Hypoxia and FSH Enhances HIF1 Activity in Rat Granulosa Cells.

Abstract

A hypoxic environment is unfavorable for growth and development for most cell types. However, within the maturing follicle, the internal granulosa cells (GCs) are exposed to an increasingly hypoxic environment. We previously reported that FSH promotes the accumulation of hypoxia inducible factor 1α (HIF1α) protein and activation of HIF1 transcriptional activity in GCs. HIF1 is a heterodimeric transcription factor composed of HIF1α and constitutively expressed HIF1β. FSH increases HIF1α protein levels by activating the phosphatidylinositol 3-kinase pathway to enhance translation of HIF1α mRNA. Our results in GCs suggest that HIF1 is a transcriptional activator not only for vascular endothelial growth factor (VEGF) but also for the luteinizing hormone/ choriogonadotropin receptor (Lhcgr) genes. We investigated the hypothesis that the FSH-stimulated increase in HIF1α accumulation as well as HIF1 activity will be greater in presence of a hypoxic versus a normoxic environment. HIF1 activity is classically activated under hypoxic conditions by the hypoxia-induced stabilization of HIF1α protein. We determined the effects of hypoxia on HIF1α protein accumulation and HIF1 activity using both using western blot technique and a HRE- Luciferase reporter assay. Our results showed that equivalent levels of HIF1α accumulated in GCs treated with FSH under both hypoxic (5% O2) and normoxic (21% O2) conditions. Because HIF1α protein accumulation is not sufficient for HIF1 activity, we also investigated HIF1 activity under normoxic versus hypoxic conditions. Results showed that FSH increased HIF1 activity approximately 5-fold under normoxia. With hypoxia, HIF1 activity increased approximately 2-fold; however, this increase was treatment independent. These results lead us to conclude that both FSH stimulation and hypoxia are necessary for maximum HIF1 activity and thus maximum target gene activation. However, we hypothesize that FSH and hypoxia enhance HIF1 activity independently. Understanding the elements that regulate HIF1 activity will allow for a greater understanding of the role of HIF1 in follicular maturation and fertility. Our results may also provide insight into the misregulation of HIF1α expression and its targets seen in many cancer cells. Supported by NIH RO1 HD065859 (to M.H.D.). (poster)