The transcriptional activator hypoxia inducible factor 2 (HIF-2/EPAS-1) regulates the oxygen-dependent expression of erythropoietin in cortical astrocytes

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-1 alpha and HIF-2 alpha in the generation of paracrine protective signals by astrocytes that modulate the survival of neurons exposed to OGD. Expression of HIF-1 alpha or HIF-2 alpha was abrogated by infecting astrocytes with lentiviral particles encoding small interference RNA specific for HIF-1 alpha or HIF-2 alpha (siHIF-1 alpha or siHIF-2 alpha). Astrocytes infected with siHIF-1 alpha showed abrogated hypoxic induction of vascular endothelial growth factor (VEGF) and lactate dehydrogenase (LDH) but normal EPO induction. In contrast, reduction of HIF-2 alpha expression by siHIF-2 alpha 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-1 alpha(mtHIF-1 alpha) (P402A/ P577A) and HIF-2 alpha( mtHIF-2 alpha) (P405A/P530A). Expression of mtHIF-2 alpha but not mtHIF-1 alpha in normoxic astrocytes resulted in a significant upregulation of EPO mRNA and protein. Accordingly, HIF-2 alpha but not HIF-1 alpha 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-2 alpha using siHIF-2 alpha. These results indicate that HIF-2 alpha mediates the transcriptional activation of EPO expression in astrocytes, and this pathway may promote astrocytic paracrinedependent neuronal survival during ischemia.