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Vascular endothelial growth factor (VEGF)-dependent angiogenesis plays a crucial role in corpus luteum formation and its functional maintenance in mammalian ovaries. We recently reported that activation of hypoxia-inducible factor (HIF)-1α signaling contributes to the regulation of VEGF expression in luteal cells (LCs) in response to human chorionic gonadotropin (HCG). We examined whether HIF prolyl-hydroxylase (PHD)-2 gene silencing induces VEGF expression in LCs and enhanced its expression induced by HCG in LCs. Using real-time polymerase chain reaction and western blot analysis, we measured the expression of PHD-2 to confirm plasmid PHD-2 shRNA transfection and protein expression and investigated the changes in HIF-1a and VEGF expression after treatment with HCG and PHD-2 shRNA transfection. After PHD-2 shRNA transfection, PHD-2 expression was significantly lower than that in control groups with or without HCG treatment, while a significant increase in VEGF mRNA was observed compared to in controls, indicating that PHD-2 plays an important role in VEGF regulation. Additionally, changes in VEGF mRNA expression were consistent with the expression levels of HIF-1a protein, not HIF- 1a mRNA, which is regulated by HIF prolyl-hydroxylase-mediated degradation. Our results indicate that PHD-2 gene silencing can induce VEGF expression in LCs and HCG-induced VEGF expression can be further enhanced by PHD-2 gene silencing through an HIF-1a-mediated mechanism in LCs. This PHD-2-mediated transcriptional activation may be important for regulating VEGF expression through HIF-1a signaling in LCs during corpus luteum development in mammals.

Effects of HIF prolyl-hydroxylase-2 gene silencing on HCG-induced vascular endothelial growth factor expression in luteal cells