Novel Regulation of Vascular Endothelial Growth Factor-A (VEGF-A) by Transforming Growth Factor β1

Vascular endothelial growth factor (VEGF) is a vital angiogenic effector, regulating key angiogenic processes. Vascular development relies on numerous signaling pathways, of which those induced by transforming growth factor-β (TGFβ) are critical. The Wnt/β-catenin signaling pathway is emerging as necessary for vascular development. Although VEGF, TGFβ, and Wnt signal transductions are well studied individually, it has not been demonstrated previously that all three can interact or be dependent on each other. We show that regulation of VEGF by TGFβ1, in human pulmonary artery smooth muscle cells (PASMCs), depends on a direct interaction between TGFβ signaling proteins, Smads, and members of the Wnt/β-catenin signaling family. VEGF promoter reporter constructs identified a region of the VEGF promoter containing two T cell factor (TCF)-binding sites as necessary for TGFβ1-induced VEGF transcription. Mutation of TCF sites and expression of dominant negative TCF4 abolished TGFβ1-induced VEGF promoter activity. Studies in Smad2 and Smad3 knock-out mouse embryonic fibroblasts demonstrated that one or both are required for VEGF regulation by TGFβ1, with transfection of dominant negative Smad2 or Smad3 into PASMCs confirming this. Chromatin immunoprecipitation assays showed in cell interactions of Smad2 and Smad3 with TCF4 and β-catenin at the VEGF promoter, whereas co-immunoprecipitation showed a direct physical interaction between Smad2 and β-catenin in the nucleus of PASMCs. Finally, we demonstrate that TGFβ1 regulates TCF by modifying β-catenin phosphorylation via regulation of glycogen synthase kinase 3β. These results provide new insight into the molecular regulation of VEGF by two interacting pathways necessary for vascular development, maintenance, and disease. Vascular endothelial growth factor (VEGF) is a vital angiogenic effector, regulating key angiogenic processes. Vascular development relies on numerous signaling pathways, of which those induced by transforming growth factor-β (TGFβ) are critical. The Wnt/β-catenin signaling pathway is emerging as necessary for vascular development. Although VEGF, TGFβ, and Wnt signal transductions are well studied individually, it has not been demonstrated previously that all three can interact or be dependent on each other. We show that regulation of VEGF by TGFβ1, in human pulmonary artery smooth muscle cells (PASMCs), depends on a direct interaction between TGFβ signaling proteins, Smads, and members of the Wnt/β-catenin signaling family. VEGF promoter reporter constructs identified a region of the VEGF promoter containing two T cell factor (TCF)-binding sites as necessary for TGFβ1-induced VEGF transcription. Mutation of TCF sites and expression of dominant negative TCF4 abolished TGFβ1-induced VEGF promoter activity. Studies in Smad2 and Smad3 knock-out mouse embryonic fibroblasts demonstrated that one or both are required for VEGF regulation by TGFβ1, with transfection of dominant negative Smad2 or Smad3 into PASMCs confirming this. Chromatin immunoprecipitation assays showed in cell interactions of Smad2 and Smad3 with TCF4 and β-catenin at the VEGF promoter, whereas co-immunoprecipitation showed a direct physical interaction between Smad2 and β-catenin in the nucleus of PASMCs. Finally, we demonstrate that TGFβ1 regulates TCF by modifying β-catenin phosphorylation via regulation of glycogen synthase kinase 3β. These results provide new insight into the molecular regulation of VEGF by two interacting pathways necessary for vascular development, maintenance, and disease. The mammalian vascular endothelial growth factor (VEGF) 2The abbreviations used are:VEGFvascular endothelial growth factorTGFβtransforming growth factor βPASMCpulmonary artery smooth muscle cellsTCFT cell factorMEFmouse embryonic fibroblastGSKglycogen synthase kinaseANOVAanalysis of varianceELISAenzyme-linked immunosorbent assayChIPchromatin immunoprecipitationDMEMDulbecco's modified Eagle's mediumRTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenaseWTwild type2The abbreviations used are:VEGFvascular endothelial growth factorTGFβtransforming growth factor βPASMCpulmonary artery smooth muscle cellsTCFT cell factorMEFmouse embryonic fibroblastGSKglycogen synthase kinaseANOVAanalysis of varianceELISAenzyme-linked immunosorbent assayChIPchromatin immunoprecipitationDMEMDulbecco's modified Eagle's mediumRTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenaseWTwild type family consists of five members as follows: VEGF-A, -B, -C, and -D and placental growth factor (1Olsson A.K. Dimberg A. Kreuger J. Claesson-Welsh L. Nat. Rev. Mol. Cell Biol. 2006; 7: 359-371Crossref PubMed Scopus (2372) Google Scholar). VEGF-A was the first of the VEGF family to be identified and has been studied in the greatest detail (2Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Nature. 2000; 407: 242-248Crossref PubMed Scopus (3241) Google Scholar). VEGF-A exists as at least six different splice variants of differing amino acid numbers, VEGF-A121, VEGF-A145, VEGF-A165, VEGF-A183, VEGF-A189, and VEGF-A206 (2Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Nature. 2000; 407: 242-248Crossref PubMed Scopus (3241) Google Scholar). VEGF-A has a central role in angiogenesis and plays an essential role in embryogenic vasculogenesis. Loss of a single VEGF allele causes embryonic lethality by embryonic day 11 because of defective vasculogenesis/angiogenesis (3McColl B.K. Stacker S.A. Achen M.G. 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PubMed Scopus Google Scholar). vascular endothelial growth factor transforming growth factor pulmonary artery smooth muscle cells T cell factor mouse embryonic glycogen synthase kinase of immunosorbent immunoprecipitation modified Eagle's type vascular endothelial growth factor transforming growth factor pulmonary artery smooth muscle cells T cell factor mouse embryonic glycogen synthase kinase of immunosorbent immunoprecipitation modified Eagle's type The of the TGFβ in vascular development, and is is by mouse in which of TGFβ angiogenesis at an in Nat. Rev. Mol. Cell Biol. PubMed Scopus Google Scholar). The TGFβ consists of three TGFβ and in to the and Although all TGFβ are in and a with TGFβ1 Nat. Rev. Mol. Cell Biol. PubMed Scopus Google Scholar). TGFβ1 is to cell and with new required for and the of smooth muscle cells or for Nat. Rev. Mol. Cell Biol. PubMed Scopus Google Scholar). 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Finally, we to was to into the at the VEGF promoter, to β-catenin and was an and showed an in with the VEGF promoter of TGFβ1 was the VEGF in the region of the promoter as TCF4 and of VEGF by TGFβ1 and signaling of TGFβ1 phosphorylation of by the kinase TGFβ type TGFβ1 pathway is in on cell PASMCs with TGFβ1 a of Smad2 phosphorylation was at all in to TGFβ1 with phosphorylation in was used as a Smad2 and Smad3 in TGFβ1 of VEGF in dominant negative of the with the VEGF promoter of the VEGF promoter reporter with the in a in with dominant negative Smad2 or dominant negative Smad3 abolished the of the VEGF promoter reporter to to TGFβ1 a role for both Smad2 and Smad3 in TGFβ1-induced VEGF however, it is that dominant negative constructs can an as with of Smad3 is Mol. 2006; PubMed Scopus Google which Smad3 phosphorylation and its with the of of TGFβ1 of VEGF to has been to on Smad2 and that Smad2 be in a TGFβ1-induced required for VEGF of Smad3 is to the a role for the and to a role for immunoprecipitation was and showed an in of and in to TGFβ1 at the VEGF was Smad2 and a for in TGFβ1-induced type Smad2 and Smad3 knock-out a used to the of and a TGFβ1 and a of with and TGFβ1, was a in VEGF with in all cell the and VEGF in the and the three cell the a TGFβ1-induced in VEGF of with TGFβ1 TGFβ1 of VEGF in the is in the is because of Smad3 to the Smad3 in and in TGFβ1-induced VEGF was by the in both type and that of Smad3 is to TGFβ1-induced VEGF in type and that of Smad2 TGFβ1-induced VEGF because of by Smad3 in Smad2 cells is the TGFβ1-induced VEGF of VEGF is in the and is of the provide for the of on VEGF the VEGF promoter reporter and the and into each of the three cell a TGFβ1 a in was in the with in with in the TGFβ1 was the TCF of the and cells with the type and constructs in the of a to TGFβ1 was to the the to TGFβ1 was in in the type in with in the type in the knock-out on TGFβ1 of the in the PASMCs and show a role for in TGFβ1-induced VEGF in both cell between of the TGFβ and and for TCF that the proteins, TCF4 interact at the region of the VEGF promoter in to direct interactions between of the Smad2 was and with β-catenin and showed direct interactions between and both β-catenin and are in PASMCs that are not by TGFβ1 immunoprecipitation and for β-catenin showed both Smad2 and an for TCF4 was however, it was because of the of the for interaction between the two pathways was by transfection of the TCF with dominant negative of with the in a in of with of dominant negative Smad2 or Smad3 with abolished the to both are required for TGFβ1 of the transcription. These results direct interaction between the two pathways in and is necessary for VEGF is the first to demonstrate that regulation of VEGF by TGFβ1 can the factor the and the kinase in to TGFβ signaling and We the that TGFβ1 is of the phosphorylation of amino of TGFβ, VEGF, and signaling are all as for vascular development. Although TGFβ1 regulation of VEGF has been studied in endothelial it is previously in vascular smooth muscle cells can angiogenesis and are not to muscle cells are to provide and Nat. 2000; PubMed Scopus Google Scholar). interaction and signaling between smooth muscle and endothelial cells a key role in showed that TGFβ1 of human pulmonary artery smooth muscle cells VEGF-A and VEGF-A TGFβ1 regulation of VEGF to via and on and required in human mouse and human S. S. Google B.K. A. A. J. PubMed Google J. Biol. 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TCF has been previously to VEGF via the β-catenin pathway in to the of pathway of by its phosphorylation by J. Biol. 2006; PubMed Scopus Google Scholar). we on to TGFβ1 was to cell β-catenin We showed that was not the and that an of glycogen synthase was to an in β-catenin that β-catenin be at or be in pulmonary artery smooth muscle cells it to is in with of β-catenin in and the of β-catenin the TGFβ1 has been previously to be of β-catenin of cell β-catenin 2006; PubMed Scopus Google Scholar). we to TGFβ1 was regulating the of β-catenin in PASMCs. 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