Src Kinase Activity Is Required for Integrin αVβ3-Mediated Activation of Nuclear Factor-κB

Abstract

Integrin adhesion to extracellular matrix proteins protects adhesion-dependent cells from suspension-induced apoptosis. Previous studies indicate that activation of the transcription factor nuclear factor-κB was necessary for the integrin αvβ3 ligand osteopontin to protect endothelial cells from apoptosis caused by serum withdrawal. In this study, β3 integrins were overexpressed in smooth muscle cells. When plated on osteopontin, cells overexpressing wild-type β3 had enhanced cell adhesion, cell spreading, and nuclear factor-κB activation compared with vector control. Removal of four amino acids (759X) from the C terminus of β3 eliminated the ability of the integrin to promote these processes. Single amino acid substitutions indicated that phosphorylation at tyrosine 759 was not required for activation of the transcription factor, however this residue appeared to play a structural role, because mutation to alanine significantly inhibited nuclear factor-κB activation. The Src family of tyrosine kinases represents important transducers during integrin signaling, and the C terminus of β3 has been implicated as the binding site for Src. Immunoprecipitations demonstrated that Src associated with wild-type β3 integrins, but Src and integrins lacking the C terminus (759X) did not form a complex. Pharmacological inhibition with the Src inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) or overexpression of kinase-dead c-Src blocked nuclear factor-κB activation. Mouse embryonic fibroblasts deficient for Src failed to activate nuclear factor-κB when plated on osteopontin, in contrast to control fibroblasts. Together, these experiments indicate that the C terminus of β3 and Src activity are required for integrin αvβ3-mediated nuclear factorκB activation. Integrin adhesion to extracellular matrix proteins protects adhesion-dependent cells from suspension-induced apoptosis. Previous studies indicate that activation of the transcription factor nuclear factor-κB was necessary for the integrin αvβ3 ligand osteopontin to protect endothelial cells from apoptosis caused by serum withdrawal. In this study, β3 integrins were overexpressed in smooth muscle cells. When plated on osteopontin, cells overexpressing wild-type β3 had enhanced cell adhesion, cell spreading, and nuclear factor-κB activation compared with vector control. Removal of four amino acids (759X) from the C terminus of β3 eliminated the ability of the integrin to promote these processes. Single amino acid substitutions indicated that phosphorylation at tyrosine 759 was not required for activation of the transcription factor, however this residue appeared to play a structural role, because mutation to alanine significantly inhibited nuclear factor-κB activation. The Src family of tyrosine kinases represents important transducers during integrin signaling, and the C terminus of β3 has been implicated as the binding site for Src. Immunoprecipitations demonstrated that Src associated with wild-type β3 integrins, but Src and integrins lacking the C terminus (759X) did not form a complex. Pharmacological inhibition with the Src inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) or overexpression of kinase-dead c-Src blocked nuclear factor-κB activation. Mouse embryonic fibroblasts deficient for Src failed to activate nuclear factor-κB when plated on osteopontin, in contrast to control fibroblasts. Together, these experiments indicate that the C terminus of β3 and Src activity are required for integrin αvβ3-mediated nuclear factorκB activation. Integrins are heterodimeric receptors composed of two type I transmembrane glycoproteins, α and β. Integrins typically contain a large extracellular domain, a transmembrane domain, and a short intracellular domain with no known enzymatic function. The integrin extracellular domain binds to extracellular matrix ligands or cell surface ligands, whereas the intracellular domain provides a link to adapter proteins and the cytoskeleton. Thus, integrins act as adhesion receptors, physically linking the extracellular matrix to the cytoskeleton. Integrin ligation also promotes intracellular signal transduction through outside-in signaling. These signaling pathways promote complex biological functions, including cell adhesion, proliferation, migration, differentiation, and cell survival. For many cell types, integrin-mediated adhesion to the extracellular matrix is required for not only cell growth but also for cell survival (1.Meredith Jr., J.E. Fazeli B. Schwartz M.A. Mol. Biol. Cell. 1993; 4: 953-961Crossref PubMed Scopus (1392) Google Scholar). Disruption of adhesion causes the cells to undergo apoptosis, a process termed anoikis. Integrin antagonists also induce apoptosis both in vitro (2.Scatena M. Almeida M. Chaisson M.L. Fausto N. Nicosia R.F. Giachelli C.M. J. Cell Biol. 1998; 141: 1083-1093Crossref PubMed Scopus (443) Google Scholar) and in vivo (3.Brooks P.C. Montgomery A.M. Rosenfeld M. Reisfeld R.A. Hu T. Klier G. Cheresh D.A. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2170) Google Scholar). It has also been suggested that non-ligated integrins can actively promote cell death (4.Stupack D.G. Puente X.S. Boutsaboualoy S. Storgard C.M. Cheresh D.A. J. Cell Biol. 2001; 155: 459-470Crossref PubMed Scopus (444) Google Scholar). The Src family of tyrosine kinases (SFKs) 1The abbreviations used are: SFK, Src family of tyrosine kinase; SYF, cells deficient for c-Src, Yes, and Fyn; NF-κB, nuclear factor-κB; IκB, inhibitor of κB; OPN, osteopontin; WT, wild-type; RAEC, rat aortic endothelial cell; WKY, Wistar Kyoto rat smooth muscle cell; PBS, phosphate-buffered saline; PDL, poly-d-lysine; KD, kinase-dead; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PP3, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine.1The abbreviations used are: SFK, Src family of tyrosine kinase; SYF, cells deficient for c-Src, Yes, and Fyn; NF-κB, nuclear factor-κB; IκB, inhibitor of κB; OPN, osteopontin; WT, wild-type; RAEC, rat aortic endothelial cell; WKY, Wistar Kyoto rat smooth muscle cell; PBS, phosphate-buffered saline; PDL, poly-d-lysine; KD, kinase-dead; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PP3, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine. represents important transducers during integrin-mediated signaling, and activated Src can protect cells from apoptosis. The Src family consists of eight members but only c-Src, Yes, and Fyn are ubiquitously expressed. SFKs show redundancy, therefore, inhibition of multiple SFKs is often required to demonstrate phenotypes. Mice deficient in the three ubiquitous kinases: c-Src, Yes, and Fyn, display developmental defects leading to embryonic lethality (5.Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (646) Google Scholar). Embryonic fibroblasts from mice lacking c-Src, Yes, and Fyn (SYF cells) have reduced protein phosphotyrosine levels following integrin ligation and reduced cell migration on fibronectin (5.Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (646) Google Scholar). Although c-Src is only modestly activated following integrin stimulation, c-Src has been shown to directly phosphorylate many downstream proteins, including FAK, Cas, and paxillin. In fibroblasts, SFKs regulate integrin-mediated cell attachment and spreading in response to fibronectin but not collagen (6.Kaplan K.B. Swedlow J.R. Morgan D.O. Varmus H.E. Genes Dev. 1995; 9: 1505-1517Crossref PubMed Scopus (295) Google Scholar). The transcription factor nuclear factor-κB (NF-κB) is another important player in cell survival. NF-κB activation protects endothelial cells, hepatocytes, B cells, osteoclasts, melanoma, and smooth muscle from apoptosis (7.Kucharczak J. Simmons M.J. Fan Y. Gelinas C. Oncogene. 2003; 22: 8961-8982Crossref PubMed Scopus (662) Google Scholar). The NF-κB family consists of homo- or heterodimeric subunits of Rel family members. In the cytoplasm, NF-κB binds to the inhibitory protein I-κB (IκB). Upon stimulation, IκB becomes phosphorylated, ubiquinated, and degraded by the proteasome, allowing NF-κB to translocate to the nucleus and promote gene expression of pro-survival genes, including but not limited to the inhibitors of apoptosis, bcl-2 family proteins, and osteoprotegrin (8.Malyankar U.M. Scatena M. Suchland K.L. Yun T.J. Clark E.A. Giachelli C.M. J. Biol. Chem. 2000; 275: 20959-20962Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). We have previously shown that integrin αvβ3 ligation in rat aortic endothelial cells (RAECs) activates NF-κB, protecting the cells from apoptosis (2.Scatena M. Almeida M. Chaisson M.L. Fausto N. Nicosia R.F. Giachelli C.M. J. Cell Biol. 1998; 141: 1083-1093Crossref PubMed Scopus (443) Google Scholar). Blocking αvβ3 ligation or preventing NF-κB activation induces cell death. Thus, NF-κB is a downstream mediator of integrin αvβ3 in endothelial cell survival. In the present study we provide evidence that the integrin αvβ3/NF-κB pathway is found in multiple cell types, perform structure/function studies to establish the region of the integrin αvβ3 required for NF-κB activation, determine that SFKs bind to integrin β3 and the association requires the C terminus of the integrin, and identify that SFK kinase activity is required for integrin αvβ3-mediated NF-κB activation. Antibodies—Rabbit polyclonal anti-integrin β3 (Chemicon, Temecula, CA) and the mouse monoclonal antibodies anti-v-Src clone 327 (Oncogene Research Products, San Diego, CA), anti-Syk N-19 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-human integrin β3 clone SZ.21 (Beckman Coulter, Brea, CA), anti-human integrin β3 clone 1 and anti-rat integrin β3 clone F11 (BD Pharmingen) were commercially available. Anti-osteopontin (OPN) antibody OP199 has been previously described (9.Liaw L. Almeida M. Hart C.E. Schwartz S.M. Giachelli C.M. Circ. Res. 1994; 74: 214-224Crossref PubMed Scopus (372) Google Scholar). Plasmid Constructs—The retroviral vector PBMN-IRES-eGFP and Phoenix packaging cell line were a gift from Gary Nolan (Stanford University, Palo Alto, CA) (10.Kinoshita S. Su L. Amano M. Timmerman L.A. Kaneshima H. Nolan G.P. Immunity. 1997; 6: 235-244Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). The retroviral NF-κB reporter pBM-SIN-κB-luciferase was a gift from Elaine Raines (University of Washington, Seattle, WA) (11.Ferri N. Garton K.J. Raines E.W. J. Biol. Chem. 2003; 278: 19757-19764Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The human integrin β3 cDNA construct was graciously donated by Mark Ginsberg (Scripps Research Institute, San Diego, CA) (12.Ylanne J. Huuskonen J. O'Toole T.E. Ginsberg M.H. Virtanen I. Gahmberg C.G. J. Biol. Chem. 1995; 270: 9550-9557Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Wild-type (WT) β3 was subcloned into the PBMN-IRES-eGFP retroviral vector by cutting with XhoI and NheI. Truncations 759X and 761X and amino acid substitutions Y759A and Y759F were produced using the Stratagene QuikChange site-directed mutagenesis kit according to manufacturer's suggested protocol using the oligonucleotides listed. Mutated plasmids were confirmed by DNA sequencing. 759X forward: 5′-CTACCTTCACCAATATCACGTAGCGGGGCACTTAATG-3′; 759X reverse: 5′-CATTAAGTGCCCCGCTACGTGATATTGGTGAAGGTAG-3′; 761X forward: 5′-CCAATATCACGTACCGGTGAACTTAATGATAAGCAGTCATCC-3′; 761X reverse: 5′-GGATGACTGCTTATCATTAAGTTCACCGGTACGTGATATTGG-3′; Y759F forward: 5′-CTACCTTCACCAATATCACGTTTCGGGGCACTTAATG3′; Y759F reverse: 5′-CATTAAGTGCCCCGAAACGTGATATTGGTGAAGGTAG-3′; Y759A forward: 5′-CTACCTTCACCAATATCACGGCCCGGGGCACTTAATG-3′; and Y759A reverse: 5′-CATTAAGTGCCCCGGGCCGTGATATTGGTGAAGGTAG-3′. Chicken c-Src constructs (WT, K295R, Y527F, and K295R/Y527F retroviral plasmids) were a gift from Jonathan Cooper (Fred Hutchinson Cancer Research Center, Seattle, WA) (13.Cary L.A. Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Mol. Cell. Biol. 2002; 22: 2427-2440Crossref PubMed Scopus (129) Google Scholar). The mammalian expression construct EMCV-Syk was a gift from Stanford Shattil (Scripps Research Institute) (14.Gao J. Zoller K.E. Ginsberg M.H. Brugge J.S. Shattil S.J. EMBO J. 1997; 16: 6414-6425Crossref PubMed Scopus (160) Google Scholar). Human Syk was subcloned into the PBMN-IRES-eGFP retroviral vector by cutting with BglII and XhoI. The PBMN-IRES-eGFP vector was also treated with calf intestinal alkaline phosphatase (New England Biolabs, Beverly, MA) to prevent self-ligation. Kinase-dead Syk (K402R) was generated with the Stratagene QuikChange site-directed mutagenesis kit using the oligonucleotides listed. Mutated plasmids were confirmed by DNA sequencing. K402R forward: 5′-CCGTGGCTGTGAGAATACTGAAAAACGAGGCC-3′; K402R reverse: 5′-GGCCTCGTTTTTCAGTATTCTCACAGCCACGG-3′. Cell Culture—RAECs were purchased from VEC Technologies Inc. and grown in MCDB 131 media (Invitrogen) supplemented with 10 mml-glutamine (Invitrogen), 10% fetal bovine serum (HyClone, Logan, UT) and 100 units/ml each of penicillin and streptomycin (Invitrogen). Medial smooth muscle cells from Wistar Kyoto rat pups were isolated as previously described (15.Lemire J.M. Covin C.W. White S. Giachelli C.M. Schwartz S.M. Am. J. Pathol. 1994; 144: 1068-1081PubMed Google Scholar) and grown in Waymouth's MB 752/a media (Invitrogen) supplemented with 10% fetal bovine serum (HyClone) and 100 units/ml each of penicillin and streptomycin (Invitrogen). Cells were passaged by immersing in 0.05% trypsin. Retroviral Infections—High titer amphotrophic retrovirus was prepared as previously described (16.Garton K.J. Ferri N. Raines E.W. BioTechniques. 2002; 32 (832, 834 passim): 830Crossref PubMed Scopus (40) Google Scholar). Briefly, Phoenix packaging cells were transiently transfected with the retroviral plasmids. Medium was removed 10 and 24 h post-transfection. At 24-h post-transfection the cells were moved to a 32 °C incubator and virus was collected between 24 and 48 h after transfection. The virus-containing media was passed through a 0.45-μm filter to remove cellular debris. Twenty-four hours prior to the infections, 105 Wistar Kyoto rat smooth muscle cells (WKY) or 2 × 105 RAECs were plated per well in 6-well tissue culture plates. Cells underwent three rounds of infection. For each infection, the medium was replaced with 2 ml of retroviral-containing medium supplemented with 4 μg/ml Polybrene (Sigma-Aldrich, St. Louis, MO) and spun at 1100 × g and 32 °C for 1 h then returned to the 37 °C incubator. After 24 h, the old medium was removed. Following the third infection, enhanced green fluorescent protein-positive cells were detected by flow cytometry when applicable. For viruses encoding Src, cells were selected with 200 μg/ml Hygromycin D (Calbiochem). Flow Cytometry—For integrin expression profiling, 1 × 106 cells were washed with phosphate-buffered saline (PBS) plus 0.2% bovine serum albumin (Sigma-Aldrich) plus 0.02% sodium azide. Cells were incubated on ice for 30 min with either non-immune mouse-IgG (Sigma-Aldrich) or the mouse monoclonal antibody SZ.21, which recognizes human β3 but not rat β3 (17.Liaw L. Lindner V. Schwartz S.M. Chambers A.F. Giachelli C.M. Circ. Res. 1995; 77: 665-672Crossref PubMed Scopus (148) Google Scholar). Cells were washed and incubated with phycoerythrin-conjugated anti-mouse IgG (Biomeda, Foster City, CA). Cells were washed and fixed in 1% paraformaldehyde. Samples were analyzed using the BD FACScan flow cytometry system and analyzed by CellQuest (BD Biosciences, San Jose, CA). Recombinant OPN—Generation of histidine-tagged full-length recombinant rat OPN has been previously described (18.Martin S.M. Ganapathy R. Kim T.K. Leach-Scampavia D. Giachelli C.M. Ratner B.D. J. Biomed. Mater. Res. 2003; 67A: 334-343Crossref Scopus (37) Google Scholar). Briefly, Escherichia coli were transformed with the pQE30 plasmid encoding recombinant rat OPN with six histidines attached to the N terminus. The bacteria were amplified, and protein expression was induced by adding 1 mm isopropyl-β-d-thiogalactopyranoside (Research Products International, Mt. Prospect, IL). The protein was collected using a nickel-nitrilotriacetic acid-agarose resin (Qiagen, Valencia, CA), and eluted using 0.2 m imidazole (Acros Organics). Protein concentration was determined using the MicroBCA assay (Pierce, Rockford, IL). Western blots and adhesion assays verified protein purity and functionality respectively. Adhesion Assays—Adhesion assays were performed as previously described (19.Liaw L. Skinner M.P. Raines E.W. Ross R. Cheresh D.A. Schwartz S.M. Giachelli C.M. J. Clin. Invest. 1995; 95: 713-724Crossref PubMed Google Scholar). Briefly, protein solutions were diluted in PBS and adsorbed to 96-well Maxisorb plates (Nalge Nunc International, Rochester, NY) at 4 °C overnight. Wells were rinsed with PBS, and nonspecific binding sites were blocked by adding 10 mg/ml bovine serum albumin and incubating at 37 °C for 1 h. Cells were trypsinized, centrifuged, and washed twice with Waymouth's media without serum. Samples were plated with 60,000 cells/well and incubated at 37 °C for 2 h. The plates were rinsed with PBS and fixed with 4% paraformaldehyde. The cells were stained with 0.5% toluidine blue (J. T. Baker, Phillipsburg, NJ) in 4% paraformaldehyde and rinsed in water. The dye was solubilized using 1% SDS. Adhesion was quantified by absorbance at 630 nm. NF-κB Activation Assays—Recombinant histidine-tagged rat OPN or poly-d-lysine (PDL) (Sigma-Aldrich) was adsorbed to 12-well tissue culture plates by incubating at 4 °C overnight. The wells were blocked with 10 mg/ml bovine serum albumin for 1 h at 37 °C. RAECs or WKY cells expressing the NF-κB-dependent luciferase reporter were trypsinized and washed in serum-free media. For inhibition studies, the cells were treated with the active pharmacological Src inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, Calbiochem) or the inactive control PP3 (4-amino-7-phenylpyrazol-[3,4-d]pyrimidine, Calbiochem) and kept in suspension for 45 min. The cells were then plated on the OPN-coated surfaces and allowed to adhere for 5–7 h. Non-adherent cells were washed away and adherent cells were lysed with Reporter Lysis Buffer from Promega (Madison, WI). Protein recovery was monitored by microBCA assay (Pierce). Luciferase activity was detected using the Promega Luciferase Assay System, and activity was normalized to total protein recovered. Western Blots—Proteins were extracted from cell monolayers in Laemmli buffer containing protease inhibitors (2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride). After lysis and boiling, protein concentration was determined by MicroBCA assay. 30 μg of lysate was loaded onto 10% SDS-polyacrylamide gels. Identical gels were stained with Pro-Blue staining solution (Owl Separation Systems, Portsmouth, NH) to verify uniform loading. Samples were transferred onto polyvinylidene difluoride membranes. Membranes were probed with the appropriate primary antibody, washed, and then horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). Bands were detected using the Western Lightning chemiluminescence kit (PerkinElmer Life Sciences). Immunoprecipitations—WKY cells overexpressing c-Src and vector control or integrin β3 subunits, WT or 759X, were grown to confluence and lysed in lysis buffer containing 1% Nonidet P-40, 150 mm NaCl, 50 mm Tris, 1 mm sodium orthovanadate, 0.5 mm NaF and protease inhibitors. 800 μg of lysate was immunoprecipitated with rabbit polyclonal anti-integrin β3 antibody or rabbit IgG control antibody and Protein A-Sepharose beads (Sigma-Aldrich). The immunoprecipitates or total cell lysate were subjected to Western blotting with the indicated detection antibodies. Previous studies showed that integrin αvβ3 ligation activated the transcription factor NF-κB in RAECs, and inhibition of αvβ3 ligation or inhibition of NF-κB activation caused endothelial cell apoptosis (2.Scatena M. Almeida M. Chaisson M.L. Fausto N. Nicosia R.F. Giachelli C.M. J. Cell Biol. 1998; 141: 1083-1093Crossref PubMed Scopus (443) Google Scholar). In the current study, we investigated the mechanism of αvβ3-mediated NF-κB activation. To determine which integrin subunit was involved in activation of NF-κB, melanoma cells overexpressing αvβ3 or αIIbβ3 were plated on the αvβ3 ligand fibronectin or the αIIbβ3 ligand fibrinogen, respectively. Both cell types activated NF-κB based on gel shift assays (data not shown), suggesting that the β3 subunit was the mediator for integrin-mediated NF-κB activation. Smooth Muscle Cell Adhesion through the Integrin αvβ3-Activated NF-κB—Medial smooth muscle cells from Wistar Kyoto rats (WKY) were identified as an excellent cell model for structure/function studies of the integrin β3 subunit, based on their high surface expression of αv and low expression of β3 (data not shown). To test NF-κB activation, WKY cells were infected with an NF-κB-dependent luciferase retroviral construct. The cells were then plated on surfaces coated with the β3 ligand OPN or PDL, which interacts with cells through a non-integrin-mediated electrostatic interaction. Luciferase readings for cells treated on PDL correspond to basal levels of NF-κB activation. Cells plated on the OPN-coated surface had enhanced luciferase activity (1.9-fold) relative to the PDL control as seen in Fig. 1, indicating that NF-κB had been activated. Activation of NF-κB was mediated by the αvβ3 integrin, because a β3-neutralizing antibody reduced OPN-mediated luciferase activity by 78%, whereas an IgG control antibody was unable to significantly reduce luciferase activity. Collagen, a ligand for β1 integrins, failed to increase NF-κB activation above basal levels (data not shown), further indicating the specificity of this pathway. Therefore, cellular adhesion to OPN through the integrin αvβ3 promoted NF-κB activation in smooth muscle cells as well as endothelial cells. The C Terminus of the β3 Subunit Was Necessary for Integrin-mediated NF-κB Activation—Retroviral vectors containing human integrin β3 constructs, either wild-type or mutated, were created (Table I) and used to stably infect WKY cells. Cells were incubated with an anti-human β3 antibody to detect surface expression of the integrin. All β3 constructs were efficiently expressed, with at least 80% of cells positive for surface expression of β3. The level of surface expression was similar for each of the β3 constructs, as shown in Fig. 2. Due to difficulties acquiring antibodies that recognize rat integrins, β3 constructs were also expressed in human smooth muscle cells and surface expression of other integrins was detected by flow cytometry. Overexpression of β3 did not significantly affect αvβ5, αvβ6, or β1 integrin expression levels (data not shown). Assuming the same trend occurs in rat smooth muscle cells, then observed phenotypes in cells infected with the β3 constructs are due to overexpression of αvβ3 integrins and not changes in the expression profile of other integrins.Table ISequences used in this studyNameCytoplasmic sequenceWild-type β3KLLITIHDRK EFAKFEEERA RAKWDTANNP LYKEATSTFT NITYRGT761XKLLITIHDRK EFAKFEEERA RAKWDTANNP LYKEATSTFT NITYRY759FKLLITIHDRK EFAKFEEERA RAKWDTANNP LYKEATSTFT NITFRGTY759AKLLITIHDRK EFAKFEEERA RAKWDTANNP LYKEATSTFT NITARGT759XKLLITIHDRK EFAKFEEERA RAKWDTANNP LYKEATSTFT NIT Open table in a new tab To test functionality of the β3 constructs, smooth muscle cells were seeded onto OPN-coated surfaces and allowed to attach for 2 h. Unattached cells were washed away, and phase contrast microscopy images of attached cells were acquired. All cell types attached to the OPN-coated surface (Fig. 3A), but substantial differences in post-receptor occupancy interactions were observed between the cell types. Overexpression of wild-type (WT) β3 dramatically increased cell spreading compared with vector control. Truncation of four amino acids from the C terminus (759X) eliminated the ability of the integrin to promote cell spreading, whereas removal of two amino acids did not impair cell spreading (761X). Substitution of tyrosine 759 with phenylalanine (Y759F) did not reduce cell spreading, but mutation to alanine (Y759A) resulted in an intermediate level of cell spreading. To quantify these post-receptor occupancy differences, adhesion assays were performed. Compared with vector control, overexpression of WT β3 significantly enhanced cellular adhesion by 3-fold or greater at all OPN concentrations tested (Fig. 3B). 759X significantly reduced adhesion compared with WT β3 (p < 0.001), whereas 761X did not impair the ability of the integrin to promote adhesion to OPN. Substitution of tyrosine 759 with phenylalanine (Y759F) did not reduce cell adhesion, indicating that phosphorylation of this residue was not required. Alanine substitution at this residue (Y759A) allowed for adhesion, but it was impaired compared with WT β3 especially at the lowest OPN concentration tested. Thus the adhesion assay results agreed with the differences in cell spreading seen in Fig. 3A. To test the ability of the integrins to mediate NF-κB activation, smooth muscle cells expressing the NF-κB-dependent luciferase reporter and the integrin β3 constructs were plated on surfaces coated with the β3 ligand OPN. After 7 h, adherent cells were lysed, and luciferase activity was measured and normalized to protein recovered. For ease of comparison, vector control samples were then normalized to a value of one. Compared with vector control cells, overexpression of full-length β3 or 761X significantly increased NF-κB activation (p < 0.01), whereas 759X was unable to increase NF-κB activation when plated on OPN (Fig. 3C). Integrins with the Y759F mutation retained the ability to activate NF-κB, but WKY cells overexpressing Y759A integrins have significantly reduced activation of NF-κB compared with WT β3 (p < 0.01). These results indicated that tyrosine 759 of integrin β3 had a critical structural role in OPN-mediated NF-κB activation, because truncation at position 759 or substitution to alanine significantly inhibited activation of the transcription factor, but tyrosine phosphorylation was not required, since the Y759F mutation retained the ability to signal. Pharmacological Inhibition of SFKs Reduced Integrin αvβ3-Mediated NF-κB Activation—Having identified that tyrosine 759 within the C terminus of β3 was required for NF-κB activation, we performed a preliminary experiment to identify proximal downstream mediators of NF-κB activation. The Src family of tyrosine kinases was recently reported to bind directly to β3 integrins through the C terminus, and its members are known proximal mediators of integrin signals (20.Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (449) Google Scholar). To test the role of SFKs in integrin-mediated NF-κB activation, RAECs were pretreated with the Src family tyrosine kinase inhibitor PP2, the inactive analog PP3, or vehicle control and plated on OPN. Treatment with 10 μm PP2 significantly inhibited integrin-mediated NF-κB activation (p < 0.01), whereas 10 μm PP3 or vehicle did not, as seen in Fig. 4. Similar results were seen when WKY smooth muscle cells were also treated with PP2 and PP3 (data not shown). Although PP2 inhibition is not complete at this dosage and higher doses of PP2 fail to yield further inhibition, 10 μm PP2 does lead to greater than 75% reduction in integrin-mediated activation of the transcription factor. These results indicate that NF-κB activation required SFK activity in this model. Overexpression of Kinase-dead c-Src Blocked Integrin αvβ3-Mediated NF-κB Activation—To further characterize the role of SFKs, RAECs cells were infected with retroviral vectors encoding WT chicken c-Src (WT), kinase-dead c-Src (K295R), activated c-Src (Y527F), or the double mutant (K295R/Y527F) (13.Cary L.A. Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Mol. Cell. Biol. 2002; 22: 2427-2440Crossref PubMed Scopus (129) Google Scholar). As seen in Fig. 5A, lane 5, Western blots of total cell lysates indicated that the RAECs expressed endogenous SFKs (the antibody was unable to distinguish Src family members). Cells infected with c-Src constructs have enhanced Src protein expression (Fig. 5A, lanes 1–4). RAECs expressing the c-Src constructs were plated on PDL- or OPN-coated surfaces. Expression of c-Src constructs did not affect cell adhesion to OPN (data not shown). RAECs infected with the control vector activated NF-κB when plated on OPN as expected (Fig. 5B). Compared with vector control, overexpression of activated c-Src (Y527F) enhanced NF-κB activation independent of substrate (p < 0.01), suggesting that c-Src was downstream of the integrin αvβ3. Overexpression of kinase-dead c-Src (K295R) acted as a dominant negative inhibitor, completely blocking OPN-mediated NF-κB activation and reducing luciferase activity to levels seen on PDL. Kinase-dead c-Src may function either by preventing c-Src from phosphorylating a downstream substrate or by preventing autophosphorylation leading to a conformational change required for recruitment of other signaling moieties necessary for NF-κB activation. The c-Src double mutant K295R/Y527F maintains an activated conformation allowing recruitment of these moieties but prevents c-Src-mediated phosphorylation of other substrates. Overexpression of the double mutant also completely eliminated NF-κB activation (Fig. 5B), thus activation of the enzyme was not able to overcome the loss of its k