17-β Estradiol Protects ARPE-19 Cells from Oxidative Stress through Estrogen Receptor-β

PURPOSE. To elucidate the mechanism of 17-beta estradiol (17 beta-E-2)-mediated protection of retinal pigment epithelium (RPE) from oxidative stress. METHODS. Cultured ARPE-19 cells were subjected to oxidative stress with t-butyl hydroxide or hydrogen peroxide in the presence or absence of 17 beta-E-2. Reactive oxygen species (ROS) were measured using H(2)DCFDA fluorescence. Apoptosis was evaluated by cell-death ELISA kit and Hoechst-3486 staining. Mitochondrial membrane potential was measured using the JC-1 assay. Cellular localization of estrogen receptor (ER) was evaluated by confocal microscopy. Gene expression and protein expression was quantified using qRT-PCR and western blotting. Superoxide dismutase and ATP levels were measured using commercial kits. RESULTS. ARPE-19 cells expressed significant amounts of ER alpha and ER beta. Pretreatment with 17 beta-E-2 protected ARPE-19 cells from oxidative stress and apoptosis. 17 beta-E-2 reduced the ROS levels and mitochondrial depolarization. The 17 beta-E-2-mediated cytoprotection was inhibited by ER antagonists ICI (ER alpha and ER beta) and THC (ER beta) but not by tamoxifen (ER alpha). Knockdown of ER beta expression by siRNA abolished the protective effects of 17 beta-E-2. Further, qRT-PCR analysis revealed that 17 beta-E-2 pretreatment upregulated the expression of ER beta and phase II cellular antioxidant genes. CONCLUSIONS. These results indicate that 17 beta-E-2 protects ARPE-19 cells from oxidative stress through an ER beta-dependent mechanism. 17 beta-E-2-mediated cytoprotection occurred through the preservation of mitochondrial function, reduction of ROS production, and induction of cellular antioxidant genes. (Invest Ophthalmol Vis Sci. 2010;51:5278-5287) DOI:10.1167/iovs.10-5316