Oxidative stress upregulates ubiquitin proteasome pathway in retinal endothelial cells

Abstract

Purpose: To establish whether oxidative stress in retinal endothelial cells upregulates the ubiquitin proteasome pathway (UPP) leading to increased protein degradation in diabetes. Methods: Retinal endothelial cells were exposed to a continuous flux of hydrogen peroxide produced by glucose oxidase. Endogenous ubiquitin conjugates were detected by western blotting. The ubiquitin conjugating activity was determined using radiolabeled ubiquitin or α-lactalbumin. The turnover of ubiquitin conjugates was determined by pulse-chase ex- periments, using radiolabeled ubiquitin. Levels of mRNA were determined by radioactive northern blot and by real time PCR. Results: The exposure of endothelial cells to physiological concentrations of hydrogen peroxide led to an increase in ubiquitin conjugating activity to both endogenous and exogenous substrates. Remarkably, the endogenous ubiquitin con- jugates did not change in response to oxidative stress presumably because the turnover of conjugates was also increased as revealed by pulse-chase experiments with radiolabeled ubiquitin. Exposure of retinal endothelial cells to oxidative stress further resulted in an increase in the levels of mRNA that encode for polyubiquitin chains or ubiquitin fused to carboxyl extension proteins. Conclusions: Oxidative stress upregulated UPP and increased turnover of ubiquitin conjugates. Upregulation of UPP may account for cell response to stress in conditions where oxidative stress is overexpressed