Characterization And Regulation Of Carrier Proteins Of Mitochondrial GSH Uptake In Human Retinal Pigment Epithelium Cells

BACKGROUND&AIMS:Since mGSH plays a significant role in cellular defense against pro-oxidants for the health of retinal pigment epithelium（RPE）,the transport of mGSH is not fully understood,the two mitochondrial membrane carriers the 2-oxoglutarate（OGC;SLC25A11）and dicarboxylate（DIC;SLC25A10）carriers localized to the inner mitochondrial membrane have been shown to function as GSH transporters mainly in liver,kidney,and lung.Our aim is to characterize the two mitochondrial membrane transporters OGC and DIC in human RPE（hRPE）and elucidate their role in the regulation of mitochondrial GSH（mGSH）uptake and cell death in oxidative stress.METHODS:The localization of OGC and DIC protein in confluent hRPE,polarized hRPE monolayers and mouse retina was assessed by immunoblotting and confocal microscopy.Time-and dose-dependent expression of the two carriers was determined after treatment of hRPE with H₂O₂,phenylsuccinate（PS）,and butyl malonate（BM）respectively for 24 hours.The effect of chemical inhibition of OGC and DIC on apoptosis（TUNEL）,mGSH level,and mtDNA was determined.Silencing of OGC by siRNA knockdown on RPE cell death was also studied.Kinetics of Caspase-3/7 activation with OGC and DIC inhibitors and attenuation of cell death by co-treatment with glutathione monoethyl ester（GSH-MEE）was determined using the IncuCyte live cell imaging.The effect of induction of polarity on the expression of carrier proteins was also assessed.RESULTS:OGC and DIC are expressed in hRPE mitochondria and exhibited a time-and dose-dependent decrease in stressed hRPE.Pharmacologic inhibition caused a decrease in OGC and DIC in mitochondria without any changes in mtDNA,and resulted in increased apoptosis and mGSH depletion;GSH-MEE prevented apoptosis through restoration of mGSH.Further,siRNA silencing of OGC exacerbated apoptotic cell death in stressed RPE which was inhibited by GSH-MEE co-treatment from increased mGSH.OGC and DIC expression were increased in polarized-RPE than that of non-polarized RPE.When OGC and DIC were inhibited by chemical inhibitors respectivley,the tight junction（ZO-1 protein）was broken in polarized-RPE,and co-treatment with GSH-MEE could improve the destruction of typical cell structure of polarized hRPE.CONCLUSION:Our findings show that the characterization and mechanism of action of two carrier proteins that mediate mGSH uptake in RPE for the first time.Regulation of OGC and DIC will be of value in devising therapeutic strategies for retinal disorders such as age related macular degeneration（AMD）.