Proteomic Exploration of Lens Epithelial Cells in Mice Null for the Antioxidant Protein, Peroxiredoxin 6, and Their Susceptibility to UV-B Exposure

Abstract

Purpose:: Oxidative stress is important in the progression of age-related cataract. The antioxidant protein Peroxiredoxin 6 (Prdx6) protects cells by removing H₂O₂ and mediating survival signaling. The present study quantified the protein changes in lens epithelial cells (LECs) that were deficient in PRDX6 and their response to UV-B induced insults.

Methods:: Prdx6^-/- and Prdx6^+/+ LECs were derived from eye lenses of Prdx6 knockout and wild type mice and maintained in complete DMEM. Cells were exposed to 400 or 800J/m² for variable times. Cell survival assay was done by MTS assay. TUNEL and DAPI staining were done to identify type of cell death. Reactive oxygen species (ROS) levels were measured with H2DCFA-DA dye. Western analysis and real-time quantitative PCR were performed to assess PRDX6 levels and mRNA expression. DIGE (Fluorescence 2-D Difference Gel Electrophoresis) with M_R’s of 10-100kDa and pI’s of 4.0-7.0 and mass spectrometry (MS) were utilized to resolve LEC protein expression profiles. Results were validated with Western analysis using specific antibodies.

Results:: After UV-B exposure, Prdx6^-/- LECs showed higher levels of ROS, were more prone to cell death, and underwent apoptosis, while Prdx6^+/+ LECs displayed reduced levels of PRDX6 protein and mRNA expression. Prdx6^+/+ LECs had more resistance than Prdx6^-/- LECs to UV-B-induced insult, suggesting a protective role for PRDX6. MS and database searching revealed a total of 321 differentially expressed protein spots, with 9 spots differentially up-regulated. The differentially expressed proteins are involved in cell differentiation (^e.g. tropomyosin 2), stress response, cell signaling, and other processes. Also bioinformatic analysis revealed altered expression levels of several proteins associated with stress-related small heat shock proteins and antioxidant enzymes. These differences may reflect mechanisms aimed at compensating for increased ROS. Alteration of protein levels may contribute to the morphological abnormalities in Prdx6^-/- LECs previously observed.

Conclusions:: These findings provide insight into oxidative stress-dependent alterations in LEC protein expression. The study may help to elucidate underlying molecular mechanisms involved in the etiology and progression of cataractogenesis.

Keywords: antioxidants • oxidation/oxidative or free radical damage • proteomics