GTx-822, an ERβ-Selective Agonist, Protects Retinal Pigment Epithelium (ARPE-19) from Oxidative Stress by Activating MAPK and PI3-K Pathways

PURPOSE. The goal of this study was to determine whether an estrogen receptor-beta (ER beta)-selective agonist (GTx-822; GTx, Inc., Memphis, TN) could prevent hydrogen peroxide (H2O2)-induced oxidative stress in ARPE-19 cells and to elucidate the molecular pathways involved in this protection. METHODS. The selectivity of GTx-822 for ER beta was determined by receptor-binding assay (RBA) and transactivation assay. Cultured ARPE-19 cells were subjected to oxidative stress with t-butyl hydroxide (t-BH) or hydrogen peroxide (H2O2) in the presence and absence of GTx-822. Reactive oxygen species (ROS) was measured by using H(2)DCFDA fluorescence. Apoptosis was evaluated by cell death ELISA. Mitochondrial membrane potential was measured with the JC-1 assay. Gene expression and protein expression and activation were quantitated with qRT-PCR and Western blot analysis. Phospho-protein arrays elucidated the activation of protein kinases. RESULTS. The RBA and transactivation assay revealed that GTx-822 is an ER beta-selective agonist (K-i = 0.53 nM). GTx-822 prevented oxidative stress in ARPE-19 cells. It preserved mitochondrial function and prevented cellular apoptosis. Pretreatment with GTx-822 increased ER beta gene and protein expression during oxidative stress. Upregulation of the phase II antioxidant genes GPx-2 and HO-1 was also seen in an ER beta-dependent mechanism. GTx-822 pretreatment induced phosphorylation of ERK1/2, PI3-K, and Bad. CONCLUSIONS. This is the first report to show that GTx-822, an ER beta agonist, can protect ARPE-19 cells from the cellular apoptosis induced by oxidative stress. GTx-822 mediated cytoprotection was mediated through induction of both genomic and nongenomic pathways. The results of this study open new avenues for the use of a selective ER beta agonist in treatment of ocular diseases like AMD where oxidative stress plays a major role in disease pathogenesis. (Invest Ophthalmol Vis Sci. 2010; 51:5934-5942) DOI:10.1167/iovs.10-5630