The Transcriptional Activator Hypoxia Inducible Factor 2 (HIF-2/EPAS-1) Regulates the Oxygen-Dependent Expression of Erythropoietin in Cortical Astrocytes

Abstract

In the ischemic or hypoxic brain, astrocytes appear to be one of the main sources of erythropoietin (EPO). In this study, we investigated the differential contribution of hypoxia inducible factor (HIF) isoforms to the regulation of hypoxic EPO expression in cultured astrocytes. In addition, using an in vitro model of oxygen-glucose deprivation (OGD), we studied the role of HIF-1alpha and HIF-2alpha in the generation of paracrine protective signals by astrocytes that modulate the survival of neurons exposed to OGD. Expression of HIF-1alpha or HIF-2alpha was abrogated by infecting astrocytes with lentiviral particles encoding small interference RNA specific for HIF-1alpha or HIF-2alpha (siHIF-1alpha or siHIF-2alpha). Astrocytes infected with siHIF-1alpha showed abrogated hypoxic induction of vascular endothelial growth factor (VEGF) and lactate dehydrogenase (LDH) but normal EPO induction. In contrast, reduction of HIF-2alpha expression by siHIF-2alpha led to a drastic decrease of EPO hypoxic expression, but it did not affect LDH or VEGF upregulation. To further test whether HIF-2 is sufficient to drive EPO upregulation, we expressed oxygen-insensitive mutant forms of HIF-1alpha (mtHIF-1alpha) (P402A/P577A) and HIF-2alpha (mtHIF-2alpha) (P405A/P530A). Expression of mtHIF-2alpha but not mtHIF-1alpha in normoxic astrocytes resulted in a significant upregulation of EPO mRNA and protein. Accordingly, HIF-2alpha but not HIF-1alpha was found to be associated with the EPO hypoxia-response element by a chromatin immunoprecipitation assay. Interestingly, conditioned medium from astrocytes challenged by sublethal OGD improved neuronal survival to OGD; however, this effect was abolished during the downregulation of astrocytic HIF-2alpha using siHIF-2alpha. These results indicate that HIF-2alpha mediates the transcriptional activation of EPO expression in astrocytes, and this pathway may promote astrocytic paracrine-dependent neuronal survival during ischemia.