IDENTIFICATION OF A HUMAN VPF VEGF 3'-UNTRANSLATED REGION MEDIATING HYPOXIA-INDUCED MESSENGER-RNA STABILITY/

Hypoxia is a prominent feature of malignant tumors that are characteri zed by angiogenesis and vascular hyperpermeability. Vascular permeabil ity factor/vascular endothelial growth factor (VPF/VEGF) has been show n to be up-regulated in the vicinity of necrotic tumor areas, and hypo xia potently induces VPF/VEGF expression in several tumor cell lines i n vitro. Here we report that hypoxia-induced VPF/VEGF expression is me diated by increased transcription and mRNA stability in human M21 mela noma cells. RNA-binding/electrophoretic mobility shift assays identifi ed a single 125-bp AU-rich element in the 3' untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expres sion of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than const ructs containing an adjacent 3' untranslated region element without RN A-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. T he 90/88-kDa and 60-kDa species were specifically competed by excess h ypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability i nduced by hypoxia is mediated, at least in part, by specific interacti ons between a defined mRNA stability sequence in the 3' untranslated r egion and distinct mRNA-binding proteins in human tumor cells.