Expression and Regulation of Methionine Sulfoxide Reductases (Msrs) in RPE

Abstract

Abstract: : Purpose:Msrs (MsrA and MsrB) play a vital role in protein repair, scavenging of free radicals and in increasing cell survival and life span. Our previous work has shown that ceramide–induced oxidative stress results in apoptosis in RPE which can be partially attenuated by antioxidants and growth factors. We have now investigated the expression, regulation and role of Msrs in protecting RPE from oxidant injury. Methods:Fetal RPE of early passages (3–4) maintained in 1% FBS overnight were exposed to H2O2 (50–200 µM for 24h) or the endoplasmic reticulum stressor tunicamycin (TM; 150–1000 ng/ml for 24h). Gene expression of MsrA and CBS–1 (the human analog of MsrB2) was determined by real–time PCR. Reactive oxygen species (ROS) production and localization of MsrA in control and 150µM H2O2 exposed RPE were determined by confocal microscopy. Effect of knockdown (silencing) of MsrA on apoptosis was performed using siRNA. Expression of MsrA in adult retina and in patients with age–related macular degeneration (AMD) was examined by immunostaining with a specific polyclonal antibody for MsrA. Results:Both H2O2 and TM caused a dose–dependent upregulation of MsrA and CBS–1 genes. The maximal increases for MsrA and CBS–1 were attained with 150 µM H2O2 (6 and 3–fold resp.) and 750ng TM (>10–fold). Translocation of MsrA to mitochondria correlated with increased mitochondrial ROS accumulation with H2O2 treatment as compared to controls. In normal adult retina, MsrA expression was found in inner nuclear layer and around the blood vessels. Focal, strong staining was observed in Drusen from AMD patients. Conclusions: MsrA and CBS–1 are upregulated in oxidative and ER stress to counteract the injury to RPE. Expression of Msr family members in AMD and Drusen suggests a critical role for Msrs in retinal protection.

Keywords: oxidation/oxidative or free radical damage • mitochondria • antioxidants