Effects of hypoxia inducible factor-1α on apoptotic inhibition and glucocorticoid receptor downregulation by dexamethasone in AtT-20 cells

Abstract

Background: Hypoxia inducible factor-1α (HIF-1α) is the central transcriptional regulator of hypoxic responses during the progression of pituitary adenomas. Although previous immunohistochemical studies revealed that HIF-1α is expressed in adreno-cortico-tropic-hormone (ACTH) pituitary adenomas, the role of HIF-1α remains unclear. Methods: AtT-20 cells were incubated under hypoxic conditions (1 % O2) for 12 h. HIF-1α mRNA and protein expression levels were measured by real-time PCR and western blotting, respectively. BrdU was used to determine the effects of hypoxia on cell viability. AtT-20 cells were transfected with siRNA targeting HIF-1α, followed by hypoxia (1 % O2) for 12 h. Apoptosis was determined by annexin V-FITC flow cytometry and Tdt-mediated dUTP nick end-labelling (TUNEL) assay. In addition, we examined interactions between HIF-1α, glucocorticoid receptor (GR), and dexamethasone under both normoxic and hypoxic conditions. Results: Hypoxia triggered the time-dependent proliferation of AtT-20 cells in association with increased HIF-1α mRNA and protein levels. However, the viability of AtT-20 cells decreased greatly when they were first transfected with HIF-1α-siRNA and then exposed to hypoxia. According to flow cytometry (annexin V-FITC and PI staining) and TUNEL analyses, a greater percentage of cells were apoptotic when transfected with HIF-1α siRNA and subsequently cultured under hypoxic conditions compared to those in the normoxia and mock groups. After AtT-20 cells were cultured in 1 % O2 and then treated with dexamethasone, HIF-1α levels significantly increased or decreased in normoxic or hypoxic conditions, respectively. Dexamethasone suppressed GR expression to a higher degree in hypoxic than normoxic conditions. Downregulation of GR by dexamethasone was greatly prevented in cells that were transfected with HIF-1α siRNA. Conclusions: These findings strongly suggest that HIF-1α exerts an antiapoptotic role and participates in the downregulation of GR by dexamethasone in hypoxic AtT-20 cells.