Triamcinolone Acetonide Decreases HIF–1 Binding to Hypoxia Response Element in Cobalt Stimulated Mueller Cells

Abstract

Abstract: : Purpose: To identify the molecular mechanism of steroid induced resolution of macular edema. Methods: Confluent cultures of human Mueller cells (MIO–M1) were serum–starved for 48 hours followed by treatment with 100 µM CoCl₂, 1 µg/ml triamcinolone acetonide (TA), or both. Vascular endothelial growth factor (VEGF) secretion was measured in the supernatants of cultures with respect to time by ELISA. VEGF mRNA was analyzed by reverse transcriptase polymerase chain reaction. The activity of the heterodimer hypoxia inducible factor–1 (HIF–1) which is composed of inducible HIF–1α and constitutive HIF–1ß subunits was measured by the relative binding of HIF–1 protein to the hypoxia response element (HRE). HIF–1α protein level was determined using Western blot. Results: TA decreased VEGF secretion by at least 50% in the presence of continuous cobalt stimulus. VEGF mRNA decreased 50–100 fold 6 hours post treatment with TA and cobalt compared to cobalt alone. HIF–1α protein peaked at 6 hours after cobalt treatment and was sustained for the entire 24 hour treatment period. HIF–1α protein level was equal in cobalt and TA/cobalt treated cells and partitioned into nuclear not cytosolic fractions. However, HIF–1 binding to the HRE was decreased by 30% in the presence of TA and cobalt compared to cobalt alone. Conclusions: TA decreases VEGF secretion and VEGF mRNA concentration in cobalt stimulated Mueller cells. No effect is seen on total HIF–1α protein level, but HIF–1 activity is decreased. TA may induce a post–translation modification of HIF–1 or availability of cofactors that does not affect nuclear translocation but does affect DNA binding.

Keywords: hypoxia • gene/expression • signal transduction: pharmacology/physiology