PTEN Modulates Vascular Endothelial Growth Factor-Mediated Signaling and Angiogenic Effects
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m110219200
ISSN1083-351X
AutoresJianhua Huang, Christopher D. Kontos,
Tópico(s)Vascular Tumors and Angiosarcomas
ResumoPhosphatidylinositol 3-kinase is activated by vascular endothelial growth factor (VEGF), and many of the angiogenic cellular responses of VEGF are regulated by the lipid products of phosphatidylinositol 3-kinase. The tumor suppressor PTEN has been shown to down-regulate phosphatidylinositol 3-kinase signaling, yet the effects of PTEN on VEGF-mediated signaling and angiogenesis are unknown. Inhibition of endogenous PTEN in cultured endothelial cells by adenovirus-mediated overexpression of a dominant negative PTEN mutant (PTEN-C/S) enhanced VEGF-mediated Akt phosphorylation, and this effect correlated with decreases in caspase-3 cleavage, caspase-3 activity, and DNA degradation after induction of apoptosis with tumor necrosis factor-α. Overexpression of PTEN-C/S also enhanced VEGF-mediated endothelial cell proliferation and migration. In contrast, overexpression of wild-type PTEN inhibited the anti-apoptotic, proliferative, and chemotactic effects of VEGF. Moreover, PTEN-C/S increased the length of vascular sprouts in the rat aortic ring assay and modulated VEGF-mediated tube formation in an in vitroangiogenesis assay, whereas PTEN-wild type inhibited these effects. Taken together, these findings demonstrate that PTEN potently modulates VEGF-mediated signaling and function and that PTEN is a viable target in therapeutic approaches to promote or inhibit angiogenesis. Phosphatidylinositol 3-kinase is activated by vascular endothelial growth factor (VEGF), and many of the angiogenic cellular responses of VEGF are regulated by the lipid products of phosphatidylinositol 3-kinase. The tumor suppressor PTEN has been shown to down-regulate phosphatidylinositol 3-kinase signaling, yet the effects of PTEN on VEGF-mediated signaling and angiogenesis are unknown. Inhibition of endogenous PTEN in cultured endothelial cells by adenovirus-mediated overexpression of a dominant negative PTEN mutant (PTEN-C/S) enhanced VEGF-mediated Akt phosphorylation, and this effect correlated with decreases in caspase-3 cleavage, caspase-3 activity, and DNA degradation after induction of apoptosis with tumor necrosis factor-α. Overexpression of PTEN-C/S also enhanced VEGF-mediated endothelial cell proliferation and migration. In contrast, overexpression of wild-type PTEN inhibited the anti-apoptotic, proliferative, and chemotactic effects of VEGF. Moreover, PTEN-C/S increased the length of vascular sprouts in the rat aortic ring assay and modulated VEGF-mediated tube formation in an in vitroangiogenesis assay, whereas PTEN-wild type inhibited these effects. Taken together, these findings demonstrate that PTEN potently modulates VEGF-mediated signaling and function and that PTEN is a viable target in therapeutic approaches to promote or inhibit angiogenesis. Vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGFvascular endothelial growth factorVEGFRVEGF receptorAdadenovirusPTENphosphatase and tensin homolog on chromosome 10C/Scatalytically inactive PTEN (C124S)EBMendothelial basal mediumEGMendothelial growth mediumeNOSendothelial nitric oxide synthaseERKextracellular signal-regulated kinaseEVempty virusFBSfetal bovine serumHUVEChuman umbilical vein endothelial cellsPI3Kphosphatidylinositol 3-kinaseTNFαtumor necrosis factor-αWTwild typeMAPmitogen-activated proteinDMEMDulbecco's modified Eagle's medium 1The abbreviations used are: VEGFvascular endothelial growth factorVEGFRVEGF receptorAdadenovirusPTENphosphatase and tensin homolog on chromosome 10C/Scatalytically inactive PTEN (C124S)EBMendothelial basal mediumEGMendothelial growth mediumeNOSendothelial nitric oxide synthaseERKextracellular signal-regulated kinaseEVempty virusFBSfetal bovine serumHUVEChuman umbilical vein endothelial cellsPI3Kphosphatidylinositol 3-kinaseTNFαtumor necrosis factor-αWTwild typeMAPmitogen-activated proteinDMEMDulbecco's modified Eagle's medium plays a key role in endothelial cell differentiation (vasculogenesis) and the sprouting of new blood vessels from preexisting ones (angiogenesis). Angiogenesis is critical for normal embryonic vascular development as well as a number of physiological and pathological conditions, including ischemic diseases, inflammation, and cancer. Binding of VEGF to VEGF receptor-2 (VEGFR-2) leads to receptor phosphorylation and subsequent activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C-γ1, Src family tyrosine kinases, and other signaling proteins (1.Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar, 2.Thakker G.D. Hajjar D.P. Muller W.A. Rosengart T.K. J. Biol. Chem. 1999; 274: 10002-10007Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 3.Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 4.Guo D. Jia Q. Song H.-Y. Warren R.S. Donner D.B. J. Biol. Chem. 1995; 270: 6729-6733Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). The key role of PI3K in VEGF-mediated signal transduction and angiogenic responses is well established (2.Thakker G.D. Hajjar D.P. Muller W.A. Rosengart T.K. J. Biol. Chem. 1999; 274: 10002-10007Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 5.Fujio Y. Walsh K. J. Biol. Chem. 1999; 274: 16349-16354Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 6.Dimmeler S. Dernbach E. Zeiher A.M. FEBS Lett. 2000; 477: 258-262Crossref PubMed Scopus (307) Google Scholar). Experimental evidence has shown that activation of PI3K is critical for VEGF-mediated endothelial cell proliferation, survival, and migration. Moreover, downstream activation of Akt by PI3K is responsible for phosphorylation and activation of endothelial nitric oxide synthase (eNOS) by VEGF (6.Dimmeler S. Dernbach E. Zeiher A.M. FEBS Lett. 2000; 477: 258-262Crossref PubMed Scopus (307) Google Scholar,7.Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. 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These 3-phosphoinositides act as potent signaling molecules to regulate many cellular responses that are important for angiogenesis, including cell adhesion, proliferation, vesicular trafficking, protein synthesis, and cellular survival (8.Cantrell D.A. J. Cell Sci. 2001; 114: 1439-1445Crossref PubMed Google Scholar, 9.Jiang B.H. Zheng J.Z. Aoki M. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1749-1753Crossref PubMed Scopus (480) Google Scholar, 10.Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (847) Google Scholar, 11.Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1216) Google Scholar). Recent studies demonstrated that the 3-phosphoinositides are important substrates for PTEN (phosphatase and tensin homology deleted from chromosome 10) both in vitro (12.Maehama T. Dixon J.E. J. Biol. 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PTEN was subsequently found to exhibit both protein tyrosine phosphatase and inositol 3′-phosphatase activity. However, its lipid phosphatase activity appears to be primarily responsible for its tumor suppressor effects, as mutation or loss of PTEN results in increased 3-phosphoinositides and downstream activation of Akt (13.Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2081) Google Scholar,16.Li J. Simpson L. Takahashi M. Miliaresis C. Myers M.P. Tonks N. Parsons R. Cancer Res. 1998; 58: 5667-5672PubMed Google Scholar, 17.Kurose K. Zhou X.P. Araki T. Cannistra S.A. Maher E.R. Eng C. Am. J. Pathol. 2001; 158: 2097-2106Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 18.Davies M.A. Lu Y. Sano T. Fang X. Tang P. LaPushin R. Koul D. Bookstein R. Stokoe D. Yung W.K. Mills G.B. Steck P.A. Cancer Res. 1998; 58: 5285-5290PubMed Google Scholar). Thus, PTEN functions in opposition to PI3K, and numerous studies have shown that overexpression of wild-type PTEN suppresses cell growth and proliferation through G1 cell cycle arrest and enhances apoptosis by down-regulating PI3K/Akt signaling (16.Li J. Simpson L. Takahashi M. Miliaresis C. Myers M.P. Tonks N. Parsons R. Cancer Res. 1998; 58: 5667-5672PubMed Google Scholar,18.Davies M.A. Lu Y. Sano T. Fang X. Tang P. LaPushin R. Koul D. Bookstein R. Stokoe D. Yung W.K. Mills G.B. Steck P.A. Cancer Res. 1998; 58: 5285-5290PubMed Google Scholar, 19.Furnari F.B. Lin H. Huang H.S. Cavenee W.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12479-12484Crossref PubMed Scopus (381) Google Scholar, 20.Weng L.P. Smith W.M. Dahia P.L. Ziebold U. Gil E. Lees J.A. Eng C. Cancer Res. 1999; 59: 5808-5814PubMed Google Scholar, 21.Gao X. Neufeld T.P. Pan D. Dev. Biol. 2000; 221: 404-418Crossref PubMed Scopus (217) Google Scholar, 22.Huang H. 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In this report, we demonstrate that inhibition of endogenous endothelial PTEN by adenovirus-mediated overexpression of a dominant negative PTEN mutant in cultured endothelial cells potently enhances a variety of VEGF-mediated cellular responses, including cell survival, mitogenesis, and migration. In contrast, these effects of VEGF are significantly inhibited by overexpression of wild-type PTEN. Moreover, overexpression of wild-type or dominant negative PTEN modulated endothelial tube formation in vitro and vascular sprouting in an ex vivo model of angiogenesis. Anti-PTEN monoclonal antibody (clone A2B1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Akt, anti-phospho-Akt (Ser-473), anti-phospho-p44/42 mitogen-activated protein (MAP) kinase (Thr-202/Tyr-204), and anti-p44/42 MAP kinase polyclonal antibodies were purchased from New England Biolabs (Beverly, MA). Anti-cleaved caspase-3 (D175) was from Cell Signaling Technologies (Beverly, MA). Recombinant human VEGF165 and tumor necrosis factor-α (TNFα) were purchased from R&D Systems (Minneapolis, MN). Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics Corp. (San Diego, CA) and were used between passages 4 and 8. HUVECs were grown on 0.1% gelatin-coated (Sigma) plates in endothelial growth medium (EGM, Clonetics Corp.) in a 37 °C, 5% CO2 incubator. EA.hy926 (25.Edgell C.-J.S. McDonald C.C. Graham J.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3734-3737Crossref PubMed Scopus (1344) Google Scholar) and Py-4-1 (26.Dubois N.A. Kolpack L.C. Wang R. Azizkhan R.G. Bautch V.L. Exp. Cell Res. 1991; 196: 302-313Crossref PubMed Scopus (66) Google Scholar) endothelial cells were gifts from Dr. Cora-Jean Edgell and Dr. Victoria Bautch, respectively (University of North Carolina, Chapel Hill, NC). EC-RF24 cells were provided by Dr. Hans Pannekoek (University of Amsterdam, The Netherlands) (27.Fontijn R. Hop C. Brinkman H.J. Slater R. Westerveld A. van Mourik J.A. Pannekoek H. Exp. Cell Res. 1995; 216: 199-207Crossref PubMed Scopus (91) Google Scholar). NIH 3T3 cells and human embryonic kidney 293 cells were from American Type Culture Collection. 3T3 cells expressing fms-Tie2 have been described (28.Kontos C.D. Stauffer T. Yang W.-P. York J.D. Huang L. Blanar M.A. Meyer T. Peters K.G. Mol. Cell. Biol. 1998; 18: 4131-4140Crossref PubMed Scopus (182) Google Scholar). cDNAs encoding wild-type (WT) and catalytically inactive PTEN, in which cysteine 124 has been mutated to serine (C/S), were generously provided by Dr. Charles Sawyers (University of California, Los Angeles). To generate adenoviruses directing the expression of these proteins, cDNAs encoding PTEN were subcloned into pShuttle-CMV then recombined with pAdEasy-1 by electroporation into BJ5183 Escherichia coli (Stratagene, La Jolla, CA) (29.He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3221) Google Scholar). The recombinant adenoviral vector DNA was transfected into human embryonic kidney 293 cells with LipofectAMINE (Invitrogen), then the viruses were serially amplified in 293 cells, purified on a CsCl density gradient by ultracentrifugation, and titered as described previously (30.Channon K.M. Blazing M.A. Shetty G.A. Potts K.E. George S.E. Cardiovasc. Res. 1996; 32: 962-972Crossref PubMed Scopus (84) Google Scholar). A control adenovirus consisting of the identical adenovirus backbone without a cDNA insert (“empty virus,” AdEV) was provided by Dr. Walter J. Koch (Duke University Medical Center, Durham, NC) (31.Shah A.S. White D.C. Emani S. Kypson A.P. Lilly R.E. Wilson K. Glower D.D. Lefkowitz R.J. Koch W.J. Circulation. 2001; 103: 1311-1316Crossref PubMed Scopus (171) Google Scholar). For most experiments, HUVECs were grown in EGM-MV (containing 5% FBS, Clonetics Corp.). When the cells were nearly confluent, the medium was changed to EGM containing 2% FBS, and viruses were added to the medium at a dilution of 1:1000 (multiplicity of infection ∼100). The cells were incubated for 16 h at 37 °C, then the medium was changed to serum-free endothelial basal medium (EBM, Clonetics Corp.), and the cells were treated as indicated. Cells were lysed in Triton lysis buffer (137 mm NaCl, 2 mm EDTA, 10% glycerol, 1% Triton X-100, 20 mm Tris-HCl, pH 8.0) containing protease inhibitors (1 mm sodium orthovanadate, 1 mmphenylmethylsulfonyl fluoride, 1 mm NaF, 1 μg/ml leupeptin, 1 μg/ml pepstatin). An aliquot of each lysate was separated by PAGE and Western-blotted with the indicated antibodies. Two assays of programmed cell death were used. To assay caspase-3 activity, HUVECs were plated in triplicate at 3 × 105 cells/well of a 6-well plate and grown overnight in EGM-MV containing 5% FBS. The following day the medium was changed to EGM containing 2% FBS, and the cells were either left uninfected or were infected with AdPTEN-WT, AdPTEN-C/S, or AdEV overnight. The next day apoptosis was induced by serum starvation in EBM and treatment with TNFα (50 ng/ml). Half of the cells were treated with VEGF (50 ng/ml), and half were left untreated, and the cells were incubated at 37 °C for 3 h. Cells were lysed, and an aliquot of each cell lysate was used in a fluorimetric assay of caspase-3 activity (EnzChek caspase-3 assay kit, Molecular Probes, E-13138) according to the manufacturer's instructions. To confirm the effects of wild-type and inactive PTEN on caspase-3 activity, apoptosis was also evaluated using the cell death detection ELISA-Plus kit (Roche Molecular Biochemicals, 1774425). HUVECs were plated in triplicate wells of a 24-well plate. The cells were left uninfected or were infected with AdPTEN-WT, AdPTEN-C/S, or AdEV overnight, and apoptosis was induced as described above in VEGF-treated and -untreated cells. Cells were lysed, and cytoplasmic histone-associated DNA fragmentation (mono- and oligonucleosomes) was detected by spectrophotometry according to the manufacturer's instructions. The migration of HUVECs was determined using a “scratch” wound assay as described previously (6.Dimmeler S. Dernbach E. Zeiher A.M. FEBS Lett. 2000; 477: 258-262Crossref PubMed Scopus (307) Google Scholar, 32.Tamura M. Gu J. Matsumoto K. Aota S. Parsons R. Yamada K.M. Science. 1998; 280: 1614-1617Crossref PubMed Scopus (1068) Google Scholar). Briefly, HUVECs were grown in triplicate in 60-mm dishes, and when confluent, they were left uninfected or were infected with AdPTEN-WT, AdPTEN-C/S, or AdEV for 16 h. The cell monolayer was scraped with a sterile rubber policeman to create a cell-free zone, then washed once with medium and treated with or without VEGF (20 ng/ml) in EGM containing 2% FBS. HUVEC migration was quantified by measuring the width of the cell-free zone (distance between the edges of the injured monolayer) at 4 distinct positions 24 h after treatment on an Olympus IX-70 inverted microscope connected to a Diagnostic Instruments Spot RT Color Camera, and data were analyzed with NIH Image, v.1.6.2. To evaluate the effect of PTEN on VEGF-mediated DNA synthesis, [3H]thymidine incorporation was assayed as described previously (33.Huang L. Sankar S. Lin C. Kontos C.D. Schroff A.D. Cha E.H. Feng S.-M. Li S.-F. Yu Z. Van Etten R.L. Blanar M.A. Peters K.G. J. Biol. Chem. 1999; 274: 38183-38188Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Briefly, uninfected, AdPTEN-WT-, AdPTEN-C/S-, or AdEV-infected HUVECs were plated at 25,000 cells/well of a 24-well plate, quiesced by incubation in EBM for 24 h, then treated with or without VEGF (20 ng/ml) for 18 h. The cells were pulse-labeled with [3H]thymidine (2 μCi/ml, Amersham Biosciences, Inc.) for 3 h, the DNA was precipitated, and the amount of [3H]thymidine incorporation was determined by liquid scintillation counting. To confirm the effects of PTEN on VEGF-mediated cellular proliferation, cell counts were performed. Uninfected, AdPTEN-WT-, AdPTEN-C/S-, and empty virus-infected HUVECs were plated in triplicate wells of 24-well plates in a volume of 500 μl of EGM-MV and incubated for 24 h at 37 °C. The following day the medium was changed to EBM with or without VEGF (20 ng/ml). After 24 h the medium was replaced with fresh EBM with or without VEGF (20 ng/ml) and incubated for an additional 24 h. After 48 h, cells were trypsinized and resuspended in EGM-MV. Cell numbers in each group were counted on a hemacytometer (Fisher) using an Olympus CK2 inverted microscope. Data were expressed as the means ± S.E. The endothelial tube formation assay, which has been described previously for the evaluation of angiogenesis in vitro (34.Madri J.A. Pratt B.M. J. Histochem. Cytochem. 1986; 34: 85-91Crossref PubMed Scopus (144) Google Scholar, 35.Pollman M.J. Naumovski L. Gibbons G.H. J. Cell. Physiol. 1999; 178: 359-370Crossref PubMed Scopus (91) Google Scholar, 36.Xin X. Yang S. Ingle G. Zlot C. Rangell L. Kowalski J. Schwall R. Ferrara N. Gerritsen M.E. Am. J. Pathol. 2001; 158: 1111-1120Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar), was performed using the in vitro angiogenesis kit (Chemicon, Temecula, CA) according to the manufacturer's instructions. HUVECs were left uninfected or were infected with AdPTEN-WT, AdPTEN-C/S, or AdEV for 16 h, then were trypsinized and resuspended in EGM containing 2% FBS. Wells of a 96-well plate were coated with ECMatrix solution, and 5 × 103 cells were plated in triplicate wells in a volume of 50 μl of EGM containing 2% FBS either with or without VEGF (25 ng/ml). The cells were incubated for 24 h at 37 °C, and tube formation was evaluated by phase-contrast microscopy using an Olympus IX-70 microscope (100× magnification) connected to a Diagnostic Instruments Spot RT Color Camera. To determine the viability of cells in these assays, cells were stained with Hoechst 33342 (5 μg/ml, Sigma) and propidium iodide (2.5 μg/ml, Sigma) for 5 min at 37 °C and analyzed by fluorescence microscopy. This ex vivo angiogenesis assay was performed essentially as described previously (33.Huang L. Sankar S. Lin C. Kontos C.D. Schroff A.D. Cha E.H. Feng S.-M. Li S.-F. Yu Z. Van Etten R.L. Blanar M.A. Peters K.G. J. Biol. Chem. 1999; 274: 38183-38188Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 37.Nicosia R.F. Lin Y.J. Hazelton D. Qian X.H. Am. J. Pathol. 1997; 151: 1379-1386PubMed Google Scholar) with some modifications. Thoracic aortas were excised from 2–3-week-old Sprague-Dawley male rats and immediately placed into cold Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. Clotted blood inside the aorta was flushed with media, and the periadventitial fibroadipose tissue was removed. Aortas were then cut into cross-sectional rings ∼1–1.5 mm in length. Aortic rings were mock-infected or infected with 2 × 1011 viral particles of AdPTEN-WT, AdPTEN-C/S, or AdEV in 1.0 ml of DMEM, 2% FBS, at room temperature for 15 min. Rings were placed into wells of a 24-well plate containing 0.4 ml of cold growth factor-reduced Matrigel (BD Biosciences) then incubated at 37 °C until the Matrigel polymerized. The wells were then overlaid with 0.5 ml of EBM without phenol red, and the rings were maintained at 37 °C for up to 10 days with medium changes every 2 days. Vascular sprouting from each ring was examined daily on an Olympus IX-70 microscope (100× magnification), and digital images were obtained. Quantitative analysis of endothelial sprouting was performed using images from day 5, and sprout length was quantified in NIH Image (v.1.6.2) using a calibrated micrometer. The greatest distance from the aortic ring body to the end of the vascular sprouts was measured at three distinct points per ring and in three different rings per treatment group. All results were expressed as the mean ± S.E. Statistical analysis was performed using the one-tailed Student's t test (two sample, unequal variance), and p < 0.05 was considered statistically significant. We first analyzed whether PTEN is expressed in endothelial cells. Lysates from several different immortalized endothelial cell lines as well as primary HUVECs and NIH 3T3 fibroblasts were analyzed by Western blotting with an antibody against PTEN. PTEN was expressed to varying degrees in both EA.hy926 cells (25.Edgell C.-J.S. McDonald C.C. Graham J.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3734-3737Crossref PubMed Scopus (1344) Google Scholar) and HUVECs, but it was not detectable in EC-RF24 cells (27.Fontijn R. Hop C. Brinkman H.J. Slater R. Westerveld A. van Mourik J.A. Pannekoek H. Exp. Cell Res. 1995; 216: 199-207Crossref PubMed Scopus (91) Google Scholar) (Fig. 1). PTEN was highly expressed in Py-4-1 cells, a murine microvascular endothelial cell line (26.Dubois N.A. Kolpack L.C. Wang R. Azizkhan R.G. Bautch V.L. Exp. Cell Res. 1991; 196: 302-313Crossref PubMed Scopus (66) Google Scholar), as well as in parental NIH 3T3 cells and 3T3 cells stably expressing chimeric fms-Tie1 or fms-Tie2 receptors (28.Kontos C.D. Stauffer T. Yang W.-P. York J.D. Huang L. Blanar M.A. Meyer T. Peters K.G. Mol. Cell. Biol. 1998; 18: 4131-4140Crossref PubMed Scopus (182) Google Scholar). The expression of PTEN in endothelial cells indicates that this protein could potentially modulate VEGF-mediated signaling. VEGF is known to activate the PI3K/Akt pathway (5.Fujio Y. Walsh K. J. Biol. Chem. 1999; 274: 16349-16354Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar); therefore, we investigated whether PTEN overexpression could alter VEGF-mediated Akt phosphorylation. Primary HUVECs were either left uninfected or were infected with recombinant adenoviruses encoding wild-type PTEN (AdPTEN-WT), a catalytically inactive mutant of PTEN in which cysteine 124 has been mutated to serine (AdPTEN-C/S), or an empty adenovirus as a control for viral infection (AdEV). After overnight virus infection, the cells were serum-starved for 6 h and then treated with or without VEGF. Endogenous PTEN was detectable at moderate levels in both uninfected and AdEV-infected HUVECs, whereas both wild-type and inactive PTEN were overexpressed after virus infection (Fig. 2). VEGF treatment increased Akt phosphorylation in uninfected and AdEV-infected cells. Importantly, overexpression of PTEN-WT abrogated this effect, whereas PTEN-C/S enhanced phosphorylation of Akt. Similar amounts of total Akt and tubulin were observed in each lane, demonstrating that the effects on Akt phosphorylation were a result of the PTEN proteins themselves. VEGF treatment also enhanced the phosphorylation of ERK1 and -2, but this was not altered by overexpression of either wild-type or inactive PTEN (Fig. 2), suggesting that VEGF-mediated ERK activation is not dependent on the production of 3-phosphoinositides. Akt is known to play a critical role in cell survival mediated by VEGF (5.Fujio Y. Walsh K. J. Biol. Chem. 1999; 274: 16349-16354Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). To determine whether the effects of PTEN on VEGF-mediated Akt phosphorylation in HUVECs correlated with the effects on apoptosis, we evaluated caspase-3 cleavage in HUVECs induced to undergo apoptosis. Caspase-3 is a key mediator of apoptosis, and cleavage of this enzyme to its active form correlates with the onset of apoptosis. HUVECs were infected with PTEN or control viruses, and the cells were serum-starved and treated with TNFα, which has been shown to induce endothelial cell apoptosis (38.Hermann C. Assmus B. Urbich C. Zeiher A.M. Dimmeler S. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 402-409Crossref PubMed Scopus (193) Google Scholar). In lysates from uninfected cells and those infected with AdPTEN-WT or AdEV, VEGF treatment blocked the TNFα-induced cleavage of caspase-3 (Fig. 3). Overexpression of PTEN-WT increased basal caspase-3 cleavage and appeared to reduce the protective effect of VEGF in these cells. In contrast, essentially no cleaved caspase-3 was detectable in cells expressing PTEN-C/S either with or without VEGF treatment. These findings indicate that overexpression of PTEN-WT enhances endothelial cell apoptosis, whereas inhibition of PTEN is protective. To confirm the effects of PTEN overexpression on endothelial cell apoptosis, we assayed both capase-3 activity and DNA fragmentation. HUVECs were infected with recombinant adenoviruses, and apoptosis was induced by serum starvation and TNFα treatment. Cell lysates were then used in a fluorimetric assay of caspase-3 activity. Consistent with the Western blotting results, VEGF treatment reduced caspase-3 activity in all cells, including those infected with AdPTEN-C/S (Fig. 4A). However, overexpression of PTEN-WT significantly increased, whereas PTEN-C/S significantly decreased caspase-3 activity compared with control cells, both with and without VEGF treatment. Similar results were obtained using a spectrophotometric assay of histone-associated DNA fragmentation, although the effects of PTEN-WT overexpression were not as pronounced (Fig. 4B). Taken together, these findings demonstrate that PTEN modulates the anti-apoptotic effects of VEGF. Although PTEN did not appear to alter VEGF-mediated MAP kinase activation, we evaluated the effects of wild-type and inactive PTEN on VEGF-mediated endothelial cell proliferation. HUVECs were either left uninfected or were infected with AdPTEN-WT, AdPTEN-C/S, or AdEV, then DNA synthesis was assayed by [3H]thymidine incorporation. As expected, VEGF induced an increase in DNA synthesis in both uninfected and empty virus-infected HUVECs (Fig. 5A). Overexpression of PTEN-C/S resulted in a significant increase in VEGF-stimulated DNA synthesis. In contrast, PTEN-WT markedly reduced both basal and VEGF-mediated thymidine incorporation. Notably, overexpression of PTEN-WT in HUVECs led to a marked increase in the number of apoptotic-appearing cells (data not shown), consistent with our earlier results demonstrating that PTEN-WT enhanced endothelial cell apoptosis. To confirm the results of thymidine incorporation assays, we performed cell counts on uninfected HUVECs and on those infected with the PTEN or control adenoviruses. Cells were treated for 48 h with or without VEGF, then trypsinized and counted in triplicate. In all g
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