Regulation of the RPE Function by Extracellular Cystine (CySS)

Abstract

Abstract: : Purpose: Our previous studies showed that variations in the ratios of the extracellular cysteine (Cys) and CySS within the physiological ranges controlled the responses of cultured human RPE (hRPE) cells to oxidant–induced apoptosis. The current study is aimed to identify molecular targets that are subject to extracellular redox regulation. Methods: ARPE–19 cells were cultured in media containing 10 to 200 µM Cys and CySS. Time–dependent changes in the extra– and intra–cellular Cys(s) contents were measured by HPLC. Differential gene expression was analyzed with microarray approach, using total RNA isolated from cells cultured under different redox conditions. The array data were verified by real–time RT–PCR and Western blot analyses. Results: At concentrations higher than 50 µM, the extracellular CySS decreased after 2hr, suggesting that CySS could be transported by cultured hRPE cells when reaching the physiological concentration as detected in human plasma. Changes in the extracellular redox controlled the mRNA levels of cell survival genes such as Akt and FKBP38. Increased CySS downregulated both the expression and activity of Akt. The effects were independent of glutathione concentration. The extracellular CySS also regulated the expression of αVß5 integrins that are associated with phagocytosis. Conclusions: RPE function may be regulated by altering concentrations of the extracellular CySS. Changes in the extracellular redox environment, which are associated with aging and smoking, control the expression and activity of cell survival genes. These observations suggest that control of extracellular redox status may be a rationale for intervention in diseases such as age–related macular degeneration.

Keywords: age-related macular degeneration • oxidation/oxidative or free radical damage • apoptosis/cell death