ACTIVATION OF VASCULAR ENDOTHELIAL GROWTH-FACTOR GENE-TRANSCRIPTION BY HYPOXIA-INDUCIBLE FACTOR-1

Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocard ial ischemia, retinal disease, and tumor growth. Hypoxia-inducible fac tor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that a ctivates transcription of the human erythropoietin gene in hypoxic cel ls. Here we demonstrate the involvement of HIF-1 in the activation of VEGF transcription. VEGF 5'-flanking sequences mediated transcriptiona l activation of reporter gene expression in hypoxic Hep3B cells. A 47- bp sequence located 985 to 939 bp 5' to the VEGF transcription initiat ion site mediated hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element that was otherwise minimally res ponsive to hypoxia. When reporters containing VEGF sequences, in the c ontext of the native VEGF or heterologous simian virus 40 promoter, we re cotransfected with expression vectors encoding HIF-1 alpha and HIF- 1 beta (ARNT [aryl hydrocarbon receptor nuclear translocator]), report er gene transcription was much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 bindi ng site was demonstrated in the 47-bp hypoxia response element, and a 3-bp substitution eliminated the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinan t HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF-1 alpha inhibited the activation of repo rter transcription in hypoxic cells in a dose-dependent manner. VEGF m RNA was not induced by hypoxia in mutant cells that do not express the HIF-1 beta (ARNT) subunit. These findings implicate HIF-1 in the acti vation of VEGF: transcription in hypoxic cells.