Sonic Hedgehog Induces Capillary Morphogenesis by Endothelial Cells through Phosphoinositide 3-Kinase

Sonic hedgehog (Shh) acts as a morphogen in many cell types. Recent studies have shown that hedgehog signaling is involved in vascular development as well as postnatal angiogenesis. However, the direct action of Shh on cultured endothelial cells has not been clearly shown. To address this issue, we examined the effect of Shh on morphological changes by murine brain capillary endothelial cells (IBE cells) and human umbilical endothelial cells (HUVECs). Shh induced capillary morphogenesis by these cells. The effect was inhibited by cyclopamine or pertussis toxin. Shh-induced capillary morphogenesis was also blocked by LY294002, a phosphoinositide 3-kinase (PI3-kinase) inhibitor. Shh rapidly increased PI3-kinase activity in IBE cells and HUVECs; this activity was inhibited by cyclopamine. Nuclear localization of Gli1 was increased in Shh-treated IBE cells, which was not affected by LY294002. Actinomycin D and cycloheximide inhibited Shh-induced capillary morphogenesis. In IBE cells expressing kinase-inactive c-Fes, Shh failed to stimulate PI3-kinase activity and capillary morphogenesis. Considered collectively, Shh induced capillary morphogenesis of endothelial cells through both rapid activation of c-Fes/PI3-kinase pathways and transcriptionally regulated pathways. Sonic hedgehog (Shh) acts as a morphogen in many cell types. Recent studies have shown that hedgehog signaling is involved in vascular development as well as postnatal angiogenesis. However, the direct action of Shh on cultured endothelial cells has not been clearly shown. To address this issue, we examined the effect of Shh on morphological changes by murine brain capillary endothelial cells (IBE cells) and human umbilical endothelial cells (HUVECs). Shh induced capillary morphogenesis by these cells. The effect was inhibited by cyclopamine or pertussis toxin. Shh-induced capillary morphogenesis was also blocked by LY294002, a phosphoinositide 3-kinase (PI3-kinase) inhibitor. Shh rapidly increased PI3-kinase activity in IBE cells and HUVECs; this activity was inhibited by cyclopamine. Nuclear localization of Gli1 was increased in Shh-treated IBE cells, which was not affected by LY294002. Actinomycin D and cycloheximide inhibited Shh-induced capillary morphogenesis. In IBE cells expressing kinase-inactive c-Fes, Shh failed to stimulate PI3-kinase activity and capillary morphogenesis. Considered collectively, Shh induced capillary morphogenesis of endothelial cells through both rapid activation of c-Fes/PI3-kinase pathways and transcriptionally regulated pathways. sonic hedgehog immortalized murine brain capillary endothelial cells human umbilical vein cord endothelial cells phosphoinositide 3-kinase patched smoothened Cubitus interuptus Sonic hedgehog (Shh)1 is a member of a family of closely related proteins consisting of Shh, indian hedgehog, and desert hedgehog. These proteins are known to regulate morphology of many kinds of tissues (1Ingham P.W. McMahon A.P. Genes Dev. 2001; 15: 3059-3087Google Scholar). Initial studies showed that hedgehog transduced signals via its receptor Patched (Ptc). It has been shown that Ptc forms complex with seven-pass membrane protein, smoothened (Smo), which exists as an inactive form. Hedgehog-bound Ptc dissociates Smo, resulting in the activation. Activated Smo allows the entry of a transcription factor, Cubitus interuptus (Ci), into nuclei, which induces the expression of a panel of downstream target molecules (2Ingham P.W. EMBO J. 1998; 17: 3505-3511Google Scholar). Vertebrate homologs of Ci are Gli1, 2, and 3. However, recent studies addressing the distribution of Ptc and Smo have presented evidence that Smo is not present in a complex containing Ptc (3Denef N. Neubuser D. Perez L. Cohen S.M. Cell. 2000; 102: 521-531Google Scholar, 4Strutt H. Thomas C. Nakano Y. Stark D. Neave B. Taylor A.M. Ingham P.W. Curr. Biol. 2000; 11: 608-613Google Scholar). Although it is still unclear how Ptc regulates Smo activity, the sterol-sensing domain of Ptc seems to be important for regulation (4Strutt H. Thomas C. Nakano Y. Stark D. Neave B. Taylor A.M. Ingham P.W. Curr. Biol. 2000; 11: 608-613Google Scholar, 5Martin V. Carrillo G. Torroja C. Guerrero I. Curr. Biol. 2001; 11: 601-607Google Scholar). The regulation of Ci depends on their phosphorylation status. Ci is first phosphorylated by protein kinase A (6Chen Y. Gallaher N. Goodman R.H. Smolik S.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2349-2354Google Scholar, 7Price M.A. Kalderon D. Development. 1999; 126: 4331-4339Google Scholar). Further phosphorylation of Ci by Shaggy/glycogen synthase kinase 3 and casein kinase 1 leads to its proteolytic degradation (8Jia J. Amanai K. Wang G. Tang J. Wang B. Jiang J. Nature. 2002; 416: 548-552Google Scholar,9Price M.A. Kalderon D. Cell. 2002; 108: 823-835Google Scholar). However, protein or lipid kinases activated by hedgehog have not been well characterized to date. It has been shown that hedgehog signaling is involved during vascular development (10Brown L.A. Rodaway A.R. Schilling T.F. Jowett T. Ingham P.W. Patient R.K. Sharrocks A.D. Mech. Dev. 2000; 90: 237-252Google Scholar, 11Dyer M.A. Farrington S.M. Mohn D. Munday J.R. Baron M.H. Development. 2001; 128: 1717-1730Google Scholar, 12Byrd N. Becker S. Maye P. Narasimhaiah R. St.-Jacques B. Zhang X. McMahon J. McMahon A. Grabel Development. 2002; 129: 361-372Google Scholar). Shh was also reported recently to induce postnatal angiogenesis (13Pola R. Ling L.E. Silver M. Corbley M.J. Kearney M. Blake Pepinsky R. Shapiro R. Taylor F.R. Baker D.P. Asahara T. Isner J.M. Nat. Med. 2001; 7: 706-711Google Scholar). Angiogenesis is composed of a series of endothelial cellular responses, and maturation of newly formed vessels is accompanied by branching, capillary-like morphology (14Folkman J. D'Amore P.A. Cell. 1996; 87: 1153-1155Google Scholar, 15Darland D.C. D'Amore P.A. J. Clin. Invest. 1999; 103: 157-158Google Scholar). In a previous report, there were no direct effects of Shh on cellular responses by cultured endothelial cells, such as proliferation, migration, and serum-deprived survival reported (13Pola R. Ling L.E. Silver M. Corbley M.J. Kearney M. Blake Pepinsky R. Shapiro R. Taylor F.R. Baker D.P. Asahara T. Isner J.M. Nat. Med. 2001; 7: 706-711Google Scholar). Because hedgehog signaling regulates morphological changes of many types of cells, we focused on the capillary morphogenesis of cultured endothelial cells mediated by Shh. We found that Shh induced capillary morphogenesis by human umbilical vein cord endothelial cells (HUVECs) as well asimmortalized murine brain capillaryendothelial (IBE) cells (16Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth Differ. 1996; 7: 383-395Google Scholar). Shh increased phosphoinositide 3- kinase (PI3-kinase) in endothelial cells, and Shh-mediated capillary morphogenesis was PI3-kinase inhibitor-sensitive. Expression of deleted mutant p85, which cannot associate with p110 catalytic subunit of PI3-kinase, and kinase-inactive c-Fes blocked Shh-mediated PI3-kinase activation as well as capillary morphogenesis. These results suggest that Shh activates PI3-kinase in endothelial cells, followed by the induction of capillary morphogenesis. Anti-phosphotyrosine (PY99) antibody, anti-FLAG goat antibody, anti-Patched-1 antibody, and anti-Gli1 antibody were purchased from Santa Cruz Biotechnologies, Santa Cruz, CA. Anti-FLAG monoclonal antibody M2 and anti-β-actin antibody were from Sigma. Anti-Akt and phospho-Akt (Ser-473) antibodies were obtained from PerkinElmer Life Sciences. Mouse recombinant Shh N-terminal peptide, human recombinant angiopoietin 2 (Ang2), human recombinant vascular endothelial growth factor-A (VEGF-A), and Tie2/Fc chimera were obtained from R & D Systems, Minneapolis, MN. The recombinant Shh protein used in this study was an N-terminal fragment corresponding to Cys-25 to Gly-198 of mouse Shh, which has been shown to be an active form (17Marti E. Bumcrot D.A. Takada R. McMahon A.P. Nature. 1995; 375: 322-325Google Scholar, 18Kanda S. Lerner E.C. Tsuda S. Shono T. Kanetake H. Smithgall T.E. J. Biol. Chem. 2000; 275: 10105-10111Google Scholar). Recombinant Shh protein was expressed in Escherichia coli and was purified to more than 97% as determined by SDS-PAGE. The contaminated endotoxin level was less than 1.0 enzyme units/μg of the Shh with the Limulus amebocyte lysate method, as determined by the manufacturer. A PI3-kinase inhibitor, LY294002, a Src family kinase inhibitor, PP2, and a mitogen-activated protein kinase/extracellular signal regulated kinase kinase inhibitor, PD98059, were from Calbiochem-Novabiochem (La Jolla, CA) and were dissolved in dimethyl sulfoxide (Me2SO) as a stock solution and stored at −30 °C until use. Stock solutions were further diluted with Me2SO and dissolved in culture medium. Final concentration of Me2SO was 0.1% in all cases. Pertussis toxin was fromCalbiochem-Novabiochem and was dissolved in Tris-buffered saline. Cyclopamine was purchased from Toronto Research Chemicals Inc. North York, ON, Canada and growth factor-reduced Matrigel was from BD Biosciences, Bedford, MA. Cycloheximide and actinomycin D were obtained from Sigma. Parental IBE cells obtained from temperature-sensitive mutant SV 40 large T transgenic mouse brain capillaries were cultured as reported previously (16Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth Differ. 1996; 7: 383-395Google Scholar). Stable IBE cell lines expressing either wild-type c-Fes or kinase-inactive c-Fes (denoted WTFes 6-8 cells and KEFes 5-15 cells, respectively; 18), and a stable cell line expressing deleted mutant p85 PI3-kinase subunit, which does not interact with p110 subunit (19Kotani K. Yonezawa K. Hara K. Ueda H. Kitamura Y. Sakaue H. Ando A. Chavanieu A. Calas B. Grigorescu F. Nishiyama M. Waterfield M.D. Kasuga M. EMBO J. 1994; 13: 2313-2321Google Scholar) (denoted Δp85-8 cells; 20), were described elsewhere. Experiments using IBE cell lines were performed at 33 °C, because at 39 °C, cells became senescent and lost responsiveness to extracellular stimuli (16Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth Differ. 1996; 7: 383-395Google Scholar). HUVECs and their culture medium were obtained from BioWhittaker, Inc., Walkersville, MD, and cells were cultured according to the protocol recommended by the manufacturer. For IBE cells, cells were suspended in Ham's F-12 medium containing 0.25% bovine serum albumin and seeded onto cultured growth factor-reduced Matrigel® in wells of 24-well plates at a density of 1.0 × 104 cells/well. Cells were treated or left untreated with indicated pharmacological inhibitors or vehicle (0.1% Me2SO) for 1 h, and then indicated growth factors were added. Twenty-four hours later, capillary morphogenesis was examined under a phase-contrast microscopic observation. For HUVECs, cells suspended in a culture medium containing 0.5% fetal bovine serum were inoculated onto growth factor-reduced Matrigel® at a density of 3.0 × 104 cells/well of 24-well plates and treated as indicated (21Kubota Y. Kleinman H.K. Martin G.R. Lawley T.J. J. Cell Biol. 1988; 107: 1589-1596Google Scholar). Cells were cultured for 24 h, and capillary morphogenesis was examined. To quantify the length of capillaries, three different phase-contrast photomicrographs (×10 objectives) per well were taken, and the length of each capillary was measured using NIH Image software (version 1.64). Capillary length was expressed as -fold increase relative to unstimulated cells. The method used for determination of PI3-kinase activity in the immunoprecipitates of anti-phosphotyrosine was described previously (20Mochizuki Y. Tsuda S. Kanetake H. Kanda S. Oncogene. 2002; 21: 7027-7033Google Scholar). In brief, serum-starved cells were either stimulated or left unstimulated by indicated cytokines and lysed in Nonidet P-40 lysis buffer, and tyrosine-phosphorylated proteins were immunoprecipitated. After extensive washing, immunoprecipitates were incubated with phosphatidylinositol and [γ-32P]ATP, and reaction products were separated by thin layer chromatography on silica Gel-60 plates. Incorporation of [γ-32P]ATP into phosphatidylinositol was measured by Image Analyzer BAS 5000 (Fuji), followed by exposure on x-ray films (Amersham Biosciences). Serum- and growth factor-starved IBE cells were either stimulated or left unstimulated with Shh in the presence of orthovanadate (50 μm) for 10 min. Cells were washed and lysed in Nonidet P-40 lysis buffer, and c-Akt was immunoprecipitated with anti-Akt antibody. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis. After electronic transfer onto polyvinylidene difluoride membranes (Millipore, Bedford, MA), the blots were probed with either anti-phospho-Akt or anti-Akt antibodies. To examine the expression of Patched protein, total cell extracts of HeLa cells, IBE cells, and HUVECs were electrophoresed, and immunoblotting was performed with anti-Patched 1 antibody followed by anti-β-actin antibody. Cells were starved, either stimulated with 5 μg/ml Shh for 8 min or left unstimulated, and lysed in the Nonidet P-40 lysis buffer. Cell lysate was separated into two portions, 90% and 10%, respectively, and FLAG-tagged c-Fes was immunoprecipitated with anti-FLAG monoclonal antibody from the 90% of each lysate. Immunoprecipitates were washed 4 times with lysis buffer, twice with Tris-buffered saline, and once with kinase buffer (25 mm HEPES, pH 7.4, supplemented with 10 mmMnCl2 and 2 mm MgCl2). Immunoprecipitates were incubated with [γ-32P]ATP at 30°C for 10 min, and reaction products were run on SDS-PAGE. Polyacrylamide gels were fixed, in 1 at for min to phosphorylated and on for the with Image Analyzer BAS 5000 (Fuji), followed by the exposure on x-ray films (Amersham Biosciences). IBE cells were cultured on and for 24 and then serum-starved for Cells were incubated with 0.1% LY294002, or cyclopamine for min, and then were treated with Shh for 2 h, 4 h, or left Cells were washed with and with at for min. Cells were washed and incubated with containing bovine serum and for min at followed by with anti-Gli1 antibody or goat at 8 μg/ml at Cells were washed and incubated with After extensive washing, localization of Gli1 protein was examined under a microscopic observation. To examine the of cells with of Gli1, more than cells were determined in each cellular responses by endothelial cells capillary morphogenesis. Shh regulates morphological changes of many cell types. we first examined the effect of Shh on morphological of IBE cells. IBE cells form capillary-like in to or (16Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth Differ. 1996; 7: 383-395Google Scholar, Y. T. Kanetake H. Kanda S. J. Cell Sci. 2002; Scholar). shown in Shh induced capillary morphogenesis of IBE cells, of which morphology was to that of or treated cells. Cells treated with Shh showed on Matrigel and each these cells, a was In the of Shh, cells failed to not induce capillary morphogenesis of IBE cells, as has been shown previously (16Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth Differ. 1996; 7: 383-395Google Scholar). have that induced capillary morphogenesis through receptor 2 B. C. M. J. A. J. 2001; Scholar, R. M. J. 2002; Scholar). receptor 2, not receptor also to and of endothelial cells J. Claesson-Welsh L. A. M. J. Biol. Chem. 1994; Scholar). Expression of receptor 2 in IBE cells was and also failed to stimulate and of IBE cells (16Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth Differ. 1996; 7: 383-395Google Scholar). It is that a level of receptor 2 expression in endothelial cells seems to be to regulate endothelial cell in to 2 that capillary morphogenesis was inhibited by the of cells with a Src family kinase inhibitor, PP2, as reported S. A. S. Kanetake H. Kanda S. 2002; Scholar). inhibited capillary morphogenesis by IBE cells. shown in 2 a that signaling via Smo D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: Scholar, J. Wang B. R.K. L. P.A. Nature. 2000; Shh-induced capillary morphogenesis. Pertussis a protein endotoxin that of protein also blocked Shh-induced capillary morphogenesis. LY294002, not or PP2, blocked Shh-mediated capillary morphogenesis. Cyclopamine and pertussis toxin not capillary morphogenesis. These results suggest that Shh activated different signaling pathways than activated by We also examined the capillary morphogenesis by 3 that as well as Shh induced capillary morphogenesis by also inhibited Shh-mediated capillary morphogenesis. not effect on capillary morphogenesis. Cyclopamine also blocked Shh-induced capillary morphogenesis by HUVECs not We examined the of transcription in Shh-induced capillary morphogenesis. for Shh, Ptc protein, was expressed in IBE cells and HUVECs 4 factor, Gli1, was in the of untreated IBE cells 4 Although not the localization of Gli1 not Shh increased localization of Gli1 in IBE cells, and cyclopamine inhibited However, localization was not inhibited by the of cells with LY294002, that Shh-mediated of Gli1 was of PI3-kinase We examined the effects of which protein or actinomycin which on Shh-induced capillary morphogenesis by IBE cells. These at a concentration of effects on IBE cells. both cycloheximide and actinomycin D inhibited Shh-induced capillary morphogenesis 4 These results suggest that Shh seems to regulate capillary morphogenesis of endothelial cells through pathways as well as In the present we not direct effects of Shh on or of cultured endothelial cells not of inhibitors on and Shh-induced capillary morphogenesis by IBE cells. capillary morphogenesis induced by was inhibited by Cells suspended in medium in the presence of 0.1% Me2SO or inhibitors were seeded onto later, cells were either stimulated or left unstimulated with 10 After 24 h, were and capillary length was measured as described in the of Shh-induced capillary morphogenesis is blocked by pertussis or PI3-kinase LY294002. Cells suspended in medium in the presence of 0.1% Me2SO or inhibitors were seeded onto later, cells were either stimulated or left unstimulated with 5 μg/ml Shh or 10 After 24 h, were and capillary length was measured as described in the of were obtained from two Image induces capillary morphogenesis by HUVECs and this morphological is also were seeded onto Matrigel in the presence of 0.1% Me2SO or 10 LY294002. later, cells were either stimulated or left unstimulated with 5 μg/ml Shh or After 24 h, were and capillary length was measured as described in the of were obtained from two Image Patched a receptor for sonic hedgehog, is expressed in HUVECs and IBE cells. cell extracts from HeLa cells a HUVECs, and IBE cells were electrophoresed, and immunoblotting was performed with goat anti-Patched 1 antibody. After the membrane was with anti-β-actin antibody. Gli1 is present in of Shh-treated IBE cells. Cells on were serum-starved and incubated with LY294002, or cyclopamine. later, cells were either stimulated or left unstimulated with 5 μg/ml Shh for 2 or 4 Gli1 was by an and cells with of Gli1 was as cells. of cell cell was were expressed as from three actinomycin D and cycloheximide Shh-induced capillary morphogenesis of IBE cells. Cells suspended in medium in the presence of 0.1% or were seeded onto later, cells were either stimulated or left unstimulated with 5 μg/ml Shh. After 24 h, were and capillary length was measured as described in the of were obtained from two Image We examined the Shh-induced activation of shown in Shh increased PI3-kinase activity in anti-phosphotyrosine immunoprecipitates of IBE cells and Stable expression of deleted mutant p85 subunit, which to p110 catalytic subunit (denoted Δp85-8 inhibited Shh-induced increase in PI3-kinase These results suggest that Shh induced phosphorylation of followed by the with p85 subunit of c-Akt was also phosphorylated at which is involved in its activation M. B. P. N. Cohen P. EMBO J. 1996; 15: and such phosphorylation was also Shh failed to c-Akt in Δp85-8 cells. c-Fes kinase is expressed in endothelial cells and cells. c-Fes is activated by the followed by In a recent we have shown that activation of PI3-kinase on c-Fes kinase activity Y. T. Kanetake H. Kanda S. J. Cell Sci. 2002; Scholar). We Shh FLAG-tagged wild-type WTFes 6-8 cells; or kinase-inactive c-Fes KEFes 5-15 cells; was and kinase activity was examined by the of [γ-32P]ATP into shown in of [γ-32P]ATP into wild-type c-Fes, not kinase-inactive c-Fes, was increased by that c-Fes to be activated by Shh We then examined the Shh-induced activation of PI3-kinase in KEFes 5-15 cells. 5 D that Shh failed to PI3-kinase in KEFes 5-15 cells. that Shh-mediated PI3-kinase activation depends on c-Fes kinase We also examined the Shh-mediated capillary morphogenesis in KEFes 5-15 cells and Δp85-8 cells. shown in as well as induced capillary morphogenesis by these cells. However, Shh failed to induce capillary morphogenesis by these cells. These results suggest that Shh-mediated capillary morphogenesis PI3-kinase activation through c-Fes in endothelial cannot induce capillary morphogenesis by cells or Δp85-8 cells. Cell were treated or left untreated with 5 μg/ml of Shh or and a and cultured on Capillary length were measured as described in the of were obtained from two Image It has previously been shown that Shh induces angiogenesis by of and (13Pola R. Ling L.E. Silver M. Corbley M.J. Kearney M. Blake Pepinsky R. Shapiro R. Taylor F.R. Baker D.P. Asahara T. Isner J.M. Nat. Med. 2001; 7: 706-711Google Scholar). direct action of Shh on proliferation, migration, and survival of cultured endothelial cells was in that study (13Pola R. Ling L.E. Silver M. Corbley M.J. Kearney M. Blake Pepinsky R. Shapiro R. Taylor F.R. Baker D.P. Asahara T. Isner J.M. Nat. Med. 2001; 7: 706-711Google Scholar). signaling is involved in the morphogenesis of We focused on the effect of Shh on morphological changes of endothelial cells and found that Shh capillary morphogenesis by cultured endothelial cells. However, we not Shh-induced or of HUVECs and IBE cells. These are with the previous (13Pola R. Ling L.E. Silver M. Corbley M.J. Kearney M. Blake Pepinsky R. Shapiro R. Taylor F.R. Baker D.P. Asahara T. Isner J.M. Nat. Med. 2001; 7: 706-711Google Scholar). HUVECs and IBE cells expressed Shh-induced capillary morphogenesis was blocked by the of cells with cyclopamine. Cyclopamine hedgehog signaling through These results that Shh induced capillary morphogenesis through Smo is a seven-pass Pertussis toxin inhibited Shh-induced morphological The also suggest that signals via Smo to be for Shh-induced capillary morphogenesis. Shh activated c-Fes/PI3-kinase in IBE cells. of PI3-kinase was rapid and was to the that Smo to be involved in this of c-Fes and p85 subunit of PI3-kinase inhibited Shh-induced capillary morphogenesis of IBE cells. These results suggest that Shh capillary morphogenesis of endothelial cells through Gli1 transcription into of Shh-treated IBE cells. of was not inhibited by LY294002, that Gli1 on In cycloheximide and actinomycin D blocked Shh-induced capillary morphogenesis. These the that followed by the of target proteins be for Shh-induced capillary morphogenesis. the IBE cells of which was measured by for murine & D However, Shh not the not The expression of in IBE cells was level (16Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth Differ. 1996; 7: 383-395Google and Shh failed to the which was examined by not induce capillary morphogenesis of IBE cells of cells with chimera & D Shh-induced capillary morphogenesis not it seems that Shh stimulated capillary morphogenesis by endothelial cells of or In the present Shh increased PI3-kinase It has been shown that synthase kinase 3 of family transcription which in allows its proteolytic degradation (8Jia J. Amanai K. Wang G. Tang J. Wang B. Jiang J. Nature. 2002; 416: 548-552Google Scholar, M.A. Kalderon D. Cell. 2002; 108: 823-835Google Scholar). of PI3-kinase, c-Akt is c-Akt which results in of activity D.A. Cohen P. M. Nature. 1995; Scholar, M. Cohen P. 416: Scholar). PI3-kinase activated by Shh activity, which in the degradation of proteins by Recent studies have also shown that c-Akt was involved in by endothelial cells M.J. S. N. M. M. T. Cell. 1999; Scholar, Y. I. A. D. K. Nat. Med. 2000; Scholar, M.J. S. N. M. M. T. Cell. 2001; Scholar). it seems that activated c-Akt to Shh-induced capillary morphogenesis of endothelial cells or through We T. M. and of the for and

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