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• W2061129733 abstract "Elevated systemic levels of the acute phase C-reactive protein (CRP) are predictors of future cardiovascular events. There is evidence that CRP may also play a direct role in atherogenesis. Here we determined whether the proinflammatory interleukin (IL)-17 stimulates CRP expression in hepatocytes (Hep3B cell line and primary hepatocytes) and coronary artery smooth muscle cells (CASMC). Our results demonstrate that IL-17 potently induces CRP expression in Hep3B cells independent of IL-1β and IL-6. IL-17 induced CRP promoter-driven reporter gene activity that could be attenuated by dominant negative IκBα or C/EBPβ knockdown and stimulated both NF-κB and C/EBP DNA binding and reporter gene activities. Targeting NF-κB and C/EBPβ activation by pharmacological inhibitors, small interfering RNA interference and adenoviral transduction of dominant negative expression vectors blocked IL-17-mediated CRP induction. Overexpression of wild type p50, p65, and C/EBPβ stimulated CRP transcription. IL-17 stimulated p38 MAPK and ERK1/2 activation, and SB203580 and PD98059 blunted IL-17-mediated NF-κB and C/EBP activation and CRP transcription. These results, confirmed in primary human hepatocytes and CASMC, demonstrate for the first time that IL-17 is a potent inducer of CRP expression via p38 MAPK and ERK1/2-dependent NF-κB and C/EBPβ activation and suggest that IL-17 may mediate chronic inflammation, atherosclerosis, and thrombosis. Elevated systemic levels of the acute phase C-reactive protein (CRP) are predictors of future cardiovascular events. There is evidence that CRP may also play a direct role in atherogenesis. Here we determined whether the proinflammatory interleukin (IL)-17 stimulates CRP expression in hepatocytes (Hep3B cell line and primary hepatocytes) and coronary artery smooth muscle cells (CASMC). Our results demonstrate that IL-17 potently induces CRP expression in Hep3B cells independent of IL-1β and IL-6. IL-17 induced CRP promoter-driven reporter gene activity that could be attenuated by dominant negative IκBα or C/EBPβ knockdown and stimulated both NF-κB and C/EBP DNA binding and reporter gene activities. Targeting NF-κB and C/EBPβ activation by pharmacological inhibitors, small interfering RNA interference and adenoviral transduction of dominant negative expression vectors blocked IL-17-mediated CRP induction. Overexpression of wild type p50, p65, and C/EBPβ stimulated CRP transcription. IL-17 stimulated p38 MAPK and ERK1/2 activation, and SB203580 and PD98059 blunted IL-17-mediated NF-κB and C/EBP activation and CRP transcription. These results, confirmed in primary human hepatocytes and CASMC, demonstrate for the first time that IL-17 is a potent inducer of CRP expression via p38 MAPK and ERK1/2-dependent NF-κB and C/EBPβ activation and suggest that IL-17 may mediate chronic inflammation, atherosclerosis, and thrombosis. C-reactive protein (CRP) 2The abbreviations used are:CRPC-reactive proteinCASMCcoronary artery smooth muscle cellsC/EBPCCAAT enhancer-binding proteindndominant negativeEMSAelectrophoretic mobility shift assayERKextracellular signal-regulated kinaseGFPgreen fluorescent proteinIκBinhibitory κBILinterleukinsiRNAsmall interfering RNAMAPKmitogen-activated protein kinaseTRAF6tumor necrosis factor receptor-associated factor 6rhrecombinant humanELISAenzyme-linked immunosorbent assayqPCRquantitative PCRGAPDHglyceraldehyde-3-phosphate dehydrogenaseRTreverse transcriptionm.o.i.multiplicity of infectionPHHprimary human hepatocytes is an acute phase reactant that is markedly increased during infection, inflammation, and tissue injury (1Liuzzo G. Biasucci L.M. Gallimore J.R. Grillo R.L. Rebuzzi A.G. Pepys M.B. Maseri A. N. Engl. J. Med. 1994; 331: 417-424Crossref PubMed Scopus (2104) Google Scholar, 2Rifai N. Ridker P.M. Clin. Chem. 2001; 47: 403-411Crossref PubMed Scopus (453) Google Scholar, 3Ridker P.M. Hennekens C.H. Buring J.E. Rifai N. N. Engl. J. Med. 2000; 342: 836-843Crossref PubMed Scopus (5039) Google Scholar, 4Koenig W. Sund M. Frohlich M. Fischer H.G. Lowel H. Doring A. Hutchinson W.L. Pepys M.B. Circulation. 1999; 99: 237-242Crossref PubMed Scopus (1777) Google Scholar, 5Rost N.S. Wolf P.A. Kase C.S. Kelly-Hayes M. Silbershatz H. Massaro J.M. D'Agostino R.B. Franzblau C. Wilson P.W. Stroke. 2001; 32: 2575-2579Crossref PubMed Scopus (678) Google Scholar). It is synthesized and secreted mainly by the liver in response to circulating inflammatory mediators (6Hurlimann J. Thorbecke G.J. Hochwald G.M. J. Exp. Med. 1966; 123: 365-378Crossref PubMed Scopus (230) Google Scholar, 7Kushner I. Feldmann G. J. Exp. Med. 1978; 148: 466-477Crossref PubMed Scopus (169) Google Scholar). Elevated serum CRP levels serve as a risk marker for cardiovascular disease and predict future cardiovascular events and mortality (8de Beer F.C. Hind C.R. Fox K.M. Allan R.M. Maseri A. Pepys M.B. Br. Heart J. 1982; 47: 239-243Crossref PubMed Scopus (321) Google Scholar, 9Ridker P.M. Cushman M. Stampfer M.J. Tracy R.P. Hennekens C.H. N. Engl. J. Med. 1997; 336: 973-979Crossref PubMed Scopus (4902) Google Scholar). C-reactive protein coronary artery smooth muscle cells CCAAT enhancer-binding protein dominant negative electrophoretic mobility shift assay extracellular signal-regulated kinase green fluorescent protein inhibitory κB interleukin small interfering RNA mitogen-activated protein kinase tumor necrosis factor receptor-associated factor 6 recombinant human enzyme-linked immunosorbent assay quantitative PCR glyceraldehyde-3-phosphate dehydrogenase reverse transcription multiplicity of infection primary human hepatocytes Data obtained both in vivo and in vitro indicate that CRP plays a role in vascular inflammation (10Inoue N. Cardiovasc. Hematol. Disord. Drug Targets. 2006; 6: 227-231Crossref PubMed Scopus (36) Google Scholar, 11Jialal I. Devaraj S. Venugopal S.K. Hypertension. 2004; 44: 6-11Crossref PubMed Scopus (499) Google Scholar, 12Mazer S.P. Rabbani L.E. J. Thromb. Thrombolysis. 2004; 17: 95-105Crossref PubMed Scopus (62) Google Scholar). CRP can be detected in human atherosclerotic plaques co-localized with modified low density lipoprotein (13Torzewski J. Torzewski M. Bowyer D.E. Frohlich M. Koenig W. Waltenberger J. Fitzsimmons C. Hombach V. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1386-1392Crossref PubMed Scopus (502) Google Scholar, 14Yasojima K. Schwab C. McGeer E.G. McGeer P.L. Am. J. Pathol. 2001; 158: 1039-1051Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar). It can also associate with the terminal complex of complement in the arterial wall, inducing its activation in plaques. CRP promotes the uptake of low density lipoprotein by macrophages (15Zwaka T.P. Hombach V. Torzewski J. Circulation. 2001; 103: 1194-1197Crossref PubMed Scopus (779) Google Scholar) and exerts a mitogenic effect on vascular smooth muscle cells (16Hattori Y. Matsumura M. Kasai K. Cardiovasc. Res. 2003; 58: 186-195Crossref PubMed Scopus (199) Google Scholar). CRP stimulates chemokine and adhesion molecule expression in vascular endothelial cells and enhances platelet adhesion to endothelial cells (17Pasceri V. Willerson J.T. Yeh E.T. Circulation. 2000; 102: 2165-2168Crossref PubMed Scopus (1730) Google Scholar). These data suggest that CRP is not just a marker of cardiovascular risk but is a risk factor in its own right, and CRP plays a causal role in atherosclerosis and thrombosis. In fact, transgenic overexpression of human CRP has been shown to promote atherosclerosis in apoE-/- mice (18Paul A. Ko K.W. Li L. Yechoor V. McCrory M.A. Szalai A.J. Chan L. Circulation. 2004; 109: 647-655Crossref PubMed Scopus (347) Google Scholar), as does chronic administration (19Griselli M. Herbert J. Hutchinson W.L. Taylor K.M. Sohail M. Krausz T. Pepys M.B. J. Exp. Med. 1999; 190: 1733-1740Crossref PubMed Scopus (448) Google Scholar). These data support an hypothesis that CRP is a proinflammatory and pro-atherogenic factor. Inflammation is an important component in all stages of atherosclerosis, with proinflammatory cytokines and chemokines playing critical roles. IL-17 is a member of a novel group of proinflammatory cytokines that is composed of six major isoforms, IL-17A, -B, -C, -D, -E (also known as IL-25), and -F (20Moseley T.A. Haudenschild D.R. Rose L. Reddi A.H. Cytokine Growth Factor Rev. 2003; 14: 155-174Crossref PubMed Scopus (750) Google Scholar). These isoforms are encoded by unique genes and share little homology with other interleukins. IL-17 signals via IL-17 receptors, products of unique genes, and includes IL-17RA, -B (also known as IL-25R), -C, -D, and -E (20Moseley T.A. Haudenschild D.R. Rose L. Reddi A.H. Cytokine Growth Factor Rev. 2003; 14: 155-174Crossref PubMed Scopus (750) Google Scholar). IL-17A is the most widely studied cytokine of the IL-17 family. It signals via IL-17RA and exerts proinflammatory, pro-apoptotic, and pro-mitogenic effects. Unlike IL-17, which is considered a T-cell-specific cytokine (21Yao Z. Painter S.L. Fanslow W.C. Ulrich D. Macduff B.M. Spriggs M.K. Armitage R.J. J. Immunol. 1995; 155: 5483-5486PubMed Google Scholar), many cell types in the body express the receptors and are therefore targets of IL-17 (22Yao Z. Fanslow W.C. Seldin M.F. Rousseau A.M. Painter S.L. Comeau M.R. Cohen J.I. Spriggs M.K. Immunity. 1995; 3: 811-821Abstract Full Text PDF PubMed Scopus (806) Google Scholar). In this study we investigated whether IL-17 stimulates CRP expression in human hepatocytes and CASMC, and we determined the signal transduction pathways involved in IL-17-mediated CRP induction. Our data show for the first time that IL-17 stimulates CRP expression in hepatocytes and coronary artery smooth muscle cells, independently of IL-1β and IL-6, and mediates CRP induction via p38 MAPK and ERK1/2-dependent NF-κB and C/EBPβ activation. These results suggest that IL-17-CRP signaling may play a role in chronic inflammatory conditions such as atherosclerosis. Materials—Recombinant human (rh) IL-1ra (280-RA/CF), IL-6 (206-IL-010), rhIL-17 (317-IL-050), IL-17R-Fc chimera (177-IR), Fc (110-HG), anti-IL-6 neutralizing antibodies (AB-206-NA), IL-6 ELISA kit (D6050), and normal goat IgG (AB-108-C) were purchased from R&D Systems. rhIL-1β (200-01B) was purchased from PeproTech, Inc. (Rocky Hill, NJ). Functional grade purified anti-human IL-17 antibodies (16-7178) and normal mouse IgG antibodies were obtained from eBioscience (San Diego, CA). Antibodies against C/EBPα (sc-61X), C/EBPβ (sc-150X), TRAF2 (sc-877), TRAF6 (sc-7221), and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p38, phospho-p38 (PhosphoPlus® p38 MAPK (Thr-180/Tyr-182) antibody kit), ERK1/2 (9102), phospho-ERK1/2 (9101S), and anti-phospho-C/EBPβ (3084S) antibodies were from Cell Signaling Technology, Inc. (Beverly, MA). SN-50 (cell-permeable peptide inhibitor of NF-κB, 50 μg/ml in phosphate-buffered saline), SN-50M (SN-50 mutant, 50 μg/ml in phosphate-buffered saline), MG-132 (a proteasomal inhibitor, 5 μm in Me2SO for 1 h), SB203580 (p38 MAPK inhibitor, 1 μm in Me2SO for 30 min), PD98059 (ERK inhibitor, 10 μm in Me2SO for 1 h), and genistein (induces ER stress and mitochondrial insult, 100 μm in Me2SO for 48 h) and Me2SO were purchased from EMD Biosciences (San Diego). All other chemicals were purchased from Sigma. Cell Culture—Human hepatoma Hep3B cells (HB-8064; ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum at 10% (complete media). At ∼70% confluency, the complete medium was replaced with media containing 0.5% bovine serum albumin. After overnight incubation to achieve quiescence, rhIL-17 was added and cultured for the indicated time periods. Culture supernatants were then collected and snap-frozen. Cells were harvested, snap-frozen, and stored at -80 °C. Primary human hepatocytes (PHH; CellzDirect, Inc., Austin, TX) were treated as described for Hep3B cells. Normal human coronary artery smooth muscle cells (CASMC) were described previously (23Chandrasekar B. Mummidi S. Mahimainathan L. Patel D.N. Bailey S.R. Imam S.Z. Greene W.C. Valente A.J. J. Biol. Chem. 2006; 281: 15099-15109Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) and were treated as described for Hep3B cells. Because IL-17 stimulates IL-6 expression (24Ruddy M.J. Wong G.C. Liu X.K. Yamamoto H. Kasayama S. Kirkwood K.L. Gaffen S.L. J. Biol. Chem. 2004; 279: 2559-2567Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), and IL-6 is a potent inducer of CRP (25Ganapathi M.K. Rzewnicki D. Samols D. Jiang S.L. Kushner I. J. Immunol. 1991; 147: 1261-1265PubMed Google Scholar), we investigated whether IL-17-stimulates IL-6 expression in hepatocytes and whether IL-17-mediated CRP expression is dependent on IL-6. Therefore, hepatocytes were treated with IL-17, IL-6 (10 ng/ml), or IL-17 + IL-6. IL-6 expression was targeted by siRNA (sense, 5′-CUCACCUCUUCAGAACGAATT-3′, 100 nm (26Labbozzetta M. Notarbartolo M. Poma P. Giannitrapani L. Cervello M. Montalto G. D'Alessandro N. Ann. N. Y. Acad. Sci. 2006; 1089: 268-275Crossref PubMed Scopus (14) Google Scholar)) or anti-IL-6 neutralizing antibodies (10 μg/ml for 1 h) prior to IL-17 addition. Normal goat/mouse IgG served as a control. Knockdown of IL-6 was confirmed by RT-qPCR (IL-6 qPCR was performed using a Cytoxpress kit, BIOSOURCE). IL-17 is also known to induce IL-1β expression (27Jovanovic D.V. Di Battista J.A. Martel-Pelletier J. Jolicoeur F.C. He Y. Zhang M. Mineau F. Pelletier J.P. J. Immunol. 1998; 160: 3513-3521PubMed Google Scholar). However, it has been reported previously that IL-1β fails to stimulate CRP expression in Hep3B cells but potentiates IL-6-mediated CRP expression (25Ganapathi M.K. Rzewnicki D. Samols D. Jiang S.L. Kushner I. J. Immunol. 1991; 147: 1261-1265PubMed Google Scholar). Therefore, we investigated whether IL-1ra blocks IL-1β, IL-6, IL-1β+IL-6, or IL-17-mediated CRP secretion. Quiescent Hep3B cells were treated with IL-1ra simultaneously with IL-1β (10 ng/ml), IL-6 (10 ng/ml), IL-1β+IL-6 (10 ng each/ml), or IL-17 (100 ng/ml) for 24 h. Hep3B cells were not pretreated with IL-1ra, and at these concentrations these cytokines did not affect cell viability (data not shown). CRP levels in culture supernatants were quantified by ELISA. Adenoviral Vectors, Propagation, and Infection—Recombinant, replication-deficient adenoviral vectors encoding green fluorescent protein (Ad-CMV-GFP), dominant negative (dn) IKKβ, and dnIκB-α (S32A/S36A) have been described (28Patel D.N. Bailey S.R. Gresham J.K. Schuchman D.B. Shelhamer J.H. Goldstein B.J. Foxwell B.M. Stemerman M.B. Maranchie J.K. Valente A.J. Mummidi S. Chandrasekar B. Biochem. Biophys. Res. Commun. 2006; 347: 1113-1120Crossref PubMed Scopus (46) Google Scholar). Cells were infected at 100 m.o.i. as described previously (28Patel D.N. Bailey S.R. Gresham J.K. Schuchman D.B. Shelhamer J.H. Goldstein B.J. Foxwell B.M. Stemerman M.B. Maranchie J.K. Valente A.J. Mummidi S. Chandrasekar B. Biochem. Biophys. Res. Commun. 2006; 347: 1113-1120Crossref PubMed Scopus (46) Google Scholar). Transient Cell Transfections and Reporter Assays—A DNA fragment containing human CRP promoter (-300/19) was amplified by PCR from human genomic DNA (Promega) using the primers sense, 5′-aga tct AGAGCTACCTCCTCCTGCCTGG-3′, and antisense, 5′-acgcgtACCCAGATGGCCACTCGTTTAATATGTTACC-3′, cloned into the pCR2.1-TOPO vector, and subcloned into the MluI/BglII sites of the pGL3-basic vector (29Voleti B. Agrawal A. J. Immunol. 2005; 175: 3386-3390Crossref PubMed Scopus (65) Google Scholar). Mutation of the NF-κB-binding site was performed by site-directed mutagenesis using the QuikChange kit (Stratagene). The κB site was mutated by converting -72AAAATT-67 to -72TTAATA-67 using the primers 5′-GCGCCACTATGTAAATTATTAACCAACATTGCTTGTTGGGGC-3′ and 5′-GCCCCAACAAGCAATGTTGGTTAATAATTTACATAGTGGCGC-3′. Mutation of the C/EBP site was performed using the primers 5′-GGAAAATTATTTACATAGTGTAGCTTACTCCCTTACTGCTTTGG-3′ and 5′-CCAAAGCAGTAAGGGAGTAAGCTACACTATGTAAATAATTTTCC-3′. All constructs were verified by restriction mapping and bidirectional sequencing. Cell Transfection and Reporter Assays—Cells were transfected with 3 μg of the CRP reporter constructs and 100 ng of the control Renilla luciferase vector pRL-TK (Promega) using Lipofectamine®. Luciferase activity was determined using the Promega Biotech™ dual-luciferase reporter assay system (23Chandrasekar B. Mummidi S. Mahimainathan L. Patel D.N. Bailey S.R. Imam S.Z. Greene W.C. Valente A.J. J. Biol. Chem. 2006; 281: 15099-15109Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Firefly luciferase data were normalized with the corresponding Renilla luciferase and expressed as mean relative stimulation ±S.E. for a representative experiment from three to six separate experiments, each performed in triplicate. Transfection efficiency of hepatocytes was determined using pEGFP-N1 vector (Clontech) and was found to be 34.3%. To investigate pathways involved in IL-17-mediated CRP expression, hepatocytes were transiently transfected with wild type or dominant negative expression vectors using Lipofectamine 2000 (Invitrogen). Wild type (CMV-C/EBPβ) and dnC/EBP-β (CMV-dnC/EBP-β) were generous gifts from Richard M. Pope (Northwestern University Medical School, Chicago). dnTRAF6 (pRK5-TRAF6-(289–522)-FLAG), dnTRAF2 (pRK5-TRAF2-(87–501)-FLAG), kdNIK (pRK7-NIK(K429A/K430A)-FLAG) were described previously (30Chandrasekar B. Mummidi S. Perla R.P. Bysani S. Dulin N.O. Liu F. Melby P.C. Biochem. J. 2003; 373: 547-558Crossref PubMed Scopus (137) Google Scholar, 31Chandrasekar B. Melby P.C. Sarau H.M. Raveendran M. Perla R.P. Marelli-Berg F.M. Dulin N.O. Singh I.S. J. Biol. Chem. 2003; 278: 4675-4686Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). pRK5 and pRK7 served as controls. To compensate for variations in transfection efficiency, cells were co-transfected with pRL-Renilla luciferase vector (pRL-TK vector; Promega, Madison, WI). C/EBPβ expression was also targeted by C/EBPβ siRNA duplex (sense, 5′-GAAGACCGUGGACAAGCACdTT-3′; 100 nm). TRAF2 expression was targeted by two siRNA duplexes (sense, 5′-AUACGAGAGCUGCCACGAAdTdT-3′, and sense, 5′-AGAGGCCAGUCAACGACAUdTdT-3′; 50 nm each) and TRAF6 by a siRNA duplex (5′-CUGUGCUGCAUCAAUGGCAdTdT-3′) as described previously (32Zhong J. Kyriakis J.M. Mol. Cell. Biol. 2004; 24: 9165-9175Crossref PubMed Scopus (28) Google Scholar). As negative control, siRNA that does not target any genes in the human genome (5′-UUCUCCGAACGUGUCACGUdTdT-3′; catalog number 1022076, Qiagen Inc.; 100 nm) was used. Gel Shift, Supershift, ELISA, and Reporter Assays—NF-κB and C/EBP DNA binding activities were assessed by EMSA. Double-stranded consensus wild type (NF-κB, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′; C/EBP, 5′-TGC AGA TTG CGC AAT CTG CA-3′) and mutant (NF-κB, 5′-AGT TGA GGC GAC TTT CCC AGG C-3′; C/EBP, 5′-TGC AGA GAC TAG TCT CTG CA-3′) oligonucleotides (Santa Cruz Biotechnology, Inc.) were used as before (23Chandrasekar B. Mummidi S. Mahimainathan L. Patel D.N. Bailey S.R. Imam S.Z. Greene W.C. Valente A.J. J. Biol. Chem. 2006; 281: 15099-15109Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 28Patel D.N. Bailey S.R. Gresham J.K. Schuchman D.B. Shelhamer J.H. Goldstein B.J. Foxwell B.M. Stemerman M.B. Maranchie J.K. Valente A.J. Mummidi S. Chandrasekar B. Biochem. Biophys. Res. Commun. 2006; 347: 1113-1120Crossref PubMed Scopus (46) Google Scholar, 30Chandrasekar B. Mummidi S. Perla R.P. Bysani S. Dulin N.O. Liu F. Melby P.C. Biochem. J. 2003; 373: 547-558Crossref PubMed Scopus (137) Google Scholar, 31Chandrasekar B. Melby P.C. Sarau H.M. Raveendran M. Perla R.P. Marelli-Berg F.M. Dulin N.O. Singh I.S. J. Biol. Chem. 2003; 278: 4675-4686Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Activation and subunit composition were determined by supershift (C/EBP) and TransAM™ NF-κB (catalog number 43296) and C/EBP α/β (catalog number 44196) transcription factor ELISA (Active Motif, Carlsbad, CA). Activation of NF-κB and C/EBP was also confirmed by reporter gene assays. Adenoviral NF-κB-luciferase vector (Ad.NFκB-Luc) was generously provided by John F. Engelhardt (University of Iowa College of Medicine, Iowa City (33Sanlioglu S. Williams C.M. Samavati L. Butler N.S. Wang G. McCray Jr., P.B. Ritchie T.C. Hunninghake G.W. Zandi E. Engelhardt J.F. J. Biol. Chem. 2001; 276: 30188-30198Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar)) and contained the luciferase gene driven by four tandem copies of the NF-κB consensus sequence fused to a TATA-like promoter from the herpes simplex virus-thymidine kinase gene. Ad.MCS-Luc (Vector Laboratories) served as a control. A 2xC/EBP-Luc reporter vector containing two canonical C/EBP-binding sites was a gift from Peter F. Johnson (Laboratory of Protein Dynamics and Signaling, NCI, Frederick, MD). pEGFP-Luc served as a control. Gene Expression—CRP transcription was analyzed by nuclear run-on assay (30Chandrasekar B. Mummidi S. Perla R.P. Bysani S. Dulin N.O. Liu F. Melby P.C. Biochem. J. 2003; 373: 547-558Crossref PubMed Scopus (137) Google Scholar). CRP mRNA expression was analyzed by quantitative real time PCR. DNA-free total RNA was extracted using RNAqueous®-4PCR kit (Ambion). RNA quality was assessed by capillary electrophoresis using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). All RNA samples used for quantitative PCR had RNA integrity numbers greater than 9.1 (scale = 1–10), as assigned by default parameters of the Expert 2100 Bioanalyzer software package (version 2.02). Real time quantitative PCR was performed as described previously by Ivashchenko et al. (34Ivashchenko Y. Kramer F. Schafer S. Bucher A. Veit K. Hombach V. Busch A. Ritzeler O. Dedio J. Torzewski J. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 186-192Crossref PubMed Scopus (31) Google Scholar) using Quanti-Tect SYBR-Green Probe RT-PCR Kit (Qiagen). Each sample was assayed in triplicate. For relative quantification, the Ct method (ratio = 2 - (Ct(CRP) - Ct(GAPDH))) was used with GAPDH as a control. For copy number determination, a calibration curve was obtained using serial dilutions of linearized GAPDH cDNA as template and the GAPDH primers 5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATGGGATTTC-3′: human CRP primer pair 1 (product size 133 bp), forward, 5′-ACTTCCTATGTATCCCTCAAAG-3′, and reverse, 5′-CTCATTGTCTTGTCTCTTGGT-3′; human CRP primer pair 2 (product size 440 bp), forward, 5′-TCGTATGCCACCAAGAGAAGACA-3′, and reverse, 5′-AACACTTCGCCTTGCACTTCATACT-3′. Primer pair 3 distinguishes between mRNA and genomic DNA (expected product size 196 bp for mRNA and 481 bp for genomic DNA): forward, 5′-TCTCATGCTTTTGGCCAGAC-3′, and reverse, 5′-CTCATTGTCTTGTCTCTTGGT-3′. ELISA—CRP levels in culture supernatants were quantified by an ELISA (IMUCLONE® High Sensitivity CRP ELISA test kit, product ID 660; American Diagnostica, Inc., Stamford, CT). IL-6 levels were quantified by human IL-6 ELISA kit (BIOSOURCE). Western Blotting—Protein extraction and Western blotting were performed as described previously (23Chandrasekar B. Mummidi S. Mahimainathan L. Patel D.N. Bailey S.R. Imam S.Z. Greene W.C. Valente A.J. J. Biol. Chem. 2006; 281: 15099-15109Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 30Chandrasekar B. Mummidi S. Perla R.P. Bysani S. Dulin N.O. Liu F. Melby P.C. Biochem. J. 2003; 373: 547-558Crossref PubMed Scopus (137) Google Scholar, 31Chandrasekar B. Melby P.C. Sarau H.M. Raveendran M. Perla R.P. Marelli-Berg F.M. Dulin N.O. Singh I.S. J. Biol. Chem. 2003; 278: 4675-4686Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 35Chandrasekar B. Mummidi S. Claycomb W.C. Mestril R. Nemer M. J. Biol. Chem. 2005; 280: 4553-4567Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) using 20–30 μg of protein/lane. Immune Complex Kinase Assays—p38 MAPK and ERK activities were determined by immune complex kinase assays (23Chandrasekar B. Mummidi S. Mahimainathan L. Patel D.N. Bailey S.R. Imam S.Z. Greene W.C. Valente A.J. J. Biol. Chem. 2006; 281: 15099-15109Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 28Patel D.N. Bailey S.R. Gresham J.K. Schuchman D.B. Shelhamer J.H. Goldstein B.J. Foxwell B.M. Stemerman M.B. Maranchie J.K. Valente A.J. Mummidi S. Chandrasekar B. Biochem. Biophys. Res. Commun. 2006; 347: 1113-1120Crossref PubMed Scopus (46) Google Scholar, 35Chandrasekar B. Mummidi S. Claycomb W.C. Mestril R. Nemer M. J. Biol. Chem. 2005; 280: 4553-4567Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) using whole cell homogenates (p38 MAPK assay kit and ERK, p44/42 MAPK assay Kit, Cell Signaling Technology, Inc.). Cell Death Assays—Quiescent hepatocytes or CASMC were treated with IL-17 (100 ng/ml) for up to 48 h. Cell death was analyzed by an ELISA (Cell Death Detection ELISAPLUS kit; Roche Diagnostics) (28Patel D.N. Bailey S.R. Gresham J.K. Schuchman D.B. Shelhamer J.H. Goldstein B.J. Foxwell B.M. Stemerman M.B. Maranchie J.K. Valente A.J. Mummidi S. Chandrasekar B. Biochem. Biophys. Res. Commun. 2006; 347: 1113-1120Crossref PubMed Scopus (46) Google Scholar, 30Chandrasekar B. Mummidi S. Perla R.P. Bysani S. Dulin N.O. Liu F. Melby P.C. Biochem. J. 2003; 373: 547-558Crossref PubMed Scopus (137) Google Scholar). Genistein, an inducer of ER stress and mitochondrial insult in hepatocytes (36Yeh T.C. Chiang P.C. Li T.K. Hsu J.L. Lin C.J. Wang S.W. Peng C.Y. Guh J.H. Biochem. Pharmacol. 2007; 73: 782-792Crossref PubMed Scopus (120) Google Scholar), was used as a positive control. Statistical Analysis—Comparisons between experimental groups were made using the unpaired t test with Bonferroni's correction for multiple comparisons, if needed. If three comparisons were made, a p value <0.025 was considered significant. For two comparisons, a p value <0.05 was considered significant. Each experiment was performed at least three times, and group data were expressed as means ± S.E. IL-17 Stimulates CRP Expression in Hep3B Cells—IL-17 functions as a proinflammatory cytokine in various models of inflammation (20Moseley T.A. Haudenschild D.R. Rose L. Reddi A.H. Cytokine Growth Factor Rev. 2003; 14: 155-174Crossref PubMed Scopus (750) Google Scholar, 21Yao Z. Painter S.L. Fanslow W.C. Ulrich D. Macduff B.M. Spriggs M.K. Armitage R.J. J. Immunol. 1995; 155: 5483-5486PubMed Google Scholar). Because CRP exerts proinflammatory effects in atherosclerosis (16Hattori Y. Matsumura M. Kasai K. Cardiovasc. Res. 2003; 58: 186-195Crossref PubMed Scopus (199) Google Scholar, 17Pasceri V. Willerson J.T. Yeh E.T. Circulation. 2000; 102: 2165-2168Crossref PubMed Scopus (1730) Google Scholar, 18Paul A. Ko K.W. Li L. Yechoor V. McCrory M.A. Szalai A.J. Chan L. Circulation. 2004; 109: 647-655Crossref PubMed Scopus (347) Google Scholar, 19Griselli M. Herbert J. Hutchinson W.L. Taylor K.M. Sohail M. Krausz T. Pepys M.B. J. Exp. Med. 1999; 190: 1733-1740Crossref PubMed Scopus (448) Google Scholar), we investigated whether IL-17 stimulates CRP expression using a hepatic cell line. Quiescent Hep3B cells were treated with rhIL-17 for 24 h, and CRP mRNA expression was quantified by RT-qPCR. IL-17 stimulated CRP mRNA expression dose-dependently with significant stimulation in CRP expression detectable at 10 ng/ml and peak levels at 100 ng/ml. Time course studies revealed that IL-17 at 100 ng/ml increased CRP expression at 24 h, with no further increases detected at 48 h (Fig. 1B). Therefore, in all subsequent experiments IL-17 was used at 100 ng/ml. IL-17 at this dose stimulated CRP secretion (6-fold, p < 0.001; Fig. 1C), and treatment with anti-IL-17 neutralizing antibodies or IL-17Fc-chimera blocked this expression (Fig. 1D). Although IL-17 is known to induce pro-apoptotic gene expression (24Ruddy M.J. Wong G.C. Liu X.K. Yamamoto H. Kasayama S. Kirkwood K.L. Gaffen S.L. J. Biol. Chem. 2004; 279: 2559-2567Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), results in Fig. 1E show that IL-17 failed to induce cell death. However, genistein, a known inducer of ER stress and mitochondrial insult in hepatocytes (36Yeh T.C. Chiang P.C. Li T.K. Hsu J.L. Lin C.J. Wang S.W. Peng C.Y. Guh J.H. Biochem. Pharmacol. 2007; 73: 782-792Crossref PubMed Scopus (120) Google Scholar), induced significant cell death. These results indicate that IL-17 is a potent inducer of CRP expression in Hep3B cells (Fig. 1). IL-17-mediated CRP Expression Is Independent of IL-1β and IL-6 in Hep3B Cells—IL-6 is a proinflammatory cytokine whose expression is increased in both subclinical and overt inflammation and potently induces CRP expression (25Ganapathi M.K. Rzewnicki D. Samols D. Jiang S.L. Kushner I. J. Immunol. 1991; 147: 1261-1265PubMed Google Scholar). Because IL-17 is known to stimulate IL-6 expression (24Ruddy M.J. Wong G.C. Liu X.K. Yamamoto H. Kasayama S. Kirkwood K.L. Gaffen S.L. J. Biol. Chem. 2004; 279: 2559-2567Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), we determined whether IL-17-mediated CRP expression is IL-6-dependent. IL-17 stimulated IL-6 mRNA expression and protein secretion (Fig. 2, A and B). As expected, IL-6 stimulated CRP expression (Fig. 2C), and this was enhanced when combined with IL-17 (Fig. 2D). However, siRNA-mediated IL-6 knockdown or pretreatment with anti-IL-6 neutralizing antibodies failed to block IL-17-mediated CRP expression (Fig. 2E). Knockdown of IL-6 was confirmed by ELISA (Fig. 2F). IL-17 is also known to induce IL-1β expression (27Jovanovic D.V. Di Battista J.A. Martel-Pelletier J. Jolicoeur F.C. He Y. Zhang M. Mineau F. Pelletier J.P. J. Immunol. 1998; 160: 3513-3521PubMed Google Scholar). However, IL-1β fails to induce CRP expression in Hep3B cells (25Ganapathi M.K. Rzewnicki D. Samols D. Jiang S.L. Kushner I. J. Immunol. 1991; 147: 1261-1265PubMed Goog" @default.
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