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Signal Transduction Pathways Involved in Rheumatoid Arthritis Synovial Fibroblast Interleukin-18-induced Vascular Cell Adhesion Molecule-1 Expression

2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês

10.1074/jbc.m206337200

ISSN

1083-351X

Autores

Jacques Morel, Christy C. Park, Kui Zhu, Pawan Kumar, Jeffrey H. Ruth, Alisa E. Koch,

Tópico(s)

Toxin Mechanisms and Immunotoxins

Resumo

Vascular cell adhesion molecule (VCAM)-1 has been implicated in interactions between leukocytes and connective tissue, including rheumatoid arthritis (RA) synovial tissue fibroblasts. Such interactions within the synovium contribute to RA inflammation. Using phosphoinositide 3-kinase (PI3-kinase) inhibitor LY294002 and Src inhibitor PP2, we show that interleukin (IL)-18-induced ERK1/2 activation is Src kinase-dependent. Antisense (AS) c-Src oligonucleotide (ODN) treatment reduced IL-18-induced ERK1/2 expression by 32% compared with control, suggesting an upstream role of Src in ERK1/2 activation. AS c-Src ODN treatment also inhibited Akt expression by 74% compared with sense control. PI3-kinase inhibitor LY294002 or AS PI3-kinase ODN inhibited Akt expression. AS c-Src ODN inhibited Akt phosphorylation, confirming Src is upstream of PI3-kinase in IL-18-induced RA synovial fibroblast signaling. IL-18 induced a time-dependent activation of c-Src, Ras, and Raf-1, suggesting this signaling cascade plays a role in ERK activation. IL-18 directly activated Src kinase by more than 4-fold over basal levels by enzymatic assay. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) is activated by IL-18 through ERK and Src but not through PI3-kinase. In an alternate pathway, inhibition of IL-1 receptor-associated kinase-1 (IRAK) with AS ODN to IRAK reduced IL-18-induced expression of nuclear factor κB (NFκB). Finally, IL-18-induced cell surface VCAM-1 expression was inhibited by treatment with AS ODNs to c-Src, IRAK, PI3-kinase, and ERK1/2 by 57, 43, 41, and 32% compared with control sense ODN treatment, respectively. These data support a role for IL-18 activation of three distinct pathways during RA synovial fibroblast stimulation: two Src-dependent pathways and the IRAK/NFκB pathway. Targeting VCAM-1 signaling mechanisms may represent therapeutic approaches to inflammatory and angiogenic diseases characterized by adhesion molecule up-regulation. Vascular cell adhesion molecule (VCAM)-1 has been implicated in interactions between leukocytes and connective tissue, including rheumatoid arthritis (RA) synovial tissue fibroblasts. Such interactions within the synovium contribute to RA inflammation. Using phosphoinositide 3-kinase (PI3-kinase) inhibitor LY294002 and Src inhibitor PP2, we show that interleukin (IL)-18-induced ERK1/2 activation is Src kinase-dependent. Antisense (AS) c-Src oligonucleotide (ODN) treatment reduced IL-18-induced ERK1/2 expression by 32% compared with control, suggesting an upstream role of Src in ERK1/2 activation. AS c-Src ODN treatment also inhibited Akt expression by 74% compared with sense control. PI3-kinase inhibitor LY294002 or AS PI3-kinase ODN inhibited Akt expression. AS c-Src ODN inhibited Akt phosphorylation, confirming Src is upstream of PI3-kinase in IL-18-induced RA synovial fibroblast signaling. IL-18 induced a time-dependent activation of c-Src, Ras, and Raf-1, suggesting this signaling cascade plays a role in ERK activation. IL-18 directly activated Src kinase by more than 4-fold over basal levels by enzymatic assay. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) is activated by IL-18 through ERK and Src but not through PI3-kinase. In an alternate pathway, inhibition of IL-1 receptor-associated kinase-1 (IRAK) with AS ODN to IRAK reduced IL-18-induced expression of nuclear factor κB (NFκB). Finally, IL-18-induced cell surface VCAM-1 expression was inhibited by treatment with AS ODNs to c-Src, IRAK, PI3-kinase, and ERK1/2 by 57, 43, 41, and 32% compared with control sense ODN treatment, respectively. These data support a role for IL-18 activation of three distinct pathways during RA synovial fibroblast stimulation: two Src-dependent pathways and the IRAK/NFκB pathway. Targeting VCAM-1 signaling mechanisms may represent therapeutic approaches to inflammatory and angiogenic diseases characterized by adhesion molecule up-regulation. Interleukin-18 (IL-18) 1The abbreviations used are: IL, interleukin; RA, rheumatoid arthritis; VCAM-1, vascular adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; IFN, interferon; NFκB, nuclear factor κB; IRAK, interleukin-1 receptor-associated kinase-1; JNK, c-Jun N-terminal kinase; AP-1, activator protein-1; STAT, signal transducer and activator of transcription; PDTC, pyrrolidine dithiocarbamate; PI3-kinase, phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; NRTK, nonreceptor tyrosine kinase; Me2SO, dimethyl sulfoxide; EMSA, electrophoretic mobility shift assay; RIPA, radioimmunoprecipitation; ODN, oligonucleotide; S, sense; AS, antisense; FACS, fluorescence-activated cell sorter; ELISA, enzyme-linked immunosorbent assay; PTK, protein-tyrosine kinase; IL-18R, IL-18 receptor; TNF, tumor necrosis factor; TRAF, TNF-receptor-associated factor 6; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; PI, phosphatidylinositol; RBD, Ras binding domain. is a proinflammatory cytokine associated with various pathological conditions including rheumatoid arthritis (RA). IL-18 induces the release of Th1 cytokines by T cells and macrophages and also stimulates the production of inflammatory mediators, such as chemokines by synovial fibroblasts or nitric oxide by macrophages and chondrocytes (1McInnes I.B. Gracie J.A. Liew F.Y. Arthritis Rheum. 2001; 44: 1481-1483Crossref PubMed Scopus (27) Google Scholar, 2Morel J.C. Park C.C. Kumar P. Koch A.E. Lab. Invest. 2001; 81: 1371-1383Crossref PubMed Scopus (80) Google Scholar). Additionally, we have shown that IL-18 acts upon endothelial cells to induce angiogenesis and cell adhesion (3Park C.C. Morel J.C. Amin M.A. Connors M.A. Harlow L.A. Koch A.E. J. Immunol. 2001; 167: 1644-1653Crossref PubMed Scopus (250) Google Scholar, 4Morel J.C. Park C.C. Woods J.M. Koch A.E. J. Biol. Chem. 2001; 276: 37069-37075Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). IL-18 is mainly produced by activated macrophages, whereas the IL-18 receptor (IL-18R) is expressed on T lymphocytes, natural killer cells, macrophages, neutrophils, and chondrocytes (1McInnes I.B. Gracie J.A. Liew F.Y. Arthritis Rheum. 2001; 44: 1481-1483Crossref PubMed Scopus (27) Google Scholar, 5Okamura H. Tsutsui H. Kashiwamura S. Yoshimoto T. Nakanishi K. Adv. Immunol. 1998; 70: 281-312Crossref PubMed Google Scholar, 6Olee T. Hashimoto S. Quach J. Lotz M. J. Immunol. 1999; 162: 1096-1100PubMed Google Scholar). The IL-18R complex is composed of two protein chains α and β. The IL-18Rα is the extracellular binding domain of the IL-18R complex, whereas the IL-18Rβ is the signal transducing chain. When IL-18 binds to the IL-18R, it induces the formation of an IL-1R-associated kinase (IRAK)/TNF receptor-associated factor 6 (TRAF-6) complex that subsequently activates nuclear factor κB (NFκB) in Th1 cells (7Matsumoto S. Tsuji-Takayama K. Aizawa Y. Koide K. Takeuchi M. Ohta T. Kurimoto M. Biochem. Biophys. Res. Commun. 1997; 234: 454-457Crossref PubMed Scopus (190) Google Scholar) and in EL4/6.1 thymoma cells (8Thomassen E. Bird T.A. Renshaw B.R. Kennedy M.K. Sims J.E. J. Interferon Cytokine Res. 1998; 18: 1077-1088Crossref PubMed Scopus (101) Google Scholar). IL-18 also operates through IRAK-independent pathways that remain to be elucidated. For instance, IL-18 mediates interferon-γ (INF-γ) production by the mitogen-activated protein kinases p38 and p42/44, also known as ERK1/2 (9Kalina U. Kauschat D. Koyama N. Nuernberger H. Ballas K. Koschmieder S. Bug G. Hofmann W.K. Hoelzer D. Ottmann O.G. J. Immunol. 2000; 165: 1307-1313Crossref PubMed Scopus (113) Google Scholar). Phosphatidylinositol 3-kinase (PI3-kinase) is a lipid kinase that consists of catalytic (p110) and regulatory (p85) subunits. PI3-kinase catalyzes the phosphorylation of the inositol phospholipids at position 3 to generate phosphatidylinositol 3-phosphates, phosphatidylinositol 3,4-biphosphates, and phosphatidylinositol 3–5-triphosphates. These phosphorylated lipid products act as second messengers, activating protein kinases such as Akt (also known as protein kinase B). PI3-kinase is activated by a large spectrum of cytokines, growth factors, and hormones (10Fry M.J. Biochim. Biophys. Acta. 1994; 1226: 237-268Crossref PubMed Scopus (174) Google Scholar). This activation of PI3-kinase is generally regulated by receptor tyrosine kinase and non-receptor tyrosine kinase (NRTK). PI3-kinase can also be activated by G-protein-coupled receptors or by the small GTPase Ras (11Kapeller R. Cantley L.C. Bioessays. 1994; 16: 565-576Crossref PubMed Scopus (553) Google Scholar, 12Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1731) Google Scholar). PI3-kinase has been implicated as a key signaling molecule for transcription factor activation, protein synthesis, angiogenesis, and cell adhesion (13Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1229) Google Scholar, 14Jiang B.H. Zheng J.Z. Aoki M. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1749-1753Crossref PubMed Scopus (485) Google Scholar). We recently showed that the PI3-kinase inhibitor LY294002 inhibited RA synovial fibroblast IL-18-induced VCAM-1 expression by 50% when used alone and by 85% when used with the NFκB inhibitor pyrrolidine dithiocarbamate (2Morel J.C. Park C.C. Kumar P. Koch A.E. Lab. Invest. 2001; 81: 1371-1383Crossref PubMed Scopus (80) Google Scholar). These results suggested the existence of at least two independent pathways involved in IL-18-induced adhesion molecule expression. Further investigation of IL-18-induced signaling mechanisms revealed involvement of Src, an NRTK that functions in ligand-induced cellular responses, such as leukocyte survival, adhesion, migration, and proliferation. Src has also been implicated in various cancers and in bone resorption (15Jeschke M. Brandi M.L. Susa M. J. Bone Miner. Res. 1998; 13: 1880-1889Crossref PubMed Scopus (21) Google Scholar). Activation of Src requires phosphorylation at its activation site Tyr-418 concomitantly with decreased phosphorylation at its negative regulatory site Tyr-529; regulation also involves phosphorylation on other residues such as Tyr-215. c-Src activation by tyrosine kinase receptors leads to translocation from the plasma membrane to the cytoskeleton, where Src interacts with a host of proteins that orchestrate cell-matrix adhesion and cell migration (16Verbeek B.S. Vroom T.M. Rijksen G. Exp. Cell Res. 1999; 248: 531-537Crossref PubMed Scopus (20) Google Scholar). Here we found that IL-18 directly activates Src with rapid kinetics, and Src activation appears to be an early event common to the PI3-kinase/Akt and ERK1/2 pathways. There is precedence for the involvement of transcription factor activation in IL-18-mediated immune and inflammatory functions. For instance, IL-18 activates AP-1 and NFκB in the Jurkat T cell leading to IL-2 expression (17Greene C.M. Meachery G. Taggart C.C. Rooney C.P. Coakley R. O'Neill S.J. McElvaney N.G. J. Immunol. 2000; 165: 4718-4724Crossref PubMed Scopus (85) Google Scholar). We therefore examined involvement of the PI3-kinase and ERK1/2 signaling pathways in AP-1 activation. Because many cytokine-mediated functions, including IL-18-induced IFN-γ expression, are regulated via NFκB activation (18Kojima H. Aizawa Y. Yanai Y. Nagaoka K. Takeuchi M. Ohta T. Ikegami H. Ikeda M. Kurimoto M. J. Immunol. 1999; 162: 5063-5069PubMed Google Scholar, 19Tsuji-Takayama K. Aizawa Y. Okamoto I. Kojima H. Koide K. Takeuchi M. Ikegami H. Ohta T. Kurimoto M. Cell. Immunol. 1999; 196: 41-50Crossref PubMed Scopus (44) Google Scholar), its significance in IL-18-induced VCAM-1 expression was examined. One approach for determining the role of the NFκB pathway is by way of IRAK involvement, because IRAK is known to be associated with NFκB upon IL-18 stimulation (20Robinson D. Shibuya K. Mui A. Zonin F. Murphy E. Sana T. Hartley S.B. Menon S. Kastelein R. Bazan F. O'Garra A. Immunity. 1997; 7: 571-581Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 21Thomas J.A. Allen J.L. Tsen M. Dubnicoff T. Danao J. Liao X.C. Cao Z. Wasserman S.A. J. Immunol. 1999; 163: 978-984PubMed Google Scholar). Furthermore, IRAK was initially cloned and characterized as a kinase associated with the IL-1 receptor (22Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar), suggesting its importance to IL-18 signaling as IL-18 is a member of the IL-1 family. Because antisense oligodeoxynucleotides (ODNs) offer a potential gene therapy strategy to block transcription or translation of specific genes, antisense ODNs to relevant signaling molecules were employed to examine RA synovial fibroblast signaling events following IL-18 stimulation. Ultimately, the treatment effect of antisense ODN on IL-18-induced VCAM-1 expression implicated specific signaling molecules involved in RA synovial fibroblast VCAM-1 expression. The ability of these antisense ODNs to inhibit IL-18-induced activation of the transcription factor NF-κB and downstream VCAM-1 expression was also assessed. We examined the signal transduction mechanisms by which IL-18 induces VCAM-1 expression in RA synovial fibroblasts. Our findings demonstrate that IL-18 induced VCAM-1 expression through Src kinase, PI3-kinase/Akt, and ERK1/2 pathways. The important role of the IRAK/NFκB pathway in VCAM-1 expression was also elucidated. Finally, we describe a new signaling cascade involving Src/Ras/Raf/ERK/AP-1 in IL-18-stimulated RA synovial fibroblasts. Recombinant human IL-18 (specific activity 4.1 × 104 units/mg) was purchased from Peprotech (Rocky Hill, NJ). Orthovanadate, para-nitrophenyl phosphate, leupeptin, aprotinin, phenylmethylsulfonyl fluoride, dimethyl sulfoxide (Me2SO), IGEPAL CA-630, protein A- and G-agarose, pertussis toxin, and phosphatidylinositol were bought from Sigma. Protease inhibitor mixture tablets were obtained from Roche Molecular Biochemicals. Modified radioimmunoprecipitation (RIPA) lysis buffer was prepared according to Upstate Biotechnology, Inc., protocol, with final concentrations being Tris-HCl (50 mm, pH 7.4), Nonidet P-40 (1%), NaCl (150 nm), EDTA (1 mm), phenylmethylsulfonyl fluoride (1 mm), aprotinin/leupeptin/pepstatin (1 μg/ml each), NaF (1 mm). The anti-Src-agarose beads, clone GD11, a mouse monoclonal IgG1, was purchased form Upstate Biotechnology, Inc. (Lake Placid, NY), as was Src kinase and the Src kinase assay kit, including Src kinase reaction buffer (SrcRB), Src substrate peptide, Src manganese/ATP mixture, and P81 phosphocellulose paper. The radioisotope [γ-32P]ATP (3000 Ci/mmol) was obtained from PerkinElmer Life Sciences. Phosphoric acid (0.75%) diluted from 85% stock solution and trichloroacetic acid was from Sigma. LY294002, PP2, SB203580, and PD98059 were purchased from Calbiochem. Monoclonal mouse anti-human VCAM-1, clone 4B9 that recognizes domain 1 of VCAM-1, was a generous gift from Dr. Roy Lobb (Biogen, Cambridge, MA); mouse IgG1 antibody (negative control) was purchased from Coulter Clone (Hialeah, FL); goat anti-mouse PE (Jackson ImmunoResearch) was used as secondary antibody for flow experiments. Mouse monoclonal anti-phosphotyrosine antibody (clone 4G10), mouse monoclonal anti-human phospho-ERK1/2 antibody, and the Ras activation detection kit were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal rabbit anti-human phospho-Src antibody (Tyr(P)418) and polyclonal rabbit anti-human phospho-Raf-1 (Tyr(P)340 and Tyr(P)341) antibody were obtained from BIOSOURCE International (Camarillo, CA). Mouse monoclonal anti-human phospho-Akt (Ser(P)473) antibody was purchased from Cell Signaling Technology (Beverly, MA) and BIOSOURCE International. Mouse polyclonal anti-human IRAK antibody was obtained from BD PharMingen, and rabbit polyclonal anti-human NFκB p65 antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal anti-tubulin antibody was obtained from Oncogene Research Products (Boston, MA). Goat anti-rabbit IgG horseradish peroxidase-conjugated antibody was purchased from Sigma. Protein estimation reagents (BCA kit) were from Pierce. Enhanced chemiluminescence Western blotting detection reagents and sheep anti-mouse IgG horseradish peroxidase conjugated antibody were obtained from Amersham Biosciences. LipofectAMINE and LipofectAMINE Plus™ Reagents were obtained from Invitrogen. Fibroblasts were isolated from synovium obtained from RA patients meeting the American College of Rheumatology criteria for RA who had undergone total joint replacement surgery or synovectomy (23Arnett F.C. Edworthy S.M. Bloch D.A. McShane D.J. Fries J.F. Cooper N.S. Healey L.A. Kaplan S.R. Liang M.H. Luthra H.S. Medsger T.A.J. Mitchell D.M. Neustadt D.H. Pinals R.S. Schaller J.G. Sharp J.T. Wilder R.L. Hunder G.G. Arthritis Rheum. 1988; 31: 315-324Crossref PubMed Scopus (18700) Google Scholar). Fresh synovial tissues were minced and digested in solution of dispase, collagenase, and DNase. Cells were used at passage 5 or older, at which time they were a homogeneous population of fibroblasts. Synovial fibroblasts were grown in 175-mm tissue culture flasks (Falcon, Franklin Lakes, NJ) at 37 °C, in a humidified atmosphere with 5% CO2. Upon confluence, cells were passaged by brief trypsinization as described previously (24Koch A.E. Polverini P.J. Leibovich S.J. Arthritis Rheum. 1986; 29: 471-479Crossref PubMed Scopus (108) Google Scholar). RA synovial fibroblasts were plated onto 6- or 10-cm Petri dishes (Falcon) at 1 × 105 cells/ml and allowed to adhere for 24 h at 37 °C in 5% CO2 atmosphere. Alternatively, for ODN experiments, fibroblasts were plated at 4 × 106cells/well on 6-well plates. Fibroblasts were serum-starved for at least 14 h before stimulation with IL-18 (10 nm) for 0, 5, 10, and 20 min. At the end of each period, supernatants were gently aspirated, and fibroblasts were lysed in extraction buffer containing 100 mm Tris, pH 7.4, 100 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm NaF, 20 mm NaP2O4, 2 mmNa3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitors (1 tablet/10 ml). For experiments with signaling inhibitors, RA synovial fibroblasts were preincubated with the respective inhibitor for 60–120 min before activation with IL-18. For the ODN experiments the cells were treated as detailed and lysed similarly. Nuclei were pelleted (1250 × g at 4 °C for 5 min), and supernatants of different samples were collected for determination of protein content using a BCA protein assay kit. Cell lysates were mixed 1:1 with Laemmli's sample buffer, boiled for 5 min, and then centrifuged at 10,000 × gfor 10 min. Equal amounts (or 15 μg) of each sample was subjected to 10% SDS-PAGE. Separated proteins were electrophoretically transferred from the gel onto nitrocellulose membranes using a semi-dry transblotting apparatus (Bio-Rad). To block nonspecific binding, membranes were incubated with 5% milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. Blots were incubated in respective primary antibody in TBST + 5% milk at 4 °C overnight. After washing with TBST, blots were incubated with horseradish peroxidase-conjugated sheep anti-mouse IgG (1:10,000) or with goat anti-rabbit IgG (1:10,000) for 1 h at room temperature. An ECL detection system (Amersham Biosciences) was used to detect specific protein bands. Different bands were then scanned and quantitated using the software UN-SCAN-IT version 5.1 (Silk Scientific, Orem, UT). Blots were subsequently stripped and restained with antibody to tubulin to determine relative band densities. RA synovial fibroblasts (8 × 105 cells) were plated in 10 cm-dishes in RPMI 1640 containing 10% FBS, 1% penicillin/streptomycin. Once cells were 80–90% confluent, they were further incubated in serum-free RPMI 1640 for at least 14 h. RA synovial fibroblasts were then stimulated with IL-18 (10 nm) for 0, 10, and 20 min at 37 °C. At the end of the incubation, cell lysate was prepared. Protein content of each sample was quantitated using a BCA protein assay kit and normalized according to the protein concentration. 500 μg of each sample in 500 μl of lysis buffer was incubated overnight at 4 °C with 5 μl of rabbit anti-PI3-kinase antibody directed against the 85-kDa regulatory subunit. 60 μl of protein A-agarose conjugate (50% slurry in PBS) was added to each sample and further incubated for 1 h at 4 °C. The immunoprecipitates were collected by centrifugation at 14,000 × g for 10 s. The immunoprecipitates were then washed 3 times with buffer A (137 mm NaCl, 20 mm Tris-HCl, pH 7.4, 1 mm CaCl2, 1 mm MgCl2, and 0.1 mm sodium orthovanadate) containing 1% nonionic detergent IGEPAL CA-630, followed by 3 washes with buffer B (0.1m Tris-HCl, pH 7.4, 5 mm lithium chloride, and 0.1 mm sodium orthovanadate) and 3 washes with TNE (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 5 mm EDTA) containing 0.1 mm sodium orthovanadate. To each sample, the following reagents were added sequentially: 50 μl of TNE, 10 μl (20 μg) of phosphatidylinositol (PI, in 10 mm Tris-HCl, pH 7.4 containing 1 mmEGTA), and 10 μl of 100 mm MgCl2. The PI reaction was initiated with addition of 5 μl of [γ-32P]ATP. The reaction mixture was incubated at 37 °C for 15 min with continuous agitation. The reaction was stopped by addition of 20 μl of 6 n HCl. Radiolabeled lipid was extracted from the reaction sample by adding 160 μl of CH3Cl/MeOH (1:1), vortexing, and separating the organic and aqueous phases by centrifugation for 10 min at 14,000 ×g. 50 μl of radiolabeled lipid containing the lower organic phase were spotted onto oxalate-treated TLC plates (Fisher) and developed in CHCl3/MeOH/H2O/NH4OH (60:47:11.3:2). TLC plates were dried and autoradiographed. Ras activation was studied using a Ras activation kit (Upstate Biotechnology, Inc.). RA synovial fibroblasts were stimulated with IL-18 (10 nm) for different times. At each time point, synovial fibroblast extracts were prepared with cell lysis buffer, and protein content in each sample was quantitated. The Ras activation assay involved two steps. In the first step, the activated form of Ras was immunoprecipitated from 800 μg of each synovial fibroblast lysate sample with an immobilized Raf-1-Ras binding domain (Raf-1-RBD) and subsequently run on 10% SDS-PAGE as described above. The presence of activated Ras in samples was then detected by probing with a specific mouse monoclonal anti-Ras antibody (1 μg/ml). Different bands were then scanned and quantitated using an imaging densitometer. Src kinase was first immunoprecipitated with agarose beads conjugated with anti-Src antibody (mouse monoclonal IgG1). RA synovial fibroblasts were plated at 1 × 106 cells on 10-cm culture dishes, adhered overnight in complete media, serum-starved overnight, and stimulated with IL-18 (10 nm) for 10 min. Cells were washed in cold PBS and lysed with 0.5 ml of RIPA buffer. The total cell lysate was diluted to 1 μg/μl with PBS, and 1 mg was mixed with 4 μg (8 μl) of anti-Src antibody-conjugated agarose beads and gently rocked at 4 °C for 2 h. Beads were collected by microcentrifuging (5 s at 14,000 × g), the supernatant was drained, and beads were then washed (3 times) with ice-cold PBS. Samples containing agarose beads, Src antibody, and Src protein in complex were then used to determine Src kinase activation. Direct activation of Src was examined with n Src kinase assay kit (Upstate Biotechnology, Inc.) to measure the ability of activated Src to act on known substrate. Stock solutions necessary for the assay were prepared as follows: Src substrate peptide (600 μm, diluted in SrcRB), purified Src kinase (p60 c-Src, 20units/10 μl/test), [γ-32P]ATP (PerkinElmer Life Sciences, 1 mCi/100 μl, 3000 Ci/mmol, further diluted to 1 μCi/μl with Src manganese/ATP mixture), and 0.075% phosphoric acid (diluted from 85% with PBS). Substrate peptide (10 μl, 150 μm final concentration) was added to 10 μl of SrcRB, to which was added 10 μl of Src (p60, c-Src, 20 units) or immunoprecipitated sample (200 μg minimum) and 10 μl of diluted [γ-32P]ATP (10 μCi) in a microcentrifuge tube. The reagent mixture was incubated for 10 min at 30 °C with agitation. To precipitate peptides, 20 μl of 40% trichloroacetic acid was added to each mixture and incubated at room temperature for 5 min. Onto the center of P81 phosphocellulose paper squares, 25 μl of each sample was spotted. Squares were washed with 0.75% phosphoric acid (5 times for 5 min) and acetone (once for 3 min). Samples were read in a scintillation counter, and counts/min of immunoprecipitated enzyme samples were compared with counts/min of the background control samples (no enzyme). This assay was performed with fibroblasts from 4 different RA donors. Sequences of the ODNs employed in this study are listed in Table I. Antisense ODNs were selected for sequence target to c-Src (25Chellaiah M. Fitzgerald C. Alvarez U. Hruska K. J. Biol. Chem. 1998; 273: 11908-11916Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), IRAK-1 (26Guo F. Wu S. Immunopharmacology. 2000; 49: 241-246Crossref PubMed Scopus (19) Google Scholar), PI3-kinase (27Guo F.K., Li, Y.L. Wu S.G. Acta Pharmacol. Sin. 2000; 21: 318-320PubMed Google Scholar, 28Skorski T. Kanakaraj P. Nieborowska-Skorska M. Ratajczak M.Z. Wen S.C. Zon G. Gewirtz A.M. Perussia B. Calabretta B. Blood. 1995; 86: 726-736Crossref PubMed Google Scholar), and ERK1/2 (29Nishibe T. Parry G. Ishida A. Aziz S. Murray J. Patel Y. Rahman S. Strand K. Saito K. Saito Y. Hammond W.P. Savidge G.F. Mackman N. Wijelath E.S. Blood. 2001; 97: 692-699Crossref PubMed Scopus (38) Google Scholar). The corresponding sense ODN was used as control for each antisense ODN. The ODNs were synthesized and purified by the Northwestern University Biotechnology Laboratory and modified with phosphorothioate. Lipofection-encapsulated ODNs were prepared using LipofectAMINE and LipofectAMINE Plus Reagent from Invitrogen. Each ODN was reconstituted to 0.4 μg/μl concentration in double distilled water and then diluted in Opti-MEM media as detailed. For transient transfection of cells, ODN/Plus reagent-LipofectAMINE complex was prepared with 40 μg of ODN/100 μl of serum-free Opti-MEM with 2.5 μl of Plus reagent and 4 μg of LipofectAMINE Reagent/100 μl of serum-free Opti-MEM and incubation at room temperature for 30 min, followed by dilution with another 800 μl of Opti-MEM serum-free medium for an additional 15-min incubation. RA synovial fibroblasts were plated at 90% confluency at 4 × 105 cells per well on 6-well plates or per 6-cm dish and allowed to adhere in RPMI, 10% FBS, 1% penicillin and streptomycin. After attachment, cells were then treated with 5 μm of antisense or sense ODN by incubation with 1 ml of ODN/Plus reagent/LipofectAMINE complex for 5 h, followed by media change to complete RPMI, 10% FBS, 1% penicillin and streptomycin overnight. The media were also changed to serum-free RPMI, 1% penicillin and streptomycin for 8–10 h prior to stimulation with IL-18 (10 nm, 10 min, for the Western blot and Src kinase experiments; 5 nm, 8 h, for VCAM-1 expression by flow cytometry experiments).Table IODN sequences used in treatmentsODNSequence (5′-3′)Antisense IRAK-1CCC CCC GGC CAT GGC TGCSense IRAK-1GCA GCC ATG GCC GGG GGGAntisense c-SrcGGG CTT GCT CTT GCT GCT CCC CATSense c-SrcATG GGG AGC AGC AAG AGC AAG CCCAntisense PI3-kinaseGTA CTG GTA CCC CTC AGC ACT CATSense PI3-kinaseATG AGT GCT GAG GGG TAC CAG TACAntisense ERK1/2GCC GCC GCC GCC GCC ATSense ERK1/2ATG GCG GCG GCG GCG GC Open table in a new tab For the chemical inhibition experiments, RA synovial fibroblasts were plated onto 6-cm Petri dishes (Falcon) at 1 × 105 cells/ml and allowed to adhere overnight, and cells were pretreated with pertussis toxin (100 ng/ml) for 12 h before stimulation with IL-18 (10 nm) for 8 h. For the ODN experiments, RA synovial fibroblasts were plated and treated as detailed above prior to IL-18 (5 nm) stimulation for 8 h. Cells were harvested with a cell scraper and transferred to fluorescence-activated cell sorting (FACS) tubes (BD PharMingen). Cells were then treated with mouse anti-VCAM-1 or isotype-matched control (5 μg/ml) as primary antibody followed by incubation for 30 min with PE-conjugated goat anti-mouse antibody (1.5 μg/ml). Samples were washed twice with PBS, 1% FBS, and then fixed with 1% paraformaldehyde. Samples were assayed using an Epics XL-MCL flow cytometer (Beckman Coulter). Prior to data acquisition, the PE channel was standardized using fluorescent beads (Rainbow Beads, Spherotech, Libertyville, IL). Isotype-matched control values were subtracted from the test results. RA ST fibroblasts were plated on 96-well tissue culture plates in RPMI 1640, 10% fetal calf serum and adhered for 14 h. Cells were preincubated with specific inhibitors or Me2SO vehicle control in RPMI 1640, 2% fetal calf serum before stimulation with IL-18 (5 nm) for 8 h. Inhibitors (LY294002, PP2, SB203580, and PD98059) or vehicle Me2SO were applied to cells for 60 min, and then cells were stimulated with IL-18 (5 nm) for 12 h. Cell viability was judged by trypan blue exclusion and was >90%. RA synovial fibroblasts were successively fixed in 3.7% formalin in PBS and blocked in PBS, 1% bovine serum albumin, 5% goat serum for 15 min. After successive incubations in mouse anti-human VCAM-1 or isotype-matched control for 2 h, goat anti-mouse IgG peroxidase-conjugated antibody was added for 1 h, and the ELISA was developed with tetramethylbenzidine substrate. The reaction was stopped with 1 n H2SO4 before reading at 450 nm with a Bio-R

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