Erythropoietin Inhibits HIF-1α Expression via Upregulation of PHD-2 Transcription and Translation in an In Vitro Model of Hypoxia-Ischemia

Hypoxia inducible factor (HIF)-1 alpha is the central transcriptional factor for the regulation of oxygen-associated genes in response to hypoxia. Erythropoietin (EPO), a hematopoietic growth factor, increases oxygen availability during hypoxia/ischemia and is associated with neuroprotection following hypoxia-ischemia in laboratory models of stroke. However, EPO has failed to translate in a clinical setting. Thus, it is critical to elucidate the key players in EPO-induced neuroprotection. Our preliminary studies have shown that EPO, as a downstream gene of HIF, inhibits HIF-1 alpha in a dose-dependent manner in an in vitro model of hypoxia-ischemia. This study is designed to elucidate the primary mediator of EPO-induced HIF-1 alpha inhibition and subsequent cell survival/neuroprotection. Oxygen and glucose deprivation (OGD) of nerve growth factor-differentiated rat pheochromocytoma (PC-12) cells were used to model hypoxia-ischemia in an in vitro environment. The profile of HIF-1 alpha, HIF-2 alpha and prolyl hydroxylase domain 2 (PHD-2) expression; HIF-1 alpha and prolyl hydroxylase (PHD-2) mRNA levels; matrix metalloproteinase (MMP)-9; and cell death was evaluated in the presence and absence of either EPO or PHD-2 inhibitor during OGD. Our findings showed that EPO treatment resulted in an increase in PHD-2 transcription and translation, inhibition of HIF-1 alpha expression, reactive oxygen species formation, and MMP-9 activity, resulting in increased cell survival after OGD. We also observed that EPO-induced cell survival/neuroprotection was reversed by siRNA silencing of PHD-2. This led to the conclusion that PHD-2 is a key mediator of EPO-induced HIF-1 alpha inhibition and subsequent neuroprotection in an in vitro model of hypoxia-ischemia.