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Weilue He, Srinivas Sripathi, Beth M. Elledge, Cameron Atkinson, Zhicong Liu, Phillip Mercier, Parrisha Louis, Wan Jin Jahng; Hypoxia and Hyperoxia Induce RPE Apoptosis through RPE65 Cleavage. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3359.
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Mutations of all-trans-retinyl ester isomerohydrolase RPE65 have been observed in inherited RPE degeneration patients. Studies on RPE65 expression and degradation will bridge the gap between retinal diseases and the exogenous environment, including oxidative stress. Our goal is to use RPE65 as a mediator to investigate the caspase dependent apoptosis process in ARPE-19 cells in abnormal oxygen concentrations; specifically, how hypoxia and hyperoxia control RPE65 turnover.
Human RPE cell line ARPE-19 was cultured in hypoxia, normoxia, and hyperoxia. RPE65 mRNA levels under different oxygen conditions were tested by real-time PCR. The bioinformatics method was applied to analyze RPE65 promoter. RPE65 cleavage and protein interactions were analyzed by Western blotting and coimmunoprecipitation (Co-IP). Caspase-3 activity in different oxygen concentrations was assessed by standardized fluorometric immunosorbent enzyme assay. RPE65 cleavage in erythropoietin (EPO) knock-down model was examined by Western blotting. Pro-apoptotic function of RPE45 and RPE20 in ARPE-19 cell was examined by cell survival assay to check cell viability. Localization of RPE65 was analyzed by confocal fluorescent microscopy.
In both hypoxia and hyperoxia, RPE65 undergoes accelerated truncation producing 45 and 20kDa fragments. Fragmentation of RPE65 induces ubiquitination shown by Co-IP. RPE65 mRNA level is increased in both hypoxia and hyperoxia, consistent with diabetes models. Predicted oxygen responding sites and transcriptional regulators were selected and examined. EPO knock-down model using siRNA showed up-regulation of RPE65 cleavage.
The expression pattern of RPE65 is oxygen dependent. RPE65 cleavage is induced by hypoxia and hyperoxia which diabetic patients are likely to experience. Formation of RPE45 and RPE20 fragment accelerates RPE cell apoptosis. RPE65 turnover pathway may suggest its role as a pro-apoptotic mediator, which can be partially redeemed by EPO. RPE65 dysfunction can be partially prevented by target protease inhibition.
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