Cooperative interaction of hypoxia-inducible factor-2α (HIF-2α) and Ets-1 in the transcriptional activation of vascular endothelial growth factor receptor-2 (Flk-1)

Abstract

Interactions between Ets family members and a variety of other transcription factors serve important functions during development and differentiation processes, e.g. in the hematopoietic system. Here we show that the endothelial basic helix-loop-helix PAS domain transcription factor, hypoxia-inducible factor-2α (HIF-2α) (but not its close relative HIF-1α), cooperates with Ets-1 in activating transcription of the vascular endothelial growth factor receptor-2 (VEGF-2) gene (Flk-1). The receptor tyrosine kinase Flk-1 is indispensable for angiogenesis, and its expression is closely regulated during development. Consistent with the hypothesis that HIF-2α controls the expression of Flk-1 in vivo, we show here that HIF-2α and Flk-1 are co-regulated in postnatal mouse brain capillaries. A tandem HIF-2α/Ets binding site was identified within the Flk-1 promoter that acted as a strong enhancer element. Based on the analysis of transgenic mouse embryos, these motifs are essential for endothelial cell-specific reporter gene expression. A single HIF-2α/Ets element conferred strong cooperative induction by HIF-2α and Ets-1 when fused to a heterologous promoter and was most active in endothelial cells. The physical interaction of HIF-2α with Ets-1 was demonstrated and localized to the HIF-2α carboxyl terminus and the autoinhibitory exon VII domain of Ets-1, respectively. The deletion of the DNA binding and carboxyl-terminal transactivation domains of HIF-2α, respectively, created dominant negative mutants that suppressed transactivation by the wild type protein and failed to synergize with Ets-1. These results suggest that the interaction between HIF-2α and endothelial Ets factors is required for the full transcriptional activation of Flk-1 in endothelial cells and may therefore represent a future target for the manipulation of angiogenesis.