Tumor Necrosis Factor-induced Nuclear Factor κB Activation Is Impaired in Focal Adhesion Kinase-deficient Fibroblasts
2003; Elsevier BV; Volume: 278; Issue: 31 Linguagem: Inglês
10.1074/jbc.m213115200
ISSN1083-351X
AutoresMegumi Funakoshi‐Tago, Yoshiko Sonoda, S. Tanaka, K. Hashimoto, Kenji Tago, Shin‐ichi Tominaga, Tadashi Kasahara,
Tópico(s)Cell death mechanisms and regulation
ResumoFocal adhesion kinase (FAK) is widely involved in important cellular functions such as proliferation, migration, and survival, although its roles in immune and inflammatory responses have yet to be explored. We demonstrate a critical role for FAK in the tumor necrosis factor (TNF)-induced activation of nuclear factor (NF)-κB, using FAK-deficient (FAK–/–) embryonic fibroblasts. Interestingly, TNF-induced interleukin (IL)-6 production was nearly abolished in FAK–/– fibroblasts, whereas a normal level of production was obtained in FAK+/– or FAK+/+ fibroblasts. FAK deficiency did not affect the three types of mitogen-activated protein kinases, ERK, JNK, and p38. Similarly, TNF-induced activation of activator protein 1 or NF-IL-6 was not impaired in FAK–/– cells. Of note, TNF-induced NF-κB DNA binding activity and activation of IκB kinases (IKKs) were markedly impaired in FAK–/– cells, whereas the expression of TNF receptor I or other signaling molecules such as receptor-interacting protein (RIP), tumor necrosis factor receptor-associated factor 2 (TRAF2), IKKα, IKKβ, and IKKγ was unchanged. Also, TNF-induced association of FAK with RIP and subsequent association of RIP with TRAF2 were not observed, resulting in a failure of RIP to recruit the IKK complex in FAK–/– cells. The reintroduction of wild type FAK into FAK–/– cells restored the interaction of RIP with TRAF2 and the IKK complex and allowed recovery of NF-κB activation and subsequent IL-6 production. Thus, we propose a novel role for FAK in the NF-κB activation pathway leading to the production of cytokines. Focal adhesion kinase (FAK) is widely involved in important cellular functions such as proliferation, migration, and survival, although its roles in immune and inflammatory responses have yet to be explored. We demonstrate a critical role for FAK in the tumor necrosis factor (TNF)-induced activation of nuclear factor (NF)-κB, using FAK-deficient (FAK–/–) embryonic fibroblasts. Interestingly, TNF-induced interleukin (IL)-6 production was nearly abolished in FAK–/– fibroblasts, whereas a normal level of production was obtained in FAK+/– or FAK+/+ fibroblasts. FAK deficiency did not affect the three types of mitogen-activated protein kinases, ERK, JNK, and p38. Similarly, TNF-induced activation of activator protein 1 or NF-IL-6 was not impaired in FAK–/– cells. Of note, TNF-induced NF-κB DNA binding activity and activation of IκB kinases (IKKs) were markedly impaired in FAK–/– cells, whereas the expression of TNF receptor I or other signaling molecules such as receptor-interacting protein (RIP), tumor necrosis factor receptor-associated factor 2 (TRAF2), IKKα, IKKβ, and IKKγ was unchanged. Also, TNF-induced association of FAK with RIP and subsequent association of RIP with TRAF2 were not observed, resulting in a failure of RIP to recruit the IKK complex in FAK–/– cells. The reintroduction of wild type FAK into FAK–/– cells restored the interaction of RIP with TRAF2 and the IKK complex and allowed recovery of NF-κB activation and subsequent IL-6 production. Thus, we propose a novel role for FAK in the NF-κB activation pathway leading to the production of cytokines. Focal adhesion kinase (FAK) 1The abbreviations used are: FAK, focal adhesion kinase; RIP, receptor-interacting protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; TRAF2, tumor necrosis factor receptor-associated factor 2; IKK, IκB kinase; EMSA, electrophoretic mobility shift assay; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; IL, interleukin; NF, nuclear factor; MAP, mitogen-activated protein; AP-1, activator protein 1; HA, hemagglutinin; PE, phycoerythrin; GST, glutathione S-transferase; RT-PCR, reverse transcription-PCR. is a non-receptor protein-tyrosine kinase implicated in controlling cellular responses to cell surface integrin with cell spreading and migration. Cellular focal adhesion contains structural and signaling proteins that in many cases are modified by tyrosine phosphorylation (reviewed in Refs. 1Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1672) Google Scholar, 2Hanks S.K. Polte T.R. Bioessays. 1997; 19: 137-145Crossref PubMed Scopus (442) Google Scholar, 3Schlaepfer D.D Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1042) Google Scholar). FAK is phosphorylated not only by integrin signaling (4Sieg D.J. Hauck C.R. Schlaepfer D.D. J. Cell Sci. 1999; 112: 2677-2691Crossref PubMed Google Scholar, 5Owen J.D. Ruest P.J. Fry D.W. Hanks S.K. Mol. Cell. Biol. 1999; 19: 4806-4818Crossref PubMed Scopus (344) Google Scholar) but also by a variety of soluble growth factors including platelet-derived growth factor and vascular endothelial growth factor as well as growth hormone (see Refs. 6Rankin S. Hooshmand-Rad R. Claesson-Welsh L. Rozengurt E. J. Biol. Chem. 1996; 271: 7829-7834Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 7Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1080) Google Scholar, 8Abedi H. Dawes K.E. Zachary I. J. Biol. Chem. 1995; 270: 11367-11376Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar and reviewed in Ref. 9Rozengurt E. Rodriguez-Fernandez J.L. Essays Biochem. 1997; 32: 73-86PubMed Google Scholar). FAK is thus implicated to play important roles in signaling pathways initiated by integrins in cell migration, survival, and cell cycle regulation. In addition, FAK has also been shown to play an essential role in the survival of anchorage-dependent cells (10Frisch S.M. Vuori K. Rouslahti E. Chan-Hui P.Y. J. Cell Biol. 1996; 134: 793-799Crossref PubMed Scopus (1008) Google Scholar) or in antiapoptotic action during growth factor deprivation-induced apoptosis in human umbilical vein endothelial cells (11Levkau B. Herren B. Koyama H. Ross R. Raines E.W. J. Exp. Med. 1998; 187: 579-586Crossref PubMed Scopus (229) Google Scholar). Overexpression of FAK in many cells induces the constitutive activation of NF-κB, which leads to the activation of survival genes as shown in human leukemic HL-60 cells, in which FAK protected cells from apoptosis caused by oxidative stress, etoposide, and ionizing radiation (12Sonoda Y. Watanabe S. Matsumoto Y. Yokota-Aizu E. Kasahara T. J. Biol. Chem. 1999; 274: 10566-10570Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 13Sonoda Y. Matsumoto Y. Funakoshi M. Yamamoto D. Hanks S.K. Kasahara T. J. Biol. Chem. 2000; 275: 16309-16315Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 14Kasahara T. Koguchi E. Funakoshi M. Yokota E. Sonoda Y. Antioxid. Redox Signal. 2002; 4: 491-499Crossref PubMed Scopus (74) Google Scholar) or UV-induced apoptosis in Madin-Darby canine kidney cells (15Chan P.C. Lai J.F. Cheng C.H. Tang M.J. Chiu C.C. Chen H.C. J. Biol. Chem. 1999; 274: 26901-26906Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). These findings imply that FAK generally has an antiapoptotic role in various cells. Furthermore, FAK triggers rapid cell cycle progression via activation of protein kinase C isoforms and cyclins (16Yamamoto D. Sonoda Y. Hasegawa M. Funakoshi-Tago M. Aizu-Yokota E. Kasahara T. Cell. Signaling. 2003; 15: 575-583Crossref PubMed Scopus (54) Google Scholar). FAK is thus phosphorylated by various stimuli and is involved in cytoplasmic signaling downstream of a variety of cell surface receptors. However, to date, no reports have described the role of FAK in inflammatory and immune responses, whereas proline-rich tyrosine kinase 2, a related adhesion focal tyrosine kinase, was found to be activated by TNF or by ultraviolet irradiation (17Tokiwa G. Dikic I. Lev S. Schlessinger J. Science. 1996; 273: 792-794Crossref PubMed Scopus (285) Google Scholar). Mechanisms of FAK activation are multiple, and other tyrosine-phosphorylated proteins such as proline-rich tyrosine kinase 2 are present in the cells (3Schlaepfer D.D Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1042) Google Scholar, 5Owen J.D. Ruest P.J. Fry D.W. Hanks S.K. Mol. Cell. Biol. 1999; 19: 4806-4818Crossref PubMed Scopus (344) Google Scholar, 18Sieg D.J. Ilic D. Jones K.C. Damsky C.H. Hunter T. Schlaepfer D.D. EMBO J. 1998; 17: 5933-5947Crossref PubMed Scopus (290) Google Scholar). Thus, we investigated embryonic fibroblasts from FAK-deficient mice to study the role of FAK because FAK deficiency in mice was embryonic lethal as a result of defective developmental gastrulation events with deficits in cell migration (18Sieg D.J. Ilic D. Jones K.C. Damsky C.H. Hunter T. Schlaepfer D.D. EMBO J. 1998; 17: 5933-5947Crossref PubMed Scopus (290) Google Scholar, 19Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1602) Google Scholar, 20Furuta Y. Ilic D. Kanazawa S. Takeda N. Yamamoto T. Aizawa S. Oncogene. 1995; 11: 1989-1995PubMed Google Scholar). Our focus was the responses of FAK–/– fibroblasts to TNF-α. TNF-α is a potent inducer of IL-6 expression in fibroblasts, and we examined whether FAK–/– fibroblasts respond normally to TNF-α and produce cytokines. We found that FAK–/– fibroblasts responded poorly to stimulation with TNF-α, and therefore we examined the underlying mechanism of their unresponsiveness to TNF-α. We present evidence for the first time that a functional FAK molecule is required for IL-6 production by TNF-α. More importantly, we have uncovered the role of FAK in TNF-α-mediated NF-κB activation through its association with receptor-interacting protein (RIP), a serine/threonine kinase. Antibodies and Reagents—Mouse monoclonal antibodies against HA-peptides (12CA5), TNFRI, and RIP were purchased from Roche, R&D Systems, and New England Biolabs Inc. (Beverly, MA), respectively. Rabbit polyclonal antibodies against FAK, TNFR-associated factor 2 (TRAF2), and RelB and goat polyclonal antibody against IκB-kinase (IKK) γ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-p65, anti-p50, and anti-c-Rel antibodies were purchased from Rockland Inc. (Gilbertsville, PA). Human recombinant TNF-α was kindly provided by Dainippon Pharmaceutical Co. (Suitashi, Osaka, Japan). Cell Culture and IL-6 Assay—Heterozygous FAK+/– and homozygous FAK–/– embryonic fibroblasts were originally established by Ilic and co-workers (19Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1602) Google Scholar, 20Furuta Y. Ilic D. Kanazawa S. Takeda N. Yamamoto T. Aizawa S. Oncogene. 1995; 11: 1989-1995PubMed Google Scholar) and were provided to us through Dr. T. Mimura (Department of Allergies, Tokyo University School of Medicine; Ref. 21Ueki K. Mimura T. Nakamoto T. Sasaki T. Aizawa S. Hirai H. Yano S. Naruse T. Nojima Y. FEBS Lett. 1998; 432: 197-201Crossref PubMed Scopus (38) Google Scholar). These cells were maintained in Dulbecco's modified Eagle's medium (Nissui Seiyaku, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum, 4 mm glutamine, 100 units/ml penicillin G, and 100 μg/ml streptomycin. The absence of FAK in the FAK–/– fibroblasts was confirmed by immunoblot analysis using anti-FAK monoclonal antibody (Transduction Laboratories, Lexington, KY; see Fig. 6). Normal FAK+/+ mouse fibroblasts were obtained from the skin of newborn BALB/c mice by trypsinization. For the measurement of secreted IL-6, cells were incubated with or without TNF-α (10 ng/ml) in Dulbecco's modified Eagle's medium containing 1% fetal bovine serum for 12–24 h. IL-6 levels in the culture supernatants were determined using a commercial ELISA kit (BIOSOURCE). All samples were assayed at least in duplicate. Transient Transfection and Luciferase Assay—HA-tagged FAK cDNA and mutant FAK cDNA subcloned into pRcCMV were originally constructed by Dr. Steven K. Hanks (22Hanks S.K. Calalb M.B. Harper M.C. Patel S.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8487-8491Crossref PubMed Scopus (735) Google Scholar) and kindly provided to us (12Sonoda Y. Watanabe S. Matsumoto Y. Yokota-Aizu E. Kasahara T. J. Biol. Chem. 1999; 274: 10566-10570Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Plasmid DNAs were transfected into FAK+/– and FAK–/– fibroblasts using LipofectAMINE TM2000 (Invitrogen). Final amounts of transfected DNA for 24-well plates were adjusted to 1 μg with empty vector, pRcCMV. 0.5 μg of pRcCMV-HA-FAK and pRcCMV-HA-FAK (K454R) was cotransfected with 0.01 μg of pRL-TK (Promega Co. Japan, Tokyo, Japan) and 0.1 μg of pNF-κB-Luc (Invitrogen). At 48 h after transfection, cells were harvested, and the luciferase activities were measured with a Lumat LB9501 (Bertold Japan, Tokyo, Japan). The efficiency of transfection was normalized with sea pansy luciferase activity as described elsewhere (23Funakoshi M. Sonoda Y. Tago K. Tominaga S.-I. Kasahara T. Int. Immunopharmcol. 2001; 1: 595-604Crossref PubMed Scopus (33) Google Scholar). Immunoprecipitation and Immunoblot Analysis—Immunoprecipitation and immunoblot analysis were performed as described previously (23Funakoshi M. Sonoda Y. Tago K. Tominaga S.-I. Kasahara T. Int. Immunopharmcol. 2001; 1: 595-604Crossref PubMed Scopus (33) Google Scholar, 24Funakoshi M. Tago K. Sonoda Y. Tominaga S. Kasahara T. Biochem. Biophys. Res. Commun. 2001; 283: 248-254Crossref PubMed Scopus (33) Google Scholar). In brief, harvested cells were lysed in lysis buffer (10 mm Tris-HCl, pH 7.4, 158 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mm EGTA, 1 mm Na3VO4, 2 μg/ml aprotinin, and 2 μg/ml leupeptin) on ice and cleaned by centrifugation to obtain whole cell extracts. Aliquots (250 μg) of cell lysate were mixed with protein G-Sepharose (Amersham Biosciences) and various antibodies overnight at 4 °C. The immune complexes were precipitated by centrifugation, washed five times with lysis buffer, and boiled in Laemmli sample buffer. Boiled samples were separated by SDS-PAGE, and the proteins were transfected to nitrocellulose membranes. Immunoblotting was performed with various antibodies and horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody and visualized using the enhanced chemiluminescence Western blotting detection system (Amersham Biosciences). Flow Cytometry—Flow cytometry using a fluorescence-activated cell sorter scan was done as follows. Cells were incubated with PE-conjugated rat anti-mouse TNFRI antibody (CD120a; IgG2a isotype; Immunotech, Beckman-Coulter, Marseilles, France) and the isotype-matched mouse control IgG, followed by PE-conjugated anti-mouse antibody. Stained cells were analyzed using FACSCalibur (Becton Dickinson). In Vitro Kinase Assay—Immunoprecipitates obtained with anti-IKKγ antibody were washed twice with lysis buffer and three times with kinase buffer (25 mm Hepes-NaOH, pH 7.5, 20 mm MgCl2, 20 mm β-glycerophosphate, 0.1 mm Na3VO4, 2 mm dithiothreitol, and 20 mm p-nitrophenylphosphate). The kinase reaction in 20 μl of kinase buffer including 10 μm ATP and [γ-32P]ATP was carried out with 1 μg of GST-IκBα (amino acids 1–60) as a substrate for 20 min at 30 °C. In some experiments, mutant GST-IκBαΔC (S32A, S36R) was used instead of GST-IκBα. Samples were resolved by 15% polyacrylamide gel electrophoresis, and phosphorylated GST-IκBα was visualized by autoradiography. RNA Isolation and PCR Amplification—Total RNA separation and RT-PCR analysis were done according to the manufacturer's protocol (Takara Shuzo) using oligo(dT)20 primer and 1 μg of total RNA for first-strand cDNA synthesis. PCR was performed at an annealing temperature of 57 °C and with 20 amplification cycles. The PCR products were resolved and electrophoresed on a 1% agarose gel in Tris borate/EDTA. Primers used were as follows: mouse IL-6, 5′-GATGCTACCAAACTGGATATAATC-3′ (upstream) and 5′-GGTCCTTAGCCACTCCTTCTGTG-3′ (downstream); and mouse glyceraldehyde-3-phosphate dehydrogenase, 5′-GAGAAACCTGCCAAGTATGA-3′ (upstream) and 5′-GCCCCTCCTGTTATTA-3′ (downstream). Reconstitution of FAK by Adenoviral Infection—Cells were plated at 1 × 106 cells/ml in 6-cm culture plates and infected with adenovirus encoding β-galactosidase (Adv-LacZ) or HA-tagged wild type FAK (Adv-FAK) for 24 h at an optimal concentration of virus (25Sakurai S. Sonoda Y. Koguchi E. Shinoura N. Hamada H. Kasahara T. Biochem. Biophys. Res. Commun. 2002; 293: 174-181Crossref PubMed Scopus (19) Google Scholar) with 1 × 102 virions/cell (i.e. a multiplicity of infection of 100). Electrophoretic Mobility Shift Assay (EMSA)—EMSA was carried out as described previously (23Funakoshi M. Sonoda Y. Tago K. Tominaga S.-I. Kasahara T. Int. Immunopharmcol. 2001; 1: 595-604Crossref PubMed Scopus (33) Google Scholar, 24Funakoshi M. Tago K. Sonoda Y. Tominaga S. Kasahara T. Biochem. Biophys. Res. Commun. 2001; 283: 248-254Crossref PubMed Scopus (33) Google Scholar). The consensus double-strand oligodeoxynucleotide probes for NF-κB, AP-1, and NF-IL-6 (Santa Cruz Biotechnology) were radioactively labeled using [γ-32P]ATP and T4 polynucleotide kinase with standard procedures. Then, 10 μg of nuclear protein prepared from cells was incubated with γ-32P-labeled double-stranded oligonucleotide probe. The binding reaction was carried out at room temperature for 30 min in a total volume of 25 μl. Bound complexes were separated on 5% gel electrophoresis in TGE (tris-glycine-EDTA) buffer, dried, and visualized by autoradiography. Defective TNF-α-induced IL-6 Production in the FAK–/– Fibroblasts—It is well documented that FAK plays important roles in the integrin signaling in cell migration, cell survival, or cell cycle regulation. We speculated that FAK might also play a role in cytokine signaling because in our preliminary studies, cytokine production was modulated in the FAK-transfected cells. In order to study the role of FAK, we obtained FAK+/– and FAK–/– embryonic fibroblasts from FAK-deficient mice, which were originally established by Ilic and co-workers (19Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1602) Google Scholar, 20Furuta Y. Ilic D. Kanazawa S. Takeda N. Yamamoto T. Aizawa S. Oncogene. 1995; 11: 1989-1995PubMed Google Scholar). First, we examined TNF-α-induced IL-6 production by the FAK+/– and FAK–/– fibroblasts as well as normal mouse embryonic skin fibroblasts (FAK+/+ cells). TNF-α induced marked IL-6 production in both FAK+/+ and FAK+/– fibroblasts, whereas only marginal IL-6 production (one-fifth or one-sixth that of FAK+/+ fibroblasts at 24 h) was observed by the FAK–/– fibroblasts, as shown in Fig. 1A. RT-PCR analysis also indicated that virtually no IL-6 mRNA was expressed in FAK–/– cells, whereas a substantial level of IL-6 mRNA was expressed in the FAK+/– and FAK+/+ cells, suggesting some critical role for FAK (Fig. 1B). This observation prompted us to further explore the role of FAK in the TNF-α-induced signaling pathway. Because significant expression of IL-6 in response to TNF-α was seen in FAK+/– as well as wild type normal FAK+/+ fibroblasts, we used FAK+/– cells and FAK–/– cells in the subsequent studies. FAK Has No Effect on the TNF-α-induced MAP Kinase Pathway or Activation of AP-1 and NF-IL-6 —TNF-α has been implicated in the activation of one or more MAP kinases in different cell types for the induction of IL-6 production (26Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (609) Google Scholar). To explore the possible involvement of the MAP kinase family in FAK function, we determined whether three types of MAP kinases (ERK, c-Jun NH2-terminal kinase (JNK), and p38) are activated (i.e. phosphorylated or not) using antibodies against each phosphorylated form. Activated forms of all types of MAP kinases were detected equally in the FAK–/– cells as well as in the FAK+/– cells in response to TNF-α (data not shown), indicating that the MAP kinase activation pathway is not involved in the critical role of FAK. AP-1, NF-IL-6, and NF-κB binding elements are known to be involved in activating the IL-6 gene (27Tuyt L.M. Dokter W.H. Birkenkamp K. Koopmans S.B. Lummen C. Kruijer W. Vellenga E. J. Immunol. 1999; 162: 4893-4902PubMed Google Scholar, 28Matsusaka T. Fujikawa K. Nishio Y. Mukaida N. Matsushima K. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10193-10197Crossref PubMed Scopus (890) Google Scholar). AP-1 can be activated directly through phosphorylation by JNK, and the expression of AP-1 components is induced through JNK- and p38-dependent pathways (29Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2351) Google Scholar). To assess the effect of FAK on AP-1 activation, we measured AP-1 activity with an EMSA, using oligonucleotide probes for AP-1. As shown in Fig. 2A, TNF-α induced marked AP-1 DNA binding activity in both FAK+/– and FAK–/– cells, which is consistent with the results on the activation of MAP kinase. Binding specificity for AP-1 was confirmed by the complete disappearance with non-radiolabeled DNA (Fig. 2B). Similarly, NF-IL-6 activation was equally observed in FAK+/– and FAK–/– cells (Fig. 2, C and D), indicating that FAK is not directly involved in the activation of NF-IL-6. Impairment of TNF-α-induced NF-κB Activation in FAK–/– Cells—Because TNF-α is a potent activator of NF-κB, which is one of the transcription factors necessary for activating the IL-6 gene, we tested the activation of NF-κB using an EMSA as well as the reporter gene assay. The EMSA indicated that TNF-α induced rapid and marked NF-κB DNA binding activity within 15 min in the FAK+/– cells, whereas only low level induction was observed in FAK–/– cells (Fig. 3A). A supershift assay revealed that the NF-κB DNA binding proteins are mostly p65 and less abundant p50 or c-Rel, as shown in Fig. 3B. This observation was also confirmed by the translocation of activated NF-κB components to the nucleus, as shown by immunoblotting. That is, whereas p65, p50, and c-Rel proteins were detected at 15 min in nuclear extracts from the TNF-α-stimulated FAK+/– cells, these proteins were not detected in FAK–/– cells (Fig. 3C). In addition, significant NF-κB luciferase activity was detected in the FAK+/– cells, whereas only minimal NF-κB activation was observed in FAK–/– cells (Fig. 3D). Thus, FAK appears to facilitate TNF-α signaling by modulating NF-κB activation, which is not dependent on the MAP kinase pathway. IKK Activity Is Severely Impaired in the FAK–/– Fibroblasts—Because the activation of IKK is a key step in the activation of NF-κB, we examined whether the activation of IKK by TNF-α differs between FAK+/– and FAK–/– cells. It was found that activation by TNF-α was severely impaired in FAK–/– cells when the IKK complex was immunoprecipitated with anti-IKKγ (NEMO) antibody, and IKK activity was measured as IκBα phosphorylation using GST-IκBαΔC as a substrate (Fig. 4A, top lane). No phosphorylation was observed when the GST-IκBαΔC (S32A, S36R) mutant was used as a substrate (Fig. 4A, bottom lane). Furthermore, when the degradation of IκBα after stimulation with TNF-α was examined by Western blotting, it was found to be reduced significantly in FAK–/– cells (Fig. 4B), confirming the impairment of IKK activation in FAK–/– cells. Because these results suggested that FAK plays essential roles in the activation of NF-κB, we further evaluated the expression levels of several upstream molecules involved in TNF-α-induced NF-κB activation including TNFRI, RIP, TRAF2, IKKα, IKKβ, and IKKγ. As shown in Fig. 4C, no significant differences between FAK+/– and FAK–/– cells were observed. Therefore, the defective NF-κB activation in FAK–/– cells was not due to altered expression of signaling molecules in the TNF-α signaling pathway. Because the TNFRI detected by Western blotting might be a non-glycosylated form of the TNFRI precursor, we also analyzed the expression of the mature form of TNFRI on the cell surface. As shown in Fig. 4D, similar levels of cell surface TNFRI were observed in both FAK+/– and FAK–/– cells, confirming that there were no significant differences between these two cell lines in terms of TNFRI expression. Critical Interaction of FAK with RIP—Because IKK activity was severely impaired in FAK–/– cells, we examined whether FAK physically interacts with RIP, which is essential for the TNFRI-mediated activation of IKKs (31Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (857) Google Scholar, 32Ilic D. Damsky C H. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar, 33Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1767) Google Scholar). Coimmunoprecipitation assay revealed that FAK associated with RIP in a TNF-α-dependent manner in FAK+/– cells, but not in FAK–/– cells (Fig. 5A). Inversely, in a pull-down assay using anti-RIP antibody, RIP also coimmunoprecipitated with FAK, confirming the physical association of FAK and RIP (Fig. 5B). The TNF-α-dependent association of TNFRI with FAK and with RIP was demonstrated clearly in FAK+/– cells but not in FAK–/– cells (Fig. 5C). In addition, TRAF2 antibody coimmunoprecipitated with RIP (Fig. 5D), which has been demonstrated by Hsu et al. (30Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (996) Google Scholar), only in FAK+/– cells. Furthermore, we found that IKKγ, which is an essential component of the IκB kinase complex (31Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (857) Google Scholar), recruited RIP in response to TNF-α (Fig. 5E), which appears to be an important step in the activation of TRAF2 (33Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1767) Google Scholar, 34Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar). It should be noted that a significant association of RIP with TRAF2 and IKKγ was observed in FAK+/– cells but not in FAK–/– cells, thus demonstrating the critical role of FAK in the physical association of RIP, TRAF2, and IKKγ. Rescue of the Deficient Phenotype in FAK–/– Fibroblasts by the Reintroduction of Wild Type FAK cDNA—To confirm that the defect in the interaction of RIP with TNFRI, TRAF2, and the IKK complex in FAK–/– cells was due to the disruption of FAK function, we examined whether the above interactions could be rescued in FAK–/– cells by the forced expression of wild type FAK. We thus reintroduced wild type FAK into FAK–/– cells using an adenovirus vector harboring FAK cDNA. As shown in Fig. 6A, FAK expression, in contrast to the Lac Z control, restored TNF-α-induced interaction between RIP and TNFRI as determined by coimmunoprecipitation assay. In addition, FAK expression also recovered the recruitment of TRAF2 and IKKγ to RIP (Fig. 6, B and C). To further ascertain the effect of FAK on the activation of NF-κB, we determined the NF-κB DNA binding activity with an EMSA. As shown in Fig. 7A, reintroduction of wild type FAK into the FAK–/– cells restored TNF-α-induced NF-κB DNA binding activity to the levels seen in FAK+/– cells (Fig. 3A). The restoration of NF-κB activation by the reintroduction of FAK was confirmed using the NF-κB reporter gene assay (Fig. 7B), as compared with that in Fig. 3D. Furthermore, introduction of FAK in FAK–/– cells resulted in a significant increase of IL-6 secretion and IL-6 mRNA expression (Fig. 7, C and D). It should be noted that mutated FAK (K454R), a kinase-defective mutant, did not restore TNF-α-induced NF-κB activation (Fig. 7B). Therefore, wild type FAK with intact kinase activity is necessary for the TNF-α-triggered FAK-associated signal pathways. In this study, we demonstrated that TNF-α-induced NF-κB activation and subsequent IL-6 gene activation were severely impaired in FAK–/– cells. These observations indicated that FAK plays a critical role in the TNF-α-mediated signal transduction pathway. Because our results demonstrated that FAK is indispensable for the TNF-α-induced IL-6 gene activation pathway, we focused on the signaling molecules with which FAK may interact. Whereas FAK is at a crossroad for multiple signaling pathways (32Ilic D. Damsky C H. Yamamoto T. J. Cell Sci. 1997; 110: 401-407Crossref PubMed Google Scholar), neither its role in the production of cytokines nor its intervention in the signals leading to the activation of cytokine genes has been studied thus far. Sieg et al. (7Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1080) Google Scholar) demonstrated that FAK integrates growth factors (platelet-derived growth factor and epidermal growth factor) and integrin signals to promote cell migration and suggested a role as a receptor-proximal link between the growth factor receptor and integrin signaling pathway. However, the role of FAK in the activation of cytokine genes had not been studied. Thus, this is the first paper describing significant involvement of FAK in cytokine production. The biological effects of TNF-α are regulated through interaction with two distinct TNFRs, TNFRI (p55) and TNFRII (p75). Upon cell stimulation with TNF-α, TNFRI recruits tumor necrosis factor receptor 1-associated death domain, an adapter protein that binds to TRAF2 and RIP, a serine/threonine kinase (33Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1767) Google Scholar,
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