Interleukin-17-induced Gene Expression in Articular Chondrocytes Is Associated with Activation of Mitogen-activated Protein Kinases and NF-κB*

This study examines intracellular signaling events associated with the activation of chondrocytes by the cytokine interleukin-17 (IL-17). Stimulation of normal human articular chondrocytes with IL-17 induced nitric oxide (NO) production, concomitant with an increase in transcripts and de novotranslation products of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) genes. Several other genes associated with inflammation and cartilage degradation, such as IL-1β, IL-6, and stromelysin, were also up-regulated in IL-17-treated chondrocytes. Among signaling events displaying early response to IL-17 in chondrocytes were the mitogen-activated protein (MAP) kinases ERK1, ERK2, JNK, and p38. DNA binding activity of NF-κB was also significantly induced. IL-17 effects on NO release, as well as iNOS, COX-2, and IL-6 protein expression, were inhibited by the anti-inflammatory drug dexamethasone. Importantly, dexamethasone blunted IL-17-dependent activation of MAP kinases, suggesting a mechanistic relationship between these activities and the aforementioned gene expression responses. Similar effects of a lesser extent were observed with the p38-specific inhibitor SB203580. These results suggest that IL-17 activation of chondrocytes is associated with and depends at least in part on the activation of MAP kinases and NF-κB.

The effector cascades mediating inflammatory responses of chondrocytes to IL-1 and TNF␣ have been elucidated in part and shown to include the mitogen-activated protein kinase (MAPK) signal transduction pathway. IL-1 and TNF␣ activate all three of the MAP kinase subgroups ERK, JNK, and p38 (7)(8)(9).
In addition to IL-1 and TNF␣, several other cytokines can be detected in arthritic joints. Among these, we focus in this study on the potential role of IL-17 in the induction of inflammatory responses in chondrocytes. IL-17 was originally identified as CTLA8, a murine cytotoxic T-cell-associated antigen, which displays significant homology to the T-lymphotropic Herpesvirus Saimiri gene 13 product (HVS13) (10,11). Subsequent studies of human IL-17 (hIL-17) demonstrated exclusive expression of this cytokine in activated T-cells, predominantly of the CD4 ϩ subtype (12,13). Expression cloning of the murine IL-17 receptor (mIL-17R) identified a type I transmembrane protein, which is ubiquitously expressed and is not related to previously cloned cytokine receptors (12). IL-17 induces NF-B activation and IL-6, IL-8, and ICAM-1 expression in murine and human fibroblasts (12,13) and the production of IL-6, IL-8, granulocyte macrophage-colony-stimulating factor, and prostaglandin E2 in bone marrow stromal cells (14). In human macrophages, IL-17 stimulates the production of proinflammatory cytokines, including IL-1␤ and TNF␣ (15). In chondrocytes from osteoarthritic human cartilage, IL-17 up-regulated the spontaneous production of nitric oxide (16). mIL-17 was shown to have mitogenic effects on mouse splenic T cells (12), although this effect has not been demonstrated with hIL-17 and human T cells (13).
In the present study, we analyze cytoplasmic and nuclear signaling events induced by IL-17 in chondrocytes. These include activation of the JNK, p38, ERK1, and ERK2 MAP kinases; induction of NF-B DNA binding activity; and glucocorticoid inhibition of both types of responses. Our data unravel IL-17 as a potential additional player in the cytokine networks involved in arthritis and underscore some of its possible effects on cartilage pathology.

EXPERIMENTAL PROCEDURES
Chondrocyte Isolation and Culture-Human cartilage was obtained at autopsy from donors without known history of joint disease. Bovine cartilage was obtained from Animal Technologies (Tyler, TX). For all experiments reported here, cartilage from the femoral condyles and tibial plateaus of the knee joints was used. Chondrocytes were isolated by collagenase digestion of cartilage and cultured as described previously (17). All experiments were performed with primary or passage 1 cells.
Preparation of Cell Lysates-For kinase and immunoblot assays, 3 ϫ 10 6 chondrocytes were cultured in 100-mm Petri dishes and serumstarved for 24 h before stimulation. After washing with phosphatebuffered saline containing 1 mM Na 3 VO 4 , cells were lysed in lysis buffer (50 mM NaCl, 50 mM Tris, pH 7.4, 0.5% Nonidet P-40, 1 mM EGTA, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, 1 * This work was supported by National Institutes of Health Grant AR43872. 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.
g/ml aprotinin, 1 g/ml leupeptin). 100 g of lysates was used for JNK assay. For immunoblot assays of the phosphorylated kinases p38, ERK1, and ERK2 and for Ib␣, 50 g of whole cell lysates were used. For iNOS and COX-2, 200 g of whole cell lysates were used. Proteins were separated on 8 -10% polyacrylamide gels.
JNK Assay-Hypotonic detergent cellular extracts were prepared, and the solid-state JNK assay was performed as described (7). The extracts were mixed with 10 l of GSH-agarose suspension (Sigma), to which glutathione S-transferase-c-Jun (1-223) was bound. The mixture was rotated at 4°C for 12-16 h, pelleted, and washed five times in lysis buffer. The beads were then resuspended in 35 l of kinase buffer (20 mM HEPES, pH 7.6, 20 mM MgCl 2 , 10 mM ␤-glycerophosphate, 0.1 mM Na 3 VO 4 , 10 mM dithiothreitol) containing 20 M ATP and 5 Ci [␥-32 P]ATP. After 30 min at 30°C, the reaction was terminated by washing with lysis buffer. Phosphorylated proteins were eluted with Laemmli buffer and resolved on 10% SDS-polyacrylamide gel electrophoresis, followed by autoradiography.
Extraction of Nuclear Proteins-Nuclear proteins were prepared according to modification of the method described by Schreiber et al. (18). Chondrocytes (3 ϫ 10 6 per condition) were washed and scraped in phosphate-buffered saline. Cells were resuspended in swelling buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 50 g/ml aprotinin) for 15 min on ice and lysed with 0.07% Nonidet P-40. The nuclei were pelleted by centrifugation, resuspended in extraction buffer, and rotated at 4°C for 120 min. After centrifugation, the supernatant containing nuclear proteins was collected, analyzed by Bradford, and stored at Ϫ80°C for electrophoretic mobility shift assay.
Electrophoretic Mobility Shift Assay-NF-B binding activities were studied by using double-stranded oligonucleotides (22-mers) 5Ј-AGTT-GAGGGGACTTTCCCAGGT-3Ј (Promega), corresponding to the NF-B site in the human light chain promoter. The double-stranded oligonucleotides (3.5 pmol) were labeled with [ 32 P]ATP in the presence of T4 polynucleotide kinase and were separated from unincorporated [ 32 P]ATP by gel filtration using Centri-sep columns (Princeton Separations, Adelphia, NJ). Nuclear proteins (5 g) were preincubated for 45 min with 1 g of poly dI:dC in NF-B binding buffer on ice. Radioactively labeled oligonucleotide (30,000 cpm) was added and incubated for 30 min at room temperature. The complexes were separated on nondenaturing 5% polyacrylamide gels.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)-Total RNA was isolated by a single step guanidinium thiocyanate-phenol chloroform method, using RNA Stat-60 (Tel-Test "B", Inc., Friendswood, TX) according to the manufacturer's protocol. Total RNA (up to 5 g) was reverse-transcribed with MoMLV-RT (Life Technologies, Inc.) for 30 -120 min at 37°C. RT reactions were subjected to PCR with Taq Western Blotting-Whole cell lysates were separated on 8 -10% SDSpolyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose membrane (S&S NC, Keene, NH). The blots were blocked in TBS/Tween with 5% milk for 1 h at room temperature and incubated with primary monoclonal antibody to iNOS (Transduction Laboratories, Lexington, KY), anti-prostaglandin H synthase II (COX-2) polyclonal antibody (Cayman Chemical Co., Ann Arbor, MI), polyclonal anti-phospho p38 antibody, polyclonal anti-phospho ERK1 and ERK2 antibody, polyclonal anti-phospho JNK antibody (New England Biolabs), or polyclonal anti-Ib␣ antibody (Santa Cruz Biotechnology). Primary antibodies were diluted in TBS/Tween with either 3% milk or 3% bovine serum albumin for 1 h at room temperature to overnight at 4°C. Following five washes with TBS/Tween (125 mM NaCl, 25 mM Tris, pH 8.0, 0.1% Tween 20), the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Jackson Immuno Research Laboratories) in TBS/Tween with 3% milk for 45 min at room temperature. After five washes with TBS/ Tween, the blots were incubated with ECL substrate solution (Amersham Pharmacia Biotech) for 1 min according to the manufacturer's instructions and exposed to x-ray film.
IL-6 Enzyme-linked Immunosorbent Assay-Conditioned media were collected from chondrocyte cultures 24 h after treatment with IL-17, p38 inhibitor, or dexamethasone. The supernatants were added to halfarea enzyme-linked immunosorbent assay plate (Costar, Cambridge, MA) that had been precoated with 25 l of 2 g/ml monoclonal mouse anti-human IL-6 antibody (R&D Systems, Minneapolis, MN) for 3 h and blocked with 3% bovine serum albumin. Samples or human IL-6 standards were incubated for 2 h followed by 1 g/ml rabbit anti-human IL-6 (R&D Systems). Biotin-labeled goat anti-rabbit Ig (Sigma) was then added. Bound IL-6 was detected with poly-horseradish peroxidase-labeled streptavidin (Accurate Chemicals, Westbury, NY) and substrate (Kirkegaard & Perry Lab, Gaithersburg, MD).
Quantification of Nitrites-The concentration of nitrites, the stable end products of cellular NO breakdown, in conditioned media from chondrocytes was determined by the Griess reaction using NaNO 2 as standard. All results are expressed as nmoles of nitrites per 100,000 cells.

Induction of Nitric Oxide Production in Normal Chondrocytes by IL-17 and Its Inhibition by a p38 Kinase Inhibitor and
Dexamethasone-We used NO production as a marker for the capacity of IL-17 to elicit cellular responses in primary normal human articular chondrocytes. As shown in Fig. 1A, IL-17 induced NO production in chondrocytes at concentrations as low as 1 ng/ml, reaching 70% of its maximal effect at 10 ng/ml. This response was comparable with that induced by IL-1 or TNF␣ in the same cells, as was the maximal NO levels induced by both cytokines (Fig. 1B). Thus, normal chondrocytes mount a significant NO response to IL-17. We therefore used this to study the signaling events evoked by IL-17 in these cells.
To gain insight into the possible IL-17 effectors mediating NO production, we evaluated the magnitude of this response in the presence of the specific p38 kinase inhibitor, SB203580 (19 -21). Fig. 1C shows that SB203580 reduced IL-17-driven NO synthesis in a dose-dependent manner, reaching a maximum of 30% inhibition at 10 M, a concentration at which it still retains full specificity toward p38 (21).
The effect of dexamethasone on IL-17-induced NO synthesis was tested next. Dexamethasone is the most commonly used anti-inflammatory drug and is an inhibitor of cytokine-induced cellular responses. Like SB203580, dexamethasone also exhibited dose-dependent inhibition of NO production, blunting this response by greater than 50% at 100 nM (Fig. 1C). A mixture of dexamethasone and SB203580 had an additive inhibitory effect. The effects reported in Fig. 1C were qualitatively similar in articular chondrocyte preparations from five different donors. Thus, NO production in response to IL-17 is mediated through a signaling cascade affected by p38 kinase and is sensitive to the anti-inflammatory drug dexamethasone.
Gene Expression in IL-17-stimulated Chondrocytes-We next analyzed the effects of IL-17 stimulation on the expression of genes associated with the inflammatory response in chondrocytes. The levels of IL-1␤, IL-6, iNOS, COX-2, and stromelysin mRNAs were assessed by performing RT-PCR on samples prepared at various time points following IL-17 administration (Fig.  2). Transcripts of all genes tested were barely detectable in the nonstimulated chondrocytes, with the exception of stromelysin, and all were substantially induced following the introduction of IL-17. Maximal induction was achieved 4 h poststimulation, and the transcripts examined maintained these maximal levels at least for 24 h. Glyceraldehyde-3-phospate dehydrogenase RT-PCR products, analyzed as internal control, were unchanged during the course of the experiments. Induction of iNOS, COX-2, and IL-6 proteins by IL-17 was consistent with the RT-PCR data ( Fig. 3) with iNOS and COX-2 displaying peak expression approximately 28 h poststimulation (Fig. 3A).
The efficacy of dexamethasone in inhibiting IL-17-augmented gene expression was then tested. As shown in Fig. 2B, the co-addition of dexamethasone with IL-17 exerted almost complete reduction of IL-1 and iNOS mRNA expression and exhibited also reduced IL-6 and COX-2 mRNAs. Likewise, nanomolar concentrations of dexamethasone also blunted the induction of the iNOS, COX-2, and IL-6 proteins (Fig. 3, B and  C). On the other hand, no inhibitory effect of dexamethasone could be registered on stromelysin mRNA.
The role of p38 kinase signal transduction cascade in the induction of the aforementioned mRNAs was assessed by using the p38-specific inhibitor SB203580. The effect of this p38 inhibitor at a concentration of 10 M was considerably less as compared with dexamethasone, with COX-2 and IL-6 mRNA induction profiles displaying the highest, but still modest, SB203580 sensitivity ( Fig. 2A). A similarly mild effect of SB203580 was observed on the protein products of iNOS and COX-2 (Fig. 3B). The most impressive inhibitory effect of SB203580 was observed on the IL-17-induced secretion of IL-6, which was lowered by approximately 65% in the presence of 10 M inhibitor. In summary, dexamethasone is a potent antagonist of multiple IL-17-mediated gene expression responses, whereas inhibition of the p38 kinase signal transduction pathway has only a modest effect on these events.
IL-17 Induces NF-B Activation in Chondrocytes-Members of the Rel/NF-B family of transcription factors are common effectors of many cytokine-regulated pathways and are known to be activated by IL-17 in fibroblasts (14). In human articular chondrocytes, IL-17 treatment caused a substantial increase in NF-B DNA binding activity (Fig. 4A). Maximal NF-B activity was observed 1-3 h following stimulation, with activity still detectable after 24 h. This response was similar to the activation of NF-B by IL-1. 2 NF-B proteins are constitutively sequestered in the cytoplasm through binding to the inhibitory protein IB. Induction of NF-B activity in response to extracellular signaling events is achieved through phosphorylation-dependent degradation of the inhibitor, which leads to the nuclear translocation of free NF-B. We therefore evaluated IB␣ degradation in response to IL-17 treatment. Fig. 4C shows that within 30 min of stimulation, IL-17 elicited a transient reduction in IB␣ levels, which returned to basal values by 5 h. Thus, IB␣ degradation and re-expression is associated with NF-B activation by IL-17 in chondrocytes.
Glucocorticoids have been established as antagonists of NF-B DNA binding activity (22)(23)(24)(25). Given the observed pleiotropic inhibitory effects of dexamethasone on IL-17-induced gene expression in chondrocytes, we determined to which extent this was related to changes in NF-B activity. As expected, 1-h co-incubation with dexamethasone reduced the levels of IL-17-induced NF-B DNA binding (Fig. 4B). However, this inhibitory effect was not as dramatic as dexamethasone effects on gene expression.

Activation of MAP Kinases in Articular Chondrocytes by IL-17-
The MAP kinase cascade is one of the pivotal intracellular pathways activated by cytokine receptors. We determined whether similar activity patterns may account for the effects of IL-17 on chondrocytes. Initial experiments in human articular chondrocytes indeed revealed activation of the MAP kinases JNK, p38, ERK1, and ERK2 in seven of nine different donors (data not shown). However, the responses varied between donors with regard to their intensity, background activity in the nonstimulated controls, and kinetics. To exclude this variability, MAP kinase activation analyses were performed with primary bovine chondrocytes.
Recombinant human IL-17 activated MAP kinases in bovine articular chondrocytes. This response was reflected both in biochemical assays measuring kinase activity (as demonstrated for JNK) as well as in direct immunodetection of the phosphorylation status, and hence the implied activation, of the MAP kinases p38, ERK1, and ERK2 (Fig. 5A). Significant activation was registered already at the earliest time point analyzed (30 min), reaching a peak at 1 h following stimulation and decaying slowly thereafter. These data clearly demonstrate that several key players of the MAP kinase cascade are activated in chondrocytes exposed to IL-17.
Targets of the MAP kinase pathway are various transcription factors activated through phosphorylation by the downstream effectors of the cascade, such as JNKs (26). These include the heterodimers of the Jun/Fos families (an activity termed collectively AP-1), ATF2, Elk-1, and TCF (27,28). In the case of AP-1, phosphorylation of Jun by JNK is a prerequisite for the ability of the complex to execute transcriptional activation (29). Consistent with an effect of MAP kinases on transcriptional activation rather than DNA binding properties of AP-1, assays addressing DNA binding activity of this complex in IL-17-stimulated chondrocytes failed to detect an effect of the cytokine (data not shown). As AP-1 is central to the transcriptional induction of most of the genes found earlier in this study to be elevated in chondrocytes in response to IL-17, we sought ways to evaluate IL-17-dependent changes in AP-1 activity other than DNA binding.
For that purpose, we took advantage of the fact that glucocorticoids and AP-1 activities are mutually inhibitory, an effect that is considered central to the immunosuppressive effects of glucocorticoids (30,31). The inhibitory effect of dexamethasone on IL-17-induced chondrocyte gene expression (see Figs. 2 and 3) is consistent with this phenomenon. Biochemical studies previously demonstrated that the inhibition is brought about, at least in part, by direct binding between AP-1 and the glucocorticoid receptor (31,32) and by competition for shared transcriptional co-activators, such as CBP/p300 (33). As downregulation of AP-1 activity could in principle be achieved also through inhibition of essential upstream kinases, we wished to determine whether this occurs in the context of IL-17-treated chondrocytes. Fig. 5, B and C, demonstrates that this was indeed the case. In IL-17-stimulated chondrocytes, pharmacological concentrations of dexamethasone elicited a significant reduction in JNK activity as well as in its phosphorylation status. This inhibitory effect most probably reflects intervention of dexamethasone with an early component of the pathway, as it could be also documented in the phosphorylation status of other parts of the pathway, represented by p38 and ERK1 and ERK2 (Fig. 5B). Dose-response studies showed that at nanomolar concentrations, dexamethasone completely inhibited the IL-17 effect on phosphorylation of JNK-1 (Fig. 5C)  inflammatory responses, in chondrocytes in particular, and in a more general sense. They can also be interpreted to infer a central role for MAP kinases as glucocorticoid-sensitive mediators of IL-17-stimulated gene expression, most likely by phosphorylating AP-1 and additional transcription factors.
Interestingly, maximal dexamethasone-mediated inhibition of MAP kinase activation was achieved when the drug was pre-incubated with the cells 1.5 h prior to IL-17 administration. Shorter pre-incubation seemed insufficient, whereas longer pre-incubation reversed the inhibitory effect of dexamethasone (Fig. 5B). The significance of this phenomenon is not completely clear. One interpretation could be that dexamethasone, through glucocorticoid receptor, induces an immediate early short-lived MAPK inhibitor, whose expression decays at later stages of the transcriptional response to the drug. Such mech-anism could be analogous to the described induction of the NF-B inhibitor IB␣ by dexamethasone (22,23), although the precise details await identification of the factor(s) involved in dexamethasone-dependent MAPK inhibition. DISCUSSION IL-17 activates chondrocytes to express genes that are associated with cartilage degradation and joint inflammation. The pattern of cellular responses induced by IL-17 is similar to IL-1, the prototypic cytokine for the induction of the catabolic program in chondrocytes. Mechanisms involved with activation of chondrocytes by IL-17 are of interest because the IL-17 receptor is not related to known cytokine receptor families. To define mechanisms of chondrocyte activation by IL-17, the present study analyzed activation of MAP kinases, NF-B, and AP-1 as intracellular signaling events and their role in IL-17 responses.
The first part of the study used SB203580, a specific inhibitor of p38 kinase, and dexamethasone to analyze signals involved with IL-17 activation of chondrocytes. Glucocorticoids are widely used in the treatment of inflammatory conditions, in part because of their inhibitory effects on cytokine expression and function. Several IL-17-inducible responses were analyzed with these inhibitors. IL-17-induced NO production was only marginally reduced by SB203580. In contrast, dexamethasone showed significant inhibition of IL-17-induced NO production. This is distinct from the relative resistance of IL-1-induced NO production to dexamethasone effects (34 -36). The IL-17-induced expression of mRNAs for IL-1␤, IL-6, iNOS, COX-2, and stromelysin was not or only moderately reduced by SB203580. Dexamethasone showed inhibitory effects on most of these mRNAs, with the exception of stromelysin, and completely prevented some IL-17 effects. Similar differences in the effects of dexamethasone and SB203580 were observed for iNOS, COX-2, and IL-6 protein levels. These results suggest that p38 is not essential for IL-17 activation of chondrocytes. On the other hand, signal transduction events that are sensitive to inhibition by dexamethasone seem to be required for IL-17 activation of chondrocytes.
The second part of the study examined activation of NF-B and MAP kinases as signaling events that are important for cytokine-induced gene expression. One of the important DNA binding proteins in many cytokine-inducible responses is NF-B. Proteins of the NF-B family are retained in the cytoplasm through heterodimerization with one of several types of IB proteins. Cytokines and LPS trigger the phosphorylation and degradation of IB, resulting in the release of NF-B to enter the nucleus, where it activates gene transcription (reviewed in Refs. 37 and 38). The results from this study demonstrate activation of NF-B by IL-17 in chondrocytes, and this is associated with a transient decrease in the levels of IB␣. Remarkably, high levels of NF-B binding activity are detectable for at least 24 h after IL-17 stimulation. IL-17-induced activation of NF-B was inhibited by dexamethasone. This inhibitory effect was significantly lower compared with dexamethasone-mediated repression of gene expression in response to the cytokine. This discrepancy suggested that NF-B is not necessarily the key transcription factor mediating IL-17 signal transduction. Moreover, it seems that in chondrocytes NF-B activation is more refractory to glucocorticoids as compared with other cell systems (24). Mechanisms of glucocorticoid action are linked to the regulation of NF-B by enhancing the expression of IB␣ (22,23). In addition, the glucocorticoid receptor is capable of antagonizing NF-B activity by physical association, which results in repression of both DNA binding and transactivation functions of NF-B (24, 25). We did not detect induction of IB␣ in dexamethasone-treated chondrocytes (data not shown), sug- gesting that in these cells glucocorticoids are unable to exercise their full potential in inhibiting NF-B function, accounting for their incomplete inhibitory effect.
Another important signaling event in response to catabolic cytokines such as IL-1 or TNF in human articular chondrocytes is MAP kinase activation (7)(8)(9). Three subgroups of the MAPK family have been identified (39 -41). These kinases are structurally related and all are phosphorylated on tyrosine and threonine and activated by upstream kinases, the MAP kinase kinases. The p44 and p42 extracellular signal-regulated kinases ERK1 and ERK2 mediate responses mainly to mitogenic stimuli, and the Jun NH 2 -terminal kinases (JNK), which include 46-kDa JNK1 and 55-kDa JNK2, and p38 mediate responses to cellular stress. We show here that IL-17 activates the three subgroups of MAP kinases in chondrocytes. The time course of this response shows a peak for all three subgroups at 30 -60 min. This is different from the pattern of IL-1-induced MAP kinase activation in chondrocytes, which is maximal after 15-30 min and declines to basal levels by 60 min (7). The prolonged MAP kinase activation by IL-17 suggests that it may either be related to a prolonged upstream signal or to the induction of a secondary protein mediator, such as IL-1, which is released by the cells.
We addressed the role of the p38 MAP kinase in the IL-17 activation of chondrocytes by using a specific inhibitor, the imidazole compound SB203580. This drug caused a relatively minor reduction in IL-17-induced NO production and protein and RNA expression, suggesting that p38 activation may not be critical for the IL-17-induced responses. We addressed the role of ERK1 and ERK2 in the IL-17 activation of chondrocytes by using PD 098059, a specific inhibitor to MEK1 (43), the kinase directly upstream to ERK1 and ERK2. This drug did not effect IL-17-induced NO production or any of the IL-17-induced RNA expression (data not shown), suggesting that ERK1 and ERK2 may not be essential for the IL-17 effects. These kinases are activated by a broad set of stimuli that not only include catabolic cytokines but also growth factors that stimulate chondrocyte proliferation and extracellular matrix synthesis (7). It is thus possible that activation of JNK may be more important in mediating the IL-17 effects.
Dexamethasone inhibited MAP kinase activation and was most effective against IL-17-induced activation of JNK. These findings further support recent reports that inhibition of JNK activation may represent an additional regulatory mechanism of glucocorticoids (44 -46). Interference with the JNK pathway by dexamethasone was shown to inhibit UV-induced AP-1, ELK-1, and ATF-2 phosphorylation (44). In macrophages, LPS induction of TNF␣ production was associated with JNK activation. These responses, but not the LPS-induced activation of p38, were inhibited by dexamethasone (45). In mast cells, ERK1 activation and tyrosine phosphorylation of Raf1, Mek1, and phospholipase A2 by antigen stimulation were inhibited by dexamethasone (47). The correlation between JNK inhibition by dexamethasone and the inhibitory effects of the latter on chondrocyte responses to IL-17 suggests that JNK activation is more central to the inflammatory response compared with the other MAP kinases.
In conclusion, the results from the present study indicate that IL-17 activates NF-B and MAP kinases in chondrocytes. Of the various MAP kinases, JNK is more likely than ERK or p38 to mediate IL-17 effects. NF-B, although activated by IL-17, does not seem as central in mediating IL-17 signal transduction.