arguments": [{"role": "Theme", "text": "expression", "start": 6725, "end": 6735}]}, {"trigger": {"text": "maintenance", "start": 6765, "end": 6776}, "arguments": [{"role": "Theme", "text": "expression", "start": 6786, "end": 6796}]}, {"trigger": {"text": "conditional", "start": 6800, "end": 6811}, "arguments": [{"role": "Theme", "text": "maintenance", "start": 6765, "end": 6776}, {"role": "Cause", "text": "IL-12", "start": 6815, "end": 6820}]}, {"trigger": {"text": "conditional", "start": 6800, "end": 6811}, "arguments": [{"role": "Theme", "text": "maintenance", "start": 6765, "end": 6776}, {"role": "Cause", "text": "IL-4", "start": 6824, "end": 6828}]}, {"trigger": {"text": "induce", "start": 7185, "end": 7191}, "arguments": [{"role": "Theme", "text": "expression", "start": 7198, "end": 7208}]}, {"trigger": {"text": "maintained", "start": 7344, "end": 7354}, "arguments": [{"role": "Theme", "text": "Expression", "start": 7243, "end": 7253}, {"role": "Cause", "text": "IL-12", "start": 7358, "end": 7363}]}, {"trigger": {"text": "dependent", "start": 7487, "end": 7496}, "arguments": [{"role": "Theme", "text": "expression", "start": 7402, "end": 7412}, {"role": "Cause", "text": "activation", "start": 7504, "end": 7514}]}, {"trigger": {"text": "dependent", "start": 7487, "end": 7496}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 7477, "end": 7482}, {"role": "Cause", "text": "activation", "start": 7504, "end": 7514}]}, {"trigger": {"text": "activation", "start": 7504, "end": 7514}, "arguments": [{"role": "Theme", "text": "ERK", "start": 7500, "end": 7503}]}, {"trigger": {"text": "essential", "start": 7609, "end": 7618}, "arguments": [{"role": "Cause", "text": "c-Maf", "start": 7579, "end": 7584}, {"role": "Theme", "text": "expression", "start": 7650, "end": 7660}]}, {"trigger": {"text": "induced", "start": 7854, "end": 7861}, "arguments": [{"role": "Theme", "text": "make", "start": 7865, "end": 7869}]}, {"trigger": {"text": "activation", "start": 7891, "end": 7901}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 7877, "end": 7881}]}, {"trigger": {"text": "activation", "start": 7891, "end": 7901}, "arguments": [{"role": "Theme", "text": "ERK2", "start": 7886, "end": 7890}]}, {"trigger": {"text": "required", "start": 7905, "end": 7913}, "arguments": [{"role": "Cause", "text": "ERK1", "start": 7877, "end": 7881}, {"role": "Theme", "text": "production", "start": 7924, "end": 7934}]}, {"trigger": {"text": "required", "start": 7905, "end": 7913}, "arguments": [{"role": "Cause", "text": "ERK2", "start": 7886, "end": 7890}, {"role": "Theme", "text": "production", "start": 7924, "end": 7934}]}, {"trigger": {"text": "induced", "start": 8075, "end": 8082}, "arguments": [{"role": "Theme", "text": "expression", "start": 8061, "end": 8071}]}, {"trigger": {"text": "amplified", "start": 8092, "end": 8101}, "arguments": [{"role": "Theme", "text": "expression", "start": 8061, "end": 8071}, {"role": "Cause", "text": "IL-12", "start": 8145, "end": 8150}]}], "regulation": [{"trigger": {"text": "provides a highly regulated feedback loop", "start": 887, "end": 928}, "arguments": [{"role": "Theme", "text": "production", "start": 770, "end": 780}, {"role": "Cause", "text": "IL-10", "start": 881, "end": 886}]}, {"trigger": {"text": "independent", "start": 3819, "end": 3830}, "arguments": [{"role": "Theme", "text": "induced", "start": 3776, "end": 3783}, {"role": "Cause", "text": "IFN-gamma", "start": 3834, "end": 3843}]}, {"trigger": {"text": "regulated", "start": 5020, "end": 5029}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 4992, "end": 4997}]}, {"trigger": {"text": "regulates", "start": 5520, "end": 5529}, "arguments": [{"role": "Theme", "text": "production", "start": 5535, "end": 5545}]}, {"trigger": {"text": "regulation", "start": 5660, "end": 5670}, "arguments": [{"role": "Cause", "text": "ERK1", "start": 5627, "end": 5631}, {"role": "Theme", "text": "production", "start": 5680, "end": 5690}]}, {"trigger": {"text": "regulation", "start": 5660, "end": 5670}, "arguments": [{"role": "Cause", "text": "ERK2", "start": 5636, "end": 5640}, {"role": "Theme", "text": "production", "start": 5680, "end": 5690}]}, {"trigger": {"text": "regulated", "start": 5934, "end": 5943}, "arguments": [{"role": "Theme", "text": "produced", "start": 5912, "end": 5920}]}, {"trigger": {"text": "role", "start": 6000, "end": 6004}, "arguments": [{"role": "Cause", "text": "activation", "start": 6023, "end": 6033}, {"role": "Theme", "text": "induction", "start": 6041, "end": 6050}]}, {"trigger": {"text": "regulation", "start": 6195, "end": 6205}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 6209, "end": 6214}]}, {"trigger": {"text": "regulated", "start": 8106, "end": 8115}, "arguments": [{"role": "Theme", "text": "expression", "start": 8061, "end": 8071}, {"role": "Cause", "text": "IL-12", "start": 8145, "end": 8150}]}, {"trigger": {"text": "regulate", "start": 8319, "end": 8327}, "arguments": [{"role": "Theme", "text": "production", "start": 8332, "end": 8342}]}, {"trigger": {"text": "regulation", "start": 8540, "end": 8550}, "arguments": [{"role": "Theme", "text": "production", "start": 8560, "end": 8570}]}]}}, "schema": []}
{"input": "Mice, Cytokines, Antibodies, and Other Reagents\nBALB/c DO11.10 mice transgenic for OVA-specific TCR WT or crossed back with Rag1-, IL-4-, IFN-gamma-, STAT4-, and STAT6-deficient mice were used as a source of antigen-specific T cells (Murphy et al., 1990; Ouyang et al., 1998; Shoemaker et al., 2006) and were bred and maintained under SPF conditions at the NIMR, London, Home Office, UK, Animals (Scientific Procedures) Act 1986 or at the Washington University School of Medicine. Female mice were used at 8-12 weeks old, and animal protocols were approved according to the Animals (Scientific Procedures) Act 1986, Home Office, UK. Reagents, including antibodies for T cell and DC preparation, purification and culture, media, cytokines, and cytokine mAbs have been described (Hosken et al., 1995; Shoemaker et al., 2006; Veldhoen et al., 2009; Veldhoen et al., 2006). LPS (S. minnesota) was from Alexis, chicken ovalbumin protein (OVA protein) was from from Sigma-Aldrich, and ovalbumin peptide323-339 (OVA) (endotoxin-free) was from Biosynthesis. U0126 was from BioMol International. PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), and CT99021 (GSK3beta inhibitor) were kind gifts from P. Cohen and N. Shpiro, University of Dundee, UK.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "deficient", "start": 168, "end": 177}, "arguments": [{"role": "Theme", "text": "Rag1", "start": 124, "end": 128}]}, {"trigger": {"text": "deficient", "start": 168, "end": 177}, "arguments": [{"role": "Theme", "text": "IL-4", "start": 131, "end": 135}]}, {"trigger": {"text": "deficient", "start": 168, "end": 177}, "arguments": [{"role": "Theme", "text": "IFN-gamma", "start": 138, "end": 147}]}, {"trigger": {"text": "deficient", "start": 168, "end": 177}, "arguments": [{"role": "Theme", "text": "STAT4", "start": 150, "end": 155}]}, {"trigger": {"text": "deficient", "start": 168, "end": 177}, "arguments": [{"role": "Theme", "text": "STAT6", "start": 162, "end": 167}]}, {"trigger": {"text": "inhibitors", "start": 1101, "end": 1111}, "arguments": [{"role": "Theme", "text": "MEK", "start": 1097, "end": 1100}]}, {"trigger": {"text": "inhibitor", "start": 1128, "end": 1137}, "arguments": [{"role": "Theme", "text": "p38", "start": 1124, "end": 1127}]}, {"trigger": {"text": "inhibitor", "start": 1162, "end": 1171}, "arguments": [{"role": "Theme", "text": "GSK3beta", "start": 1153, "end": 1161}]}]}}, "schema": []}
{"input": "Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation\nT cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25-, or CD4+CD44loCD25- to >98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009). In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained. Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture. Purified DO11.10 CD4+ T cells (1 x 105 cells/ml) were cultured as before (Hosken et al., 1995), in a total volume of 1 ml cRPMI medium in a 48-well plate, with splenic DCs (2 x 104 cells/ml), and varying amounts of OVA and of IL-12. APC-independent differentiation of naive CD4+ T cells into Th1 and Th2 cells used stimulation with anti-CD3 and anti-CD28 and appropriate cytokine conditions, and control Th1 and Th2 cells were cultured as described before (Hosken et al., 1995; Shoemaker et al., 2006). Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006). Importantly, Th1 and Th2 cells could be differentiated in cRPMI or IMDM (Hosken et al., 1995; Shoemaker et al., 2006; Veldhoen et al., 2006), but Th17 cells were only differentiated optimally in IMDM (Veldhoen et al., 2009). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3beta inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "inhibitors", "start": 1375, "end": 1385}, "arguments": [{"role": "Theme", "text": "MEK", "start": 1371, "end": 1374}]}, {"trigger": {"text": "inhibitor", "start": 1402, "end": 1411}, "arguments": [{"role": "Theme", "text": "p38", "start": 1398, "end": 1401}]}, {"trigger": {"text": "inhibitor", "start": 1432, "end": 1441}, "arguments": [{"role": "Theme", "text": "GSK3beta", "start": 1423, "end": 1431}]}]}}, "schema": []}
{"input": "Cytokine Detection by ICS and ELISA\nAt day 5 or 7, cells were restimulated with immobilized anti-CD3 (2 mug/ml) and anti-CD28 (2 mug/ml) (4 hr with BrefeldinA [10 mug/ml] in the last 2 hr) or with PdBU and Ionomycin (5 hr with BrefeldinA [1 mug/ml]). After ICS FACS, data were collected on a FACSCalibur (Becton Dickinson) and analyzed with FlowJo (Tree Star). For ELISA, cells were similarly restimulated for 48 hr and supernatant was collected and analyzed for IL-4, IL-10, and IFN-gamma as described before (Shoemaker et al., 2006).", "output": {"json_structures": {}}, "schema": []}
{"input": "In Vivo Studies\nBALB/c mice were injected intravenously (i.v.) with a red blood cell-depleted single-cell spleen suspension (2.5 x 107 cells) (Castro et al., 2000) from DO11.10 WT or STAT-deficient mice. After 48 hr, they were injected subcutaneously with PBS or with OVA protein (5 mg) plus LPS (5 mug). The inguinal lymph nodes were removed 48 hr later. A single-cell suspension (1 x 106 cells) was restimulated for 24 or 48 hr with 1 muM or 3 muM of OVA and with BrefeldinA for the last 6 hr. Half of the supernatant was removed before the addition of BrefeldinA for use in an ELISA assay. The cells were fixed and stained as before. Those positive for KJ1-26-Bio and for CD4-PerCP were gated, and IFN-gamma and IL-10 staining was examined for this population and analyzed as before.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "deficient", "start": 188, "end": 197}, "arguments": [{"role": "Theme", "text": "STAT", "start": 183, "end": 187}]}]}}, "schema": []}
{"input": "Real-Time Quantitative RT-PCR\nCells were harvested and restimulated in the presence of immobilized anti-CD3 (2 mug/ml) plus anti-CD28 (2 mug/ml) for 3 hr or immediately lysed. RNA was extracted and reverse-transcribed and cDNA was analyzed for the expression of cytokines and transcription factors by real-time PCR assay as before (Shoemaker et al., 2006). Target gene mRNA expression was quantified either with SYBR Green (Applied Biosystems) or with Master Mix (Applied Biosystems) and normalized to ubiquitin or HPRT mRNA levels, respectively.", "output": {"json_structures": {"transcription": [{"trigger": {"text": "mRNA levels", "start": 520, "end": 531}, "arguments": [{"role": "Theme", "text": "ubiquitin", "start": 502, "end": 511}]}, {"trigger": {"text": "mRNA levels", "start": 520, "end": 531}, "arguments": [{"role": "Theme", "text": "HPRT", "start": 515, "end": 519}]}]}}, "schema": []}
{"input": "Immunoblotting\nDifferentiated CD4+ T cells were rested for 5 hr in 1% FCS-containing medium and restimulated as described for specific experiments. Cell lysates were prepared, equal amounts of protein were separated by SDS-PAGE, and phosphorylated or total ERK and actin were detected as described before (Beinke et al., 2004).", "output": {"json_structures": {}}, "schema": []}
{"input": "Differential cytokine regulation by NF-kappaB and AP-1 in Jurkat T-cells\nBackground\nActivator protein (AP)-1 and nuclear factor (NF)-kappaB largely control T-cell activation, following binding of foreign antigens to the T-cell receptor leading to cytokine secretion. Elevated levels of pro-inflammatory cytokines and chemokines such as TNF, IL-6 and CXCL8 are associated with several human diseases including cystic fibrosis, pulmonary fibrosis and AIDS. The aim of this study was to investigate the role of the transcription factors, AP-1 and NF-kappaB, in IL-6 and CXCL8 regulation in Jurkat T-cells.\nResults\nPhorbol myristate acetate (PMA) exposure resulted in an up-regulation of AP-1 and down-regulation of NF-kappaB activity, however, exposure to heat killed (HK) Escherichia. coli MG1655 resulted in a dose-dependent increase in NF-kappaB activity without affecting AP-1. The cytokine profile revealed an up-regulation of the chemokine CXCL8 and the pro-inflammatory cytokines TNF, IL-2 and IL-6 following treatment with both PMA and HK E. coli, while the levels of the anti-inflammatory cytokine IL-10 were not affected by PMA but were significantly down-regulated by HK E. coli. AP-1 activation was significantly increased 2 h after PMA exposure and continued to increase thereafter. In contrast, NF-kappaB responded to PMA exposure by a rapid up-regulation followed by a subsequent down-regulation. Increased intracellular Ca2+ concentrations countered the down-regulation of NF-kappaB by PMA, while similar treatment with calcium ionophore resulted in a reduced NF-kappaB activity following induction with HK E. coli. In order to further study NF-kappaB activation, we considered two up-stream signalling proteins, PKC and Bcl10. Phosphorylated-PKC levels increased in response to PMA and HK E. coli, while Bcl10 levels significantly decreased following PMA treatment. Using an NF-kappaB activation inhibitor, we observed complete inhibition of IL-6 expression while CXCL8 levels only decreased by 40% at the highest concentration. Treatment of Jurkat T-cells with PMA in the presence of JNK-inhibitor suppressed both CXCL8 and IL-6 while PKC-inhibitor primarily decreased CXCL8 expression.\nConclusion\nThe present study shows that NF-kappaB regulated IL-6 but not CXCL8. This complex regulation of CXCL8 suggests that there is a need to further evaluate the signalling pathways in order to develop new treatment for diseases with elevated CXCL8 levels, such as AIDS and autoimmune diseases.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 1961, "end": 1971}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1956, "end": 1960}]}, {"trigger": {"text": "expression", "start": 2190, "end": 2200}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 2184, "end": 2189}]}], "negative regulation": [{"trigger": {"text": "down-regulated", "start": 1158, "end": 1172}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1104, "end": 1109}]}, {"trigger": {"text": "decreased", "start": 1845, "end": 1854}, "arguments": [{"role": "Theme", "text": "Bcl10", "start": 1818, "end": 1823}]}, {"trigger": {"text": "inhibition", "start": 1942, "end": 1952}, "arguments": [{"role": "Theme", "text": "expression", "start": 1961, "end": 1971}]}, {"trigger": {"text": "decreased", "start": 1996, "end": 2005}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 1978, "end": 1983}]}, {"trigger": {"text": "suppressed", "start": 2113, "end": 2123}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 2129, "end": 2134}]}, {"trigger": {"text": "suppressed", "start": 2113, "end": 2123}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 2139, "end": 2143}]}, {"trigger": {"text": "decreased", "start": 2174, "end": 2183}, "arguments": [{"role": "Theme", "text": "expression", "start": 2190, "end": 2200}]}], "positive regulation": [{"trigger": {"text": "up-regulation", "start": 912, "end": 925}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 943, "end": 948}]}, {"trigger": {"text": "up-regulation", "start": 912, "end": 925}, "arguments": [{"role": "Theme", "text": "IL-2", "start": 989, "end": 993}]}, {"trigger": {"text": "up-regulation", "start": 912, "end": 925}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 998, "end": 1002}]}, {"trigger": {"text": "elevated", "start": 2441, "end": 2449}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 2450, "end": 2455}]}], "regulation": [{"trigger": {"text": "role", "start": 500, "end": 504}, "arguments": [{"role": "Theme", "text": "regulation", "start": 573, "end": 583}]}, {"trigger": {"text": "regulation", "start": 573, "end": 583}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 558, "end": 562}]}, {"trigger": {"text": "regulation", "start": 573, "end": 583}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 567, "end": 572}]}, {"trigger": {"text": "affected", "start": 1119, "end": 1127}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 1104, "end": 1109}]}, {"trigger": {"text": "regulated", "start": 2252, "end": 2261}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 2262, "end": 2266}]}, {"trigger": {"text": "regulated", "start": 2252, "end": 2261}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 2275, "end": 2280}]}, {"trigger": {"text": "regulation", "start": 2295, "end": 2305}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 2309, "end": 2314}]}]}}, "schema": []}
{"input": "Cytokines and chemokines are important in immune cell recruitment and in regulation of inflammatory responses [1]. T-cells produce a broad range of inflammatory mediators, including IL-2, IL-6, TNF and CXCL8, all of which are important in cell proliferation, differentiation, communication and initiation of inflammatory responses [2]. Elevated levels of pro-inflammatory cytokines and chemokines, such as TNF, IL-6 and CXCL8, are associated with several human diseases including cystic fibrosis [3-5], pulmonary fibrosis [6,7] and AIDS [8,9]. Induction of CXCL8 has been suggested to be mediated through NF-kappaB in cooperation with AP-1 [10,11], however the precise mechanism is not fully elucidated, and treatment strategies aimed at inhibiting CXCL8 have failed [12]. Persistent production of IL-6 and CXCL8 leads to chronic inflammation and enhanced survival of lymphocytes increasing serum cytokine/chemokine levels. This forms the basis of several autoimmune disorders including plasmacytosis and hyperplasia [13]. To develop viable CXCL8 based treatment strategies, it is necessary to identify the signalling pathways regulating CXCL8 and determine how this is coupled to NF-kappaB, AP-1 and IL-6.\nThe signalling pathways leading to NF-kappaB and AP-1 activation are overlapping, where both are involved in the induction and regulation of cytokines/chemokines. NF-kappaB is activated in response to stress, such as oxidative stress, bacterial toxins, viruses and UV light [14], and is essential for differentiation, proliferation and survival of many cell types including T-lymphocytes [15]. AP-1 activation requires Fos (c-Fos, FosB, Fra-1, Fra-2) and Jun (c-Jun, v-Jun, JunB, JunD) through the formation of homo- and hetero-dimers [16,17], and regulates transcription of a broad range of genes involved in immune responses [18-21]. Both AP-1 and NF-kappaB binding sites have been identified in the promoter region of IL-6 and CXCL8 [12,22], however, the mechanism by which these interleukins are regulated in T-cells is still not clear. CXCL8 is a C-X-C chemokine with properties enabling it to recruit T-cells and basophils and to activate neutrophils and monocytes [23]. IL-6 is a cytokine that possesses both pro- and anti-inflammatory characteristics and that plays a key role in haematopoiesis and acute-phase responses [24,25].\nThe present study suggests that the regulation of CXCL8 and IL-6 is uncoupled. Using Jurkat T-cells exposed to PMA and heat killed (HK) Escherichia coli MG1655 in combination with inhibitors of NF-kappaB, JNK and PKC, we demonstrated that NF-kappaB regulates IL-6 expression while the regulation of CXCL8 more closely correlated to AP-1 activity. These results indicate that inhibition of NF-kappaB is not an effective strategy in countering the high CXCL8 activities in diseases such as cystic fibrosis, AIDS and pulmonary fibrosis.", "output": {"json_structures": {"binding": [{"trigger": {"text": "formation of homo- and hetero-dimers", "start": 1705, "end": 1741}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 1631, "end": 1636}, {"role": "Theme2", "text": "c-Jun", "start": 1667, "end": 1672}]}, {"trigger": {"text": "formation of homo- and hetero-dimers", "start": 1705, "end": 1741}, "arguments": [{"role": "Theme", "text": "FosB", "start": 1638, "end": 1642}, {"role": "Theme2", "text": "c-Jun", "start": 1667, "end": 1672}]}, {"trigger": {"text": "formation of homo- and hetero-dimers", "start": 1705, "end": 1741}, "arguments": [{"role": "Theme", "text": "Fra-1", "start": 1644, "end": 1649}, {"role": "Theme2", "text": "c-Jun", "start": 1667, "end": 1672}]}, {"trigger": {"text": "formation of homo- and hetero-dimers", "start": 1705, "end": 1741}, "arguments": [{"role": "Theme", "text": "Fra-2", "start": 1651, "end": 1656}, {"role": "Theme2", "text": "c-Jun", "start": 1667, "end": 1672}]}, {"trigger": {"text": "formation of homo- and hetero-dimers", "start": 1705, "end": 1741}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 1631, "end": 1636}, {"role": "Theme2", "text": "v-Jun", "start": 1674, "end": 1679}]}, {"trigger": {"text": "formation of homo- and hetero-dimers", "start": 1705, "end": 1741}, "arguments": [{"role": "Theme", "text": "FosB", "start": 1638, "end": 1642}, {"role": "Theme2", "text": "v-Jun", "start": 1674, "end": 1679}]}, {"trigger": {"text": "formation of homo- and hetero-dimers", "start": 1705, "end": 1741}, "arguments": [{"role": "Theme", "text": "Fra-1", "start": 1644, "end": 1649}, {"role": "Theme2", "text": "v-Jun", "start": 1674, "end": 1679}]}, {"trigger": {"text": "formation of homo- and hetero-dimers", "start": 1705, "end": 1741}, "arguments": [{"role": "Theme", 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"Theme", "text": "CXCL8", "start": 2395, "end": 2400}]}, {"trigger": {"text": "regulation", "start": 2381, "end": 2391}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 2405, "end": 2409}]}, {"trigger": {"text": "regulates", "start": 2594, "end": 2603}, "arguments": [{"role": "Theme", "text": "expression", "start": 2609, "end": 2619}]}, {"trigger": {"text": "regulation", "start": 2630, "end": 2640}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 2644, "end": 2649}]}]}}, "schema": []}
{"input": "Regulation of AP-1 and NF-kappaB activation\nThe transcription factors NF-kappaB and AP-1 play key roles in the initiation of an inflammatory response by inducing the expression and secretion of chemokines and cytokines that attract and activate immune cells. However, the signal transduction pathways and subsequent inflammatory cytokine induction by these transcription factors is not fully elucidated. The present study is aimed at determining the involvement of AP-1 and NF-kappaB in cytokine induction and regulation. PMA treatment resulted in an up-regulation of AP-1 after 2 h exposure and continued to increase throughout the analysis period (figure 1a). HK E. coli treatment did not affect AP-1 activation in Jurkat T-cells (figure 1b). To determine the involvement of associated pathways, we exposed cells to Ca2+ ionophore with or without PMA and observed a modest involvement of Ca2+ in PMA-dependent AP-1 activation (figure 1c) while Ca2+ alone did not alter AP-1 activity (data not shown). Furthermore, AP-1 activity decreased in a TCR-deficient Jurkat cell line when exposed to PMA compared to the parent cell line indicating that regulation of AP-1 was only partially T-cell receptor dependent (figure 1d).\nNF-kappaB levels showed a transient increase at 1 min after exposure to PMA (figure 2a). However, 1 h after exposure the NF-kappaB levels began to drop reaching the lowest levels by 6 h, after which they increased again by 24 h. Exposure of Jurkat T-cells to HK E. coli resulted in a dose-dependent NF-kappaB activation, with the highest activity observed at a relative concentration of 5 x 107 CFU/ml (figure 2b). The time-dependent activation of NF-kappaB by HK E. coli was assessed further using the optimal concentration obtained from figure 2b and showed that the NF-kappaB activity increased after 3 h of exposure (figure 2c). Furthermore, increased intracellular Ca2+ reversed the PMA dependent NF-kappaB inhibition (figure 2d) and reduced the HK E. coli -dependent NF-kappaB activation (figure 2e).", "output": {"json_structures": {}}, "schema": []}
{"input": "Induction of inflammatory responses\nThe ability of PMA and HK E. coli to induce an inflammatory response in Jurkat T-cells was evaluated using a multiplex cytokine assay following 24 h stimulation. The cytokine profile revealed an enhanced induction of the pro-inflammatory cytokines IL-2, IL-6, TNF and the chemokine CXCL8. The levels of the anti-inflammatory cytokine IL-10 were unaffected by PMA but were significantly decreased by HK E. coli (Table 1). These results confirmed that PMA and HK E. coli induced an inflammatory response in the Jurkat T-cells. It is interesting to note that PMA was 120-fold more effective at inducing CXCL8 than HK E. coli. PMA-dependent induction of AP-1 and down-regulation of NF-kappaB suggests an involvement of AP-1 in CXCL8 regulation. Determination of the time course of cytokine induction in response to PMA showed that CXCL8 was already released between 2-6 h, while TNF and IL-6 were released between 6-24 h (figure 3). These results indicated that the cytokines were differentially regulated and that the release was not associated with the early transient induction of NF-kappaB. The temporal induction of AP-1 correlated to the CXCL8 levels and preceded the TNF and IL-6 release. This suggests an association between CXCL8 release and AP-1 signalling.", "output": {"json_structures": {"localization": [{"trigger": {"text": "released", "start": 881, "end": 889}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 863, "end": 868}]}, {"trigger": {"text": "released", "start": 929, "end": 937}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 919, "end": 923}]}, {"trigger": {"text": "release", "start": 1219, "end": 1226}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1214, "end": 1218}]}, {"trigger": {"text": "release", "start": 1271, "end": 1278}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 1265, "end": 1270}]}], "negative regulation": [{"trigger": {"text": "decreased", "start": 422, "end": 431}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 370, "end": 375}]}], "positive regulation": [{"trigger": {"text": "enhanced", "start": 231, "end": 239}, "arguments": [{"role": "Theme", "text": "induction", "start": 240, "end": 249}]}, {"trigger": {"text": "induction", "start": 240, "end": 249}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 318, "end": 323}]}, {"trigger": {"text": "effective", "start": 614, "end": 623}, "arguments": [{"role": "Theme", "text": "inducing", "start": 627, "end": 635}]}, {"trigger": {"text": "inducing", "start": 627, "end": 635}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 636, "end": 641}]}], "regulation": [{"trigger": {"text": "unaffected", "start": 381, "end": 391}, "arguments": [{"role": "Theme", "text": "IL-10", "start": 370, "end": 375}]}, {"trigger": {"text": "involvement", "start": 736, "end": 747}, "arguments": [{"role": "Theme", "text": "regulation", "start": 765, "end": 775}]}, {"trigger": {"text": "regulation", "start": 765, "end": 775}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 759, "end": 764}]}, {"trigger": {"text": "regulated", "start": 1028, "end": 1037}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 863, "end": 868}]}, {"trigger": {"text": "regulated", "start": 1028, "end": 1037}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 919, "end": 923}]}]}}, "schema": []}
{"input": "Cooperative induction of cytokines by AP-1 and NF-kappaB\nTo further characterize the involvement of NF-kappaB in cytokine regulation, we treated cells with an NF-kappaB activation inhibitor (NAI). The results showed that NAI selectively down-regulated NF-kappaB activation (figures 4a and 4b) and did not alter AP-1 activity (figures 4c and 4d). Exposure of Jurkat T-cells to NAI resulted in a modest reduction of CXCL8 following PMA exposure, while it did not alter the CXCL8 release following HK E. coli exposure (figure 5a). NAI did not affect TNF expression (figure 5b) indicating that NF-kappaB is not the main regulator of CXCL8 or TNF following either PMA or HK E. coli exposure in Jurkat T-cells. In contrast, NAI resulted in a complete inhibition of IL-6 following PMA exposure and a 45% inhibition following HK E. coli exposure (figure 5c), suggesting an involvement of NF-kappaB in IL-6 regulation.\nCa2+ was observed to increase AP-1 activity (figure 1c) and reduce NF-kappaB activity (figure 2e); therefore, we exposed T-cells to a PKC inhibitor together with PMA to determine its effect on cytokine expression. Inhibition of PKC reduced CXCL8 release from 7 ng/ml to 3 ng/ml while it had a modest effect on IL-6 and TNF (figure 6a-c). This prompted us to test the effect of JNK inhibition on PMA-induced cytokine expression. JNK is involved in the regulation of a multitude of different transcription factors, including the phosphorylation and activation of c-Jun, c-Fos and p53, leading to cellular apoptosis [26]. Inhibition of the JNK pathways resulted in a down-regulation of both CXCL8 and IL-6, while no clear effect was observed on TNF expression (figure 6a-c). Analysis of mRNA levels using RT-qPCR (table 2) showed that PMA induced both il-6 and cxcl8 mRNA (5.1-fold and 111.8 fold respectively). Addition of the NF-kappaB inhibitor NAI and the JNK inhibitor reduced the il-6 expression below basal levels. In contrast, while the cxcl8 levels were suppressed by the same treatments the levels remained elevated above basal level.", "output": {"json_structures": {"localization": [{"trigger": {"text": "release", "start": 477, "end": 484}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 471, "end": 476}]}, {"trigger": {"text": "release", "start": 1156, "end": 1163}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 1150, "end": 1155}]}], "negative regulation": [{"trigger": {"text": "reduction", "start": 401, "end": 410}, "arguments": [{"role": "Theme", "text": "following", "start": 420, "end": 429}]}, {"trigger": {"text": "inhibition", "start": 745, "end": 755}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 759, "end": 763}]}, {"trigger": {"text": "inhibition", "start": 797, "end": 807}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 759, "end": 763}]}, {"trigger": {"text": "reduced", "start": 1142, "end": 1149}, "arguments": [{"role": "Theme", "text": "release", "start": 1156, "end": 1163}]}, {"trigger": {"text": "down-regulation", "start": 1574, "end": 1589}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 1598, "end": 1603}]}, {"trigger": {"text": "down-regulation", "start": 1574, "end": 1589}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 1608, "end": 1612}]}, {"trigger": {"text": "reduced", "start": 1881, "end": 1888}, "arguments": [{"role": "Theme", "text": "expression", "start": 1898, "end": 1908}]}, {"trigger": {"text": "suppressed", "start": 1970, "end": 1980}, "arguments": [{"role": "Theme", "text": "levels", "start": 1958, "end": 1964}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1437, "end": 1452}, "arguments": [{"role": "Theme", "text": "c-Jun", "start": 1471, "end": 1476}]}, {"trigger": {"text": "phosphorylation", "start": 1437, "end": 1452}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 1478, "end": 1483}]}, {"trigger": {"text": "phosphorylation", "start": 1437, "end": 1452}, "arguments": [{"role": "Theme", "text": "p53", "start": 1488, "end": 1491}]}], "positive regulation": [{"trigger": {"text": "following", "start": 420, "end": 429}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 414, "end": 419}]}, {"trigger": {"text": "following", "start": 485, "end": 494}, "arguments": [{"role": "Theme", "text": "release", "start": 477, "end": 484}]}, {"trigger": {"text": "following", "start": 642, "end": 651}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 629, "end": 634}]}, {"trigger": {"text": "following", "start": 764, "end": 773}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 759, "end": 763}]}, {"trigger": {"text": "following", "start": 808, "end": 817}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 759, "end": 763}]}, {"trigger": {"text": "activation", "start": 1457, "end": 1467}, "arguments": [{"role": "Theme", "text": "c-Jun", "start": 1471, "end": 1476}]}, {"trigger": {"text": "activation", "start": 1457, "end": 1467}, "arguments": [{"role": "Theme", "text": "c-Fos", "start": 1478, "end": 1483}]}, {"trigger": {"text": "activation", "start": 1457, "end": 1467}, "arguments": [{"role": "Theme", "text": "p53", "start": 1488, "end": 1491}]}, {"trigger": {"text": "resulted", "start": 1560, "end": 1568}, "arguments": [{"role": "Theme", "text": "down-regulation", "start": 1574, "end": 1589}]}, {"trigger": {"text": "induced", "start": 1746, "end": 1753}, "arguments": [{"role": "Theme", "text": "mRNA", "start": 1774, "end": 1778}]}, {"trigger": {"text": "elevated", "start": 2024, "end": 2032}, "arguments": [{"role": "Theme", "text": "levels", "start": 1958, "end": 1964}]}], "regulation": [{"trigger": {"text": "alter", "start": 461, "end": 466}, "arguments": [{"role": "Theme", "text": "following", "start": 485, "end": 494}]}, {"trigger": {"text": "regulator", "start": 616, "end": 625}, "arguments": [{"role": "Theme", "text": "following", "start": 642, "end": 651}]}, {"trigger": 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{"trigger": {"text": "levels", "start": 1958, "end": 1964}, "arguments": [{"role": "Theme", "text": "cxcl8", "start": 1952, "end": 1957}]}]}}, "schema": []}
{"input": "NF-kappaB inhibition due to PKC-dependent Bcl10 degradation\nWestern blot analysis revealed an up-regulation of phosphorylated-PKC after 24 h treatment of Jurkat T-cells with PMA or HK E. coli (figure 7a), while IkappaBbeta levels remained unaffected (figure 7b). Bcl10 is a signalling protein that acts upstream of NF-kappaB in concert with CARMA1 and MALT1 and has been suggested to directly regulate NF-kappaB activity in T-cells [27]. Therefore, Bcl10 activation was evaluated in both control and PMA stimulated cells after 10 min, 1 h, 6 h, and 24 h using western blot analysis. The Bcl10 levels decreased following treatment with PMA, while in control cells, Bcl10 returned to higher levels by 24 h (figure 7c). This suggests that Bcl10 is involved in the PMA dependent inhibition of NF-kappaB activation.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "decreased", "start": 600, "end": 609}, "arguments": [{"role": "Theme", "text": "Bcl10", "start": 587, "end": 592}]}], "positive regulation": [{"trigger": {"text": "activation", "start": 455, "end": 465}, "arguments": [{"role": "Theme", "text": "Bcl10", "start": 449, "end": 454}]}, {"trigger": {"text": "returned to higher", "start": 670, "end": 688}, "arguments": [{"role": "Theme", "text": "Bcl10", "start": 664, "end": 669}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 48, "end": 59}, "arguments": [{"role": "Theme", "text": "Bcl10", "start": 42, "end": 47}]}], "regulation": [{"trigger": {"text": "dependent", "start": 32, "end": 41}, "arguments": [{"role": "Theme", "text": "degradation", "start": 48, "end": 59}]}, {"trigger": {"text": "unaffected", "start": 239, "end": 249}, "arguments": [{"role": "Theme", "text": "IkappaBbeta", "start": 211, "end": 222}]}]}}, "schema": []}
{"input": "NF-kappaB and AP-1 are critical regulators of inflammatory responses, proliferation and differentiation of T-cells [28-30], however, the signal transduction and subsequent cytokine/chemokine expression is not fully understood. The aim of the present study was to investigate IL-6 and CXCL8 regulation by NF-kappaB and AP-1 in Jurkat T-cells.\nOur results demonstrated that PMA induced AP-1 activation, indicating a specific activation of the MAPK pathway. Furthermore, we demonstrated that PMA dependent AP-1 activation in T-cells was delayed (>2 h) and increased following long-term treatment. MAPK is one of the main signalling pathways in T-cells that regulate cell- and transcriptional activation [31,32]. Several studies [20,33,34] have indicated the importance of AP-1 in T-cell activation and the induction of inflammatory responses [35], including pro-inflammatory cytokine release. In contrast to AP-1, NF-kappaB activity rapidly increased (1 min) during PMA exposure followed by a down-regulation to the lowest levels at 6 h. This is in line with Park and colleagues [36] who demonstrated a rapid increase in NF-kappaB activity following short-term stimulation with PMA, but prolonged challenge resulted in a persistent inhibition of NF-kappaB. They showed that the inhibition of NF-kappaB was due to PKC-dependent degradation of IkappaB kinase beta and gamma in response to PMA. Interestingly, the HK E. coli exposure induced NF-kappaB activation without affecting AP-1 activity. Wang and colleagues [37] reported an elevated inflammatory response by obtaining expression of IL-6 following the exposure of T-cells to peptidoglycan. Reduction in NF-kappaB by calcium ionophore following HK E. coli stimulation may be due to the Ca2+ binding protein calmodulin (CaM), which has been shown to negatively regulate c-Rel when activated [38]. The regulation of NF-kappaB and AP-1 observed in the present study was in agreement with earlier studies.\nT-cells produce a broad range of pro- and anti-inflammatory cytokines, including IL-2, IL-6, CXCL8, TNF and IL-10, in response to infections or other stress factors [39]. The assessment of Jurkat T-cell inflammatory responses (Table 1) revealed an enhanced IL-2 expression upon exposure to PMA or HK E. coli due to PKC activation [40]. In addition to the transcription factor NF-kappaB, AP-1 binding sites have been identified in the IL-6 promoter region, indicating multiple regulation [22]. AP-1 and NF-kappaB have also been demonstrated to regulate CXCL8 expression during induction of inflammatory responses in T-cells [12]. Furthermore, IL-6 and CXCL8 gene-expression is associated with an early immune response in Jurkat T-cells [41]. In the present study, both PMA and HK E. coli resulted in comparable increases in IL-6 while PMA was more potent at activating CXCL8 release. PMA and HK E. coli treatment also induced TNF expression. TNF is one of the first cytokines induced by T-cells [42] and its expression is regulated by calcineurin, NFAT and ATF-2/Jun [20]. However, PMA-stimulated Jurkat T-cells showed no difference in IL-10 expression indicating an induced inflammatory response. HK E. coli treatment resulted in a significant reduction of IL-10 expression. The anti-inflammatory cytokine IL-10 is known to inhibit T-cell activation, proliferation and the expression of pro-inflammatory cytokines, such as IL-2, IL-5 and INF-gamma [39,43] and regulate inflammatory responses by inducing T-cell anergy [44].\nThe time course analysis of cytokine expression showed a correlation between AP-1 and the chemokine CXCL8 where CXCL8 expression was significantly elevated already at 2-6 h after PMA exposure. The CXCL8 expression did not correlate with the early NF-kappaB activation (1 min) or with the down-regulation of NF-kappaB at 2 h post exposure. Both IL-6 and TNF expression were up regulated between 6-24 h. During this period, NF-kappaB increased from its minimum level at 6 h. PKC has been shown to be associated with the activation of AP-1, but not with NF-kappaB activation of the IL-2 promoter [45]. Mutation of the NF-kappaB site did not affect IL-2 expression, whereas mutation of the AP-1 site or PKC depletion almost revoked IL-2 release. These observations indicate that the MAPK pathway and the transcription factor AP-1 play an important role in the induction of inflammatory responses in Jurkat T-cells [12,20,22]. The obtained results signify that CXCL8 was primarily regulated through the MAPK pathway.\nThe NF-kappaB activation inhibitor (NAI) showed specificity against NF-kappaB and resulted in a complete IL-6 inhibition following induction with PMA and significant IL-6 reduction after HK E. coli treatment. CXCL8 was highly up regulated by PMA and the addition of NAI resulted in a minor reduction in CXCL8 expression. Furthermore, CXCL8 expression was not affected by NAI following HK E. coli treatment, indicating a lack of correlation between CXCL8 and NF-kappaB. Further analysis of CXCL8 expression revealed a down-regulation by PKC- and JNK- inhibitors, suggesting an involvement of AP-1 via PKC and JNK, respectively. Analysis of gene expression further confirmed that il-6 and cxcl8 were upregulated by PMA. Expression of il-6 dropped below basal levels following inhibition of NF-kappaB and JNK whereas cxcl8 remained elevated above basal levels (16.5-fold and 7.1-fold respectively) following the same treatment. Furthermore, inhibition of PKC did not result in a significant decrease of il-6 or cxcl8, which is in accordance with protein data (figure 6). These results suggest that NF-kappaB is involved in IL-6 regulation and release while it is not required for the expression of CXCL8 in Jurkat T cells.\nThe transcription factor NF-kappaB is responsible for a rapid immune response which is followed by an increase in transcription of IkappaB thus inhibiting NF-kappaB [46]. Activation of NF-kappaB in Jurkat T-cells is dependent on Bcl10 activation, which in turn is regulated by PKC. Recent studies have established the importance of a protein complex consisting of CARMA1, Bcl10 and MALT1 (CBM), in the induction of NF-kappaB. Investigating Bcl10, Scharschmidt and colleagues [47] demonstrated that it is a critical regulator of NF-kappaB activity. Down-regulation of Bcl10 from signals transduced via the TCR/CD28 and PKC resulted in a concomitant down-regulation of NF-kappaB. They suggested that Bcl10 is initially activated by TCR/PKC but that continued activation (>1 h) promotes its degradation. Narayan and colleagues [27] suggested that deletion in any of the three CBM complex proteins impairs antigen-receptor dependent activation of NF-kappaB. They showed that NF-kappaB activation via Akt requires CARMA1 and acts in cooperation with PKC following short-term exposure (30 min) of Jurkat T-cells with PMA. Akt phosphorylates and thus activates Bcl10. These studies indicate that PKC is crucial for NF-kappaB activation following short-term treatment through signals via membrane bound receptors such as the TCR and the co-stimulatory receptor CD28. Thus, the CBM complex proteins play a key role in this signalling process. PMA diffuses into the cytosol and directly activates PKC since it is an analogue to diacylglycerol [48]. Several studies have demonstrated that inhibition of PKC blocks NF-kappaB and AP-1 activity, suggesting a direct regulation of these transcription factors by PKC [49,50]. PKC is activated at an early stage following T-cell stimulation and is therefore an important regulator of downstream inflammatory signalling pathways leading to cytokine expression [50]. We have shown that phosphorylated-PKC is up regulated in response to PMA and HK E. coli, indicating an association between PKC and the transcription factors AP-1 and NF-kappaB. Bcl10 levels were down-regulated following extended treatment of Jurkat T-cells with PMA. In the control groups, a loss of Bcl10 occurred after 6 h, followed by an increase after 24 h, which is in accordance with the observed NF-kappaB activity (figure 7). These results are supported by an earlier study [47], demonstrating that prolonged PKC activation by PMA leads to an inhibition of Bcl10 and NF-kappaB.\nIt has been shown that NF-kappaB is an important transcription factor complex involved in almost every aspect of cell regulation including apoptosis, differentiation, proliferation and initiation of immune responses [51-53]. NF-kappaB is constitutively active in many human malignancies, which makes it an attractive therapeutic target [54]. Elevated CXCL8 levels during chronic inflammation result in an enhanced recruitment of immune cells to the site of infection, which may lead to the development of autoimmune diseases following secretion of pro-inflammatory cytokines. 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{"input": "In the present study, IL-6 release was found to be associated with NF-kappaB activity while CXCL8 release more closely correlated with AP-1 activity. Treatment of Jurkat T-cells with PMA was more potent than HK E. coli at elevating the CXCL8 levels. PMA induced AP-1 activation and down-regulated NF-kappaB while HK E. coli up-regulated NF-kappaB without affecting AP-1 activity. In addition, the temporal induction pattern of AP-1 correlated to the release of CXCL8 while IL-6 followed the NF-kappaB activity. Likewise, blocking NF-kappaB activation resulted in a complete inhibition of IL-6 while the CXCL8 levels remained elevated as shown both at the protein and mRNA level. Furthermore, the CXCL8 release was down-regulated by inhibition of JNK activity. The present study indicates that in Jurkat T-cells, IL-6 is regulated through NF-kappaB while CXCL8 regulation is independent of NF-kappaB and closely associated with AP-1 activation.", "output": {"json_structures": {"localization": [{"trigger": {"text": "release", "start": 27, "end": 34}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 22, "end": 26}]}, {"trigger": {"text": "release", "start": 98, "end": 105}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 92, "end": 97}]}, {"trigger": {"text": "release", "start": 450, "end": 457}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 461, "end": 466}]}, {"trigger": {"text": "release", "start": 702, "end": 709}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 696, "end": 701}]}], "negative regulation": [{"trigger": {"text": "inhibition", "start": 574, "end": 584}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 588, "end": 592}]}, {"trigger": {"text": "remained", "start": 616, "end": 624}, "arguments": [{"role": "Theme", "text": "elevated", "start": 625, "end": 633}]}, {"trigger": {"text": "down-regulated", "start": 714, "end": 728}, "arguments": [{"role": "Theme", "text": "release", "start": 702, "end": 709}]}], "positive regulation": [{"trigger": {"text": "elevating", "start": 222, "end": 231}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 236, "end": 241}]}, {"trigger": {"text": "resulted", "start": 551, "end": 559}, "arguments": [{"role": "Theme", "text": "inhibition", "start": 574, "end": 584}]}, {"trigger": {"text": "elevated", "start": 625, "end": 633}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 603, "end": 608}]}], "regulation": [{"trigger": {"text": "regulated", "start": 820, "end": 829}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 812, "end": 816}]}, {"trigger": {"text": "regulation", "start": 860, "end": 870}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 854, "end": 859}]}, {"trigger": {"text": "independent", "start": 874, "end": 885}, "arguments": [{"role": "Theme", "text": "regulation", "start": 860, "end": 870}]}]}}, "schema": []}
{"input": "Chemicals\nThe following chemicals were used in the present study: PMA (Phorbol 12-myristate 13-acetate, (Sigma #P1585, USA)); NF-kappaB activation inhibitor (NAI), (InSolution(TM) NF-kappaB Activation Inhibitor, Calbiochem #481407, USA); JNK inhibitor, (InSolution(TM) JNK Inhibitor II, Calbiochem #420128, USA); PKC Inhibitor, (InSolution(TM) Bisindolylmaleimide I, Calbiochem #203293, USA); Calcium Ionophore, (Calcium Ionophore A23187 mixed calcium magnesium salt, Sigma #C5149, USA).", "output": {"json_structures": {}}, "schema": []}
{"input": "Heat killed (HK) Escherichia coli\nE. coli MG1655 were grown on Luria-Bertani (LB) agar and incubated at 37degreesC overnight. One colony was inoculated into 10 ml LB broth and incubated on a shaker (200 rpm) at 37degreesC overnight. The bacteria were centrifuged for 10 min at 3000 x g, washed with 3 ml phosphate buffered saline (PBS; 8 g NaCl, 1.16 g Na2HPO4, 0.2 g KH2PO4, 0.2 g KCl, pH7) and resuspended in 50 mul PBS. The bacteria were killed by heating to 70degreesC for 1 h. To ensure that the bacteria were killed; 10 mul of the heat-killed suspension was spread on a LB plate and incubated overnight at 37degreesC.", "output": {"json_structures": {}}, "schema": []}
{"input": "Cell culturing, transfection and stimulation\nJurkat T-cells (wild type and TCR deficient- TCR-/-) were maintained in 90% RPMI 1640 medium (PAA laboratories, Austria) with 1.5 mM L-glutamine (Invitrogen, USA), 10% foetal bovine serum (Invitrogen, USA) and 1% antibiotic-antimycotic (Invitrogen, USA) and incubated in a stable environment of 5% CO2 at 37degreesC.\nThe cells were centrifuged at 1000 x g for 8 min and resuspended in fresh media to a final cell density of 1.6 x 107 cells/ml in a 24-well plate. Reporter plasmid (pNFkappaB-Luc, pAP1 (PMA)-TA-Luc, pNFkappaB-SEAP), internal control plasmid (pRL) (Promega, USA) and lipofectamine 2000 (Invitrogen, USA) were added to each well at 0.54 mug/well, 0.06 mug/well and 1.5 mul/well, respectively. Initially, the reporter plasmid and pRL were mixed separately with OptiMEM (Gibco, USA). After 5 min of incubation at room temperature, lipofectamine 2000 was added and the mixture was incubated further for 20 min at room temperature. The transfection was allowed to proceed overnight at 37degreesC, after which, the cells were centrifuged, the media removed and fresh pre-warmed media added. The cells were pre-incubated with NF-kappaB, JNK and PKC inhibitors and stimulated in 24-well plates with different concentrations of PMA, HK E. coli MG1655 and Calcium Ionophore A23187.\nThe cells were lysed and luciferase activity (NF-kappaB and AP-1) was measured using the Dual-Luciferase(R) reporter assay system (Promega, USA) according to the manufacturer's instructions on a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). Secreted alkaline phosphatase (NFkappaB-SEAP, figure 4a, b) levels were measured using Great EscAPe(TM) SEAP Detection Kit (Clontech, USA).", "output": {"json_structures": {"positive regulation": [{"trigger": {"text": "activity", "start": 1368, "end": 1376}, "arguments": [{"role": "Theme", "text": "luciferase", "start": 1357, "end": 1367}]}]}}, "schema": []}
{"input": "Multiplex cytokine assay\nQuantification of the levels of cytokines IL-2, IL-6, IL-10 and TNF and the chemokine CXCL8 was performed on culture supernatants using multiplexed biomarker immunoassay kits according to manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA). A Bio-Plex(TM) 200 readout System was used (Bio-Rad), which utilizes Luminex(R) xMAP(TM) fluorescent bead-based technology (Luminex Corp., Austin). Levels were automatically calculated from standard curves using Bio-Plex Manager software (v.4.1.1, Bio-Rad).", "output": {"json_structures": {}}, "schema": []}
{"input": "Enzyme-linked immunosorbent assay (ELISA)\nELISA was performed on supernatants from challenged Jurkat T-cells to quantify IL-6, CXCL8 and TNF (BD OptEIA Human IL-6 Elisa Set, BD OptEIA Human CXCL8 Elisa Set and BD OptEIA Human TNF Elisa Set, Biosciences, USA) according to the manufacturer's instructions. Briefly, Jurkat T-cells were stimulated with PMA (162 nM) for 1 h, centrifuged (1000 x g, 8 min) and the supernatants were collected and stored at -80degreesC until use. Following centrifugation, the cells were resuspended in 1 h aged media, where cells have been grown in, containing PMA. The same procedure was performed to collect media after 2 h and 6 h. The final collection of media was performed after 24 h.", "output": {"json_structures": {}}, "schema": []}
{"input": "Western blot analysis\nFollowing stimulation, Jurkat T-cells were centrifuged at 1000 x g for 8 min and lysed on ice for 2 h using sodium hydroxide with the addition of a protease inhibitor cocktail (Roche, Mannheim). The cells were further centrifuged at 8000 x g, 4degreesC for 10 min and the supernatants were transferred to new tubes. Cytoplasmic proteins (~8 mug) were separated by SDS-PAGE (10%) followed by western blotting using anti-IkappaBbeta, phospho-PKC (pan)(zeta Thr410)(190D10), anti-Bcl10 (Cell Signalling Technology, Boston) and beta-actin (Abcam, Cambridge). Detection was performed following incubation with ECL(TM) Anti-rabbit IgG, horseradish peroxidase linked whole antibodies (Amersham Biosciences, Buckinghamshire) and developed using ECL(TM) Western Blotting Detection Reagents (GE Healthcare, UK).", "output": {"json_structures": {}}, "schema": []}
{"input": "RNA extraction\nJurkat T-cells were treated with NF-kappaB, JNK and PKC inhibitors for 2 h in 6-well plates followed by stimulation with 162 nM PMA for 24 h. At sampling the cells were pelleted followed by RNA extraction using 100 mul TRI-reagent (Sigma, USA). This was followed by addition of 100 mul chloroform/isoamylalcohol (24/1). The solutions were mixed by vortexing followed by centrifugation at 12,000 rpm for 15 min at 4degreesC. The upper phase was transferred to a new tube followed by addition of 100 mul iso-propanol and incubated at room temperature for 10 min. RNA was then pelleted by centrifugation at 12,000 rpm for 15 min at 4degreesC and washed with 70% ethanol. The RNA pellet was dissolved in 25 mul RNase free water and the yield and ratio (A260/A280) was determined using NanoVue (GE Healthcare, UK). The samples were stored at -80degreesC until further use.", "output": {"json_structures": {}}, "schema": []}
{"input": "Reverse transcription quantitative PCR (RT-qPCR)\nRT-qPCR was used to determine gene expression levels of il-6 and cxcl8 in response to PMA following inhibition of NF-kappaB, JNK and PKC. The following primer sequences were used, il-6: forward- TGTGAAAGCAGCAAAGAGGCACTG, reverse- ACAGCTCTGGCTTGTTCCTCACTA; cxcl8: forward- ACCACACTGCGCCAACACAGAAAT, reverse- AAACTTCTCCACAACCCTCTGCAC. Thermocycling conditions for CYBR Green (Quanta, USA) consisted of a denaturation step for 10 min at 95 degreesC followed by 60 cycles of 95degreesC for 1s and 60degreesC for 30s. Gene expression was analysed using Stratagene (Mx3000p(TM)) (AH diagnostics). The obtained Ct values were normalized against 18S. Initially, all measured 18S Ct values were used to calculate a mean Ct value that was used to determine the deltaCt values for each sample. Gene expression patterns for il-6 and cxcl8 were then normalized with regard to the samples 18S deltaCt.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 84, "end": 94}, "arguments": [{"role": "Theme", "text": "il-6", "start": 105, "end": 109}]}, {"trigger": {"text": "expression", "start": 84, "end": 94}, "arguments": [{"role": "Theme", "text": "cxcl8", "start": 114, "end": 119}]}, {"trigger": {"text": "expression", "start": 837, "end": 847}, "arguments": [{"role": "Theme", "text": "il-6", "start": 861, "end": 865}]}, {"trigger": {"text": "expression", "start": 837, "end": 847}, "arguments": [{"role": "Theme", "text": "cxcl8", "start": 870, "end": 875}]}], "positive regulation": [{"trigger": {"text": "in response to", "start": 120, "end": 134}, "arguments": [{"role": "Theme", "text": "expression", "start": 84, "end": 94}]}, {"trigger": {"text": "following", "start": 139, "end": 148}, "arguments": [{"role": "Theme", "text": "in response to", "start": 120, "end": 134}]}]}}, "schema": []}
{"input": "Statistical analysis\nStatistical significant differences were determined using two-tailed Student's t-test (*p < 0.05; **p < 0.01; ***p < 0.001).", "output": {"json_structures": {}}, "schema": []}
{"input": "AP-1 activation following long-term exposure of Jurkat T-cells to PMA. Jurkat T-cells were transfected with luciferase reporter plasmids containing the AP-1 cis-elements. (A) Time-dependent AP-1 activation in response to PMA. (B) HK E. coli does not activate AP-1. (C) Dose-dependent activation of AP-1 were performed using PMA alone (grey bars) and in combination with calcium ionophore (CaI 955 muM, black bars). (D) TCR-/- deficient cells responded to PMA (162 nM) by up-regulating AP-1 activity. Statistical significance from the control was determined using Student's t-test. (n = 4). Controls were arbitrarily set to 1.", "output": {"json_structures": {}}, "schema": []}
{"input": "NF-kappaB down-regulation by PMA and up-regulation by heat killed E. coli MG1655 following long-term stimulation. Transfection of Jurkat T-cells was performed using luciferase reporter plasmids containing NF-kappaB cis-elements. (A) Time-dependent stimulation of Jurkat T-cells using PMA. NF-kappaB activation was evaluated using HK E. coli in a (B) dose- and (C) time-dependant manner. Calcium ionophore increased NF-kappaB activity following PMA exposure (E) and resulted in a negative regulation in response to HK E. coli stimulation (D). Statistical significance from the control was determined using Student's t-test. (n = 4). Controls were arbitrarily set to 1.", "output": {"json_structures": {}}, "schema": []}
{"input": "CXCL8, IL-6 and TNF expression following long-term stimulation with PMA. Jurkat T-cells were either treated with media (white bars) or stimulated with PMA (black bars) and incubated for 1 h, 2 h, 6 h and 24 h. Aged media was added following each centrifugation step representative to the stimulation time (see Materials and methods). Cytokine levels (A) CXCL8, (B) IL-6 and (C) TNF were detected by ELISA. Statistical significance from the control was determined using Student's t-test. (n = 3).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 20, "end": 30}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 0, "end": 5}]}, {"trigger": {"text": "expression", "start": 20, "end": 30}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 7, "end": 11}]}], "positive regulation": [{"trigger": {"text": "following", "start": 31, "end": 40}, "arguments": [{"role": "Theme", "text": "expression", "start": 20, "end": 30}]}]}}, "schema": []}
{"input": "Inhibition of NF-kappaB activity by NAI. Jurkat T-cells were transfected with luciferase reporter plasmids containing either NF-kappaB or AP-1 cis-acting elements. The cells were incubated with NF-kappaB activation inhibitor (NAI) for 1 h followed by stimulation with PMA (162 nM, A and C) or HK E. coli (5 x 107 CFU/ml, B and D) for 24 h. (A, B) NF-kappaB, but not (C, D) AP-1 was inhibited. Statistical significance from the positive control (PMA/HK E. coli) was determined using Student's t-test. (n = 4). Controls were set to 1.", "output": {"json_structures": {}}, "schema": []}
{"input": "Involvement of NF-kappaB in cytokine regulation. Cytokine/chemokine levels were determined using ELISA following incubation of Jurkat T-cells with NAI (1 h) and stimulation (24 h). (A) CXCL8 expression was partially inhibited following PMA stimulation (grey bars), whereas the levels were not altered following stimulation with HK E. coli (black bars), this indicates an induction mainly regulated by AP-1. (B) TNF expression was not affected by NAI. (C) IL-6 release was completely inhibited by NAI following PMA exposure, indicating regulation through NF-kappaB since IL-6 expression was significantly increased in response to HK E. coli than PMA. Statistical significance from the positive control (PMA/HK E. coli) was determined using Student's t-test. (n = 3).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 191, "end": 201}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 185, "end": 190}]}, {"trigger": {"text": "expression", "start": 575, "end": 585}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 570, "end": 574}]}], "localization": [{"trigger": {"text": "release", "start": 460, "end": 467}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 455, "end": 459}]}], "negative regulation": [{"trigger": {"text": "inhibited", "start": 216, "end": 225}, "arguments": [{"role": "Theme", "text": "expression", "start": 191, "end": 201}]}, {"trigger": {"text": "inhibited", "start": 483, "end": 492}, "arguments": [{"role": "Theme", "text": "release", "start": 460, "end": 467}]}], "positive regulation": [{"trigger": {"text": "induction", "start": 371, "end": 380}, "arguments": [{"role": "Theme", "text": "expression", "start": 191, "end": 201}]}, {"trigger": {"text": "increased", "start": 604, "end": 613}, "arguments": [{"role": "Theme", "text": "expression", "start": 575, "end": 585}]}], "regulation": [{"trigger": {"text": "altered", "start": 293, "end": 300}, "arguments": [{"role": "Theme", "text": "expression", "start": 191, "end": 201}]}, {"trigger": {"text": "regulated", "start": 388, "end": 397}, "arguments": [{"role": "Theme", "text": "induction", "start": 371, "end": 380}]}, {"trigger": {"text": "regulation", "start": 535, "end": 545}, "arguments": [{"role": "Theme", "text": "release", "start": 460, "end": 467}]}]}}, "schema": []}
{"input": "Involvement of PKC and JNK in cytokine expression. Jurkat T-cells were incubated with PKC- and JNK inhibitors for 1 h followed by stimulation with PMA (162 nM) for 24 h. (A) CXCL8 expression was inhibited in a dose-dependent manner by both inhibitors. (B) JNK-I revealed a stronger inhibitory effect on IL-6 expression than the PKC-I. (C) TNF expression was not affected by either inhibitor. Statistical significance from the positive control (PMA) was determined using Student's t-test. (n = 3).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 180, "end": 190}, "arguments": [{"role": "Theme", "text": "CXCL8", "start": 174, "end": 179}]}, {"trigger": {"text": "expression", "start": 308, "end": 318}, "arguments": [{"role": "Theme", "text": "IL-6", "start": 303, "end": 307}]}], "negative regulation": [{"trigger": {"text": "inhibited", "start": 195, "end": 204}, "arguments": [{"role": "Theme", "text": "expression", "start": 180, "end": 190}]}, {"trigger": {"text": "inhibitory effect", "start": 282, "end": 299}, "arguments": [{"role": "Theme", "text": "expression", "start": 308, "end": 318}]}], "regulation": [{"trigger": {"text": "dependent", "start": 215, "end": 224}, "arguments": [{"role": "Theme", "text": "inhibited", "start": 195, "end": 204}]}]}}, "schema": []}
{"input": "NF-kappaB inhibition by PMA correlated to PKC-dependent Bcl10 degradation. Levels of intracellular protein were assessed following 24 h stimulation with PMA (162 nM) or HK E. coli (5 x 107 CFU/ml). (A) Phospho-PKC increased in response to PMA and HK E. coli stimulation. (B) IkappaB decreased following stimulation with HK E. coli indicating NF-kappaB activation. (C) Bcl10 activation was inhibited following long-term stimulation with PMA, which explains the inhibitory effect of PMA on NF-kappaB activation. beta-actin was used as a loading control. (n = 3).", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "inhibited", "start": 389, "end": 398}, "arguments": [{"role": "Theme", "text": "activation", "start": 374, "end": 384}]}], "positive regulation": [{"trigger": {"text": "activation", "start": 374, "end": 384}, "arguments": [{"role": "Theme", "text": "Bcl10", "start": 368, "end": 373}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 62, "end": 73}, "arguments": [{"role": "Theme", "text": "Bcl10", "start": 56, "end": 61}]}], "regulation": [{"trigger": {"text": "dependent", "start": 46, "end": 55}, "arguments": [{"role": "Theme", "text": "degradation", "start": 62, "end": 73}]}]}}, "schema": []}
{"input": "Jurkat T-cells were stimulated with 162 nM PMA and 5 x 107 CFU/ml HK E. coli for 24 h.", "output": {"json_structures": {}}, "schema": []}
{"input": "Jurkat T-cells were treated with 10 nM NAI, 10 muM JNK-I or10 nM PKC-I for 2 h followed by induction with 162 nM PMA for 24 h.", "output": {"json_structures": {}}, "schema": []}
{"input": "HOIL-1L Interacting Protein (HOIP) Is Essential for CD40 Signaling\nCD40 is a cell surface receptor important in the activation of antigen-presenting cells during immune responses. In macrophages and dendritic cells, engagement of CD40 by its ligand CD154 provides signals critical for anti-microbial and T cell-mediated immune responses, respectively. In B cells, CD40 signaling has a major role in regulating cell proliferation, antibody production, and memory B cell development. CD40 engagement results in the formation of a receptor-associated complex that mediates activation of NF-kappaB, stress-activated protein kinases, and other signaling molecules. However, the mechanisms that link CD40 to these signaling events have been only partially characterized. Known components of the CD40 signaling complex include members of the TNF receptor-associated factor (TRAF) family of proteins. We previously showed that the TRAF family member TRAF2 mediates recruitment of HOIL-1L-interacting protein (HOIP) to the cytoplasmic domain of CD40, suggesting that HOIP has a role in the CD40 signaling pathway. To determine the role of HOIP in CD40 signaling, we used somatic cell gene targeting to generate mouse B cell lines deficient in HOIP. We found that the CD40-induced upregulation of CD80 and activation of germline immunoglobulin epsilon transcription were defective in HOIP-deficient cells. We also found that the CD40-mediated activation of NF-kappaB and c-Jun kinase was impaired. Recruitment of IkappaB kinase proteins to the CD40 signaling complex was undetectable in HOIP-deficient cells, potentially explaining the defect in NF-kappaB activation. Restoration of HOIP expression reversed the defects in cellular activation and signaling. These results reveal HOIP as a key component of the CD40 signaling pathway.", "output": {"json_structures": {"binding": [{"trigger": {"text": "engagement", "start": 216, "end": 226}, "arguments": [{"role": "Theme", "text": "CD40", "start": 230, "end": 234}, {"role": "Theme2", "text": "CD154", "start": 249, "end": 254}]}, {"trigger": {"text": "engagement", "start": 487, "end": 497}, "arguments": [{"role": "Theme", "text": "CD40", "start": 482, "end": 486}]}, {"trigger": {"text": "recruitment", "start": 957, "end": 968}, "arguments": [{"role": "Theme", "text": "HOIL-1L-interacting protein", "start": 972, "end": 999}, {"role": "Site2", "text": "cytoplasmic domain", "start": 1014, "end": 1032}, {"role": "Theme2", "text": "CD40", "start": 1036, "end": 1040}]}], "gene expression": [{"trigger": {"text": "expression", "start": 1678, "end": 1688}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1673, "end": 1677}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 1221, "end": 1230}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1234, "end": 1238}]}, {"trigger": {"text": "defective", "start": 1361, "end": 1370}, "arguments": [{"role": "Theme", "text": "induced", "start": 1263, "end": 1270}]}, {"trigger": {"text": "deficient", "start": 1379, "end": 1388}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1374, "end": 1378}]}, {"trigger": {"text": "deficient", "start": 1582, "end": 1591}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1577, "end": 1581}]}], "positive regulation": [{"trigger": {"text": "mediates", "start": 948, "end": 956}, "arguments": [{"role": "Cause", "text": "TRAF2", "start": 942, "end": 947}, {"role": "Theme", "text": "recruitment", "start": 957, "end": 968}]}, {"trigger": {"text": "induced", "start": 1263, "end": 1270}, "arguments": [{"role": "Cause", "text": "CD40", "start": 1258, "end": 1262}, {"role": "Theme", "text": "upregulation", "start": 1271, "end": 1283}]}, {"trigger": {"text": "upregulation", "start": 1271, "end": 1283}, "arguments": [{"role": "Theme", "text": "CD80", "start": 1287, "end": 1291}]}, {"trigger": {"text": "Restoration", "start": 1658, "end": 1669}, "arguments": [{"role": "Theme", "text": "expression", "start": 1678, "end": 1688}]}]}}, "schema": []}
{"input": "CD40 signaling in professional antigen-presenting cells, including B cells, macrophages, and dendritic cells, is critical for the efficient activation of humoral and cell-mediated immune responses [1], [2], [3]. CD40 signaling is activated in a T cell-dependent manner, as the ligand for CD40, CD154, is expressed primarily by activated T cells. CD40 engagement leads to the activation of various signaling molecules, including stress-activated protein kinases and the transcription factor NF-kappaB, which upregulate the expression of cytokines and other factors that promote immune responses. The mechanism by which CD40 induces these signaling pathways has not been completely defined. The cytoplasmic domain of CD40 does not appear to have intrinsic enzymatic activity, but is able to mediate signaling through the recruitment of several intracellular proteins. Members of the TNF receptor-associated factor (TRAF) family, including TRAF1, TRAF2, TRAF3, and TRAF6, appear to be particularly important for the initiation and regulation of CD40 signaling [4]. These proteins function in part as adaptor molecules, binding to the cytoplasmic tail of CD40 and recruiting other proteins to the receptor-associated complex. Some of the TRAFs also function as E3 ubiquitin ligases, and this enzymatic activity may contribute to signal propagation and regulation. Among the multiple TRAFs that associate with CD40, TRAF3 can function as a negative regulator of signaling, while TRAF2 and TRAF6 promote the activation of downstream signaling pathways [4].\nWe recently demonstrated that HOIL-1L interacting protein (HOIP), a ubiquitin ligase that can catalyze the assembly of linear polyubiquitin chains [5], is recruited to CD40 in a TRAF2-dependent manner following engagement of CD40 by agonistic antibody [6]. These and other findings led us to hypothesize that HOIP functions downstream of TRAF2 in the CD40 signaling pathway and that HOIP is necessary for the activation of NF-kappaB and possibly other signaling molecules. To test this hypothesis, we employed somatic cell gene targeting to ablate expression of HOIP in a mouse B cell line that has proven to be a useful model for B cell CD40 signaling [7], [8], [9]. We found that the CD40-induced upregulation of CD80 (a costimulatory molecule for T cells) was defective in HOIP-deficient cells. Similarly, the CD40 and IL-4 driven production of germline transcripts from the immunoglobulin epsilon heavy chain locus, an event that precedes immunoglobulin gene rearrangement and isotype switching, was defective in the absence of HOIP. We also found that the CD40-mediated activation of NF-kappaB and the stress-activated protein kinase c-Jun kinase (JNK) was defective in HOIP-deficient cells. Consistent with impaired NF-kappaB activation, association of the IkappaB kinase (IKK) complex with CD40 was undetectable in HOIP-deficient cells. Together, our results indicate that HOIP plays a critical role in the activation of signaling pathways that regulate cellular responses to CD40 engagement.", "output": {"json_structures": {"binding": [{"trigger": {"text": "engagement", "start": 351, "end": 361}, "arguments": [{"role": "Theme", "text": "CD40", "start": 346, "end": 350}]}, {"trigger": {"text": "recruitment", "start": 819, "end": 830}, "arguments": [{"role": "Site", "text": "cytoplasmic domain", "start": 693, "end": 711}, {"role": "Theme", "text": "CD40", "start": 715, "end": 719}]}, {"trigger": {"text": "binding", "start": 1116, "end": 1123}, "arguments": [{"role": "Theme", "text": "TRAF1", "start": 937, "end": 942}, {"role": "Site2", "text": "cytoplasmic tail", "start": 1131, "end": 1147}, {"role": "Theme2", "text": "CD40", "start": 1151, "end": 1155}]}, {"trigger": {"text": "binding", "start": 1116, "end": 1123}, "arguments": [{"role": "Theme", "text": "TRAF2", "start": 944, "end": 949}, {"role": "Site2", "text": "cytoplasmic tail", "start": 1131, "end": 1147}, {"role": "Theme2", "text": "CD40", "start": 1151, "end": 1155}]}, {"trigger": {"text": "binding", "start": 1116, "end": 1123}, "arguments": [{"role": "Theme", "text": "TRAF3", "start": 951, "end": 956}, {"role": "Site2", "text": "cytoplasmic tail", "start": 1131, "end": 1147}, {"role": "Theme2", "text": "CD40", "start": 1151, "end": 1155}]}, {"trigger": {"text": "binding", "start": 1116, "end": 1123}, "arguments": [{"role": "Theme", "text": "TRAF6", "start": 962, "end": 967}, {"role": "Site2", "text": "cytoplasmic tail", "start": 1131, "end": 1147}, {"role": "Theme2", "text": "CD40", "start": 1151, "end": 1155}]}, {"trigger": {"text": "associate", "start": 1390, "end": 1399}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1405, "end": 1409}, {"role": "Theme2", "text": "TRAF3", "start": 1411, "end": 1416}]}, {"trigger": {"text": "associate", "start": 1390, "end": 1399}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1405, "end": 1409}, {"role": "Theme2", "text": "TRAF2", "start": 1474, "end": 1479}]}, {"trigger": {"text": "associate", "start": 1390, "end": 1399}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1405, "end": 1409}, {"role": "Theme2", "text": "TRAF6", "start": 1484, "end": 1489}]}, {"trigger": {"text": "recruited", "start": 1706, "end": 1715}, "arguments": [{"role": "Theme", "text": "HOIL-1L interacting protein", "start": 1581, "end": 1608}, {"role": "Theme2", "text": "CD40", "start": 1719, "end": 1723}]}, {"trigger": {"text": "engagement", "start": 1762, "end": 1772}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1776, "end": 1780}]}, {"trigger": {"text": "association", "start": 2795, "end": 2806}, "arguments": [{"role": "Theme", "text": "CD40", "start": 2848, "end": 2852}]}, {"trigger": {"text": "engagement", "start": 3039, "end": 3049}, "arguments": [{"role": "Theme", "text": "CD40", "start": 3034, "end": 3038}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 304, "end": 313}, "arguments": [{"role": "Theme", "text": "CD154", "start": 294, "end": 299}]}, {"trigger": {"text": "expression", "start": 2099, "end": 2109}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2113, "end": 2117}]}], "negative regulation": [{"trigger": {"text": "ablate", "start": 2092, "end": 2098}, "arguments": [{"role": "Theme", "text": "expression", "start": 2099, "end": 2109}]}, {"trigger": {"text": "defective", "start": 2314, "end": 2323}, "arguments": [{"role": "Theme", "text": "induced", "start": 2242, "end": 2249}]}, {"trigger": {"text": "deficient", "start": 2332, "end": 2341}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2327, "end": 2331}]}, {"trigger": {"text": "absence", "start": 2572, "end": 2579}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2583, "end": 2587}]}, {"trigger": {"text": "deficient", "start": 2731, "end": 2740}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2726, "end": 2730}]}, {"trigger": {"text": "deficient", "start": 2878, "end": 2887}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2873, "end": 2877}]}], "positive regulation": [{"trigger": {"text": "following", "start": 1752, "end": 1761}, "arguments": [{"role": "Theme", "text": "dependent", "start": 1735, "end": 1744}, {"role": "Cause", "text": "engagement", "start": 1762, "end": 1772}]}, {"trigger": {"text": "functions downstream", "start": 1865, "end": 1885}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 1860, "end": 1864}, {"role": "Theme", "text": "TRAF2", "start": 1889, "end": 1894}]}, {"trigger": {"text": "induced", "start": 2242, "end": 2249}, "arguments": [{"role": "Cause", "text": "CD40", "start": 2237, "end": 2241}, {"role": "Theme", "text": "upregulation", "start": 2250, "end": 2262}]}, {"trigger": {"text": "upregulation", "start": 2250, "end": 2262}, "arguments": [{"role": "Theme", "text": "CD80", "start": 2266, "end": 2270}]}], "regulation": [{"trigger": {"text": "dependent", "start": 1735, "end": 1744}, "arguments": [{"role": "Theme", "text": "recruited", "start": 1706, "end": 1715}, {"role": "Cause", "text": "TRAF2", "start": 1729, "end": 1734}]}]}}, "schema": []}
{"input": "Generation of HOIP-deficient B cells via targeted disruption of Rnf31, the gene encoding HOIP\nIn our previous study, we demonstrated that HOIP is recruited to the CD40 signaling complex in two mouse B cell lines [6]. Similar results were obtained with mouse splenocytes, indicating that the interaction of CD40 with HOIP is not limited to transformed B cell lines (unpublished observations). We also found that the recruitment of HOIP to CD40 was TRAF2-dependent and that overexpression of a truncated HOIP mutant partially inhibited CD40-mediated NF-kappaB activation. These results support the hypothesis that HOIP plays a role in CD40 signal transduction. To further define the role and evaluate the importance of HOIP in CD40 signaling, we used somatic cell gene targeting to disrupt the gene encoding HOIP in the mouse B cell line A20.2J. This cell line has been particularly useful in the characterization of CD40 signaling mechanisms due to the relative ease with which its genome can be modified by homologous recombination [7], [8], [9]. We used a targeting vector capable of undergoing homologous recombination with Rnf31 (the gene encoding HOIP) to disrupt the coding sequence of the gene in exon 5 (Fig. 1A). Following introduction of the vector, the neomycin-resistant clones that arose were screened by PCR amplification of genomic DNA to identify cells containing a disrupted Rnf31 allele. To remove the selectable marker gene cassette from the disrupted Rnf31 allele, recombinant cell lines were transiently transfected with a plasmid that encodes Cre recombinase. This step allowed us to perform a second round of targeting and drug selection, generating cells in which both copies of Rnf31 were disrupted. Two independent clonal cell lines were chosen for further analysis. HOIP protein expression was undetectable in both cell lines, as determined by Western blot analysis of cell lysates (Fig. 1B), demonstrating that the targeting process was successful. In the text that follows, the two gene-targeted cell lines are referred to as HOIP-deficient cells.\nTo enable us to confirm that any signaling or functional defects observed in HOIP-deficient cells were due to disruption of the Rnf31 gene, both HOIP-deficient cell lines were transduced with a retrovirus encoding FLAG-tagged wild-type HOIP, thus restoring HOIP protein expression (Fig. 1B). These cells are referred to as HOIP-reconstituted cells. In the experiments that follow, responses by these cells were compared to those of A20.2J cells and HOIP-deficient cells transduced with a retroviral vector (pMIP) lacking a cDNA insert (empty vector).", "output": {"json_structures": {"binding": [{"trigger": {"text": "recruited", "start": 146, "end": 155}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 138, "end": 142}]}, {"trigger": {"text": "interaction", "start": 291, "end": 302}, "arguments": [{"role": "Theme", "text": "CD40", "start": 306, "end": 310}, {"role": "Theme2", "text": "HOIP", "start": 316, "end": 320}]}, {"trigger": {"text": "recruitment", "start": 415, "end": 426}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 430, "end": 434}, {"role": "Theme2", "text": "CD40", "start": 438, "end": 442}]}], "gene expression": [{"trigger": {"text": "expression", "start": 1805, "end": 1815}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1792, "end": 1796}]}, {"trigger": {"text": "expression", "start": 2346, "end": 2356}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2333, "end": 2337}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 19, "end": 28}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 14, "end": 18}]}, {"trigger": {"text": "disruption", "start": 50, "end": 60}, "arguments": [{"role": "Theme", "text": "Rnf31", "start": 64, "end": 69}]}, {"trigger": {"text": "disrupt", "start": 780, "end": 787}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 806, "end": 810}]}, {"trigger": {"text": "disrupted", "start": 1381, "end": 1390}, "arguments": [{"role": "Theme", "text": "Rnf31", "start": 1391, "end": 1396}]}, {"trigger": {"text": "disrupted", "start": 1460, "end": 1469}, "arguments": [{"role": "Theme", "text": "Rnf31", "start": 1470, "end": 1475}]}, {"trigger": {"text": "disrupted", "start": 1713, "end": 1722}, "arguments": [{"role": "Theme", "text": "Rnf31", "start": 1702, "end": 1707}]}, {"trigger": {"text": "deficient", "start": 2059, "end": 2068}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2054, "end": 2058}]}, {"trigger": {"text": "deficient", "start": 2158, "end": 2167}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2153, "end": 2157}]}, {"trigger": {"text": "disruption", "start": 2186, "end": 2196}, "arguments": [{"role": "Theme", "text": "Rnf31", "start": 2204, "end": 2209}]}, {"trigger": {"text": "deficient", "start": 2226, "end": 2235}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2221, "end": 2225}]}, {"trigger": {"text": "deficient", "start": 2530, "end": 2539}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2525, "end": 2529}]}], "positive regulation": [{"trigger": {"text": "restoring", "start": 2323, "end": 2332}, "arguments": [{"role": "Theme", "text": "expression", "start": 2346, "end": 2356}]}, {"trigger": {"text": "reconstituted", "start": 2404, "end": 2417}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2399, "end": 2403}]}], "regulation": [{"trigger": {"text": "dependent", "start": 453, "end": 462}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 415, "end": 426}, {"role": "Cause", "text": "TRAF2", "start": 447, "end": 452}]}]}}, "schema": []}
{"input": "HOIP is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\nCD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLepsilon) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLepsilon transcription in parental A20.2J cells as expected. In contrast, only low levels of GLepsilon expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLepsilon expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.", "output": {"json_structures": {"binding": [{"trigger": {"text": "Engagement", "start": 102, "end": 112}, "arguments": [{"role": "Theme", "text": "CD40", "start": 116, "end": 120}]}, {"trigger": {"text": "engagement", "start": 386, "end": 396}, "arguments": [{"role": "Theme", "text": "CD40", "start": 400, "end": 404}]}], "gene expression": [{"trigger": {"text": "expression", "start": 144, "end": 154}, "arguments": [{"role": "Theme", "text": "CD80", "start": 158, "end": 162}]}, {"trigger": {"text": "expression", "start": 1843, "end": 1853}, "arguments": [{"role": "Theme", "text": "GLepsilon", "start": 1833, "end": 1842}]}, {"trigger": {"text": "expression", "start": 1990, "end": 2000}, "arguments": [{"role": "Theme", "text": "GLepsilon", "start": 1980, "end": 1989}]}], "negative regulation": [{"trigger": {"text": "reduced", "start": 574, "end": 581}, "arguments": [{"role": "Theme", "text": "upregulation", "start": 536, "end": 548}, {"role": "Cause", "text": "deficient", "start": 594, "end": 603}]}, {"trigger": {"text": "deficient", "start": 594, "end": 603}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 589, "end": 593}]}, {"trigger": {"text": "defect", "start": 714, "end": 720}, "arguments": [{"role": "Theme", "text": "upregulation", "start": 729, "end": 741}, {"role": "Cause", "text": "deficient", "start": 754, "end": 763}]}, {"trigger": {"text": "deficient", "start": 754, "end": 763}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 749, "end": 753}]}, {"trigger": {"text": "disruption", "start": 785, "end": 795}, "arguments": [{"role": "Theme", "text": "Rnf31", "start": 799, "end": 804}]}, {"trigger": {"text": "deficient", "start": 1876, "end": 1885}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1871, "end": 1875}]}], "positive regulation": [{"trigger": {"text": "required", "start": 8, "end": 16}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 0, "end": 4}, {"role": "Theme", "text": "mediated", "start": 26, "end": 34}]}, {"trigger": {"text": "mediated", "start": 26, "end": 34}, "arguments": [{"role": "Theme", "text": "upregulation", "start": 40, "end": 52}]}, {"trigger": {"text": "mediated", "start": 26, "end": 34}, "arguments": [{"role": "Theme", "text": "activation", "start": 57, "end": 67}]}, {"trigger": {"text": "upregulation", "start": 40, "end": 52}, "arguments": [{"role": "Theme", "text": "CD80", "start": 35, "end": 39}]}, {"trigger": {"text": "activation", "start": 57, "end": 67}, "arguments": [{"role": "Theme", "text": "transcription", "start": 88, "end": 101}]}, {"trigger": {"text": "upregulates", "start": 132, "end": 143}, "arguments": [{"role": "Cause", "text": "Engagement", "start": 102, "end": 112}, {"role": "Theme", "text": "expression", "start": 144, "end": 154}]}, {"trigger": {"text": "upregulation", "start": 354, "end": 366}, "arguments": [{"role": "Theme", "text": "CD80", "start": 370, "end": 374}]}, {"trigger": {"text": "induced", "start": 375, "end": 382}, "arguments": [{"role": "Theme", "text": "upregulation", "start": 354, "end": 366}, {"role": "Cause", "text": "engagement", "start": 386, "end": 396}]}, {"trigger": {"text": "reconstituted", "start": 450, "end": 463}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 445, "end": 449}]}, {"trigger": {"text": "stimulated", "start": 525, "end": 535}, "arguments": [{"role": "Cause", "text": "CD40", "start": 520, "end": 524}, {"role": "Theme", "text": "upregulation", "start": 536, "end": 548}]}, {"trigger": {"text": "upregulation", "start": 536, "end": 548}, "arguments": [{"role": "Theme", "text": "CD80", "start": 552, "end": 556}]}, {"trigger": {"text": "upregulation", "start": 631, "end": 643}, "arguments": [{"role": "Theme", "text": "CD80", "start": 647, "end": 651}]}, {"trigger": {"text": "reconstituted", "start": 673, "end": 686}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 668, "end": 672}]}, {"trigger": {"text": "upregulation", "start": 729, "end": 741}, "arguments": [{"role": "Theme", "text": "CD80", "start": 724, "end": 728}]}, {"trigger": {"text": "due", "start": 774, "end": 777}, "arguments": [{"role": "Theme", "text": "defect", "start": 714, "end": 720}, {"role": "Cause", "text": "disruption", "start": 785, "end": 795}]}, {"trigger": {"text": "induced", "start": 1731, "end": 1738}, "arguments": [{"role": "Theme", "text": "transcription", "start": 1749, "end": 1762}]}, {"trigger": {"text": "induced", "start": 2006, "end": 2013}, "arguments": [{"role": "Theme", "text": "expression", "start": 1990, "end": 2000}]}, {"trigger": {"text": "reconstituted", "start": 2022, "end": 2035}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2017, "end": 2021}]}], "transcription": [{"trigger": {"text": "transcription", "start": 88, "end": 101}, "arguments": [{"role": "Theme", "text": "germline epsilon", "start": 71, "end": 87}]}, {"trigger": {"text": "production", "start": 1607, "end": 1617}, "arguments": [{"role": "Theme", "text": "germline epsilon", "start": 1621, "end": 1637}]}, {"trigger": {"text": "transcription", "start": 1749, "end": 1762}, "arguments": [{"role": "Theme", "text": "GLepsilon", "start": 1739, "end": 1748}]}]}}, "schema": []}
{"input": "HOIP mediates CD40-stimulated NF-kappaB and JNK activation\nThe results described above show that HOIP plays an important role in CD40-mediated effector functions of B cells. It follows that HOIP is likely a key mediator of CD40 signaling. To test this hypothesis, we stimulated A20.2J and HOIP-deficient cells with CD154 (CD40 ligand) expressed by HI5 insect cells [7], [8] and measured activation of the NF-kappaB and JNK pathways, two of the major transcriptional regulators activated by CD40 [4]. Cell-associated CD154 was used as the stimulus in these experiments as it typically provides more robust, and therefore more readily detected, activation signals than does anti-CD40 antibody. Activation of the canonical NF-kappaB pathway is initiated with the phosphorylation of IkappaB proteins by the IkappaB kinase complex (IKK). In resting cells, IkappaB proteins are responsible for sequestering NF-kappaB subunits in the cytoplasm. Phosphorylation by the IKK complex targets IkappaB proteins for ubiquitination and degradation, allowing NF-kappaB to enter the nucleus and activate gene expression. CD40-mediated phosphorylation and degradation of IkappaBalpha in HOIP-deficient cells was dramatically impaired relative to that observed in parental A20.2J cells (Fig. 4). We also assayed activation of the stress-activated protein kinase JNK in response to CD40 engagement (Fig. 4). CD40-mediated JNK activation in HOIP-deficient cells was impaired as measured by phosphorylation of Thr183 and Tyr185 in JNK. CD40-induced activation of NF-kappaB and JNK in HOIP-reconstituted cells was normal, demonstrating that the defects observed in gene-deficient cells were due to the absence of HOIP expression.", "output": {"json_structures": {"binding": [{"trigger": {"text": "associated", "start": 505, "end": 515}, "arguments": [{"role": "Theme", "text": "CD154", "start": 516, "end": 521}]}, {"trigger": {"text": "engagement", "start": 1367, "end": 1377}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1362, "end": 1366}]}], "gene expression": [{"trigger": {"text": "expressed", "start": 335, "end": 344}, "arguments": [{"role": "Theme", "text": "CD154", "start": 315, "end": 320}]}, {"trigger": {"text": "expression", "start": 1695, "end": 1705}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1690, "end": 1694}]}], "negative regulation": [{"trigger": {"text": "sequestering", "start": 888, "end": 900}, "arguments": [{"role": "Theme", "text": "NF-kappaB subunits", "start": 901, "end": 919}]}, {"trigger": {"text": "deficient", "start": 1174, "end": 1183}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1169, "end": 1173}]}, {"trigger": {"text": "impaired", "start": 1207, "end": 1215}, "arguments": [{"role": "Theme", "text": "mediated", "start": 1109, "end": 1117}]}, {"trigger": {"text": "deficient", "start": 1425, "end": 1434}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1420, "end": 1424}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1118, "end": 1133}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1153, "end": 1165}]}], "positive regulation": [{"trigger": {"text": "mediated", "start": 1109, "end": 1117}, "arguments": [{"role": "Cause", "text": "CD40", "start": 1104, "end": 1108}, {"role": "Theme", "text": "phosphorylation", "start": 1118, "end": 1133}]}, {"trigger": {"text": "mediated", "start": 1109, "end": 1117}, "arguments": [{"role": "Cause", "text": "CD40", "start": 1104, "end": 1108}, {"role": "Theme", "text": "degradation", "start": 1138, "end": 1149}]}, {"trigger": {"text": "reconstituted", "start": 1567, "end": 1580}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1562, "end": 1566}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 1138, "end": 1149}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1153, "end": 1165}]}], "regulation": [{"trigger": {"text": "responsible", "start": 872, "end": 883}, "arguments": [{"role": "Theme", "text": "sequestering", "start": 888, "end": 900}]}]}}, "schema": []}
{"input": "HOIP is necessary for association of the IKK complex with CD40\nThe marked defects in CD40-mediated cell activation and signaling displayed by HOIP-deficient cells suggested that HOIP mediates recruitment of critical components of the CD40 signaling apparatus to the receptor. Previously, we demonstrated that HOIP is recruited to the CD40 signaling complex in a TRAF2-dependent manner, suggesting that HOIP functions downstream of TRAF2 [6]. Studies by others suggest that the TRAF2-associated proteins cIAP1 and cIAP2 play a role in the recruitment of HOIP to TNFR1 and CD40 [14]. Therefore, we determined whether the association of HOIP with CD40 in A20.2J cells was altered by treatment of cells with an inhibitor of cIAP activity, a membrane-permeable peptide derived from the apoptosis regulator SMAC [15]. We found that pretreatment of cells with the SMAC peptide dramatically reduced the amount of cIAP1 associated with the CD40 signaling complex in cells stimulated with anti-CD40 antibody-coated beads (Fig. 5A). SMAC peptide treatment also resulted in a slight but reproducible decrease in the amount of the major HOIP form recovered by CD40 immunoprecipitation, along with an apparent increase in higher molecular weight species recognized by anti-HOIP antibody. In contrast, treatment with the SMAC peptide did not alter the amount or molecular weight of HOIP present in cell lysates. These data suggest that SMAC peptide treatment specifically alters the characteristics of CD40-associated HOIP rather than the entire cellular pool of this protein. Together, these results support the idea that the cIAP proteins influence the recruitment and post-translational modification state of CD40-associated HOIP.\nTo test the possibility that HOIP is responsible for the recruitment of other critical signaling proteins to CD40, we immunoprecipitated CD40 signaling complexes from HOIP-deficient and HOIP-reconstituted cells (Fig. 5B). As described previously, CD40 signaling complexes immunoprecipitated from A20.2J cells contained HOIP as well as TRAF2, TRAF3, IKKalpha/beta, and IKKgamma [6]. The amounts of TRAF2, TRAF3, and cIAP1 in CD40 immunoprecipitates from HOIP-deficient cells were similar to those from parental A20.2J cells, indicating that HOIP is not required for the association of these proteins with CD40. In contrast, IKKalpha/beta and IKKgamma were not detectable in CD40 immunoprecipitates recovered from HOIP-deficient cells (Fig. 5B,C). The amounts of IKKalpha/beta and IKKgamma in CD40 immunoprecipitates from HOIP-reconstituted cells were similar to those detected in samples prepared from parental cells, demonstrating that the defects in IKK recruitment we observed were due specifically to the absence of HOIP expression. These data indicate that HOIP is required for recruitment of the NF-kappaB-activating complex to the CD40 signaling complex.\nAs shown here and in our previous study [6], IKKgamma present in CD40 immunoprecipitates appears to have a higher molecular weight than that found in cell lysates. To confirm that this higher molecular weight species was indeed IKKgamma, we generated A20.2J cell lines containing a stably integrated retroviral vector that encoded an epitope-tagged version of mouse IKKgamma. Cell lysates and CD40 immunoprecipitates prepared from these cells were analyzed by Western blotting for the epitope tag (Fig. 5C). This analysis produced a pattern of bands that was essentially the same as that obtained using an antibody specific for native IKKgamma (compare panels B and C in Fig. 5). Moreover, analysis of cells expressing epitope tagged-IKKgamma confirmed that recruitment of IKKgamma to CD40 requires HOIP. To determine what was responsible for the increased molecular weight of IKKgamma in CD40 immunoprecipitates, the protein samples were incubated with lambda phosphatase, which removes phosphates attached to tyrosine, threonine, or serine residues in proteins. This treatment reduced much of CD40-associated IKKgamma to an apparent molecular weight similar to that of the protein in cell lysates (Fig. 5C). However, at least one band of significantly higher molecular weight remained after phosphatase treatment, suggesting that CD40-associated IKKgamma is subject to at least one other modification in addition to phosphorylation. Overall, these data demonstrate that HOIP is required for the association of IKKgamma with CD40, and suggest that this event is coupled to post-translational modifications of IKKgamma.", "output": {"json_structures": {"binding": [{"trigger": {"text": "association", "start": 22, "end": 33}, "arguments": [{"role": "Theme", "text": "CD40", "start": 58, "end": 62}]}, {"trigger": {"text": "recruited", "start": 317, "end": 326}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 309, "end": 313}]}, {"trigger": {"text": "associated", "start": 483, "end": 493}, "arguments": [{"role": "Theme", "text": "TRAF2", "start": 477, "end": 482}, {"role": "Theme2", "text": "cIAP1", "start": 503, "end": 508}]}, {"trigger": {"text": "associated", "start": 483, "end": 493}, "arguments": [{"role": "Theme", "text": "TRAF2", "start": 477, "end": 482}, {"role": "Theme2", "text": "cIAP2", "start": 513, "end": 518}]}, {"trigger": {"text": "recruitment", "start": 538, "end": 549}, "arguments": [{"role": 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1128}]}, {"trigger": {"text": "associated", "start": 1492, "end": 1502}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1487, "end": 1491}, {"role": "Theme2", "text": "HOIP", "start": 1503, "end": 1507}]}, {"trigger": {"text": "recruitment", "start": 1640, "end": 1651}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1713, "end": 1717}]}, {"trigger": {"text": "associated", "start": 1702, "end": 1712}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1697, "end": 1701}, {"role": "Theme2", "text": "HOIP", "start": 1713, "end": 1717}]}, {"trigger": {"text": "recruitment", "start": 1776, "end": 1787}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1828, "end": 1832}]}, {"trigger": {"text": "association", "start": 2288, "end": 2299}, "arguments": [{"role": "Theme", "text": "TRAF2", "start": 2116, "end": 2121}, {"role": "Theme2", "text": "CD40", "start": 2323, "end": 2327}]}, {"trigger": {"text": "association", "start": 2288, "end": 2299}, "arguments": [{"role": "Theme", "text": "TRAF3", "start": 2123, "end": 2128}, {"role": "Theme2", "text": "CD40", "start": 2323, "end": 2327}]}, {"trigger": {"text": "association", "start": 2288, "end": 2299}, "arguments": [{"role": "Theme", "text": "cIAP1", "start": 2134, "end": 2139}, {"role": "Theme2", "text": "CD40", "start": 2323, "end": 2327}]}, {"trigger": {"text": "recruitment", "start": 3638, "end": 3649}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 3653, "end": 3661}, {"role": "Theme2", "text": "CD40", "start": 3665, "end": 3669}]}, {"trigger": {"text": "associated", "start": 3980, "end": 3990}, "arguments": [{"role": "Theme", "text": "CD40", "start": 3975, "end": 3979}, {"role": "Theme2", "text": "IKKgamma", "start": 3991, "end": 3999}]}, {"trigger": {"text": "associated", "start": 4217, "end": 4227}, "arguments": [{"role": "Theme", "text": "CD40", "start": 4212, "end": 4216}, {"role": "Theme2", "text": "IKKgamma", "start": 4228, "end": 4236}]}, {"trigger": {"text": "association", "start": 4377, "end": 4388}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 4392, "end": 4400}, {"role": "Theme2", "text": "CD40", "start": 4406, "end": 4410}]}], "gene expression": [{"trigger": {"text": "expression", "start": 2743, "end": 2753}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2738, "end": 2742}]}, {"trigger": {"text": "expressing", "start": 3588, "end": 3598}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 3614, "end": 3622}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 147, "end": 156}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 142, "end": 146}]}, {"trigger": {"text": "reduced", "start": 883, "end": 890}, "arguments": [{"role": "Theme", "text": "associated", "start": 911, "end": 921}]}, {"trigger": {"text": "decrease", "start": 1088, "end": 1096}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1124, "end": 1128}]}, {"trigger": {"text": "deficient", "start": 1891, "end": 1900}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1886, "end": 1890}]}, {"trigger": {"text": "deficient", "start": 2436, "end": 2445}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2431, "end": 2435}]}, {"trigger": {"text": "absence", "start": 2727, "end": 2734}, "arguments": [{"role": "Theme", "text": "expression", "start": 2743, "end": 2753}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 4298, "end": 4313}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 4228, "end": 4236}]}], "positive regulation": [{"trigger": {"text": "necessary", "start": 8, "end": 17}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 0, "end": 4}, {"role": "Theme", "text": "association", "start": 22, "end": 33}]}, {"trigger": {"text": "resulted", "start": 1050, "end": 1058}, "arguments": [{"role": "Theme", "text": "decrease", "start": 1088, "end": 1096}]}, {"trigger": {"text": "resulted", "start": 1050, "end": 1058}, "arguments": [{"role": "Theme", "text": "increase", "start": 1196, "end": 1204}]}, {"trigger": {"text": "increase", "start": 1196, "end": 1204}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1124, "end": 1128}]}, {"trigger": {"text": "reconstituted", "start": 1910, "end": 1923}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1905, "end": 1909}]}, {"trigger": {"text": "required", "start": 2271, "end": 2279}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 2259, "end": 2263}, {"role": "Theme", "text": "association", "start": 2288, "end": 2299}]}, {"trigger": {"text": "reconstituted", "start": 2544, "end": 2557}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2539, "end": 2543}]}, {"trigger": {"text": "requires", "start": 3670, "end": 3678}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 3638, "end": 3649}, {"role": "Cause", "text": "HOIP", "start": 3679, "end": 3683}]}, {"trigger": {"text": "required", "start": 4360, "end": 4368}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 4352, "end": 4356}, {"role": "Theme", "text": "association", "start": 4377, "end": 4388}]}, {"trigger": {"text": "coupled", "start": 4443, "end": 4450}, "arguments": [{"role": "Cause", "text": "association", "start": 4377, "end": 4388}, {"role": "Theme", "text": "post-translational modifications", "start": 4454, "end": 4486}]}], "protein modification": [{"trigger": {"text": "post-translational modification", "start": 1656, "end": 1687}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1713, "end": 1717}]}, {"trigger": {"text": "post-translational modifications", "start": 4454, "end": 4486}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 4490, "end": 4498}]}], "regulation": [{"trigger": {"text": "dependent", "start": 368, "end": 377}, "arguments": [{"role": "Theme", "text": "recruited", "start": 317, "end": 326}, {"role": "Cause", "text": "TRAF2", "start": 362, "end": 367}]}, {"trigger": {"text": "functions downstream", "start": 407, "end": 427}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 402, "end": 406}, {"role": "Cause", "text": "TRAF2", "start": 431, "end": 436}]}, {"trigger": {"text": "role", "start": 526, "end": 530}, "arguments": [{"role": "Cause", "text": "cIAP1", "start": 503, "end": 508}, {"role": "Theme", "text": "recruitment", "start": 538, "end": 549}]}, {"trigger": {"text": "role", "start": 526, "end": 530}, "arguments": [{"role": "Cause", "text": "cIAP2", "start": 513, "end": 518}, {"role": "Theme", "text": "recruitment", "start": 538, "end": 549}]}, {"trigger": {"text": "altered", "start": 669, "end": 676}, "arguments": [{"role": "Theme", "text": "association", "start": 619, "end": 630}]}, {"trigger": {"text": "alter", "start": 1327, "end": 1332}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1367, "end": 1371}]}, {"trigger": {"text": "alters", "start": 1457, "end": 1463}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1503, "end": 1507}]}, {"trigger": {"text": "influence", "start": 1626, "end": 1635}, "arguments": [{"role": "Cause", "text": "cIAP", "start": 1612, "end": 1616}, {"role": "Theme", "text": "recruitment", "start": 1640, "end": 1651}]}, {"trigger": {"text": "influence", "start": 1626, "end": 1635}, "arguments": [{"role": "Cause", "text": "cIAP", "start": 1612, "end": 1616}, {"role": "Theme", "text": "post-translational modification", "start": 1656, "end": 1687}]}, {"trigger": {"text": "responsible", "start": 1756, "end": 1767}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 1748, "end": 1752}, {"role": "Theme", "text": "recruitment", "start": 1776, "end": 1787}]}]}}, "schema": []}
{"input": "Our results indicate that the protein HOIP is critical for CD40-induced signals that regulate B cell function. Our data show that HOIP-dependent cellular responses include CD40-mediated upregulation of CD80 expression and synthesis of germline RNA transcripts from the immunoglobulin heavy chain locus, two events that are important for T-cell-dependent antibody-mediated immune responses. At the molecular level, our data indicate that HOIP functions downstream of TRAF2 in the CD40 signaling pathway and that HOIP has a key role in promoting the recruitment of the IKK complex to CD40. Consistent with this, CD40-induced activation of NF-kappaB is dependent on the presence of HOIP. In addition, our data show that HOIP facilitates the activation of JNK in response to CD40 engagement. Together, our findings provide support for the conclusion that HOIP is a key component of the CD40 signaling pathway. Given the importance of CD40 signaling in both cellular and humoral immune responses, our results indicate that HOIP has a critical role in the regulation of the immune system.\nThe functional properties of HOIP have only been partially characterized. Initial studies showed that HOIP and the related protein HOIL-1 are components of a large (~600 kDa) protein complex capable of synthesizing linear polyubiquitin chains [5]. Subsequent studies showed that a HOIP-containing complex can interact with IKKgamma and facilitate activation of NF-kappaB via the canonical pathway [16]. These data, considered together with ours, suggest that a HOIP-containing complex mediates recruitment of IKKgamma to the CD40 signaling complex. In addition, CD40-associated HOIP could play a role in activating IKKgamma after its recruitment to the signaling complex [16]. The higher molecular weight forms of IKKgamma we observed in CD40 immunoprecipitates would be consistent with the presence of post-transcriptional modifications including phosphorylation and ubiquitination, which have been suggested to reduce or enhance IKKgamma activity, respectively [16], [17].\nThe mechanisms by which HOIP mediates recruitment of the IKK complex to CD40 and by which HOIP is recruited to CD40 remain unclear. A previous study indicates that HOIP may mediate direct contacts with the IKK complex [16], suggesting that it functions as an adaptor for the recruitment of the IKK complex to CD40. However, the ubiquitin ligase activity of HOIP suggests that it is more than a simple adapter molecule. As IKKgamma appears capable of binding linear polyubiquitin [18], [19], it is possible that HOIP directs formation of linear polyubiquitin chains on a CD40-associated factor, and it is these chains that serve to recruit IKKgamma to the CD40 signaling complex. The molecular interactions necessary for recruitment of HOIP itself to the CD40 signaling complex also remain to be fully characterized. We previously showed that the recruitment of HOIP to the signaling complex is TRAF2-dependent [8]. Potentially, TRAF2 and HOIP directly interact, but it is possible that the ubiquitin ligase activity of TRAF2 [20] (or TRAF2-associated proteins, such as the cIAPs [21]) generates K63-linked polyubiquitin chains to which HOIP can bind and thus associate with the CD40 signaling complex [22].\nWhile our previous work indicated a potentially important link between TRAF2 and HOIP in CD40 signaling, the signals and functions tested here are dependent upon TRAF6 as well as TRAF2. In previous experiments with TRAF-deficient A20.2J cells, we found that the activation of NF-kappaB by CD40 could be mediated by either TRAF2 or TRAF6, while activation of JNK by CD40 was largely dependent on TRAF6 alone [8]. HOIP deficiency compromises the CD40-mediated activation of both NF-kappaB and JNK, indicating that signals mediated by both TRAF2 and TRAF6 likely pass through HOIP. Our previous work also demonstrated that the CD40-mediated activation of NF-kappaB and JNK, while TRAF6-dependent, was not compromised by the disruption of the binding site for TRAF6 in the cytoplasmic domain of CD40 or deletion of the receptor binding domain (the TRAF-C domain) in TRAF6 [8]. These observations indicate that TRAF6 need not directly bind CD40 in order to mediate certain signals, suggesting the assembly of a signaling complex not directly associated with the receptor. If such a complex exists, our results indicate that the absence of HOIP compromises its function as well.\nAlthough the experiments presented here focus on CD40, our results and those of other groups [16], [22] support the possibility that HOIP is important in many signaling pathways in which TRAF2 or TRAF6 are involved, including those associated with various members of the TNF receptor superfamily and the Toll-like receptors. The potential importance of HOIP in immune function and TNFR family signaling is further supported by the recent discovery that HOIP interacts with a protein known as SHARPIN (SHANK-associated RH domain interacting protein in postsynaptic density), which appears capable of working together with HOIP and HOIL to mediate the assembly of linear polyubiquitin [14], [23], [24]. Interestingly, mice with a spontaneous mutation in the gene encoding SHARPIN (chronic proliferative dermatitis (cpdm) mice) exhibit chronic inflammation of the skin and internal organs, defective development of secondary lymphoid tissue, and defects in the production of switched immunoglobulin isotypes [17], [25]. The apparently intimate functional link between SHARPIN and HOIP strongly suggests that at least part of the cpdm phenotype stems from defects in the regulation or function of HOIP.", "output": {"json_structures": {"binding": [{"trigger": {"text": "recruitment", "start": 548, "end": 559}, "arguments": [{"role": "Theme", "text": "CD40", "start": 582, "end": 586}]}, {"trigger": {"text": "engagement", "start": 776, "end": 786}, "arguments": [{"role": "Theme", "text": "CD40", "start": 771, "end": 775}]}, {"trigger": {"text": "interact", "start": 1392, "end": 1400}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 1406, "end": 1414}]}, {"trigger": {"text": "recruitment", "start": 1577, "end": 1588}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 1592, "end": 1600}]}, {"trigger": {"text": "associated", "start": 1650, "end": 1660}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1645, "end": 1649}, {"role": "Theme2", "text": "HOIP", "start": 1661, "end": 1665}]}, {"trigger": {"text": "recruitment", "start": 1717, "end": 1728}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1661, "end": 1665}]}, {"trigger": {"text": "recruitment", "start": 2096, "end": 2107}, "arguments": [{"role": "Theme", "text": "CD40", "start": 2130, "end": 2134}]}, {"trigger": {"text": "recruited", "start": 2156, "end": 2165}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2148, "end": 2152}, {"role": "Theme2", "text": "CD40", "start": 2169, "end": 2173}]}, {"trigger": {"text": "recruitment", "start": 2333, "end": 2344}, "arguments": [{"role": "Theme", "text": "CD40", "start": 2367, "end": 2371}]}, {"trigger": {"text": "binding", "start": 2508, "end": 2515}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 2480, "end": 2488}]}, {"trigger": {"text": "recruit", "start": 2689, "end": 2696}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 2697, "end": 2705}]}, {"trigger": {"text": "recruitment", "start": 2778, "end": 2789}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2793, "end": 2797}]}, {"trigger": {"text": "recruitment", "start": 2904, "end": 2915}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 2919, "end": 2923}]}, {"trigger": {"text": "interact", "start": 3010, "end": 3018}, "arguments": [{"role": "Theme", "text": "TRAF2", "start": 2986, "end": 2991}, {"role": "Theme2", "text": "HOIP", "start": 2996, "end": 3000}]}, {"trigger": {"text": "bind", "start": 3203, "end": 3207}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 3194, "end": 3198}]}, {"trigger": {"text": "associate", "start": 3217, "end": 3226}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 3194, "end": 3198}]}, {"trigger": {"text": "bind", "start": 4195, "end": 4199}, "arguments": [{"role": "Theme", "text": "TRAF6", "start": 4171, "end": 4176}, {"role": "Theme2", "text": "CD40", "start": 4200, "end": 4204}]}, {"trigger": {"text": "interacts", "start": 4896, "end": 4905}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 4891, "end": 4895}, {"role": "Theme2", "text": "SHARPIN", "start": 4930, "end": 4937}]}, {"trigger": {"text": "link", "start": 5490, "end": 5494}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 5503, "end": 5510}, {"role": "Theme2", "text": "HOIP", "start": 5515, "end": 5519}]}], "gene expression": [{"trigger": {"text": "expression", "start": 207, "end": 217}, "arguments": [{"role": "Theme", "text": "CD80", "start": 202, "end": 206}]}], "negative regulation": [{"trigger": {"text": "reduce", "start": 1996, "end": 2002}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 1931, "end": 1946}, {"role": "Theme", "text": "IKKgamma", "start": 2014, "end": 2022}]}, {"trigger": {"text": "reduce", "start": 1996, "end": 2002}, "arguments": [{"role": "Cause", "text": "ubiquitination", "start": 1951, "end": 1965}, {"role": "Theme", "text": "IKKgamma", "start": 2014, "end": 2022}]}, {"trigger": {"text": "deficiency", "start": 3682, "end": 3692}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 3677, "end": 3681}]}, {"trigger": {"text": "disruption", "start": 3986, "end": 3996}, "arguments": [{"role": "Site", "text": "binding site", "start": 4004, "end": 4016}, {"role": "Theme", "text": "CD40", "start": 4056, "end": 4060}]}, {"trigger": {"text": "deletion", "start": 4064, "end": 4072}, "arguments": [{"role": "Site", "text": "receptor binding domain", "start": 4080, "end": 4103}, {"role": "Theme", "text": "TRAF6", "start": 4127, "end": 4132}]}, {"trigger": {"text": "absence", "start": 4388, "end": 4395}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 4399, "end": 4403}]}, {"trigger": {"text": "defects", "start": 5590, "end": 5597}, "arguments": [{"role": "Theme", "text": "regulation", "start": 5605, "end": 5615}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1931, "end": 1946}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 1797, "end": 1805}]}], "positive regulation": [{"trigger": {"text": "mediated", "start": 177, "end": 185}, "arguments": [{"role": "Cause", "text": "CD40", "start": 172, "end": 176}, {"role": "Theme", "text": "upregulation", "start": 186, "end": 198}]}, {"trigger": {"text": "upregulation", "start": 186, "end": 198}, "arguments": [{"role": "Theme", "text": "expression", "start": 207, "end": 217}]}, {"trigger": {"text": "promoting", "start": 534, "end": 543}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 548, "end": 559}]}, {"trigger": {"text": "mediates", "start": 1568, "end": 1576}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 1577, "end": 1588}]}, {"trigger": {"text": "activating", "start": 1687, "end": 1697}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 1698, "end": 1706}]}, {"trigger": {"text": "enhance", "start": 2006, "end": 2013}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 1931, "end": 1946}, {"role": "Theme", "text": "IKKgamma", "start": 2014, "end": 2022}]}, {"trigger": {"text": "enhance", "start": 2006, "end": 2013}, "arguments": [{"role": "Cause", "text": "ubiquitination", "start": 1951, "end": 1965}, {"role": "Theme", "text": "IKKgamma", "start": 2014, "end": 2022}]}, {"trigger": {"text": "mediates", "start": 2087, "end": 2095}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 2082, "end": 2086}, {"role": "Theme", "text": "recruitment", "start": 2096, "end": 2107}]}, {"trigger": {"text": "functions as an adaptor", "start": 2301, "end": 2324}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 2222, "end": 2226}, {"role": "Theme", "text": "recruitment", "start": 2333, "end": 2344}]}, {"trigger": {"text": "serve", "start": 2680, "end": 2685}, "arguments": [{"role": "Theme", "text": "recruit", "start": 2689, "end": 2696}]}, {"trigger": {"text": "necessary", "start": 2764, "end": 2773}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 2778, "end": 2789}]}], "regulation": [{"trigger": {"text": "dependent", "start": 135, "end": 144}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 130, "end": 134}, {"role": "Theme", "text": "mediated", "start": 177, "end": 185}]}, {"trigger": {"text": "downstream", "start": 452, "end": 462}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 437, "end": 441}, {"role": "Cause", "text": "TRAF2", "start": 466, "end": 471}]}, {"trigger": {"text": "role", "start": 526, "end": 530}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 511, "end": 515}, {"role": "Theme", "text": "promoting", "start": 534, "end": 543}]}, {"trigger": {"text": "role", "start": 1679, "end": 1683}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 1661, "end": 1665}, {"role": "Theme", "text": "activating", "start": 1687, "end": 1697}]}, {"trigger": {"text": "dependent", "start": 2958, "end": 2967}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 2904, "end": 2915}, {"role": "Cause", "text": "TRAF2", "start": 2952, "end": 2957}]}, {"trigger": {"text": "regulation", "start": 5605, "end": 5615}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 5631, "end": 5635}]}], "ubiquitination": [{"trigger": {"text": "ubiquitination", "start": 1951, "end": 1965}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 1797, "end": 1805}]}]}}, "schema": []}
{"input": "Cell lines\nThe mouse B cell line A20.2J has been previously described [26], [27]. Cells were grown in RPMI 1640, 10% FCS, 10 microM 2-ME, 2 mM L-glutamine, and antibiotics. HI-5 insect cells (Invitrogen) expressing CD154 have been described [28].", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressing", "start": 204, "end": 214}, "arguments": [{"role": "Theme", "text": "CD154", "start": 215, "end": 220}]}]}}, "schema": []}
{"input": "Antibodies\nMouse anti-birch profilin antibody (4A6 [29]), rat anti-mouse CD40 (1C10 [30]) and a rat isotype control antibody (mAb72, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) were isolated from hybridoma supernatants. FITC-labeled anti-mouse CD80 and an isotype control Ab were from eBiosciences. Rabbit anti-HOIP Ab [6] was the kind gift of Dr. Betty Eipper (University of Connecticut Health Center, Farmington, Connecticut). Goat anti-rat IgG, and HRP-labeled secondary Abs were from Jackson ImmunoResearch Laboratories, Inc. All other antibodies used were described previously [6].", "output": {"json_structures": {}}, "schema": []}
{"input": "Somatic cell gene targeting\nThe generation of HOIP-deficient cells was accomplished using a homologous recombination approach described previously [7], [8]. Segments of Rnf31 gene sequence used in the targeting construct (Fig. 1A) were amplified by PCR from A20.2J genomic DNA. The oligonucleotide primers used to generate the 5' flank (1224 bp) were 5'-ttttctagagcggtggcttaagtgaccc-3' and 5'-tattctagatgcagcatctgagaaagcaagc-3'. The 3' flank (6032 bp) primers were 5'-aaaaccggtgtatgcttctttacgggagaaaaatattag-3' and 5'-tataccggtatgaagccaaaggaacactgagag-3'. Restriction endonuclease sites in the oligonucleotide primers allowed insertion of the PCR products into the targeting vector. A20.2J cells were transfected (by electroporation [7]) with the targeting construct and subcloned in medium containing 600 microg/ml G418 sulfate. Homologous recombination in G418-resistant clones was detected by PCR of genomic DNA, as described [7]. Oligonucleotide primers used for screening were 5'-cttcctgatctcagctttaccgtcac-3' (homologous to genomic sequence; approximate position noted in Fig. 1) and 5'-caatccatcttgttcagccat-3' (homologous to sequence in NeoR). Clones in which one copy of Rnf31 had been disrupted were transiently transfected with an expression plasmid encoding Cre, in order to remove NeoR. G418-sensitive subclones were subjected to a second round of targeting to disrupt the remaining copy of Rnf31. G418-resistant clones were tested for homologous recombination by PCR and HOIP protein expression by Western blot.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 1498, "end": 1508}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1485, "end": 1489}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 51, "end": 60}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 46, "end": 50}]}, {"trigger": {"text": "disrupted", "start": 1195, "end": 1204}, "arguments": [{"role": "Theme", "text": "Rnf31", "start": 1180, "end": 1185}]}]}}, "schema": []}
{"input": "Retroviral transduction\nA retroviral vector (pMIP) was used for the stable transduction of cells with HOIP and IKKgamma cDNA constructs. pMIP was constructed by replacing the IRES and GFP encoding sequences in pMIG [31] with the IRES and puromycin resistance gene from pIRESpuro2 (Clontech). FLAG-tagged mouse HOIP cDNA was inserted into the multiple cloning site of pMIP. A pMIP construct encoding mouse IKKgamma with an amino terminal birch profilin peptide (BP)-tag was also prepared. Retroviral particles were generated by transient transfection of 293T cells with pMIP, pMIP-HOIP, or pMIP-IKKgamma and the packaging vector pCL-Eco [32]. Two days after transfection, culture supernatants were harvested and filtered. A20.2J or HOIP-deficient cells were added to 24-well plates (2.5x105 cells/well) with 1 ml virus-containing supernatant and 8 microg/ml polybrene. Plates were centrifuged at 500 x g for 2 hrs at room temperature. Cells were cultured in B cell culture medium for 48 hrs, after which the transduced cells were selected in 3 microg/ml puromycin.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "deficient", "start": 736, "end": 745}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 731, "end": 735}]}]}}, "schema": []}
{"input": "CD80 upregulation and flow cytometry\nCells (5x104 in 2 ml) were cultured in 24-well plates for 3 days with 5 microg/ml anti-CD40 or an isotype control antibody. Cells were stained for flow cytometry with FITC-anti-CD80 or an appropriate control antibody as described [28]. Data acquired with a FACScan flow cytometer (BD Biosciences) were analyzed with WinMDI 2.8 (Scripps Research Institute, San Diego, CA).", "output": {"json_structures": {"positive regulation": [{"trigger": {"text": "upregulation", "start": 5, "end": 17}, "arguments": [{"role": "Theme", "text": "CD80", "start": 0, "end": 4}]}]}}, "schema": []}
{"input": "GLepsilon transcript assay\nThe CD40-simulated activation of GLepsilon transcription was evaluated as previously reported [33]. Briefly, 1x106 cells were stimulated overnight with anti-CD40 antibody (10 microg/ml) or an isotype control antibody, with or without 500 U/ml mouse IL-4 (BD Biosciences). RNA was isolated using Trizol (Invitrogen), and reverse-transcribed (Superscript III kit, Invitrogen). Quantitative PCR for GLepsilon and Hprt1 was performed using SYBR GREEN master mix (Applied Biosystems), and an Applied Biosystems 7900HT Fast Real-Time PCR instrument. Expression of GLepsilon in each sample was normalized to the expression of Hprt1.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "Expression", "start": 571, "end": 581}, "arguments": [{"role": "Theme", "text": "GLepsilon", "start": 585, "end": 594}]}, {"trigger": {"text": "expression", "start": 632, "end": 642}, "arguments": [{"role": "Theme", "text": "Hprt1", "start": 646, "end": 651}]}], "positive regulation": [{"trigger": {"text": "simulated", "start": 36, "end": 45}, "arguments": [{"role": "Cause", "text": "CD40", "start": 31, "end": 35}, {"role": "Theme", "text": "activation", "start": 46, "end": 56}]}, {"trigger": {"text": "activation", "start": 46, "end": 56}, "arguments": [{"role": "Theme", "text": "transcription", "start": 70, "end": 83}]}], "transcription": [{"trigger": {"text": "transcription", "start": 70, "end": 83}, "arguments": [{"role": "Theme", "text": "GLepsilon", "start": 60, "end": 69}]}]}}, "schema": []}
{"input": "NF-kappaB and JNK activation assays\nFor activation of CD40 signaling, 5x104 HI5 insect cells or HI5 insect cells expressing mouse CD154 (CD40 ligand) were added to 1x106 B cells. Cells were centrifuged for 1 minute at 400 x g (to promote contact between ligand cells and B cells) then incubated at 37degreesC for times indicated in Fig. 4. Following stimulation, cells were chilled on ice for 2 minutes. Cell pellets were dissolved in 2X SDS-PAGE loading buffer, sonicated, and heated for 5 minutes at 95degreesC. Total cell lysates (1x105 cell equivalents per lane) were fractionated by SDS-PAGE and blotted to polyvinylidine fluoride (PVDF) membranes. Membranes were probed with antibodies as indicated in Fig. 4. Chemiluminescent detection (Pierce Biotechnology) was used for the visualization of bands on Western blots. Images of blots were recorded with a low-light imaging system (LAS4000, Fuji Medical Systems) and on X-ray film.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressing", "start": 113, "end": 123}, "arguments": [{"role": "Theme", "text": "CD154", "start": 130, "end": 135}]}]}}, "schema": []}
{"input": "Immunoprecipitations\nImmunoprecipitation of CD40 was performed by activated receptor capture, as previously described [6]. Briefly, 3x107 cells (A20.2J and derivatives) were incubated for 60 minutes at room temperature with 10 microl magnetic protein G beads (Dynal) pre-coated with 10 microg goat anti-rat IgG (Jackson), and 10 microg anti-CD40 (1C10) or an isotype control antibody (mAb72). Cells/beads were then pelleted by centrifugation and lysed in buffer containing 1% Triton X100. Beads were washed with lysis buffer to remove unbound material. In some experiments, material associated with the beads was dephosphorylated with lambda phosphatase (New England BioLabs) as per manufacturer's instructions. Beads were resuspended in 2X SDS-PAGE sample buffer and heated for 5 minutes at 95degreesC. Material eluted from the beads was fractionated by SDS-PAGE and transferred to PVDF membranes for Western blotting. In some experiments, cells were cultured for 6 hrs with 25 microM antennapedia-linked SMAC-N7 peptide (Calbiochem) or an appropriate volume of the solvent used for the peptide (DMSO). After incubation, cells (in peptide- or DMSO-containing medium) were stimulated with antibody-coated beads as outlined above.", "output": {"json_structures": {}}, "schema": []}
{"input": "HOIP gene targeting.\n(A) Regions of Rnf31 gene sequence (bottom) used in the HOIP gene targeting vector (top) are shown. Arrows (F and R) indicate approximate positions of sequences homologous to oligonucleotides used for PCR-mediated detection of homologous recombination. Homologous recombination of the vector with Rnf31 resulted in a small chromosomal deletion and the in-frame insertion of neomycin phosphotransferase (NeoR) into the amino-terminal HOIP coding sequence. A diphtheria toxin (DT) cassette in the vector facilitates the negative selection of cells in which random chromosomal insertion of the vector takes place. LoxP sequences allow the Cre-mediated deletion of the NeoR coding sequence. The SV40pA sequence helps to ensure disruption of gene expression after deletion of the NeoR sequence. (B) Anti-HOIP and anti-FLAG Western blots of cell lysates from A20.2J cells and HOIP-deficient (HOIP-/-) clones. A partial decrease in HOIP protein expression is evident in cells following disruption of one copy of Rnf31 (HOIP-/+). HOIP expression in clones reconstituted with an empty retroviral vector (pMIP) or a retroviral vector encoding FLAG-tagged HOIP is also shown. Approximate molecular weight of HOIP is 120 kD.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 959, "end": 969}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 946, "end": 950}]}, {"trigger": {"text": "expression", "start": 1048, "end": 1058}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1043, "end": 1047}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 896, "end": 905}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 891, "end": 895}]}, {"trigger": {"text": "decrease", "start": 934, "end": 942}, "arguments": [{"role": "Theme", "text": "expression", "start": 959, "end": 969}]}, {"trigger": {"text": "disruption", "start": 1000, "end": 1010}, "arguments": [{"role": "Theme", "text": "Rnf31", "start": 1026, "end": 1031}]}], "positive regulation": [{"trigger": {"text": "following", "start": 990, "end": 999}, "arguments": [{"role": "Theme", "text": "decrease", "start": 934, "end": 942}, {"role": "Cause", "text": "disruption", "start": 1000, "end": 1010}]}]}}, "schema": []}
{"input": "CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 193, "end": 203}, "arguments": [{"role": "Theme", "text": "CD80", "start": 207, "end": 211}]}], "negative regulation": [{"trigger": {"text": "defective", "start": 35, "end": 44}, "arguments": [{"role": "Theme", "text": "mediated", "start": 5, "end": 13}]}, {"trigger": {"text": "deficient", "start": 53, "end": 62}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 48, "end": 52}]}, {"trigger": {"text": "deficient", "start": 360, "end": 369}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 355, "end": 359}]}, {"trigger": {"text": "reversed", "start": 424, "end": 432}, "arguments": [{"role": "Theme", "text": "defective", "start": 433, "end": 442}]}, {"trigger": {"text": "defective", "start": 433, "end": 442}, "arguments": [{"role": "Theme", "text": "upregulation", "start": 448, "end": 460}]}], "positive regulation": [{"trigger": {"text": "mediated", "start": 5, "end": 13}, "arguments": [{"role": "Cause", "text": "CD40", "start": 0, "end": 4}, {"role": "Theme", "text": "upregulation", "start": 19, "end": 31}]}, {"trigger": {"text": "upregulation", "start": 19, "end": 31}, "arguments": [{"role": "Theme", "text": "CD80", "start": 14, "end": 18}]}, {"trigger": {"text": "upregulation", "start": 448, "end": 460}, "arguments": [{"role": "Theme", "text": "CD80", "start": 443, "end": 447}]}]}}, "schema": []}
{"input": "GLepsilon transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLepsilon transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "defective", "start": 27, "end": 36}, "arguments": [{"role": "Theme", "text": "transcription", "start": 10, "end": 23}]}, {"trigger": {"text": "deficient", "start": 61, "end": 70}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 56, "end": 60}]}, {"trigger": {"text": "deficient", "start": 94, "end": 103}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 89, "end": 93}]}, {"trigger": {"text": "deficient", "start": 211, "end": 220}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 206, "end": 210}]}, {"trigger": {"text": "deficient", "start": 813, "end": 822}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 808, "end": 812}]}], "transcription": [{"trigger": {"text": "transcription", "start": 10, "end": 23}, "arguments": [{"role": "Theme", "text": "GLepsilon", "start": 0, "end": 9}]}, {"trigger": {"text": "transcripts", "start": 522, "end": 533}, "arguments": [{"role": "Theme", "text": "GLepsilon", "start": 512, "end": 521}]}, {"trigger": {"text": "transcripts", "start": 582, "end": 593}, "arguments": [{"role": "Theme", "text": "Hprt1", "start": 576, "end": 581}]}]}}, "schema": []}
{"input": "CD40-induced activation of NF-kappaB and JNK is defective in HOIP-deficient cells.\nCD40-mediated signaling was defective in HOIP-deficient cells (HOIP-/- + pMIP), but intact in parental cells transduced with an empty retroviral vector (A20.2J + pMIP) and in HOIP-deficient cells transduced with a retroviral vector encoding HOIP (HOIP-/- + HOIP). Cells were activated with CD154-expressing insect cells for the times indicated. As a negative control, cells were stimulated with insect cells lacking CD154 (5 minute time point only). Western blots of whole-cell lysates were probed with the indicated antibodies. Phospho-IkappaBalpha (pIkappaBalpha) and phospho-JNK (pJNK) blots were stripped and reprobed for total IkappaBalpha and total JNK, respectively (anti-JNK antibodies recognize p46 and p54 isoforms). IkappaBalpha blots were also reprobed for actin to demonstrate equal lane loading. Molecular weights are indicated at right. Similar results were obtained in a second experiment and in two additional experiments with a second HOIP-deficient clone.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressing", "start": 379, "end": 389}, "arguments": [{"role": "Theme", "text": "CD154", "start": 373, "end": 378}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 66, "end": 75}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 61, "end": 65}]}, {"trigger": {"text": "deficient", "start": 129, "end": 138}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 124, "end": 128}]}, {"trigger": {"text": "deficient", "start": 263, "end": 272}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 258, "end": 262}]}, {"trigger": {"text": "lacking", "start": 491, "end": 498}, "arguments": [{"role": "Theme", "text": "CD154", "start": 499, "end": 504}]}, {"trigger": {"text": "deficient", "start": 1041, "end": 1050}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1036, "end": 1040}]}]}}, "schema": []}
{"input": "IKK recruitment to the CD40 signaling complex is defective in HOIP-deficient cells.\n(A) SMAC peptide treatment reduces recruitment of cIAP1 to CD40 and may modify HOIP recruitment. A20.2J cells were incubated for six hours with membrane-permeable SMAC-N7 peptide or 1.5% DMSO (solvent used for the peptide). Following the incubation, cell lysates were prepared, fractionated by SDS-PAGE, and evaluated by Western blot (lanes 1 and 2). Cells incubated with DMSO or SMAC-N7 were also stimulated with magnetic beads coated with anti-CD40 or an isotype control antibody. Immunoprecipitated (IP) material bound to the beads was loaded in lanes 3-5. Samples of the cell lysates after immunoprecipitation appear in lanes 6-8. Western blots were probed with antibodies specific for cIAP1, TRAF2, TRAF3, and HOIP (approximate molecular weights indicated on left). Similar results were obtained in two additional experiments. (B) CD40 was isolated by immunoprecipitation (as in (A)) from A20.2J cells and HOIP-deficient cells transduced with an empty retroviral vector (pMIP) or a retroviral vector encoding HOIP. Material immunoprecipitated with an isotype control antibody (isotype) or anti-CD40 antibody was examined by Western blotting for CD40, TRAF2, TRAF3, cIAP1, HOIP, IKKalpha/beta, and IKKgamma (right panels). HOIP expression was required for coprecipitation of IKK proteins with CD40. Cell lysates from unstimulated cells are shown in the left panels. Similar results were obtained in a second experiment and in two experiments with a second HOIP-deficient clone. (C) To further evaluate HOIP-dependent recruitment of IKKgamma to CD40, A20.2J cells or HOIP-deficient (HOIP-/-) A20.2J cells were transduced with an empty retroviral vector or a retroviral construct encoding BP epitope-tagged IKKgamma (noted in the figure as pMIP and IKKgamma, respectively). Lysates (lanes 1-3) and immunoprecipitation (IP) samples (lanes 4-11) from the cell lines were fractionated by SDS-PAGE and evaluated by Western blotting with antibodies to the BP tag (IKKgamma, upper panel) and TRAF2. The anti-CD40 IP sample in lane 11 was treated with lambda phosphatase; the sample in lane 10 was mock-treated. Protein samples (minus those treated with phosphatase) were also fractionated on a separate gel (lower acrylamide concentration) for the evaluation of TRAF3 and HOIP (bottom two panels). Similar results were obtained in two additional experiments.", "output": {"json_structures": {"binding": [{"trigger": {"text": "recruitment", "start": 119, "end": 130}, "arguments": [{"role": "Theme", "text": "cIAP1", "start": 134, "end": 139}, {"role": "Theme2", "text": "CD40", "start": 143, "end": 147}]}, {"trigger": {"text": "recruitment", "start": 168, "end": 179}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 163, "end": 167}]}, {"trigger": {"text": "recruitment", "start": 1605, "end": 1616}, "arguments": [{"role": "Theme", "text": "IKKgamma", "start": 1620, "end": 1628}, {"role": "Theme2", "text": "CD40", "start": 1632, "end": 1636}]}], "gene expression": [{"trigger": {"text": "expression", "start": 1316, "end": 1326}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1311, "end": 1315}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 67, "end": 76}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 62, "end": 66}]}, {"trigger": {"text": "reduces", "start": 111, "end": 118}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 119, "end": 130}]}, {"trigger": {"text": "deficient", "start": 1000, "end": 1009}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 995, "end": 999}]}, {"trigger": {"text": "deficient", "start": 1549, "end": 1558}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1544, "end": 1548}]}, {"trigger": {"text": "deficient", "start": 1659, "end": 1668}, "arguments": [{"role": "Theme", "text": "HOIP", "start": 1654, "end": 1658}]}], "regulation": [{"trigger": {"text": "modify", "start": 156, "end": 162}, "arguments": [{"role": "Theme", "text": "recruitment", "start": 168, "end": 179}]}, {"trigger": {"text": "dependent", "start": 1595, "end": 1604}, "arguments": [{"role": "Cause", "text": "HOIP", "start": 1590, "end": 1594}, {"role": "Theme", "text": "recruitment", "start": 1605, "end": 1616}]}]}}, "schema": []}
{"input": "M-CSF Induces Monocyte Survival by Activating NF-kappaB p65 Phosphorylation at Ser276 via Protein Kinase C\nMacrophage colony-stimulating factor (M-CSF) promotes mononuclear phagocyte survival and proliferation. The transcription factor Nuclear Factor-kappaB (NF-kappaB) is a key regulator of genes involved in M-CSF-induced mononuclear phagocyte survival and this study focused at identifying the mechanism of NF-kappaB transcriptional activation. Here, we demonstrate that M-CSF stimulated NF-kappaB transcriptional activity in human monocyte-derived macrophages (MDMs) and the murine macrophage cell line RAW 264.7. The general protein kinase C (PKC) inhibitor Ro-31-8220, the conventional PKCalpha/beta inhibitor Go-6976, overexpression of dominant negative PKCalpha constructs and PKCalpha siRNA reduced NF-kappaB activity in response to M-CSF. Interestingly, Ro-31-8220 reduced Ser276 phosphorylation of NF-kappaBp65 leading to decreased M-CSF-induced monocyte survival. In this report, we identify conventional PKCs, including PKCalpha as important upstream kinases for M-CSF-induced NF-kappaB transcriptional activation, NF-kappaB-regulated gene expression, NF-kappaB p65 Ser276 phosphorylation, and macrophage survival. Lastly, we find that NF-kappaB p65 Ser276 plays an important role in basal and M-CSF-stimulated NF-kappaB activation in human mononuclear phagocytes.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "overexpression", "start": 725, "end": 739}, "arguments": [{"role": "Theme", "text": "dominant negative PKCalpha", "start": 743, "end": 769}]}], "negative regulation": [{"trigger": {"text": "inhibitor", "start": 706, "end": 715}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 692, "end": 700}]}, {"trigger": {"text": "inhibitor", "start": 706, "end": 715}, "arguments": [{"role": "Theme", "text": "beta", "start": 701, "end": 705}]}, {"trigger": {"text": "reduced", "start": 875, "end": 882}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 890, "end": 905}]}], "phosphorylation": [{"trigger": {"text": "Phosphorylation", "start": 60, "end": 75}, "arguments": [{"role": "Theme", "text": "p65", "start": 56, "end": 59}, {"role": "Site", "text": "Ser276", "start": 79, "end": 85}]}, {"trigger": {"text": "phosphorylation", "start": 890, "end": 905}, "arguments": [{"role": "Site", "text": "Ser276", "start": 883, "end": 889}, {"role": "Theme", "text": "p65", "start": 918, "end": 921}]}, {"trigger": {"text": "phosphorylation", "start": 1186, "end": 1201}, "arguments": [{"role": "Theme", "text": "p65", "start": 1175, "end": 1178}, {"role": "Site", "text": "Ser276", "start": 1179, "end": 1185}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 1082, "end": 1089}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1186, "end": 1201}]}], "regulation": [{"trigger": {"text": "important", "start": 1045, "end": 1054}, "arguments": [{"role": "Theme", "text": "induced", "start": 1082, "end": 1089}]}]}}, "schema": []}
{"input": "Monocytes are produced in the bone marrow and circulate in blood for 24-48 hours [1]. In the absence of serum, monocytes die via apoptosis [1], [2]. Macrophage colony-stimulating factor (M-CSF) stimulates mononuclear phagocyte survival and differentiation [3]. Importantly, M-CSF-mediated cell survival and activation is associated with a variety of human diseases, including atherosclerosis, transplant vascular sclerosis and breast cancer metastasis [4], [5], [6].\nWe previously identified that NF-kappaB activation is important in M-CSF-induced monocyte survival [7]. In addition to its role in mononuclear phagocyte survival, the transcription factor NF-kappaB regulates numerous genes that play important roles in cellular signaling, stress response, cell growth, survival, differentiation and inflammation [8]. There are five members in the NF-kappaB family: RelA/p65, p50, p52, c-Rel and RelB. The most common activating complex is the p50/p65 heterodimer, driven by the activation domain in the NF-kappaB p65 subunit. NF-kappaB p65 regulates important developmental processes [9], [10]. Mice lacking NF-kappaB p65 die in utero and have extensive liver damage via enhanced apoptosis [9]. Embryonic macrophages from NF-kappaB p65 null mice are susceptible to TNFalpha-induced apoptosis which is rescued by overexpressing the NF-kappaB p65 subunit [10]. Moreover, inhibiting NF-kappaB induces cell death in many cell types and cytokine-independent survival is mediated by constitutively active NF-kappaB in murine macrophages [11].\nIn monocytes and macrophages, NF-kappaB is an important transcriptional factor for expression of cytokines and cell surface receptors [12]. However, unlike resting T-cells, NF-kappaB is constitutively present in the nuclei of primary monocytes and monocytic cell lines in the absence of exogenous stimuli as demonstrated by mobility shift analysis [13]. Similarly, constitutively active NF-kappaB was observed in human alveolar macrophages [14].\nIn the classic/canonical pathway, the NF-kappaB p50/p65 complex is sequestered in the cytosol by IkappaBalpha [15]. Upon stimulation by cytokines or UV radiation, IkappaBalpha is phosphorylated, ubiquitinated, and degraded, releasing NF-kappaB p50/p65 to translocate to the nucleus and transactivate target genes. After its release from IkappaBalpha, NF-kappaB p65 can undergo post-translational modification to activate gene transcription. The role of NF-kappaB p65 phosphorylation on NF-kappaB transcriptional activity varies by stimulus, time of stimulation and cell type [16]. Previous research shows that phosphorylation of NF-kappaB p65 at Ser276, Ser529 or Ser536 plays an important role in regulating transcriptional activity of NF-kappaB [17]. In TNFalpha-treated murine fibroblasts, Ser276 of NF-kappaB p65 is phosphorylated by MSK1 to enhance NF-kappaB transcriptional activity [18]. In macrophages treated with endotoxin, NF-kappaB transcription activity is associated with phosphorylation on Ser276 and Ser536 that is regulated through protein kinase A (PKA) and IKKbeta respectively[16], [19]. In human T cells, NF-kappaB p65 is constitutively phosphorylated on Ser536 to facilitate NF-kappaB p65 nuclear translocation following cellular stimulation [20]. Accumulating evidence reveals that NF-kappaB p65 phosphorylation at Ser276 is crucial for its transcriptional activity. Upon nuclear translocation, phosphorylation of Ser276 on NF-kappaB p65 by PKA recruits the transcription co-activator, p300 to potentiate NF-kappaB-regulated gene transcription [21]. However, other studies show that PKA inhibits NF-kappaB-regulated gene expression by stabilizing IkappaBalpha [22], [23]. Interestingly, the serine/threonine kinase Pim-1 directly phosphorylates NF-kappaB p65 at Ser276 by stabilizing to prevent ubiquitin-proteasome degradation [24]. Several other phosphorylation sites are also described to enhance NF-kappaB gene transactivation [25].\nHere, we investigate the role of protein kinase C (PKC) in M-CSF-stimulated NF-kappaB activation. PKC proteins are multifunctional kinases that differ in structure, function and co-factor requirement [26]. PKCs are involved in diverse cell responses, including cell growth, survival, differentiation and development [27]. The 12 closely related enzymes of the PKC family are divided into three classes: conventional (cPKCs: alpha betaI betaII and gamma require Ca2+ and diacylglycerol (DAG); novel (nPKCs: delta, epsilon, eta, theta and mu) require DAG; and atypical (aPKCs: xi, iota and lambda) require neither Ca2+ nor DAG. Monocytes and macrophages predominantly express conventional PKC isoforms (PKCalpha PKCbetaI and PKCbetaII) and novel PKCs (PKCdelta and PKCepsilon). Conventional PKCs regulate differentiation of the human promyelocytic leukemia cell line HL60 to macrophages [28]. PKCalpha induces IL-1alpha, iNOS and TNFalpha mRNA production after lipopolysaccharide (LPS) exposure [29]. In addition, accumulating evidence suggests that conventional PKCs like PKCalpha have anti-apoptotic functions. For example, PKCalpha is overexpressed in a variety of tumor cells including gliomas, liver, and lung [30], [31]. In epithelial cells, inhibition of PKCalpha induces PKCdelta-dependent apoptosis [31]. Interestingly, in human monocytes and premonocytic THP-1 leukemia cells, novel PKCs like PKCdelta have the opposite effect on cell survival, Voss et al showed that PKCdelta directly phosphorylates caspase-3 and promotes etoposide-induced apoptosis [32]. Moreover, knockout mouse studies suggest that another novel PKC, PKCepsilon is critically involved in early LPS-mediated signaling in activated macrophages [33].\nPreviously, we reported that M-CSF promotes monocyte survival through the activation of the PI3-K/AKT pathway [3]. In addition to AKT activation, M-CSF stimulates PKC in human monocytes and increases NF-kappaB DNA binding [34]. However, whether PKC and/or NF-kappaB activation is critical in M-CSF-stimulated mononuclear phagocyte survival and/or differentiation is unclear. In other cells, PKC plays an important role in NF-kappaB activation and cell survival [35], however the specific mechanisms of this activation and the biological effects on cellular phenotype are not known. Therefore, we focused at understanding the role of PKC in the regulation of NF-kappaB activation and M-CSF-induced monocyte survival.\nHere we demonstrates that M-CSF upregulated the NF-kappaB transcription and cell survival in human and mouse macrophages. This activity was reduced by the conventional, but not novel, PKC inhibitors, dominant negative PKCalpha constructs or PKCalpha siRNA. Conventional PKC regulated NF-kappaB-regulated gene expression and phosphorylation of Ser276 of NF-kappaB p65 occurred in an M-CSF-dependent fashion correlating with its maximal transcriptional activity. Furthermore, PKCalpha-regulated phosphorylation of Ser276 of NF-kappaB p65 plays an important role in regulating its activity in mononuclear phagocytes and murine embryonic fibroblasts.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "overexpressing", "start": 1312, "end": 1326}, "arguments": [{"role": "Theme", "text": "p65", "start": 1341, "end": 1344}]}], "localization": [{"trigger": {"text": "release", "start": 2307, "end": 2314}, "arguments": [{"role": "Theme", "text": "p65", "start": 2344, "end": 2347}]}, {"trigger": {"text": "translocation", "start": 3202, "end": 3215}, "arguments": [{"role": "Theme", "text": "p65", "start": 3190, "end": 3193}, {"role": "ToLoc", "text": "nuclear", "start": 3194, "end": 3201}]}], "negative regulation": [{"trigger": {"text": "lacking", "start": 1100, "end": 1107}, "arguments": [{"role": "Theme", "text": "p65", "start": 1118, "end": 1121}]}, {"trigger": {"text": "null", "start": 1236, "end": 1240}, "arguments": [{"role": "Theme", "text": "p65", "start": 1232, "end": 1235}]}, {"trigger": {"text": "inhibition", "start": 5189, "end": 5199}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 5203, "end": 5211}]}], "phosphorylation": [{"trigger": {"text": "phosphorylated", "start": 2162, "end": 2176}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 2146, "end": 2158}]}, {"trigger": {"text": "phosphorylation", "start": 2450, "end": 2465}, "arguments": [{"role": "Theme", "text": "p65", "start": 2446, "end": 2449}]}, {"trigger": {"text": "phosphorylation", "start": 2593, "end": 2608}, "arguments": [{"role": "Theme", "text": "p65", "start": 2622, "end": 2625}, {"role": "Site", "text": "Ser276", "start": 2629, "end": 2635}]}, {"trigger": {"text": "phosphorylation", "start": 2593, "end": 2608}, "arguments": [{"role": "Theme", "text": "p65", "start": 2622, "end": 2625}, {"role": "Site", "text": "Ser529", "start": 2637, "end": 2643}]}, {"trigger": {"text": "phosphorylation", "start": 2593, "end": 2608}, "arguments": [{"role": "Theme", "text": "p65", "start": 2622, "end": 2625}, {"role": "Site", "text": "Ser536", "start": 2647, "end": 2653}]}, {"trigger": {"text": "phosphorylated", "start": 2803, "end": 2817}, "arguments": [{"role": "Site", "text": "Ser276", "start": 2776, "end": 2782}, {"role": "Theme", "text": "p65", "start": 2796, "end": 2799}, {"role": "Cause", "text": "MSK1", "start": 2821, "end": 2825}]}, {"trigger": {"text": "phosphorylation", "start": 2969, "end": 2984}, "arguments": [{"role": "Theme", "text": "p65", "start": 2796, "end": 2799}, {"role": "Site", "text": "Ser276", "start": 2988, "end": 2994}]}, {"trigger": {"text": "phosphorylation", "start": 2969, "end": 2984}, "arguments": [{"role": "Theme", "text": "p65", "start": 2796, "end": 2799}, {"role": "Site", "text": "Ser536", "start": 2999, "end": 3005}]}, {"trigger": {"text": "phosphorylated", "start": 3141, "end": 3155}, "arguments": [{"role": "Theme", "text": "p65", "start": 3119, "end": 3122}, {"role": "Site", "text": "Ser536", "start": 3159, "end": 3165}]}, {"trigger": {"text": "phosphorylation", "start": 3302, "end": 3317}, "arguments": [{"role": "Theme", "text": "p65", "start": 3298, "end": 3301}, {"role": "Site", "text": "Ser276", "start": 3321, "end": 3327}]}, {"trigger": {"text": "phosphorylation", "start": 3401, "end": 3416}, "arguments": [{"role": "Site", "text": "Ser276", "start": 3420, "end": 3426}, {"role": "Theme", "text": "p65", "start": 3440, "end": 3443}]}, {"trigger": {"text": "phosphorylates", "start": 3736, "end": 3750}, "arguments": [{"role": "Cause", "text": "Pim-1", "start": 3721, "end": 3726}, {"role": "Theme", "text": "p65", "start": 3761, "end": 3764}, {"role": "Site", "text": "Ser276", "start": 3768, "end": 3774}]}, {"trigger": {"text": "phosphorylates", "start": 5437, "end": 5451}, "arguments": [{"role": "Cause", "text": "PKCdelta", "start": 5419, "end": 5427}, {"role": "Theme", "text": "caspase-3", "start": 5452, "end": 5461}]}, {"trigger": {"text": "phosphorylation", "start": 6711, "end": 6726}, "arguments": [{"role": "Site", "text": "Ser276", "start": 6730, "end": 6736}, {"role": "Theme", "text": "p65", "start": 6750, "end": 6753}]}, {"trigger": {"text": "phosphorylation", "start": 6880, "end": 6895}, "arguments": [{"role": "Site", "text": "Ser276", "start": 6899, "end": 6905}, {"role": "Theme", "text": "p65", "start": 6919, "end": 6922}]}], "positive regulation": [{"trigger": {"text": "Upon", "start": 2099, "end": 2103}, "arguments": [{"role": "Theme", "text": "phosphorylated", "start": 2162, "end": 2176}]}, {"trigger": {"text": "Upon", "start": 2099, "end": 2103}, "arguments": [{"role": "Theme", "text": "degraded", "start": 2197, "end": 2205}]}, {"trigger": {"text": "by", "start": 3444, "end": 3446}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 3401, "end": 3416}]}, {"trigger": {"text": "stabilizing", "start": 3641, "end": 3652}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 3653, "end": 3665}]}, {"trigger": {"text": "induces", "start": 4843, "end": 4850}, "arguments": [{"role": "Cause", "text": "PKCalpha", "start": 4834, "end": 4842}, {"role": "Theme", "text": "production", "start": 4885, "end": 4895}]}, {"trigger": {"text": "overexpressed", "start": 5079, "end": 5092}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 5067, "end": 5075}]}], "protein catabolism": [{"trigger": {"text": "degraded", "start": 2197, "end": 2205}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 2146, "end": 2158}]}], "protein modification": [{"trigger": {"text": "post-translational modification", "start": 2360, "end": 2391}, "arguments": [{"role": "Theme", "text": "p65", "start": 2344, "end": 2347}]}], "regulation": [{"trigger": {"text": "regulated", "start": 3014, "end": 3023}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 2969, "end": 2984}, {"role": "Cause", "text": "IKKbeta", "start": 3059, "end": 3066}]}, {"trigger": {"text": "regulated", "start": 6661, "end": 6670}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 6711, "end": 6726}]}, {"trigger": {"text": "dependent", "start": 6775, "end": 6784}, "arguments": [{"role": "Theme", "text": "regulated", "start": 6661, "end": 6670}]}, {"trigger": {"text": "regulated", "start": 6870, "end": 6879}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 6880, "end": 6895}]}], "transcription": [{"trigger": {"text": "production", "start": 4885, "end": 4895}, "arguments": [{"role": "Theme", "text": "IL-1alpha", "start": 4851, "end": 4860}]}, {"trigger": {"text": "production", "start": 4885, "end": 4895}, "arguments": [{"role": "Theme", "text": "iNOS", "start": 4862, "end": 4866}]}, {"trigger": {"text": "production", "start": 4885, "end": 4895}, "arguments": [{"role": "Theme", "text": "TNFalpha", "start": 4871, "end": 4879}]}]}}, "schema": []}
{"input": "M-CSF Induces NF-kappaB Transcriptional Activity in Human Monocyte-Derived Macrophages (MDMs) and Mouse Macrophage Cell Line, RAW 264.7\nTo determine if M-CSF induced NF-kappaB DNA binding in human macrophages, we performed EMSA analysis on nuclear lysates from M-CSF-treated MDMs. Similar to previous reports [11], nuclear NF-kappaB constitutively bound DNA in non-stimulated monocytes (Figure 1A). Interestingly, adding M-CSF did not alter NF-kappaB DNA binding by EMSA. In contrast, after transiently transfecting human MDMs with pNF-kappaB-SEAP constructs containing four NF-kappaB consensus binding sequences, M-CSF treatment of the transfected cells resulted in a 2.3-fold increase in SEAP release in the culture media compared to PBS (vehicle)-treated transfected MDMs (Figure 1B). As a control, the pTAL-SEAP construct lacking NF-kappaB binding sites was used. Cells transfected with the pTAL-SEAP construct did not produce SEAP in the absence or presence of M-CSF (Figure 1B).\nWe next investigated whether M-CSF induced NF-kappaB activity in the mouse macrophage cell line, RAW 264.7. RAW 264.7 cells were transfected with either the NF-kappaB-SEAP reporter or control pTAL-SEAP construct. As shown in Figure 1C, M-CSF treatment of RAW 264.7 cells increased NF-kappaB reporter activity by 2.5-fold over that of non-treated cells. Together, our data demonstrate that M-CSF induced NF-kappaB transcriptional activity in macrophages.", "output": {"json_structures": {"positive regulation": [{"trigger": {"text": "increased", "start": 1256, "end": 1265}, "arguments": [{"role": "Theme", "text": "activity", "start": 1285, "end": 1293}]}, {"trigger": {"text": "activity", "start": 1285, "end": 1293}, "arguments": [{"role": "Theme", "text": "NF-kappaB", "start": 1266, "end": 1275}]}]}}, "schema": []}
{"input": "PKC Inhibition Reduces NF-kappaB Activity in Human MDMs and RAW 264.7 Cells\nSince NF-kappaB is activated by PKC in several cell types [36], we next determined if M-CSF-induced NF-kappaB transcriptional activity was dependent on PKC activation and if calcium, a co-factor for conventional PKC isoform activation, was important. In addition, because of the number of conventional PKCs existing within mononuclear cells, pharmacological inhibitors of PKC family activation was used to determine the relationship of PKC activation to M-CSF-induced cellular survival. MDMs were transfected with the pNF-kappaB-SEAP reporter and then treated with the general PKC inhibitor, Ro-31-8220; the conventional PKCalpha/beta inhibitor Go-6976; or the intracellular calcium blocker BAPTA/AM. Ro-31-8220 significantly suppressed M-CSF-induced NF-kappaB activity compared to cells treated with M-CSF alone (Figure 2A). In addition, Go-6976 and BAPTA/AM also blocked NF-kappaB activity in M-CSF-treated MDMs. Trypan blue exclusion analysis did not indicate cell death suggesting that this suppression was not due to non-specific toxicity. These data indicate that M-CSF mediated NF-kappaB activation through calcium-dependent conventional PKC activation.\nWe next investigated whether PKC inhibition affected NF-kappaB activity in the mouse macrophage cell line, RAW 264.7. Similar to MDMs, PKC inhibitors Ro-31-8220, Go-6976 and BAPTA/AM reduced M-CSF-induced NF-kappaB activity in a dose-dependent manner in RAW 264.7 macrophages (Figure 2B). To ensure that PKC specifically regulated NF-kappaB p65 and not the closely related family member, c-Rel, we co-transfected c-Rel and NF-kappaB-SEAP constructs into the Raw 264.7 cell line and measured NF-kappaB activity in response to M-CSF. There was no increased NF-kappaB activity in cells expressing c-Rel (Figure S1). These observations indicate that PKC functioned upstream of NF-kappaB p65 in MDMs and RAW 264.7 cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressing", "start": 1820, "end": 1830}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 1831, "end": 1836}]}], "negative regulation": [{"trigger": {"text": "inhibitor", "start": 711, "end": 720}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 697, "end": 705}]}, {"trigger": {"text": "inhibitor", "start": 711, "end": 720}, "arguments": [{"role": "Theme", "text": "beta", "start": 706, "end": 710}]}], "regulation": [{"trigger": {"text": "regulated", "start": 1558, "end": 1567}, "arguments": [{"role": "Theme", "text": "p65", "start": 1578, "end": 1581}]}, {"trigger": {"text": "regulated", "start": 1558, "end": 1567}, "arguments": [{"role": "Theme", "text": "c-Rel", "start": 1625, "end": 1630}]}, {"trigger": {"text": "functioned", "start": 1887, "end": 1897}, "arguments": [{"role": "Theme", "text": "p65", "start": 1920, "end": 1923}]}]}}, "schema": []}
{"input": "NF-kappaB and PKC(s) Mediate Human MDM Survival in Response to M-CSF Stimulation\nSince PKC and NF-kappaB are critical in cell survival [37], [38], we hypothesized that M-CSF promoted cell survival through PKC in human MDMs. To detect apoptosis, the expression of cleaved caspase-3, a marker of apoptosis, was analyzed in the presence or absence of M-CSF and PKC inhibitors. In MDMs pretreated with PKC inhibitors (Ro-31-8220, Go-6976, or BAPTA/AM) and stimulated with M-CSF, cleaved caspase-3 was elevated to levels of cells treated with vehicle alone (Figure 3A). In contrast, cells incubated with M-CSF and vehicle had less cleaved caspase-3 than cells incubated with vehicle alone or M-CSF with PKC inhibitors. These data supported the hypothesis that M-CSF-induced NF-kappaB activity was regulated by conventional PKCs, not novel PKCs.\nTo confirm that PKC inhibition decreased cell survival, cells were treated with M-CSF in the absence or presence of PKC inhibitors and then also examined for apoptosis by Annexin V/PI staining. PKC inhibitors in the presence of M-CSF reduced the number of Annexin V/PI negative MDMs compared to cells treated with M-CSF alone (Figure 3B). These observations further suggested that MDM survival is promoted by M-CSF and critically involves conventional PKCs and NF-kappaB activity.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "less", "start": 621, "end": 625}, "arguments": [{"role": "Theme", "text": "cleaved", "start": 626, "end": 633}]}, {"trigger": {"text": "negative", "start": 1109, "end": 1117}, "arguments": [{"role": "Theme", "text": "Annexin V", "start": 1096, "end": 1105}]}], "positive regulation": [{"trigger": {"text": "expression", "start": 249, "end": 259}, "arguments": [{"role": "Theme", "text": "cleaved", "start": 263, "end": 270}]}, {"trigger": {"text": "elevated", "start": 497, "end": 505}, "arguments": [{"role": "Theme", "text": "cleaved", "start": 475, "end": 482}]}], "protein catabolism": [{"trigger": {"text": "cleaved", "start": 263, "end": 270}, "arguments": [{"role": "Theme", "text": "caspase-3", "start": 271, "end": 280}]}, {"trigger": {"text": "cleaved", "start": 475, "end": 482}, "arguments": [{"role": "Theme", "text": "caspase-3", "start": 483, "end": 492}]}, {"trigger": {"text": "cleaved", "start": 626, "end": 633}, "arguments": [{"role": "Theme", "text": "caspase-3", "start": 634, "end": 643}]}]}}, "schema": []}
{"input": "M-CSF-Induces PKCalpha Kinase Activity\nSince our data suggested that conventional PKCs were involved in activating NF-kappaB in response to M-CSF in primary human macrophages, we next investigated whether the conventional PKC, PKCalpha was a downstream target of M-CSF in human MDMs. PKCalpha was immunoprecipitated from human MDMs at the indicated time points (Figure 4), and then a PKC kinase assay was performed using a fluorescein-tagged peptide as a substrate. As shown in Figure 4 (upper panel), the peptide substrate was maximally phosphorylated within 10 minutes of stimulation (1.8-fold over resting cells, lower panel), and then returned to basal levels by 15 minutes. This effect was not seen in M-CSF-stimulated monocytes when isogenic antibodies (IgG) were used for immunoprecipitation. Western blots of identical samples using an antibody recognizing PKCalpha demonstrated that equal amounts of PKCalpha were assayed (Figure 4, middle panel).", "output": {"json_structures": {"regulation": [{"trigger": {"text": "downstream target", "start": 242, "end": 259}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 227, "end": 235}]}]}}, "schema": []}
{"input": "M-CSF-Dependent Activation of PKC Does Not Regulate the Classical NF-kappaB p65 Activation Pathway\nCanonical activation of NF-kappaB occurs via phosphorylation and degradation of IkappaBalpha leading to the release and nuclear translocation of the NF-kappaB p50/p65 heterodimer to transactivate target genes [37], [39]. Since conventional PKC activity was important in regulating M-CSF-induced NF-kappaB activation, we next investigated whether IkappaBalpha degradation was regulated by PKC. Cells were treated with cyclohexamide (CHX) to inhibit protein synthesis of IkappaBalpha, and its degradation was followed. As shown in Figure 5A, PKC inhibition with Ro-31-8220 did not alter M-CSF-induced IkappaBalpha degradation, suggesting that M-CSF-induced PKC activity augmented NF-kappaB transcriptional activity by an alternative pathway, like post-translational modification of NF-kappaB p65.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "synthesis", "start": 555, "end": 564}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 568, "end": 580}]}], "negative regulation": [{"trigger": {"text": "inhibit", "start": 539, "end": 546}, "arguments": [{"role": "Theme", "text": "synthesis", "start": 555, "end": 564}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 144, "end": 159}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 179, "end": 191}]}], "positive regulation": [{"trigger": {"text": "Activation", "start": 80, "end": 90}, "arguments": [{"role": "Theme", "text": "p65", "start": 76, "end": 79}]}, {"trigger": {"text": "induced", "start": 690, "end": 697}, "arguments": [{"role": "Theme", "text": "degradation", "start": 711, "end": 722}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 164, "end": 175}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 179, "end": 191}]}, {"trigger": {"text": "degradation", "start": 458, "end": 469}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 445, "end": 457}]}, {"trigger": {"text": "degradation", "start": 711, "end": 722}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 698, "end": 710}]}], "protein modification": [{"trigger": {"text": "post-translational modification", "start": 844, "end": 875}, "arguments": [{"role": "Theme", "text": "p65", "start": 889, "end": 892}]}], "regulation": [{"trigger": {"text": "Regulate", "start": 43, "end": 51}, "arguments": [{"role": "Theme", "text": "Activation", "start": 80, "end": 90}]}, {"trigger": {"text": "regulated", "start": 474, "end": 483}, "arguments": [{"role": "Theme", "text": "degradation", "start": 458, "end": 469}]}, {"trigger": {"text": "alter", "start": 678, "end": 683}, "arguments": [{"role": "Theme", "text": "induced", "start": 690, "end": 697}]}, {"trigger": {"text": "augmented", "start": 767, "end": 776}, "arguments": [{"role": "Theme", "text": "post-translational modification", "start": 844, "end": 875}]}]}}, "schema": []}
{"input": "M-CSF Induces Phosphorylation of NF-kappaB Ser276 in a PKC-dependent Fashion\nSince M-CSF did not regulate NF-kappaB activation by influencing IkappaBalpha, we next sought to determine if M-CSF affected NF-kappaB p65 by post-translational mechanisms. Thus, we examined the phosphorylation of NF-kappaB p65 with specific phospho-NFkappaB p65 (Ser276 and Ser536) antibodies. M-CSF induced the phosphorylation of Ser276 but not Ser536 of NF-kappaB p65 in MDMs. Compared to vehicle, the general PKC inhibitor Ro-31-8220 reduced Ser276 phosphorylation, but not Ser536, phosphorylation in M-CSF-stimulated cells (Figure 5B). Furthermore, M-CSF-stimulated NF-kappaB p65 phosphorylation at residue Ser276 in RAW 264.7 cells was also PKC dependent (Figure 5C). These studies suggested that PKC(s) regulated Ser276 phosphorylation but not Ser536 in both human MDMs and mouse macrophages after M-CSF stimulation.\nWe next performed cellular fractionation to identify the cellular location of phosphorylated NF-kappaB p65 in Raw 264.7 cells. Non-phosphorylated NF-kappaB p65 was located in both cytosolic and nuclear fractions, but phosphorylated Ser276 and Ser536 NF-kappaB p65 was primarily located in nuclear fraction after M-CSF stimulation (Figure 5D). Notably, constitutive phosphorylation of Ser536 NF-kappaB p65 was found in these cells. Importantly, Ro-31-8220 reduced M-CSF-induced Ser276 phosphorylation of NF-kappaB p65 in both the cytosolic and nuclear fractions, while M-CSF-induced NF-kappaB p65 Ser536 phosphorylation was present in the nucleus regardless of PKC inhibition. These observations indicate that M-CSF-induced Ser276 and Ser536 are regulated differently by conventional PKC activation in mononuclear phagocytes. The purity of the cytosol and nuclear cell fractions was confirmed by immunoblotting with GAPDH and Lamin B, respectively (Figure 5D).", "output": {"json_structures": {"localization": [{"trigger": {"text": "location", "start": 967, "end": 975}, "arguments": [{"role": "Theme", "text": "p65", "start": 1004, "end": 1007}]}, {"trigger": {"text": "located", "start": 1065, "end": 1072}, "arguments": [{"role": "Theme", "text": "p65", "start": 1057, "end": 1060}, {"role": "ToLoc", "text": "both cytosolic and nuclear fractions", "start": 1076, "end": 1112}]}, {"trigger": {"text": "located", "start": 1179, "end": 1186}, "arguments": [{"role": "Theme", "text": "p65", "start": 1161, "end": 1164}, {"role": "ToLoc", "text": "nuclear", "start": 1190, "end": 1197}]}], "negative regulation": [{"trigger": {"text": "reduced", "start": 515, "end": 522}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 530, "end": 545}]}, {"trigger": {"text": "reduced", "start": 515, "end": 522}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 563, "end": 578}]}, {"trigger": {"text": "reduced", "start": 1356, "end": 1363}, "arguments": [{"role": "Theme", "text": "induced", "start": 1370, "end": 1377}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 272, "end": 287}, "arguments": [{"role": "Theme", "text": "p65", "start": 301, "end": 304}]}, {"trigger": {"text": "phosphorylation", "start": 390, "end": 405}, "arguments": [{"role": "Site", "text": "Ser276", "start": 409, "end": 415}, {"role": "Theme", "text": "p65", "start": 444, "end": 447}]}, {"trigger": {"text": "phosphorylation", "start": 390, "end": 405}, "arguments": [{"role": "Site", "text": "Ser536", "start": 424, "end": 430}, {"role": "Theme", "text": "p65", "start": 444, "end": 447}]}, {"trigger": {"text": "phosphorylation", "start": 530, "end": 545}, "arguments": [{"role": "Theme", "text": "p65", "start": 444, "end": 447}, {"role": "Site", "text": "Ser276", "start": 523, "end": 529}]}, {"trigger": {"text": "phosphorylation", "start": 563, "end": 578}, "arguments": [{"role": "Theme", "text": "p65", "start": 444, "end": 447}, {"role": "Site", "text": "Ser536", "start": 555, "end": 561}]}, {"trigger": {"text": "phosphorylation", "start": 662, "end": 677}, "arguments": [{"role": "Theme", "text": "p65", "start": 658, "end": 661}, {"role": "Site", "text": "Ser276", "start": 689, "end": 695}]}, {"trigger": {"text": "phosphorylation", "start": 804, "end": 819}, "arguments": [{"role": "Theme", "text": "p65", "start": 658, "end": 661}, {"role": "Site", "text": "Ser276", "start": 797, "end": 803}]}, {"trigger": {"text": "phosphorylation", "start": 804, "end": 819}, "arguments": [{"role": "Theme", "text": "p65", "start": 658, "end": 661}, {"role": "Site", "text": "Ser536", "start": 828, "end": 834}]}, {"trigger": {"text": "phosphorylated", "start": 979, "end": 993}, "arguments": [{"role": "Theme", "text": "p65", "start": 1004, "end": 1007}]}, {"trigger": {"text": "phosphorylated", "start": 1032, "end": 1046}, "arguments": [{"role": "Theme", "text": "p65", "start": 1057, "end": 1060}]}, {"trigger": {"text": "phosphorylated", "start": 1118, "end": 1132}, "arguments": [{"role": "Site", "text": "Ser276", "start": 1133, "end": 1139}, {"role": "Theme", "text": "p65", "start": 1161, "end": 1164}]}, {"trigger": {"text": "phosphorylated", "start": 1118, "end": 1132}, "arguments": [{"role": "Site", "text": "Ser536", "start": 1144, "end": 1150}, {"role": "Theme", "text": "p65", "start": 1161, "end": 1164}]}, {"trigger": {"text": "phosphorylation", "start": 1266, "end": 1281}, "arguments": [{"role": "Site", "text": "Ser536", "start": 1285, "end": 1291}, {"role": "Theme", "text": "p65", "start": 1302, "end": 1305}]}, {"trigger": {"text": "phosphorylation", "start": 1385, "end": 1400}, "arguments": [{"role": "Site", "text": "Ser276", "start": 1378, "end": 1384}, {"role": "Theme", "text": "p65", "start": 1414, "end": 1417}]}, {"trigger": {"text": "phosphorylation", "start": 1504, "end": 1519}, "arguments": [{"role": "Theme", "text": "p65", "start": 1493, "end": 1496}, {"role": "Site", "text": "Ser536", "start": 1497, "end": 1503}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 378, "end": 385}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 390, "end": 405}]}, {"trigger": {"text": "stimulated", "start": 637, "end": 647}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 662, "end": 677}]}, {"trigger": {"text": "stimulation", "start": 888, "end": 899}, "arguments": [{"role": "Theme", "text": "regulated", "start": 787, "end": 796}]}, {"trigger": {"text": "induced", "start": 1370, "end": 1377}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1385, "end": 1400}]}, {"trigger": {"text": "induced", "start": 1475, "end": 1482}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1504, "end": 1519}]}, {"trigger": {"text": "induced", "start": 1616, "end": 1623}, "arguments": [{"role": "Theme", "text": "p65", "start": 1493, "end": 1496}, {"role": "Site", "text": "Ser276", "start": 1624, "end": 1630}]}, {"trigger": {"text": "induced", "start": 1616, "end": 1623}, "arguments": [{"role": "Theme", "text": "p65", "start": 1493, "end": 1496}, {"role": "Site", "text": "Ser536", "start": 1635, "end": 1641}]}], "protein modification": [{"trigger": {"text": "post-translational mechanisms", "start": 219, "end": 248}, "arguments": [{"role": "Theme", "text": "p65", "start": 212, "end": 215}]}], "regulation": [{"trigger": {"text": "affected", "start": 193, "end": 201}, "arguments": [{"role": "Theme", "text": "post-translational mechanisms", "start": 219, "end": 248}]}, {"trigger": {"text": "dependent", "start": 728, "end": 737}, "arguments": [{"role": "Theme", "text": "stimulated", "start": 637, "end": 647}]}, {"trigger": {"text": "regulated", "start": 787, "end": 796}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 804, "end": 819}]}, {"trigger": {"text": "regardless", "start": 1547, "end": 1557}, "arguments": [{"role": "Theme", "text": "induced", "start": 1475, "end": 1482}]}, {"trigger": {"text": "regulated", "start": 1646, "end": 1655}, "arguments": [{"role": "Theme", "text": "induced", "start": 1616, "end": 1623}]}]}}, "schema": []}
{"input": "M-CSF-dependent PKC Regulates NF-kappaB-targeted Genes\nNF-kappaB induces a number of downstream genes, including the IkappaB family. Among the IkappaB molecules, IkappaBalpha is highly induced by NF-kappaB activation [40]. Having shown that PKC regulated NF-kappaB activity in M-CSF-stimulated MDMs, we next determined whether inhibition of PKC activity decreased expression of NF-kappaB-regulated genes. We treated both MDMs and RAW 264.7 cells with the PKC inhibitor Ro-31-8220 for 30 minutes and then stimulated with M-CSF. IkappaBalpha gene was measured by qRT-PCR. As shown in Figures 6A and 6B, M-CSF enhanced IkappaBalpha gene expression and PKC inhibition by Ro-31-8220 decreased IkappaBalpha gene expression in both MDMs and RAW 264.7 cells (p<0.01), demonstrating that PKC affected NF-kappaB-regulated gene expression in macrophages.\nTo further define the role of PKC in mediating human MDM survival in response to M-CSF, we examined the expression of the anti-apoptotic gene BCL-xL, which is also regulated by NF-kappaB. As shown in Figure 6C, Ro-31-8220 reduced M-CSF-stimulated BCL-xL expression compared to cells treated with M-CSF and the vehicle control DMSO (p<0.05).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 634, "end": 644}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 616, "end": 628}]}, {"trigger": {"text": "expression", "start": 706, "end": 716}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 688, "end": 700}]}, {"trigger": {"text": "expression", "start": 948, "end": 958}, "arguments": [{"role": "Theme", "text": "BCL-xL", "start": 986, "end": 992}]}, {"trigger": {"text": "expression", "start": 1098, "end": 1108}, "arguments": [{"role": "Theme", "text": "BCL-xL", "start": 1091, "end": 1097}]}], "negative regulation": [{"trigger": {"text": "decreased", "start": 678, "end": 687}, "arguments": [{"role": "Theme", "text": "expression", "start": 706, "end": 716}]}, {"trigger": {"text": "reduced", "start": 1066, "end": 1073}, "arguments": [{"role": "Theme", "text": "stimulated", "start": 1080, "end": 1090}]}], "positive regulation": [{"trigger": {"text": "induced", "start": 185, "end": 192}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 162, "end": 174}]}, {"trigger": {"text": "enhanced", "start": 607, "end": 615}, "arguments": [{"role": "Theme", "text": "expression", "start": 634, "end": 644}]}, {"trigger": {"text": "stimulated", "start": 1080, "end": 1090}, "arguments": [{"role": "Theme", "text": "expression", "start": 1098, "end": 1108}]}], "regulation": [{"trigger": {"text": "regulated", "start": 1008, "end": 1017}, "arguments": [{"role": "Theme", "text": "BCL-xL", "start": 986, "end": 992}]}]}}, "schema": []}
{"input": "Identification of PKCalpha as the Upstream Activator of NF-kappaB in Myeloid Cells\nEven though the PKC family consists of 10 members, finding that PKCalpha/beta inhibitors and intracellular calcium inhibitors reduced M-CSF-induced NF-kappaB activity, suggested PKCalpha was involved in NF-kappaB activation after M-CSF treatment. To confirm the role of PKCalpha in NF-kappaB activation in macrophages, constructs for either wildtype (WT)-PKCalpha or kinase-deficient (KD)-PKCalpha was co-transfected with the pNF-kappaB-SEAP reporter gene and SEAP secretion was measured. As shown in Figure 7A, MDMs co-transfected with pNF-kappaB-SEAP and WT-PKCalpha had a 1.8-fold increase in NF-kappaB transcriptional activity after M-CSF activation compared with NS cells (p = 0.05), similar to M-CSF-treated cells expressing only pNF-kappaB-SEAP. Transfecting human macrophages with the KD-PKCalpha construct significantly reduced M-CSF-induced NF-kappaB activity compared to WT-PKCalpha transfected cells (p = 0.016). Similarly, RAW 264.7 cells transfected with WT-PKCalpha had 2.5-fold more NF-kappaB transcriptional activity after M-CSF activation compared to unstimulated RAW 264.7 cells (NS) transfected with WT-PKCalpha (Figure 7B). Expression of the KD-PKCalpha construct into RAW 264.7 cells reduced M-CSF-induced NF-kappaB activity to 1.5-fold (p = 0.045) compared to cells transfected with WT PKCalpha.\nCell survival was also examined in MDMs expressing either WT-PKCalpha or KD-PKCalpha constructs by Annexin V-FITC and PI staining. As expected, M-CSF increased MDM survival as measured by the percent of Annexin V/PI negative cells. Similarly, expression of WT-PKCalpha protected cells from apoptosis. In contrast, expression of KD-PKCalpha decreased M-CSF-induced cell survival (p<0.01) (Figure 7C).\nNext, we examined the effect of expressing the PKCalpha constructs on NF-kappaB phosphorylation. As shown in Figure 7D, expression of KD-PKCalpha in RAW 264.7 cells did not affect the constitutive phosphorylation at Ser536 of NF-kappaB p65, but attenuated the phosphorylation at Ser276. Expression of WT-PKCalpha did not effect the phosphorylation of either residue with or without M-CSF stimulation. These observations demonstrate that PKCalpha is important in M-CSF-regulated cell survival and NF-kappaB activation and likely regulated through phosphorylation of Ser276 of NF-kappaB p65.\nTo further validate the impact that PKCalpha played in M-CSF-induced NF-kappaB transcriptional activity, we next employed PKCalpha siRNA treatment of MDM or RAW cells. A pool of specific PKCalpha siRNA were transfected into MDM or Raw 264.7 cells in the presence or absence or M-CSF. Reducing native PKCalpha expression decreased M-CSF-induced NF-kappaB transcriptional activity in both MDM (Figure 7E) (p = 0.012) and Raw 264.7 cells (Figure 7F) (p = 0.01). We also examined cell survival of the MDMs by Annexin V-FITC and PI staining after PKCalpha siRNA transfection. As shown in Figure 7G, M-CSF-induced MDM survival was reduced in the cells transfected with PKCalpha siRNA compared with cells transfected with control siRNA (p = 0.047). In Figure 7H, we confirmed that PKCalpha siRNA transfection decreased PKCalpha protein expression in both MDM and Raw 264.7 cells. Our results indicated that PKCalpha regulated NF-kappaB activation and M-CSF-regulated cell survival.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "co-transfected", "start": 485, "end": 499}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 438, "end": 446}]}, {"trigger": {"text": "co-transfected", "start": 600, "end": 614}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 643, "end": 651}]}, {"trigger": {"text": "transfected", "start": 977, "end": 988}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 968, "end": 976}]}, {"trigger": {"text": "transfected", "start": 1035, "end": 1046}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1055, "end": 1063}]}, {"trigger": {"text": "transfected", "start": 1186, "end": 1197}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1206, "end": 1214}]}, {"trigger": {"text": "transfected", "start": 1372, "end": 1383}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1392, "end": 1400}]}, {"trigger": {"text": "expressing", "start": 1442, "end": 1452}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1463, "end": 1471}]}, {"trigger": {"text": "expression", "start": 1645, "end": 1655}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1662, "end": 1670}]}, {"trigger": {"text": "expressing", "start": 1834, "end": 1844}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1849, "end": 1857}]}, {"trigger": {"text": "Expression", "start": 2089, "end": 2099}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 2106, "end": 2114}]}, {"trigger": {"text": "expression", "start": 2701, "end": 2711}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 2692, "end": 2700}]}, {"trigger": {"text": "expression", "start": 3221, "end": 3231}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 3204, "end": 3212}]}], "negative regulation": [{"trigger": {"text": "inhibitors", "start": 161, "end": 171}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 147, "end": 155}]}, {"trigger": {"text": "inhibitors", "start": 161, "end": 171}, "arguments": [{"role": "Theme", "text": "beta", "start": 156, "end": 160}]}, {"trigger": {"text": "kinase-deficient", "start": 450, "end": 466}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 472, "end": 480}]}, {"trigger": {"text": "KD", "start": 876, "end": 878}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 879, "end": 887}]}, {"trigger": {"text": "KD", "start": 1246, "end": 1248}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1249, "end": 1257}]}, {"trigger": {"text": "KD", "start": 1475, "end": 1477}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1478, "end": 1486}]}, {"trigger": {"text": "negative", "start": 1618, "end": 1626}, "arguments": [{"role": "Theme", "text": "Annexin V", "start": 1605, "end": 1614}]}, {"trigger": {"text": "KD", "start": 1730, "end": 1732}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1733, "end": 1741}]}, {"trigger": {"text": "KD", "start": 1936, "end": 1938}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1939, "end": 1947}]}, {"trigger": {"text": "attenuated", "start": 2047, "end": 2057}, "arguments": [{"role": "Cause", "text": "KD", "start": 1936, "end": 1938}, {"role": "Theme", "text": "phosphorylation", "start": 2062, "end": 2077}]}, {"trigger": {"text": "Reducing", "start": 2676, "end": 2684}, "arguments": [{"role": "Theme", "text": "expression", "start": 2701, "end": 2711}]}, {"trigger": {"text": "decreased", "start": 3194, "end": 3203}, "arguments": [{"role": "Cause", "text": "PKCalpha siRNA", "start": 3166, "end": 3180}, {"role": "Theme", "text": "expression", "start": 3221, "end": 3231}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1999, "end": 2014}, "arguments": [{"role": "Site", "text": "Ser536", "start": 2018, "end": 2024}, {"role": "Theme", "text": "p65", "start": 2038, "end": 2041}]}, {"trigger": {"text": "phosphorylation", "start": 2062, "end": 2077}, "arguments": [{"role": "Theme", "text": "p65", "start": 2038, "end": 2041}, {"role": "Site", "text": "Ser276", "start": 2081, "end": 2087}]}, {"trigger": {"text": "phosphorylation", "start": 2134, "end": 2149}, "arguments": [{"role": "Site", "text": "Ser536", "start": 2018, "end": 2024}, {"role": "Theme", "text": "p65", "start": 2038, "end": 2041}]}, {"trigger": {"text": "phosphorylation", "start": 2134, "end": 2149}, "arguments": [{"role": "Theme", "text": "p65", "start": 2038, "end": 2041}, {"role": "Site", "text": "Ser276", "start": 2081, "end": 2087}]}, {"trigger": {"text": "phosphorylation", "start": 2348, "end": 2363}, "arguments": [{"role": "Site", "text": "Ser276", "start": 2367, "end": 2373}, {"role": "Theme", "text": "p65", "start": 2387, "end": 2390}]}], "regulation": [{"trigger": {"text": "affect", "start": 1975, "end": 1981}, "arguments": [{"role": "Cause", "text": "KD", "start": 1936, "end": 1938}, {"role": "Theme", "text": "phosphorylation", "start": 1999, "end": 2014}]}, {"trigger": {"text": "effect", "start": 2123, "end": 2129}, "arguments": [{"role": "Cause", "text": "Expression", "start": 2089, "end": 2099}, {"role": "Theme", "text": "phosphorylation", "start": 2134, "end": 2149}]}, {"trigger": {"text": "with or without", "start": 2168, "end": 2183}, "arguments": [{"role": "Theme", "text": "effect", "start": 2123, "end": 2129}]}]}}, "schema": []}
{"input": "PKC is Essential in Regulating NF-kappaB Activity\nM-CSF-dependent PKC activity facilitated NF-kappaB p65 phosphorylation at Ser276 but not Ser536. To confirm the role of PKC and p65 Ser276 phosphorylation on NF-kappaB activity, we co-transfected the NF-kappaB-SEAP construct with either WT NF-kappaB p65 or NF-kappaB p65 276S/A constructs in Raw 264.7 cells, then treated cells with the PKC inhibitor Ro-31-8220 in the absence or presences of M-CSF. As expected in cells transfected with vector control, inhibiting PKC reduced M-CSF-stimulated NF-kappaB activity compared to cells treated with M-CSF and the vehicle DMSO (p = 0.001) (Figure 8A). In contrast, expressing WT NF-kappaB p65 (p65 WT) increased NF-kappaB activity, while the general PKC inhibitor Ro-31-8220 decreased this NF-kappaB activity (p = 0.001). Notably, the introduction of NF-kappaB p65 276 S/A construct significantly reduced NF-kappaB activity compared with the WT NF-kappaB p65 construct (p = 0.005) and M-CSF treatment was unable to overcome this inhibition.\nFurthermore, we co-transfected the NF-kappaB-SEAP construct along with either the WT NF-kappaB p65, NF-kappaB p65 276S/A or NF-kappaB p65 536S/A constructs into a NF-kappaB p65-/- murine fibroblast cell line and measured NF-kappaB activity. As predicted, expression of WT NF-kappaB p65 (p65 WT) in NF-kappaB p65-/- cell line constitutively activated NF-kappaB compared to cells transfected with vector control without any stimulation (Figure 8B). In comparison, transfecting the NF-kappaB p65 276S/A construct reduced NF-kappaB activity by 5-fold (p = 0.006, WT NF-kappaB p65 vs. NF-kappaB p65 276S/A), while expressing the NF-kappaB p65 536S/A construct increased NF-kappaB activity in the p65-/- cells to levels similar to WT NF-kappaB p65 transfected cells.\nSince M-CSF-induced PKC activation regulated NF-kappaB activity via Ser276 residue of NF-kappaB p65 in primary human MDMs and RAW 264.7 cells, we next examined if this occurred in NF-kappaB p65-/- fibroblasts in response to a native stimulus for these cells, TNFalpha. NF-kappaB activity was measured in the WT NF-kappaB p65 or NF-kappaB p65 276S/A transfected cells treated with TNFalpha in the absence or presence of Ro-31-8220 (Figure 8C). As expected, TNFalpha increased NF-kappaB activity in NF-kappaB p65-/- cells expressing WT p65 and (p = 0.001) PKC inhibitors decreased NF-kappaB activity to the non-stimulated (NS) level (p = 0.007). Introducing NF-kappaB p65 276 S/A constructs significantly reduced NF-kappaB activity compared with the WT NF-kappaB p65 construct (p = 0.001). Notably, TNFalpha failed to increase NF-kappaB activity in the NF-kappaB p65-/- cells expressing NF-kappaB p65 276S/A constructs. These observations are similar to macrophages overexpressing the NF-kappaB p65 276S/A (Figure 8A). Our data demonstrate that Ser276 of NF-kappaB p65 is essential in regulating NF-kappaB activity and suggests that PKC regulates NF-kappaB activity by modulating the phosphorylation of NF-kappaB p65 at Ser276 residue.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "co-transfected", "start": 231, "end": 245}, "arguments": [{"role": "Theme", "text": "p65", "start": 300, "end": 303}]}, {"trigger": {"text": "co-transfected", "start": 231, "end": 245}, "arguments": [{"role": "Theme", "text": "p65", "start": 317, "end": 320}]}, {"trigger": {"text": "expressing", "start": 659, "end": 669}, "arguments": [{"role": "Theme", "text": "p65", "start": 683, "end": 686}]}, {"trigger": {"text": "introduction", "start": 829, "end": 841}, "arguments": [{"role": "Theme", "text": "p65", "start": 855, "end": 858}]}, {"trigger": {"text": "co-transfected", "start": 1051, "end": 1065}, "arguments": [{"role": "Theme", "text": "p65", "start": 1145, "end": 1148}]}, {"trigger": {"text": "co-transfected", "start": 1051, "end": 1065}, "arguments": [{"role": "Theme", "text": "p65", "start": 1169, "end": 1172}]}, {"trigger": {"text": "-/-", "start": 1211, "end": 1214}, "arguments": [{"role": "Theme", "text": "p65", "start": 1208, "end": 1211}]}, {"trigger": {"text": "expression", "start": 1290, "end": 1300}, "arguments": [{"role": "Theme", "text": "p65", "start": 1317, "end": 1320}]}, {"trigger": {"text": "-/-", "start": 1346, "end": 1349}, "arguments": [{"role": "Theme", "text": "p65", "start": 1343, "end": 1346}]}, {"trigger": {"text": "transfecting", "start": 1497, "end": 1509}, "arguments": [{"role": "Theme", "text": "p65", "start": 1524, "end": 1527}]}, {"trigger": {"text": "expressing", "start": 1644, "end": 1654}, "arguments": [{"role": "Theme", "text": "p65", "start": 1669, "end": 1672}]}, {"trigger": {"text": "-/-", "start": 1729, "end": 1732}, "arguments": [{"role": "Theme", "text": "p65", "start": 1726, "end": 1729}]}, {"trigger": {"text": "transfected", "start": 1777, "end": 1788}, "arguments": [{"role": "Theme", "text": "p65", "start": 1773, "end": 1776}]}, {"trigger": {"text": "-/-", "start": 1989, "end": 1992}, "arguments": [{"role": "Theme", "text": "p65", "start": 1986, "end": 1989}]}, {"trigger": {"text": "transfected", "start": 2145, "end": 2156}, "arguments": [{"role": "Theme", "text": "p65", "start": 2117, "end": 2120}]}, {"trigger": {"text": "transfected", "start": 2145, "end": 2156}, "arguments": [{"role": "Theme", "text": "p65", "start": 2134, "end": 2137}]}, {"trigger": {"text": "-/-", "start": 2306, "end": 2309}, "arguments": [{"role": "Theme", "text": "p65", "start": 2303, "end": 2306}]}, {"trigger": {"text": "expressing", "start": 2316, "end": 2326}, "arguments": [{"role": "Theme", "text": "p65", "start": 2330, "end": 2333}]}, {"trigger": {"text": "Introducing", "start": 2440, "end": 2451}, "arguments": [{"role": "Theme", "text": "p65", "start": 2462, "end": 2465}]}, {"trigger": {"text": "-/-", "start": 2660, "end": 2663}, "arguments": [{"role": "Theme", "text": "p65", "start": 2657, "end": 2660}]}, {"trigger": {"text": "expressing", "start": 2670, "end": 2680}, "arguments": [{"role": "Theme", "text": "p65", "start": 2691, "end": 2694}]}, {"trigger": {"text": "overexpressing", "start": 2760, "end": 2774}, "arguments": [{"role": "Theme", "text": "p65", "start": 2789, "end": 2792}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 105, "end": 120}, "arguments": [{"role": "Theme", "text": "p65", "start": 101, "end": 104}, {"role": "Site", "text": "Ser276", "start": 124, "end": 130}]}, {"trigger": {"text": "phosphorylation", "start": 105, "end": 120}, "arguments": [{"role": "Theme", "text": "p65", "start": 101, "end": 104}, {"role": "Site", "text": "Ser536", "start": 139, "end": 145}]}, {"trigger": {"text": "phosphorylation", "start": 189, "end": 204}, "arguments": [{"role": "Theme", "text": "p65", "start": 178, "end": 181}, {"role": "Site", "text": "Ser276", "start": 182, "end": 188}]}, {"trigger": {"text": "phosphorylation", "start": 2978, "end": 2993}, "arguments": [{"role": "Theme", "text": "p65", "start": 3007, "end": 3010}, {"role": "Site", "text": "Ser276", "start": 3014, "end": 3020}]}], "positive regulation": [{"trigger": {"text": "facilitated", "start": 79, "end": 90}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 105, "end": 120}]}], "regulation": [{"trigger": {"text": "modulating", "start": 2963, "end": 2973}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 2978, "end": 2993}]}]}}, "schema": []}
{"input": "NF-kappaB transcriptional activity is important in regulating intracellular signaling, stress response, proliferation, survival, differentiation and inflammation [8]. To regulate gene transcription, NF-kappaB p50/p65 heterodimers translocate to the nucleus from the cytoplasm, however, their translocation is not sufficient to activate gene transcription. Post-translational modification of NF-kappaB heterodimers such as phosphorylation or acetylation also contributes to its transcriptional activity [41], [42], [43]. Because NF-kappaB constitutively translocates to the nucleus in monocytic lineage [13] and activates gene transcription and survival in murine macrophages [11], we sought to determine how M-CSF affected NF-kappaB transcriptional activity in human MDMs and whether this activation regulated their survival. The present study demonstrated that M-CSF stimulated PKCalpha kinase upstream of NF-kappaB transcriptional activity in primary human MDMs and RAW 264.7 cells. We showed inhibition of conventional PKCs by PKC inhibitors, kinase deficient PKCalpha or PKCalpha siRNA blocked M-CSF-induced cell survival and NF-kappaB-regulated gene expression. This block correlated with a reduction in M-CSF-induced phosphorylation of NF-kappaB p65 at Ser276. Consistent with these findings the dominant negative PKCalpha constructs also inhibited NF-kappaB p65 phosphorylation at Ser276 but not at Ser536 resulting in reduced NF-kappaB transcriptional activation and M-CSF-induced MDM survival. A simplified proposed cartoon model in Figure 9 demonstrates that M-CSF induced monocytes survival is regulated by activating NF-kappaB p65 phosphorylation at Ser276 via PKC. Finally, in a NF-kappaB p65-/- fibroblast cell line, we confirmed that the Ser276 residue of NF-kappaB p65 is important and essential for PKC modulation of NF-kappaB activity. More compelling is that we observed a similar regulation between two different cell lineages in our study.\nPost-translational modification of NF-kappaB p65 regulates its activating or repressing effects on gene expression. Among the seven reported putative NF-kappaB p65 phosphorylation sites, five increase nuclear translocation, DNA binding and NF-kappaB transcriptional activity [17]. Similar to our finding, Zhong et al. found that in response to LPS, NF-kappaB p65 is phosphorylated at the highly conserved Ser276 residue by the catalytic subunit of PKA [16]. In addition to PKA, Ser276 can be phosphorylated by MSK1 in the nucleus upon TNFalpha treatment [18]. Phosphorylation of NF-kappaB p65 at Ser536 occurs in response to many inflammatory stimuli as well as kinases, IKKalpha, IKKbeta and RSK1 [17], [18], [19], [20], [21]. Notably, in addition to phosphorylating Ser276 of NF-kappaB, our study revealed that M-CSF also modulated the phosphorylation of the NF-kappaB p65 subunit at Ser536, however, PKC inhibitors or kinase deficient PKCalpha constructs did not affect this phosphorylation event. Moreover, mutant constructs containing a point mutation at Ser536 of NF-kappaB p65 did not reduce NF-kappaB activity in cells lacking endogenous NF-kappaB p65, while the NF-kappaB p65 276S/A construct did reduce NF-kappaB activity in these cells. Therefore, specific post-translational modification of NF-kappaB p65 is likely to regulate transcriptional activation in response to specific stimuli [15], [43].\nIt is notable that M-CSF activated NF-kappaB p65 Ser276 phosphorylation and transcriptional activation in a PKCalpha-dependent manner. PKCalpha is a member of the conventional PKC family of protein kinases that are critical for cell growth, differentiation and cell death. PKCalpha primarily modulates anti-apoptotic and proliferation signals following cytokine treatment in various cell types [42]. Expression of PKCalpha attenuates apoptosis in many different cell types, while PKCalpha inhibition generally potentiates apoptosis [42]. In our studies using conventional PKC inhibitors and dominant negative PKCalpha constructs, PKCalpha was critical in M-CSF-induced macrophage survival by inducing Ser276 phosphorylation of NF-kappaB p65 in both primary human MDMs and Raw 264.7 cells. We also demonstrated PKC inhibition downregulated NF-kappaB-induction of the anti-apoptotic gene BCL-xL. Consistent with our finding, others reported NF-kappaB regulated the gene expression of many anti-apoptotic proteins including other BCL-2 family members and inhibitor of apoptosis (IAP) proteins which are regulators of apoptosis and function upstream of caspases [44]. It has been shown that phosphorylation of p65 at Ser276 prevents its degradation by ubiquitin-mediated proteolysis and promotes cell survival in HeLa cells [24]. In addition to BCL-xL expression as examined in this study, we are evaluating the possibility that PKCalpha-regulated NF-kappaB activity upregulates additional anti-apoptotic genes in an M-CSF-dependent fashion to modulate cell survival.\nThe use of inhibitors and siRNA in our study has not ruled out that other conventional PKCs might also be important in M-CSF-induced NF-kappaB activation and macrophage survival. In contrast to the actions of PKCalpha activation of other conventional PKC isoforms, like PKCbetaI/betaII, appear necessary for macrophage apoptosis. The PKCbetaI/betaII isoforms are expressed in apoptotic U937 myelomonocytic cells [45]. The human myeloid cell line HL-525 which is devoid of endogenous PKCbetaII is resistant to TNFalpha-induced apoptosis [46]. Re-expression of PKCbetaII in HL-525 cells restores their susceptibility to TNFalpha-induced apoptosis implying that PKCbetaII is pro-apoptotic and may be required to induce macrophage apoptosis.\nRecent studies demonstrated that NF-kappaB transcriptional activity can be regulated by phosphorylation of the NF-kappaB p65 subunit in the absence of IkappaBalpha degradation [47]. By this pathway, phosphorylation of NF-kappaB regulates its interaction with other components of the basal transcriptional machinery without affecting its capability to bind DNA [21]. Since NF-kappaB can bind the promoters of numerous gene targets, post-translational modification of NF-kappaB p65 may affect its interaction with other proteins, refining the expression of gene targets. We did not find that PKC was involved in IkappaBalpha degradation or NF-kappaB p65 nuclear translocation induced by M-CSF, but that M-CSF-induced phosphorylation of NF-kappaB p65 at Ser276 was dramatically reduced by the conventional PKC inhibitor and kinase deficient PKCalpha and by siRNA towards PKCalpha. Current studies are underway to delineate the protein complexes formed by activated NF-kappaB and other transcriptional co-activators in response to M-CSF and PKC activation. Recently studies indicated that the non-canonical pathway was activated during human monocyte-macrophage differentiation [48]. During this process, expression of IKKalpha among other proteins, is elevated leading to increased p52 NF-kappaB (relB) expression through partial proteolytic degradation of the p100 NF-kappaB protein. Thus, it is possible that M-CSF regulates monocyte survival through both the canonical and non-canonical NF-kappaB pathways, which will be a focus in future research.\nWe previously showed that M-CSF reduced caspase-3 and -9 activity and prevented monocyte apoptosis [3]. This study reveals that treatment of monocytes with conventional PKC inhibitors, or overexpression of kinase-deficient PKCalpha blocked M-CSF-induced cell survival. Our data revealed that conventional PKCs are upstream of NF-kappaB activation in response to M-CSF and mediate post-translational activation of NF-kappaB p65 by phosphorylating Ser276 to regulate gene transcription and cell survival. Thus, our observation may provide insight in potential therapeutic targets for inflammatory diseases.", "output": {"json_structures": {"binding": [{"trigger": {"text": "interaction", "start": 6168, "end": 6179}, "arguments": [{"role": "Theme", "text": "p65", "start": 6149, "end": 6152}]}], "gene expression": [{"trigger": {"text": "-/-", "start": 1705, "end": 1708}, "arguments": [{"role": "Theme", "text": "p65", "start": 1702, "end": 1705}]}, {"trigger": {"text": "Expression", "start": 3771, "end": 3781}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 3785, "end": 3793}]}, {"trigger": {"text": "expression", "start": 4719, "end": 4729}, "arguments": [{"role": "Theme", "text": "BCL-xL", "start": 4712, "end": 4718}]}, {"trigger": {"text": "expressed", "start": 5298, "end": 5307}, "arguments": [{"role": "Theme", "text": "PKCbetaI", "start": 5269, "end": 5277}]}, {"trigger": {"text": "expressed", "start": 5298, "end": 5307}, "arguments": [{"role": "Theme", "text": "betaII", 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"start": 2882, "end": 2898}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 2899, "end": 2907}]}, {"trigger": {"text": "lacking", "start": 3088, "end": 3095}, "arguments": [{"role": "Theme", "text": "p65", "start": 3117, "end": 3120}]}, {"trigger": {"text": "inhibition", "start": 3860, "end": 3870}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 3851, "end": 3859}]}, {"trigger": {"text": "downregulated", "start": 4196, "end": 4209}, "arguments": [{"role": "Theme", "text": "induction", "start": 4220, "end": 4229}]}, {"trigger": {"text": "prevents", "start": 4591, "end": 4599}, "arguments": [{"role": "Cause", "text": "phosphorylation", "start": 4558, "end": 4573}, {"role": "Theme", "text": "by", "start": 4616, "end": 4618}]}, {"trigger": {"text": "absence", "start": 5813, "end": 5820}, "arguments": [{"role": "Theme", "text": "degradation", "start": 5837, "end": 5848}]}, {"trigger": {"text": "reduced", "start": 6448, "end": 6455}, "arguments": [{"role": 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1223, "end": 1238}, "arguments": [{"role": "Theme", "text": "p65", "start": 1252, "end": 1255}, {"role": "Site", "text": "Ser276", "start": 1259, "end": 1265}]}, {"trigger": {"text": "phosphorylation", "start": 1369, "end": 1384}, "arguments": [{"role": "Theme", "text": "p65", "start": 1365, "end": 1368}, {"role": "Site", "text": "Ser276", "start": 1388, "end": 1394}]}, {"trigger": {"text": "phosphorylation", "start": 1369, "end": 1384}, "arguments": [{"role": "Theme", "text": "p65", "start": 1365, "end": 1368}, {"role": "Site", "text": "Ser536", "start": 1406, "end": 1412}]}, {"trigger": {"text": "phosphorylation", "start": 1643, "end": 1658}, "arguments": [{"role": "Theme", "text": "p65", "start": 1639, "end": 1642}, {"role": "Site", "text": "Ser276", "start": 1662, "end": 1668}]}, {"trigger": {"text": "phosphorylated", "start": 2327, "end": 2341}, "arguments": [{"role": "Theme", "text": "p65", "start": 2320, "end": 2323}, {"role": "Site", "text": "Ser276", "start": 2366, "end": 2372}]}, {"trigger": {"text": "phosphorylated", "start": 2453, "end": 2467}, "arguments": [{"role": "Theme", "text": "p65", "start": 2320, "end": 2323}, {"role": "Site", "text": "Ser276", "start": 2439, "end": 2445}, {"role": "Cause", "text": "MSK1", "start": 2471, "end": 2475}]}, {"trigger": {"text": "Phosphorylation", "start": 2521, "end": 2536}, "arguments": [{"role": "Theme", "text": "p65", "start": 2550, "end": 2553}, {"role": "Site", "text": "Ser536", "start": 2557, "end": 2563}]}, {"trigger": {"text": "phosphorylation", "start": 2799, "end": 2814}, "arguments": [{"role": "Theme", "text": "p65", "start": 2832, "end": 2835}, {"role": "Site", "text": "Ser536", "start": 2847, "end": 2853}]}, {"trigger": {"text": "phosphorylation", "start": 3427, "end": 3442}, "arguments": [{"role": "Theme", "text": "p65", "start": 3416, "end": 3419}, {"role": "Site", "text": "Ser276", "start": 3420, "end": 3426}]}, {"trigger": {"text": "phosphorylation", "start": 4079, "end": 4094}, "arguments": [{"role": "Site", "text": "Ser276", "start": 4072, "end": 4078}, {"role": "Theme", "text": "p65", "start": 4108, "end": 4111}]}, {"trigger": {"text": "phosphorylation", "start": 4558, "end": 4573}, "arguments": [{"role": "Theme", "text": "p65", "start": 4577, "end": 4580}, {"role": "Site", "text": "Ser276", "start": 4584, "end": 4590}]}, {"trigger": {"text": "phosphorylation", "start": 5761, "end": 5776}, "arguments": [{"role": "Theme", "text": "p65", "start": 5794, "end": 5797}]}, {"trigger": {"text": "phosphorylation", "start": 6388, "end": 6403}, "arguments": [{"role": "Theme", "text": "p65", "start": 6417, "end": 6420}, {"role": "Site", "text": "Ser276", "start": 6424, "end": 6430}]}, {"trigger": {"text": "phosphorylating", "start": 7652, "end": 7667}, "arguments": [{"role": "Theme", "text": "p65", "start": 7645, "end": 7648}, {"role": "Site", "text": "Ser276", "start": 7668, "end": 7674}]}], "positive regulation": [{"trigger": {"text": "stimulated", "start": 868, "end": 878}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 879, "end": 887}]}, {"trigger": {"text": "induced", "start": 1215, "end": 1222}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1223, "end": 1238}]}, {"trigger": {"text": "activating", "start": 1618, "end": 1628}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1643, "end": 1658}]}, {"trigger": {"text": "in response to", "start": 2290, "end": 2304}, "arguments": [{"role": "Theme", "text": "phosphorylated", "start": 2327, "end": 2341}]}, {"trigger": {"text": "upon", "start": 2491, "end": 2495}, "arguments": [{"role": "Theme", "text": "phosphorylated", "start": 2453, "end": 2467}, {"role": "Cause", "text": "TNFalpha", "start": 2496, "end": 2504}]}, {"trigger": {"text": "in response to", "start": 2571, "end": 2585}, "arguments": [{"role": "Theme", "text": "Phosphorylation", "start": 2521, "end": 2536}]}, {"trigger": {"text": "activated", "start": 3396, "end": 3405}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 3427, "end": 3442}]}, {"trigger": {"text": "inducing", "start": 4063, "end": 4071}, "arguments": [{"role": "Cause", "text": "PKCalpha", "start": 4001, "end": 4009}, {"role": "Theme", "text": "phosphorylation", "start": 4079, "end": 4094}]}, {"trigger": {"text": "induction", "start": 4220, "end": 4229}, "arguments": [{"role": "Theme", "text": "BCL-xL", "start": 4257, "end": 4263}]}, {"trigger": {"text": "by", "start": 4616, "end": 4618}, "arguments": [{"role": "Theme", "text": "degradation", "start": 4604, "end": 4615}, {"role": "Cause", "text": "mediated", "start": 4629, "end": 4637}]}, {"trigger": {"text": "mediated", "start": 4629, "end": 4637}, "arguments": [{"role": "Cause", "text": "ubiquitin", "start": 4619, "end": 4628}, {"role": "Theme", "text": "proteolysis", "start": 4638, "end": 4649}]}, {"trigger": {"text": "induced", "start": 6347, "end": 6354}, "arguments": [{"role": "Theme", "text": "translocation", "start": 6333, "end": 6346}]}, {"trigger": {"text": "induced", "start": 6380, "end": 6387}, "arguments": [{"role": "Theme", "text": "induced", "start": 6347, "end": 6354}]}, {"trigger": {"text": "elevated", "start": 6922, "end": 6930}, "arguments": [{"role": "Theme", "text": "expression", "start": 6874, "end": 6884}]}, {"trigger": {"text": "increased", "start": 6942, "end": 6951}, "arguments": [{"role": "Cause", "text": "expression", "start": 6874, "end": 6884}, {"role": "Theme", "text": "expression", "start": 6973, "end": 6983}]}, {"trigger": {"text": "through", "start": 6984, "end": 6991}, "arguments": [{"role": "Theme", "text": "increased", "start": 6942, "end": 6951}, {"role": "Cause", "text": "degradation", "start": 7012, "end": 7023}]}, {"trigger": {"text": "mediate", "start": 7594, "end": 7601}, "arguments": [{"role": "Theme", "text": "activation", "start": 7621, "end": 7631}]}, {"trigger": {"text": "activation", "start": 7621, "end": 7631}, "arguments": [{"role": "Theme", "text": "p65", "start": 7645, "end": 7648}, {"role": "Cause", "text": "phosphorylating", "start": 7652, "end": 7667}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 4604, "end": 4615}, "arguments": [{"role": "Theme", "text": "p65", "start": 4577, "end": 4580}]}, {"trigger": {"text": "proteolysis", "start": 4638, "end": 4649}, "arguments": [{"role": "Theme", "text": "p65", "start": 4577, "end": 4580}]}, {"trigger": {"text": "degradation", "start": 5837, "end": 5848}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 5824, "end": 5836}]}, {"trigger": {"text": "degradation", "start": 6296, "end": 6307}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 6283, "end": 6295}]}, {"trigger": {"text": "degradation", "start": 7012, "end": 7023}, "arguments": [{"role": "Theme", "text": "p100", "start": 7031, "end": 7035}]}], "protein modification": [{"trigger": {"text": "Post-translational modification", "start": 1961, "end": 1992}, "arguments": [{"role": "Theme", "text": "p65", "start": 2006, "end": 2009}]}, {"trigger": {"text": "post-translational modification", "start": 3229, "end": 3260}, "arguments": [{"role": "Theme", "text": "p65", "start": 3274, "end": 3277}]}, {"trigger": {"text": "post-translational modification", "start": 6104, "end": 6135}, "arguments": [{"role": "Theme", "text": "p65", "start": 6149, "end": 6152}]}], "regulation": [{"trigger": {"text": "modulated", "start": 2785, "end": 2794}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 2799, "end": 2814}]}, {"trigger": {"text": "point mutation", "start": 3003, "end": 3017}, "arguments": [{"role": "Site", "text": "Ser536", "start": 3021, "end": 3027}, {"role": "Theme", "text": "p65", "start": 3041, "end": 3044}]}, {"trigger": {"text": "dependent", "start": 3488, "end": 3497}, "arguments": [{"role": "Theme", "text": "activated", "start": 3396, "end": 3405}, {"role": "Cause", "text": "PKCalpha", "start": 3479, "end": 3487}]}, {"trigger": {"text": "affect", "start": 6157, "end": 6163}, "arguments": [{"role": "Cause", "text": "post-translational modification", "start": 6104, "end": 6135}, {"role": "Theme", "text": "interaction", "start": 6168, "end": 6179}]}, {"trigger": {"text": "involved", "start": 6271, "end": 6279}, "arguments": [{"role": "Theme", "text": "degradation", "start": 6296, "end": 6307}]}, {"trigger": {"text": "involved", "start": 6271, "end": 6279}, "arguments": [{"role": "Theme", "text": "induced", "start": 6347, "end": 6354}]}]}}, "schema": []}
{"input": "Materials\nRecombinant human M-CSF was purchased from R&D Systems (Minneapolis, MN). Ro-31-8220, Go-6976, Rotterin, and BAPTA/AM were obtained from Calbiochem (San Diego, CA). Low endotoxin RPMI and X-vivo serum free medium were obtained from Lonza Walkersville Inc (Walkersville, MD). Fetal bovine serum (FBS, certified <0.06 endotoxin units/ml endotoxin levels) was purchased from Atlanta Biological (Lawrenceville, GA). All other cell culture reagents were obtained through Invitrogen (Carlsbad, CA). PKCalpha NF-kappaB p65, Lamin B, beta-actin, and GAPDH antibodies, anti-mouse and rabbit IgG-HRP conjugated antibodies, and PKCalpha siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-NFkappaB p65 (Ser276 or Ser536) antibodies were purchased from Cell Signaling Technology (Danvers, MA). DNA constructs pTAL-SEAP and pNF-kappaB-SEAP were obtained from Clontech (Mountian View, CA). Expression vectors containing CMV-p65 WT, CMV-p65 276S/A and CMV-p65 536S/A were cloned in pBa5e Puro vector or pFLAG-CMV-2 vector as described previously [49]. Kinase deficient CMV-PKCalpha (KD-PKCalpha) or wild type pCMV-PKCalpha (WT-PKCalpha) were generous gifts from Alexandra C. Newton (University of California, San Diego, CA). IkappaBalpha (NFKBIA) and GAPDH primers for real-time RT-PCR were obtained from SABiosciences (Frederick, MD). A pool of mouse or human PKCalpha specific siRNA were purchased from Santa Cruz Biotechnology (Santa cruz, CA).", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "Kinase deficient", "start": 1071, "end": 1087}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1092, "end": 1100}]}, {"trigger": {"text": "KD", "start": 1102, "end": 1104}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 1105, "end": 1113}]}]}}, "schema": []}
{"input": "Cell Culture\nThe murine macrophage cell line RAW 264.7 was purchased from ATCC (Manassas, VA). RAW 264.7 cells were maintained in RPMI supplemented with 5% FBS and antibiotic-antimycotic (1000 U/ml penicillin, 1000 microg/ml streptomycin sulfate, and 250 ng/ml amphotericin B) at 37degreesC. Mouse embryonic fibroblasts (MEFs) cell line lacking specific NFkappaB signaling subunits NF-kappaB p65 (p65-/- cell line) [50] were cultured in DMEM medium supplemented with 10% FBS and antibiotic-antimycotic solution.", "output": {"json_structures": {}}, "schema": []}
{"input": "Purification of Peripheral Blood Monocytes and Monocyte-Derived Macrophages (MDMs)\nMonocytes were isolated from source leukocyte packs obtained from the American Red Cross as described previously [51]. Monocytes used in real time PCR experiments and transfection experiments were purified by positive selection using CD14 Monocyte Isolation Kit from Miltenyi Biotech (Auburn, CA) (>90% pure). In some experiments, monocytes were isolated by clumping method (70% pure). To obtain monocyte-derived macrophages (MDMs), monocytes were cultured in RPMI-1640 medium containing 10% FBS, and 10 microg/ml polymyxin B and 20 ng/ml M-CSF.", "output": {"json_structures": {}}, "schema": []}
{"input": "Electrophoresis of the Mobility Shift Analysis (EMSA)\nNuclear extracts were isolated from MDMs by using a nuclear extraction kit according to the manufacturer's instruction from Active Motif (Carlsbad, CA). Briefly, MDMs were lysed in hypotonic lysis buffer (20 mM Hepes, pH 7.5; 5 mM NaF, 10 microM Na2MoO4 0.5% NP-40 and 0.1 mM EDTA), and then nuclei were resuspended in lysis buffer supplemented with 0.5 mM DTT and 0.2 mM PMSF. The NF-kappaB consensus oligonucleotides (sense: AGTTGAGGGGACTTTCCCAGGC; antisense: GCCTGGGAAAGTCCCCTCAACT) labeled with 32P by T4 polynucleokinase (Promega, Madison, WI) were incubated with nuclear extracts in binding buffer (10 mM Tris pH 7.6, 1 mM DTT, 0.5 mM EDTA, 2 microg polydI-dC and 10% Glycerol) at 30degreesC for 30 minutes. The free DNA and DNA-protein mixtures were resolved in 5% native polyacrylamide gels in 0.5x TBE buffer (45 mM Tris, 45 mM boric acid and 1 mM EDTA, pH 8.3) by electrophoresis. The gel was dried and subjected to autoradiography analysis.", "output": {"json_structures": {}}, "schema": []}
{"input": "Transient Transfection of RAW 264.7 Cells\nRAW 264.7 cells were seeded in 12-well plates in RPMI medium containing 5% FBS one day prior to transfection, Secreted alkaline phosphatase (SEAP) reporter plasmids pTAL-SEAP or pNF-kappaB-SEAP were transfected into cells using Qiagene Effectene or Attractene transfection kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. For co-transfection studies, PKCalpha or NF-kappaB p65 constructs were mixed at a ratio of 5:1 with the reporter plasmid. For siRNA transfection, 100 nM of PKCalpha siRNA or control siRNA were used. Cells were incubated with the transfection reagents for 16-24 hours, washed and starved in RPMI without FBS for 4 hours. Samples were then stimulated with 100 ng/ml M-CSF for 4 hours at which point the medium was collected for the SEAP analysis. For inhibition studies, cells were pre-incubated with inhibitors for 30 minutes before M-CSF treatment. On average, we realized 30-40% transfection efficiency as confirmed by GFP expression in Raw 264.7 cells.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "co-transfection", "start": 388, "end": 403}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 413, "end": 421}]}, {"trigger": {"text": "co-transfection", "start": 388, "end": 403}, "arguments": [{"role": "Theme", "text": "p65", "start": 435, "end": 438}]}, {"trigger": {"text": "expression", "start": 1008, "end": 1018}, "arguments": [{"role": "Theme", "text": "GFP", "start": 1004, "end": 1007}]}]}}, "schema": []}
{"input": "Transient Transfection of Primary Human Monocytes\nMonocytes were transfected using the human monocyte Amaxa nucleofector kit (Lonza Walkersville Inc, Walkersville, MD) as previously described [51]. Briefly, monocytes were resuspended in Amaxa Nucleofactor solution at a density of 20x106 cells/ml, and 2 microg of total plasmid DNA 100 nM of PKCalpha siRNA or control siRNA was added and transfected using program Amaxa Y-01. For co-transfection studies, PKCalpha or NF-kappaB p65 constructs were mixed at a ratio of 5:1 with the reporter plasmid. Immediately after transfection, cells were washed with 1 ml of RPMI medium containing 2 mM glutamine and 10% FBS or serum free LGM medium (Lonza Walkersville Inc.) then plated at 4x105 cells/well in 24-well plates. The original medium was replaced by serum free LGM medium without or with M-CSF (100 ng/ml) one hour later. The next day, cell-free supernatants were collected for the SEAP analysis. Cells were stained with Annexin V/propidium iodide (PI). For the inhibition studies, cells were pre-incubated with inhibitor for 30 minutes in X-vivo medium prior to the addition of M-CSF.", "output": {"json_structures": {}}, "schema": []}
{"input": "Secreted Alkaline Phosphatase (SEAP) Analysis\nSecreted alkaline phosphatase activity was analyzed by GreatEscape SEAP kit according to the manufacturer's instruction. Briefly, medium was diluted in the dilution buffer and heated to 65degreesC to inactivate endogenous phosphatases then incubated with the substrate for 30 minutes. The chemiluminence signal was recorded using a luminometer.", "output": {"json_structures": {}}, "schema": []}
{"input": "Annexin V/PI Apoptosis Assay\nCell apoptosis assay was performed as described previously [51] using the Annexin V-FITC apoptosis detection kit (BD PharMingen, San Diego, CA). Briefly, human monocytes (5x106) were incubated with inhibitors for 30 minutes in X-vivo medium and restimulated with 100 ng/ml M-CSF overnight. The cells were removed from the culture dish using Accutase (eBioscience, San Diego, CA) and stained with Annexin V-FITC and PI and analyzed by flow cytometry (FACSCalibur; BD PharMingen). Annexin V-FITC and PI double negative cells were considered non-apoptotic cells for statistical analysis.", "output": {"json_structures": {}}, "schema": []}
{"input": "Western Blot Analysis\nCells were washed with PBS and resuspended in cell lysis buffer. (Cell Signaling Technology) and incubated for 10 minutes on ice then centrifuged to remove the insoluble fraction. The protein concentration was determined by the BCA protein assay (Bio-Rad, Hercules, CA). Cell lysates were separated by SDS-PAGE on 10% polyacrylamide gels and then transferred onto nitrocellulose membranes and subjected to Western blotting. The immunoblotted proteins were detected by ECL reagent (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).", "output": {"json_structures": {}}, "schema": []}
{"input": "Analysis of PKCalpha Kinase Activity\nHuman MDMs were starved for 2 hours before stimulation with M-CSF (0-60 minutes), then washed with cold PBS and removed from the plates. The cells were resuspended in lysis buffer (20 mM Tris, 5 mM MgCl2, 1 mM EGTA, 20 mM beta-glycerol phosphate, 1 mM PMSF 2 microg/ml aprotinin and 2 microg/ml leupeptin, pH 7.5), sonicated and centrifuged to obtain whole cell lysates, which were then immunoprecipitated with anti-PKCalpha antibody and protein G-agarose (Invitrogen). Immune complexes were washed twice, and PKCalpha activity was analyzed by non-radioactive Peptag assay kit (Promega). Briefly, kinase buffer, activator, peptide protection solution and Peptag peptide were incubated with the immune complexes at 30degreesC for 30 minutes. Reactions were stopped by boiling for 10 minutes and samples were separated on 0.8% agarose gel. Phosphorylated peptide migrated toward the anode (+), while non-phosphorylated peptide migrated toward the cathode (-). Fluorescein-tagged peptides were visualized by UV light.", "output": {"json_structures": {}}, "schema": []}
{"input": "RNA Isolation and Quantitative Real-time PCR\nMDMs or RAW 264.7 cells were serum-starved overnight or for 2 hours, respectively, prior to incubating with inhibitors for 30 minutes. The cells were restimulated with 100 ng/ml M-CSF and total mRNA was extracted from cells with Trizol (Invitrogen) and 1-2 microg of total RNA was used to synthesized cDNA using SuperScript III (Invitrogen). Quantitative real-time (qRT)-PCR was performed using SYBR Green Master Mix (Applied BioSystems, Carlsbad, CA). The reactions were performed using an ABI PRIZM 7700 machine with software Sequence Detector version 1.7 (Applied Biosystems). The target gene values were normalized to the values of GAPDH as a housekeeping gene and expressed as relative fold increase 2(-deltadeltaCt) over the non-stimulated samples (NS).", "output": {"json_structures": {}}, "schema": []}
{"input": "Statistical Analysis\nStatistical comparisons were performed using analysis of variance testing (Minitab software, State Park, PA or SPSS16 software, SPSS Inc. Chicago, IL). Statistical significance was defined as p<=0.05.", "output": {"json_structures": {}}, "schema": []}
{"input": "Ethics Statement\nAll research involving human blood (Buffy coat) were ordered from American Red cross and have been approved by the Ohio State University review board (ARC IRB Protocol #2001-26).", "output": {"json_structures": {}}, "schema": []}
{"input": "M-CSF induces NF-kappaB transcriptional activity in macrophages.\n(A) Nuclei were extracted from human MDMs treated without or with M-CSF for 15 or 30 minutes. NF-kappaB DNA binding activity was analyzed by EMSA Shown is a representative blot from three independent experiments. NS: non-stimulated. (B) Human MDMs transiently transfected with pTAL-SEAP or pNF-kappaB-SEAP constructs were treated with M-CSF in X-vivo medium and incubated for 6 hours before the collection of medium. NF-kappaB activity was analyzed by measuring the amount of SEAP secreted into the medium and data are expressed as fold increase of SEAP activity over that in pTAL-SEAP transfected resting cells. (C) RAW 264.7 cells were transiently transfected with pTAL-SEAP or pNF-kappaB-SEAP construct. Cells were serum starved for 4 hours prior to 2 hours stimulation with mouse recombinant M-CSF (100 ng/ml). Culture media was collected to measure SEAP production. Data are expressed mean +/- S.E.M, for three independent experiments.", "output": {"json_structures": {}}, "schema": []}
{"input": "Inhibition of PKC reduces NF-kappaB activity in M-CSF stimulated macrophages.\n(A) Human MDMs transfected with pNF-kappaB-SEAP were pre-incubated with inhibitors; Ro-31-8220, Go-6976, or BAPTA/AM for 30 minutes prior to 6 hours M-CSF stimulation. (B) RAW 264.7 cells transfected with the pNF-kappaB-SEAP construct were pre-incubated with inhibitors for 30 minutes prior to 2 hours of stimulation with mouse recombinant M-CSF. The NF-kappaB activity was analyzed by measuring SEAP production in the medium and data are expressed as fold increase of SEAP activity over that in pTAL-SEAP transfected resting cells. The graph represents mean +/- S.E.M for three independent experiments. *The p-values of cells treated with inhibitors/M-CSF compared to vehicle/M-CSF were <=0.05.", "output": {"json_structures": {}}, "schema": []}
{"input": "Inhibition of PKC or NF-kappaB induces apoptosis in MDMs.\n(A) MDMs were pre-incubated in RPMI medium containing inhibitors (Ro-31-8220: 5 microM, Go-6976: 5 microM, BAPTA/AM: 2.5 microM) for 30 minutes prior to the addition of M-CSF. As a control, untreated cells were incubated with dimethyl sulfoxide (DMSO). Cell lysates were resolved by SDS-PAGE and immunoblotted with antibody recognizing the active cleaved form of caspase-3. The blots were reblotted with beta-actin that served as a loading control. The ratio of active caspase-3 bands (17 kD and 19 kD) to beta-actin control was determined by densitometry analysis (bottom panel). Data represents the mean +/- S.E.M from two independent donors. (B) Apoptosis of the treated MDMs was also measured by Annexin V-FITC and propidium iodine (PI) staining and analyzed by flow cytometry. The percentage of surviving cells (Annexin V/PI negative) for the cells treated with vehicle/M-CSF was arbitrarily set as 100. Data shown represent the mean +/- S.E.M from three independent experiments. *The p-values of inhibitors/M-CSF compared to vehicle/M-CSF were <=0.05.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "negative", "start": 888, "end": 896}, "arguments": [{"role": "Theme", "text": "Annexin V", "start": 875, "end": 884}]}], "positive regulation": [{"trigger": {"text": "active", "start": 520, "end": 526}, "arguments": [{"role": "Theme", "text": "caspase-3", "start": 527, "end": 536}]}]}}, "schema": []}
{"input": "M-CSF activates PKCalpha in human MDMs.\nMDMs treated with M-CSF (100 ng/ml) for varying amounts of time were lysed and immunoprecipitated using anti-PKC antibody or control IgG antibody. One half of the samples was used to analyze PKC kinase activity using a fluorescein tagged peptide and visualized by agarose gel electrophoresis (top panel), while the other half was subjected to Western blot analysis to confirm equal amounts of PKCalpha were immunoprecipitated from each sample (middle panel). The kinase assay was quantitated using Quantity One software (Bio-Rad) (bottom panel). Data represents the average fold increase of PKCalpha activity in non-stimulated samples compared to M-CSF-treated MDM +/- S.E.M for three independent experiments. NS: non-stimulated. * The p-values of M-CSF stimulated compared to non-stimulated were <=0.05.", "output": {"json_structures": {"positive regulation": [{"trigger": {"text": "activates", "start": 6, "end": 15}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 16, "end": 24}]}]}}, "schema": []}
{"input": "M-CSF-induced PKC activity does not regulate IkappaBalpha degradation but regulates the phosphorylation of NF-kappaB p65 at Ser276.\n(A) MDMs were pretreated with cycloheximide (CHX) in the absence or presence of Ro-31-8220 for 30 minutes prior to M-CSF stimulation for the indicated times. Whole cell lysates were subjected to Western blotting with anti-IkappaBalpha antibody. Data are representative of three independent experiments. (B) MDMs were pretreated with either vehicle or Ro-31-8220 for 30 minutes before the addition of M-CSF for 10 minutes. Whole cell lysates were resolved by SDS-PAGE and phospho-Ser276 or phospho-Ser536 of NF-kappaB p65 was detected using phospho-specific antibodies to either residue of NF-kappaB p65. (C) Whole cell lysates of RAW 264.7 cells treated with vehicle control or Ro-31-8220 in the absence or presence of M-CSF were subjected to Western blot analysis with phospho-Ser276 or phospho-Ser536 NF-kappaB p65 antibodies. (D) Cytosolic and nuclear fractions of RAW 264.7 were obtained from the treated cells and immunoblotted for phospho-NF-kappaB. The purity of the cytosolic and nuclear fractions was confirmed by immunoblotting with GAPDH and Lamin B, respectively. Shown are representative blots from three independent experiments.", "output": {"json_structures": {"phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 88, "end": 103}, "arguments": [{"role": "Theme", "text": "p65", "start": 117, "end": 120}, {"role": "Site", "text": "Ser276", "start": 124, "end": 130}]}, {"trigger": {"text": "phospho", "start": 603, "end": 610}, "arguments": [{"role": "Site", "text": "Ser276", "start": 611, "end": 617}, {"role": "Theme", "text": "p65", "start": 649, "end": 652}]}, {"trigger": {"text": "phospho", "start": 621, "end": 628}, "arguments": [{"role": "Site", "text": "Ser536", "start": 629, "end": 635}, {"role": "Theme", "text": "p65", "start": 649, "end": 652}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 58, "end": 69}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 45, "end": 57}]}], "regulation": [{"trigger": {"text": "regulate", "start": 36, "end": 44}, "arguments": [{"role": "Theme", "text": "degradation", "start": 58, "end": 69}]}, {"trigger": {"text": "regulates", "start": 74, "end": 83}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 88, "end": 103}]}]}}, "schema": []}
{"input": "Inhibition of M-CSF-induced PKC reduces NF-kappaB-regulated genes in both MDMs and RAW 264.7 cells.\nMDMs (A and C) and RAW 264.7 (B) cells were pretreated with Ro-31-8220 or solvent control DMSO for 30 minutes prior to M-CSF stimulation for the indicated times. Total RNA was isolated and converted to cDNA. Real-time RT PCR was performed using primers for IkappaBalpha, BCL-xl or GAPDH. Data are expressed as relative fold increase of IkappaBalpha or BCL-xl gene expression upon treatment over non-stimulated cells. Data represent the mean +/- S.E.M for three independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 464, "end": 474}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 436, "end": 448}]}, {"trigger": {"text": "expression", "start": 464, "end": 474}, "arguments": [{"role": "Theme", "text": "BCL-xl", "start": 452, "end": 458}]}], "positive regulation": [{"trigger": {"text": "increase", "start": 424, "end": 432}, "arguments": [{"role": "Theme", "text": "expression", "start": 464, "end": 474}]}]}}, "schema": []}
{"input": "PKCalpha regulates phosphorylation of NF-kappaB p65 at Ser276.\n(A) MDM or (B) RAW 264.7 cells were transiently transfected with pNF-kappaB-SEAP along with either WT-PKCalpha or the kinase-deficient (KD)-PKCalpha construct at a 1:5 ratio. Cells were serum starved and stimulated with M-CSF and then SEAP secretion in the medium was measured. Data is from of three independent experiments. The p-value of cells transfected with KD compared to those transfected with WT was 0.05. (C) MDMs were removed from the plate using accutase and apoptosis of MDMs was measured by flow cytometry using Annexin V-FITC and propidium iodine (PI). (D) Whole cell lysates from the transfected RAW 264.7 cells were subjected to Western blot analysis with phospho-Ser276 or phospho-Ser536 NF-kappaB p65 antibodies. Blots were immunoblotted with PKCalpha to determine equal protein expression for the PKCalpha constructs. beta-actin served as a loading control. Shown is a representative blot from three independent experiments. (E) MDM or (F) RAW 264.7 cells were transiently transfected with a pNF-kappaB-SEAP along with either 100 nM PKCalpha siRNA or control siRNA for 20-24 hours. Cells were serum starved for 2-4 hours and stimulated with 100 ng/ml M-CSF for 6 hours for MDM or RAW 264.7 for 2 hours and then SEAP secretion in the medium was measured. Shown is data of three independent experiments. (G) MDMs were removed from the plate using accutase and apoptosis of MDMs was measured by Annexin V-FITC and propidium iodine (PI) staining and analyzed by flow cytometry. (H) Whole cell lysates from the transfected MDM and RAW 264.7 cells were subjected to Western blot analysis with PKCalpha antibody. beta-actin served as a loading control. Shown is a representative blot from at least three independent experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "transfected", "start": 111, "end": 122}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 165, "end": 173}]}, {"trigger": {"text": "transfected", "start": 111, "end": 122}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 203, "end": 211}]}, {"trigger": {"text": "expression", "start": 860, "end": 870}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 879, "end": 887}]}, {"trigger": {"text": "transfected", "start": 1055, "end": 1066}, "arguments": [{"role": "Theme", "text": "PKCalpha siRNA", "start": 1115, "end": 1129}]}], "negative regulation": [{"trigger": {"text": "kinase-deficient", "start": 181, "end": 197}, "arguments": [{"role": "Theme", "text": "PKCalpha", "start": 203, "end": 211}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 19, "end": 34}, "arguments": [{"role": "Theme", "text": "p65", "start": 48, "end": 51}, {"role": "Site", "text": "Ser276", "start": 55, "end": 61}]}], "regulation": [{"trigger": {"text": "regulates", "start": 9, "end": 18}, "arguments": [{"role": "Cause", "text": "PKCalpha", "start": 0, "end": 8}, {"role": "Theme", "text": "phosphorylation", "start": 19, "end": 34}]}]}}, "schema": []}
{"input": "NF-kappaB p65 Ser276 is essential in regulating NF-kappaB activity.\n(A) Raw 264.7 cell line was transiently transfected with pNF-kappaB-SEAP along with empty vector or plasmid encoding either NF-kappaB p65 WT or NF-kappaB p65 276S/A. The cells were transfected for 18-24 hours, serum starved for 4 hours, and then incubated with 10 microM of Ro-31-8220 for 30 minutes prior to treatment with 100 ng/ml of M-CSF for 2 hours and SEAP secretion in the medium was measured. (B) NF-kappaB p65-/- cell line was transiently transfected with pNF-kappaB-SEAP along with empty vector or plasmid encoding either NF-kappaB p65 WT, NF-kappaB p65 276S/A, or NF-kappaB p65 536S/A. The cells were cultured for 24 hours and then serum starved for 4 hours. Cells were then incubated in fresh DMEM medium for 2 hours and SEAP secretion in the medium was measured. The results shown are fold change over empty vector + pTAL-SEAP. (C) NF-kappaB p65-/- cell line was transiently transfected with pNF-kappaB-SEAP with either empty vector or plasmid encoding either NF-kappaB p65 WT or NF-kappaB p65 276S/A. The cells were transfected for 24 hours and serum starved for 4 hours, and then incubated with 10 microM of Ro-31-8220 for 30 minutes prior to treatment with 10 ng/ml of TNFalpha. The supernatant were collected after 2 hours of treatment and SEAP secretion in the medium was measured. The results shown are the fold change over empty vector + pNF-kappaB-SEAP. Data shown are mean +/- S.E.M for at least three independent experiments performed in duplicate.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "transfected", "start": 108, "end": 119}, "arguments": [{"role": "Theme", "text": "p65", "start": 202, "end": 205}]}, {"trigger": {"text": "transfected", "start": 108, "end": 119}, "arguments": [{"role": "Theme", "text": "p65", "start": 222, "end": 225}]}, {"trigger": {"text": "transfected", "start": 517, "end": 528}, "arguments": [{"role": "Theme", "text": "p65", "start": 611, "end": 614}]}, {"trigger": {"text": "transfected", "start": 517, "end": 528}, "arguments": [{"role": "Theme", "text": "p65", "start": 629, "end": 632}]}, {"trigger": {"text": "transfected", "start": 517, "end": 528}, "arguments": [{"role": "Theme", "text": "p65", "start": 654, "end": 657}]}, {"trigger": {"text": "transfected", "start": 957, "end": 968}, "arguments": [{"role": "Theme", "text": "p65", "start": 1052, "end": 1055}]}, {"trigger": {"text": "transfected", "start": 957, "end": 968}, "arguments": [{"role": "Theme", "text": "p65", "start": 1072, "end": 1075}]}]}}, "schema": []}
{"input": "Proposed model for M-CSF-induced monocyte survival via PKC regulation by activating NF-kappaB p65 phosphorylation at Ser276.", "output": {"json_structures": {"phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 98, "end": 113}, "arguments": [{"role": "Theme", "text": "p65", "start": 94, "end": 97}, {"role": "Site", "text": "Ser276", "start": 117, "end": 123}]}], "positive regulation": [{"trigger": {"text": "activating", "start": 73, "end": 83}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 98, "end": 113}]}]}}, "schema": []}
{"input": "SHARPIN Is Essential for Cytokine Production, NF-kappaB Signaling, and Induction of Th1 Differentiation by Dendritic Cells\nSpontaneous mutations of the Sharpin (SHANK-associated RH domain-interacting protein, other aliases: Rbckl1, Sipl1) gene in mice result in systemic inflammation that is characterized by chronic proliferative dermatitis and dysregulated secretion of T helper1 (Th1) and Th2 cytokines. The cellular and molecular mechanisms underlying this inflammatory phenotype remain elusive. Dendritic cells may contribute to the initiation and progression of the phenotype of SHARPIN-deficient mice because of their pivotal role in innate and adaptive immunity. Here we show by flow cytometry that SHARPIN- deficiency did not alter the distribution of different DC subtypes in the spleen. In response to TOLL-like receptor (TLR) agonists LPS and poly I:C, cultured bone marrow-derived dendritic cells (BMDC) from WT and mutant mice exhibited similar increases in expression of co-stimulatory molecules CD40, CD80, and CD86. However, stimulated SHARPIN-deficient BMDC had reduced transcription and secretion of pro-inflammatory mediators IL6, IL12P70, GMCSF, and nitric oxide. Mutant BMDC had defective activation of NF-kappaB signaling, whereas the MAPK1/3 (ERK1/2) and MAPK11/12/13/14 (p38 MAP kinase isoforms) and TBK1 signaling pathways were intact. A mixed lymphocyte reaction showed that mutant BMDC only induced a weak Th1 immune response but stimulated increased Th2 cytokine production from allogeneic naive CD4+ T cells. In conclusion, loss of Sharpin in mice significantly affects the immune function of DC and this may partially account for the systemic inflammation and Th2-biased immune response.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "secretion", "start": 1106, "end": 1115}, "arguments": [{"role": "Theme", "text": "IL6", "start": 1146, "end": 1149}]}, {"trigger": {"text": "secretion", "start": 1106, "end": 1115}, "arguments": [{"role": "Theme", "text": "GMCSF", "start": 1160, "end": 1165}]}], "negative regulation": [{"trigger": {"text": "mutations", "start": 135, "end": 144}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 152, "end": 159}]}, {"trigger": {"text": "deficient", "start": 593, "end": 602}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 585, "end": 592}]}, {"trigger": {"text": "deficiency", "start": 716, "end": 726}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 707, "end": 714}]}, {"trigger": {"text": "deficient", "start": 1061, "end": 1070}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 1053, "end": 1060}]}, {"trigger": {"text": "reduced", "start": 1080, "end": 1087}, "arguments": [{"role": "Cause", "text": "deficient", "start": 1061, "end": 1070}, {"role": "Theme", "text": "transcription", "start": 1088, "end": 1101}]}, {"trigger": {"text": "reduced", "start": 1080, "end": 1087}, "arguments": [{"role": "Cause", "text": "deficient", "start": 1061, "end": 1070}, {"role": "Theme", "text": "secretion", "start": 1106, "end": 1115}]}, {"trigger": {"text": "loss", "start": 1554, "end": 1558}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 1562, "end": 1569}]}], "transcription": [{"trigger": {"text": "transcription", "start": 1088, "end": 1101}, "arguments": [{"role": "Theme", "text": "IL6", "start": 1146, "end": 1149}]}, {"trigger": {"text": "transcription", "start": 1088, "end": 1101}, "arguments": [{"role": "Theme", "text": "GMCSF", "start": 1160, "end": 1165}]}]}}, "schema": []}
{"input": "SHARPIN was originally identified in post-synaptic densities of excitatory synapses in the brain of rats [1], but this protein is widely expressed in a variety of tissues [2]. Two allelic, autosomal recessive mutations in the Sharpin gene occurred spontaneously in two inbred strains of mice, C57B/KaLawRij-Sharpincpdm/Sharpincpdm and CBy.Ocb3/Dem-Sharpincpdm-Dem/Sharpincpdm-Dem, resulting in premature termination of mRNA synthesis and absence of a functional protein product [2]. Despite different genetic backgrounds, both mutations cause similar inflammatory disease with severe chronic progressive dermatitis and defective development of secondary lymphoid organs [2]-[4]. The dermatitis becomes clinically apparent at about four weeks of age. There are accumulations of eosinophils, neutrophils and macrophages in the skin of Sharpincpdm/Sharpincpdm mutant mice (hereafter referred to as cpdm mice) associated with increased expression of Th2 cytokines in the skin and in the supernatants of activated splenocytes [5]. The mice have an impaired delayed type hypersensitivity response and decreased secretion of IFNgamma [5], indicating a defect in Th1 immune responses and a bias towards a Th2 immune response. Systemic treatment of cpdm mice with recombinant IL12 caused complete remission of the dermatitis [5]. Neutralization of IL5 by antibody treatment or crosses with IL5-deficient mice reduced the number of circulating and cutaneous eosinophils, but failed to reduce the onset and severity of the dermatitis [6].\nRecently, three independent groups identified SHARPIN as an essential component of the linear ubiquitin chain assembly complex (LUBAC) that regulates TNFalpha-induced canonical NF-kappaB signaling [7]-[9]. SHARPIN-deficient mouse embryonic fibroblast (MEF) were sensitized to TNFalpha-induced apoptosis and cell death was implicated as a factor in the dermatitis of cpdm mice [7]-[9].\nDendritic cells (DC) have a sentinel role in sensing pathogen or danger signals and initiate and direct activation of the adaptive immune response [10]. Activated and mature DC can carry processed antigenic peptides, migrate to lymphoid organs, and induce T-cell-mediated immune responses or tolerance. DC direct the differentiation of CD4+ T cells, and hence the type of immune response, through the selective secretion of cytokines. We hypothesized that defective cytokine secretion by DC contributed to the Th2-biased inflammatory phenotype in SHARPIN-deficient mice. The studies reported here found that lack of SHARPIN protein in BMDC caused defective expression of pro-inflammatory mediators and impaired NF-kappaB activation upon ligand stimulation. The ability of cpdm BMDC to stimulate Th1 cytokine production in allogeneic CD4+ T cells was compromised. Taken together, these results reveal that SHARPIN is a novel regulatory molecule in DC biology and suggest that the dysregulated function of SHARPIN-deficient DC plays a role in the cpdm phenotype.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expressed", "start": 137, "end": 146}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 0, "end": 7}]}, {"trigger": {"text": "secretion", "start": 1105, "end": 1114}, "arguments": [{"role": "Theme", "text": "IFNgamma", "start": 1118, "end": 1126}]}], "negative regulation": [{"trigger": {"text": "recessive mutations", "start": 199, "end": 218}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 226, "end": 233}]}, {"trigger": {"text": "termination", "start": 404, "end": 415}, "arguments": [{"role": "Theme", "text": "synthesis", "start": 424, "end": 433}]}, {"trigger": {"text": "absence", "start": 438, "end": 445}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 226, "end": 233}]}, {"trigger": {"text": "decreased", "start": 1095, "end": 1104}, "arguments": [{"role": "Theme", "text": "secretion", "start": 1105, "end": 1114}]}, {"trigger": {"text": "Neutralization", "start": 1321, "end": 1335}, "arguments": [{"role": "Theme", "text": "IL5", "start": 1339, "end": 1342}]}, {"trigger": {"text": "deficient", "start": 1385, "end": 1394}, "arguments": [{"role": "Theme", "text": "IL5", "start": 1381, "end": 1384}]}, {"trigger": {"text": "deficient", "start": 1742, "end": 1751}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 1734, "end": 1741}]}, {"trigger": {"text": "deficient", "start": 2468, "end": 2477}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 2460, "end": 2467}]}, {"trigger": {"text": "lack", "start": 2521, "end": 2525}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 2529, "end": 2536}]}, {"trigger": {"text": "deficient", "start": 2925, "end": 2934}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 2917, "end": 2924}]}], "positive regulation": [{"trigger": {"text": "resulting", "start": 381, "end": 390}, "arguments": [{"role": "Cause", "text": "recessive mutations", "start": 199, "end": 218}, {"role": "Theme", "text": "termination", "start": 404, "end": 415}]}, {"trigger": {"text": "resulting", "start": 381, "end": 390}, "arguments": [{"role": "Cause", "text": "recessive mutations", "start": 199, "end": 218}, {"role": "Theme", "text": "absence", "start": 438, "end": 445}]}], "transcription": [{"trigger": {"text": "synthesis", "start": 424, "end": 433}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 226, "end": 233}]}]}}, "schema": []}
{"input": "Characterization of Sharpin\nOrthologs of SHARPIN protein are found in various species, including human, mouse and rat. Motif prediction programming, using COILS [11] and MotifScan [12], suggests that SHARPIN contains a coiled-coil (CC) domain, a ubiquitin-like (UBL) domain, and a zinc-finger Ran-binding protein 2 (ZFRBP) domain. These functional motifs constitute similar domain profiles that are present in the SHARPIN protein of all three origins (Fig. 1A), suggesting that SHARPIN exerts highly conserved functions across species. Spontaneous mutations in the mouse Sharpin gene results in a complex inflammatory phenotype characterized by severe dermatitis (Fig. 1B), systemic inflammation and an enlarged spleen (Fig. 1C) caused by extramedullary hematopoiesis [3]. The endogenous expression of Sharpin mRNA in BMDC was determined by quantitative real time-PCR (qRT-PCR) following culture in medium only or after stimulation with LPS. Sharpin mRNA was present in BMDC generated from WT mice (Fig. 1D) and its level was modestly decreased by LPS stimulation. There was a significant reduction of Sharpin mRNA (6-7-fold) in BMDC generated from cpdm mice. Transfection of Flag-tagged Sharpin in fibroblasts (NIH3T3) and macrophages (RAW264.7) indicated cytoplasmic localization of the SHARPIN protein (Fig. 1E).", "output": {"json_structures": {"localization": [{"trigger": {"text": "localization", "start": 1269, "end": 1281}, "arguments": [{"role": "ToLoc", "text": "cytoplasmic", "start": 1257, "end": 1268}, {"role": "Theme", "text": "SHARPIN", "start": 1289, "end": 1296}]}], "negative regulation": [{"trigger": {"text": "mutations", "start": 548, "end": 557}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 571, "end": 578}]}, {"trigger": {"text": "decreased", "start": 1035, "end": 1044}, "arguments": [{"role": "Theme", "text": "present", "start": 959, "end": 966}]}, {"trigger": {"text": "reduction", "start": 1089, "end": 1098}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 1102, "end": 1109}]}], "transcription": [{"trigger": {"text": "expression", "start": 788, "end": 798}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 802, "end": 809}]}, {"trigger": {"text": "present", "start": 959, "end": 966}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 942, "end": 949}]}]}}, "schema": []}
{"input": "Phenotyping splenic DC and BMDC from WT and cpdm mice\nDC are heterogeneous and can be categorized into multiple subtypes based on surface markers [13]. To determine if the Sharpin mutation affects DC development in lymphoid tissues, mouse spleens were examined for the distribution of conventional DC (cDC; CD11c+CD8alpha+ and CD11c+CD8alpha-) [13] and plasmacytoid DC (pDC; CD11c-PDCA-1+) [14]. The percentages for splenic cDC and pDC were both reduced in cpdm mice when compared with WT controls (Fig. 2A). However, when gated on CD11c+ cDC, the percentages of CD8alpha+ and CD8alpha- cells were not affected by SHARPIN deficiency (Fig. 2A). Since the spleen of cpdm mice is markedly enlarged and contains three times as many cells (Fig. 1C), the different percentages of splenic cDC and pDC between WT and mutant mice reflect the increased number of total spleen cells rather than a reduction in cDC and pDC numbers. These data indicate that the Sharpin mutation does not affect the distribution of the examined DC subsets in the spleen.\nBMDC from in vitro cultures functionally resemble non-lymphoid tissue DC and monocyte-derived inflammatory DC [15], [16]. The yields of BMDC from WT and cpdm mice were similar. BMDC were CD11c+ and MHC II+ with low expression of co-stimulatory molecules CD40, CD80, and CD86. The TLR3 ligand poly I:C and the TLR4 ligand LPS each activate overlapping but different signaling pathways and were used to induce DC maturation [17], [18]. Incubation with the TLR agonists for 24 hours resulted in increased expression of CD40, CD80, and CD86 on BMDC; however, there was no difference in the expression levels of these markers between WT and cpdm BMDC (Fig. 2B). Thus, SHARPIN deficiency did not influence the expression of co-stimulatory molecules by BMDC.", "output": {"json_structures": {"binding": [{"trigger": {"text": "ligand", "start": 1326, "end": 1332}, "arguments": [{"role": "Theme", "text": "TLR3", "start": 1321, "end": 1325}]}, {"trigger": {"text": "ligand", "start": 1355, "end": 1361}, "arguments": [{"role": "Theme", "text": "TLR4", "start": 1350, "end": 1354}]}], "gene expression": [{"trigger": {"text": "expression", "start": 1543, "end": 1553}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1557, "end": 1561}]}, {"trigger": {"text": "expression", "start": 1543, "end": 1553}, "arguments": [{"role": "Theme", "text": "CD80", "start": 1563, "end": 1567}]}, {"trigger": {"text": "expression", "start": 1543, "end": 1553}, "arguments": [{"role": "Theme", "text": "CD86", "start": 1573, "end": 1577}]}, {"trigger": {"text": "expression", "start": 1627, "end": 1637}, "arguments": [{"role": "Theme", "text": "CD40", "start": 1557, "end": 1561}]}, {"trigger": {"text": "expression", "start": 1627, "end": 1637}, "arguments": [{"role": "Theme", "text": "CD80", "start": 1563, "end": 1567}]}, {"trigger": {"text": "expression", "start": 1627, "end": 1637}, "arguments": [{"role": "Theme", "text": "CD86", "start": 1573, "end": 1577}]}], "negative regulation": [{"trigger": {"text": "mutation", "start": 180, "end": 188}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 172, "end": 179}]}, {"trigger": {"text": "deficiency", "start": 622, "end": 632}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 614, "end": 621}]}, {"trigger": {"text": "mutation", "start": 957, "end": 965}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 949, "end": 956}]}, {"trigger": {"text": "deficiency", "start": 1712, "end": 1722}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 1704, "end": 1711}]}], "positive regulation": [{"trigger": {"text": "resulted", "start": 1521, "end": 1529}, "arguments": [{"role": "Theme", "text": "increased", "start": 1533, "end": 1542}]}, {"trigger": {"text": "increased", "start": 1533, "end": 1542}, "arguments": [{"role": "Theme", "text": "expression", "start": 1543, "end": 1553}]}], "regulation": [{"trigger": {"text": "difference", "start": 1609, "end": 1619}, "arguments": [{"role": "Theme", "text": "expression", "start": 1627, "end": 1637}]}]}}, "schema": []}
{"input": "Production of proinflammatory mediators by cpdm BMDC is impaired\nIncubation with LPS or poly I:C resulted in secretion of IL6, IL12P70, and GMCSF from both WT and cpdm BMDC; however, BMDC from cpdm mice produced significantly less of all three cytokines compared with WT BMDC (Fig. 3A-C, E-F). The amount of nitric oxide generated by mutant BMDC was also significantly reduced compared with WT cells (Fig. 3D), indicating severely disrupted production of proinflammatory mediators from Sharpin-deficient BMDC. In vivo complementation with a Sharpin gene-containing BAC reversed the phenotype of the mutant mice [2], and BMDC generated from these rescued mice secreted significantly more IL12P70 than BMDC from cpdm mice (Fig. 3G), supporting a necessary role of SHARPIN for the production of IL12P70. In addition, the transcript levels of the inflammatory cytokines Il6, Il12p40, Gmcsf, and Ifnb were examined, and these were all significantly reduced in stimulated cpdm BMDC when compared with WT controls (Fig. 4).", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "secretion", "start": 109, "end": 118}, "arguments": [{"role": "Theme", "text": "IL6", "start": 122, "end": 125}]}, {"trigger": {"text": "secretion", "start": 109, "end": 118}, "arguments": [{"role": "Theme", "text": "GMCSF", "start": 140, "end": 145}]}, {"trigger": {"text": "produced", "start": 203, "end": 211}, "arguments": [{"role": "Theme", "text": "IL6", "start": 122, "end": 125}]}, {"trigger": {"text": "produced", "start": 203, "end": 211}, "arguments": [{"role": "Theme", "text": "GMCSF", "start": 140, "end": 145}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 494, "end": 503}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 486, "end": 493}]}], "positive regulation": [{"trigger": {"text": "resulted", "start": 97, "end": 105}, "arguments": [{"role": "Theme", "text": "secretion", "start": 109, "end": 118}]}, {"trigger": {"text": "less", "start": 226, "end": 230}, "arguments": [{"role": "Theme", "text": "produced", "start": 203, "end": 211}]}]}}, "schema": []}
{"input": "Impaired cytokine production by cpdm BMDC is correlated with selective defects in NF-kappaB signaling\nThere are a number of possible explanations for the defective cytokine secretion in stimulated cpdm BMDC, including 1) reduced surface expression of the LPS receptor complex, 2) increased production of anti-inflammatory mediators, 3) increased expression of negative regulators of TLR pathways, and 4) impaired TLR-induced signaling activation.\nWe determined the surface expression of the LPS receptor complex that comprises TLR4, the accessory proteins CD14 and myeloid differentiation factor 2 (MD2/LY96) [19]. Flow cytometric analysis shows that the expression levels of CD14 and TLR4/MD2 between WT and cpdm BMDC were similar (Fig. 5A). We then quantified the secretion of the suppressive cytokines IL10 that can inhibit IL12 secretion in an autocrine manner [20], [21]. The supernatants from LPS-stimulated cpdm BMDC contained significantly lower levels of IL10 than stimulated WT BMDC (Fig. 5B), suggesting that IL10 was not responsible for decreased secretion of IL12P70 by cpdm BMDC. Increased expression of a negative regulator of TLR signaling such as A20 [22] may also suppress cytokine secretion. However, the transcript level of A20 was lower in LPS-activated cpdm BMDC than WT controls (Fig. 5C), thereby ruling out overexpression of A20 as a factor in the reduced cytokine production.\nThe transcription of TLR3/4-induced proinflammatory intermediates is tightly regulated by cellular signaling pathways, in particular NF-kappaB, TBK1/IRF3, and MAPK [23]-[27]. We next determined if disrupted NF-kappaB, TBK1/IRF3, and/or MAPK signaling may underlie the impaired cytokine production from stimulated Sharpin-deficient BMDC. Stimulus-induced phosphorylation of the IkappaB kinase (IKK1/2) is an essential step in NF-kappaB signaling, allowing phosphorylation and proteasome-mediated degradation of the NF-kappaB inhibitor IkappaBalpha to release the NF-kappaB transcription factors into the nucleus. The amount of phosphorylated IKK1/2 (p-IKK1/2) and IkappaBalpha (p-IkappaBalpha) following incubation with LPS or poly I:C was severely decreased in cpdm BMDC as compared with WT controls (Fig. 6). The cpdm BMDC exhibited similar levels of TBK1, ERK1/2, and p38 phosphorylation to those of WT cells (Fig. 6). These results indicate that the absence of functional SHARPIN decreased NF-kappaB activation but did not affect TBK1/IRF3, ERK1/2, and p38 signaling in BMDC.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 655, "end": 665}, "arguments": [{"role": "Theme", "text": "CD14", "start": 676, "end": 680}]}, {"trigger": {"text": "expression", "start": 655, "end": 665}, "arguments": [{"role": "Theme", "text": "TLR4", "start": 685, "end": 689}]}, {"trigger": {"text": "expression", "start": 655, "end": 665}, "arguments": [{"role": "Theme", "text": "MD2", "start": 690, "end": 693}]}, {"trigger": {"text": "secretion", "start": 766, "end": 775}, "arguments": [{"role": "Theme", "text": "IL10", "start": 805, "end": 809}]}, {"trigger": {"text": "levels", "start": 954, "end": 960}, "arguments": [{"role": "Theme", "text": "IL10", "start": 964, "end": 968}]}, {"trigger": {"text": "expression", "start": 1104, "end": 1114}, "arguments": [{"role": "Theme", "text": "A20", "start": 1164, "end": 1167}]}], "negative regulation": [{"trigger": {"text": "deficient", "start": 1723, "end": 1732}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 1715, "end": 1722}]}, {"trigger": {"text": "decreased", "start": 2150, "end": 2159}, "arguments": [{"role": "Theme", "text": "phosphorylated", "start": 2028, "end": 2042}]}, {"trigger": {"text": "absence", "start": 2355, "end": 2362}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 2377, "end": 2384}]}], "phosphorylation": [{"trigger": {"text": "phosphorylation", "start": 1857, "end": 1872}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1936, "end": 1948}]}, {"trigger": {"text": "phosphorylated", "start": 2028, "end": 2042}, "arguments": [{"role": "Theme", "text": "IKK1", "start": 2043, "end": 2047}]}, {"trigger": {"text": "phosphorylated", "start": 2028, "end": 2042}, "arguments": [{"role": "Theme", "text": "2", "start": 2048, "end": 2049}]}, {"trigger": {"text": "phosphorylated", "start": 2028, "end": 2042}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 2065, "end": 2077}]}, {"trigger": {"text": "phosphorylation", "start": 2276, "end": 2291}, "arguments": [{"role": "Theme", "text": "TBK1", "start": 2254, "end": 2258}]}, {"trigger": {"text": "phosphorylation", "start": 2276, "end": 2291}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 2260, "end": 2264}]}, {"trigger": {"text": "phosphorylation", "start": 2276, "end": 2291}, "arguments": [{"role": "Theme", "text": "2", "start": 2265, "end": 2266}]}], "positive regulation": [{"trigger": {"text": "lower", "start": 948, "end": 953}, "arguments": [{"role": "Theme", "text": "levels", "start": 954, "end": 960}]}, {"trigger": {"text": "Increased", "start": 1094, "end": 1103}, "arguments": [{"role": "Theme", "text": "expression", "start": 1104, "end": 1114}]}, {"trigger": {"text": "lower", "start": 1252, "end": 1257}, "arguments": [{"role": "Theme", "text": "transcript level", "start": 1224, "end": 1240}]}, {"trigger": {"text": "overexpression", "start": 1332, "end": 1346}, "arguments": [{"role": "Theme", "text": "A20", "start": 1350, "end": 1353}]}, {"trigger": {"text": "allowing", "start": 1848, "end": 1856}, "arguments": [{"role": "Theme", "text": "phosphorylation", "start": 1857, "end": 1872}]}, {"trigger": {"text": "allowing", "start": 1848, "end": 1856}, "arguments": [{"role": "Theme", "text": "degradation", "start": 1897, "end": 1908}]}, {"trigger": {"text": "mediated", "start": 1888, "end": 1896}, "arguments": [{"role": "Theme", "text": "degradation", "start": 1897, "end": 1908}]}], "protein catabolism": [{"trigger": {"text": "degradation", "start": 1897, "end": 1908}, "arguments": [{"role": "Theme", "text": "IkappaBalpha", "start": 1936, "end": 1948}]}], "transcription": [{"trigger": {"text": "transcript level", "start": 1224, "end": 1240}, "arguments": [{"role": "Theme", "text": "A20", "start": 1244, "end": 1247}]}]}}, "schema": []}
{"input": "Th2-biased immunogenicity of stimulated cpdm BMDC\nThe defective IL12 production (Fig. 3A) and Th2-dominant cytokine profile in cpdm mice [5] suggest that the absence of SHARPIN affects the ability of cpdm BMDC to induce T cell differentiation into effector cells. Co-culture of allogeneic naive CD4+ T cells with WT BMDC stimulated with LPS or poly I:C elicited robust IFNgamma production, whereas the concentration of IFNgamma in cpdm BMDC-T cell cultures was significantly lower after LPS stimulation (Fig. 7A), indicating impaired Th1-polarizing abilities of cpdm BMDC. In addition to TLR3/4 agonists, the TLR2 ligand Pam3CYS was used since it has been shown to induce both Th1 and Th2 responses [28]-[30]. Pam3CYS-matured WT BMDC induced robust IFNgamma production at a significantly higher level than cpdm BMDC (Fig. 7A). The reduced Th1 differentiation following Pam3CYS stimulation is consistent with the recent report of decreased IL12 production in cpdm macrophages following TLR2 stimulation [31]. In contrast, more Th2-specific IL4 cytokine was produced in the cpdm BMDC co-cultures than the WT control (Fig. 7B), suggesting Th2-skewed immunogenicity of cpdm BMDC. The production of Th17-specific cytokine IL17A following LPS stimulation of dendritic cells was similar between stimulated WT and cpdm BMDC cocultures (data not shown). Despite the distinct Th1- and Th2-stimulating abilities of WT and cpdm BMDC, they were equally effective in IL2 production from BMDC-T cell co-cultures except for poly I:C stimulation where WT BMDC induced more IL2 than cpdm BMDC (Fig. 7C). The Th2-biased stimulating capability of cpdm BMDC when co-cultured with allogeneic naive CD4+ T cells is consistent with the Th2-dominant cytokine phenotype observed in cpdm mice.", "output": {"json_structures": {"binding": [{"trigger": {"text": "ligand", "start": 614, "end": 620}, "arguments": [{"role": "Theme", "text": "TLR2", "start": 609, "end": 613}, {"role": "Theme2", "text": "Pam3CYS", "start": 621, "end": 628}]}], "gene expression": [{"trigger": {"text": "production", "start": 378, "end": 388}, "arguments": [{"role": "Theme", "text": "IFNgamma", "start": 369, "end": 377}]}, {"trigger": {"text": "concentration", "start": 402, "end": 415}, "arguments": [{"role": "Theme", "text": "IFNgamma", "start": 419, "end": 427}]}, {"trigger": {"text": "production", "start": 758, "end": 768}, "arguments": [{"role": "Theme", "text": "IFNgamma", "start": 749, "end": 757}]}, {"trigger": {"text": "produced", "start": 1056, "end": 1064}, "arguments": [{"role": "Theme", "text": "IL4", "start": 1039, "end": 1042}]}, {"trigger": {"text": "production", "start": 1180, "end": 1190}, "arguments": [{"role": "Theme", "text": "IL17A", "start": 1217, "end": 1222}]}, {"trigger": {"text": "production", "start": 1457, "end": 1467}, "arguments": [{"role": "Theme", "text": "IL2", "start": 1453, "end": 1456}]}], "negative regulation": [{"trigger": {"text": "absence", "start": 158, "end": 165}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 169, "end": 176}]}], "positive regulation": [{"trigger": {"text": "elicited", "start": 353, "end": 361}, "arguments": [{"role": "Theme", "text": "production", "start": 378, "end": 388}]}, {"trigger": {"text": "lower", "start": 475, "end": 480}, "arguments": [{"role": "Theme", "text": "concentration", "start": 402, "end": 415}]}, {"trigger": {"text": "agonists", "start": 595, "end": 603}, "arguments": [{"role": "Theme", "text": "TLR3", "start": 588, "end": 592}]}, {"trigger": {"text": "agonists", "start": 595, "end": 603}, "arguments": [{"role": "Theme", "text": "4", "start": 593, "end": 594}]}, {"trigger": {"text": "induced", "start": 734, "end": 741}, "arguments": [{"role": "Cause", "text": "Pam3CYS", "start": 710, "end": 717}, {"role": "Theme", "text": "production", "start": 758, "end": 768}]}, {"trigger": {"text": "following", "start": 1223, "end": 1232}, "arguments": [{"role": "Theme", "text": "production", "start": 1180, "end": 1190}]}, {"trigger": {"text": "induced", "start": 1543, "end": 1550}, "arguments": [{"role": "Theme", "text": "IL2", "start": 1556, "end": 1559}]}, {"trigger": {"text": "more", "start": 1551, "end": 1555}, "arguments": [{"role": "Theme", "text": "induced", "start": 1543, "end": 1550}]}]}}, "schema": []}
{"input": "The present report showed that null mutations of the mouse Sharpin gene did not affect the steady-state distribution of splenic DC subsets nor the development and phenotype of BMDC. However, loss of SHARPIN significantly diminished the capacity of BMDC to secrete inflammatory cytokines and nitric oxide. The attenuated cytokine production was not due to the presence of anti-inflammatory inhibitors, and can be largely explained by selective inactivation of NF-kappaB signaling. Stimulated cpdm BMDC exhibited Th2-biased T cell-polarizing capabilities, consistent with the Th2 cytokine-dominant phenotype in cpdm mice. Together, these results indicate an indispensible role of SHARPIN in regulating DC immunological functions, disruption of which may contribute to the development of immune diseases.\nSince WT and cpdm mice are both specific pathogen-free, the nature of the trigger of the severe inflammation in cpdm mice is not obvious. One such initiating factor could be endogenous apoptotic and/or necrotic cells that can release danger-associated molecular patterns (DAMPs) to launch and amplify an inflammatory response [32]. Such 'sterile' inflammation could be initiated and take place in all organs and tissues affected in cpdm mice, thus causing multi-organ inflammatory disorders. This hypothesis is supported by several recent studies. Fibroblasts of cpdm mice are highly sensitive to TNFalpha-induced cell death and the cpdm phenotype can be partially rescued by deletion of TNF [7], [8], suggesting that deficiency of SHARPIN compromises the anti-apoptotic mechanisms in cpdm mice resulting in cell death-induced inflammatory disease. Apoptosis of keratinocytes is a prominent feature of the skin lesions in cpdm mice [33] and this is mediated by caspase-dependent mitochondrial pathways [34]. These induced and/or intrinsic apoptotic cells can release various types of DAMPs that exert their pro-inflammatory properties by activating DC through pattern recognition receptors [32], [35], such as HMGB1 recognized by TLR2/4 [36], [37].\nConsistent with the impaired Th1 immune response in cpdm mice [5], stimulated BMDC weakly polarized Th1 differentiation, but strongly supported the development of Th2 effector cells. Combined with the dramatic effect of IL12 treatment on the phenotype of these mice [5], this suggests that the Th2-biased systemic inflammation in cpdm mice is caused by reduced IL12P70 production from DC. The importance of IL12P70 in regulating Th1 and Th2 immune responses in mice was clearly demonstrated in IL12P35- and IL12P40-deficient mice [38], [39] and through clinical studies in human patients [40]-[42].\nNF-kappaB, TBK/IRF3, and MAPK signaling are important pathways activated by LPS or poly I:C and disruption of either pathway may lead to decreased cytokine expression. NF-kappaB activation was selectively inhibited in LPS and poly I:C stimulated cpdm BMDC compared to wild type, while activation of TBK1/IRF3, ERK1/2, or p38 was not, indicating that disrupted NF-kappaB signaling in cpdm DC is responsible for the defective cytokine expression. These results seem to contradict a recent study that found increased levels of NF-kappaB activation and IL1 transcription in cpdm mice [43]. This difference can probably be attributed to the different cells and tissues used in these studies, and may point to cell- and tissue- type specific functions of SHARPIN. This is consistent with recent reports of tissue-specific effects of NF-kappaB signaling [44]. Ubiquitous activation of NF-kappaB by removing inhibitors such as A20 and ITCH through genetic manipulation results in widespread inflammation, consistent with the role of NF-kappaB in the production of pro-inflammatory mediators [45], [46]. However, selective inhibition of NF-kappaB activation in parenchymal cells of the skin, liver, and intestine results in chronic inflammation driven by NF-kappaB competent leukocytes [47]-[50]. This indicates that a balance between the pro-inflammatory role of NF-kappaB in leukocytes and the anti-inflammatory role in parenchymal cells is critical in the maintenance of tissue homeostasis. Experiments with mice with cell- and tissue-specific deletion of Sharpin, currently under way, will help to elucidate the role of SHARPIN in inflammation.\nDespite defective NF-kappaB activation in the absence of Sharpin expression, there was no significant change in splenic DC populations or expression of co-stimulatory molecules on BMDC. Different NF-kappaB subunits involved in the canonical and non-canonical branches of the NF-kappaB signaling pathway have distinct functions to control specific aspects of DC development and function [51], [52]. The non-canonical pathway (p100 processing to produce p52) appears to be intact in SHARPIN-deficient cells [7], [9] suggesting that the NF-kappaB heterodimer p52/RELB is sufficient to maintain the normal regulation of DC homeostasis and maturation.\nThe molecular basis by which the Sharpin mutation causes reduced NF-kappaB activation in BMDC remains to be determined. LPS and poly I:C used here are well-defined ligands that specifically engage TLR4 and TLR3, respectively. The expression profile of surface TLR4 complexes is similar between WT and cpdm BMDC suggesting that defective NF-kappaB activation is not a result of differential TLR expression on target cells. LPS engages TLR4 to activate MYD88-dependent and TRIF-dependent pathways, whereas TLR3 stimulated by poly I:C only triggers TRIF-dependent signaling [53]. The defective NF-kappaB activation by both stimuli suggests that the Sharpin mutation interferes with the protein adaptors or kinases shared by both signaling pathways, such as RIP1 and TRAF6 [18]. Recent studies demonstrated that SHARPIN interacts with HOIP to form LUBAC that exerts its linear-ubiquitin-chain-ligase activity on NF-kappaB signaling players RIP1 and NEMO [7], an essential step for intact TNFalpha-stimulated NF-kappaB activation. The Sharpin null mutation disrupts the ubiquitylation process and abrogates the TNFalpha-induced NF-kappaB signaling pathway. Since TNFR and TLR partially share their downstream signaling cascades, a similar ubiquitin-mediated regulation may hold true for SHARPIN in LPS- and poly I:C-induced NF-kappaB activation.\nIn summary, the present study identified an indispensible role of SHARPIN in the production of pro-inflammatory mediators and TLR-induced NF-kappaB signaling. The impaired Th1-stimulating ability of Sharpin-deficient DC may account for the Th2-dominant inflammatory phenotype of cpdm mice. The balance between Th1 and Th2 differentiation is critical for immune homeostasis. A better understanding of how such balance is maintained will help design cytokine treatment for human diseases with Th2-biased cytokine secretion similar to the mouse cpdm, such as allergies and hypereosinophilic syndromes.", "output": {"json_structures": {"binding": [{"trigger": {"text": "recognized", "start": 2018, "end": 2028}, "arguments": [{"role": "Theme", "text": "HMGB1", "start": 2012, "end": 2017}, {"role": "Theme2", "text": "TLR2", "start": 2032, "end": 2036}]}, {"trigger": {"text": "engage", "start": 5127, "end": 5133}, "arguments": [{"role": "Theme", "text": "TLR4", "start": 5134, "end": 5138}]}, {"trigger": {"text": "engage", "start": 5127, "end": 5133}, "arguments": [{"role": "Theme", "text": "TLR3", "start": 5143, "end": 5147}]}, {"trigger": {"text": "engages", "start": 5363, "end": 5370}, "arguments": [{"role": "Theme", "text": "TLR4", "start": 5371, "end": 5375}]}, {"trigger": {"text": "interacts", "start": 5753, "end": 5762}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 5745, "end": 5752}, {"role": "Theme2", "text": "HOIP", "start": 5768, "end": 5772}]}], "gene expression": [{"trigger": {"text": "expression", "start": 4355, "end": 4365}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 4347, "end": 4354}]}], "negative regulation": [{"trigger": {"text": "null mutations", "start": 31, "end": 45}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 59, "end": 66}]}, {"trigger": {"text": "loss", "start": 191, "end": 195}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 199, "end": 206}]}, {"trigger": {"text": "disruption", "start": 728, "end": 738}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 678, "end": 685}]}, {"trigger": {"text": "deletion", "start": 1478, "end": 1486}, "arguments": [{"role": "Theme", "text": "TNF", "start": 1490, "end": 1493}]}, {"trigger": {"text": "deficiency", "start": 1520, "end": 1530}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 1534, "end": 1541}]}, {"trigger": {"text": "deficient", "start": 2566, "end": 2575}, "arguments": [{"role": "Theme", "text": "IL12P35", "start": 2545, "end": 2552}]}, {"trigger": {"text": "deficient", "start": 2566, "end": 2575}, "arguments": [{"role": "Theme", "text": "IL12P40", "start": 2558, "end": 2565}]}, {"trigger": {"text": "was not", "start": 2975, "end": 2982}, "arguments": [{"role": "Theme", "text": "activation", "start": 2935, "end": 2945}]}, {"trigger": {"text": "deletion", "start": 4188, "end": 4196}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 4200, "end": 4207}]}, {"trigger": {"text": "absence", "start": 4336, "end": 4343}, "arguments": [{"role": "Theme", "text": "expression", "start": 4355, "end": 4365}]}, {"trigger": {"text": "deficient", "start": 4779, "end": 4788}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 4771, "end": 4778}]}, {"trigger": {"text": "mutation", "start": 4978, "end": 4986}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 4970, "end": 4977}]}, {"trigger": {"text": "mutation", "start": 5591, "end": 5599}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 5583, "end": 5590}]}, {"trigger": {"text": "null mutation", "start": 5975, "end": 5988}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 5967, "end": 5974}]}, {"trigger": {"text": "disrupts", "start": 5989, "end": 5997}, "arguments": [{"role": "Theme", "text": "linear-ubiquitin-chain-ligase activity", "start": 5803, "end": 5841}, {"role": "Cause", "text": "null mutation", "start": 5975, "end": 5988}]}, {"trigger": {"text": "deficient", "start": 6485, "end": 6494}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 6477, "end": 6484}]}], "positive regulation": [{"trigger": {"text": "activation", "start": 2935, "end": 2945}, "arguments": [{"role": "Theme", "text": "TBK1", "start": 2949, "end": 2953}]}, {"trigger": {"text": "activation", "start": 2935, "end": 2945}, "arguments": [{"role": "Theme", "text": "IRF3", "start": 2954, "end": 2958}]}, {"trigger": {"text": "activation", "start": 2935, "end": 2945}, "arguments": [{"role": "Theme", "text": "ERK1", "start": 2960, "end": 2964}]}, {"trigger": {"text": "activation", "start": 2935, "end": 2945}, "arguments": [{"role": "Theme", "text": "2", "start": 2965, "end": 2966}]}, {"trigger": {"text": "stimulated", "start": 5446, "end": 5456}, "arguments": [{"role": "Theme", "text": "TLR3", "start": 5441, "end": 5445}]}, {"trigger": {"text": "exerts", "start": 5792, "end": 5798}, "arguments": [{"role": "Theme", "text": "linear-ubiquitin-chain-ligase activity", "start": 5803, "end": 5841}]}], "ubiquitination": [{"trigger": {"text": "linear-ubiquitin-chain-ligase activity", "start": 5803, "end": 5841}, "arguments": [{"role": "Cause", "text": "SHARPIN", "start": 5745, "end": 5752}, {"role": "Theme", "text": "RIP1", "start": 5873, "end": 5877}]}, {"trigger": {"text": "linear-ubiquitin-chain-ligase activity", "start": 5803, "end": 5841}, "arguments": [{"role": "Cause", "text": "SHARPIN", "start": 5745, "end": 5752}, {"role": "Theme", "text": "NEMO", "start": 5882, "end": 5886}]}]}}, "schema": []}
{"input": "Ethics Statement\nAll mouse work was carried out in strict accordance with protocols approved by the Institutional Animal Care and Use Committees.", "output": {"json_structures": {}}, "schema": []}
{"input": "Mice\nSpecific-pathogen free colonies of C57BL/KaLawRij-Sharpincpdm/RijSunJ (JR#7599) and WT mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained in a barrier facility. Normal littermate controls were either +/+ or +/Sharpincpdm. These control animals were phenotypically indistinguishable and are referred to as WT. Sex-matched WT and mutant mice were used at 6-10 weeks of age. For some experiments, cpdm mice were crossed with transgenic mice with a bacterial artificial chromosome (BAC) containing the Sharpin gene (FVB/NJ-Tg(RP24-173I23)1Sun/Sun, JR#8279). These mice were backcrossed onto the C57BL/KaLawRij-Sharpincpdm/RijSunJ background and N4 mice were used in the experiments reported here. All mouse work was carried out in strict accordance with the approved protocols by the Institutional Animal Care and Use Committee.", "output": {"json_structures": {}}, "schema": []}
{"input": "Constructs and transfection\nThe complementary DNA (cDNA) of the mouse Sharpin gene was cloned and amplified from RAW264.7 RNA extracts. The primer sequences were forward, 5'-CC ATG GCG ATG TCG CCG CCC GCC GGC GGT; reverse, 5'- AAG CTT CTA GGT GGA AGC TGC AGC AAG A. The Sharpin cDNA was cloned into expression vector pFLAG-CMV-2. Murine fibroblasts and macrophages were were used to express recombinant SHARPIN protein. Cells (2x104) were seeded in 96-well treated plates the day before transfection. After overnight incubation, 200 ng pFLAG-SHARPIN plasmids were transfected with 0.5 ul Lipofectamine 2000. 24 hours later, cells were incubated with fresh culture medium. After another 24 hours, cells were lysed to confirm the FLAG-SHARPIN expression by immunoblots with anti-FLAG. Cells with no transfection and transfected with empty vector pFLAG-CMV-2 were used as negative control in all transfection experiments.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "express", "start": 383, "end": 390}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 403, "end": 410}]}, {"trigger": {"text": "expression", "start": 741, "end": 751}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 733, "end": 740}]}]}}, "schema": []}
{"input": "Generation of BMDC\nBMDC were developed as previously reported [54], [55]. Cells were collected after 10-12 days of culture. The cell yield was 2-3x107 cells/mouse with 80-95% BMDC.", "output": {"json_structures": {}}, "schema": []}
{"input": "Phenotype of BMDC and splenic DC subsets\nThe phenotype of BMDC was determined before and after 24 hour culture in the presence of 100 ng/mL LPS. The cells were incubated in PBS with 0.1% NaN3, 1% BSA and 10% normal rabbit serum for 20 minutes on ice, washed and incubated for 30 minutes on ice with Alex Fluor-labeled anti-CD11c (MCD11C20, CALTAG) in combination with PE- anti-CD40 (3/23, BD Biosciences), PE-anti-CD80 (16-10A1, eBioscience), PE-anti-CD86 (P03.1, eBioscience), PE-anti-CD14 (Sa2.8, eBioscience) and biotinylated anti-TLR4 (BioLegend). The cells incubated with biotinylated anti-TLR4/MD-2 were washed twice and incubated for 30 minutes on ice with avidin-PE. To isolate splenic DC, spleens were collagenase digested and subject to Percoll gradient centrifugation. The bands at the 35-55% interface were collected to stain with PE-anti-CD11c (HL3, BioLegend), APC-anti-PDCA-1 (927, BioLegend) and FITC-anti-CD8alpha (53-6.7, BD BioScience). The cells were washed twice, fixed in 2% paraformaldehyde, and stored at 4degreesC until analysis. Flow cytometry was performed in an Excel (Coulter) instrument. Dead cells were omitted from the analysis by gating on forward and 90degrees light scatter, and 10,000 cells were analyzed by FlowJo software.", "output": {"json_structures": {}}, "schema": []}
{"input": "BMDC-T cell in vitro interaction\nWT and cpdm BMDC (5x104) were stimulated with medium, 1 microg/mL LPS, 25 microg/mL and 5 microg/mL Pam3CYS. 24 hours later, cells were collected and washed with PBS. Allogeneic naive CD4+ T cells were isolated from spleens of BALB/c mice by negative selection kit (Invitrogen) and were then added at 2.5x105 and co-cultured with activated BMDC. After 5 days, supernatant was collected and the secretion of IFNgamma, IL4, IL2, and IL17A measured by ELISA. Negative controls are 1) stimulated BMDC without co-culture with allogeneic CD4+ T cells; 2) allogeneic CD4+ T cells without co-culture with stimulated BMDC. Both negative controls show no production of aforementioned cytokines.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "secretion", "start": 427, "end": 436}, "arguments": [{"role": "Theme", "text": "IFNgamma", "start": 440, "end": 448}]}, {"trigger": {"text": "secretion", "start": 427, "end": 436}, "arguments": [{"role": "Theme", "text": "IL4", "start": 450, "end": 453}]}, {"trigger": {"text": "secretion", "start": 427, "end": 436}, "arguments": [{"role": "Theme", "text": "IL2", "start": 455, "end": 458}]}, {"trigger": {"text": "secretion", "start": 427, "end": 436}, "arguments": [{"role": "Theme", "text": "IL17A", "start": 464, "end": 469}]}, {"trigger": {"text": "production", "start": 678, "end": 688}, "arguments": [{"role": "Theme", "text": "IFNgamma", "start": 440, "end": 448}]}, {"trigger": {"text": "production", "start": 678, "end": 688}, "arguments": [{"role": "Theme", "text": "IL4", "start": 450, "end": 453}]}, {"trigger": {"text": "production", "start": 678, "end": 688}, "arguments": [{"role": "Theme", "text": "IL2", "start": 455, "end": 458}]}, {"trigger": {"text": "production", "start": 678, "end": 688}, "arguments": [{"role": "Theme", "text": "IL17A", "start": 464, "end": 469}]}]}}, "schema": []}
{"input": "RNA expression by BMDC\nBMDC were cultured at 106 cells/mL in 10 mL of RPMI-1640 complete medium in the presence or absence of 100 ng/mL LPS or 25 microg/mL poly I:C. After 1 and 2 hours, RNA was isolated with TRI-reagent (Sigma) according to the manufacturer's instructions. The expression of Il6, Il12p40, Gmcsf, and Sharpin mRNA was determined by qRT-PCR. Primers and probes were purchased from Applied Biosystems. Reverse transcription was performed at 42degreesC for 60 minutes with the final denaturation step at 90degreesC for 5 minutes in 30 microl containing 0.5 microg of total RNA. dNTPs, oligo(dT)15 primer, recombinant RNasin Ribonuclease Inhibitor, and M-MLV Reverse Transcriptase (all from Promega, Madison, WI) were used according to the manufacturer's instruction. Reverse transcription was done in a PTC-200 Peltier Thermal Cycler (MJ Research, Watertown, MA). qRT-PCR was performed in ABI Prism 7700 Sequence Detection System with TaqMan(R) Gene Expression Assays (Applied Biosystems, Foster City, CA) for mouse Actb, IL6, IL12p40, Ifnbeta, Gmcsf, and Sharpin according to the manufacturer's protocol. The endogenous standard for normalization of the target gene was beta-actin. Relative gene expression was calculated using the 2-deltadeltaCt method [56].", "output": {"json_structures": {"transcription": [{"trigger": {"text": "expression", "start": 279, "end": 289}, "arguments": [{"role": "Theme", "text": "Il6", "start": 293, "end": 296}]}, {"trigger": {"text": "expression", "start": 279, "end": 289}, "arguments": [{"role": "Theme", "text": "Il12p40", "start": 298, "end": 305}]}, {"trigger": {"text": "expression", "start": 279, "end": 289}, "arguments": [{"role": "Theme", "text": "Gmcsf", "start": 307, "end": 312}]}, {"trigger": {"text": "expression", "start": 279, "end": 289}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 318, "end": 325}]}]}}, "schema": []}
{"input": "In vitro cytokine secretion by BMDC\nThe BMDC were cultured in triplicate wells of 24-well or 96-well plates at 106 or 105 cells/mL, respectively, in RPMI-1640 complete medium. The cells were washed and stimulated with 100 ng/mL LPS or 25 microg/mL poly I:C. After 24 hours, supernatants were harvested for ELISA analysis. The presence of nitrite in the supernatants was determined using the Griess reagent.", "output": {"json_structures": {}}, "schema": []}
{"input": "Immunofluorescence\nFibroblast and macrophage cells were transfected with pFLAG-SHARPIN. 48 hours later, cells were washed briefly with PBS, then fixed in cold methanol at -20degreesC for 10 minutes and cold acetone at -20degreesC for 1 minute. Incubate cells with PBS+1% BSA for 15 minutes to block non-specific binding. Fixed cells were then incubated with anti-FLAG (1:100) as the primary antibody at room temperature for 1 hour, followed by three 5-minute washes. Cells were further incubated with goat anti-rabbit IgG-FITC (1:100) as the secondary antibody at room temperature for 30 minutes, followed by three 5-minute washes. Cells were then examined using inverse fluorescence microscope.", "output": {"json_structures": {}}, "schema": []}
{"input": "Immunoblots\nAfter adding stimulating ligands, 100 ng/mL LPS or 25 microg/mL poly I:C, BMDC were collected and lysed at 0-, 15-, 30-, and 60-minute time points. Immunoblots were performed with antibodies (Cell Signaling Technology) against p-IKK1/2(#2697), p-IkappaBalpha (#9246), IkappaBalpha (#4814), p-TBK1(#5483), p-p38 (#9216), p38 (#9212), p-ERK1/2 (#4376), and ERK1/2 (#4695). Beta-actin (sc-47778, Santa Cruz Biotechnology) was used as loading control.", "output": {"json_structures": {}}, "schema": []}
{"input": "Statistical analysis\nData are expressed as mean +/- SD. The statistical significance of differences of means between experimental groups was determined by Students' t-test.", "output": {"json_structures": {}}, "schema": []}
{"input": "In vivo and in vitro features of SHARPIN.\n(A) COILS and MotifScan programs were used to predict the presence of CC (coiled-coil) domain, UBL (ubiquitin-like) domain and ZFRBP (zinc-finger Ran-Binding protein 2) domain which form similar motif patterns in the SHARPIN protein of human, mouse and rat origins. (B) Eight week-old females of WT and cpdm mice. The mutant mice (above) develop progressive skin inflammation starting at about four weeks. (C) Extramedullary hematopoiesis causes marked enlargement of the spleen of cpdm mice. (D) BMDC were incubated in the absence or presence of 100 ng/ml LPS for 4 hours. The mRNA level of Sharpin was measured by qRT-PCR and presented relative to the mRNA expression in non-stimulated WT BMDC. Bars represent the mean +/- s.d. of 3 mice. * P<0.05; ** P<0.001. (E) Fibroblast (NIH3T3) and macrophages (RAW264.7) cells were transfected with the expression plasmid pFLAG-SHARPIN. After 48 hours, cells were fixed and probed with anti-FLAG and FITC-conjugated secondary antibody. Nuclei were stained with DAPI. In both transfected cell lines, FLAG-SHARPIN was found to be cytoplasm-localized.", "output": {"json_structures": {"localization": [{"trigger": {"text": "localized", "start": 1123, "end": 1132}, "arguments": [{"role": "Theme", "text": "SHARPIN", "start": 1089, "end": 1096}, {"role": "ToLoc", "text": "cytoplasm", "start": 1113, "end": 1122}]}], "transcription": [{"trigger": {"text": "mRNA level", "start": 620, "end": 630}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 634, "end": 641}]}]}}, "schema": []}
{"input": "Effect of Sharpin mutation on DC subpopulations and maturation.\n(A) Spleens from WT and cpdm mice were isolated and subject to collagenase and DNase digestion. The obtained splenic homogenates were centrifuged over a Percoll gradient (35% and 55% density) for 15 minutes. The bands at the 35%-medium and the 35-55% interface were pooled, washed and stained with a combination of various antibodies to stain different DC subsets, conventional CD11c+CD8alpha+, CD11c+CD8alpha- and plasmacytoid DC (CD11c-PDCA-1+).The top panels were gated on FSChiSSClo cells to show separate populations of CD11c+PDCA-1- and CD11c-PDCA-1+ cells. Further gating on the CD11c+PDCA-1- subpopulation gave the bottom panel that showed two distinct pools of CD11c+CD8alpha+ and CD11c+CD8alpha- cells. Percentages were calculated based on the parental population and were additionally shown as bar graphs (n = 2) (B). (C) WT and cpdm BMDC (5x105) cells were stimulated with medium, 100 ng/ml LPS or 25 microg/ml poly I:C for 24 hours. The cells were labeled with PE-labeled anti-CD40, anti-CD80, and anti-CD86, and subjected to flow cytometry analysis. The populations shown in histograms were gated on CD11c+ cells. Unstained cells served as negative controls. Results are representative of two independent experiments.", "output": {"json_structures": {"negative regulation": [{"trigger": {"text": "mutation", "start": 18, "end": 26}, "arguments": [{"role": "Theme", "text": "Sharpin", "start": 10, "end": 17}]}]}}, "schema": []}
{"input": "Defective production of pro-inflammatory mediators from stimulated cpdm BMDC.\n(A-D) Cultured WT and cpdm BMDC (1x105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 microg/ml poly I:C for 24 hours. Supernatants were collected for ELISA of IL12P70, IL6, and GMCSF, and for quantification of nitric oxide (NO). (E-F) Gradient numbers (1x104, 2x104, and 4x104 cells in 0.1 mL complete medium) of BMDC were used for100 ng/mL LPS stimulation. After 24 hours, the amounts of IL12P70 and IL6 from the supernatant were measured. (G) The cpdm mice rescued by Sharpin-containing BAC had complete remission of the inflammatory phenotype [2]. BMDC developed from cpdm and rescued cpdm mice were plated (1x106 cells in 0.3 mL) and stimulated with 100 ng/mL LPS. After 24 hours, supernatants were collected for analysis of IL12P70 production. Data are representative of three independent experiments. * P<0.01; ** P<0.005.", "output": {"json_structures": {}}, "schema": []}
{"input": "Decreased mRNA levels of inflammatory cytokines from cpdm BMDC.\nCultured WT and cpdm BMDC (5x105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS (A,C,E,G) or 25 microg/ml poly I:C (B,D,F,H). At 0, 1 and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the expression of Il12p40 (A,B), Il6 (C,D), Gmcsf (E,F), and Ifnb (G,H) mRNA. Bars represent mean +/- SD. Data are representative of two independent experiments.", "output": {"json_structures": {"transcription": [{"trigger": {"text": "expression", "start": 305, "end": 315}, "arguments": [{"role": "Theme", "text": "Il12p40", "start": 319, "end": 326}]}, {"trigger": {"text": "expression", "start": 305, "end": 315}, "arguments": [{"role": "Theme", "text": "Il6", "start": 334, "end": 337}]}, {"trigger": {"text": "expression", "start": 305, "end": 315}, "arguments": [{"role": "Theme", "text": "Gmcsf", "start": 345, "end": 350}]}, {"trigger": {"text": "expression", "start": 305, "end": 315}, "arguments": [{"role": "Theme", "text": "Ifnb", "start": 362, "end": 366}]}]}}, "schema": []}
{"input": "Normal TLR4 and MD2 expression and decreased IL10 secretion and A20 expression by cpdm BMDC.\n(A) Unstimulated WT and cpdm BMDC (5x105) were labeled with PE-labeled anti-TLR4/MD2 or anti-CD14 and then subject to flow cytometry analysis. Unstained cells serve as negative controls. (B) Cultured WT and cpdm BMDC (1x105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 microg/ml poly I:C for 24 hours. Supernatants were collected for ELISA of IL10. (C) Cultured WT and cpdm BMDC (5x105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS or 25 microg/ml poly I:C. At 0, 1, and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the production of A20.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "expression", "start": 20, "end": 30}, "arguments": [{"role": "Theme", "text": "MD2", "start": 16, "end": 19}]}, {"trigger": {"text": "expression", "start": 20, "end": 30}, "arguments": [{"role": "Theme", "text": "TLR4", "start": 7, "end": 11}]}, {"trigger": {"text": "secretion", "start": 50, "end": 59}, "arguments": [{"role": "Theme", "text": "IL10", "start": 45, "end": 49}]}, {"trigger": {"text": "expression", "start": 68, "end": 78}, "arguments": [{"role": "Theme", "text": "A20", "start": 64, "end": 67}]}, {"trigger": {"text": "production", "start": 716, "end": 726}, "arguments": [{"role": "Theme", "text": "A20", "start": 730, "end": 733}]}], "negative regulation": [{"trigger": {"text": "Normal", "start": 0, "end": 6}, "arguments": [{"role": "Theme", "text": "expression", "start": 20, "end": 30}]}, {"trigger": {"text": "decreased", "start": 35, "end": 44}, "arguments": [{"role": "Theme", "text": "secretion", "start": 50, "end": 59}]}, {"trigger": {"text": "decreased", "start": 35, "end": 44}, "arguments": [{"role": "Theme", "text": "expression", "start": 68, "end": 78}]}]}}, "schema": []}
{"input": "Inhibition of NF-kappaB signaling in cpdm BMDC.\nWT and cpdm BMDC (2x106 cells in 0.5 mL complete medium) were stimulated with 100 ng/mL LPS (A) or 25 microg/mL poly I:C (B). At 0, 15, 30, and 60 minutes, whole-cell lysates were obtained and subject to immunoblots with antibodies against proteins involved in NF-kappaB, TBK1/IRF3, ERK1/2, and p38 signaling pathways. Beta-actin was used as loading control. (C) Cellular levels of p-IKK1/2 and p-IkappaBalpha in LPS- or poly I:C-stimulated BMDC were quantitated with ImageJ (NIH) and presented as trend lines. Results are representative of at least two independent experiments.", "output": {"json_structures": {}}, "schema": []}
{"input": "Stimulated cpdm BMDC induced Th2-biased cytokine production from naive CD4+ T cells.\nWT and cpdm BMDC (5x104 cells in 0.1 mL complete medium; MHC haplotype: H-2b) were incubated with medium, 100 ng/mL LPS, 25 microg/mL poly I:C or 5 microg/mL Pam3CYS. After 24 hours, cells were washed with PBS and incubated with freshly isolated allogeneic naive CD4+ T cells (2.5x105 cells in 0.1 mL complete medium; MHC haplotype: H-2d). After 5 days, supernatants were collected and the secretion of IFNgamma, IL4, and IL2 were measured by ELISA. Negative controls are 1) stimulated BMDC without co-culture with allogeneic CD4+ T cells; 2) allogeneic CD4+ T cells without co-culture with stimulated BMDC. Samples from both negative controls had no detectable production of the aforementioned cytokines (not shown). Results are analyzed based on 2-4 mice per group. * P<0.05.", "output": {"json_structures": {"gene expression": [{"trigger": {"text": "secretion", "start": 475, "end": 484}, "arguments": [{"role": "Theme", "text": "IFNgamma", "start": 488, "end": 496}]}, {"trigger": {"text": "secretion", "start": 475, "end": 484}, "arguments": [{"role": "Theme", "text": "IL4", "start": 498, "end": 501}]}, {"trigger": {"text": "secretion", "start": 475, "end": 484}, "arguments": [{"role": "Theme", "text": "IL2", "start": 507, "end": 510}]}, {"trigger": {"text": "production", "start": 747, "end": 757}, "arguments": [{"role": "Theme", "text": "IFNgamma", "start": 488, "end": 496}]}, {"trigger": {"text": "production", "start": 747, "end": 757}, "arguments": [{"role": "Theme", "text": "IL4", "start": 498, "end": 501}]}, {"trigger": {"text": "production", "start": 747, "end": 757}, "arguments": [{"role": "Theme", "text": "IL2", "start": 507, "end": 510}]}]}}, "schema": []}