ERK 1/2- and JNKs-dependent Synthesis of Interleukins 6 and 8 by Fibroblast-like Synoviocytes Stimulated with Protein I/II, a Modulin from Oral Streptococci, Requires Focal Adhesion Kinase
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m212065200
ISSN1083-351X
AutoresL. Neff, Mirjam B. Zeisel, Vanessa A. Druet, Ken Takeda, Jean‐Paul Klein, Jean Sibilia, Dominique Wachsmann,
Tópico(s)Peptidase Inhibition and Analysis
ResumoProtein I/II, a pathogen-associated molecular pattern from oral streptococci, is a potent inducer of interleukin-6 (IL-6) and IL-8 synthesis and release from fibroblast-like synoviocytes (FLSs), cells that are critically involved in joint inflammation. This synthesis implicates ERK 1/2 and JNKs as well as AP-1-binding activity and nuclear translocation of NF-κB. The mechanisms by which protein I/II activates MAPKs remain, however, elusive. Because focal adhesion kinase (FAK) was proposed to play a role in signaling to MAPKs, we examined its ability to contribute to the MAPKs-dependent synthesis of IL-6 and IL-8 in response to protein I/II. We used FAK–/– fibroblasts as well as FAK+/+ fibroblasts and FLSs transfected with FRNK, a dominant negative form of FAK. The results demonstrate that IL-6 and IL-8 release in response to protein I/II was strongly inhibited in both protein I/II-stimulated FAK–/– and FRNK-transfected cells. Cytochalasin D, which inhibits protein I/II-induced phosphorylation of FAK (Tyr-397), had no effect either on activation of ERK 1/2 and JNKs or on IL-6 and IL-8 release. Taken together, these results indicate that IL-6 and IL-8 release by protein I/II-activated FLSs is regulated by FAK independently of Tyr-397 phosphorylation. Protein I/II, a pathogen-associated molecular pattern from oral streptococci, is a potent inducer of interleukin-6 (IL-6) and IL-8 synthesis and release from fibroblast-like synoviocytes (FLSs), cells that are critically involved in joint inflammation. This synthesis implicates ERK 1/2 and JNKs as well as AP-1-binding activity and nuclear translocation of NF-κB. The mechanisms by which protein I/II activates MAPKs remain, however, elusive. Because focal adhesion kinase (FAK) was proposed to play a role in signaling to MAPKs, we examined its ability to contribute to the MAPKs-dependent synthesis of IL-6 and IL-8 in response to protein I/II. We used FAK–/– fibroblasts as well as FAK+/+ fibroblasts and FLSs transfected with FRNK, a dominant negative form of FAK. The results demonstrate that IL-6 and IL-8 release in response to protein I/II was strongly inhibited in both protein I/II-stimulated FAK–/– and FRNK-transfected cells. Cytochalasin D, which inhibits protein I/II-induced phosphorylation of FAK (Tyr-397), had no effect either on activation of ERK 1/2 and JNKs or on IL-6 and IL-8 release. Taken together, these results indicate that IL-6 and IL-8 release by protein I/II-activated FLSs is regulated by FAK independently of Tyr-397 phosphorylation. Integrins are a family of heterodimeric transmembrane proteins that bind a variety of ligands, including proteins of the extracellular matrix, intercellular adhesion molecules, plasma proteins, and complement factors (1Van der Flier A. Sonnenberg A. Cell Tissue Res. 2001; 305: 285-298Crossref PubMed Scopus (818) Google Scholar). Furthermore, integrins act as receptors for many pathogenic bacteria or viruses and are presently referred to as pattern recognition receptors as they recognize molecular structures called pathogen-associated molecular patterns (PAMPs) 1The abbreviations used are: PAMPs, pathogen-associated molecular patterns; MAPKs, mitogen-activated protein kinases; ERK 1/2, extracellular-regulated kinases; JNKs, receptor Jun N-terminal kinases; FAK, focal adhesion kinase; PI, phosphatidyl inositol; FRNK, FAK-related non-kinase; FAT, focal adhesion targeting; GFP, green fluorescent protein; FLSs, fibroblast-like synoviocytes; IL-6, interleukin-6; EGF, epidermal growth factor; FCS, fetal calf serum; BES, 2-[bis(2-hydroxyethyl)-amino]ethanesulfonic acid; TBS, Tris-buffered saline; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay. on pathogens. For example, α5β1 integrin is the known receptor of several bacterial PAMPs such as invasin of Yersinia pseudotuberculosis (2Gustavsson A. Armulik A. Brakebusch C. Fassler R. Johansson S. Fallman M. J. Cell Sci. 2002; 115: 2669-2678PubMed Google Scholar), fimbrillin A of Porphyromonas gingivalis (3Yilmaz O. Watanabe K. Lamont R.J. Cell. Microbiol. 2002; 4: 305-314Crossref PubMed Scopus (182) Google Scholar), ipa proteins of Shigella spp. (4Watarai M. Funato S. Sasakawa C. J. Exp. Med. 1996; 183: 991-999Crossref PubMed Scopus (182) Google Scholar), filamentous hemagglutinin of Bordetella pertussis (5Ishibashi Y. Relman D.A. Nishikawa A. Microb. Pathog. 2001; 30: 279-288Crossref PubMed Scopus (54) Google Scholar), and protein I/II of Streptococcus mutans (6Al-Okla S. Chatenay-Rivauday C. Klein J.P. Wachsmann D. Cell. Microbiol. 1999; 1: 157-168Crossref PubMed Scopus (25) Google Scholar). In addition, several other observations have demonstrated that integrin recognition of fibronectin promotes adhesion and internalization of bacteria expressing fibronectin-binding proteins, for example, protein M1 of Streptococcus pyogenes (7Cue D. Southern S.O. Southern P.O. Prabhakar J. Lorelli W. Smalheer J.M. Mousa S.A. Cleary P.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 14: 2858-2863Crossref Scopus (90) Google Scholar) or fibronectin-binding protein A of Staphylococcus aureus (8Sinha B. Francois P. Que Y.A. Hussain M. Heilmann C. Moreillon P. Lew D. Krause K.H. Peters G. Ferrmann M. Infect. Immun. 2000; 68: 6871-6878Crossref PubMed Scopus (202) Google Scholar). Because integrins are known to control cellular processes as diverse as proliferation, differentiation, apoptosis, and cell migration, it is likely that their interactions with pathogens will have an important impact on host cell responses as well as on microbial pathogenesis. There are many outside-in signaling pathways that have been identified downstream from integrins, notably, the MAPKs pathway, which converts extracellular stimuli to intracellular signals and which is central to many cellular functions. MAPKs belong to one of the major pathways transmitting signals to early genes implicated in the regulation of cytokine responses. Numerous data demonstrate that pro-inflammatory cytokine synthesis in response to bacteria or bacterial components (e.g. lipopolysaccharide, polyosides, lipoteichoic acids, and proteins), after binding to their cognate receptors on different eukaryotic cells, is controlled by the MAPKs pathway and that this synthesis may play an important role in innate immunity as well as in various inflammatory disorders (9Rawadi G. Ramez V. Lemercier B. Roman-Roman S. J. Immunol. 1998; 160: 1330-1339PubMed Google Scholar, 10Scherle P.A. Jones E.A. Favata M.F. Daulerio A.J. Covington M.B. Nurnberg S.A. Mafolda R.L. Trzaskos J.M. J. Immunol. 1998; 161: 5681-5686PubMed Google Scholar). Using protein I/II, a PAMP from oral streptococci, we reported previously that interaction of this cell wall component with fibroblast-like synoviocytes (FLSs), cells that are critically involved in rheumatoid arthritis-associated joint inflammation, triggers the production and release of inflammatory mediators such as IL-6 and IL-8 (11Müller-Ladner U. Gay R.E. Gay S. Curr. Opin. Rheumatol. 2000; 12: 186-194Crossref PubMed Scopus (52) Google Scholar, 12Gourieux B. AL-Okla S. Schöller M. Klein J.P. Sibilia J. Wachsmann D. FEMS Immunol. Med. Microbiol. 2000; 1280: 1-7Google Scholar). This cytokine synthesis involves ERK 1/2 and JNKs as well as AP-1-binding activity and nuclear translocation of NF-κB (13Neff L. Zeisel M. Sibilia J. Schöller-Guinard M. Klein J.P. Wachsmann D. Cell. Microbiol. 2001; 3: 703-712Crossref PubMed Scopus (62) Google Scholar). However, the mechanisms by which integrins initiate the MAPKs pathway are generally not fully understood. There is increasing evidence that FAK is critical in linking integrins to this pathway insofar as FAK, which colocalizes with integrins in focal adhesions, is associated with different signaling, adaptor, or structural proteins, including Src family protein-tyrosine kinases, phospholipase C-γ, PI 3-kinase, p130Cas, Shc, Grb2, and paxillin. Several mechanisms can be used by FAK to activate ERK 1/2. For example, autophosphorylation of FAK at Tyr-397 generates a binding site for Src family protein-tyrosine kinases (14Calalb M. Polte T. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar), and Src-mediated phosphorylation of FAK Tyr-925 allows the binding of the SH2 domain of Grb2 and the formation of a Grb2·SoS complex, which activates the Ras/MAPKs cascade. In addition, interaction with Src leads to the phosphorylation of FAK Tyr-576 and Tyr-577 and full kinase activity. The Ras/MAPKs pathway can also be activated by recruitment and phosphorylation of p130Cas, which promotes the binding of the adaptor proteins Crk, Nck, and SoS (15Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1995; 372: 786-791Crossref Scopus (1448) Google Scholar, 16Polte T.R. Hanks S.K. J. Biol. Chem. 1997; 272: 5501-5509Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 17Ilic D. Damsky C. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar, 18Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1036) Google Scholar, 19Schaller M.D. Biochim. Biophys. Acta. 2001; 1540: 1-2Crossref PubMed Scopus (505) Google Scholar). Many integrins use more than one mechanism to activate the ERK pathway, and some of them seem to be independent of FAK and cell-specific. One group has provided evidence that caveolin-1 and the adaptor protein Shc play a role in relaying signals from integrins to ERK in primary cells, but FAK·Src complexes might control the temporal response of ERK initiated by Shc, in B-Raf-expressing cells (20Wary K.K. Mariotti A. Zurzolo C. Giancotti F.G. Cell. 1998; 94: 625-626Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 21Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3829) Google Scholar, 22Barberis L. Wary K.K. Fiucci G. Liu F. Hirsch E. Brancaccio M. Altruda F. Tarone G. Giancotti F.G. J. Biol. Chem. 2000; 275: 36532-36540Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In addition, FAK, independently of tyrosine phosphorylation and kinase activity, was proposed to regulate integrin-dependent activation of JNKs by a mechanism involving paxillin and the small GTP-binding proteins of the Rho family (23Igishi T. Fukuhara S. Patel V. Katz B.Z. Yamada K. Gutkind J.S. J. Biol. Chem. 1999; 274: 30738-30746Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Another linkage was suggested, occurring through association of FAK with Src and p130Cas and the recruitment of Crk and Dock 180 (24Oktay M. Wary K.K. Dans M. Bireg R.B. Giancotti F. J. Cell Biol. 1999; 145: 1461-1469Crossref PubMed Scopus (248) Google Scholar). Recent findings have also demonstrated that FAK coordinates MAPKs signaling, following costimulation of integrins and growth factors receptors for EGF or platelet-derived growth factor, through interactions mediated by FAK-C-terminal and -N-terminal domain connections to the respective transmembrane receptors (25Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Damsky C.H. Schlaepfer D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1068) Google Scholar, 26Hauck C.R. Sieg D.J. Hsia D.J. Loftus J.C. Gaarde W.A. Monia B.P. Schlaepfer D. Cancer Res. 2001; 61: 7079-7090PubMed Google Scholar). Based on these observations, it can be hypothesized that FAK may participate in various biochemical routes linking integrins to MAPKs cascades. This work was thus undertaken to examine the ability of FAK to contribute to signaling events leading to ERK 1/2- and JNKs-dependent synthesis of proinflammatory cytokines, in response to protein I/II. Our results indicate that FAK is critical for IL-6 and IL-8 release by protein I/II-activated FLSs but that Tyr-397, the major site of autophosphorylation that promotes the assembly of a number of signaling complexes, is not essential for this process. Material—Cell culture media (RPMI 1640 and M199), fetal calf serum (FCS), penicillin, streptomycin, amphotericin B, Taq DNA polymerase, dNTPs, Moloney murine leukemia virus, RNase inhibitor, IL-6, and β actin primers were from Invitrogen (Cergy-Pontoise, France). Cell culture media had an endotoxin content that never exceeded 0.04 ng/ml, as tested by the Limulus chromogenic assay. Polymyxin B, Tri reagent, type XI collagenase, wortmannin, cytochalasin D, and Geneticin were obtained from Sigma (Saint-Quentin-Fallavier, France). Protease inhibitor mixture was from Roche Applied Science (Meylan, France). Hexanucleotide mix was from Roche Applied Science (Mannheim, Germany). The enzyme immunoassay kits for human IL-8 and IL-6 detection were from Endogen (Interchim, Montluçon, France) and for mouse IL-6 detection from R&D (Minneapolis, MN). Purified α5β1 integrins were obtained from Valbiotech (Paris, France). Rabbit anti-FAK (pY397), anti-Shc (pY317) polyclonal antibodies were from BIOSOURCE (Cliniscience, Montrouge, France), rat anti-integrin β1 chain, clone 9EG7 monoclonal antibodies, and rabbit anti-FAK polyclonal antibodies were from BD Pharmingen (Cliniscience). Rabbit anti-active ERK 1/2 and anti-active JNKs polyclonal antibodies were from Promega (Charbonnières, France), rabbit anti-ERK 1/2, anti-JNKs, and anti-Shc polyclonal antibodies were from Upstate Biotechnology (Euromedex, Souffelweyersheim, France). Horseradish peroxidase-conjugated goat anti-mouse IgG polyclonal antibodies, anti-rabbit IgG polyclonal antibodies, and the chemiluminescence kit were from Amersham Biosciences (Saclay, France). A universal tyrosine kinase assay kit was from Takara Biomedicals (Genevilliers, France). The FAK-related non-kinase plasmid (FRNK-YCam) has been previously described (27Giannone G. Rondé P. Guire M. Haiech J. Takeda K. J. Biol. Chem. 2002; 277: 26364-26371Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), the pRk5-ShcY317F plasmid was a generous gift from Dr. Filippo G. Giancotti (Memorial Sloane-Kettering Cancer Center, New York, NY). The Nucleofector™ kit was from Amaxa (Köln, Germany). Throughout this study, buffers were prepared with apyrogenic water obtained from Fandre (Ludres, France). Cell Culture and Transfections—Human FLSs were isolated from RA synovial tissues from three different patients, at the time of knee joint arthroscopic synovectomy as described previously (28Dechanet J. Taupin J.L. Chomarat M.C. Moreau J.F. Banchereau J. Miossec P. Eur. J. Immunol. 1994; 24: 3222-3228Crossref PubMed Scopus (63) Google Scholar). The diagnoses conformed to the revised criteria of the American College of Rheumatology (29Arnett F. Edworthy S.M. Bloch D.A. McShane D.J. Fires J.F. Cooper N.S. et al.Arthritis Rheum. 1988; 3: 315-324Crossref Scopus (18700) Google Scholar). Briefly, tissues were minced, digested with 1 mg/ml collagenase in serum-free RPMI 1640 for 3 h at 37 °C, centrifuged (130 × g, 10 min, 4 °C), and resuspended in M199-RPMI 1640 (1:1) containing 2 mm l-glutamine, penicillin (100 IU/ml), streptomycin (100 μg/ml), amphotericin B (0.25 μg/ml), and 20% heat-inactivated FCS (complete medium). After overnight culture, non-adherent cells were removed, and adherent cells were cultured in complete medium. At confluence, cells were trypsinized and passaged in 75-cm2 culture flasks in complete medium containing 10% heat-inactivated FCS. Between the third and the tenth passages, during which time cultures were a homogeneous population of fibroblastic cells, negative for CD16 as determined by fluorescence-activated cell sorting analysis, cells (5 × 103 cells per well) were grown to confluence in 96-well plates (7–10 days). Cells were deprived of serum for 24 h, before addition of the appropriate stimuli, and diluted in serum-free RPMI 1640 with antibiotics. Cell number and cell viability were examined by the MTT test (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test) as described elsewhere (30Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (46884) Google Scholar). FAK+/+ and FAK–/– primary mouse embryo fibroblasts were a generous gift from Dr. Dusko Ilic (Department of Medicine, University of California, San Francisco). Cells were cultured in Dulbecco's modified Eagle's medium containing penicillin (100 IU/ml), streptomycin (100 μg/ml), amphotericin B (0.25 μg/ml), β-mercaptoethanol (0.1 mm), non-essential amino acids (1%), sodium pyruvate (1%), and 10% heat-inactivated FCS. FAK+/+ cells were transfected with FRNK-YCam (27Giannone G. Rondé P. Guire M. Haiech J. Takeda K. J. Biol. Chem. 2002; 277: 26364-26371Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) by the calcium phosphate method. Briefly, 15 μg of plasmidic DNA in 1 ml of BES-buffered saline (borate-buffered saline, pH 6.95, containing 2.5 mm of CaCl2) was added to 5 × 105 cells for 20 h at 37 °C (3% CO2). Following transfection, cells were rinsed and then cultured in complete Dulbecco's modified Eagle's medium containing Geneticin (1.5 mg/ml) for 2 weeks. The antibiotic-resistant cells were then pooled and used for further analysis. Green fluorescent protein (GFP) was used to determine the transfection efficiency. Transient transfection of FLSs was performed using the Nucleofector™ kit. 2 μg of plasmidic DNA was added to 5 × 105 FLSs suspended in 100 μl of human dermal fibroblast Nucleofector™ solution. The program U-23 was selected for a high density of transfection according to the manufacturer's instructions. Cells were then plated in 96-well plates (5 × 104 cells per well) and serum-starved for 24 h before activation experiments. Purification of Protein I/II—Recombinant protein I/II of S. mutans OMZ 175 was purified from pHBsr-1-transformed Escherichia coli cell extract by gel filtration and immunoaffinity chromatography as previously described (31Chatenay-Rivauday C. Yamodo I. Sciotti M. Ogier J.A. Klein J.P. Mol. Microbiol. 1998; 29: 39-48Crossref PubMed Scopus (26) Google Scholar). The purity of the protein was checked by SDS-PAGE after staining with Coomassie Blue. Protein I/II migrated as a single band having an apparent molecular mass of 195 kDa. Activation of Cells—FLSs were preincubated with 100 μl of various inhibitors diluted in serum-free RPMI 1640 with antibiotics: for 40 min at 37 °C with cytochalasin D (0.5, 1 and 2 μm), for 1 h at 37 °C with anti-integrin β1 chain mAbs (5, 20, and 40 μg/ml), with wortmannin (50, 100, and 200 nm), and then incubated with 100 μl of serum-free RPMI 1640 containing protein I/II (125 pm final concentration). Protein I/II (125 pm, 200 μl) was also preincubated for2hat4 °C with purified α5β1 integrins (1, 5, and 10 μg/ml) and then used to stimulate FLSs. After a 20-h incubation period, culture supernatants were harvested and used to estimate IL-6 and IL-8 release by a heterologous two-site sandwich ELISA as previously described (32Vernier A. Diab M. Soell M. Haan-Archipoff G. Beretz A. Wachsmann D. Klein J.P. Infect. Immun. 1996; 64: 3016-3022Crossref PubMed Google Scholar). To confirm that the observed effects were not due to possible lipopolysaccharide contamination, all the experiments were performed in presence of polymyxin B (2 μg/ml). Detection of IL-6-mRNA—Total RNA was extracted from 106 FAK+/+ or FAK–/– cells activated with 125 pm protein I/II for 1 h, using 1 ml of Tri reagent and reverse-transcribed for 45 min at 42 °C. 100 ng of total RNA was mixed with 5 units of Moloney murine leukemia virus, 10 units of RNase inhibitor, 10 nm of dNTP, 50 pm of hexanucleotide mix, 100 nm dithiothreitol, 2.5 mm MgCl2, 500 mm KCl, and 100 mm Tris-HCl, pH 8.3. PCR was performed in a volume of 50 μl containing 1 μl of the reverse transcription mixture, 100 mm Tris-HCl, pH 8.3, 500 mm KCl, 1.25 mm MgCl2, 20 pm of each primer, 20 mm of dNTPs, and 2.5 units of Taq DNA polymerase, in a 9600 PerkinElmer Life Sciences cycler set for 30 cycles. The PCR temperatures used were 94 °C for 1 min (denaturing), 60 °C for 1 min (annealing), and 72 °C for 1 min (polymerization) followed by an extension of 10 min at 72 °C. PCR fragments were then separated on 1.5% agarose gels and visualized with ethidium bromide. The specific primers for IL-6 and β-actin were selected based on published mouse IL-6 and β actin cDNA sequences. The oligonucleotide primers used were for IL-6: 5′-TTCCTCTCTGCAAGAGACT-3′ and 5′-TCAGGAAGTCTCTCTATGT-3′, and for β-actin: 5′-ATGGATGACGATATCGCT-3′ and 5′-TGGACTGTCTGATGGAGTA-3′. Western Blot Detection of Tyrosine Phosphorylation—106 cells were incubated for various times in 100 μl of serum-free RPMI 1640 supplemented with antibiotics with or without 125 pm of protein I/II, in the presence or absence of cytochalasin D (1 μm). After stimulation, cells were centrifuged (130 × g for 10 min at 4 °C), and the pellets were suspended for 20 min in 100 μl of ice-cold lysis buffer (1% Triton X-100, 20 mm Tris-HCl, pH 8.0, 137 mm NaCl, 10% glycerol, 1 mm sodium orthovanadate, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitors) (33Tachado S.D. Gerold P. McConvill M.J. Baldwin T. Quilici D. Schwarz R.T. Schofield L. J. Immunol. 1996; 156: 1897-1907PubMed Google Scholar). The Triton X-100-soluble proteins were separated by centrifugation (14,000 × g for 10 min at 4 °C), and the supernatant was subjected to SDS-PAGE and transferred electrophoretically to nitrocellulose membranes. Membranes were blocked using 1% bovine serum albumin in TBS (20 mm Tris, pH 7.5, 150 mm NaCl) for 1 h at 25 °C. The blots were then incubated with various antibodies: anti-FAK (pY397), anti-Shc (pY317), anti-active ERK 1/2, and anti-active JNKs in TBS-Tween (0.1% Tween 20) for 2 h at 25 °C, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG polyclonal antibodies (1 h at 25 °C) and detected by enhanced chemiluminescence according to the manufacturer's instructions. To confirm the presence of equal amounts of FAK, ERK 1/2, JNK, and Shc proteins, bound antibodies were removed from the membrane by incubation in 62.5 mm Tris, pH 6.7, 100 mm β-mercaptoethanol, 2% SDS for 30 min at 50 °C and probed again with either anti-FAK, anti-ERK 1/2, anti-JNKs, or anti-Shc polyclonal antibodies. FAK Tyrosine Kinase Assay—4 × 106 cells were incubated for 15 min in serum-free RPMI 1640 supplemented with antibiotics with or without 125 pm protein I/II, in the presence or absence of cytochalasin D (1 μm). Cells activated for 15 min with fibronectin (10 μg/ml) were used as control. Cells were lysed as described above, and FAK was immunoprecipitated with anti-FAK polyclonal antibodies. Protein A-Sepharose CL-4B (50%, 100 μl) was then added for 90 min at 4 °C. After centrifugation (10,000 × g, 1 min, 4 °C), the pellet was used for a non-radioactive tyrosine kinase assay according to the manufacturer's instructions. FAK kinase activity was assessed using protein-tyrosine kinase standards included in the assay kit. Exposure of FLSs to Protein I/II Causes Phosphorylation of FAK—Because previous observations from our laboratory indicated that, in endothelial cells, protein I/II interactions with α5β1 integrins increased the tyrosine phosphorylation of FAK and lead to IL-8 secretion (6Al-Okla S. Chatenay-Rivauday C. Klein J.P. Wachsmann D. Cell. Microbiol. 1999; 1: 157-168Crossref PubMed Scopus (25) Google Scholar), we first investigated the role of α5β1 integrins in the FLSs activation process. The results show that addition of either increasing amounts of purified α5β1 integrins or anti-integrin β1 chain antibodies induced inhibition of protein I/II-stimulated IL-6 and IL-8 release (Fig. 1). An inhibition of cytokine release of about 80% was obtained with 10 μg/ml α5β1 integrins and 45% with 40 μg/ml anti-integrin β1 chain antibodies (p < 0.05). As observed in endothelial cells, this suggests that α5β1 integrins are implicated in the activation process leading to IL-6 and IL-8 release from protein I/II-activated FLSs. We next examined the capacity of protein I/II to stimulate phosphorylation of FAK in FLSs. Cell lysates were analyzed directly by blotting with specific anti-FAK (pY397) antibodies. Stimulation of FLSs with protein I/II for various times (1, 5, 15, 30, and 60 min) resulted in an increased amount of phosphorylated FAK (Fig. 2), which was detectable within 5 min and remained elevated for at least 30 min. Fibronectin was used as a positive control. These results demonstrate that interaction of protein I/II with FLSs induces phosphorylation of FAK at Tyr-397. Protein I/II-induced Signaling to MAPKs and IL-6 and IL-8 Release Occurs via an FAK-dependent Pathway—Recently, Neff et al. (13Neff L. Zeisel M. Sibilia J. Schöller-Guinard M. Klein J.P. Wachsmann D. Cell. Microbiol. 2001; 3: 703-712Crossref PubMed Scopus (62) Google Scholar) studied the eventual role of bacterial components such as protein I/II in promoting joint inflammation and reported that NF-κB and the MAPKs/AP-1 pathways are both involved in IL-6 and IL-8 release from FLSs stimulated with protein I/II. Thus, we next asked whether FAK could participate in the signaling events leading to IL-6 and IL-8 release from activated cells via the MAPKs pathway. In one set of studies and as preliminary experiments, we used FAK+/+ and FAK–/– primary mouse embryo fibroblasts. FAK+/+ and FAK–/– cells were incubated with protein I/II (125 pm) for 30 min, and then immunoblotting experiments were performed. As shown in Fig. 3A, stimulation of FAK+/+ fibroblasts with protein I/II increased FAK as well as ERK 1/2 tyrosine phosphorylation, however, protein I/II failed to stimulate tyrosine phosphorylation of ERK 1/2 in FAK–/– cells. These results raise the possibility that FAK participates in MAPKs activation in protein I/II-stimulated cells. We thus explored the cytokine response of FAK–/– fibroblasts stimulated with protein I/II, using FAK+/+ fibroblasts as positive controls. As illustrated in Fig. 3 (B and C), protein I/II increased IL-6 mRNA production and IL-6 release from FAK+/+ fibroblasts, but protein I/II-induced IL-6 production was completely inhibited in FAK–/– fibroblasts (p < 0.01). This suggests that FAK is involved in protein I/II-induced release of IL-6. To rule out the possibility that the observed inhibition could be due to a non-specific inhibition of other cellular functions, FAK+/+ fibroblasts were transfected with FRNK, the C-terminal region of FAK, which localizes to focal adhesions but does not contain the FAK kinase domain and which is known to block activation of FAK when overexpressed. As shown in Fig. 3A, protein I/II treatment did not increase tyrosine phosphorylation of FAK and ERK 1/2 in FRNK-transfected FAK+/+ fibroblasts. Moreover, overexpression of FRNK inhibited IL-6 release from activated cells as compared with wild-type cells (p < 0.01, Fig 3C). These observations correlate with the results obtained with FAK–/– fibroblasts. To further demonstrate the requirement of FAK in cytokine release from protein I/II-activated fibroblasts, the cytokine response of FLSs transiently transfected with FRNK was examined. FLSs transfected with a GFP-expressing vector were used as controls. Wild-type FLSs and transfected FLSs were then incubated with 125 pm protein I/II for 20 h at 37 °C. As seen in Fig. 4, overexpression of FRNK inhibited significantly IL-6 and IL-8 release from protein I/II-activated FLSs. In a complementary approach, we have also evaluated the contribution of the adaptor protein Shc to this pathway. It is known that a subset of integrins, including α5β1, activates the Ras/ERK pathway by a mechanism implicating the membrane protein caveolin and tyrosine phosphorylation of Shc by the tyrosine kinase Fyn (20Wary K.K. Mariotti A. Zurzolo C. Giancotti F.G. Cell. 1998; 94: 625-626Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). This last event is necessary and sufficient to activate MAPKs as demonstrated by results from dominant negative studies and from mouse embryos deficient in Shc (18Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1036) Google Scholar). Two tyrosine phosphorylation sites have been identified on Shc (34Ravichandran K.S. Oncogene. 2001; 20: 6322-6330Crossref PubMed Scopus (334) Google Scholar): Tyr-239/240, which has been linked to c-Myc activation and Tyr-317, which appears to be critical for MAPKs activation in response to integrins and growth factor receptors. Western blotting analysis using specific anti-Shc (pY317) antibodies showed that two isoforms of Shc, p46 and p52, were constitutively phosphorylated in control FLSs and that protein I/II did not increase the level of Shc phosphorylation (Fig. 5A). EGF was used as a positive control. To further demonstrate that Shc is not involved in cytokine synthesis, FLSs were transiently transfected with a dominant negative version of Shc (pRk5-Shc-Y317F), in which the tyrosine residue that is phosphorylated and binds Grb2 is replaced by a phenylalanine. FLSs transfected with a GFP-expressing vector were used to control transfection. Wild-type and transfected cells were then incubated with 125 pm protein I/II for 20 h at 37 °C. As shown in Fig. 5B, overexpression of a dominant negative version of Shc had no effect on IL-6 and IL-8 release from protein I/II-activated FLSs, as compared with activated wild-type FLSs, indicating that IL-6 and IL-8 release from protein I/II-activated FLSs does not require integrin-mediated Shc (Y317) signaling. Taken together, these results indicate that FAK plays a predominant role in protein I/II-induced IL-6 and IL-8 release from activated FLSs. FAK Tyr-397 Phosphorylation Is Not Implicated in Either Signaling to MAPKs or Release of IL-6 and IL-8 —To further study the mechanisms by which FAK is required for protein I/II-induced IL-6 and IL-8 release, we used cytochalasin D, which has been shown to prevent FAK Tyr-397 phosphorylation by disrupting the actin cytoskeleton (35Chen Q. Kinch M. Lin T. Burrid
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