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Originally published In Press as doi:10.1074/jbc.M703250200 on July 25, 2007

J. Biol. Chem., Vol. 282, Issue 37, 27229-27238, September 14, 2007
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Interleukin-17 Stimulates C-reactive Protein Expression in Hepatocytes and Smooth Muscle Cells via p38 MAPK and ERK1/2-dependent NF-{kappa}B and C/EBPbeta Activation*

Devang N. Patel{ddagger}, Carter A. King{ddagger}, Steven R. Bailey{ddagger}, Jeffrey W. Holt{ddagger}, Kaliyamurthi Venkatachalam{ddagger}, Alok Agrawal§, Anthony J. Valente{ddagger}, and Bysani Chandrasekar{ddagger}1

From the Department of Veterans Affairs, South Texas Veterans Health Care System, San Antonio, Texas 78229-4404, the {ddagger}Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900, and the §Department of Pharmacology, East Tennessee State University, Johnson City, Tennessee 37614

Received for publication, April 17, 2007 , and in revised form, July 9, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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-1beta and IL-6. IL-17 induced CRP promoter-driven reporter gene activity that could be attenuated by dominant negative I{kappa}B{alpha} or C/EBPbeta knockdown and stimulated both NF-{kappa}B and C/EBP DNA binding and reporter gene activities. Targeting NF-{kappa}B and C/EBPbeta 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/EBPbeta stimulated CRP transcription. IL-17 stimulated p38 MAPK and ERK1/2 activation, and SB203580 and PD98059 blunted IL-17-mediated NF-{kappa}B 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-{kappa}B and C/EBPbeta activation and suggest that IL-17 may mediate chronic inflammation, atherosclerosis, and thrombosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
C-reactive protein (CRP)2 is an acute phase reactant that is markedly increased during infection, inflammation, and tissue injury (1–5). It is synthesized and secreted mainly by the liver in response to circulating inflammatory mediators (6, 7). Elevated serum CRP levels serve as a risk marker for cardiovascular disease and predict future cardiovascular events and mortality (8, 9).

Data obtained both in vivo and in vitro indicate that CRP plays a role in vascular inflammation (10–12). CRP can be detected in human atherosclerotic plaques co-localized with modified low density lipoprotein (13, 14). 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 (15) and exerts a mitogenic effect on vascular smooth muscle cells (16). CRP stimulates chemokine and adhesion molecule expression in vascular endothelial cells and enhances platelet adhesion to endothelial cells (17). 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 (18), as does chronic administration (19). 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 (20). 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 (20).

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 (21), many cell types in the body express the receptors and are therefore targets of IL-17 (22). 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-1beta and IL-6, and mediates CRP induction via p38 MAPK and ERK1/2-dependent NF-{kappa}B and C/EBPbeta activation. These results suggest that IL-17-CRP signaling may play a role in chronic inflammatory conditions such as atherosclerosis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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-1beta (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/EBP{alpha} (sc-61X), C/EBPbeta (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/EBPbeta (3084S) antibodies were from Cell Signaling Technology, Inc. (Beverly, MA). SN-50 (cell-permeable peptide inhibitor of NF-{kappa}B, 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 (23) and were treated as described for Hep3B cells.

Because IL-17 stimulates IL-6 expression (24), and IL-6 is a potent inducer of CRP (25), 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 (26)) 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-1beta expression (27). However, it has been reported previously that IL-1beta fails to stimulate CRP expression in Hep3B cells but potentiates IL-6-mediated CRP expression (25). Therefore, we investigated whether IL-1ra blocks IL-1beta, IL-6, IL-1beta+IL-6, or IL-17-mediated CRP secretion. Quiescent Hep3B cells were treated with IL-1ra simultaneously with IL-1beta (10 ng/ml), IL-6 (10 ng/ml), IL-1beta+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) IKKbeta, and dnI{kappa}B-{alpha} (S32A/S36A) have been described (28). Cells were infected at 100 m.o.i. as described previously (28).

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 (29). Mutation of the NF-{kappa}B-binding site was performed by site-directed mutagenesis using the QuikChange kit (Stratagene). The {kappa}B 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 BiotechTM dual-luciferase reporter assay system (23). 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/EBPbeta) and dnC/EBP-beta (CMV-dnC/EBP-beta) 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 (30, 31). 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).


Figure 1
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  		FIGURE 1.
IL-17 stimulates CRP expression in Hep3B cells. A, dose-dependent effects of IL-17 on CRP mRNA expression. Quiescent Hep3B cells were treated with rhIL-17. Cells were harvested at 24 h; DNA-free total RNA was isolated, and CRP mRNA expression was quantified by RT-qPCR. GAPDH was used as an internal control, and the results are presented as a ratio of IL-17 to GAPDH. B, kinetics of IL-17-mediated CRP induction. Quiescent Hep3B cells were treated with rhIL-17 (100 ng/ml). At the indicated time periods, RNA was isolated, and CRP mRNA expression was quantified. C, IL-17 stimulates CRP secretion. Hep3B cells were treated with IL-17 (100 ng/ml) for 24 h, and CRP levels in culture supernatants were quantified by ELISA. D, neutralizing IL-17 or IL-17R antibodies block IL-17-mediated CRP secretion. Quiescent Hep3B cells were treated with neutralizing anti-IL-17 antibodies or IL-17R/Fc chimera (10 µg/ml) for 1 h prior to IL-17 treatment for 24 h. CRP levels in culture supernatants were quantified by ELISA. Normal mouse IgG and Fc served as controls. E, IL-17 does not induce Hep3B apoptosis. Quiescent Hep3B cells were treated with IL-17 (100 ng/ml for 24 h), and cell death was quantified by an ELISA that measures mono- and oligonucleosomal fragmented DNA in the cytoplasmic extracts. Genistein was used as a positive control. A, *, p < 0.05; **, p < 0.001 versus control (C); B, *, p < 0.01, **, p < 0.001 versus control (C); C, *, p < 0.001 versus untreated; D, *, p < 0.001 versus untreated; {dagger}, p < 0.001 versus IL-17; E, *, p < 0.001 versus IL-17. DMSO, Me2SO.

 
C/EBPbeta expression was also targeted by C/EBPbeta 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 (32). 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-{kappa}B and C/EBP DNA binding activities were assessed by EMSA. Double-stranded consensus wild type (NF-{kappa}B, 5'-AGT TGA GGG GAC TTT CCC AGG C-3'; C/EBP, 5'-TGC AGA TTG CGC AAT CTG CA-3') and mutant (NF-{kappa}B, 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 (23, 28, 30, 31). Activation and subunit composition were determined by supershift (C/EBP) and TransAMTM NF-{kappa}B (catalog number 43296) and C/EBP {alpha}/beta (catalog number 44196) transcription factor ELISA (Active Motif, Carlsbad, CA). Activation of NF-{kappa}B and C/EBP was also confirmed by reporter gene assays. Adenoviral NF-{kappa}B-luciferase vector (Ad.NF{kappa}B-Luc) was generously provided by John F. Engelhardt (University of Iowa College of Medicine, Iowa City (33)) and contained the luciferase gene driven by four tandem copies of the NF-{kappa}B 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 (30). 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. (34) 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).


Figure 2
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  		FIGURE 2.
IL-17-mediated CRP expression in Hep3B is not dependent on IL-6. A, IL-17 induces IL-6 expression. Quiescent Hep3B cells were treated with rhIL-17. Total RNA was isolated, and IL-6 mRNA expression was quantified by real time qPCR. B, IL-17 stimulates IL-6 secretion. Quiescent Hep3B cells were treated as in A, and 24 h later IL-6 levels in culture supernatants were quantified by ELISA. C, IL-6 stimulates CRP secretion. Quiescent Hep3B cells were treated with rhIL-6 for 24 h, and CRP levels in culture supernatants were quantified by ELISA. D, IL-6 potentiates IL-17 effects on CRP secretion. Quiescent Hep3B cells were treated with IL-17 (100 ng/ml), IL-6 (10 ng/ml), or IL-17+IL-6 for 24 h. CRP levels in culture supernatants were quantified by ELISA. E, IL-17-mediated CRP expression is not dependent on IL-6. Quiescent Hep3B cells were treated with IL-6 siRNA (100 nM for 48 h) or neutralizing IL-6 antibodies (10 µg/ml for 1 h) prior to IL-17 treatment. Control siRNA (100 nM) or normal goat IgG (10 µg/ml) served as controls. CRP secretion at 24 h was quantified by ELISA. F, IL-6 knockdown significantly inhibited IL-17-mediated IL-6 secretion. Quiescent Hep3B cells were treated with IL-6-specific siRNA (50 nM) for 48 h and then stimulated with IL-17 for an additional 24 h. IL-6 levels in culture supernatants were quantified by ELISA. G, IL-1beta + IL-6-, but not IL-6 or IL-17, induced CRP secretion is attenuated by IL-1ra. Quiescent Hep3B cells were treated for 24 h with IL-1beta (10 ng/ml), IL-6 (10 ng/ml), IL-1b + IL-6 (10 ng each/ml), or IL-17 (100 ng/ml) along with IL-1ra (500 ng/ml). IL-1ra was added simultaneously with the cytokines and not pretreated with IL-1ra. CRP levels in culture supernatants were quantified by ELISA. A, *, p < 0.05; **, p < 0.01 versus control (C); B, *, p < 0.001 versus control (C); C, *, p < 0.001 (C); D, *, p < at least 0.01; **, p < 0.001 versus untreated; E, p < 0.001 versus untreated; {dagger}, p < 0.01 versus IL-17; F, *, p < 0.001 versus untreated; {dagger}, p < 0.001 versus IL-17; G, *, p < 0.01 versus untreated; §, p < 0.01 versus IL-1beta or IL-6; {dagger}, p < 0.01 versus IL-1beta + IL-6.

 
Western Blotting—Protein extraction and Western blotting were performed as described previously (23, 30, 31, 35) using 20–30 µg of protein/lane.

Immune Complex Kinase Assays—p38 MAPK and ERK activities were determined by immune complex kinase assays (23, 28, 35) 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) (28, 30). Genistein, an inducer of ER stress and mitochondrial insult in hepatocytes (36), 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
IL-17 Stimulates CRP Expression in Hep3B Cells—IL-17 functions as a proinflammatory cytokine in various models of inflammation (20, 21). Because CRP exerts proinflammatory effects in atherosclerosis (16–19), 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 (24), 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 (36), 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-1beta 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 (25). Because IL-17 is known to stimulate IL-6 expression (24), 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-1beta expression (27). However, IL-1beta fails to induce CRP expression in Hep3B cells (25) but has been shown to potentiate IL-6-mediated CRP expression in Hep3B cells (25). Therefore, we investigated whether IL-17-mediated CRP expression is dependent on IL-1beta or IL-1beta + IL-6 expression. Results in Fig. 2G showed that IL-1beta had no significant stimulatory effects on CRP secretion. However, IL-1beta potentiated IL-6-induced CRP secretion. Importantly, simultaneous treatment with IL-1ra blocked IL-1beta + IL-6-mediated but not IL-17-stimulated CRP secretion. IL-1ra did not affect basal CRP secretion. These results demonstrate that IL-17 stimulates CRP expression in Hep3B cells independent of IL-1beta and IL-6 (Fig. 2).


Figure 3
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  		FIGURE 3.
IL-17 induces CRP transcription in NF-{kappa}B and C/EBP-dependent manner. A, IL-17 induces CRP transcription. Quiescent Hep3B cells were treated with IL-17 for 3 h. CRP transcription was analyzed by nuclear run-on using RNA isolated from the nuclei. GAPDH served as a control. TOPO2.1 vector servedas a negative control. B, IL-17 stimulates CRP promoter-reporter activity. Hep3B cells were transiently transfected with CRP promoter construct or the promoter constructs mutated in NF-{kappa}B- or C/EBP-binding sites. pGL3-Basic vector served as a control. Cells were co-transfected with pRL-TK vector. 24 h later, cells were treated with IL-17 (100 ng/ml for 7 h). Firefly and Renilla luciferase activities were determined. C, ectopic expression of NF-{kappa}B p50, p65, or C/EBPbeta activates CRP promoter-reporter activity. Hep3B cells were transfected with CRP (-300/-1) and the p50, p65, or C/EBPbeta expression vector together with the pRL-TK control vector. After 48 h, cell lysates were analyzed for reporter gene activities. Data are the means ± S.E. of four independent experiments. D, IL-17 induces NF-{kappa}B activation. Quiescent Hep3B cells were treated with IL-17. At the indicated time periods, nuclear protein was isolated and analyzed by EMSA for NF-{kappa}B DNA binding activity. E, anti-IL-17 neutralizing antibodies block IL-17-mediated NF-{kappa}B DNA binding activity. Quiescent Hep3B cells were treated with anti-IL-17-neutralizing antibodies (10 µg/ml) for 1 h prior to IL-17 treatment. NF-{kappa}B activation at 1 h was measured by EMSA. F, p50, p65, and c-Rel contribute to IL-17-mediated NF-{kappa}B activation. Quiescent Hep3B cells treated as in B were analyzed for NF-{kappa}B subunits in the nuclear protein extracts by ELISA. G, IL-17 stimulates NF-{kappa}B-dependent reporter gene activity. Hep3B cells transduced with adenoviral NF-{kappa}B-Luc vector (50 m.o.i.) were treated with IL-17 (100 ng/ml). Cells were co-transfected with Ad-beta-gal (50 m.o.i.). 7 h later, firefly luciferase and beta-galactosidase activities were measured, and the results were presented as a ratio of firefly to beta-galactosidase activity. Adenoviral transduction of MCS-Luc vector (50 m.o.i.) served as a control. Arrows in D and E denote NF-{kappa}B-specific DNA-protein complexes. B, *, p < 0.001 versus untreated; {dagger}, p < 0.01 versus CRP (-300/-1); C, *, p < 0.01; **, p < 0.001 versus untreated. F, *, p < 0.01 versus corresponding untreated; G, *, p < 0.001 versus Ad-MCS-Luc.

 
IL-17 Stimulates CRP Transcription via NF-{kappa}B and C/EBP in Hep3B Cells—Because IL-17 induced CRP expression, we next investigated whether IL-17-mediated CRP expression is regulated at transcriptional level. IL-17 induced CRP transcription (Fig. 3A) and potently stimulated CRP promoter (-300/-1)-reporter activity in Hep3B cells (Fig. 3B), and mutation of the NF-{kappa}B or C/EBP sites blunted this response, indicating that IL-17 induces CRP transcription via NF-{kappa}B and C/EBP. Furthermore, ectopic expression of NF-{kappa}B p50, p65, or C/EBPbeta all significantly stimulated CRP transcription in Hep3B (Fig. 3C), indicating that IL-17 stimulates CRP transcription in hepatic cells through NF-{kappa}B and C/EBPbeta (Fig. 3).

IL-17 Stimulates NF-{kappa}B Activation in Hep3B Cells—We have demonstrated that IL-17-induced CRP promoter-driven reporter gene activity is attenuated when NF-{kappa}B core DNA-binding sequence is mutated (Fig. 3B). Conversely, ectopic expression of wild type NF-{kappa}B p50 or p65 stimulated CRP promoter-reporter activity (Fig. 3C). Therefore, we investigated whether IL-17 induces NF-{kappa}B activation in Hep3B cells. Our results show that IL-17 potently stimulated NF-{kappa}B DNA binding activity within 1 h (Fig. 3D, lane 6) and was attenuated by preincubation with anti-IL-17 neutralizing antibodies (Fig. 3E, lane 8). Furthermore, IL-17 increased the levels of p65, p50, and c-Rel proteins in the nucleus (Fig. 3F) and stimulated NF-{kappa}B-driven luciferase activity (Fig. 3G). Together, these results indicate that IL-17 is a potent inducer of NF-{kappa}B activation in Hep3B cells (Fig. 3).

IL-17 Induces NF-{kappa}B Activation and CRP Expression via TRAF6, NIK, IKK, and I{kappa}B-{alpha}—We next investigated the pathway involved in IL-17-mediated NF-{kappa}B activation and CRP gene expression. IL-17-mediated NF-{kappa}B activation was inhibited by adenoviral transduction of dnIKKbeta (Fig. 4A, lane 10) and dnI{kappa}B-{alpha} (lane 11). Furthermore, treatment with SN-50, a peptide inhibitor of NF-{kappa}B activation, blunted IL-17-mediated NF-{kappa}B activation (Fig. 4B, lane 10), as did the proteasomal inhibitor MG-132 (Fig. 4B, lane 12). Similarly, transient overexpression of dnTRAF6 (Fig. 4C, lane 12), but not dnTRAF2 (lane 13), and kinase-deficient NIK (lane 14) attenuated IL-17-mediated NF-{kappa}B activation. Importantly, both overexpression of dnTRAF6 and TRAF6 knockdown (Fig. 4D, right-hand panels) blunted IL-17-mediated CRP mRNA expression. IL-17-mediated CRP expression was also inhibited by pretreatment with SN-50 and MG-132 or adenoviral transduction of dnIKKbeta or dnI{kappa}B-{alpha} (Fig. 4E). Our results also show that transient transfection with kdNIK blunts IL-17-mediated CRP induction. NIK activates NF-{kappa}BinIKKbeta-dependent manner. Because kdNIK attenuated IL-17-mediated NF-{kappa}B activation, it is possible that transient overexpression of kdNIK might have inhibited IKKbeta phosphorylation and NF-{kappa}B activation. However, it has also been reported that kdNIK may inhibit NF-{kappa}B activation via pathways that do not involve NIK per se but by sequestering IKKs (37). Thus our results do not exclude the possibility that kdNIK may inhibit IL-17-mediated CRP induction via multiple mechanisms. Together, these results demonstrate that IL-17 induces CRP expression in Hep3B cells via TRAF6-IKKbeta-NF-{kappa}B-dependent signaling (Fig. 4).


Figure 4
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  		FIGURE 4.
IL-17 induces CRP expression via TRAF6-dependent NF-{kappa}B activation. A, adenoviral transduction of dnIKKbeta or dnI{kappa}B-{alpha} blocks IL-17-mediated NF-{kappa}B activation. Hep3B cells were transduced with Ad.dnIKKbeta or Ad.dnI{kappa}B-{alpha} (50 m.o.i.), and 24 h later treated with IL-17 (100 ng/ml for 1 h). Nuclear extracts were isolated and analyzed for NF-{kappa}B activation by EMSA. Adenoviral transduction of GFP served as a control. B, peptide inhibitor SN-50 or the proteasomal inhibitor MG-132 blocks IL-17-mediated NF-{kappa}B activation. Quiescent Hep3B cells were treated with SN-50 or MG-132 prior to IL-17 treatment. SN-50 M and Me2SO (DMSO) served as controls. NF-{kappa}B DNA binding activity was analyzed by EMSA. C, IL-17 induces NF-{kappa}B activation via TRAF6. Hep3B cells were transiently transfected with dnTRAF2 or dnTRAF6 in pRK5 vector. 24 h later, cells were treated with IL-17 for 1 h. NF-{kappa}B DNA binding activity was analyzed by EMSA. D, IL-17 stimulates CRP mRNA expression in TRAF6-dependent manner. Hep3B cells transiently transfected with dnTRAF2 or dnTRAF6 in pRK5 vector for 24 h or treated with TRAF2 (75 nM each of a two siRNA duplexes) or TRAF6 (100 nM) siRNA for 48 h were treated with IL-17 for an additional 24 h. CRP mRNA expression was analyzed by RT-qPCR. Knockdown of TRAF2 and TRAF6 was confirmed by Western blotting (the two panels on the right). Actin served as a control. E, IL-17 induces CRP expression via NF-{kappa}B activation. Hep3B cells were treated with SN-50, MG-132 for 1 h, or transduced with adenoviral dnIKKbeta, dnI{kappa}B-{alpha} for 24 h, or transiently transfected with kdNIK for 48 h prior to IL-17 treatment. 24 h later, CRP mRNA expression was analyzed by RT-qPCR. Arrows in A–C denote NF-{kappa}B-specific DNA-protein complexes. D, *, p < 0.001 versus untreated; {dagger}, p < 0.05 versus IL-17; E, *, p < 0.001 versus untreated; {dagger}, p < 0.01 versus IL-17.

 
IL-17 Induces C/EBP Activation and CRP Expression in TRAF6-dependent Manner—We have demonstrated that IL-17-induced CRP promoter-driven reporter gene activity is attenuated when C/EBP core DNA binding sequence is mutated (Fig. 3B). Conversely, ectopic expression of wild type C/EBPbeta stimulated CRP promoter-reporter activity (Fig. 3C). Therefore, we next investigated whether IL-17 stimulates C/EBP activation in Hep3B cells. Our results show that IL-17 stimulated C/EBP DNA binding activity with high levels at 2 h (Fig. 5A, lane 6), and supershift assays revealed predominantly C/EBPbeta in the protein-DNA complexes (Fig. 5A, lane 8). Our results also show that anti-IL-17 antibodies blunt IL-17-mediated C/EBP activation (Fig. 5B). Confirming the EMSA results (Fig. 5A), ELISA of nuclear protein extracts revealed a significant increase in C/EBPbeta and a small, but statistically insignificant, increase in C/EBP{alpha} levels following IL-17 treatment (Fig. 5C). IL-17 also induced C/EBP-driven luciferase activity (Fig. 5D) and C/EBPbeta phosphorylation (Fig. 5E). Furthermore, transient overexpression of dnTRAF6, but not TRAF2, or TRAF6 knockdown attenuated IL-17-mediated C/EBP activation (Fig. 5E) and CRP mRNA expression (Fig. 5F), as was C/EBPbeta knockdown (Fig. 5G; knockdown of C/EBPbeta by Western blotting is shown on the right). Together, these results indicate that IL-17 induces CRP mRNA expression via TRAF6-dependent C/EBPbeta activation (Fig. 5).

IL-17 Induces NF-{kappa}B and C/EBPbeta Activation via TRAF6 and TRAF-6-dependent p38 MAPK and ERK1/2 Activation—Because IL-17-mediated NF-{kappa}B and C/EBPbeta activation is TRAF6-dependent, and IL-17 is known to activate MAPKs (38, 39), we investigated the role of MAPKs in TRAF6-dependent transcription factor activation following IL-17 treatment. IL-17 induced SB203580-inhibitable p38 MAPK activation (Fig. 6A). Immune complex kinase assays confirmed IL-17-mediated p38 MAPK activity (Fig. 6B). Similarly, IL-17 induced PD98059-inhibitable ERK1/2 phosphorylation (Fig. 6C) and activity (Fig. 6D). Importantly, TRAF6, but not TRAF2, knockdown abrogated IL-17-mediated p38 MAPK (Fig. 6E) and ERK1/2 (Fig. 6F) activities. Finally, inhibition of p38 MAPK and ERK1/2 attenuated IL-17-mediated NF-{kappa}B p65 (Fig. 6G) and C/EBPbeta (Fig. 6H) activation and CRP mRNA expression (Fig. 6I). Together, these results indicate that IL-17 induces CRP mRNA expression via TRAF6-dependent p38 MAPK and ERK1/2 activation.

IL-17 Stimulates CRP Expression via p38 MAPK and ERK1/2-dependent NF-{kappa}B and C/EBPbeta Activation in Primary Human Hepatocytes and Coronary Artery Smooth Muscle Cells—We have demonstrated that IL-17 stimulates CRP expression in Hep3B cells via TRAF6-dependent p38 MAPK and ERK1/2-mediated NF-{kappa}B and C/EBP activation. Because Hep3B cells are derived from human hepatoma, we investigated whether IL-17 exerts similar effects in PHH. Our results show that IL-17 is a potent inducer of CRP mRNA expression in PHH (Fig. 7A), and knockdown of TRAF6, C/EBPbeta, and adenoviral transduction of dnI{kappa}B-{alpha} or pretreatment with SB203580 or PD98059 attenuate IL-17-mediated CRP mRNA expression (Fig. 7A). Furthermore, pretreatment with MG-132, SN-50, SB203580, or PD98059 significantly attenuated IL-17-mediated CRP secretion in PHH. However, SN-50M and Me2SO had no modulatory effects. Similar to its effects on PHH, IL-17 induced CRP mRNA expression in CASMC via similar signaling pathways (Fig. 7C). Together, these results indicate that IL-17 is a potent inducer of CRP expression in primary hepatocytes and CASMC, and IL-17-mediated CRP induction is dependent on TRAF6, p38 MAPK, ERK1/2, NF-{kappa}B, and C/EBPbeta (Fig. 7).


Figure 5
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  		FIGURE 5.
IL-17 stimulates CRP expression in C/EBP-dependent manner in Hep3B cells. A, IL-17 induces C/EBP activation. Quiescent Hep3B cells were treated with IL-17. C/EBP DNA binding activity (lanes 1–6) and C/EBP subunit composition (lanes 7–9) were analyzed by EMSA and supershift assays. Normal rabbit IgG served as a control. B, anti-IL-17 neutralizing antibodies block IL-17-mediated C/EBP activation. Quiescent Hep3B cells were treated with anti-IL-17 neutralizing antibodies (10 µg/ml) for 1 h followed by IL-17 for an additional 2 h. Normal mouse IgG served as a control. C/EBP DNA binding activity was determined by EMSA. C, IL-17-induced C/EBP activation was confirmed by ELISA. Quiescent Hep3B cells were treated with IL-17 for 2 h. Nuclear protein was extracted and analyzed for C/EBP subunits by ELISA. D, IL-17 stimulates C/EBP-dependent reporter gene activity. Hep3B cells were transiently transfected with C/EBP reporter vector. pEGFP-Luc served as a control. Cells were co-transfected with the pRL-TK vector. 24 h after transfection, cells were treated with IL-17. 7 h later, luciferase activities were determined. E, IL-17 induces C/EBPbeta phosphorylation. Quiescent Hep3B cells treated as in A were analyzed for C/EBPbeta phosphorylation by Western blotting. F, IL-17 induces C/EBP activation via TRAF6. Hep3B cells were treated with TRAF2 or TRAF6 siRNA (100 nM for 48 h) or transiently transfected with dnTRAF2 or dnTRAF6 in pRK5 vector for 24 h, followed IL-17 treatment for 2 h. C/EBP DNA binding activity was determined by EMSA. G, IL-17 induces CRP mRNA expression in C/EBPbeta and NF-{kappa}B-dependent manner. Hep3B cells were treated with C/EBPbeta siRNA for 24 h, transduced with Ad-dnI{kappa}B-{alpha} for an additional 24 h, and then treated with IL-17. Total RNA was isolated after 24 h and analyzed for CRP mRNA expression by RT-qPCR. Solid arrows in A, B, and F denote C/EBP-specific DNA-protein complexes. Open arrow in A denotes supershifted C/EBPbeta band. C and D, p < 0.001 versus untreated; G, *, p < 0.001 versus untreated; {dagger}, p < 0.05 versus IL-17.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The major novel finding of this study is that the proinflammatory cytokine interleukin-17 stimulates CRP expression in a human hepatoma cell line, primary human hepatocytes, and human coronary artery smooth muscle cells. In all of these cells, IL-17 stimulates CRP expression via p38 MAPK and ERK1/2-dependent NF-{kappa}B and C/EBPbeta activation (Fig. 7D). These results suggest that IL-17-CRP signaling may play a role in chronic inflammatory conditions.

The IL-17 family of proinflammatory cytokine contains six members (A–F) that share little to no homology with other interleukins (21, 22). IL-17 has been shown to play a role in various models of inflammation and autoimmune diseases, including rheumatoid arthritis (21, 22, 40, 41). IL-17 is reported to stimulate a variety of genes, including chemokines, cytokines, and transcription factors (42), and the stimulatory effects of IL-17 are enhanced when combined with suboptimal doses of tumor necrosis factor-{alpha} (43). In co-cultures of mouse bone marrow cells and osteoblasts, IL-17 is reported to increase osteoclast formation in a dose-dependent manner (40). Here we demonstrate that IL-17 stimulates CRP and IL-6 expression in hepatocytes. However targeting of IL-6 expression by neutralizing antibodies, antisense oligonucleotides, and siRNA-mediated knockdown all failed to block IL-17-mediated CRP induction. Therefore IL-6 is not required for IL-17-mediated CRP expression. IL-6 however, potentiated the IL-17 effects. Potentiating effects of IL-6 have also been reported previously on CRP induction in hepatocytes treated with IL-1 (29). In that study, although IL-1 failed to stimulate CRP expression, a significant induction of CRP was observed when IL-1 was combined with IL-6 (29). Because inflammation is characterized by the up-regulation of various cytokines that stimulate CRP induction, it is possible that IL-17 may act in synergy with other proinflammatory cytokines in stimulating CRP expression in vivo in liver and other tissues.


Figure 6
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  		FIGURE 6.
IL-17 stimulates CRP expression via p38 MAPK and ERK1/2 in Hep3B cells. A, IL-17 induces p38 MAPK activation. Quiescent Hep3B cells were treated with SB203580 (SB;1 µM in Me2SO (DMSO) for 1 h) prior to IL-17 treatment. Total and phospho-p38 MAPK levels at 30 min were analyzed by Western blotting using activation-specific antibodies. B, IL-17 stimulates p38 MAPK activity. Quiescent Hep3B cells treated as in A were analyzed for p38 MAPK activity by immune complex kinase assay using ATF-2 as a substrate. C, IL-17 induces ERK1/2 activation. Quiescent Hep3B cells were treated with PD98059 (PD;10 µM for 1 h) prior to IL-17 addition. Total and phospho-ERK1/2 levels were analyzed at 30 min by Western blotting. D, IL-17 stimulates ERK1/2 activity. Quiescent Hep3B cells treated as in C were analyzed for ERK1/2 activity by immune complex kinase assay using Elk as a substrate. E, TRAF6 knockdown blocks IL-17-mediated p38 MAPK activation. Hep3B cells were treated with TRAF6, TRAF2, or control siRNA for 48 h and then treated with IL-17 for 30 min. p38 MAPK activity was determined as in B. F, Hep3B cells treated as in E were analyzed for ERK1/2 activity as described in D. G, inhibition of p38 MAPK and ERK1/2 attenuates IL-17-mediated NF-{kappa}B activation. Quiescent Hep3B cells were treated with SB203580 (1 µM) or PD98059 (10 µM) for 1 h prior to IL-17 addition. Nuclear protein was extracted at 1 h and analyzed for p65 levels by ELISA. H, inhibition of p38 MAPK and ERK1/2 attenuate IL-17-mediated C/EBPbeta activation. Quiescent Hep3B cells were treated with SB203580 or PD98059 (10 µM for 1 h) prior to IL-17 addition. Nuclear protein was extracted at 2 h and analyzed for C/EBPbeta levels by ELISA. I, inhibition of p38 MAPK and ERK1/2 attenuates IL-17-mediated CRP mRNA expression. Quiescent Hep3B cells were treated with SB203580 (1 µM) or PD98059 (10 µM) for 1 h prior to IL-17 addition. CRP mRNA expression was analyzed at 24 h by RT-qPCR. G–I, *, p < 0.001 versus untreated; {dagger}, p < 0.01 versus IL-17.

 
Our results also show that IL-17-mediated CRP induction is dependent on p38 MAPK and ERK1/2-dependent C/EBPbeta and NF-{kappa}B activation. While targeting C/EBPbeta or NF-{kappa}B each reduced CRP activity, their combined significantly attenuated, but not abrogated, CRP transcription. These results suggest that both C/EBPbeta and NF-{kappa}B play critical roles in IL-17-CRP signaling but that other transcriptional elements are also involved in CRP induction. In a series of well executed studies, Voleti and Agrawal (29) have demonstrated that IL-6 stimulates CRP expression in hepatocytes via synergistic activation of C/EBPbeta and NF-{kappa}B. These authors also demonstrated that Oct-1 and Stat3 also contribute to basal and induced CRP expression (44), suggesting that multiple transcriptional regulatory elements contribute to CRP induction in agonist and cell type-dependent manner. Studies are in progress to investigate if similar interactions occur in hepatocytes treated with IL-17. Because CRP is also known to stimulate NF-{kappa}B activation (42), IL-17-CRP signaling may play a role in vascular inflammation via activation of signal transduction pathways that converge at NF-{kappa}B.

Our results also show that IL-17 stimulates CRP expression in human coronary artery smooth muscle cells through NF-{kappa}B and C/EBP activation. Although hepatocytes are reported to be the major source of circulating CRP (6, 7), CRP expression has also been detected in human atherosclerotic lesions and is associated with calcification and plaque rupture (13, 14). In coronary vessels, CRP is localized to macrophages and smooth muscle cells (17) and mediates SMC proliferation (45, 46). CRP also promotes endothelial dysfunction (11), a hallmark of atherosclerosis. In endothelial progenitor cells, CRP stimulates reactive oxygen species generation, inhibits antioxidative enzyme levels, inactivates telomerase, and promotes cell death (47). These reports indicate that CRP may differentially affect various cell types in a vessel wall, resulting in the development and progression of atherosclerosis.

Recently CRP has been shown to be involved in the pathogenesis of obesity and its metabolic complications. CRP binds leptin and prevents its effects on food intake, body weight, blood glucose, and lipid metabolism (48). Because obesity plays a significant role in coronary artery and cardiovascular diseases, it appears that CRP acts on several cellular targets (endothelial cells, smooth muscle cells, hepatocytes, and adipocytes) to regulate energy metabolism and promote atherosclerosis. CRP is also known to stimulate the expression of various cytokines, chemokines, and adhesion molecules (49). However, it is not known whether CRP stimulates IL-17 expression in SMC. Because CRP is known to activate NF-{kappa}B and AP-1 (16), and as IL-17 is a NF-{kappa}B- and AP-1-responsive gene (50), it is logical to speculate that CRP may stimulate IL-17 expression in SMC. Interestingly, it has been demonstrated that IL-17 expression increases with aging (51). In that study, although coronary vessels from young animals showed relatively low levels of IL-17 mRNA and protein expression, coronary vessels from aged Fisher 344 rats showed a 2.5-fold increase in IL-17 expression, with IL-17 expression localized to SMC (51). These studies suggest that the cross-talk between locally expressed IL-17 and CRP may further amplify the inflammatory cascade in the vessel wall promoting atherosclerosis.


Figure 7
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  		FIGURE 7.
IL-17 stimulates CRP mRNA expression in PHH (A) and primary human CASMC (B) via TRAF6, p38 MAPK, ERK1/2, NF-{kappa}B, and C/EBPbeta. A, PHH were transduced with adenoviral dnI{kappa}B-{alpha} (50 m.o.i.; GFP served as a control), treated with TRAF6, C/EBPbeta, or control siRNA (100 nM for 48 h), SB203580 (SB), or PD98059 (PD) for 1 h prior to IL-17 addition (100 ng/ml for 24 h). CRP mRNA expression was analyzed by RT-qPCR. B, PHH were pretreated with MG-132, SN-50, SB203580, or PD98059 prior to IL-17 (100 ng/ml) addition. SN-50M and Me2SO (DMSO) served as controls. CRP levels in culture supernatants were quantified by ELISA. C, CASMC treated similarly as in A were analyzed for CRP mRNA expression by RT-qPCR. D, schematic depicting the signaling pathway involved in IL-17-mediated CRP induction in hepatocytes (PHH and Hep3B cell line) and CASMC. A, *, p < 0.001 versus untreated; {dagger}, p < 0.01 versus IL-17; B, *, p < 0.001 versus untreated, {dagger}, p < 0.01 versus IL-17; C, *, p < 0.001 versus untreated; {dagger}, p < 0.01 versus IL-17.

 
In contrast to its proinflammatory and pro-atherogenic effects, CRP has been shown to exert vasoprotective effects (52–55). Whether CRP is pro-atherogenic or vasoprotective depends on its conformation. Serum CRP or native CRP is a pentamer composed of five identical globular subunits arranged in an annular disk. This pentameric structure can dissociate into monomers that can only exist in tissues as membrane-associated forms (mCRP) and exhibit altered solubility and antigenicity (56–58). Whether the ratio of pentameric to monomeric forms in the vessel wall differentially affects atherogenesis and whether IL-17 differentially regulates their expression and confirmation are not known.

Our studies have several important implications as follows: (i) IL-17 can mediate chronic inflammation and increase CRP expression in hepatocytes and smooth muscle cells, and by doing so enhance atherosclerosis; (ii) IL-17 may enhance myocardial inflammation and injury via up-regulation of IL-6 and other proinflammatory and pro-apoptotic cytokines; (iii) IL-17 may enhance atherogenesis and plaque rupture by stimulating the expression of pro-atherogenic cytokines (e.g. tumor necrosis factor), chemokines, and extracellular matrix-degrading matrix metalloproteinases through NF-{kappa}B activation. Thus the IL-17-CRP signaling pathway may be a significant inflammatory component in atherogenesis and cardiovascular diseases.


   FOOTNOTES
 
* This work was supported in part by the Research Service of the Department of Veterans Affairs and NHLBI Grant HL68020 from the National Institutes of Health (to B. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Medicine/Cardiology, the University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: 210-567-4598; Fax: 210-567-6960; E-mail: chandraseka{at}uthscsa.edu.

2 The abbreviations used are: CRP, C-reactive protein; CASMC, coronary artery smooth muscle cells; C/EBP, CCAAT enhancer-binding protein; dn, dominant negative; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; I{kappa}B, inhibitory {kappa}B; IL, interleukin; siRNA, small interfering RNA; MAPK, mitogen-activated protein kinase; TRAF6, tumor necrosis factor receptor-associated factor 6; rh, recombinant human; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; m.o.i., multiplicity of infection; PHH, primary human hepatocytes. Back


   ACKNOWLEDGMENTS
 
We thank Jie Li for excellent technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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