Thymidine Phosphorylase and 2-Deoxyribose Stimulate Human Endothelial Cell Migration by Specific Activation of the Integrins α5β1 and αVβ3
2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês
10.1074/jbc.m212670200
ISSN1083-351X
AutoresKylie A. Hotchkiss, Anthony W. Ashton, Edward L. Schwartz,
Tópico(s)HER2/EGFR in Cancer Research
ResumoThymidine phosphorylase is an angiogenic factor that is frequently overexpressed in solid tumors, in rheumatoid arthritis, and in response to inflammatory cytokines. Our previous studies showed that cells expressing thymidine phosphorylase stimulated endothelial cell migration in vitro. This was a consequence of the intracellular metabolism of thymidine by thymidine phosphorylase and subsequent extracellular release of 2-deoxyribose. The mechanisms by which 2-deoxyribose might mediate thymidine phosphorylase-induced cell migration in vitro, however, are obscure. Here we show that both thymidine phosphorylase and 2-deoxyribose stimulated the formation of focal adhesions and the tyrosine 397 phosphorylation of focal adhesion kinase in human umbilical vein endothelial cells. Although similar actions occurred upon treatment with the angiogenic factor vascular endothelial growth factor (VEGF), thymidine phosphorylase differed from VEGF in that its effect on endothelial cell migration was blocked by antibodies to either integrin α5β1 or αvβ3, whereas VEGF-induced endothelial cell migration was only blocked by the αvβ3 antibody. Further, thymidine phosphorylase and 2-deoxyribose, but not VEGF, increased the association of both focal adhesion kinase and the focal adhesion-associated protein vinculin with integrin α5β1 and, in intact cells, increased the co-localization of focal adhesion kinase with α5β1. Thymidine phosphorylase and 2-deoxyribose-induced focal adhesion kinase phosphorylation was blocked by the antibodies to α5β1 and αvβ3, directly linking the migration and signaling components of thymidine phosphorylase and 2-deoxyribose action. Cell surface expression of α5β1 was also increased by thymidine phosphorylase and 2-deoxyribose. These experiments are the first to demonstrate a direct effect of thymidine phosphorylase and 2-deoxyribose on signaling pathways associated with endothelial cell migration. Thymidine phosphorylase is an angiogenic factor that is frequently overexpressed in solid tumors, in rheumatoid arthritis, and in response to inflammatory cytokines. Our previous studies showed that cells expressing thymidine phosphorylase stimulated endothelial cell migration in vitro. This was a consequence of the intracellular metabolism of thymidine by thymidine phosphorylase and subsequent extracellular release of 2-deoxyribose. The mechanisms by which 2-deoxyribose might mediate thymidine phosphorylase-induced cell migration in vitro, however, are obscure. Here we show that both thymidine phosphorylase and 2-deoxyribose stimulated the formation of focal adhesions and the tyrosine 397 phosphorylation of focal adhesion kinase in human umbilical vein endothelial cells. Although similar actions occurred upon treatment with the angiogenic factor vascular endothelial growth factor (VEGF), thymidine phosphorylase differed from VEGF in that its effect on endothelial cell migration was blocked by antibodies to either integrin α5β1 or αvβ3, whereas VEGF-induced endothelial cell migration was only blocked by the αvβ3 antibody. Further, thymidine phosphorylase and 2-deoxyribose, but not VEGF, increased the association of both focal adhesion kinase and the focal adhesion-associated protein vinculin with integrin α5β1 and, in intact cells, increased the co-localization of focal adhesion kinase with α5β1. Thymidine phosphorylase and 2-deoxyribose-induced focal adhesion kinase phosphorylation was blocked by the antibodies to α5β1 and αvβ3, directly linking the migration and signaling components of thymidine phosphorylase and 2-deoxyribose action. Cell surface expression of α5β1 was also increased by thymidine phosphorylase and 2-deoxyribose. These experiments are the first to demonstrate a direct effect of thymidine phosphorylase and 2-deoxyribose on signaling pathways associated with endothelial cell migration. Angiogenesis, the process of generating new capillaries from preexisting blood vessels, is a fundamental, tightly regulated process in reproduction and wound healing (1Folkman J. Singh Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar). It is well established that the process of angiogenesis is essential for tumorigenesis and the growth of both primary and metastatic tumors (1Folkman J. Singh Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar). Endothelial cell (EC) 1The abbreviations used are: EC, endothelial cell(s); 2dR, 2-deoxyribose; FAK, focal adhesion kinase; HUVEC, human umbilical vein endothelial cell(s); TP, thymidine phosphorylase; VEGF, vascular endothelial growth factor; ECM, extracellular matrix; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate. migration is a key component of the angiogenic process, accompanied by and coordinated with EC proliferation and redifferentiation (2Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7282) Google Scholar). EC migration is regulated by factors that are produced and secreted by tumor cells and tumor-infiltrating stromal cells and is further dependent on the interaction of the endothelial cells with the extracellular matrix (ECM) (3Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3320) Google Scholar). The integrin family protein heterodimers are the major cellular receptors for ECM and mediate cell-matrix interactions that activate multiple signaling pathways important for regulating endothelial cell responses, including cell attachment, adhesion, migration, survival, proliferation, and angiogenesis (4Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3869) Google Scholar). The engagement of the integrins with their extracellular ligands triggers the formation of focal adhesions, large, stable structures that are sites of adhesion and through which signals are transduced (5Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5273) Google Scholar, 6Parsons J.T. Martin K.H. Slack J.K. Taylor J.M. Weed S.A. Oncogene. 2000; 19: 5606-5613Crossref PubMed Scopus (571) Google Scholar). The integrin-matrix interaction is critical to the angiogenic process, and its disruption forms the basis for several experimental therapeutic approaches to the treatment of cancer (7Kerbel R. Folkman J. Nat. Rev. 2002; 2: 727-739Crossref Scopus (1433) Google Scholar). Integrins function as both outside-in and inside-out mediators of cell signaling due to their ability to regulate the activities of cytoplasmic kinases, growth factor receptors, and ion channels (reviewed in Refs. 4Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3869) Google Scholar, 8Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1672) Google Scholar, and 9Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9139) Google Scholar). The cytoplasmic tails of integrins lack enzymatic activity; therefore, they transduce signals by stimulating the association of adapter proteins that link cytoplasmic and transmembrane kinases with the cytoskeleton. Interactions with other cellular signaling pathways have also been described. The multiple complex and overlapping integrin-ECM interactions are a consequence of the 20 or more different α and β subunits that dimerize in many different combinations and are differentially expressed on various cell types (9Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9139) Google Scholar). Different components of the ECM also serve as substrates for single or in some cases multiple integrins. Expression of specific integrins can be up-regulated by cytokines and angiogenic factors, thereby altering the nature of the interaction of the cell with the ECM. Integrins also appear to be necessary for the optimal activation of growth factor and angiogenic factor receptors, including receptors for VEGF, insulin, fibroblast growth factor, tumor necrosis factor-α, and platelet-derived growth factor (4Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3869) Google Scholar, 10Friedlander M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1227) Google Scholar, 11Soldi R. Mitola S. Strasly M. Defilippi P. Tarone G. Bussolino F. EMBO J. 1999; 18: 882-892Crossref PubMed Scopus (539) Google Scholar). As such, cross-talk between integrins and growth factor receptors amplifies the angiogenic response on extracellular matrices that engage those integrins. Upon binding to ECM, integrins directly activate cell signaling pathways (4Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3869) Google Scholar, 8Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1672) Google Scholar, 12Hanks S.K. Polte R. BioEssays. 1997; 19: 137-145Crossref PubMed Scopus (442) Google Scholar). Focal adhesion kinase (FAK) is a 125-kDa nonreceptor protein-tyrosine kinase that is recruited to focal adhesions by integrin engagement with ECM (6Parsons J.T. Martin K.H. Slack J.K. Taylor J.M. Weed S.A. Oncogene. 2000; 19: 5606-5613Crossref PubMed Scopus (571) Google Scholar, 13Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Crossref PubMed Scopus (1296) Google Scholar). Concurrent with these events, FAK is tyrosine-phosphorylated, and thereby its own kinase activity is increased (14Calalb M.B. Polte T.R. Hanks S.K. Mol. Cell Biol. 1995; 15: 954-963Crossref PubMed Google Scholar). This activation is an early event associated with the assembly of focal adhesions and is accompanied by the recruitment of other cytoplasmic and cytoskeletal proteins to the focal adhesions, including talin, paxillin, vinculin, and ultimately actin (6Parsons J.T. Martin K.H. Slack J.K. Taylor J.M. Weed S.A. Oncogene. 2000; 19: 5606-5613Crossref PubMed Scopus (571) Google Scholar). It is these interactions that place FAK in a central role in mediating cell attachment and migration. The angiogenic factor platelet-derived endothelial cell growth factor is identical to human thymidine phosphorylase (TP), an enzyme that catalyzes the reversible conversion of thymidine to thymine and 2-deoxyribose-1-phosphate; the latter is subsequently converted to 2-deoxyribose (2dR) (15Furukawa T. Yoshimura A. Sumizawa T. Haraguchi M. Akiyama S-I. Fukui K. Ishizawa M. Yamada Y. Nature. 1992; 356: 668Crossref PubMed Scopus (413) Google Scholar, 16Moghaddam A. Bicknell R. Biochemistry. 1992; 31: 12141-12146Crossref PubMed Scopus (210) Google Scholar, 17Sumizawa T. Furukawa T. Haraguchi M. Yoshimura A. Takeysu A. Ishizawa M. Yamada Y. Akiyama S-I. J. Biochem. (Tokyo). 1993; 114: 9-14Crossref PubMed Scopus (167) Google Scholar, 18Finnis C. Dodsworth N. Pollitt C.E. Carr G. Sleep D. Eur. J. Biochem. 1993; 212: 201-210Crossref PubMed Scopus (71) Google Scholar). TP is chemotactic for endothelial cells and has angiogenic activity in several in vivo assays, although it did not directly stimulate endothelial cell proliferation (16Moghaddam A. Bicknell R. Biochemistry. 1992; 31: 12141-12146Crossref PubMed Scopus (210) Google Scholar, 17Sumizawa T. Furukawa T. Haraguchi M. Yoshimura A. Takeysu A. Ishizawa M. Yamada Y. Akiyama S-I. J. Biochem. (Tokyo). 1993; 114: 9-14Crossref PubMed Scopus (167) Google Scholar, 18Finnis C. Dodsworth N. Pollitt C.E. Carr G. Sleep D. Eur. J. Biochem. 1993; 212: 201-210Crossref PubMed Scopus (71) Google Scholar, 19Haraguchi M. Miyadera K. Uemura K. Sumizawa T. Furukawa T. Yamada K. Akiyama S-I. Nature. 1994; 368: 198-199Crossref PubMed Scopus (389) Google Scholar, 20Ishikawa F. Miyazono K. Hellman U. Drexler H. Wernstedt C. Usuki K. Takaku F. Risau W. Heldin C-H. Nature. 1989; 338: 557-562Crossref PubMed Scopus (713) Google Scholar). Studies have established a role for TP in experimental and clinical tumor angiogenesis, including transfection studies in which the TP gene increased the vascularization and growth of tumors growing in nude mice (20Ishikawa F. Miyazono K. Hellman U. Drexler H. Wernstedt C. Usuki K. Takaku F. Risau W. Heldin C-H. Nature. 1989; 338: 557-562Crossref PubMed Scopus (713) Google Scholar, 21Moghaddam A. Zhang H-T. Fan T-P.D. Hu D-E. Lees V.C. Turley H. Fox S.B. Gatter K.C. Harris A.L. Bicknell R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 998-1002Crossref PubMed Scopus (419) Google Scholar), and immunohistochemical studies of primary human solid tumors, in which TP was often found to be elevated in the tumors when compared with the corresponding nonneoplastic regions of the same organs (22Luccioni C. Beaumatin J. Bardot V. Lefrancois D. Int. J. Cancer. 1994; 58: 517-522Crossref PubMed Scopus (59) Google Scholar, 23Takabayashi Y. Akiyama S. Akiba S. Yamada K. Miyadera K. Sumizawa T. Yamada Y. Murata F. Aikou T. J. Natl. Cancer Inst. 1996; 88: 1110-1117Crossref PubMed Scopus (394) Google Scholar, 24Takahasi Y. Bucana C.D. Liu W. Yoneda J. Kitadai Y. Cleary K.R. Ellis L.M. J. Natl. Cancer Inst. 1996; 88: 1146-1151Crossref PubMed Scopus (262) Google Scholar, 25Relf M. LeJeune S. Scott P.A.E. Fox S. Smith K. Leek R. Moghaddam A. Whitehouse R. Bicknell R. Harris A.L. Cancer Res. 1997; 57: 963-969PubMed Google Scholar, 26van Triest B. Pinedo H.M. Blaauwgeers J.L.G. van Diest P.J. Schoenmakers P.S. Voorn D.A. Smid K. Koekman K. Hoitsma H.F.W. Peters G.F. Clin. Cancer Res. 2000; 6: 1063-1072PubMed Google Scholar). The mechanism by which TP mediates angiogenesis is unknown; we have used endothelial cell migration as an in vitro model to address this question. No cell surface receptor for TP has been identified; indeed, the protein lacks a signal sequence required for cell secretion (20Ishikawa F. Miyazono K. Hellman U. Drexler H. Wernstedt C. Usuki K. Takaku F. Risau W. Heldin C-H. Nature. 1989; 338: 557-562Crossref PubMed Scopus (713) Google Scholar). Our previous studies of the mechanisms by which tumor cells expressing high levels of TP induce EC migration have demonstrated that it is mediated by the intracellular synthesis and extracellular release of the thymidine metabolite 2dR to form a chemotactic gradient; the effects of 2dR on EC were identical to those of TP (27Hotchkiss K.A. Ashton A.W. Klein R.S. Lenzi M.L. Zhu G.H. Schwartz E.L. Cancer Res. 2003; 63: 527-533PubMed Google Scholar). That the angiogenic actions of TP are mediated by a diffusible metabolite clearly distinguishes it from other angiogenic factors (e.g. VEGF), which work, as do most cytokines, by specific binding to cell surface receptors. That TP and 2dR can stimulate endothelial cell chemotaxis indicates that they must engage, in an unknown manner, intracellular signaling pathways that lead to migration. The aim of the present study was to determine the effect of TP/2dR on focal adhesion formation, to define the role of extracellular matrix proteins and their cognate integrin receptors in TP/2dR-mediated migration, and, in the absence of a known receptor for TP/2dR, to identify specific signaling pathways engaged by TP/2dR to mediate endothelial cell migration and focal adhesion formation. This report documents these events and also provides evidence that there are both similarities and differences in aspects of the activation of cell surface integrins and focal adhesion formation in EC between TP and VEGF. Materials—A human TP cDNA (kindly provided by C.-H. Heldin) was used to prepare recombinant TP. VEGF was from R&D Systems, and 2dR was from Sigma. Antibodies used for neutralization, immunoprecipitation, and Western blotting were all commercially available and obtained from the indicated sources. All other reagents were obtained from Sigma unless otherwise stated. Isolation of Endothelial Cells from Human Umbilical Veins—Human venous endothelial cells (HUVEC) were isolated from umbilical cords as previously described (28Ashton A.W. Yokota R. John G. Zhao S. Suadicani S.O. Spray D.C. Ware J.A. J. Biol. Chem. 1999; 274: 35562-35570Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Culture medium consisted of Medium 199 (M199) supplemented with 20% (v/v) newborn calf serum, 5% (v/v) pooled human serum, 2 mm l-glutamine, 5 units/ml penicillin G, 5 μg/ml streptomycin sulfate, 10 units/ml heparin, and 7.5 μg/ml EC growth supplement (Invitrogen). Primary cultures of HUVEC were passaged with 0.05% trypsin, 0.02% EDTA. Confluent HUVEC monolayers (passages 1–4) were used in the experiments described below. Boyden Chamber Assay of Endothelial Cell Migration—Confluent HUVEC monolayers were cultured with media lacking growth supplement for 48 h prior to harvesting with cell dissociation solution (Sigma). Harvested cells were suspended at 106/ml in M199 with 1% serum, and 105 cells were seeded into precoated transwell inserts (8-μm pore; Costar). Inserts containing HUVEC were placed into a 24-well plate containing M199 with 1% serum and incubated for 1 h at 37 °C. HUVEC migration was stimulated by the addition of a purified factor (TP, VEGF, or 2dR) to the lower chamber. After 5 h, HUVEC were stained with 10 μm Cell Tracker Green (Molecular Probes, Inc., Eugene, OR) for 30 min at 37 °C, and the upper membrane of the insert was swabbed to remove nonmigrated cells. Inserts were washed three times with PBS, fixed in 3.7% formaldehyde in PBS for 10 min at room temperature, and mounted on microscope slides. HUVEC migration was quantitated by counting the number of cells in three random fields (×100 total magnification) per insert. The data are expressed as the average number of cells/field. Where indicated, migration of HUVEC on alternate substrates was examined by coating the inserts with 0.2% (w/v) gelatin or fibronectin, thrombospondin, or vitronectin (10 μg/ml). In addition, the involvement of integrins in TP-induced migration was determined through inclusion of monoclonal antibodies. For these experiments, cells were treated for 15 min with antibodies (10 μg/ml) against specific integrin subunits and heterodimers (Chemicon International) prior to plating (along with the antibody) for 1 h in the Boyden chamber. Antibody was also added to the lower wells upon the addition of the chemotactic stimulus. Nonspecific IgG was used as the control in these experiments. Immunoprecipitation and Western Blots—Confluent HUVEC, seeded on fibronectin, were treated with TP (100 ng/ml), VEGF (10 ng/ml), or 2dR (1 μm) for up to 4 h. Monolayers were washed twice in PBS containing 1 mm phenylmethylsulfonyl fluoride and scraped into immunoprecipitation lysis buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1% (v/v) Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin). Cell suspensions were incubated on ice for 30 min and clarified by centrifugation. For immunoprecipitation, protein content was determined, and 300 μg of total protein was incubated overnight at 4 °C with protein G-agarose beads coated with saturating amounts of antibodies to integrins α3, αvβ3, and α5β1 (Chemicon International, CA) or p125FAK (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The resulting immune complexes were recovered after centrifugation by boiling for 10 min in SDS-PAGE loading buffer. For immunoblotting, aliquots of whole cell lysates (30 μg) or isolated immunocomplexes were separated by SDS-PAGE under reducing conditions using 10% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membrane and analyzed by immunoblotting as previously described (28Ashton A.W. Yokota R. John G. Zhao S. Suadicani S.O. Spray D.C. Ware J.A. J. Biol. Chem. 1999; 274: 35562-35570Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) using antibodies against phosphotyrosine, FAK, integrin α5 (Santa Cruz Biotechnology), vinculin (Sigma), integrin β1 (Chemicon), and FAK phosphorylated on various tyrosine residues (including Tyr397, Tyr407, Tyr576, Tyr861, and Tyr925) (Biosource International). Antibodies against α-tubulin and unphosphorylated FAK were used to control for loading when using whole cell lysates, whereas antibodies against integrins β1, β3, and α3 were used to quantitate protein loading for the immunoprecipitation reactions. Immunofluorescent Staining for Focal Adhesion Complexes—HUVEC were seeded on Fn and stimulated with TP (100 ng/ml), 2dR (1 μm), VEGF (10 ng/ml), or media (control) for 4 h. HUVEC were fixed with 4% paraformaldehyde in PBS for 10 min and rendered permeable by incubation with 0.1% Triton X-100 in PBS for 20 min. Cells were washed three times with 1% bovine serum albumin in PBS (PBS-B) and blocked with 3% preimmune goat serum in PBS-B for 90 min at room temperature. For staining focal adhesions, cells were incubated with antibodies against vinculin (Sigma), FAK (Santa Cruz Biotechnology), or integrin α5β1 (Chemicon) for 1 h. Staining was detected with either Cy3-or FITC-conjugated second antibodies, and images were collected with a Bio-Rad MRC 600 krypton/argon laser-scanning confocal microscope on a Nikon Eclipse 200 microscope with a ×60 numerical aperture 1.4 planapo infinity corrected objective. Controls were imaged to ensure that there was no background fluorescence and no cross-talk from the FITC channel to the Cy3 channel. For co-localization analysis, Cy3 and FITC images were overlaid using Photoshop 6.0 imaging software (Adobe Systems). The analysis of focal adhesion size and number was performed using automated image analysis software (Scion Image). Flow Cytometric Analysis of Cell Surface Integrin Expression—Confluent HUVEC monolayers on fibronectin-coated plates were treated with media alone (control), TP (100 ng/ml), VEGF (10 ng/ml), or 2dR (1 μm) for up to 4 h. Cells were washed twice with PBS and harvested using cell dissociation solution. Cells were suspended in PBS-B for 30 min at 4 °C. At the end of the incubation, antibodies to integrin α5β1 or αvβ3 (5 μg/ml; Chemicon) were added. Negative controls were stained with preimmune mouse serum. Cells were incubated with antibodies for 90 min at 4 °C and washed three times with PBS-B before incubation with an FITC-conjugated secondary antibody (1:100 in PBS-B) for 60 min. Analysis of integrin surface expression was performed on a flow cytometer using an argon ion laser (λex 488 nm). Data are expressed as-fold change in mean fluorescence intensity (Gm) of stimulated HUVEC compared with control unstimulated HUVEC. Statistical Analysis—Data were pooled, and statistical analysis was performed using the Mann-Whitney U test. TP and VEGF Have Additive Effects on HUVEC Migration— Induction of migration of EC is one of the cellular effects associated with the neovascularization actions of angiogenic factors. Both VEGF and TP have been previously shown to be chemoattractants for HUVEC, although mechanistic studies have only been extensively reported for VEGF. To assess potential interactions between the two chemotactic agents in inducing endothelial cell migration, HUVEC were stimulated with TP and VEGF, alone and in combination, in increasing concentrations. As seen in Fig. 1, concentrations of 2.5 ng/ml TP and 1 ng/ml VEGF produced only modest (i.e. less than 25% of maximal) induction of migration. When tested together at these concentrations, the extent of migration induced was close to that expected for the addition of the individual agents. A similar additive effect was observed when the two agents were combined at concentrations that individually were approximately half-maximal for induction of migration (10 and 5 ng/ml for TP and VEGF, respectively (Fig. 1)), and the combined effect of the two agents was equivalent to the maximal effect seen with either agent alone (not shown). Increasing the concentration of either TP or VEGF above this point did not further increase the extent of migration. Thus, TP and VEGF appear to be primary mediators of HUVEC migration whose chemotactic effects are additive in nature. The simplest, although not exclusive, explanation for these observations is that TP and VEGF utilize the same or substantially overlapping pathways to induce HUVEC migration. These pathways are not likely to be identical in all aspects, however, as VEGF acts by binding to a family of well defined, high affinity cell surface receptors with which TP most likely does not interact with in a similar manner. TP, 2dR, and VEGF All Induce Focal Adhesion Formation and FAK Phosphorylation in HUVEC in Vitro—Whereas the effects of VEGF on EC are well described, the signaling mechanisms induced by TP/2dR to elicit a migratory response are undocumented. Thus, we examined the signaling pathways used by VEGF to compare them with those of TP/2dR. One of the primary mechanisms used by VEGF to induce migration is the activation of integrins and their respective signaling pathways. We first examined whether TP/2dR could enhance focal adhesion formation in HUVEC. Focal adhesion content of EC monolayers stimulated for 4 h with TP (100 ng/ml), 2dR (1 μm), or VEGF (10 ng/ml) was examined using vinculin immunostaining (Fig. 2). Focal adhesions in HUVEC in media alone (control) were few in number and small in size. Focal adhesion formation was significantly induced by treatment with either TP or 2dR and was equivalent to the induction by VEGF (Fig. 2A). In all cases, the number of focal adhesions/cell was increased 3.5-fold, and the average size of the focal adhesions was increased 2-fold in HUVEC treated with the chemotactic agents (Fig. 2, B and C). Since FAK is important for the formation of focal adhesions by multiple growth factors (6Parsons J.T. Martin K.H. Slack J.K. Taylor J.M. Weed S.A. Oncogene. 2000; 19: 5606-5613Crossref PubMed Scopus (571) Google Scholar), we assessed whether FAK Tyr phosphorylation, as a marker of activation, was elevated in response to TP/2dR. FAK was immunoprecipitated from lysates of HUVEC stimulated with TP (100 ng/ml), VEGF (10 ng/ml), or 2dR (1 μm), and the levels of Tyr phosphorylation were examined by immunoblotting (Fig. 3A). Total Tyr phosphorylation of FAK was stimulated by all factors with similar kinetics. For each stimulus, FAK phosphorylation was increased at 1 h and maximally stimulated at 4 h, and phosphorylation persisted for 24 h (Fig. 3A). FAK can be phosphorylated on multiple Tyr residues, and we next defined which residues were involved using site-specific antibodies. As can be seen in Fig. 3B, TP and 2dR induced significant phosphorylation of residues Tyr397, the autophosphorylation site, and Tyr925. VEGF also induced phosphorylation of these sites. In contrast, TP and 2dR suppressed the phosphorylation of Tyr576 of FAK, which remained unaffected by VEGF stimulation. Thus, TP and 2dR appear to activate FAK to regulate focal adhesion formation and enhance endothelial cell migration. TP-and VEGF-stimulated HUVEC Migration Was Optimal on Fibronectin and Mediated through Two Distinct Integrins— The observed increase in focal adhesion formation and FAK activation by TP and 2dR was strong evidence that these agents activated signaling pathways that regulate cell attachment and migration. Focal adhesions and, in particular, their integrin constituents are the point at which extracellular and intracellular events interface. Integrin heterodimers mediate the attachment of cells to different extracellular matrix components via their extracellular domains, with the attachment to each matrix protein mediated through a specific subset of integrins (29Eliceiri B.P. Cheresh D.A. Curr. Opin. Cell Biol. 2001; 13: 563-568Crossref PubMed Scopus (263) Google Scholar). To begin to explore the nature of the activation of these processes, we next determined the matrix protein(s) on which the response of HUVEC to TP and 2dR was optimal. Transwell inserts were coated with either fibronectin, vitronectin, thrombospondin, or gelatin (10 μg/ml), and HUVEC migration in response to TP, 2dR, and VEGF was examined (Fig. 4). Stimulation of HUVEC migration with TP was optimal on fibronectin, compared with the other matrix proteins examined. Whereas the induction of migration by VEGF was also maximal on fibronectin, in contrast to TP, optimal migration by VEGF was also observed when HUVEC were plated on vitronectin. HUVEC contain a distinct complement of integrin receptors. Of the 18 α and 11 β integrin subunits identified to date, HUVEC express α subunits α2, α3, α5, α6, αV, αL, αM, and αα and β subunits β1, β3, β5, and ββ (9Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9139) Google Scholar, 30Xu Y. Swerlick R.A. Sepp N. Bosse D. Ades E.W. Lawley T.J. J. Invest. Dermatol. 1994; 102: 833-837Abstract Full Text PDF PubMed Google Scholar). Using neutralizing antibodies against individual integrin subunits, chemotaxis in response to TP on fibronectin-coated inserts was found to be dependent upon the integrins α5, αV, β1, and β3 but not integrins α2, α3, and α6 (Fig. 5A). The migration of VEGF was only sensitive to inhibition by αV and β3 antibodies, and in contrast to TP, it was not inhibited by antibodies to α5 or β1 (Fig. 5A). Of the integrins known to mediate attachment to fibronectin, HUVEC potentially contain α5β1, αvβ3, αVβ1, and α3 heterodimers, of which α5β1 and αVβ3 would be predicted to be involved in mediating the chemotactic effects. Using heterodimer-specific antibodies, we confirmed the data obtained using antibodies to individual integrin subunits by observing that the migration of HUVEC in response to TP (100 ng/ml) was inhibited by antibodies to either α5β1 or αVβ3 integrins (Fig. 5B). As expected, chemotaxis in response to 2dR was inhibited by antibodies to the same integrins. In contrast, VEGF-induced migration was only sensitive to the inhibition of αVβ3 (Fig. 5B). TP and 2dR Stimulated Increased Association of Integrins α5β1and αvβ3with FAK and Vinculin—To further substantiate the involvement of t
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