Novel regulation of vascular endothelial growth factor-A (VEGF-A) by transforming growth factor (beta)1: requirement for Smads, (beta)-CATENIN, AND GSK3(beta).

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-beta (TGFbeta) are critical. The Wnt/beta-catenin signaling pathway is emerging as necessary for vascular development. Although VEGF, TGFbeta, 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 TGFbeta(1), in human pulmonary artery smooth muscle cells (PASMCs), depends on a direct interaction between TGFbeta signaling proteins, Smads, and members of the Wnt/beta-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 TGFbeta(1)-induced VEGF transcription. Mutation of TCF sites and expression of dominant negative TCF4 abolished TGFbeta(1)-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 TGFbeta(1), 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 beta-catenin at the VEGF promoter, whereas co-immunoprecipitation showed a direct physical interaction between Smad2 and beta-catenin in the nucleus of PASMCs. Finally, we demonstrate that TGFbeta(1) regulates TCF by modifying beta-catenin phosphorylation via regulation of glycogen synthase kinase 3beta. These results provide new insight into the molecular regulation of VEGF by two interacting pathways necessary for vascular development, maintenance, and disease.