Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNAbinding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3Ј untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3Ј untranslated region element without RNA-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. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3Ј untranslated region and distinct mRNA-binding proteins in human tumor cells.
INTRODUCTIONVascular permeability factor/vascular endothelial growth factor (VPF/VEGF) 1 is a potent activator of microvascular permeability in vivo and an endothelial cell-specific mitogen in vitro Ferrara and Henzel, 1989;Gospodarowicz et al., 1989;Keck et al., 1989;Leung et al., 1989;Levy et al., 1989;Conn et al., 1990;Senger et al., 1990; Ferrara et al., 1991a,b;Dvorak et al., 1992). VPF/VEGF expression has been closely associated with the pathological angiogenesis observed in malignant tumors (Plate et al., 1992; Brown et al., 1993a,b;Weindel et al., 1994; Brown et al., 1995a,b), diabetic retinopathy (Adamis et al., 1994;Aiello et al., 1994), retinopathy of prematurity Pierce et al., 1995;Stone et al., 1995), rheumatoid arthritis (Fava et al., 1994), and coronary artery disease (Sabri et al., 1991;Ladoux and Frelin, 1993;Hashimoto et al., 1994). Tissue hypoxia is a common feature in many of these diseases and VPF/VEGF expression is dramatically up-regulated in human solid tumors adjacent to sites of focal necrosis (Plate et al., 1992;Shweiki et al., 1992; Brown et al., 1993).Hypoxia-stimulated VPF/VEGF expression has been attributed to increases in both transcriptional and posttranscriptional mechanisms (Ikeda et al., 1995;Stein et al., 1995; Levy et al., 1996a,b). The transcriptional up-regulation of VPF/VEGF under hypoxia appears to be a common mechanism in a multitude of † Corresponding author. 1 Abbreviations used: ARE, adenylate-uridylate-rich region;EMSA, electrophoretic mobility shift assay; GM-CSF, granulocyte macrophage-colony stimulating factor; HSR, hypoxia stab...
