Heterotrimeric Gαq/Gα11 Proteins Function Upstream of Vascular Endothelial Growth Factor (VEGF) Receptor-2 (KDR) Phosphorylation in Vascular Permeability Factor/VEGF Signaling
2003; Elsevier BV; Volume: 278; Issue: 23 Linguagem: Inglês
10.1074/jbc.m209712200
ISSN1083-351X
AutoresHuiyan Zeng, Dezheng Zhao, Suping Yang, Kaustubh Datta, Debabrata Mukhopadhyay,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoVascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor-tyrosine kinases, Flt-1 (VEGF receptor (VEGFR)-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell proliferation and migration, whereas Flt-1 down-modulates KDR-mediated endothelial cell proliferation. Our most recent works show that pertussis toxin-sensitive G proteins and Gβγ subunits are required for Flt-1-mediated down-regulation of human umbilical vein endothelial cell (HUVEC) proliferation and that Gq/11 proteins are required for KDR-mediated RhoA activation and HUVEC migration. In this study, we demonstrate that Gq/11 proteins are also required for VPF/VEGF-stimulated HUVEC proliferation. Our results further indicate that Gq/11 proteins specifically mediate KDR signaling such as intracellular Ca2+ mobilization rather than Flt-1-induced CDC42 activation and that a Gq/11 antisense oligonucleotide completely inhibits MAPK phosphorylation induced by KDR but has no effect on Flt-1-induced MAPK activation. More importantly, we demonstrate that Gq/11 proteins interact with KDR in vivo, and the interaction of Gq/11 proteins with KDR does not require KDR tyrosine phosphorylation. Surprisingly, the Gq/11 antisense oligonucleotide completely inhibits VPF/VEGF-stimulated KDR phosphorylation. Expression of a constitutively active mutant of G11 but not Gq can cause phosphorylation of KDR and MAPK. In addition, a Gβγ minigene, hβARK1(495), inhibits VPF/VEGF-stimulated HUVEC proliferation, MAPK phosphorylation, and intracellular Ca2+ mobilization but has no effect on KDR phosphorylation. Taken together, this study demonstrates that Gq/11 proteins mediate KDR tyrosine phosphorylation and KDR-mediated HUVEC proliferation through interaction with KDR. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor-tyrosine kinases, Flt-1 (VEGF receptor (VEGFR)-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell proliferation and migration, whereas Flt-1 down-modulates KDR-mediated endothelial cell proliferation. Our most recent works show that pertussis toxin-sensitive G proteins and Gβγ subunits are required for Flt-1-mediated down-regulation of human umbilical vein endothelial cell (HUVEC) proliferation and that Gq/11 proteins are required for KDR-mediated RhoA activation and HUVEC migration. In this study, we demonstrate that Gq/11 proteins are also required for VPF/VEGF-stimulated HUVEC proliferation. Our results further indicate that Gq/11 proteins specifically mediate KDR signaling such as intracellular Ca2+ mobilization rather than Flt-1-induced CDC42 activation and that a Gq/11 antisense oligonucleotide completely inhibits MAPK phosphorylation induced by KDR but has no effect on Flt-1-induced MAPK activation. More importantly, we demonstrate that Gq/11 proteins interact with KDR in vivo, and the interaction of Gq/11 proteins with KDR does not require KDR tyrosine phosphorylation. Surprisingly, the Gq/11 antisense oligonucleotide completely inhibits VPF/VEGF-stimulated KDR phosphorylation. Expression of a constitutively active mutant of G11 but not Gq can cause phosphorylation of KDR and MAPK. In addition, a Gβγ minigene, hβARK1(495), inhibits VPF/VEGF-stimulated HUVEC proliferation, MAPK phosphorylation, and intracellular Ca2+ mobilization but has no effect on KDR phosphorylation. Taken together, this study demonstrates that Gq/11 proteins mediate KDR tyrosine phosphorylation and KDR-mediated HUVEC proliferation through interaction with KDR. Pathological angiogenesis is a hallmark of cancer and various ischemic and inflammatory diseases. Many different cytokines and growth factors, such as vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), 1The abbreviations used are: VPF, vascular permeability factor; VEGF, vascular endothelial growth factor; HUVEC, human umbilical vein endothelial cell(s); EC, endothelial cell(s); MAPK, mitogen-activated protein kinase; PLC, phospholipase C; bFGF, basic fibroblast growth factor; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; EGDR, the fusion receptor of EGFR and KDR; EGLT, the fusion receptor of EGFR and Flt-1. basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and transforming growth factor-β, have an angiogenic activity (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7235) Google Scholar, 2Folkman J. D'Amore P.A. Cell. 1996; 87: 1153-1155Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar, 3Folkman J. EXS. 1997; 79: 1-8PubMed Google Scholar). Among these, VPF/VEGF stands out because of its potency and selectivity for vascular endothelium. VPF/VEGF is not only involved in several steps of angiogenesis but is also the only angiogenic factor recognized to date that renders microvessels hyperpermeable to circulating macromolecules (4Dvorak H.F. Prog. Clin. Biol. Res. 1990; 354A: 317-330PubMed Google Scholar, 5Dvorak H.F. Orenstein N.S. Carvalho A.C. Churchill W.H. Dvorak A.M. Galli S.J. Feder J. Bitzer A.M. Rypysc J. Giovinco P. J. Immunol. 1979; 122: 166-174PubMed Google Scholar, 6Dvorak H.F. Senger D.R. Dvorak A.M. Dev. Oncol. 1984; 22 (1984): 96-114Google Scholar, 7Senger D.R. Galli S.J. Dvorak A.M. Perruzzi C.A. Harvey V.S. Dvorak H.F. Science. 1983; 219: 983-985Crossref PubMed Scopus (3428) Google Scholar, 8Senger D.R. Perruzzi C.A. Feder J. Dvorak H.F. Cancer Res. 1986; 46: 5629-5632PubMed Google Scholar). VPF/VEGF extensively reprograms endothelial cell expression of proteases, integrins, and glucose transporters; stimulates endothelial cell migration and division; and protects endothelial cells from apoptosis and senescence (9Leung D.W. Cachianes G. Kuang W.J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4466) Google Scholar, 10Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4867) Google Scholar, 11Dvorak H.F. Nagy J.A. Feng D. Brown L.F. Dvorak A.M. Curr. Top. Microbiol. Immunol. 1999; 237: 97-132Crossref PubMed Scopus (650) Google Scholar, 12Ferrara N. Curr. Top. Microbiol. Immunol. 1999; 237: 1-30Crossref PubMed Scopus (510) Google Scholar). Most VPF/VEGF biological activities are mediated by its interaction with two high affinity receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2, FLK-1 in mice) (13Fong G.H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2224) Google Scholar, 14Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Meller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1764) Google Scholar, 15Quinn T.P. Peters K.G. De Vries C. Ferrara N. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7533-7537Crossref PubMed Scopus (673) Google Scholar, 16Shalaby F. Ho J. Stanford W.L. Fischer K.D. Schuh A.C. Schwartz L. Bernstein A. Rossant J. Cell. 1997; 89: 981-990Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar, 17Terman B. Dougher-Vermazen M. Carrion M. Dimitrov D. Armellino D. Gospodarowicz D. Bohlen P. Biochem. Biophys. Res. Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1405) Google Scholar). A third receptor, neuropilin, which binds to VEGF165 but not VEGF121, has been recognized, but less is known about neuropilin's capacity to initiate endothelial cell signaling (18Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2088) Google Scholar, 19Gagnon M.L. Bielenberg D.R. Gechtman Z. Miao H.Q. Takashima S. Soker S. Klagsbrun M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2573-2578Crossref PubMed Scopus (258) Google Scholar). A large body of work has demonstrated that KDR, not Flt-1, is responsible for VPF/VEGF-stimulated cell proliferation and migration in cultured EC and for microvascular permeability (20Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 21Bernatchez P.N. Soker S. Sirois M.G. J. Biol. Chem. 1999; 274: 31047-31054Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 22Gille H. Kowalski J. Li B. LeCouter J. Moffat B. Zioncheck T.F. Pelletier N. Ferrara N. J. Biol. Chem. 2001; 276: 3222-3230Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar, 23Rahimi N. Dayanir V. Lashkari K. J. Biol. Chem. 2000; 275: 16986-16992Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). However, Flt-1 functions to down-regulate KDR-mediated cultured EC proliferation as shown by two different VPF/VEGF receptor chimeric fusion systems in which the N-terminal domains of KDR and Flt-1 were replaced by the N-terminal domain of either epidermal growth factor receptor or colony-stimulating factor-1 receptor (23Rahimi N. Dayanir V. Lashkari K. J. Biol. Chem. 2000; 275: 16986-16992Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Further studies indicated that the signaling pathway required for Flt-1-mediated down-regulation of EC proliferation involves activation of phosphatidylinositol 3-kinase, small GTPase Rac1, CDC42, and, surprisingly, pertussis toxin-sensitive G proteins (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 25Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The signal transduction pathways mediated by KDR involve KDR phosphorylation (20Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 21Bernatchez P.N. Soker S. Sirois M.G. J. Biol. Chem. 1999; 274: 31047-31054Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 22Gille H. Kowalski J. Li B. LeCouter J. Moffat B. Zioncheck T.F. Pelletier N. Ferrara N. J. Biol. Chem. 2001; 276: 3222-3230Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar, 23Rahimi N. Dayanir V. Lashkari K. J. Biol. Chem. 2000; 275: 16986-16992Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), phospholipase C (PLC) activation (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 26Guo D. Jia Q. Song H. Warren R. Donner D. J. Biol. Chem. 1995; 270: 6729-6733Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 27Petrova T.V. Makinen T. Alitalo K. Exp. Cell Res. 1999; 253: 117-130Crossref PubMed Scopus (241) Google Scholar, 28Takahashi T. Shibuya M. Oncogene. 1997; 14: 2079-2089Crossref PubMed Scopus (273) Google Scholar, 29Takahashi T. Ueno H. Shibuya M. Oncogene. 1999; 18: 2221-2230Crossref PubMed Scopus (482) Google Scholar, 30Wu L.W. Mayo L.D. Dunbar J.D. Kessler K.M. Ozes O.N. Warren R.S. Donner D.B. J. Biol. Chem. 2000; 275: 6059-6062Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), inositol 1,4,5-trisphosphate accumulation (31Brock T.A. Dvorak H.F. Senger D.R. Am. J. Pathol. 1991; 138: 213-221PubMed Google Scholar), intracellular Ca2+ mobilization (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 32Kanno S. Oda N. Abe M. Terai Y. Ito M. Shitara K. Tabayashi K. Shibuya M. Sato Y. Oncogene. 2000; 19: 2138-2146Crossref PubMed Scopus (257) Google Scholar), and protein kinase C and MAPK activation (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 27Petrova T.V. Makinen T. Alitalo K. Exp. Cell Res. 1999; 253: 117-130Crossref PubMed Scopus (241) Google Scholar, 28Takahashi T. Shibuya M. Oncogene. 1997; 14: 2079-2089Crossref PubMed Scopus (273) Google Scholar, 29Takahashi T. Ueno H. Shibuya M. Oncogene. 1999; 18: 2221-2230Crossref PubMed Scopus (482) Google Scholar, 30Wu L.W. Mayo L.D. Dunbar J.D. Kessler K.M. Ozes O.N. Warren R.S. Donner D.B. J. Biol. Chem. 2000; 275: 6059-6062Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Whereas PLC activation is involved in VPF/VEGF-induced HUVEC proliferation and migration, intracellular Ca2+ mobilization and MAPK are required for VPF/VEGF-induced HUVEC proliferation but not migration (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Our recent studies have shown that the Gq/11 family of the heterotrimeric GTP-binding proteins and Gβγ subunits mediate VPF/VEGF-induced HUVEC migration through the small GTPase RhoA (33Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). However, it is not clear whether G protein Gq/11 proteins are involved in VPF/VEGF-stimulated proliferation. In this study, we show for the first time that inhibition of Gq/11 protein expression by a Gq/11-specific antisense oligonucleotide blocked VPF/VEGF-stimulated HUVEC proliferation and activation of signaling molecules in VPF/VEGF-stimulated HUVEC that are mediated by KDR, not by Flt-1. Moreover, Gq/11 proteins are not activated by tyrosine phosphorylation but through physical interaction with KDR. Surprisingly, the interaction of Gq/11 proteins with KDR does not require KDR tyrosine phosphorylation, and the Gq/11-specific antisense oligonucleotide blocks the interaction of Gq/11 with KDR and inhibits KDR phosphorylation. Furthermore, the Gα11 constitutively active mutant, Gα11(G209L), not the Gαq constitutively active mutant, Gαq(G209L), activates phosphorylation of KDR and MAPK. However, expression of the Gβγ minigene, hβARK1(495), inhibits VPF/VEGF-stimulated HUVEC proliferation, MAPK phosphorylation, and intracellular Ca2+ mobilization but has no effect on KDR phosphorylation. Materials—Recombinant VPF/VEGF was obtained from R&D Systems (Minneapolis, MN). The EGM-MV Bullet kit, trypsin-EDTA, and trypsin neutralization solution were obtained from Clonetics (San Diego, CA). Vitrogen 100 was purchased from Collagen Biomaterials (Palo Alto, CA). Rabbit polyclonal antibodies against the KDR C-terminal domain, Gαq/11 and Gαi/o/t/z were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-phosphotyrosine antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The antiphospho-p42/p44 MAPK antibody was obtained from New England Biolabs (Beverly, MA). [3H]thymidine was obtained from PerkinElmer Life Sciences. Fura-2/AM and Pluronic F-127 were obtained from Molecular Probes, Inc. (Eugene, Oregon). Pertussis toxin was obtained from Calbiochem. Cell Culture—Primary human umbilical vein endothelial cells (HUVEC; obtained from Clonetics) were cultured with or without transduced with retroviruses as described (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Only cells from passage 3 or 4 that were ∼80% confluent were used for experiments. Transduction of HUVEC with EGDR- or EGLT-bearing retroviruses was carried out as described previously (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 33Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Overexpression of Proteins in HUVEC—Retrovirus preparation and HUVEC infection with retrovirus were carried out as described (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 33Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Briefly, 293T cells were seeded at a density of 6 × 106 cells/100-mm plate 24 h before transfection. DNA transfection was carried out with the EffecteneTM transfection reagent (Qiagen, Valencia, CA). Two μg of each target gene (pMMP-EGDR, pMMP-LacZ, etc.), 1.5 μg of pMD.MLV gag.pol, and 0.5 μg of pMD.G DNA, encoding the cDNAs of the proteins that are required for virus packaging (kindly provided by Dr. Mulligan), were mixed in 300 μl of EC buffer. 32 μl of enhancer was added to the DNA mixture. After incubation at room temperature for 2 min, 30 μl of Effectene was added to the DNA mixture and incubated at room temperature for 5 min. The DNA mixture was added dropwise to 293T cells. The medium was changed after 16 h. The retrovirus was isolated 48 h after transfection and used immediately for infection or frozen at -70 °C. 24 h before infection, HUVECs were seeded at a density of 0.3 × 106 cells/100-mm plate. 1 ml of retrovirus solution (∼2 × 107 plaque-forming units/ml) and 5 ml of fresh medium were added to cells with 10 μg/ml polybrene. The medium was changed after 16 h, and cells were ready for experiments 48 h after infection. The expression plasmids containing Gαq, Gαq(Q209L), Gα11, and Gα11(209L) cDNAs were obtained from Guthrie cDNA Resource Center, Guthrie Research Institute (Sayre, PA). Transfection of plasmids to HUVEC was carried out as described (34Paik J.H. Chae S. Lee M.J. Thangada S. Hla T. J. Biol. Chem. 2001; 276: 11830-11837Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Synthesis and Transfection of Antisense Oligonucleotides—3′-End FITC-labeled phosphorothioated Gαq/11 antisense oligonucleotide (ODN-Gq/11), 5′-CCATGCGGTTCTCATTGTCTG-3′, and a 3′-end fluorescein isothiocyanate-labeled phosphorothioated random oligonucleotide (ODN-RD), 5′-CCCTTATTTACTACTTTCGC-3′ (35Sanchez-Blazquez P. Garzon J. J. Pharmacol. Exp. Ther. 1998; 285: 820-827PubMed Google Scholar), were synthesized by Genemed Synthesis (Genemed Synthesis, South San Francisco, CA). Proliferation Assays—Assays were carried out as described (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 33Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Briefly, HUVEC (with or without oligonucleotide transfection) were serum-starved (0.1% serum) for 24 h and then stimulated with 10 ng/ml VEGF for 20 h. 1 μCi/ml [3H]thymidine was added to each well, and 4 h later the cells were washed, fixed, and lysed. Data were expressed as -fold activation with the stimulated cells compared with control treated cells. The data shown represent the means with S.D. of triplicate determinations per experimental condition, and the experiments were repeated at least three times. Intracellular Ca2+Release—Serum-starved HUVEC with or without transfection were loaded with Fura-2 AM and stimulated with 10 ng/ml VPF/VEGF. The assay was carried out as described (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 33Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). All experiments were repeated at least three times. CDC42 Activation Assays—The CDC42 activity assay was carried out as described (25Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Briefly, 24-h serum-starved HUVEC with or without oligonucleotide transfection were stimulated with 10 ng/ml VPF/VEGF for 1 min. Cells were washed and lysed. Cell lysates were centrifuged at 14,000 rpm for 3 min. The supernatant was incubated with 50 μl of freshly prepared GST-Pak-CRIB beads at 4 °C for 45 min. The proteins bound to beads were washed and analyzed by SDS-PAGE with antibodies against CDC42. All experiments were repeated at least three times. Immunoprecipitation and Immunoblotting—HUVEC, with or without virus infection or oligonucleotide transfection, were serum-starved for 24 h and stimulated with 10 ng/ml VPF/VEGF or EGF as indicated for various time periods. Stimulation was halted by the addition of ice-cold phosphate-buffered saline, and cells were washed three times with ice-cold phosphate-buffered saline and lysed with cold radioimmune precipitation buffer (20 mm Tris-HCl, pH 7.5, 0.15 m NaCl, 1% Triton X-100, 1 μm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 1 mm EGTA, 1 μg/ml leupeptin, 0.5% aprotinin, 2 μg/ml pepstatin A). Cell lysates were collected after centrifugation at 14,000 × g for 15 min at 4 °C. One mg of lysate protein was incubated with antibodies as indicated for 1 h, and with 50 μl of protein A-conjugated agarose beads at 4 °C for another 1 h. For immunoprecipitation with phosphorylated tyrosine, cellular extracts were incubated with agarose-conjugated antiphosphorylated tyrosine for 2 h. Beads were washed with radioimmune precipitation buffer three times, and immunoprecipitates were resuspended in 2× SDS sample buffer for Western blot analysis. For the experiments with pertussis toxin, this was added 16 h before stimulation. All experiments were repeated at least three times. Requirement of Gq/11Proteins for DNA Synthesis in VPF/VEGF-stimulated HUVEC—In order to examine whether Gq/11 is involved in VPF/VEGF-induced HUVEC DNA synthesis, we utilized a Gq/11-specific oligonucleotide that has been previously shown by us to inhibit VPF/VEGF-induced HUVEC migration (33Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The Gq/11 antisense oligonucleotide specifically blocks expression of both Gαq and Gα11, as shown by Western blot analysis using an antibody that recognizes both Gαq and Gα11 (Fig. 1a, top panel). However, this has no effect on the expression of the Gi/o family of G proteins (Fig. 1a, middle panels). To rule out the possibility that any effect of the Gq/11 antisense on KDR signaling is due to inhibition of KDR expression, equal amounts of cellular extracts from HUVEC with or without transfection of ODN-Gq/11 or ODN-RD were subjected to immunoblot analysis with an antibody against KDR. The data show that ODN-Gq/11 has no effect on KDR expression (Fig. 1a, bottom panel). Then we determined the effect of the Gq/11 antisense oligonucleotide on KDR-mediated DNA synthesis in HUVEC. HUVEC were transfected with fluorescein isothiocyanate-labeled phosphorothioate ODN-Gq/11 with a method that gave out almost 100% transfection yield as described before (36Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 46791-46798Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and subjected to a DNA synthesis assay with 10 ng/ml VPF/VEGF or bFGF stimulation. As shown in Fig. 1b, antisense oligonucleotide ODN-Gq/11 almost completely inhibits VPF/VEGF-stimulated but has no effect on bFGF-stimulated DNA synthesis in HUVEC. However, the control oligonucleotide, OND-RD (36Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 46791-46798Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), has no effect. These data clearly indicate that Gq/11 proteins are required for VPF/VEGF-stimulated DNA synthesis in HUVEC. Gq/11Proteins Are Required for the KDR-mediated, Not Flt-1-mediated, Signaling Pathway in VPF/VEGF-stimulated HUVEC—Next, we examined the effect of ODN-Gq/11 on the activation of signaling molecules mediated by KDR and Flt-1. As shown in Fig. 2a, ODN-Gq/11, not the control ODN-RD, completely inhibits intracellular Ca2+ mobilization that is mediated by KDR but not Flt-1 (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). We have previously shown that Flt-1, but not KDR, can activate CDC42, a member of the Rho family of small GTPases (25Zeng H. Zhao D. Mukhopadhyay D. J. Biol. Chem. 2002; 277: 4003-4009Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Therefore, we examined the effect of ODN-Gq/11 on CDC42 activation. To do this, serumstarved HUVEC that were transfected with ODN-Gq/11 or nontransfected were stimulated with VPF/VEGF for 1 min, and cellular extracts were subjected to a CDC42 activation assay as described under “Experimental Procedures.” The data indicate that ODN-Gq/11 has no effect on CDC42 activation (Fig. 2b). It is known that MAPK phosphorylation is required for VPF/VEGF-stimulated HUVEC proliferation (24Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 27Petrova T.V. Makinen T. Alitalo K. Exp. Cell Res. 1999; 253: 117-130Crossref PubMed Scopus (241) Google Scholar, 28Takahashi T. Shibuya M. Oncogene. 1997; 14: 2079-2089Crossref PubMed Scopus (273) Google Scholar, 29Takahashi T. Ueno H. Shibuya M. Oncogene. 1999; 18: 2221-2230Crossref PubMed Scopus (482) Google Scholar, 30Wu L.W. Mayo L.D. Dunbar J.D. Kessler K.M. Ozes O.N. Warren R.S. Donner D.B. J. Biol. Chem. 2000; 275: 6059-6062Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Therefore, we tested whether Gq/11 proteins are required for VPF/VEGF-stimulated MAPK phosphorylation in HUVEC. Serum-starved HUVEC, which were transfected with either ODN-Gq/11 or ODN-RD or nontransfected, were stimulated with VPF/VEGF for 10 min. Cellular extracts were subjected to Western blot analysis using an antibody specifically against phosphorylated MAPK. Surprisingly, ODN-Gq/11 partially inhibits VPF/VEGF-stimulated MAPK phosphorylation in HUVEC, whereas ODN-RD has no effect (Fig. 3a). Similar results were also obtained when cells were treated with VPF/VEGF for 15 and 30 min (data not shown). Because both KDR and Flt-1 are present in HUVEC, in order to further characterize the partial inhibition of MAPK phosphorylation by the Gq/11 antisense oligonucleotide, we used the recently developed chimeric receptors, EGDR and EGLT, in which the extracellular domains of KDR and Flt-1 were replaced with that of epidermal growth factor receptor. Serum-starved HUVEC with or without transduction of LacZ as a control, EGDR, or EGLT was stimulated with VPF/VEGF or EGF for the indicated time. Cellular extracts were used to determine the levels of phosphorylated MAPK. As shown in Fig. 3b, both EGDR and EGLT can mediate MAPK phosphorylation; however, HUVEC transduced with LacZ do not show any detectable MAPK phosphorylation. Furthermore, we used this receptor chimera system to further confirm whether ODN-Gq/11 inhibits only KDR-mediated and not Flt-1-mediated MAPK phosphorylation. EGDR/HUVEC and EGLT/HUVEC, transfected with either ODN-Gq/11 or pertussis toxin, were stimulated with EGF for 10 min. Cellular extracts were used to determine MAPK phosphorylation. The data show that ODN-Gq/11 completely inhibits EGDR-mediated MAPK phosphorylation but has no effect on MAPK phosphorylation mediated by EGLT (Fig. 3c). Moreover, our data also demonstrate that pertussis toxin completely inhibits Flt-1-mediated MAPK phosphorylation but has no effect on KDR-mediated MAPK phosphorylation (Fig. 3c). These results indicate that Gq/11 proteins are required for KDR-mediated signaling pathways and that pertussis toxin-sensitive Gi/o proteins are required for Flt-1 signaling. VPF/VEGF Stimulates Interaction between KDR and Gq/11but Not Tyrosine Phosphorylation of Gq/11—Recently, it was reported that platelet-derived growth factor (PDGF) receptor stimulation leads to tyrosine phosphorylation of Gi protein (37Alderton F. Rakhit S. Kong K.C. Palmer T. Sambi B. Pyne S. Pyne N.J. J. Biol. Chem. 2001; 276: 28578-28585Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Therefore, we tested whether VPF/VEGF could stimulate Gq/11 phosphorylation. Serum-starved HUVEC were stimulated with VPF/VEGF for 20 s, 40 s, 1 min, and 2 min as indicated. Cellular extracts were immunoprecipitated with an antibody against phosphorylated tyrosine, and the immunoprecipitates were then subjected to immunoblot analysis using antibodies against KDR and Gq/11. The results show that Gq/11 is not phosphorylated (Fig. 4, bottom panel), whereas KDR is phosphorylated at 1 min (Fig. 4, top panel). These results indicate that Gq/11 proteins are not activated by tyrosine phosphorylation. In G protein-coupling receptor signaling pathways, G proteins are activated by its interaction with the G protein-coupling receptor. Therefore, we tested whether Gq/11 proteins interact with KDR in VPF/VEGF-stimulated HUVEC. HUVEC were stimulated with 10 ng/ml VPF/VEGF for 5, 10, and 20 s. Cellular extracts were immunoprecipitated with an antibody against Gαq/11, and the immunoprecipitates were then immunoblotted with an antibody against KDR. The data show that VPF/VEGF stimulates interaction between KDR and Gαq/11 as early as 5 s after treatment and that this interaction is transient (Fig. 5a). Furthermore, when cellular extracts from serum-starved HUVEC transfected with ODN-Gq/11 or ODN-RD were immunoprecipitated with an antibody against Gq/11 and then immunoblotted with an antibody a
Referência(s)