The Hypoxia-Inducible Factor 1/NOR-1 Axis Regulates the Survival Response of Endothelial Cells to Hypoxia

Abstract

Hypoxia induces apoptosis but also triggers adaptive mechanisms to ensure cell survival. Here we show that the prosurvival effects of hypoxia-inducible factor 1 (HIF-1) in endothelial cells are mediated by neuron-derived orphan receptor 1 (NOR-1). The overexpression of NOR-1 decreased the rate of endothelial cells undergoing apoptosis in cultures exposed to hypoxia, while the inhibition of NOR-1 increased cell apoptosis. Hypoxia upregulated NOR-1 mRNA levels in a time- and dose-dependent manner. Blocking antibodies against VEGF or SU5614 (a VEGF receptor 2 inhibitor) did not prevent hypoxia-induced NOR-1 expression, suggesting that NOR-1 is not induced by the autocrine secretion of VEGF in response to hypoxia. The reduction of HIF-1α protein levels by small interfering RNAs, or by inhibitors of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway or mTOR, significantly counteracted hypoxia-induced NOR-1 upregulation. Intracellular Ca²⁺ was involved in hypoxia-induced PI3K/Akt activation and in the downstream NOR-1 upregulation. A hypoxia response element mediated the transcriptional activation of NOR-1 induced by hypoxia as we show by transient transfection and chromatin immunoprecipitation assays. Finally, the attenuation of NOR-1 expression reduced both basal and hypoxia-induced cIAP2 (cellular inhibitor of apoptosis protein 2) mRNA levels, while NOR-1 overexpression upregulated cIAP2. Therefore, NOR-1 is a downstream effector of HIF-1 signaling involved in the survival response of endothelial cells to hypoxia.

ACKNOWLEDGMENTS

We thank Naganari Ohkura for generously providing the pNORα/-1703 construct and L. Eric Huang for the pCDNA3-HIF-1α expression vector. We also thank Esther Peña for her help with confocal microscopy.

This work was supported by the Ministerio de Educación y Ciencia (SAF2006-07378 and SAF2006-1009), Red Temática de Investigación Cardiovascular (RECAVA; RD06/0014/0027), PI061480 from the Ministerio de Sanidad-Instituto de Salud Carlos III, and Fundación Española de Arteriosclerosis-Sociedad Española de Arteriosclerosis (FEA-SEA 2006). M.G. is a recipient of a research fellowship (FI) from Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) de la Generalitat de Catalunya.