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Stromal Cell-derived Factor-1α Induces Tube-like Structure Formation of Endothelial Cells through Phosphoinositide 3-Kinase

2002; Elsevier BV; Volume: 278; Issue: 1 Linguagem: Inglês

10.1074/jbc.m204771200

1083-351X

Shigeru Kanda, Yasushi Mochizuki, Hiroshi Kanetake,

Glycosylation and Glycoproteins Research

Stromal cell-derived factor-1α (SDF-1α) is a CXC chemokine, which induces tube formation of endothelial cells. Although SDF-1α transduces signals via CXC receptor 4 (CXCR4), resulting in activating a panel of downstream signaling molecules, such as phosphoinositide 3-kinase (PI3-kinase), little is known about the SDF-1α-mediated signaling pathways leading to tube formation. Here we examined the signal transduction pathway involved in SDF-1α-mediated tube formation by primary human umbilical endothelial cells and murine brain capillary endothelial cell line (IBE (immortalized murine brain capillary endothelial) cells). SDF-1α stimulated tube formation by IBE cells, which was blocked by LY294002 and pertussis toxin, suggesting that PI3-kinase and Giprotein were involved in this process. SDF-1 also stimulated tube formation of human umbilical endothelial cells, and the response was LY294002-sensitive. SDF-1α activated PI3-kinase in IBE cells. In stable IBE cell lines expressing either the mutant p85 subunit of PI3-kinase (denoted Δp85-8 cells), which lacks association with the p110 subunit, or kinase-inactive c-Fes (denoted KEFes 5-15 cells), SDF-1α failed to activate PI3-kinase and to stimulate tube formation. SDF-1α-induced tube formation was inhibited by an antibody against murine vascular endothelial cadherin. The antibody as well as LY294002 attenuated SDF-1α-mediated compact cell-cell contact, which proceeded to tube formation. Taken together, SDF-1α induces compact cell-cell contact through PI3-kinase, resulting in tube formation of endothelial cells. Stromal cell-derived factor-1α (SDF-1α) is a CXC chemokine, which induces tube formation of endothelial cells. Although SDF-1α transduces signals via CXC receptor 4 (CXCR4), resulting in activating a panel of downstream signaling molecules, such as phosphoinositide 3-kinase (PI3-kinase), little is known about the SDF-1α-mediated signaling pathways leading to tube formation. Here we examined the signal transduction pathway involved in SDF-1α-mediated tube formation by primary human umbilical endothelial cells and murine brain capillary endothelial cell line (IBE (immortalized murine brain capillary endothelial) cells). SDF-1α stimulated tube formation by IBE cells, which was blocked by LY294002 and pertussis toxin, suggesting that PI3-kinase and Giprotein were involved in this process. SDF-1 also stimulated tube formation of human umbilical endothelial cells, and the response was LY294002-sensitive. SDF-1α activated PI3-kinase in IBE cells. In stable IBE cell lines expressing either the mutant p85 subunit of PI3-kinase (denoted Δp85-8 cells), which lacks association with the p110 subunit, or kinase-inactive c-Fes (denoted KEFes 5-15 cells), SDF-1α failed to activate PI3-kinase and to stimulate tube formation. SDF-1α-induced tube formation was inhibited by an antibody against murine vascular endothelial cadherin. The antibody as well as LY294002 attenuated SDF-1α-mediated compact cell-cell contact, which proceeded to tube formation. Taken together, SDF-1α induces compact cell-cell contact through PI3-kinase, resulting in tube formation of endothelial cells. stromal cell-derived factor-1-α angiopoietin 2 human umbilical vein endothelial cells immortalized murine brain capillary endothelial phosphoinositide 3-kinase vascular endothelial cadherin fibroblast growth factor 2 mitogen-activated protein kinase CXC chemokines are involved in angiogenesis (1Strieter R.M. Polverini P.J. Kunkel S.L. Arenberg D.A. Burdick M.D. Kasper J. Dzuiba J. Van Damme J. Walz A. Marriott D. Chan S.-Y. Roczniak S. Shanafelt B. J. Biol. Chem. 1995; 270: 27348-27357Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar, 2Moore B.B. Arenberg D.A. Addison C.L. Keane M.P. Strieter M.R. J. Lab. Clin. Med. 1998; 132: 97-103Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 3Horuk R. Cytokine Growth Factor Rev. 2001; 12: 313-335Crossref PubMed Scopus (352) Google Scholar, 4Dias S. Choy M. Rafii S. Cancer Invest. 2001; 19: 732-738Crossref PubMed Scopus (20) Google Scholar). They are divided into two groups, angiogenic stimulators, which share a consensus Glu-Leu-Arg-motif preceding the first cysteine residue, and inhibitors that lack this motif in their sequence. Among angiogenic CXC chemokines, stromal cell-derived factor-1-α (SDF-1α)1 plays pivotal roles in inflamed immune responses and angiogenesis. Targeted disruption of a gene encoding SDF-1α in mouse resulted in impaired hematopoiesis and lymphopoiesis (5Nagasawa t. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (2020) Google Scholar). SDF-1α transduces signals via its receptor CXCR4. CXCR4 knockout in mice demonstrated impaired vasculogenesis in intestine, suggesting that signals via CXCR4 may contribute to vasculogenesis and angiogenesis. In fact, CXCR4 is expressed in vascular endothelial cells (6Gupta S.K. Lysko P.G. Pillarisetti K. Ohlstein E. Stadel J.M. J. Biol. Chem. 1998; 273: 4282-4287Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 7Volin M.V. Joseph L. Shockley M.S. Davies P.F. Biochem. Biophys. Res. Commun. 1998; 242: 46-53Crossref PubMed Scopus (120) Google Scholar), and its expression is up-regulated by angiogenic growth factors, fibroblast growth factor-2, and vascular endothelial growth factor (8Feil C. Augustin H.G. Biochem. Biophys. Res. Commun. 1998; 247: 38-45Crossref PubMed Scopus (161) Google Scholar, 9Salcedo R. Wasserman K. Young H.A. Grimm M.C. Howard O.M. Anver M.R. Kleinman H.K. Murphy W.J. Oppenheim J.J. Am. J. Pathol. 1999; 154: 1125-1135Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). Furthermore, treatment of endothelial cells by SDF-1α induces tube-like structure formation and migration (10Mirshahi F. Pourtau J. Li H. Muraine M. Trochon V. legrand E. Vannier J.-P. Soria J. Vasse M. Soria C. Thromb. Res. 2000; 99: 587-594Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 11Molino M. Woolkails M.J. Prevost N. Pratico D. Barnathan E.S. Taraboletti G. Haggarty B.S. Hesselgesser J. Horuk R. Hoxie J.A. Brass L.F. Biochim. Biophys. Acta. 2000; 1500: 227-240Crossref PubMed Scopus (51) Google Scholar). Activation of CXCR4 in lymphatic cells resulted in activation of Gi protein, the Ras/mitogen-activated protein kinase (MAPK) pathway, phosphoinositide 3-kinase (PI3-kinase), focal adhesion kinase, SHP2, c-Fyn, c-Lyn, and Pyk2 (12Davis C.B. Dikic I. Unutmaz D. Hill C.M. Arthos J. Siani M. Thompson D.A. Schlessinger J. Littman D.R. J. Exp. Med. 1997; 186: 1793-1798Crossref PubMed Scopus (344) Google Scholar, 13Chen W.J. Jayawickreme C. Watson C. Wolfe L. Holmes W. Ferris R. Armour S. Dallas W. Chen G. Boone L. Luther M. Kenakin T. Mol. Pharmacol. 1998; 53: 177-181Crossref PubMed Scopus (21) Google Scholar, 14Ganju R.K. Brubaker S.A. meyer J. Dutt P. Yang Y. Qin S. Newman W. Groopman J.E. J. Biol. Chem. 1998; 273: 23169-23175Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 15Chemock R.D. Cherla R.P. Ganju R.K. Blood. 2001; 97: 608-615Crossref PubMed Scopus (72) Google Scholar, 16Weber K.S.C. Ostermann G. Zernecke A. Schroder A. Klickstein L.B. Weber C. Mol. Biol. Cell. 2001; 12: 3074-3086Crossref PubMed Scopus (43) Google Scholar). SDF-1α activated PI3-kinase and SHP2 have been implicated in migration of lymphocytes and leukemia cells (14Ganju R.K. Brubaker S.A. meyer J. Dutt P. Yang Y. Qin S. Newman W. Groopman J.E. J. Biol. Chem. 1998; 273: 23169-23175Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 15Chemock R.D. Cherla R.P. Ganju R.K. Blood. 2001; 97: 608-615Crossref PubMed Scopus (72) Google Scholar). However, little is known about the roles of these signaling molecules in SDF-1α-mediated angiogenic responses of endothelial cells to date. Previously, we have established a murine brain capillary endothelial cell line from ts-A58-H-2Kb transgenic mice, denoted IBE (Immortomouse Brain Endothelial) cells. IBE cells can form lumen-containing tube-like structures in response to fibroblast growth factor 2 (FGF-2) and angiopoietin 2 (Ang2) treatment (17Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth & Differ. 1996; 7: 383-395PubMed Google Scholar, 18Rahmanian M. Pertoft H. Kanda S. Christofferson R. Claesson-Welsh L. Heldin P. Exp. Cell Res. 1997; 237: 223-230Crossref PubMed Scopus (65) Google Scholar, 19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar). Using this culture model, we have shown previously that expression of dominant negative c-Fes caused impaired chemotaxis toward FGF-2 and Ang2 (19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar, 20Kanda S. Lerner E.C. Tsuda S. Shono T. Kanetake H. Smithgall T.E. J. Biol. Chem. 2000; 275: 10105-10111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). It has also been shown that Ang2-induced PI3-kinase activation depended on c-Fes (19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar). Furthermore, expression of dominant negative c-Fyn inhibited FGF-2- and Ang2-mediated tube formation (19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar, 21Tsuda S. Ohtsuru A. Yamashita S. Kanetake H. Kanda S. Biochem. Biophys. Res. Commun. 2002; 290: 1354-1360Crossref PubMed Scopus (40) Google Scholar). In the present study, we examined the signal transduction pathways leading to SDF-1α-mediated tube-like structure formation of IBE cells and human umbilical cord vein endothelial cells (HUVECs). SDF-1α stimulated tube formation of both cells, and PI3-kinase inhibitor LY294002 blocked tube formation, suggesting that PI3-kinase might be involved in this process by these cells. PI3-kinase was activated by SDF-1α-treatment in IBE cells, and the activation was dependent on c-Fes kinase activity. These results suggest that PI3-kinase is an important signaling molecule of SDF-1α-induced tube-like structure formation of endothelial cells. Anti-phosphotyrosine (PY99) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rat monoclonal antibody against mouse vascular endothelial cadherin (VE-cadherin) was from Pharmingen. Mouse recombinant SDF-1α was obtained from R&D Systems (Minneapolis, MN). LY294002, PP2, PD98059, and pertussis toxin were from Calbiochem-Novabiochem and were dissolved in dimethyl sulfoxide (Me2SO) as a stock solution, except pertussis toxin, and stored at −30 °C until use. Stock solutions were further diluted with Me2SO and dissolved in culture medium. Final concentration of Me2SO was 0.1% in all cases. Pertussis toxin was suspended in Tris-buffered saline and was kept at 4 °C until use. HUVECs and their culture medium were purchased from BioWhittaker, Inc. (Walkersville, MD) and cultured in endothelial cell basal medium supplemented with endothelial cell growth supplement, dexamethasone, fetal bovine serum, insulin, and epidermal growth factor as described in the protocol provided by the manufacturer. Parental IBE cells obtained from temperature-sensitive mutant SV40 large T transgenic mouse brain capillaries were cultured in Ham's F-12 medium containing fetal bovine serum, endothelial cell growth supplement, insulin, interferon-γ, and epidermal growth factor as has been described previously (17Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth & Differ. 1996; 7: 383-395PubMed Google Scholar). Stable IBE cell lines expressing kinase-inactive (dominant negative) c-Fes (denoted KEFes 5-15 cells) were described elsewhere (20Kanda S. Lerner E.C. Tsuda S. Shono T. Kanetake H. Smithgall T.E. J. Biol. Chem. 2000; 275: 10105-10111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). A stable cell line expressing deleted mutant p85 PI3-kinase subunit, which does not interact with p110 catalytic subunit (22Kotani K. Yonezawa K. Hara K. Ueda H. Kitamura Y. Sakaue H. Ando A. Chavanieu A. Calas B. Grigorescu F. Nishiyama M. Waterfield M.D. Kasuga M. EMBO J. 1994; 13: 2313-2321Crossref PubMed Scopus (328) Google Scholar) (denoted Δp85-8 cells), was established, which demonstrated the dominant negative effect on epidermal growth factor-mediated PI3-kinase activation (23Mochizuki Y. Tsuda S. Kanetake H. Kanda S. Oncogene. 2002; 21: 7027-7033Crossref PubMed Scopus (17) Google Scholar). Experiments using IBE cell lines were performed at 33 °C rather than at 39 °C because at the latter temperature, cells became senescent and lost responsiveness to extracellular stimuli (17Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth & Differ. 1996; 7: 383-395PubMed Google Scholar). For IBE cells, cells were cultured between two layers of type I collagen gels in Ham's F-12 medium containing 0.25% bovine serum albumin with or without indicated samples as described previously (17Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth & Differ. 1996; 7: 383-395PubMed Google Scholar). For HUVECs, cells suspended in endothelial cell basal medium containing 0.5% fetal bovine serum were inoculated onto growth factor-reduced Matrigel® (BD Biosciences) with or without indicated samples and cultured for 24 h as described previously (24Dawson D.W. Pearce S.F. Zhong R. Silverstein R.L. Frazier W.A. Bouck N.P. J. Cell Biol. 1997; 138: 707-717Crossref PubMed Scopus (552) Google Scholar). To quantify the length of newly formed tubes, three random phase-contrast photomicrographs (×10 objectives) per well were taken, and the length of each tube was measured using NIH Image software (version 1.64). Tube length obtained from FGF-2-stimulated cells or SDF-1α-stimulated cells was set to 100 as described in the respective figure legends. The method used for determination of PI3-kinase activity in immunoprecipitates of anti-phosphotyrosine was described previously (25Kanda S. Hodgkin M.N. Woodfield R.J. Wakelam M.J.O. Thomas G. Claesson-Welsh L. J. Biol. Chem. 1997; 272: 23347-23353Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In brief, serum-starved cells were lysed in Nonidet P-40 lysis buffer and incubated with anti-phosphotyrosine antibody followed by absorption with protein A-agarose beads. After extensive washing, immunoprecipitates were incubated with phosphatidylinositol and [γ-32P]ATP, and reaction products were separated by thin layer chromatography on silica gel 60 plates. Incorporation of [γ-32P]ATP into phosphatidylinositol was measured by Image Analyzer BAS 5000 (Fuji) followed by exposure on x-ray films (Amersham Biosciences). Serum- and growth factor-starved IBE cells were either stimulated or left unstimulated with 500 ng/ml SDF-1α in the presence of orthovanadate (50 μm) for 10 min. c-Akt was immunoprecipitated with anti-Akt antibody followed by immunoblotting with either anti-phospho-Akt or anti-Akt antibodies. SDF-1α induces tube-like structure formation of primary human endothelial cells (10Mirshahi F. Pourtau J. Li H. Muraine M. Trochon V. legrand E. Vannier J.-P. Soria J. Vasse M. Soria C. Thromb. Res. 2000; 99: 587-594Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 11Molino M. Woolkails M.J. Prevost N. Pratico D. Barnathan E.S. Taraboletti G. Haggarty B.S. Hesselgesser J. Horuk R. Hoxie J.A. Brass L.F. Biochim. Biophys. Acta. 2000; 1500: 227-240Crossref PubMed Scopus (51) Google Scholar). To examine whether SDF-1α also stimulates tube formation of IBE cells, we tested the effect of SDF-1α on morphological changes of the cells. IBE cells respond to FGF-2 and angiopoietin 2 (Ang2) to form lumen-containing tube-like structures (18Rahmanian M. Pertoft H. Kanda S. Christofferson R. Claesson-Welsh L. Heldin P. Exp. Cell Res. 1997; 237: 223-230Crossref PubMed Scopus (65) Google Scholar, 19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar). As shown in Fig. 1 A, FGF-2 induced multicellular aggregates of IBE cells followed by sprouting and fusion of aggregates, resulting in branching tube-like structures (26Kanda S. Tomasini-Johansson B. Rubin K. Claesson-Welsh L. Exp. Cell Res. 1999; 248: 203-213Crossref PubMed Scopus (54) Google Scholar). SDF-1α induced the formation of the tube-like structures of IBE cells, which was similar to the effect of FGF-2. CXCR4 is a G protein-coupled receptor. We then examined the effect of pertussis toxin on SDF-1α-induced tube formation. As shown in Fig. 1 B, pertussis toxin at 50 ng/ml blocked SDF-1α-induced tube formation, suggesting that Gi protein was involved in this process. We then tested the effects of pharmacological inhibitors on SDF-1α-induced tube formation. Neither Src family inhibitor PP2 nor mitogen-activated protein kinase/extracellular signal regulated kinase kinase inhibitor PD98059 inhibited SDF-1α-induced tube formation of IBE cells (data not shown). Treatment of cells with PI3-kinase inhibitor LY294002 blocked SDF-1α-induced tube formation (Fig. 1 C). PI3-kinase inhibitor could not block FGF-2- and Ang2-mediated tube formation (19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar, 25Kanda S. Hodgkin M.N. Woodfield R.J. Wakelam M.J.O. Thomas G. Claesson-Welsh L. J. Biol. Chem. 1997; 272: 23347-23353Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). FGF-2- and Ang2-mediated tube formation was dependent on c-Fyn kinase activity (19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar, 21Tsuda S. Ohtsuru A. Yamashita S. Kanetake H. Kanda S. Biochem. Biophys. Res. Commun. 2002; 290: 1354-1360Crossref PubMed Scopus (40) Google Scholar). Ang2 could not further stimulate FGF-2-induced tube formation (19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar), suggesting that Ang2 and FGF-2 utilized a common signaling pathway leading to tube formation, such as c-Fyn. To test whether SDF-1α-induced tube formation requires different signaling pathways activated by FGF-2 and Ang2, we examined the additive effect of SDF-1α on FGF-2- and Ang2-induced tube formation. As shown in Fig. 1 D, SDF-1α further stimulated FGF-2- and Ang2-induced tube formation. These results suggest that SDF-1α-induced tube formation of IBE cells may be dependent on PI3-kinase, which is not involved in FGF-2- and Ang2-mediated tube formation. We also examined the formation of tube-like structures by primary HUVECs on Matrigel. Fig. 2 A shows that FGF-2 as well as SDF-1α induced the formation of tube-like structures. In addition, LY294002 markedly inhibited SDF-1α-mediated formation of tube-like structures of HUVECs (Fig. 2 B). LY294002 showed little effect on FGF-2-induced formation of tube-like structures of HUVECs. 2S. Kanda, unpublished observations. Considered collectively, PI3-kinase may be involved commonly in SDF-1α-mediated tube formation by endothelial cells from different origins.Figure 2As shown in A, SDF-1α-induces tube-like structure formation of HUVECs. HUVECs were cultured on to the surface of Matrigel in the presence or absence of indicated growth factors. Tube length obtained from FGF-2-stimulated cells was set to 100. As shown in B, the SDF-1α-induced tube-like structure formation of HUVECs is LY294002-sensitive. HUVECs were seeded onto the surface of Matrigel in the presence of either 0.1% Me2SO (DMSO) or 10 μm LY294002 and cultured for 24 h. Tube length obtained from SDF-1α-stimulated cells was set to 100. Data shown are reproduced from two experiments.Bar, 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next examined the effect of SDF-1α on PI3-kinase activity. IBE cells were treated with either Me2SO (vehicle) or LY294002 for 30 min and then stimulated with SDF-1α. Cells were lysed, and PI3-kinase activity in anti-phosphotyrosine immunoprecipitates was examined by thin layer chromatography. As shown in Fig. 3 A, PI3-kinase activity in immunoprecipitates of anti-phosphotyrosine antibody from IBE cells was increased by SDF-1α treatment, suggesting that SDF-1α induced tyrosine phosphorylation of particular proteins followed by association with PI3-kinase. SDF-1α also increased the PI3-kinase activity in anti-phosphotyrosine immunoprecipitates of HUVECs. c-Fes tyrosine kinase is exclusively expressed in endothelial cells and hematopoietic cells. Activation of c-Fes by extracellular stimuli requires oligomerization by its coiled-coil domain followed by autophosphorylation (27Smithgall T.E. Rogers J.A. Peters K.L., Li, J. Briggs S.D. Lionberger J.M. Cheng H. Shibata A. Scholtz B. Schreiner S. Dunham N. Crit. Rev. Oncog. 1998; 9: 43-62Crossref PubMed Scopus (61) Google Scholar). c-Fes tyrosine kinase is expressed in both HUVECs and IBE cells (20Kanda S. Lerner E.C. Tsuda S. Shono T. Kanetake H. Smithgall T.E. J. Biol. Chem. 2000; 275: 10105-10111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In a recent study, we showed that Ang2-mediated activation of PI3-kinase was dependent on c-Fes (19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar). We then examined the effect of SDF-1α on PI3-kinase in KEFes 5-15 cells. As shown in Fig. 3 B, SDF-1α could not elevate the PI3-kinase activity in anti-phosphotyrosine immunoprecipitates of KEFes 5-15 cells. This result suggests that SDF-1α–mediated tyrosine phosphorylation of particular proteins, which subsequently associate with PI3-kinase, may be c-Fes-dependent. SDF-1α also failed to increase the PI3-kinase activity in Δp85-8 cells, suggesting that the association of the p110 catalytic subunit of PI3-kinase with tyrosine phosphorylated proteins was required in this reaction. c-Akt/protein kinase B is one of the important downstream targets of PI3-kinase and is involved in cell survival. We examined the activation of c-Akt by SDF-1α. As shown in Fig. 3 C, phosphorylation of c-Akt was increased by SDF-1α-treatment in IBE cells, and the increase was not observed in KEFes 5-15 cells. Considered collectively, these data suggest that SDF-1α utilized c-Fes, resulting in activation of PI3-kinase and c-Akt in IBE cells. We also examined the SDF-1α-mediated tube formation in KEFes 5-15 and Δp85-8 cells. As shown in Fig. 4, SDF-1α failed to stimulate tube formation in KEFes 5-15 cells as well as Δp85-8 cells. These results strongly suggest that SDF-1α-mediated tube-like structure formation by endothelial cells requires PI3-kinase activation, possibly through c-Fes.Figure 4SDF-1α cannot induce tube-like structure formation by KEFes 5-15 cells (A) or Δp85-8 cells (B).Indicated IBE cell lines were treated or untreated with 500 ng/ml of SDF-1α or FGF-2 (as a positive control) and cultured between two layers of collagen gels. Tube length obtained from FGF-2-stimulated cells was set to 100. Bar, 100 μm. Data shown are representative of two experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although distinct methods are employed to assess the ability of cultured endothelial cells to form tube-like structures, common behavior involved in this process includes cell-cell contact. Endothelial cells contact each other to form cellular aggregates followed by lumen formation between aggregated cells (26Kanda S. Tomasini-Johansson B. Rubin K. Claesson-Welsh L. Exp. Cell Res. 1999; 248: 203-213Crossref PubMed Scopus (54) Google Scholar, 28Montesano R. Orci L. Vassalli P. J. Cell Biol. 1983; 97: 1648-1652Crossref PubMed Scopus (513) Google Scholar, 29Ingber D.E. Folkman J. J. Cell Biol. 1989; 109: 317-330Crossref PubMed Scopus (723) Google Scholar, 30Matsumoto T. Turesson I. Book M. Gerwins P. Claesson-Welsh L. J. Cell Biol. 2002; 156: 149-160Crossref PubMed Scopus (177) Google Scholar). We then examined the effect of SDF-1α on endothelial cell-cell contact. When treated with SDF-1α, IBE cells adhered to neighboring cells and formed compact aggregation at 8 h, which is characterized by the disappearance of borders between aggregated cells (Fig. 5 A). This tight cell-cell contact, denoted cell compaction, is observed in cadherin-dependent cell-cell aggregation (31Vestweber, D., and Kemler, R. EMBO J. 4,3393–3398Google Scholar, 32Matsubara, S., and Ozawa, M. J. Cell Biol. 154, 573–584Google Scholar). Tight cell-cell contact was not observed in untreated IBE cells (Fig. 5 A). As shown in Fig. 5 B, cell compaction was inhibited by anti-VE-cadherin antibody, suggesting that VE-cadherin was required for cell compaction. Consequently, anti-mouse VE-cadherin antibody inhibited SDF-1α-induced tube formation (Fig. 5 C), suggesting that VE-cadherin-mediated cell-cell contact may be involved in this process. As shown in Fig. 6, compaction was hardly observed in SDF-1α–treated IBE cells in the presence of LY294002. Only loose cell-cell contact was observed in SDF-1α–treated KEFes 5-15 cells and in Δp85-8 cells as well. These results suggest that SDF-1α-induced tube formation may be regulated by PI3-kinase-dependent tight cell-cell contact. Considered collectively, PI3-kinase and its downstream target molecules, such as c-Akt, seem to be required for SDF-1α-induced tube formation. In fact, recent reports have demonstrated that c-Akt was involved in tube formation of endothelial cells (33Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar, 34Kureishi Y. Luo Z. Shiojima I. Bialik A. Fulton D. Lefer D.J. Sessa W.C. Walsh K. Nat. Med. 2000; 6: 1004-1010Crossref PubMed Scopus (1332) Google Scholar, 35Lee M.J. Thangada S. Paik J.H. Sapkota G.P. Ancellin N. Chae S.S., Wu, M. Morales-Ruiz M. Sessa W.C. Alessi D.R. Hla T. Mol. Cell. 2001; 8: 693-704Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Although FGF-2 induced cell compaction of IBE cells as well, PI3-kinase inhibitor did not inhibit the cell compaction (data not shown). FGF-2 sufficiently promoted tube formation by KEFes 5-15 cells and Δp85-8 cells. FGF-2 activates PI3-kinase through activated Ras but not through binding to tyrosine phosphorylated proteins (23Mochizuki Y. Tsuda S. Kanetake H. Kanda S. Oncogene. 2002; 21: 7027-7033Crossref PubMed Scopus (17) Google Scholar). Ang2 activated PI3-kinase through binding to tyrosine phosphorylated proteins, and the activation was dependent on c-Fes kinase activity (19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar). Ang2-induced PI3-kinase activity was involved in chemotaxis of IBE cells. Treatment of cells with LY294002 did not inhibit Ang2-induced tube formation (19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar), suggesting that PI3-kinase does not seem to be involved in this response. On the other hand, FGF-2 and Ang2 activated c-Fyn in IBE cells (19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar, 21Tsuda S. Ohtsuru A. Yamashita S. Kanetake H. Kanda S. Biochem. Biophys. Res. Commun. 2002; 290: 1354-1360Crossref PubMed Scopus (40) Google Scholar). Treatment of cells with PP2 or expression of kinase-inactive c-Fyn inhibited FGF-2- or Ang2-promoted tube formation of IBE cells, suggesting that signals through receptor tyrosine kinases leading to tube formation seem to require c-Fyn but not PI3-kinase (19Mochizuki Y. Nakamura T. Kanetake H. Kanda S. J. Cell Sci. 2002; 115: 175-183Crossref PubMed Google Scholar, 21Tsuda S. Ohtsuru A. Yamashita S. Kanetake H. Kanda S. Biochem. Biophys. Res. Commun. 2002; 290: 1354-1360Crossref PubMed Scopus (40) Google Scholar). A previous study has shown that c-Fyn was activated by SDF-1α treatment in Jurkat T cells (15Chemock R.D. Cherla R.P. Ganju R.K. Blood. 2001; 97: 608-615Crossref PubMed Scopus (72) Google Scholar). However, we could not detect SDF-1α-induced c-Fyn activation in IBE cells (data not shown). Additionally, PP2 failed to inhibit SDF-1α-induced tube formation. Since induction of chemotaxis involves dissociation of cells, Ang2-mediated PI3-kinase activation would not be involved in cell compaction. Conversely, SDF-1α-induced PI3-kinase activation was required for cell compaction. A number of protein kinases are activated by lipid products of PI3-kinase (36Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1323) Google Scholar, 37Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (876) Google Scholar). Depending on the culture condition (i.e. two dimensional culture for migration assay and three-dimensional culture for tube formation), qualitatively different signaling molecules may be accumulated into focal adhesion complexes or cell-cell contacts, where cross-talk between growth factor signaling and cell adhesion signaling exist. Therefore, PI3-kinase activated by Ang2 or SDF-1α may regulate distinct sets of downstream signaling molecules. In conclusion, we have shown in the present study that SDF-1α induced tube formation of endothelial cells through c-Fes-dependent activation of PI3-kinase, and formation of cadherin-dependent tight cell-cell contact seemed to be involved in this process.Figure 6Treatment of cells with LY294002 or cells expressing kinase-inactive c-Fes and mutant p85 subunit of PI3-kinase reduces tight cell-cell contact mediated by 500 ng/ml SDF-1α. Parental IBE cells were incubated with 0.1% Me2SO (DMSO) or 10 μm LY294002. One 1 h later, SDF-1α was added, and after 3 h, cells were covered with the second layer of collagen gels. KEFes 5-15 cells or Δp85-8 cells were either stimulated with 500 ng/ml SDF-1α or left unstimulated for 4 h. Cells were covered with collagen gels, and 8 h later, photographs were taken.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We are grateful to T. Shimogama, M. Yoshimoto, and members of the Nagasaki University Radioisotope Center for excellent outstanding help.