Vascular Permeability Factor (VPF)/Vascular Endothelial Growth Factor (VEGF) Receptor-1 Down-modulates VPF/VEGF Receptor-2-mediated Endothelial Cell Proliferation, but Not Migration, through Phosphatidylinositol 3-Kinase-dependent Pathways
2001; Elsevier BV; Volume: 276; Issue: 29 Linguagem: Inglês
10.1074/jbc.m103213200
ISSN1083-351X
AutoresHuiyan Zeng, Harold F. Dvorak, Debabrata Mukhopadhyay,
Tópico(s)Coronary Interventions and Diagnostics
ResumoVascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) achieves its multiple functions by activating two receptor tyrosine kinases, Flt-1 (VEGF receptor-1) and KDR (VEGF receptor-2), both of which are selectively expressed on primary vascular endothelium. To dissect the respective signaling pathways and biological functions mediated by these receptors in primary endothelial cells with these two receptors intact, we developed a chimeric receptor system in which the N terminus of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular domain of KDR (EGDR) and Flt-1 (EGLT). We observed that KDR, but not Flt-1, was responsible for VPF/VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration. Moreover, Flt-1 showed an inhibitory effect on KDR-mediated proliferation, but not migration. We also demonstrated that the inhibitory function of Flt-1 was mediated through the phosphatidylinositol 3-kinase (PI-3K)-dependent pathway because inhibitors of PI-3K as well as a dominant negative mutant of p85 (PI-3K subunit) reversed the inhibition, whereas a constitutively activated mutant of p110 introduced the inhibition to HUVEC-EGDR. We also observed that, in VPF/VEGF-stimulated HUVECs, the Flt-1/EGLT-mediated down-modulation of KDR/EGDR signaling was at or before intracellular Ca2+mobilization, but after KDR/EGDR phosphorylation. By mutational analysis, we further identified that the tyrosine 794 residue of Flt-1 was essential for its antiproliferative effect. Taken together, these studies contribute significantly to our understanding of the signaling pathways and biological functions triggered by KDR and Flt-1 and describe a unique mechanism in which PI-3K acts as a mediator of antiproliferation in primary vascular endothelium. Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) achieves its multiple functions by activating two receptor tyrosine kinases, Flt-1 (VEGF receptor-1) and KDR (VEGF receptor-2), both of which are selectively expressed on primary vascular endothelium. To dissect the respective signaling pathways and biological functions mediated by these receptors in primary endothelial cells with these two receptors intact, we developed a chimeric receptor system in which the N terminus of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular domain of KDR (EGDR) and Flt-1 (EGLT). We observed that KDR, but not Flt-1, was responsible for VPF/VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration. Moreover, Flt-1 showed an inhibitory effect on KDR-mediated proliferation, but not migration. We also demonstrated that the inhibitory function of Flt-1 was mediated through the phosphatidylinositol 3-kinase (PI-3K)-dependent pathway because inhibitors of PI-3K as well as a dominant negative mutant of p85 (PI-3K subunit) reversed the inhibition, whereas a constitutively activated mutant of p110 introduced the inhibition to HUVEC-EGDR. We also observed that, in VPF/VEGF-stimulated HUVECs, the Flt-1/EGLT-mediated down-modulation of KDR/EGDR signaling was at or before intracellular Ca2+mobilization, but after KDR/EGDR phosphorylation. By mutational analysis, we further identified that the tyrosine 794 residue of Flt-1 was essential for its antiproliferative effect. Taken together, these studies contribute significantly to our understanding of the signaling pathways and biological functions triggered by KDR and Flt-1 and describe a unique mechanism in which PI-3K acts as a mediator of antiproliferation in primary vascular endothelium. vascular permeability factor vascular endothelial growth factor human umbilical vein endothelial cell phosphatidylinositol 3-kinase endothelial cell phospholipase C PAE, porcine aortic endothelial epidermal growth factor epidermal growth factor receptor polymerase chain reaction fluorescence-activated cell-sorting phosphate-buffered saline the fusion receptor of EGFR N terminus with KDR C terminus the fusion receptor of EGFR N terminus with Flt-1 C-terminus To grow beyond minimal size, tumors must generate a new vascular supply for purposes of gas exchange, cell nutrition, and waste disposal (1Folkman J. Sci. Am. 1996; 275: 150-154Crossref PubMed Scopus (315) Google Scholar, 2Folkman J. N. Engl. J. 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In addition, VPF/VEGF is the only angiogenic cytokine identified thus far that renders microvessels hyperpermeable to circulating macromolecules, a characteristic feature of angiogenic blood vessels (8Senger D.R. Perruzzi C.A. Feder J. Dvorak H.F. Cancer Res. 1986; 46: 5629-5632PubMed Google Scholar, 10Dvorak H.F. Prog. Clin. Biol. Res. 1990; 354A: 317-330PubMed Google Scholar, 11Dvorak 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, 12Dvorak H.F. Senger D.R. Dvorak A.M. Dev. Oncol. 1984; 22: 96-114Google Scholar, 16Senger D.R. Galli S.J. Dvorak A.M. Perruzzi C.A. Harvey V.S. Dvorak H.F. Science. 1983; 219: 983-985Crossref PubMed Scopus (3362) Google Scholar). Most of the biological activities of VPF/VEGF are thought to be mediated by its interaction with two high-affinity receptor tyrosine kinases, Flt-1 (VEGF receptor-1) and KDR (VEGF receptor-2; flk-1 in mice) (17Fong G.H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2185) Google Scholar, 18Millauer 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 (1742) Google Scholar, 19Quinn 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 (666) Google Scholar, 20Shalaby 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 (736) Google Scholar, 21Terman B. Dougher-Vermazen M. Carrion M. Dimitrov D. Armellino D. Gospodarowicz D. Bohlen P. Biochem. Biophys. Res. Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1387) Google Scholar). A third receptor, neuropilin, has been recognized, but little is known about its capacity to initiate endothelial cell signaling (22Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2049) Google Scholar, 23Gagnon 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 (248) Google Scholar). Both Flt-1 and KDR are selectively expressed on vascular endothelium but bind VPF/VEGF with different affinities; thus, Flt-1 binds VPF/VEGF with a K d of ∼10 pm, whereas the K d for KDR binding is 400–900 pm (24Joukov V. Sorsa T. Kumar V. Jeltsch M. Claesson-Welsh L. Cao Y. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (634) Google Scholar, 25Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). Both receptors possess tyrosine kinase domains, potential ATP-binding sites, and long kinase insert regions that contain phosphorylation sites with binding capacity for different signaling molecules. Flt-1 and KDR also have different ligand specificities. Thus, Flt-1 interacts with VPF/VEGF (also known as VEGF-A) and with two other members of the VPF/VEGF family, PlGF and VEGF-B. KDR, on the other hand, interacts with VEGF-C and VEGF-D, in addition to VPF/VEGF (26Petrova T.V. Makinen T. Alitalo K. Exp. Cell Res. 1999; 253: 117-130Crossref PubMed Scopus (238) Google Scholar). Both Flt-1 and KDR are essential for normal vascular development (17Fong G.H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2185) Google Scholar, 20Shalaby 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 (736) Google Scholar). At present, the signaling cascades following VPF/VEGF interaction with cultured endothelial cells (ECs) are only partially understood but are known to involve a series of protein phosphorylations, beginning with receptor phosphorylation and subsequently with tyrosine phosphorylation of phospholipase C-γ (PLC-γ) and phosphatidylinositol 3-kinase (PI-3K) (for review, see Refs. 26Petrova T.V. Makinen T. Alitalo K. Exp. Cell Res. 1999; 253: 117-130Crossref PubMed Scopus (238) Google Scholar and 27English J. Pearson G. Wilsbacher J. Swantek J. Karandikar M. Xu S. Cobb M.H. Exp. Cell Res. 1999; 253: 255-270Crossref PubMed Scopus (377) Google Scholar). Like other endothelial cell agonists such as thrombin and histamine, VPF/VEGF activates protein kinase C, increases [Ca2+]i, and stimulates inositol-1,4,5-triphosphate accumulation (28Brock T.A. Dvorak H.F. Senger D.R. Am. J. Pathol. 1991; 138: 213-221PubMed Google Scholar). Because most cultured endothelial cells express both Flt-1 and KDR, it has been difficult to delineate the distinct signaling pathways and biological functions triggered individually by each, and much of our current information comes from studies with cell lines, particularly porcine aortic endothelial (PAE) cells, which do not normally express detectable levels of either KDR or Flt-1 and do not respond to VPF/VEGF. However, when PAE cells were engineered to express KDR, VPF/VEGF induced striking changes in cell morphology and behavior including actin reorganization, membrane ruffling, cell division, and chemotaxis (25Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). Less is known about the consequences of VPF/VEGF interaction with Flt-1. In PAE cells engineered to express Flt-1 but not KDR, VPF/VEGF stimulated tissue factor expression but not cell migration or proliferation (25Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). Most recently, in PAE cells overexpressing KDR and Flt-1, it was reported that Flt-1 repressed KDR-mediated proliferation (29Rahimi N. Dayanir V. Lashkari K. J. Biol. Chem. 2000; 275: 16986-16992Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). By inhibiting the expression of either receptor with antisense oligonucleotides, it was also found that KDR was required for HUVEC proliferation (30Bernatchez P.N. Soker S. Sirois M.G. J. Biol. Chem. 1999; 274: 31047-31054Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). A similar result was also obtained with KDR- or Flt-1-specific antibodies that blocked receptor interaction with VPF/VEGF (31Kanno S. Oda N. Abe M. Terai Y. Ito M. Shitara K. Tabayashi K. Shibuya M. Sato Y. Oncogene. 2000; 19: 2138-2146Crossref PubMed Scopus (249) Google Scholar) and VPF/VEGF mutants that specifically bind to KDR (32Gille 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 (541) Google Scholar). Available data suggest that KDR and Flt-1 have different and perhaps complementary roles in vasculogenesis and angiogenesis. However, many of the data have been obtained from immortalized cell lines that may differ significantly in behavior from early-passage cells derived from primary endothelial cultures. Therefore, to elucidate the respective roles of KDR and Flt-1 in early-passage HUVECs expressing both KDR and Flt-1, we engineered chimeric constructs of both receptors, replacing the extracellular domain of each with the extracellular domain of epidermal growth factor receptor (EGFR). We used retroviral vectors to express these chimeric receptors in HUVECs that expressed both KDR and Flt-1 but not EGFR. Using this system, we demonstrated that HUVEC proliferation and migration were mediated exclusively through the KDR signaling pathway, a conclusion consistent with that of others (25Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar,30Bernatchez P.N. Soker S. Sirois M.G. J. Biol. Chem. 1999; 274: 31047-31054Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 31Kanno S. Oda N. Abe M. Terai Y. Ito M. Shitara K. Tabayashi K. Shibuya M. Sato Y. Oncogene. 2000; 19: 2138-2146Crossref PubMed Scopus (249) Google Scholar, 32Gille 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 (541) Google Scholar). Interestingly, however, Flt-1 activation mediated a distinctive inhibitory signaling pathway through PI-3K that down-regulated the cell proliferation pathway triggered by KDR. Recombinant VPF/VEGF was obtained from R&D Systems (Minneapolis, MN). 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). Mouse monoclonal antibodies against the KDR C-terminal domain and against the EGFR N-terminal domain and a rabbit polyclonal antibody against the Flt-1 C-terminal domain and p85α were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phosphotyrosine antibody was obtained from Upstate Biotechnology (Lake Placid, NY). [3H]Thymidine was obtained from PerkinElmer Life Sciences. Sulfinpyrazone, collagenase, and FITC-conjugated anti-mouse IgG were from Sigma. Transwell plate inserts were from Fisher Scientific. CyQuant, Fura-2/AM, and Pluronic F-127 were obtained from Molecular Probes (Eugene, OR). Primary HUVECs were obtained from Clonetics. Cells were grown on plates coated with 30 µg/ml vitrogen in EGM-MV Bullet Kit (5% fetal bovine serum in endothelial cell basic medium with 12 µg/ml bovine brain extract, 1 µg/ml hydrocortisone, 1 µl/ml GA-1000, and human EGF). HUVECs transduced with EGDR or EGLT were grown in the same medium without human EGF. HUVECs (passage 3 or 4) that were ∼80% confluent were used for most experiments. Cells were serum-starved in 0.1% fetal bovine serum in endothelial cell basic medium for 24 h before testing. Chimeric receptors were engineered that consisted of the extracellular domain of EGFR and the transmembrane and intracellular domains of either KDR or Flt-1. The N-terminal portion of EGF receptor was generated by PCR, using EGFR cDNA kindly provided by Dr. Alex Ulrich as a template. A SalI enzyme restriction sequence was inserted in the 5′ primer (GGGGGGTCGACCAGCATGGGACCCTCCGGGACGGCCGGG) just before the translation start site (ATG); also, the fourth nucleotide C was changed to G to create a kozak sequence that resulted in conversion of Arg to Gly. In the 3′ fusion primer (GCGCGGTACCTACTAGAATAATGATGGCGATGGACGGGATCTTAGGCCCATT) of EGFR, the nucleotide sequence ending at amino acid 647 was fused to a sequence of KDR beginning at amino acid 744, in which aKpnI restriction site had been created by two silent mutations (GGC to GGT and ACG to ACC). The C-terminal portion of KDR was generated by reverse transcription-PCR with RNA isolated from HUVECs. The 5′ primer (GGGGGGTACCACGGTGATTGCCATGTTC) began at amino acid 744 of KDR with two silent mutations to create a KpnI restriction site (GGC to GGT and ACG to ACC). In the 3′ primer (GCGCGCGCGCGGCCGCTTAAACAGGAGGAGAGCTCAGTGT), a NotI site was created immediately after the stop codon. The PCR products were subcloned into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) to create pTOPO-EGFR and pTOPO-KDR. Both of these PCR products were sequenced. These two receptor fragments were then subcloned into retroviral vector pMMP (kindly provided by Dr. Richard A. Mulligan) to create EGDR(EGFR/KDR), the EGFR N terminus and KDR C terminus fusion receptor. EGLT, the EGFR N terminus and Flt-1 C terminus fusion receptor, was created by two rounds of PCR. In the first round, two DNA fragments were generated using EGFR and Flt-1 cDNA (kindly provided by Dr. Masabumi Shibuya) as templates, with oligonucleotides a (5′-GCGTCTCTTGCCGGAATGT-3′) and b (5′-CTCTTTCAATCGTTGGACAGCCTTCAAGACC-3′) and c (5′-CTGTCCAACGATTGAAAGAGTCACAGAAGAGG-3′) and d (5′-AAATGCTGATGCTTGAACC-3′) as primers, respectively. The second-round PCR was performed with both of the first-round PCR products as template and oligonucleotides a and d as primers. pUC18-EGLT was created by replacement of the SalI/HpaI fragment of pUC18-Flt-1 with the N-terminal fragment of EGFR created by digestion of pTOPO-EGFR with SalI and BsmI restriction enzymes, and the second-round PCR product was digested withBsmI and HpaI restriction enzymes. The EGLT was subcloned to retroviral vector pMMP to express protein in HUVECs. The EGLT(793stop) and EGLT(824stop) mutants were obtained by PCR with stop codons introduced at amino acids 794 and 825, respectively. EGLT(Y794F) was created by mutation of TAC to TTC. The dominant negative mutant of p85 (p85(DN)), the subunit of PI-3K, and the constitutively activated form of PI-3K (p110CAAX; kindly provided by Dr. Alex Toker) were subcloned to retroviral vector pMMP to express protein in HUVECs. To prepare the retrovirus, 293T cells were seeded at 6 × 106 cells/100-mm plate 24 h before transfection. 20 µg of targeted genes (pMMP-EGDR, pMMP-EGLT, or pMMP-LacZ, etc.), 15 µg of pMD.MLV gag.pol, and 5 µg of pMD.G DNA encoding the cDNAs of the proteins required for virus packaging (kindly provided by Dr. Richard A. Mulligan) were mixed in 500 µl of water with 62 µl of 2m CaCl2. 500 µl of 2× HBS (280 mm NaCl, 10 mm KCl, 1.5 mmNa2HPO4, 12 mm dextrose, and 50 mm HEPES, pH 7.05) was added to the DNA mixture and incubated at room temperature for 20 min. The DNA mixture was added dropwise to 293T cells. Medium was changed after 16 h. Retrovirus was isolated 48 h after transfection and used immediately for infection. 24 h before infection, HUVECs were seeded at a density of 0.3 × 106 cells/100-mm plate. 5 ml of retrovirus solution and 5 ml of fresh medium (∼1 × 107 plaque-forming units/ml) were added to cells with 10 µg/ml Polybrene. Medium was changed after 16 h and cells were ready for experiment 48 h after infection. Serum-starved HUVECs (with or without retrovirus infection) were washed twice with PBS and incubated with 4 ml of collagenase solution (0.2 µg/ml collagenase, 0.2 µg/ml soybean trypsin inhibitor, 1 µg/ml bovine serum albumin, and 2 mm EDTA in PBS) at 37 °C for 30 min. Cells were detached by gentle scraping and centrifuged at 1100 rpm for 3 min. Cell pellets were washed twice with cold PBS containing 0.1% bovine serum albumin and resuspended in the same buffer at 0.5 × 106cells/ml. Then, aliquots (1 × 105 cells in 200 µl) were added to 96-well plates and pelleted by centrifugation at 1300 rpm for 3 min. Cells were resuspended in 10 µl of the same buffer containing 1 µg of mouse anti-EGFR-N antibody or mouse IgG and incubated at 4 °C for 1 h. Cells were centrifuged at 1300 rpm for 3 min. Cell pellets were washed twice with the same buffer, resuspended in 10 µl of the same buffer containing 2.5 µg/ml FITC-conjugated anti-mouse IgG antibody, and incubated at 4 °C for 0.5 h. Cells were then washed twice and resuspended in 400 µl of the same buffer. FACS analysis was carried out in a FACScalibur instrument (Becton Dickinson) with Cellquest software. 2 × 103 HUVECs/well (with or without retrovirus infection) were seeded in 24-well plates. After 2 days, cells were serum-starved (0.1% serum) for 24 h and then stimulated with 10 ng/ml VEGF or EGF for 20 h. 1 µCi/ml [3H]thymidine was added to each well, and 4 h later, cells were washed three times with cold PBS, fixed with 100% cold methanol for 15 min at 4 °C, precipitated with 10% cold trichloroacetic acid for 15 min at 4 °C, washed with water three times, and lysed with 200 µl of 0.1 n NaOH for 30 min at room temperature. [3H]Thymidine incorporation was measured in scintillation solution. For growth inhibition experiments, various inhibitors (always in final Me2SO concentrations ≤ 0.1%) were added 15 min before VPF/VEGF or EGF stimulation. Data are expressed as the mean ± SD of triplicate values. Serum-starved HUVECs (with or without retrovirus infection) were detached from tissue culture plates as described in FACS analysis, washed twice with endothelial cell basic medium containing 0.1% fetal bovine serum, and seeded (1 × 105 cells/well) into the transwells coated with vitrogen (30 µg/ml), and the transwells were inserted in a 24-well plate containing 1 ml of the same medium. Cells over a range of 3 × 103 to 1 × 105 cells/well were seeded in a 96-well plate for standard curve. Cells were incubated at 37 °C for 1 h to allow the cells to attach, and then VPF/VEGF or EGF was added at a final concentration of 10 ng/ml. After incubation for an additional 2 h, cells remaining on the upper surface of the transwell filter membrane were wiped off with a cotton tip. The whole transwell membrane was cut out and placed in an individual well of the 96-well plate that contained the cells for standard curve. 200 µl of Cyquant DNA stain was added to each well containing cells or membrane, and the plate was kept at 4 °C overnight. After warming to room temperature, stained cells were counted in a spectrofluorometer (SpectraFluor; TECAN) with Delta Soft 3 software. Data are expressed as the mean ± SD of quadruplicate values. Serum-starved HUVECs (with or without retrovirus infection) were detached from plates as described in FACS analysis. Cell pellets were resuspended in 2 ml of Ca2+ buffer (5 mm KCl, 140 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, 5.6 mm glucose, 0.1% bovine serum albumin, 0.25 mm sulfinpyrazone, and 10 mm HEPES, pH 7.5) and centrifuged again at the same speed. Cell pellets were resuspended in 2 ml of Ca2+ buffer containing 1 µg/ml Fura-2 and 0.02% Pluronic F-127 and incubated at 37 °C for 0.5 h. Cells were collected by centrifugation at 1100 rpm for 3 min and resuspended in 2 ml of Ca2+ buffer for stimulation with VPF/VEGF or EGF. In some experiments, cells were preincubated with inhibitors at 30 °C before VPF/VEGF stimulation. Intracellular Ca2+concentrations were measured with the DeltaScan Illumination System (Photon Technology International) using Felix software. Serum-starved cells were stimulated with 10 ng/ml VPF/VEGF or EGF for different lengths of time, as indicated. Stimulation was halted by the addition of ice-cold PBS, and cells were washed three times with ice-cold PBS and lysed with cold radioimmune precipitation buffer (20 mmTris-HCl, pH 7.5, 0.15 m NaCl, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 1 mmNa3VO4, 1 mm EGTA, 1 µg/ml leupeptin, 0.5% aprotinin, and 2 µg/ml pepstatin A). Cell lysates were collected after centrifugation for 15 min at 4 °C. 500 µg of lysate protein was incubated with 1 µg of different antibodies (as indicated) for 1 h and with protein A-conjugated agarose beads at 4 °C for another hour. Beads were washed three times with radioimmune precipitation buffer, and immunoprecipitates were resuspended in 2× SDS sample buffer for Western blot analysis. Cell proliferation and migration data were subjected to analysis of variance and to post hoc testing with the Tukey-Kramer multiple comparison test. To elucidate the specific signaling events and biological activities mediated by KDR and Flt-1 in early-passage HUVECs, we prepared two chimeric receptors, EGDR and EGLT, by fusing the EGFR extracellular domain to the transmembrane and cytoplasmic domains of either KDR or Flt-1 (Fig. 1 a). Each receptor (or both receptors in some experiments) was then transduced into HUVECs with a retroviral expression vector (pMMP). Cells transduced with chimeric receptors or with LacZ as a control were designated EGDR/HUVEC, EGLT/HUVEC, LacZ/HUVEC, and EGDR-EGLT/HUVEC, respectively. Each cell type was then stimulated with EGF or VPF/VEGF, allowing us to study separately the signaling pathways triggered by each chimeric receptor and to compare the results obtained with those that followed stimulation of the wild-type receptors. This approach was possible because HUVECs did not express detectable endogenous EGFR by Western blotting or FACS analysis and did not respond to EGF under the conditions of our experiments (see below). Immunoblots performed directly on EGDR/HUVEC or EGLT/HUVEC lysates demonstrated clear specific bands with antibodies to the C terminus of KDR (mouse monoclonal antibody) or Flt-1 (rabbit polyclonal antibody), respectively (Fig. 1 b, A). The blotting antibodies specifically recognized EGDR and EGLT without detectable cross-reactivity. Neither EDGR nor EGLT was recognized in LacZ/HUVEC. To test whether these proteins contained the EGFR N terminus, lysates of transduced HUVECs were immunoprecipitated with antibodies to the EGFR N terminus before immunoblotting with antibodies against the C terminus of KDR or Flt-1, respectively (Fig. 1 b, B). The chimeric proteins expressed in HUVECs reacted specifically with antibodies to KDR and Flt-1, and both of them contained the N terminus of EGFR. To determine the fraction of transduced cells that expressed these proteins on their surface, we performed FACS analysis with a mouse monoclonal antibody specific for the N terminus of EGFR. Fig.1 c shows that more than 80% of EGDR/HUVEC and EGLT/HUVEC expressed the expected receptor on their surface. EGFR expression was undetectable in parental HUVECs and in LacZ/HUVEC. Stimulation of HUVECs with VPF/VEGF led to time-dependent phosphorylation of both KDR and Flt-1, but with different kinetics (Fig. 1 d, A). Phosphorylation of KDR was maximal and equivalent at 5 and 10 min; however, phosphorylation of Flt-1 was maximal and equivalent at 1 and 5 min and had markedly decreased by 10 min. The decline in Flt-1 phosphorylation was not attributable to unequal protein loading because Flt-1 levels were found to be equal when blots were stripped and reprobed with antibody against Flt-1. The differences in phosphorylation therefore likely reflect differences in the kinetics of
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