p66shc Regulates Redox–Sensitive NF–B Activation and Oxidative Stress–Induced Apoptosis in Human RPE Cells

Abstract

Abstract: : Purpose: The p66^shc protein has been implicated as a possible signal involved in pathways activated by oxidative stress. In this study, we investigated the effect of siRNA–induced knock–down of p66shc in cultured RPE cells or retina exposed to oxidative stress. Methods: ARPE–19 cells were transfected using oligofectamine with p66^shc siRNA or GFP siRNA (controls). The specificity and efficiency of p66shc knock–down was analyzed by western blots. Cell viability and apoptosis were assessed by trypan blue exclusion and TdT–dUTP terminal nick–end labeling (TUNEL) assay, respectively. Intracellular reactive oxygen species (ROS) were visualized by the redox–sensitive flurophore 2’, 7’– dichloroflurescein diacetate (DCFDA). Levels of mRNA for several antioxidant defense genes including SODs, GPX, GST and catalase were measured by real–time PCR. NF–ΚB activity was determined using the dual luciferase report assay system after co–transfection of pNF–ΚB–luc with siRNA. Retinal function was evaluated by electroretinography (ERG) after intravitreous injection of paraquat followed by subretinal injection of either p66^shc siRNA or GFP siRNA. Results: There was a 60% reduction in p66^shc in RPE cells 72 hours after incubation with p66^shc siRNA compared to GFP siRNA. Exposure of RPE cells to H₂O₂ or paraquat resulted in enhanced DCFDA–induced cellular fluorescence and a striking increase in TUNEL–positive cells. Incubation with p66^shc siRNA, but not GFP siRNA, prior to exposure to H₂O₂ or paraquat resulted in reduction of DCFDA–induced fluorescence and a 10–fold decrease in TUNEL–positive cells. The mRNAs for several antioxidant genes were significantly increased by incubation of RPE cells with p66^shc siRNA and there was a 2–fold increase in NF–ΚB activity. Compared to mice injected with GFP siRNA, those injected with p66^shc siRNA had less paraquat–induced reduction in ERG amplitudes. Conclusions: Paraquat– or H₂O₂–induced oxidative damage in RPE and retinal cells is at least partially dependent on p66^shc and it may be a good target for treatment of diseases such as AMD in which oxidative damage has been implicated.

Keywords: oxidation/oxidative or free radical damage • signal transduction • gene/expression