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

Biology of hypoxia-inducible factor-2α in development and disease

“…31,46 Local synthesis of Epo by astrocytes in the central nervous system also appears to be regulated by HIF-2a and may be important for survival of neurons during periods of ischemia. 47 O 2 transport by red blood cells is mediated by reversible binding to iron present in the hemoglobin molecule. Therefore, organisms must coordinate erthyropoiesis with iron availability.…”

Targeting Astrocytes for Stroke Therapy

“…[85][86][87][88] Recent reports suggest that another HIF family member, HIF-2, is the major inducer of EPO in hypoxia. 39,89 Not only does EPO production by astrocytes have local effects, but ablation of HIF-2 function selectively in astrocytes causes anemia during hypoxia-ischemia, demonstrating the importance of astrocyte EPO production for erythropoiesis. 90 Several studies suggest a neuroprotective role for EPO (FIG.…”

“…92 Astrocytes contribute to the EPO-mediated neuroprotection, as demonstrated by experiments in which conditioned media taken from hypoxic astrocyte cultures is protective to neurons exposed to OGD. 39 To reduce ischemic damage in the brain, neuroprotectants would need to reduce cell death of both neurons and astrocytes. On this note, it is interesting that not only does EPO protect neurons, but it also enhances viability of astrocytes exposed to NO or experiencing oxidative stress.…”

Cited2, a Transcriptional Modulator Protein, Regulates Metabolism in Murine Embryonic Stem Cells

“…HIF-1 is responsible for controlling the rate of glycolysis in ESC lines with gain-or loss-of-function of HIF-1␣. In HIF-1␣ Ϫ/Ϫ ESCs, the induced expression of genes (including Glut1, HK1, HK2, LDHA, and PGK1) involved in the synthesis of ATP by the conversion of extracellular glucose to intracellular lactate was abrogated in response to hypoxia and hypoglycemic stresses (55,56). Stabilization of HIF-1␣ in ESCs reduces mitochondrial respiration, whereas the rate of extracellular acidification is not significantly affected (35).…”