INCREASED EXPRESSION OF GLUTAREDOXIN 1 (Grx1) PROTECTS HUMAN RETINAL PIGMENT EPITHELIAL CELLS FROM OXIDATIVE DAMAGE

Oxidative stress is believed to contribute to the pathogenesis of many diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial (RPE) cells are considered as major targets. It is widely accepted that the RPE cells have enormous number of thiol-containing proteins, which can undergo modifications to change retinal protein functions. In contrast, the mechanism of thiol redox regulation in the retina and its association with AMD are still very poorly understood. In particular, the function of glutaredoxin 1 (Grx1), a thiol repair enzyme in cytosol, is virtually unknown. This project seeks to address this paucity in a comprehensive and physiological relevant fashion, and therefore is both novel and innovative. Furthermore, the ability to identify novel therapeutic targets for further research is the first critical step in finding new treatments for AMD. The overall success of this project will raise new hope that Grx1 or its mimic may be a potential therapeutic agent for AMD, and perhaps for other ocular diseases induced by oxidative stress. Purpose (a): The retina is constantly exposed to oxidative stress, which is countered by well-designed antioxidant systems present in retinal pigment epithelial (RPE) cells. Disruption of these systems may lead to the development of age-related macular degeneration (AMD). In this study, we explored the strategy of overexpressing glutaredoxin 1 (Grx1), a component of the endogenous antioxidant defense system, to combat oxidative damage in RPE cells. Methods (b): Human retinal pigment epithelial (ARPE-19) cells were transfected with either a Grx1-containing plasmid or an empty vector. Normal ARPE-19 cells and transfected cells were treated with or without 200 µM H2O2 for 24 h. Grx1 protein expression was detected by western blots and enzyme activity was measured by spectrophotometry. Cell viability was measured by a colorimetric assay with WST8. The morphology of nuclear chromatin was assessed by staining with Hoechst 33342. Apoptosis was quantitatively analyzed by flow cytometry. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Results (c): Grx1 protein level and enzyme activity in Grx1 transfected cells were significantly increased as compared to non-transfected and vector transfected cells. Grx1 overexpression protected ARPE-19 cells from H2O2-induced cell viability loss. Assessment of apoptosis indicated that cells transfected with Grx1 were relatively more resistant to H2O2 with fewer cells undergoing apoptosis as compared to vector control or non-transfected cells. Furthermore, PSSG accumulation was also dramatically attenuated by Grx1 overexpression. Conclusions (d): Grx1 can protect human retinal pigment epithelial cells against H2O2-induced cell death. The mechanism of this protection is likely associated with its ability to prevent lethal accumulation of PSSG.