Interleukin-17-induced Gene Expression in Articular Chondrocytes Is Associated with Activation of Mitogen-activated Protein Kinases and NF-κB
1998; Elsevier BV; Volume: 273; Issue: 42 Linguagem: Inglês
10.1074/jbc.273.42.27467
ISSN1083-351X
AutoresTali Shalom‐Barak, Jacqueline Quach, Martin Lotz,
Tópico(s)Natural product bioactivities and synthesis
ResumoThis 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. 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. interleukin 1 mitogen-activated protein kinase extracellular signal-regulated kinase c-Jun NH2-terminal kinase nitric oxide inducible NO synthase tumor necrosis factor α cyclooxygenase-2 lipopolysaccharide reverse transcriptase-polymerase chain reaction Tris-buffered saline. Arthritic diseases are characterized by synovial inflammation and cartilage destruction (1Lotz M. Int. Med. 1992; 13: 55-66Google Scholar). Cytokines such as interleukin-1 (IL-1)1 and tumor necrosis factor α (TNFα) have been shown to play a pivotal role in these pathologies (2Westacott C.I. Sharif M. Semin. Arthritis Rheum. 1996; 25: 254-272Crossref PubMed Scopus (240) Google Scholar). IL-1 and TNFα promote extracellular matrix degradation through the induction of matrix metalloproteinases, such as collagenase and stromelysin. In addition, these inflammatory cytokines inhibit proteoglycan synthesis and induce inflammation-mediating enzymes like cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) (3Lotz M. Blanco F.J. von Kempis J. Dudler J. Maier R. Villiger P.M. Geng Y. J. Rheumatol. Suppl. 1995; 43: 104-108PubMed Google Scholar). Human articular chondrocytes stimulated with IL-1, TNFα, and bacterial lipopolysaccharides (LPS) produce high levels of prostaglandins and nitric oxide (NO) (4Stadler J. Stefanovic-Racic M. Billiar T. Curran R. McIntyre L. Georgescu H. Simmons R. Evans C. J. Immunol. 1991; 147: 3915-3920PubMed Google Scholar, 5Rediske J. Koehne C. Zhang B. Lotz M. Osteoarthritis Cartilage. 1994; 2: 199-206Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 6Palmer R.M. Hickery M.S. Charles I.G. Moncada S. Bayliss M.T. Biochem. Biophys. Res. Commun. 1993; 193: 398-405Crossref PubMed Scopus (279) Google Scholar). The effector cascades mediating inflammatory responses of chondrocytes to IL-1 and TNFα have been elucidated in part and shown to include the mitogen-activated protein kinase (MAPK) signal transduction pathway. IL-1 and TNFα activate all three of the MAP kinase subgroups ERK, JNK, and p38 (7Geng Y. Valbracht J. Lotz M. J. Clin. Invest. 1996; 98: 2425-2430Crossref PubMed Scopus (200) Google Scholar, 8Lo Y.Y.C. Wong J.M.S. Cruz T.F. J. Biol. Chem. 1996; 271: 15703-15707Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 9Scherle P.A. Pratta M.A. Feeser W.S. Tancula E.J. Arner E.C. Biochem. Biophys. Res. Commun. 1997; 230: 573-577Crossref PubMed Scopus (46) Google Scholar). In addition to IL-1 and TNFα, 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) (10Rouvier E. Luciani M.F. Mattei M.G. Denizot F. Golstein P. J. Immunol. 1993; 150: 5445-5456PubMed Google Scholar, 11Albrecht J.C. Nicholas J. Biller D. Cameron K.R. Biesinger B. Newman C. Wittmann S. Craxton M.A. Coleman H. Fleckenstein B. et al.J. Virol. 1992; 66: 5047-5058Crossref PubMed Google Scholar). Subsequent studies of human IL-17 (hIL-17) demonstrated exclusive expression of this cytokine in activated T-cells, predominantly of the CD4+ subtype (12Yao Z. Painter S.L. Fanslow W.C. Ulrich D. Macduff B.M. Spriggs M.K. Armitage R.J. J. Immunol. 1995; 155: 5483-5486PubMed Google Scholar, 13Fossiez F. Djossou O. Chomarat P. Flores-Romo L. Ait-Yahia S. Maat C. Pin J.J. Garrone P. Garcia E. Saeland S. Blanchard D. Gaillard C. Das Mahapatra B. Rouvier E. Golstein P. Banchereau J. Lebecque S. J. Exp. Med. 1996; 183: 2593-2603Crossref PubMed Scopus (1251) Google Scholar). 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 (12Yao Z. Painter S.L. Fanslow W.C. Ulrich D. Macduff B.M. Spriggs M.K. Armitage R.J. J. Immunol. 1995; 155: 5483-5486PubMed Google Scholar). IL-17 induces NF-κB activation and IL-6, IL-8, and ICAM-1 expression in murine and human fibroblasts (12Yao Z. Painter S.L. Fanslow W.C. Ulrich D. Macduff B.M. Spriggs M.K. Armitage R.J. J. Immunol. 1995; 155: 5483-5486PubMed Google Scholar, 13Fossiez F. Djossou O. Chomarat P. Flores-Romo L. Ait-Yahia S. Maat C. Pin J.J. Garrone P. Garcia E. Saeland S. Blanchard D. Gaillard C. Das Mahapatra B. Rouvier E. Golstein P. Banchereau J. Lebecque S. J. Exp. Med. 1996; 183: 2593-2603Crossref PubMed Scopus (1251) Google Scholar) and the production of IL-6, IL-8, granulocyte macrophage-colony-stimulating factor, and prostaglandin E2 in bone marrow stromal cells (14Yao Z. Fanslow W.C. Seldin M.F. Rousseau A.M. Painter S.L. Comeau M.R. Cohen J.I. Spriggs M.K. Immunity. 1995; 3: 811-821Abstract Full Text PDF PubMed Scopus (806) Google Scholar). In human macrophages, IL-17 stimulates the production of proinflammatory cytokines, including IL-1β and TNFα (15Jovanovic D.V. Di Battista J.A. Martel-Pelletier J. Jolicoeur F.C. He Y. Zhang M. Mineau F. Pelletier J.P. J Immunol. 1998; 160: 3513-3521PubMed Google Scholar). In chondrocytes from osteoarthritic human cartilage, IL-17 up-regulated the spontaneous production of nitric oxide (16Attur M.G. Patel R.N. Abramson S.B. Amin A.R. Arthritis Rheum. 1997; 40: 1050-1053Crossref PubMed Scopus (171) Google Scholar). mIL-17 was shown to have mitogenic effects on mouse splenic T cells (12Yao Z. Painter S.L. Fanslow W.C. Ulrich D. Macduff B.M. Spriggs M.K. Armitage R.J. J. Immunol. 1995; 155: 5483-5486PubMed Google Scholar), although this effect has not been demonstrated with hIL-17 and human T cells (13Fossiez F. Djossou O. Chomarat P. Flores-Romo L. Ait-Yahia S. Maat C. Pin J.J. Garrone P. Garcia E. Saeland S. Blanchard D. Gaillard C. Das Mahapatra B. Rouvier E. Golstein P. Banchereau J. Lebecque S. J. Exp. Med. 1996; 183: 2593-2603Crossref PubMed Scopus (1251) Google Scholar). 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-κB 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. 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 (17Blanco F.J. Ochs R.L. Schwarz H. Lotz M. Am. J. Pathol. 1995; 146: 75-85PubMed Google Scholar). All experiments were performed with primary or passage 1 cells. For kinase and immunoblot assays, 3 × 106 chondrocytes were cultured in 100-mm Petri dishes and serum-starved for 24 h before stimulation. After washing with phosphate-buffered saline containing 1 mmNa3VO4, cells were lysed in lysis buffer (50 mm NaCl, 50 mm Tris, pH 7.4, 0.5% Nonidet P-40, 1 mm EGTA, 1 mmNa3VO4, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin). 100 μg of lysates was used for JNK assay. For immunoblot assays of the phosphorylated kinases p38, ERK1, and ERK2 and for Iκbα, 50 μg of whole cell lysates were used. For iNOS and COX-2, 200 μg of whole cell lysates were used. Proteins were separated on 8–10% polyacrylamide gels. Hypotonic detergent cellular extracts were prepared, and the solid-state JNK assay was performed as described (7Geng Y. Valbracht J. Lotz M. J. Clin. Invest. 1996; 98: 2425-2430Crossref PubMed Scopus (200) Google Scholar). The extracts were mixed with 10 μl 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 μl of kinase buffer (20 mm HEPES, pH 7.6, 20 mmMgCl2, 10 mm β-glycerophosphate, 0.1 mm Na3VO4, 10 mmdithiothreitol) containing 20 μm ATP and 5 μCi [γ-32P]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. Nuclear proteins were prepared according to modification of the method described by Schreiberet al. (18Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3917) Google Scholar). Chondrocytes (3 × 106 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 μg/ml leupeptin, and 50 μg/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 −80 °C for electrophoretic mobility shift assay. NF-κB binding activities were studied by using double-stranded oligonucleotides (22-mers) 5′-AGTTGAGGGGACTTTCCCAGGT-3′ (Promega), corresponding to the NF-κB site in the human κ light chain promoter. The double-stranded oligonucleotides (3.5 pmol) were labeled with [32P]ATP in the presence of T4 polynucleotide kinase and were separated from unincorporated [32P]ATP by gel filtration using Centri-sep columns (Princeton Separations, Adelphia, NJ). Nuclear proteins (5 μg) were preincubated for 45 min with 1 μg of poly dI:dC in NF-κB 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. 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 μg) was reverse-transcribed with MoMLV-RT (Life Technologies, Inc.) for 30–120 min at 37 °C. RT reactions were subjected to PCR with Taq DNA polymerase (Boehringer Mannheim). PCR conditions were within the linear range of amplification of the target cDNAs. PCR primers used were IL-1β (387-bp product): sense, GAGCTCGCCAGTGAAATGATGGC; antisense, CAAGCTTTTTTGCTGTGAGTCCCG (35 cycles); IL-6 (465-bp product): sense, CACAGACAGCCACTCACCTCTTC; antisense, GCTGCGCAGAATGAGATGAGTTGT (28 cycles); iNOS (236-bp product): sense, ACATTGATCAGAAGCTGTCCCAC; antisense, CAAAGGCTGTGAGTCCTGCAC (28 cycles); COX-2 (337-bp product): sense, TTGTCCCAGACAAGCAGGC; antisense, CATTCCTACCACCAGCAACC (35 cycles); stromelysin (504-bp product): sense, TGGACAAAGGATACAACAGGGA; antisense, AGCTCGTACCTCATTTCCTCTG (23 cycles); and glyceraldehyde-3-phospate dehydrogenase (190-bp product): sense, TGGTATCGTGGAAGGACTCATGAC; antisense, ATGCCAGTGAGCTTCCCGTTCAGC (23 cycles). Whole cell lysates were separated on 8–10% SDS-polyacrylamide 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-Iκbα 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. Conditioned media were collected from chondrocyte cultures 24 h after treatment with IL-17, p38 inhibitor, or dexamethasone. The supernatants were added to half-area enzyme-linked immunosorbent assay plate (Costar, Cambridge, MA) that had been precoated with 25 μl of 2 μg/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 μg/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). The concentration of nitrites, the stable end products of cellular NO breakdown, in conditioned media from chondrocytes was determined by the Griess reaction using NaNO2 as standard. All results are expressed as nmoles of nitrites per 100,000 cells. Recombinant human IL-17 and recombinant human TNFα were purchased from R&D Systems (Minneapolis, MN). Recombinant human IL-1β was purchased from Intergen (Purchase, NY). p38 kinase inhibitor, SB203580, and dexamethasone were purchased from Sigma. MEK1 inhibitor, PD98059, was purchased from New England Biolabs. GlutathioneS-transferase-c-Jun (1–223) was a gift of Dr. Michael Karin (University of California, San Diego). 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. 1 A, 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 TNFα in the same cells, as was the maximal NO levels induced by both cytokines (Fig. 1 B). 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.Figure 1Inhibition of NO release in IL-17-treated chondrocytes. Primary cultures of human articular chondrocytes were cultured in media supplemented with 1% fetal bovine serum alone or stimulated with different concentrations of IL-17 (A) or IL-1 or TNFα (B). C, cells were stimulated with IL-17 (20 ng/ml) in the presence of the indicated concentrations of SB203580 (SB) or dexamethasone (DEX). Conditioned media were collected after 48 h for measurement of NO levels by the Griess reaction. Results represent mean values ± S.E. A total of five experiments was performed to test the effects of dexamethasone and SB203580.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (19Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. et al.Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar, 20Lee J.C. Badger A.M. Griswold D.E. Dunnington D. Truneh A. Votta B. White J.R. Young P.R. Bender P.E. Ann. N. Y. Acad. Sci. 1993; 696: 149-170Crossref PubMed Scopus (154) Google Scholar, 21Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1980) Google Scholar). Fig. 1 C shows that SB203580 reduced IL-17-driven NO synthesis in a dose-dependent manner, reaching a maximum of 30% inhibition at 10 μm, a concentration at which it still retains full specificity toward p38 (21Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1980) Google Scholar). 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. 1 C). A mixture of dexamethasone and SB203580 had an additive inhibitory effect. The effects reported in Fig. 1 C 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. 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-1β, 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. 3 A).Figure 3iNOS, COX-2, and IL-6 protein synthesis.Chondrocytes were cultured in media or stimulated with 10 ng/ml IL-17 for the time points indicated (A). B, cells were stimulated with 10 ng/ml IL-17 for 24 h in the presence of the indicated concentrations of dexamethasone (DEX) or SB203580. Cell lysates were analyzed by Western blotting for iNOS or COX-2.C, conditioned media from 24 h cultures of chondrocytes that were treated with the indicated combinations of IL-17, dexamethasone, or SB203580 were analyzed for IL-6 content by enzyme-linked immunosorbent assay. Data represent mean ± S.E. from two experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The efficacy of dexamethasone in inhibiting IL-17-augmented gene expression was then tested. As shown in Fig. 2 B, 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 μm 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. 2 A). A similarly mild effect of SB203580 was observed on the protein products of iNOS and COX-2 (Fig. 3 B). 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 μm 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. Members of the Rel/NF-κB family of transcription factors are common effectors of many cytokine-regulated pathways and are known to be activated by IL-17 in fibroblasts (14Yao Z. Fanslow W.C. Seldin M.F. Rousseau A.M. Painter S.L. Comeau M.R. Cohen J.I. Spriggs M.K. Immunity. 1995; 3: 811-821Abstract Full Text PDF PubMed Scopus (806) Google Scholar). In human articular chondrocytes, IL-17 treatment caused a substantial increase in NF-κB DNA binding activity (Fig. 4 A). Maximal NF-κB activity was observed 1–3 h following stimulation, with activity still detectable after 24 h. This response was similar to the activation of NF-κB by IL-1. 2T. Shalom-Barak and M. Lotz, unpublished data. NF-κB proteins are constitutively sequestered in the cytoplasm through binding to the inhibitory protein IκB. Induction of NF-κB 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-κB. We therefore evaluated IκBα degradation in response to IL-17 treatment. Fig. 4 C shows that within 30 min of stimulation, IL-17 elicited a transient reduction in IκBα levels, which returned to basal values by 5 h. Thus, IκBα degradation and re-expression is associated with NF-κB activation by IL-17 in chondrocytes. Glucocorticoids have been established as antagonists of NF-κB DNA binding activity (22Scheinman R.I. Cogswell P.C. Lofquist A.K. Baldwin Jr., A.S. Science. 1995; 270: 283-286Crossref PubMed Scopus (1597) Google Scholar, 23Auphan N. DiDonato J.A. Rosette C. Helmberg A. Karin M. Science. 1995; 270: 286-290Crossref PubMed Scopus (2163) Google Scholar, 24Scheinman R.I. Gualberto A. Jewell C.M. Cidlowski J.A. Baldwin Jr., A.S. Mol. Cell. Biol. 1995; 15: 943-953Crossref PubMed Google Scholar, 25Ray A. Prefontaine K.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 752-756Crossref PubMed Scopus (917) Google Scholar). 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-κB activity. As expected, 1-h co-incubation with dexamethasone reduced the levels of IL-17-induced NF-κB DNA binding (Fig. 4 B). However, this inhibitory effect was not as dramatic as dexamethasone effects on gene expression. 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. 5 A). 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 (26Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2414) Google Scholar). These include the heterodimers of the Jun/Fos families (an activity termed collectively AP-1), ATF2, Elk-1, and TCF (27Karin M. Hunter T. Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 28Stein B. Anderson A.D. Annu. Rep. Med. Chem. 1996; 31: 289-298Google Scholar). In the case of AP-1, phosphorylation of Jun by JNK is a prerequisite for the ability of the complex to execute transcriptional activation (29Westwick J.K. Weitzel C. Minden A. Karin M. Brenner D.A. J. Biol. 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Herrlich P. EMBO J. 1992; 11: 2241-2246Crossref PubMed Scopus (233) Google Scholar). 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 (31Konig H. Ponta H. Rahmsdorf H.J. Herrlich P. EMBO J. 1992; 11: 2241-2246Crossref PubMed Scopus (233) Google Scholar, 32Schule R. Rangarajan P. Kliewer S. Ransone L.J. Bolado J. Yang N. Verma I.M. Evans R.M. Cell. 1990; 62: 1217-1226Abstract Full Text PDF PubMed Scopus (1038) Google Scholar) and by competition for shared transcriptional co-activators, such as CBP/p300 (33Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1926) Google Scholar). As down-regulation 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
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