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• W2034771296 abstract "Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that promotes cell migration, survival, and gene expression. Here we show that FAK signaling is important for tumor necrosis factor-α (TNFα)-induced interleukin 6 (IL-6) mRNA and protein expression in breast (4T1), lung (A549), prostate (PC-3), and neural (NB-8) tumor cells by FAK short hairpin RNA knockdown and by comparisons of FAK-null (FAK–/–) and FAK+/+ mouse embryo fibroblasts. FAK promoted TNFα-stimulated MAPK activation needed for maximal IL-6 production. FAK was not required for TNFα-mediated nuclear factor-κB or c-Jun N-terminal kinase activation. TNFα-stimulated FAK catalytic activation and IL-6 production were inhibited by FAK N-terminal but not FAK C-terminal domain overexpression. Analysis of FAK–/– fibroblasts stably reconstituted with wild type or various FAK point mutants showed that FAK catalytic activity, Tyr-397 phosphorylation, and the Pro-712/713 proline-rich region of FAK were required for TNFα-stimulated MAPK activation and IL-6 production. Constitutively activated MAPK kinase-1 (MEK1) expression in FAK–/– and A549 FAK short hairpin RNA-expressing cells rescued TNFα-stimulated IL-6 production. Inhibition of Src protein-tyrosine kinase activity or mutation of Src phosphorylation sites on FAK (Tyr-861 or Tyr-925) did not affect TNFα-stimulated IL-6 expression. Moreover, analyses of Src–/–, Yes–/–, and Fyn–/– fibroblasts showed that Src expression was inhibitory to TNFα-stimulated IL-6 production. These studies provide evidence for a novel Src-independent FAK to MAPK signaling pathway regulating IL-6 expression with potential importance to inflammation and tumor progression. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that promotes cell migration, survival, and gene expression. Here we show that FAK signaling is important for tumor necrosis factor-α (TNFα)-induced interleukin 6 (IL-6) mRNA and protein expression in breast (4T1), lung (A549), prostate (PC-3), and neural (NB-8) tumor cells by FAK short hairpin RNA knockdown and by comparisons of FAK-null (FAK–/–) and FAK+/+ mouse embryo fibroblasts. FAK promoted TNFα-stimulated MAPK activation needed for maximal IL-6 production. FAK was not required for TNFα-mediated nuclear factor-κB or c-Jun N-terminal kinase activation. TNFα-stimulated FAK catalytic activation and IL-6 production were inhibited by FAK N-terminal but not FAK C-terminal domain overexpression. Analysis of FAK–/– fibroblasts stably reconstituted with wild type or various FAK point mutants showed that FAK catalytic activity, Tyr-397 phosphorylation, and the Pro-712/713 proline-rich region of FAK were required for TNFα-stimulated MAPK activation and IL-6 production. Constitutively activated MAPK kinase-1 (MEK1) expression in FAK–/– and A549 FAK short hairpin RNA-expressing cells rescued TNFα-stimulated IL-6 production. Inhibition of Src protein-tyrosine kinase activity or mutation of Src phosphorylation sites on FAK (Tyr-861 or Tyr-925) did not affect TNFα-stimulated IL-6 expression. Moreover, analyses of Src–/–, Yes–/–, and Fyn–/– fibroblasts showed that Src expression was inhibitory to TNFα-stimulated IL-6 production. These studies provide evidence for a novel Src-independent FAK to MAPK signaling pathway regulating IL-6 expression with potential importance to inflammation and tumor progression. Focal adhesion kinase (FAK) 2The abbreviations used are: FAK, focal adhesion kinase; CA, constitutively activated; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; FAK–/–, FAK-null; FRNK, FAK-related non-kinase; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; IL-6, interleukin 6; MEK1, MAPK kinase-1; MAPK, mitogen-activated protein kinase; mAbs, monoclonal antibodies; NF-κB, nuclear factor-κB; RT, reverse transcription; Scr, scrambled; shRNA, short-hairpin RNA; TNFR1, tumor necrosis factor receptor-1; TNFα, tumor necrosis factor-α; VEGF, vascular endothelial growth factor; and WT, wild type; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; IVK, in vitro kinase; IP, immunoprecipitate; RIP, receptor interacting protein; Ad, adenoviral; SH, Src homology; SYF, Src-Yes-Fyn null. is best known for its role as an integrin-stimulated protein-tyrosine kinase (1Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1141) Google Scholar). FAK is recruited to sites of integrin clustering via interactions of its C-terminal domain with integrin-associated proteins such as talin and paxillin. FAK contains a central kinase domain, C-terminal proline-rich regions that serve as binding sites for Src homology 3 (SH3) domain-containing proteins, and an N-terminal band 4.1, ezrin, radixin, moesin homology (FERM) domain that acts to regulate FAK kinase activity through an auto-inhibitory mechanism (2Ceccarelli D.F. Song H.K. Poy F. Schaller M.D. Eck M.J. J. Biol. Chem. 2006; 281: 252-259Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 3Cooper L.A. Shen T.L. Guan J.L. Mol. Cell. Biol. 2003; 23: 8030-8041Crossref PubMed Scopus (145) Google Scholar). Integrin clustering promotes FAK activation, results in FAK phosphorylation at Tyr-397 (Tyr(P)-397), promotes Src family protein-tyrosine kinase binding to the FAK Tyr(P)-397 site, and facilitates the formation of the FAK-Src signaling complex that results in the secondary phosphorylation of FAK at Tyr-861 and Tyr-925 (4Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1033) Google Scholar, 5Schlaepfer D.D. Mitra S.K. Ilic D. Biochim. Biophys. Acta. 2004; 1692: 77-102Crossref PubMed Scopus (370) Google Scholar). The FAK-Src complex can promote the tyrosine phosphorylation of various targets linked to either growth, survival, or motility-associated signaling pathways (6Cox B.D. Natarajan M. Stettner M.R. Gladson C.L. J. Cell. Biochem. 2006; 99: 35-52Crossref PubMed Scopus (239) Google Scholar, 7Mitra S.K. Schlaepfer D.D. Curr. Opin. Cell Biol. 2006; 18: 516-523Crossref PubMed Scopus (1194) Google Scholar). Canonical FAK-Src integrin signaling can be blocked by overexpression of the FAK C-terminal domain termed FRNK (1Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1141) Google Scholar). FAK can also be activated by growth factor receptors, G-protein-linked, and cytokine stimulation of cells (5Schlaepfer D.D. Mitra S.K. Ilic D. Biochim. Biophys. Acta. 2004; 1692: 77-102Crossref PubMed Scopus (370) Google Scholar), but how FAK gets activated by non-integrin stimuli remains incompletely defined. FAK also participates in immune and inflammatory response signaling by regulating cytokine production (8Watanabe Y. Tamura M. Osajima A. Anai H. Kabashima N. Serino R. Nakashima Y. Kidney Int. 2003; 64: 431-440Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). FAK is important for lipopolysaccharide-induced interleukin-6 (IL-6) secretion by human and murine fibroblasts (9Zeisel M.B. Druet V.A. Sibilia J. Klein J.P. Quesniaux V. Wachsmann D. J. Immunol. 2005; 174: 7393-7397Crossref PubMed Scopus (56) Google Scholar). IL-6 and IL-8 expression by human synoviocytes stimulated with oral streptococci protein I/II also requires FAK (10Neff L. Zeisel M. Druet V. Takeda K. Klein J.P. Sibilia J. Wachsmann D. J. Biol. Chem. 2003; 278: 27721-27728Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, the molecular mechanism(s) whereby FAK promotes IL-6 expression remain largely undefined. IL-6 is an important mediator of immune responses, inflammation, and tumor progression (11Hodge D.R. Hurt E.M. Farrar W.L. Eur. J. Cancer. 2005; 41: 2502-2512Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar). IL-6 expression is largely regulated by gene transcription, and the tumor necrosis factor-α (TNFα) cytokine is a potent stimulator of IL-6 expression (12MacEwan D.J. Cell Signal. 2002; 14: 477-492Crossref PubMed Scopus (527) Google Scholar). TNFα binds to two distinct cell surface receptors (TNFR) 1 and TNFR2, which can activate a variety of intracellular signaling cascades (12MacEwan D.J. Cell Signal. 2002; 14: 477-492Crossref PubMed Scopus (527) Google Scholar). TNFα can activate FAK, and this is linked to enhanced cell motility (13Corredor J. Yan F. Shen C.C. Tong W. John S.K. Wilson G. Whitehead R. Polk D.B. Am. J. Physiol. 2003; 284: C953-C961Crossref PubMed Scopus (104) Google Scholar), gene expression (14Funakoshi-Tago M. Sonoda Y. Tanaka S. Hashimoto K. Tago K. Tominaga S. Kasahara T. J. Biol. Chem. 2003; 278: 29359-29365Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 15Mon N.N. Hasegawa H. Thant A.A. Huang P. Tanimura Y. Senga T. Hamaguchi M. Cancer Res. 2006; 66: 6778-6784Crossref PubMed Scopus (61) Google Scholar), and survival (16Huang D. Khoe M. Befekadu M. Chung S. Takata Y. Ilic D. Bryer-Ash M. Am. J. Physiol. Cell Physiol. 2006; 292: C1339-C1352Crossref PubMed Scopus (66) Google Scholar, 17Takahashi R. Sonoda Y. Ichikawa D. Yoshida N. Eriko A.Y. Tadashi K. Biochim. Biophys. Acta. 2006; 1770: 518-526Crossref PubMed Scopus (24) Google Scholar). TNFα-stimulated genes include IL-1α, IL-1β, IL-6, IL-8, IL-18, cycloxygenase-2, matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) (18Aggarwal B.B. Shishodia S. Sandur S.K. Pandey M.K. Sethi G. Biochem. Pharmacol. 2006; 72: 1605-1621Crossref PubMed Scopus (1097) Google Scholar). FAK promotes matrix metalloproteinase-9 and VEGF mRNA expression by generating signals leading to the activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), respectively (19Hsia D.A. Mitra S.K. Hauck C.R. Streblow D.N. Nelson J.A. Ilic D. Huang S. Li E. Nemerow G.R. Leng J. Spencer K.S. Cheresh D.A. Schlaepfer D.D. J. Cell Biol. 2003; 160: 753-767Crossref PubMed Scopus (459) Google Scholar, 20Mitra S.K. Mikolon D. Molina J.E. Hsia D.A. Hanson D.A. Chi A. Lim S.T. Bernard-Trifilo J.A. Ilic D. Stupack D.G. Cheresh D.A. Schlaepfer D.D. Oncogene. 2006; 25: 5969-5984Crossref PubMed Scopus (135) Google Scholar). In addition, the transcription factor nuclear factor-κB (NF-κB) is an important mediator of TNFα signaling, and NF-κB activation is a major regulator of TNFα-stimulated IL-6 expression (21Vanden Berghe W. Vermeulen L. De Wilde G. De Bosscher K. Boone E. Haegeman G. Biochem. Pharmacol. 2000; 60: 1185-1195Crossref PubMed Scopus (266) Google Scholar). As FAK has been linked to both TNFα-stimulated NF-κB activation (22Zhang H.M. Keledjian K.M. Rao J.N. Zou T. Liu L. Marasa B.S. Wang S.R. Ru L. Strauch E.D. Wang J.Y. Am. J. Physiol. 2006; 290: C1310-C1320Crossref PubMed Scopus (36) Google Scholar) and TNFα-stimulated IL-6 expression (14Funakoshi-Tago M. Sonoda Y. Tanaka S. Hashimoto K. Tago K. Tominaga S. Kasahara T. J. Biol. Chem. 2003; 278: 29359-29365Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), we have assessed the molecular mechanism(s) of how FAK promotes TNFα-stimulated IL-6 expression in human tumor cells and normal mouse fibroblasts. We employed FAK–/– and FAK-reconstituted fibroblasts as well as a FAK short hairpin RNA (shRNA) knockdown to examine the role of FAK in TNFα-stimulated IL-6 expression. Surprisingly, TNFα-stimulated NF-κB and JNK activation were normal in FAK–/– cells, yet TNFα did not function to promote IL-6 expression in the absence of FAK or in FAK shRNA knockdown cells. We found that TNFα-stimulated FAK catalytic activation, FAK Tyr-397 phosphorylation, and the Pro-712/713 proline-rich region of FAK were required for TNFα-stimulated ERK2/mitogen-activated protein kinase (MAPK) activation and IL-6 production. Inhibition of Src activity or disruption of Src phosphorylation sites on FAK at Tyr-861 or Tyr-925 did not affect TNFα-stimulated IL-6 expression. However, constitutively activated (CA) MAPK kinase-1 (MEK1) expression in FAK–/– or FAK shRNA cells rescued IL-6 production in response to TNFα stimulation. As TNFα-stimulated FAK activation and IL-6 expression were inhibited by overexpression of the FAK N-terminal FERM but not C-terminal integrin-association domain (termed FRNK), these studies have identified a novel Src-independent pathway whereby FAK kinase activity promotes signaling leading to ERK activation and IL-6 cytokine expression. Cell Culture—FAK+/+, FAK–/–, FAK-reconstituted (clone DP3), SYF (Src–/–, c-Yes–/–, Fyn–/–), and SYF + c-Src murine embryonic fibroblasts were used as described (23Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Aizawa S. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1587) Google Scholar, 24Sieg D.J. Hauck C.R. Schlaepfer D.D. J. Cell Sci. 1999; 112: 2677-2691Crossref PubMed Google Scholar, 25Hsia D.A. Lim S.T. Bernard-Trifilo J.A. Mitra S.K. Tanaka S. den Hertog J. Streblow D.N. Ilic D. Ginsberg M.H. Schlaepfer D.D. Mol. Cell. Biol. 2005; 25: 9700-9712Crossref PubMed Scopus (72) Google Scholar). 4T1 breast carcinoma cells, A549 lung adenocarcinoma cells, and PC-3 prostate carcinoma cells were from ATCC (Manassas, VA). NB-8 neuroblastoma cells were generously provided by Dwayne Stupack (University of California, San Diego) (26Stupack D.G. Teitz T. Potter M.D. Mikolon D. Houghton P.J. Kidd V.J. Lahti J.M. Cheresh D.A. Nature. 2006; 439: 95-99Crossref PubMed Scopus (228) Google Scholar). 4T1 FAK shRNA and scrambled shRNA control cells were generated as described (27Mitra S.K. Lim S.T. Chi A. Schlaepfer D.D. Oncogene. 2006; 25: 4429-4440Crossref PubMed Scopus (83) Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). Antibodies and Reagents—IκBα polyclonal, NF-κB p65 monoclonal, and FAK Tyr(P)925 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies (mAbs) to FAK (clone 4.47) and to phosphotyrosine (4G10) were from Upstate (Charlottesville, VA). Pyk2 and MEK1 mAbs were from BD Transduction Laboratories (San Diego). mAbs to β-actin (AC-74) were from Sigma. Myc tag (9E10) and GFP tag antibodies were from Covance (Berkeley, CA). ERK1/2 Thr(P)-185-Tyr(P)-187, ERK1/2, JNK1/2 Thr(P)-183-Tyr(P)-185, FAK Tyr(P)-397, and FAK Tyr(P)-861 polyclonal antibodies were from BIOSOURCE. FAK Tyr(P)-576/Tyr(P)-577 and MEK1/2 Ser(P)-217/221 antibodies were from Cell Signaling Technologies (Danvers, MA). Phycoerythrin-conjugated anti-mouse TNFR1 and TNFR2 antibodies were from BioLegend (San Diego). Rabbit polyclonal anti-FAK (5904) directed against N-terminal residues 8–27 was generated as described (24Sieg D.J. Hauck C.R. Schlaepfer D.D. J. Cell Sci. 1999; 112: 2677-2691Crossref PubMed Google Scholar). Recombinant TNFα and IL-6 detection kits were from eBioscience (San Diego). Pharmacological inhibitors PP2, PD98059, SB203580, SP600125, and wortmannin were from Calbiochem. Viral Expression—The pLentiLox shRNA vector was used to create stable knockdown of FAK expression in NB8, PC3, and A549 carcinoma cells as described (20Mitra S.K. Mikolon D. Molina J.E. Hsia D.A. Hanson D.A. Chi A. Lim S.T. Bernard-Trifilo J.A. Ilic D. Stupack D.G. Cheresh D.A. Schlaepfer D.D. Oncogene. 2006; 25: 5969-5984Crossref PubMed Scopus (135) Google Scholar). Targeting sequences were designed to nucleotides 394–415 of mouse FAK and nucleotides 1089–1107 of human FAK. A scrambled shRNA oligonucleotide forward 5′-tgtctccgaacgtgtcacgtttcaagagaacgtgacacgttcggagacttttttc-3′ and reverse 5′-tcgagaaaaaagtctccgaacgtgtcacgttctcttgaaacgtgacacgttcggagaca-3′ was used as a control. For murine Pyk2 shRNA, the oligonucleotides forward 5′-tgaagtagttcttaaccgcattcaagagatgcggttaagaactacttcttttttc-3′ and reverse 5′-tcgagaaaaaagaagtagttcttaaccgcatctcttgaatgcggttaagaactacttca-3′ were cloned into pLentiLox. Cells were sorted for GFP expression, and the shRNA efficacy was verified by immunoblotting. Rabbit CA MEK1 (S231E/S235E) was a kind gift from Jiahuai Han (The Scripps Research Institute). The 1.2-kb MEK1 cDNA was amplified by PCR, subcloned into pCMV6M to add an N-terminal Myc tag, and the DNA sequence verified. Myc-tagged CA-MEK1 was subcloned into the lentiviral vector pCDH-puro (System BioSciences, Mountain View, CA), and recombinant lentiviruses were produced as described (20Mitra S.K. Mikolon D. Molina J.E. Hsia D.A. Hanson D.A. Chi A. Lim S.T. Bernard-Trifilo J.A. Ilic D. Stupack D.G. Cheresh D.A. Schlaepfer D.D. Oncogene. 2006; 25: 5969-5984Crossref PubMed Scopus (135) Google Scholar). FAK–/– fibroblasts and A549 carcinoma cells were selected in puromycin (5 μg/ml), and CA-MEK1 expression was verified by immunoblotting. HA-tagged FRNK and FRNK Ser-1034 were created and used as described (27Mitra S.K. Lim S.T. Chi A. Schlaepfer D.D. Oncogene. 2006; 25: 4429-4440Crossref PubMed Scopus (83) Google Scholar). Briefly, cells were infected with five plaque-forming units/cell for Ad-TA (mock control) or with five Ad-TA plus 50 plaque-forming units/cell Ad-FRNK constructs. GFP FAK FERM-(1–402) was generated by PCR with HindIII and BamHI ends and cloned into pEGFP-C1. Quickchange (Stratagene, La Jolla, CA) site-directed was used to create the double R177A/R178A mutation within pEGFP-C1 FAK FERM. For adenoviral expression, NheI-MluI fragments of pEGFP-C1 FAK FERM-(1–402) were cloned into the EcoRV site of pCMV-Shuttle (Stratagene), and virus was produced using the AdEasy system (Stratagene). Cells were infected with 50 plaque-forming units/cell GFP or GFP-FAK FERM constructs and analyzed after 48 h. GFP-FAK-reconstituted FAK–/– Fibroblasts—GFP-FAK wild type (WT), kinase-inactive (Arg-454), and Phe-925 FAK in the retroviral pBabepuro vector were created as described (20Mitra S.K. Mikolon D. Molina J.E. Hsia D.A. Hanson D.A. Chi A. Lim S.T. Bernard-Trifilo J.A. Ilic D. Stupack D.G. Cheresh D.A. Schlaepfer D.D. Oncogene. 2006; 25: 5969-5984Crossref PubMed Scopus (135) Google Scholar). FAK mutants (Phe-397, Ala-712/Ala-713, and Phe-861) in pCDNA3 (24Sieg D.J. Hauck C.R. Schlaepfer D.D. J. Cell Sci. 1999; 112: 2677-2691Crossref PubMed Google Scholar) were subcloned into pEGFP-C1 as KpnI-XbaI fragments, and 5′-untranslated sequences from FAK were removed by BspEI/PvuI digestion and replaced by a 275-bp sequence from WT FAK cloned in pEGFP-C1 (28Ilic D. Almeida E.A. Schlaepfer D.D. Dazin P. Aizawa S. Damsky C.H. J. Cell Biol. 1998; 143: 547-560Crossref PubMed Scopus (436) Google Scholar). All GFP-FAK constructs also contain a triple HA tag at the FAK C terminus and were subcloned into pBabepuro for retrovirus production. Early passage FAK–/– fibroblasts were infected with GFP-FAK retrovirus, selected in puromycin (2 μg/ml), and pooled populations of cells enriched by fluorescence-activated cell sorting. Upon expansion, GFP-FAK expression was not stably maintained, and therefore, clonal cell lines were obtained by further sorting, expanded, and GFP-FAK expression verified by immunoblotting. Three or more clonal cell lines for each GFP-FAK construct were pooled together and include the GFP-FAK-reconstituted cells used in this study. IL-6 ELISA—2 × 106 cells were plated and allowed to spread for 4 h and then stimulated with or without TNFα (10 ng/ml) in Dulbecco's modified Eagle's medium containing 10% FBS for 24 h. IL-6 levels in conditioned media were measured using mouse or human IL-6 ELISA kits (eBioscience) according to the manufacturer's instructions. All samples were analyzed in duplicate. Fluorescence-activated Cell Sorting Analysis—FAK+/+ and FAK–/– fibroblasts were trypsinized, fixed with 3.7% formaldehyde solution for 10 min at room temperature, and incubated with phycoerythrin-conjugated anti-tumor necrosis factor receptor-1 (TNFR1) or TNFR2 antibodies. After extensive phosphate-buffered saline washes, cell surface TNFR1 and TNFR2 levels were analyzed by flow cytometry and compared with IgG control. Total RNA Preparation and Reverse Transcription (RT)-PCR—Total RNA was isolated from cells using TRIzol reagent (Invitrogen). First strand synthesis was conducted using Superscript First Strand Synthesis kit (Invitrogen) with random primers and 5 μg of total RNA as template. PCR was performed using TaqPro Complete (Denville Scientific, Metuchen, NJ). IL-6 and β-actin were co-amplified with specific primer pairs (IL-6 forward, 5′-gatgctaccaaactggatataatc; IL-6 reverse, 5′-ggtccttagccactccttctgtg; β-actin forward, 5′-tgtgatggtgggaatgggtcag; β-actin reverse, 5′-tttgatgtcacgcacgatttcc). Cycling conditions were 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. IL-6 was amplified for 31 cycles, with β-actin primers present in the last 18 cycles as an internal control. Protein Extracts—Total cell protein was prepared using RIPA lysis buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 10% glycerol, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 10 mm sodium pyrophosphate, 100 mm NaF, 1 mm sodium orthovanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin). Cytoplasmic and nuclear extracts were isolated as described (29Hou S. Guan H. Ricciardi R.P. J. Biol. Chem. 2003; 278: 45994-45998Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Electrophoretic Mobility Shift Assay (EMSA)—Double-stranded oligonucleotides were labeled with [α-32P]dCTP (PerkinElmer Life Sciences) and Klenow DNA polymerase. EMSA was performed as described previously (29Hou S. Guan H. Ricciardi R.P. J. Biol. Chem. 2003; 278: 45994-45998Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Wild type NF-κB oligonucleotides were 5′-acgtgtgggattttccatg (forward) and 5′-tcgacatgggaaaatcccac (reverse), whereas mutant oligonucleotides contained the sequences 5′-acgtgttacattttcccatg (forward) and 5′-tcgacatgggaaaatgtaac (reverse) with mutations preventing NF-κB binding in boldface type. Transfection and Luciferase Assay—FAK+/+ and FAK–/– fibroblasts were grown in 6-well plates and co-transfected with 5 μg of NF-κB firefly luciferase reporter (from Qilin Pan, The Scripps Research Institute) and 1 μg of TK-Renilla luciferase (Promega, San Luis Obispo) using Lipofectamine 2000 (Invitrogen). After 24 h, transfected cells were stimulated with 10 ng/ml TNFα for an additional 24 h. Cellular proteins were extracted in passive lysis buffer and analyzed according to the manufacturer's instructions for dual luciferase reporter assay (Promega). Immunofluorescence—FAK–/– or A549 cells growing on glass coverslips were fixed with 3.7% formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 and 0.05% Tween 20 detergents for 6 min. Cells were stained with 0.2 ng/ml Texas Red-conjugated phalloidin (Invitrogen) for 15 min. Photographs were taken using a monochrome CCD camera (Hamamatsu ORCA ER) and processed using OpenLab software (Improvision, Lexington MA). Phase pictures were taken at ×20 (Olympus, NA = 0.5). FAK in Vitro Kinase (IVK) Assay—FAK immunoprecipitates (5904 polyclonal and protein A-agarose beads) were washed once in RIPA buffer, twice in HNTG buffer (20 mm HEPES, pH 7.4, 150 mm NaCl, 0.1% Triton, and 10% glycerol), and once in kinase buffer (20 mm HEPES, pH 7.4, 10% glycerol, 10 mm MgCl2, 10 mm MnCl2, 150 mm NaCl). FAK IVK activity was measured by addition of 25 μCi of [γ-32P]ATP (6000 Ci/mmol; PerkinElmer Life Sciences) for 15 min at 30 °C. The assay was stopped by adding 2× Laemmli sample buffer, resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membrane. Labeled FAK was visualized by autoradiography and quantified using a PhosphorImager (GE Healthcare). Equal immunoprecipitation was verified by anti-FAK blotting. TNFα-stimulated IL-6 Expression Is Dependent on FAK—Pro- and anti-inflammatory cytokine production can influence tumor progression (30Coussens L.M. Werb Z. Nature. 2002; 420: 860-867Crossref PubMed Scopus (11276) Google Scholar). FAK expression and activity promote 4T1 breast carcinoma tumor growth and metastasis in BALB/c mice (20Mitra S.K. Mikolon D. Molina J.E. Hsia D.A. Hanson D.A. Chi A. Lim S.T. Bernard-Trifilo J.A. Ilic D. Stupack D.G. Cheresh D.A. Schlaepfer D.D. Oncogene. 2006; 25: 5969-5984Crossref PubMed Scopus (135) Google Scholar, 27Mitra S.K. Lim S.T. Chi A. Schlaepfer D.D. Oncogene. 2006; 25: 4429-4440Crossref PubMed Scopus (83) Google Scholar). As 4T1 tumors also induce a leukemoid reaction (31Dupre S.A. Hunter Jr., K.W. Exp. Mol. Pathol. 2006; 82: 12-24Crossref PubMed Scopus (192) Google Scholar) and metastatic 4T1 lung tumors were associated with lung edema and immune cell infiltration (27Mitra S.K. Lim S.T. Chi A. Schlaepfer D.D. Oncogene. 2006; 25: 4429-4440Crossref PubMed Scopus (83) Google Scholar), we tested whether FAK signaling was involved in promoting IL-6 expression as this cytokine promotes inflammation in breast cancer (32Bachelot T. Ray-Coquard I. Menetrier-Caux C. Rastkha M. Duc A. Blay J.Y. Br. J. Cancer. 2003; 88: 1721-1726Crossref PubMed Scopus (249) Google Scholar). Basal levels of secreted IL-6 were reduced >10-fold in 4T1 FAK shRNA compared with scrambled (control) shRNA-expressing 4T1 cells as determined by ELISA (Fig. 1A). TNFα is a strong promoter of IL-6 expression in many cell types (33Philip M. Rowley D.A. Schreiber H. Semin. Cancer Biol. 2004; 14: 433-439Crossref PubMed Scopus (499) Google Scholar). TNFα promoted 3-fold increased IL-6 secretion from 4T1 control cells whereas in 4T1 FAK shRNA cells, IL-6 levels remained below the basal level of 4T1 controls (Fig. 1A). To test whether FAK facilitates TNFα-stimulated IL-6 expression in human tumor cells, we analyzed NB8 neuroblastoma, A549 lung carcinoma, and PC3 prostate carcinoma cells stably expressing either scrambled or FAK shRNA (Fig. 1B). In all three cell lines, 70–90% reduction in FAK expression was accompanied by the inhibition of TNFα-stimulated IL-6 production. As fibroblasts in the tumor stroma can also contribute to the production of inflammatory cytokines (30Coussens L.M. Werb Z. Nature. 2002; 420: 860-867Crossref PubMed Scopus (11276) Google Scholar), we compared the responses of murine FAK+/+p53–/– and FAK–/–p53–/– embryonic fibroblasts (MEFs) to TNFα stimulation. In wild type FAK+/+ MEFs, TNFα stimulation resulted in a dose-dependent increase of IL-6 protein secretion (Fig. 1C). In contrast, FAK–/– MEFs produced only marginal amounts of IL-6 in response to TNFα. Analysis of TNFα receptors TNFR1 and TNFR2 by flow cytometry showed equivalent surface expression in FAK+/+ and FAK–/– MEFs (Fig. 1D), indicating that the defect in TNFα-initiated IL-6 production is associated with the loss of intracellular FAK signaling. These results support the notion that FAK promotes TNFα-induced IL-6 expression in both normal fibroblasts and carcinoma cells of murine and human origin. FAK Promotes IL-6 mRNA Expression—Since TNFα can promote IL-6 mRNA transcription, we performed semi-quantitative RT-PCR analyses on TNFα-treated FAK+/+, FAK–/–, and FAK-reconstituted FAK–/– MEFs (clone DP3) to determine whether IL-6 expression in FAK–/– MEFs was blocked at the mRNA level. By normalizing the IL-6-amplified band to the internal actin control in all reactions, RT-PCR analysis revealed that basal and TNFα-stimulated IL-6 mRNA levels were reduced ∼3-fold in FAK–/– compared with FAK+/+ and FAK-reconstituted MEFs (Fig. 2A). One caveat of using FAK–/– MEFs for signaling studies is that the expression of the FAK-related kinase Pyk2 is elevated in these cells (34Sieg D.J. Ilic D. Jones K.C. Damsky C.H. Hunter T. Schlaepfer D.D. EMBO J. 1998; 17: 5933-5947Crossref PubMed Scopus (289) Google Scholar). To confirm that the inhibition of TNFα-stimulated IL-6 expression was due to lack of FAK and not due to interference by Pyk2, FAK expression in FAK+/+ MEFs was inhibited via lentiviral anti-FAK shRNA (Fig. 2B). Stable knockdown (∼85%) of FAK levels in FAK+/+ MEFs resulted in a 4–5-fold reduction in both basal and TNFα-stimulated IL-6 mRNA levels compared with scrambled shRNA-expressing FAK+/+ MEFs (Fig. 2C). Notably, stable FAK knockdown did not promote an alteration in cell morphology compared with scrambled (Scr) shRNA-expressing MEFs (Fig. 2D), and there was no compensatory change in Pyk2 levels in FAK shRNA-expressing FAK+/+ p53–/– MEFs (data not shown). These results support the conclusion that TNFα effects on IL-6 expression are dependent on FAK and occur in part through effects on IL-6 mRNA expression. FAK Is Not Required for TNFα-induced NF-κB Activation—A major signaling pathway promoting TNFα-induced IL-6 mRNA transcription is through NF-κB activation (12MacEwan D.J. Cell Signal. 2002; 14: 477-492Crossref PubMed Scopus (527) Google Scholar). As a previous study showed that TNFα-induced NF-κB activation may be impaired in FAK–/– fibroblasts (14Funakoshi-Tago M. Sonoda Y. Tanaka S. Hashimoto K. Tago K. Tominaga S. Kasahara T. J. Biol. Chem. 2003; 278: 29359-29365Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), we investigated the regulation of NF-κB in FAK+/+ and FAK–/– fibroblasts (Fig. 3). In unstimulated cells, NF-κB is cytoplasmically localized and kept inactive in part through an association with the inhibitory protein IκB. TNFα enhances the phosphorylation of IκBα that triggers its rapid degradation, thus releasing NF-κB and allowing for NF-κB nuclear translocation. In both FAK+/+ and FAK–/– cells, TNFα induced a rapid and identical reduction of IκBα protein levels within 5 min followed by IκBα resynthesis after 60 min (Fig. 3A). Similar results were obtained with anti-FAK shRNA-expressing FAK+/+ cells (Fig. 3B) and Pyk2 shRNA-expressing FAK–/– cells (Fig. 3, C and D). To determine whether IκBα degradation corresponded to NF-κB nuclear translocation, control and TNFα-treated (30 min) cells were separated into cytoplasmic and nuclear fractions. TNFα equally stimulated the degradation of IκBα and corresponding to the nuclear translocation of the NF-κB p65 subunit in FAK+/+, FAK shRNA-expressing FAK+/+, FAK–/–, and Pyk2 shRNA-expressing FAK–/– MEFs (Fig. 3D). T" @default.
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• W2034771296 title "Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression" @default.
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