Stem Cell Factor/c-kit Signaling Promotes the Survival, Migration, and Capillary Tube Formation of Human Umbilical Vein Endothelial Cells
2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês
10.1074/jbc.m311643200
ISSN1083-351X
AutoresJunji Matsui, Toshiaki WAKABAYASHI, Makoto Asada, Kentaro Yoshimatsu, Masayuki Okada,
Tópico(s)Axon Guidance and Neuronal Signaling
Resumoc-kit receptor tyrosine kinase is a marker of progenitor cells, which differentiate into blood and/or vascular endothelial cells, and has an important role in the amplification/mobilization of progenitor cells. c-kit is expressed in mature endothelial cells, but its role there is unclear. Stem cell factor, a c-kit ligand, dose-dependently promoted survival, migration, and capillary tube formation of human umbilical vein endothelial cells. These effects mimicked those of vascular endothelial growth factor, except that stem cell factor did not sufficiently support proliferation of these cells. After exposing cells to this factor, Akt, Erk1/2, and c-kit were immediately (≤5 min) and dose-dependently tyrosinephosphorylated. STI-571, a c-kit inhibitor, dose-dependently attenuated these phosphorylations and inhibited stem cell factor-promoted survival and capillary tube formation over the same dose range. Wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase, and PD98059, an inhibitor of MEK, abrogated survival and capillary tube formation, indicating that Akt and Erk1/2 should promote survival and capillary tube formation of these endothelial cells at a locus downstream to stem cell factor/c-kit signaling. Akt was more strongly phosphorylated, whereas Erk1/2 and p38 were more weakly phosphorylated with stem cell factor than with vascular endothelial growth factor. Phospholipase Cγ was phosphorylated only with the latter, indicating that stem cell factor/c-kit signaling is somewhat different. c-kit receptor tyrosine kinase is a marker of progenitor cells, which differentiate into blood and/or vascular endothelial cells, and has an important role in the amplification/mobilization of progenitor cells. c-kit is expressed in mature endothelial cells, but its role there is unclear. Stem cell factor, a c-kit ligand, dose-dependently promoted survival, migration, and capillary tube formation of human umbilical vein endothelial cells. These effects mimicked those of vascular endothelial growth factor, except that stem cell factor did not sufficiently support proliferation of these cells. After exposing cells to this factor, Akt, Erk1/2, and c-kit were immediately (≤5 min) and dose-dependently tyrosinephosphorylated. STI-571, a c-kit inhibitor, dose-dependently attenuated these phosphorylations and inhibited stem cell factor-promoted survival and capillary tube formation over the same dose range. Wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase, and PD98059, an inhibitor of MEK, abrogated survival and capillary tube formation, indicating that Akt and Erk1/2 should promote survival and capillary tube formation of these endothelial cells at a locus downstream to stem cell factor/c-kit signaling. Akt was more strongly phosphorylated, whereas Erk1/2 and p38 were more weakly phosphorylated with stem cell factor than with vascular endothelial growth factor. Phospholipase Cγ was phosphorylated only with the latter, indicating that stem cell factor/c-kit signaling is somewhat different. Angiogenesis, the formation of new blood vessels from preexisting vessels, occurs not only in physiological processes but also in several pathological conditions especially the growth and maintenance of tumors (1Folkman J. Watson K. Ingber D. Hanahan D. Nature. 1989; 339: 58-61Google Scholar, 2Hanahan D. Folkman J. Cell. 1996; 86: 353-364Google Scholar, 3Carmeliet P. Jain R.K. Nature. 2000; 407: 249-257Google Scholar, 4Folkman J. Nat. Med. 1995; 1: 27-31Google Scholar). Recent evidence indicates that, in addition to the sprouting and co-opting of adjacent, pre-existing vessels, the mobilization and incorporation of bone marrow-derived progenitor cells, which are circulating ECPs, 1The abbreviations used are: ECP, endothelial progenitor cell; SCF, stem cell factor; EGF, epidermal growth factor; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; HGF, hepatocyte growth factor; PDGF, platelet-derived growth factor; Ang-1, angiopoietin-1; SDF-1α, stromal derived factor 1α; HUVEC, human umbilical vein endothelial cell; HSC, hematopoietic stem cell; KDR, kinase insert domain receptor; Flk-1, fetal liver kinase; Flt-1, fms-like tyrosine kinase; Erk1/2, extracellular signal-regulated kinase; PI3K, phosphoinositide 3-kinase; MEK, mitogen-activated protein kinase kinase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; CXCR4, CXC receptor 4; SFM, serum-free medium; TF, tube formation; sTF, sandwich tube formation; RT, reverse transcriptase; MTT, 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium; PLCγ, phospholipase Cγ. and HSCs can contribute to this process, especially to tumor angiogenesis (5Isner J.M. 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Cancer. 2002; 2: 826-835Google Scholar). These markers disappear according to the differentiation of the cells into mature blood cells of most hematopoietic lineages, whereas their expression is still highly maintained in mature endothelial cells (14Gill M. Dias S. Hattori K. Rivera M.L. Hicklin D. Witte L. Girardi L. Yurt R. Himel H. Rafii S. Circ. Res. 2001; 88: 167-174Google Scholar, 15Peichev M. Naiyer A.J. Pereira D. Zhu Z. Lane W.J. Williams M. Oz M.C. Hicklin D.J. Witte L. Moore M.A. Rafii S. Blood. 2000; 95: 952-958Google Scholar). Among them, c-kit, a receptor tyrosine kinase, is known to play important roles in amplification and recruitment of progenitor cells from bone marrow into the circulation (16Linnekin D. Int. J. Biochem. Cell Biol. 1999; 31: 1053-1074Google Scholar, 17Heissig B. Hattori K. Dias S. Friedrich M. Ferris B. Hackett N.R. Crystal R.G. Besmer P. Lyden D. Moore M.A. Werb Z. Rafii S. Cell. 2002; 109: 625-637Google Scholar, 18Hattori K. Dias S. Heissig B. 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Miyazaki S. Br. J. Haematol. 1996; 94: 606-611Google Scholar). Proliferation, migration, and capillary tube formation of endothelial cells are essential processes of angiogenesis and directed by several angiogenic cytokines, such as VEGF (23Ferrara N. J. Mol. Med. 1999; 77: 527-543Google Scholar), bFGF (24Klagsbrun M. D'Amore P.A. Annu. Rev. Physiol. 1991; 53: 217-239Google Scholar), and HGF (25Grant D.S. Kleinman H.K. Goldberg I.D. Bhargava M.M. Nickoloff B.J. Kinsella J.L. Polverini P. Rosen E.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1937-1941Google Scholar). VEGF is known to be the master stimulus of angiogenesis through binding to two receptor tyrosine kinases, Flt-1 and KDR, which belong to the PDGF receptor superfamily (23Ferrara N. J. Mol. Med. 1999; 77: 527-543Google Scholar). KDR is a major positive signal transducer and has a stronger tyrosine kinase activity compared with Flt-1 (23Ferrara N. J. Mol. Med. 1999; 77: 527-543Google Scholar). VEGF/KDR signaling in endothelial cells is not fully elucidated, but at least protein kinase C-dependent activation of the MAPK pathway involving Erk1/2 (26Takahashi T. Ueno H. Shibuya M. Oncogene. 1999; 18: 2221-2230Google Scholar) and activation of the PI3K-Akt (27Gerber H.P. McMurtrey A. Kowalski J. Yan M. Keyt B.A. Dixit V. Ferrara N. J. Biol. Chem. 1998; 273: 30336-30343Google Scholar) pathway should be important for proliferation and survival of the endothelial cell. Since c-kit is also a member of the PDGF receptor superfamily (28Yarden Y. Kuang W.J. Yang-Feng T. Coussens L. Munemitsu S. Dull T.J. Chen E. Schlessinger J. Francke U. Ullrich A. EMBO J. 1987; 6: 3341-3351Google Scholar), and SCF/c-kit signaling in hematopoietic cell lines are reported to involve Erk1/2, and Akt (29Kapur R. Chandra S. Cooper R. McCarthy J. Williams D.A. Blood. 2002; 100: 1287-1293Google Scholar, 30Sui X. Krantz S.B. Zhao Z.J. Br. J. Haematol. 2000; 110: 63-70Google Scholar), we studied whether SCF/c-kit signaling has similar roles to VEGF/KDR signaling in endothelial cells or not. In this report we show that SCF/c-kit signaling can promote the survival, migration, and capillary tube formation of HUVEC. This signaling involves Akt and Erk1/2, as VEGF signaling does, but the overall signaling pathways is different. Reagents—HMVECs were purchased from Cambrex BioScience Walkersville, Inc. (Walkersville, MD). Type I collagen coated dishes were purchased from Sumitomo Bakelite Co., Ltd. (Tokyo, Japan). type I collagen gel solution, ×5 RPMI 1640, and reconstitution buffer were purchased from Nitta Gelatin Inc. (Osaka, Japan). SCF was purchased from PEPRO TECH EC., Ltd. (London, UK). Recombinant human VEGF was purchased from Genzyme Tech (Minneapolis, MN). Recombinant human bFGF, EGF, human endothelial SFM basal growth medium, and TRIzol reagent were purchased from Invitrogen (Tokyo, Japan). Bullet kit EGM-2 containing 2% heat-inactivated fetal bovine serum, hFGF-B, VEGF, R3-IGF-1, ascorbic acid, heparin, hEGF, gentamicin, and amphotericin-B were purchased from Sanko Junyaku Co., Ltd. (Tokyo, Japan). Anti-human SCF neutralizing antibody and VEGF neutralizing antibody were purchased from R & D systems (Minneapolis, MN). Anti-c-kit, KDR, and anti-goat IgG-horseradish peroxidase antibody were purchased form Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-c-kit antibody, phospho-KDR antibody, Erk1/2 antibody, phospho-Erk1/2 antibody, Akt antibody, phospho-Akt anti-body, p38 antibody, phospho-p38 antibody, and anti-rabbit IgG (H&L) HRP-linked antibody were purchased form Cell Signaling Technology (Beverly, MA). Anti-β-actin antibody was purchased from Sigma. Anti-mouse IgG HRP-linked antibody was purchased from Amersham Biosciences (Tokyo). PD98059, a specific inhibitor of MEK, and wortmannin and LY294002, specific inhibitors of PI 3-kinase, were purchased from Calbiochem. The chemical identity of STI-571 was supported by nuclear magnetic response and mass spectroscopy data. Cell Culture—HUVECs were isolated from human umbilical cords by a method described previously (31Bussolino F. Di Renzo M.F. Ziche M. Bocchietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. J. Cell Biol. 1992; 119: 629-641Google Scholar). Briefly, the inside of an umbilical vein was rinsed with sterile saline and incubated with trypsin-EDTA at 37 °C for 30 min. Then, endothelial cells that come off the vessel were obtained and cultured on type I collagen-coated plates at 37 °C with 5% CO2 in EGM-2. In this study we used three to six passage cells. The mRNA level of Prox-1, which is a marker of lymphangio endothelial cells, was checked by real time RT-PCR. HMVECs were used as a control, about half of which were reported to be lymphangio endothelial cells (32Makinen T. Veikkola T. Mustjoki S. Karpanen T. Catimel B. Nice EC. Wise L. Mercer A. Kowalski H. Kerjaschki D. Stacker SA. Achen M.G. Alitalo K. EMBO J. 2001; 20: 4762-4773Google Scholar). Endothelial cell purity was measured as CD31-positive cells by a flow cytometer (FACS Calibur) and determined to be more than 99%. sTF Assay—The sTF assay (a collagen three-dimensional culture of HUVECs) was performed according to Deroanne et al. (33Deroanne C.F. Colige A.C. Nusgens B.V. Lapiere C.M. Exp. Cell Res. 1996; 224: 215-223Google Scholar) with a small modification. Briefly, HUVECs were maintained by changing culture medium every day and used in subconfluence. Cells were harvested by trypsin-EDTA and counted. Seven volumes of type I collagen solution were mixed with 2 volumes of 5× RPMI 1640 and 1 volume of reconstitution buffer on ice. An aliquot (0.4 ml) of the collagen gel mixture was added to each well of 24-well plates and allowed to gel at 37 °C. HUVECs were plated onto the gel at 1.5 × 105 cells per well with SFM containing 10 ng/ml EGF (assay medium) and incubated overnight at 37 °C in a 5% CO2 atmosphere. Medium was removed, and 0.4 ml of type I collagen gel mixture was added upon the cells and allowed to gel for 4 h at 37 °C. An aliquot (0.5 ml) of vehicle/test compounds/neutralizing antibody (three times final concentration) and 1 ml of assay medium plus SCF/VEGF/bFGF/HGF-containing solution (1.5 times final concentration) were added to each well. HUVECs sandwiched in collagen gel were incubated at 37 °Cina5%CO2 atmosphere for 3 days. The medium was removed, and 0.4 ml of MTT solutions (3.3 mg/ml 3-(4,5-dimethlthiazol-2)-2,5-diphenyltetrazolium (Sigma) in PBS) were added to each well and incubated for another 4 h. Photomicrographs of capillaries were taken with a light microscope. Tube length of each capillary was measured using image analysis (Angiogenesis Image Analyzer, version 1.0, Kurabo, Osaka, Japan). Assays were performed in duplicate. Survival Assay—HUVECs (1 × 104/50 μl/well) were cultured in collagen type I-coated 96-well multiplates under EGM-2. After 6 h of culturing, culture medium was exchanged for SFM (assay medium). After overnight culture, assay medium containing several concentrations of growth factor and/or a neutralizing antibody, a compound was added to each well, and cells were cultured at 37 °C for 72 h. After incubation, 0.01 ml/well of the WST-1 reagent was added in each well and incubated for another 4 h. Absorbance of each well was measured in a microplate reader (Spectra Max 250, Molecular Devices Corp., Sunnyvale, CA) at 415 nm (reference wavelength at 660 nm) (34Ishiyama M. Tominaga H. Shiga M. Sasamoto K. Ohkura Y. Ueno K. Biol. Pharm. Bull. 1996; 19: 1518-1520Google Scholar). Assays were performed in duplicate. Would Healing Assay—HUVECs were maintained by changing culture medium every day and used in subconfluence. Cells were harvested by trypsin-EDTA and counted. HUVEC were seeded in collagen-coated 12-well multiplates (2.2 × 105/ml/well) and incubated at 37 °C and 5% CO2 in EGM-2 medium. When cells adhered (2 h), medium was aspirated, and SCF (100 ng/ml), VEGF (20 ng/ml), FGF-2 (20 ng/ml) in SFM was added (1 ml/well). After 6-h incubation, confluent monolayers of HUVEC were wounded with a pipette chip and incubated in the same medium (35Pepper M.S. Sappino A.P. Montesano R. Orci L. Vassali J.D. J. Cell. Physiol. 1992; 153: 129-139Google Scholar). After 24 h, the cells that had migrated across the edge of the wound were observed. Quantitative RT-PCR Assay—Using TRIzol reagent, total RNA was extracted from HUVECs, human microvascular endothelial cells (HMVECs), and H526 cells. Reactions of cDNA (0.0375 ng/well) were prepared for assay. Real time RT-PCR was performed using TaqMan Gold RT-PCR kit and an ABI PRISM 7900 sequence detection system (Applied Biosystems, Foster City, CA) according to the manufacturer's instruction. A set of primers and TaqMan probe for each gene were purchased from Applied Biosystems. c-kit and Prox-1 were Assay on Demand™ gene expression products: ID numbers Hs00174029 and Hs00174029. Flow Cytometric Analysis—Cells in assay medium condition were washed with PBS and detached with trypsin-EDTA. After centrifugation the cell pellet was suspended at 1–3 × 105 cells/ml in 50 μl of PBS containing 1% bovine serum albumin and 0.05% NaN3 and incubated with 1 μg of primary antibody (anti-CD31 or anti-c-kit antibody) for 30 min at 4 °C. After washing with PBS, cells were incubated in 50 μl of anti-conjugated second antibody diluted 1:50 in PBS for 30 min at 4 °C. The control sample (for background) was incubated in PBS, and cells were analyzed by flow cytometry using FACS Calibur (BD Biosciences) to quantify staining intensity. The expression of CD31 or c-kit was calculated using the mean fluorescence intensity of each sample as determined by flow cytometry: relative expression = mean fluorescence intensity of sample/mean fluorescence intensity of background. Western Blotting Analysis—Confluent-grown HUVECs in 6-well multiplates were cultured with SFM medium containing 0.5% FBS for 24 h. Cells were treated with the indicated concentrations of compounds for 60 min and then with SCF or VEGF stimulation (20 ng/ml) for the indicated time. Cells were washed with cold PBS twice, collected in 100 μl of lysis buffer (50 mm Hepes, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EDTA, 100 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 50 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 mm Na3VO4), and allowed to lyse for 10 min on ice. Samples were clarified by centrifugation for 30 min at 15,000 rpm at 4 °C. Western blotting was performed as described previously (36Kanno S. Oda N. Abe M. Terai Y. Ito M. Shitara K. Tabayashi K. Shibuya M. Sato Y. Oncogene. 2000; 19: 2138-2146Google Scholar). A sample (20 μg) of lysate protein was subjected to SDS-PAGE under reducing conditions and immunoblotting. Briefly, samples were loaded onto an SDS-PAGE gel and run at 30 mA for 2 h. The proteins were then transferred onto polyvinylidene difluoride membrane at 350 mA for 4.5 h, followed by Western blot analysis. Blots were blocked with Trisbuffered saline containing 0.05% Tween 20 and either 5% skim milk or 5% bovine serum albumin for 1 h at room temperature. The primary antibodies were added at a dilution of 1:1000 overnight at 4 °C. After washing, the appropriate secondary antibodies were added at a dilution of 1:1000 for 1 h at room temperature. After extensive washing, blots were developed with Super Signal enhanced chemiluminescence kits. Immunoreactive bands were visualized by chemiluminescence with Image Master. The intensity of each band was measured by using an image analyzer (1D Image Analysis Software, Eastman Kodak Co.). SCF Drives the Capillary Tube Formation of HUVECs— Capillary tube formation is one of the most unique features of mature endothelial cells. We initially examined whether SCF can drive capillary tube formation of HUVEC in collagen three-dimensional culture. As shown in Fig. 1A, HUVEC formed capillary tube networks in collagen gel with an assay medium as described under “Experimental Procedures” containing 100 ng/ml SCF, and the network structure was maintained for at least 3 days. The capillary tube formation did not proceed well in the gel with assay medium only (Fig. 1A), and the total length of tube structure depended on the dose of SCF (Fig. 1B), indicating that SCF effectively drives the capillary tube formation of HUVEC. Fig. 1A also shows the capillary tube network driven by VEGF, bFGF, and HGF, the amounts of which were determined as those giving maximal effects. These angiogenic factors are known to induce angiogenesis in this model. Structures of the capillary tube network were basically the same, but detailed morphologies were characteristic of each angiogenic factor. Effects of Anti-SCF, Ant-VEGF, Anti-bFGF, and Anti-HGF Antibodies—Since the amount of SCF (100 ng/ml) that fully drove the capillary tube formation of HUVECs was relatively high, it is possible that the effects of SCF are indirect, such as stimulation of HUVECs to produce some angiogenic factors. To exclude this possibility, we investigated the effects of neutralizing antibody against known angiogenic factors on capillary tube formation of HUVECs. As shown in Fig. 2, capillary tube formation of HUVECs driven by SCF was not inhibited by neutralizing antibody against either VEGF, bFGF, or HGF, whereas those driven by VEGF, bFGF, or HGF were markedly inhibited by each specific antibody (data not shown). These results indicate that production of VEGF, bFGF, and HGF does not contribute to the effects of SCF. SCF Promotes Cell Survival and Migration of HUVECs—It is known that promotion of cell mobility and maintenance of cell survival signaling are essential for capillary tube formation. Thus, we next investigated whether HUVECs proliferate and/or survive in SFM containing SCF. Subconfluent HUVECs were starved in SFM containing 0.5% FBS for 24 h, and the medium was changed to SFM containing various amounts of SCF. As shown in Fig. 3A, the number of viable cells after 3 days in culture depended on the concentration of SCF. Generally, the number of viable cells using 100 ng/ml SCF was the same as the initial number of inoculated cells. When the medium had been exchanged to a fresh one containing 100 ng/ml SCF every 3 days, HUVECs survived at least 1 week (data not shown). These results indicate that SCF promotes cell survival but not the proliferation of HUVECs. On the other hand, HU-VECs proliferated in SFM containing 50 ng/ml VEGF (Fig. 3A), confirming that VEGF promotes both survival and proliferation of HUVEC. It is known that HUVEC themselves produce SCF (5Isner J.M. Asahara T. J. Clin. Invest. 1999; 103: 1231-1236Google Scholar), but the contribution of SCF production to the survival of HUVECs might be small in our culture system, since almost all the cells died in the basal medium without adding SCF after 3 days in culture. Fig. 3B shows the migration of HUVECs in the wound healing assay. The cell-free zone made by scratching using a micropipette tip was filled with migrated HUVECs within 24 h in assay medium containing SCF, VEGF, bFGF, or HGF. A picture of migrating cells was taken 12 h after scratching. The migration speed depended on the concentration of SCF and reached a plateau at 100 ng/ml SCF. VEGF and FGF-2 also promoted the migration of HUVECs, and both effects reached a plateau at 20 ng/ml. These doses were well correlated to the doses driving capillary tube formation of HUVECs. Effects Downstream of SCF/c-kit Signaling—To confirm the direct effect of SCF on HUVECs, we first measured the expression of its receptor, c-kit in HUVEC at both mRNA and protein level by real time RT-PCR and flow cytometric analysis. H526 cells (lung cancer), which are well studied as c-kit-expressing cells (37Krystal G.W. DeBerry C.S. Linnekin D. Litz J. Cancer Res. 1998; 58: 4660-4666Google Scholar, 38Abrams T.J. Lee L.B. Murray L.J. Pryer N.K. Cherrington J.M. Mol. Cancer Ther. 2003; 2: 471-478Google Scholar), were used as an expression control. The expression levels of c-kit mRNA and protein in the assay medium were 25 and 27% of that of H526 cells, respectively, confirming that HUVECs actually express considerable amounts of c-kit. Next we analyzed tyrosine autophosphorylation of the c-kit by SCF. As shown in Fig. 4, c-kit was also expressed in HUVECs by protein level, and the level was comparable with that of H526 cells, which respond well to SCF (data not shown). After adding SCF to HUVECs, c-kit was immediately tyrosine-phosphorylated (within 5 min), showing that SCF directly stimulates its specific receptor. The phosphorylation gave a peak at 5–10 min and persisted for 30 min. Protein levels of c-kit were not changed during this period. Akt and Erk1/2 were also tyrosine-phosphorylated simultaneously with the phosphorylation of c-kit. STI-571, an inhibitor of c-kit (39Tuveson D.A. Willis N.A. Jacks T. Griffin J.D. Singer S. Fletcher C.D. Fletcher J.A. Demetri G.D. Oncogene. 2001; 20: 5054-5058Google Scholar, 40Attoub S. Rivat C. Rodrigues S. Van Bocxlaer S. Bedin M. Bruyneel E. Louvet C. Kornprobst M. Andre T. Mareel M. Mester J. Gespach C. Cancer Res. 2002; 62: 4879-4883Google Scholar), clearly inhibited the phosphorylation of Akt and Erk1/2 as well as c-kit, dose-dependently (Fig. 5C). STI-571 also inhibited SCF-driven capillary tube formation and cell survival at the same dose (Fig. 5, A and B), suggesting that Akt and Erk1/2 should promote survival and tube formation downstream of SCF/c-kit signaling.Fig. 5Effects of STI-571 (c-kit tyrosine kinase inhibitor) on SCF-induced phenotypes of HUVECs. A, effect of STI-571 on SCF or VEGF driven capillary tube formation. HUVECs were three-dimensionally cultured for 3 days in type I collagen gels containing the assay medium with SCF or VEGF and increasing amounts of STI-571. B, effects of STI-571 on SCF- or VEGF-driven cell survival. HUVECs were cultured for 3 days in the assay medium with SCF or VEGF and increasing amounts of STI-571 on 96-well plates coated with collagen type I. C, effects of STI-571 on SCF-driven phosphorylation of c-kit, Erk1/2, and Akt. Confluent monolayers of HUVECs were cultured in the assay medium containing 0.5% FBS for 24 h on dishes coated with collagen type I. Cells were then treated with increasing amounts of STI-571 for 60 min, followed by SCF stimulation (100 ng/ml) for 10 min. Cells were lysed and analyzed by Western blotting. All of experiments were done at least in duplicate and repeated three times.View Large Image Figure ViewerDownload (PPT) Comparison of Events Downstream of SCF c-kit and VEGF/KDR Signaling—It is well known that VEGF induces tyrosine phosphorylation of several signal transducers including Erk1/2 and Akt (26Takahashi T. Ueno H. Shibuya M. Oncogene. 1999; 18: 2221-2230Google Scholar, 27Gerber H.P. McMurtrey A. Kowalski J. Yan M. Keyt B.A. Dixit V. Ferrara N. J. Biol. Chem. 1998; 273: 30336-30343Google Scholar). Our preliminary studies showed that their maximum phosphorylation could be observed at about 10 min after treatment of VEGF and SCF. Therefore, we next compared the tyrosine phosphorylation profile of several signal transducers in HUVECs treated with VEGF and SCF for 10 min. As shown in Fig. 6, VEGF induced phosphorylation of Akt, Erk1/2, p38, and PLCγ as well as its receptor, KDR, in a dose-dependent manner. SCF also induced phosphorylation of Akt, Erk1/2, p38, and c-kit dose-dependently, confirming the results shown in Fig. 4, whereas it did not induce the phosphorylation of PLCγ. Furthermore, maximal tyrosine phosphorylation levels of each molecule in HUVECs with SCF and those with VEGF were different. Akt is more strongly phosphorylated, whereas Erk1/2, p38, and PLCγ were more weakly phosphorylated with SCF than with VEGF. Protein levels of all these molecules were not changed in these analyses. These results indicate that their overall signaling pathways are partly different and that although the same molecules such as Erk1/2, Akt, and P38 are involved downstream of SCF/c-kit and VEGF/KDR, their share of signaling in each downstream pathway are different. Contribution of Akt and Erk1/2 to Capillary Tube Formation in HUVECs Stimulated with SCF or VEGF—Since tyrosine phosphorylation profiles of Akt and Erk1/2 were different in HUVECs treated with SCF and VEGF, we then evaluated the contributions of Akt and Erk1/2 in the cells using two kinds of tyrosine kinase inhibitors. Wortmannin and LY294002, inhibitors of PI3K, inhibit the tyrosine phosphorylation of Akt, and PD98059, an inhibitor of MEK, inhibits the tyrosine phosphorylation of Erk1/2 (41Thakker G.D. Hajjar D.P. Muller W.A. Rosengart T.K. J. Biol. Chem. 1999; 274: 10002-10007Google Scholar, 42Surapisitchat J. Hoefen R.J. Pi X. Yoshizumi M. Yan C. Berk B.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6476-6481Google Scholar). As shown in Fig. 7D, wortmannin (0.1 μm) and LY294002 (4 μm) inhibited both SCF- and VEGF-dependent tyrosine phosphorylation of Akt, and this dose of wortmannin and LY294002 completely blocked SCF-driven capillary tube formation (Fig. 7, A and B). However, they could not inhibit VEGF-driven capillary tube formation (Fig. 7, A and B). Higher concentrations of the inhibitors were not studied,
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