The Role of Phosphatidylinositol 3-Kinase in Vascular Endothelial Growth Factor Signaling

Vascular endothelial growth factor (VEGF) receptor Flk-1/KDR in endothelial cells is activated during vasculogenesis and angiogenesis upon ligand-receptor interaction. Activated Flk-1/KDR has been shown to recruit Src homology 2 domain-containing signaling molecules that are known to serve as links to the activation of the mitogen-activated protein (MAP) kinase signaling pathway. To define the functional significance of phosphatidylinositol (PI) 3-kinase in VEGF signaling, we have examined its role in human umbilical vein endothelial cell (HUVEC) cycle progression. We show herein that p85, the regulatory subunit of PI 3-kinase, is constitutively associated with Flk-1/KDR. The treatment of HUVECs with VEGF promoted tyrosine autophosphorylation of Flk-1/KDR and also induced phosphorylation of p85. This was followed by an increase in the PI 3-kinase activity, which was sensitive to wortmannin, a potent PI 3-kinase inhibitor. VEGF also induced a striking activation of MAP kinase in a time-dependent manner. Inhibition studies with both a dominant-negative p85 mutant and the PI 3-kinase inhibitor, wortmannin, were employed to show for the first time that VEGF-stimulated PI 3-kinase modulates MAP kinase activation and nuclear events such as transcription from c-fos promoter and entry into the synthesis (S)-phase. Our data demonstrate the importance of PI 3-kinase as a necessary signaling component of VEGF-mediated cell cycle progression. Vascular endothelial growth factor (VEGF) receptor Flk-1/KDR in endothelial cells is activated during vasculogenesis and angiogenesis upon ligand-receptor interaction. Activated Flk-1/KDR has been shown to recruit Src homology 2 domain-containing signaling molecules that are known to serve as links to the activation of the mitogen-activated protein (MAP) kinase signaling pathway. To define the functional significance of phosphatidylinositol (PI) 3-kinase in VEGF signaling, we have examined its role in human umbilical vein endothelial cell (HUVEC) cycle progression. We show herein that p85, the regulatory subunit of PI 3-kinase, is constitutively associated with Flk-1/KDR. The treatment of HUVECs with VEGF promoted tyrosine autophosphorylation of Flk-1/KDR and also induced phosphorylation of p85. This was followed by an increase in the PI 3-kinase activity, which was sensitive to wortmannin, a potent PI 3-kinase inhibitor. VEGF also induced a striking activation of MAP kinase in a time-dependent manner. Inhibition studies with both a dominant-negative p85 mutant and the PI 3-kinase inhibitor, wortmannin, were employed to show for the first time that VEGF-stimulated PI 3-kinase modulates MAP kinase activation and nuclear events such as transcription from c-fos promoter and entry into the synthesis (S)-phase. Our data demonstrate the importance of PI 3-kinase as a necessary signaling component of VEGF-mediated cell cycle progression. vascular endothelial growth factor mitogen-activated protein kinase extracellular-regulated kinase, HUVEC, human umbilical vein endothelial cell phosphoinositide 3-kinase platelet-derived growth factor platelet-derived growth factor receptor serum response element radioimmunoprecipitation assay luciferase cytomegalovirus polyacrylamide gel electrophoresis dominant negative hemagglutinin 5′-bromodeoxyuridine Src homology-2 and -3 myelin basic protein protein kinase C growth factor receptor binding protein Src homology/collagen 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside Vascular endothelial growth factor (VEGF)1 is a secreted glycoprotein specific for endothelial cells (1Leung D.W. Cachianes G. Kuang W.-J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4396) Google Scholar, 2Keck P.J. Hauser S.D. Krivi G. Sanzo K. Warren T. 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Rossant J. Yamaguchi T.P. Gertenstein M. Wu X.-F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3308) Google Scholar). Flk-1/KDR and Flt-1 are the two receptor tyrosine kinases that regulate the actions of VEGF and are expressed in endothelial cells (9de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1875) Google Scholar, 10Terman B.I. Dougher-Vermazen M. Carrion M.E. Dimitrov D. Armellino D.C. Gospodarowicz D. Bohlen P. Biochem. Biophys. Res. Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1387) Google Scholar, 11Oberg C. Waltenberger J. Claesson-Welsh L. Welsh M. Growth Factors. 1994; 10: 115-126Crossref PubMed Scopus (88) Google Scholar, 12Clauss M. Weich H. Breier G. Knies U. Rockl W. Waltenberger J. Risau W. J. Biol. Chem. 1996; 271: 17629-17634Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar, 13Brown L.F. Detmar M. Tognazzi K. Abu-Jawdeh G. Iruela-Arispe M.L. Lab. Invest. 1997; 76: 245-255PubMed Google Scholar), while the related receptor, Flt-4, is found on lymphatic endothelium. The expression pattern of Flt-4 suggests it may play a role during lymphangiogenesis (14Kaipainen A. Korhonen J. Mustonen T. van Hinsbergh V.W.M. Fang G.-H. Dumont D. Breitman M. Alitalo K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3566-3570Crossref PubMed Scopus (1167) Google Scholar). All of the three VEGF receptors belong to the PDGFR-β family of receptor tyrosine kinases (15Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar). Upon activation, these receptors dimerize and/or oligomerize, following which autophosphorylation and transphosphorylation of their tyrosine residues in the intracellular domain occurs. There are four putative tyrosine phosphorylation sites (Tyr-951, Tyr-996, Tyr-1054, and Tyr-1059) in the KDR intracellular domain (16Dougher-Vermazen M. Hulmes J.D. Bohlen P. Terman B.I. Biochem. Biophys. Res. Commun. 1994; 205: 728-738Crossref PubMed Scopus (108) Google Scholar). These phosphorylated tyrosine molecules act as docking sites for adaptor signaling molecules and non-receptor tyrosine kinases, thereby generating a signal cascade that culminates in a cellular response. The signal transduction pathways involved in mediating the various biological functions of VEGF on endothelial cells such as migration, proliferation, differentiation, or survival remain to be completely defined. The Ras-MAP (mitogen-activated protein) kinase pathway is a key component in the transduction of signals leading to growth and transformation. It consists of a linear cascade of protein kinases, Raf, MAP kinase kinase, and MAP kinase, which are also called extracellular-regulated kinases (Erks). Erk-1 and Erk-2 are acutely activated upon growth factor stimulation (17Malarkey K. Belham C.M. Paul A. Graham A. McLees A. Scott P.H. Plevin R. Biochem. J. 1995; 309: 361-375Crossref PubMed Scopus (264) Google Scholar). Phosphatidylinositol (PI) 3-kinase, a heterodimer of an 85-kDa (p85) adaptor subunit and a 100-kDa (p110) catalytic subunit (18Hiles I.D. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz L.F. Thompson A. Totty N.F. Hsuan J.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1992; 70: 419-429Abstract Full Text PDF PubMed Scopus (538) Google Scholar, 19Klippel A. Escobedo J.A. Hu Q. Williams L.T. Mol. Cell. Biol. 1993; 13: 5560-5566Crossref PubMed Scopus (87) Google Scholar, 20Dhand R. Hara K. Hiles I. Bax B. Gout I. Panayotou G. Fry M.J. Yonezawa K. Kasuga M. Waterfield M.D. EMBO J. 1994; 13: 511-521Crossref PubMed Scopus (295) Google Scholar, 21Hu P. Schlessinger J. Mol. Cell. Biol. 1994; 14: 2577-2583Crossref PubMed Scopus (47) Google Scholar), is activated by most growth factors and has been implicated as a critical factor in the control of cell proliferation and cell survival. PI 3-kinase phosphorylates the D-3 position of the inositol ring of phosphoinositides, which in turn act as second messengers. The p85 subunit contains two Src homology 2 (SH2) domains, which bind to tyrosine-phosphorylated receptors after stimulation of cells with growth factors and in this manner recruit p110 into the complex at the cell membrane. The region between the two SH2 domains, the iSH2 region, mediates the association with p110, and this interaction is required for the enzymatic activity of p110 (22Klippel A. Escobeda J.A. Hirano M. Williams L.T. Mol. Cell. Biol. 1994; 14: 2675-2685Crossref PubMed Scopus (127) Google Scholar). Phosphorylation of the p85 subunit of PI 3-kinase upon VEGF stimulation (23Guo D. Jia Q. Song H.Y. Warren R.S. Donner D. J. Biol. Chem. 1995; 270: 6729-6733Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar) is suggestive of a potential role for PI 3-kinase in VEGF-mediated signaling. Given this observation, we hypothesized that PI 3-kinase might play a critical role in VEGF signaling, including the Ras-MAP kinase pathway. In this report, we demonstrate for the first time the functional significance of PI 3-kinase in VEGF signaling from Flk-1/KDR leading to MAP kinase activation, followed by transcriptional activation of the c-Fos serum response element that eventually culminates in endothelial cell proliferation. Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (San Diego, CA) and were passaged in medium M199 (Life Technologies, Inc.) containing 20% fetal bovine serum, 4 mml-glutamine, penicillin, streptomycin, and 2 ng/ml basic fibroblast growth factor on 80-mm diameter dishes coated with 0.2% gelatin (Sigma). HUVECs were not used after the sixth passage. For experimental purposes, HUVECs were plated on 0.2% gelatin-coated dishes and allowed to form a monolayer. Cultures were serum-starved with unsupplemented medium M199 containing 0.5% fetal bovine serum for 16–18 h. Serum-starved cultures were left untreated or treated with VEGF 165 at the indicated concentrations and time in 5.0 ml of medium M199 at 37 °C and harvested in appropriate lysis buffer at 4 °C. Anti-Flk-1, Flt-1, and Erk-2 polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-phospho-Erk-1/2 polyclonal antibodies were from New England Biolabs, and anti-p85 and anti-phosphotyrosine antibodies (4G10) were from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-hemagglutinin (HA) antibody (12C5A), β-galactosidase and luciferase assay kits, and cell proliferation assay kits containing BrdUrd and anti-BrdUrd antibody were purchased from Boehringer Mannheim. Wortmannin was purchased from Sigma, and PD98059 was bought from Calbiochem. PI 3-kinase substrate, phosphatidylinositol (sodium salt), was bought from Avanti Polar Lipids, Inc. (Alabaster, AL). The ECL reagent was obtained from Amersham Pharmacia Biotech. VEGF 165 was procured from R PubMed Scopus Google Scholar). Serum-starved HUVECs were treated with VEGF for antibody was used for at a of 4 of PI 3-kinase assay was in kinase assay buffer and containing phosphatidylinositol and of for min at °C. were by the addition of of The were with of The was by and using a were with and in lysis buffer and of was in to the were with antibodies that phosphorylation of For serum-starved cells were with the indicated concentrations of wortmannin or PD98059 for min prior to VEGF For of antibody was used for The of cell used in the was for β-galactosidase activity to for Erk-2 complex were by of kinase assay buffer containing of and of bovine myelin basic protein The kinase complex was in and by as an HUVECs with and plasmids were with wortmannin at the in the and with Cultures were harvested in lysis buffer and for luciferase and β-galactosidase HUVECs were plated on gelatin-coated in at a of allowed to for and serum-starved for h. of in serum-free medium M199 with ng/ml VEGF containing 5′-bromodeoxyuridine cells were and with anti-BrdUrd antibody and For DN cells with and DN p85 or DN Ras were serum-starved for and in medium M199 containing BrdUrd and ng/ml VEGF for h. were and with followed by anti-BrdUrd as The of cells that BrdUrd was using a was by are expressed as of the In to the response to VEGF in a we to the response in HUVECs cell antibodies not KDR from we these cells with a cDNA the of in HUVECs from 20% to and of HUVECs not their We demonstrate the of Flk-1/KDR with p85 from VEGF and human endothelial cells following with anti-p85 antibodies In to these the antibodies not p85 We from these that p85 and constitutively with Flk-1/KDR. p85 has been shown to with Flt-1 in a S.A. J. Biol. Chem. 1995; 270: PubMed Scopus Google Scholar). We for Flt-1 association with p85 and Flk-1/KDR in the by with Flt-1 specific experimental we not Flt-1 in p85 or Flk-1 not We that which has kinase activity, was tyrosine-phosphorylated as a of VEGF treatment 2 We found in addition to two with of and were also phosphorylation of the p85 subunit of PI 3-kinase in response to VEGF stimulation was by in assay following with a specific anti-p85 antibody 2 We to p85 phosphorylation in response to VEGF treatment was to regulate the catalytic of the PI 3-kinase We found VEGF treatment in increase in PI 3-kinase activity examined by an in vitro kinase assay using phosphatidylinositol as a and This increase in PI 3-kinase activity be endothelial cells were with wortmannin and a that is a potent of PI 3-kinase Wortmannin also the PI 3-kinase at MAP kinase a role in signals for growth from most growth factor receptor tyrosine We at the of MAP kinase activation in VEGF-stimulated endothelial cells and found a time-dependent which was at min not To the role of PI 3-kinase in MAP kinase activation by cells were with concentrations of wortmannin prior to VEGF wortmannin in a manner activation 4 that PI 3-kinase is involved in MAP kinase activation by of MAP kinase activation was with wortmannin to PI 3-kinase The MAP kinase kinase PD98059, was used a control 4 The was and with Erk-2 antibodies to of protein 4 To that the PI 3-kinase pathway was for MAP kinase in response to cells were with Erk-2 and concentrations of a DN form of p85 This DN p85 the in the iSH2 domain and not bind p110, the catalytic subunit of PI 3-kinase P. P.H. A. D. P. Waterfield M.D. A. Downward J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of p85 with Erk-2 was necessary to the of dominant negative in the stimulation with VEGF an activation of the of a DN p85 mutant activation in a manner. of and DN p85 at a Erk-2 activation regulate transcription from the c-Fos serum response element by the complex factors and which control of R. EMBO J. 1995; 14: PubMed Scopus Google Scholar). To PI 3-kinase was for the nuclear events following VEGF HUVECs were with a containing the promoter element to the luciferase treatment with VEGF in a increase in transcription from the Wortmannin at a of the transcriptional activity by wortmannin not the transcriptional activity These that PI 3-kinase activation following VEGF stimulation the transcription of c-Fos from and To the biological of PI 3-kinase signaling events were to BrdUrd as a of VEGF of the serum HUVECs into the as by with anti-BrdUrd Wortmannin BrdUrd by at and at of wortmannin were by studies of cell cycle with DN p85. DN p85 in a manner entry of the cells into DN Ras was used as DN p85 and DN Ras to cells into the of the cell cycle in containing 20% serum not To of the signal transduction pathways from VEGF receptor, we have the functional importance of PI 3-kinase in VEGF signaling from Flk-1/KDR. It has been shown that p85, the adaptor subunit of PI 3-kinase, is of with the VEGF receptor (23Guo D. Jia Q. 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