Opposite functions of HIF-α isoforms in VEGF induction by TGF-β1 under non-hypoxic conditions

Transforming growth factor (TGF)-beta 1 has biphasic functions in prostate tumorigenesis, having a growth-inhibitory effect in the early stages, but in the late stages promoting tumor angiogenesis and metastasis. We demonstrate here that tumor-producing TGF-beta 1 induces vascular endothelial growth factor (VEGF) in prostate cancer cells, and hypoxia-inducible factor (HIF)-1 alpha and HIF-2 alpha has opposite functions in TGF-beta 1 regulation of VEGF expression under non-hypoxic conditions. The promoter response of VEGF to TGF-beta 1 was upregulated by the transfection of HIF-2 alpha or siHIF-1 alpha but downregulated by HIF-1 alpha and siHIF-2 alpha. Both HIF-1 alpha and HIF-2 alpha were induced by TGF-beta 1 at mRNA and protein levels, however, their nuclear translocation was differentially regulated by TGF-beta 1, suggesting its association with their opposite effects. VEGF induction by TGF-beta 1 occurred in a Smad3-dependent manner, and the Smad-binding element 2 (SBE2, -992 to -986) and hypoxia response element (-975 to -968) in the VEGF promoter were required for the promoter response to TGF-beta 1. Smad3 cooperated with HIF-2 alpha in TGF-beta 1 activation of VEGF transcription and Smad3 binding to the SBE2 site was greatly impaired by knockdown of HIF-2 alpha expression. Moreover, the VEGF promoter response to TGF-beta 1 was synergistically elevated by co-transfection of Smad3 and HIF-2 alpha but attenuated by HIF-1 alpha in a dose-dependent manner. Additionally, TGF-beta 1 was found to increase the stability of VEGF transcript by facilitating the cytoplasmic translocation of a RNA-stabilizing factor HuR. Collectively, our data show that tumor-producing TGF-beta 1 induces VEGF at the both transcription and post-transcriptional levels through multiple routes including Smad3, HIF-2 alpha and HuR. This study thus suggests that autocrine TGF-beta 1 production may contribute to tumor angiogenesis via HIF-2 alpha signaling under non-hypoxic conditions, providing a selective growth advantage for prostate tumor cells. Oncogene (2011) 30, 1213-1228; doi:10.1038/onc.2010.498; published online 8 November 2010