Hypoxia induces vascular endothelial growth factor gene transcription in human osteoblast-like cells through the hypoxia-inducible factor-2 alpha

VEGF is produced by osteoblasts and has been postulated to function as an a ngiogenic stimulus during normal skeletal development and in fracture repai r. In this study, we characterized the molecular mechanisms by which experi mental hypoxia increases VEGF mRNA in human MG63 osteoblast-like cells. Exp osure of MG63 cells to 1% O-2 for 24 h resulted in a four-fold increase in VEGF mRNA. Immunoblotting of nuclear extracts demonstrated a time-dependent increase in the level of the Hif-2 alpha protein, which preceded the rise in VEGF mRNA. To determine the effect of hypoxia on VEGF gene transcription , MG63 cells were transiently transfected with a segment of the VEGF promot er construct fused to luciferase and then exposed to 1% O-2 Hypoxia induced VEGF promoter activity five-fold by 24h. Forced expression of Hif-2a, but not Hif-1 alpha, increased both basal and hypoxia induced VEGF promoter act ivity. By contrast, the ability of the VEGF reporter to respond to hypoxia or recombinant Hif-2a was abolished in cells transfected with a VEGF promot er construct containing a mutation in the hypoxia response element. In summ ary, exposure of osteoblast-like cells to hypoxia induces VEGF expression v ia induction of Hif-2 alpha and transcriptional activation of the VEGF prom oter.