Interleukin-17 Stimulates C-reactive Protein Expression in Hepatocytes and Smooth Muscle Cells via p38 MAPK and ERK1/2-dependent NF-κB and C/EBPβ Activation*

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.

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 Ϫ/Ϫ 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-1␤ and IL-6, and mediates CRP induction via p38 MAPK and ERK1/2dependent NF-B and C/EBP␤ activation. These results suggest that IL-17-CRP signaling may play a role in chronic inflammatory conditions such as atherosclerosis.
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 ϳ70% 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 Ϫ80°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.
Cell Transfection and Reporter Assays-Cells were transfected with 3 g 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 TM dual-luciferase reporter assay system (23). Firefly luciferase data were normalized with the corresponding Renilla luciferase and expressed as mean relative stimulation ϮS.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%.
Gel Shift, Supershift, ELISA, and Reporter Assays-NF-B and C/EBP DNA binding activities were assessed by EMSA. Double-stranded consensus wild type (NF-B, 5Ј-AGT TGA GGG GAC TTT CCC AGG C-3Ј; C/EBP, 5Ј-TGC AGA TTG CGC AAT CTG CA-3Ј) and mutant (NF-B, 5Ј-AGT TGA GGC GAC TTT CCC AGG C-3Ј; C/EBP, 5Ј-TGC AGA GAC TAG TCT CTG CA-3Ј) oligonucleotides (Santa Cruz Biotechnology, Inc.) were used as before (23,28,30,31). Activation and subunit composition were determined by supershift (C/EBP) and TransAM TM NF-B (catalog number 43296) and C/EBP ␣/␤ (catalog number 44196) transcription factor ELISA (Active Motif, Carlsbad, CA). Activation of NF-B and C/EBP was also confirmed by reporter gene assays. Adenoviral NF-B-luciferase vector (Ad.NFB-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-B consensus sequence fused to a TATAlike 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.
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 ELISA PLUS 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 Ͻ0.025 was considered significant. For two comparisons, a p value Ͻ0.05 was considered significant. Each experiment was performed at least three times, and group data were expressed as means Ϯ S.E.

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 Ͻ 0.001; Fig.  1C), and treatment with anti-IL-17 neutralizing antibodies or IL-17Fcchimera 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 Stimulates CRP Transcription via NF-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 (Ϫ300/Ϫ1)reporter activity in Hep3B cells (Fig. 3B), and mutation of the NF-B or C/EBP sites blunted this response, indicating that IL-17 induces CRP transcription via NF-B and C/EBP. Furthermore, ectopic expression of NF-B p50, p65, or C/EBP␤ all significantly stimulated CRP transcription in Hep3B (Fig. 3C), indicating that IL-17 stimulates CRP transcription in hepatic cells through NF-B and C/EBP␤ (Fig. 3).

IL-17 Stimulates NF-B Activation in Hep3B
Cells-We have demonstrated that IL-17-induced CRP promoter-driven reporter gene activity is attenuated when NF-B core DNAbinding sequence is mutated (Fig. 3B). Conversely, ectopic expression of wild type NF-B p50 or p65 stimulated CRP pro-moter-reporter activity (Fig. 3C). Therefore, we investigated whether IL-17 induces NF-B activation in Hep3B cells. Our results show that IL-17 potently stimulated NF-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-Bdriven luciferase activity (Fig. 3G). Together, these results indicate that IL-17 is a potent inducer of NF-B activation in Hep3B cells (Fig. 3).

IL-17 Stimulates CRP Expression via p38 MAPK and ERK1/ 2-dependent NF-B and C/EBP␤ 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-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/EBP␤, and adenoviral transduction of dnIB-␣ 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 Me 2 SO 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-B, and C/EBP␤ (Fig. 7).

DISCUSSION
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 ERK1/2dependent NF-B and C/EBP␤ 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-␣ (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-regu- lation 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.
Our results also show that IL-17-mediated CRP induction is dependent on p38 MAPK and ERK1/2-dependent C/EBP␤ and NF-B activation. While targeting C/EBP␤ or NF-B each reduced CRP activity, their combined significantly attenuated, but not abrogated, CRP transcription. These results suggest that both C/EBP␤ and NF-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/EBP␤ and NF-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-B activation (42), IL-17-CRP signaling may play a role in vascular inflammation via activation of signal transduction pathways that converge at NF-B.
Our results also show that IL-17 stimulates CRP expression in human coronary artery smooth muscle cells through NF-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 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 M 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-B activation. Quiescent Hep3B cells were treated with SB203580 (1 M) or PD98059 (10 M) 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-17mediated C/EBP␤ activation. Quiescent Hep3B cells were treated with SB203580 or PD98059 (10 M for 1 h) prior to IL-17 addition. Nuclear protein was extracted at 2 h and analyzed for C/EBP␤ 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 M) or PD98059 (10 M) for 1 h prior to IL-17 addition. CRP mRNA expression was analyzed at 24 h by RT-qPCR. G-I, *, p Ͻ 0.001 versus untreated; †, p Ͻ 0.01 versus IL-17. 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-B and AP-1 (16), and as IL-17 is a NF-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.
In contrast to its proinflammatory and pro-atherogenic effects, CRP has been shown to exert vasoprotective effects (52)(53)(54)(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-B activation. Thus the IL-17-CRP signaling pathway may be a significant inflammatory component in atherogenesis and cardiovascular diseases.