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Research Article

Protein Expression Profiling of Lens Epithelial Cells from Prdx6-depleted Mice and Their Vulnerability to UV Radiation Exposure

¹University of Fukui ²University of Nebraska Medical Center

Submitted 24 July 2009 ; revised 8 October 2009 ; accepted in final form 29 October 2009

Oxidative stress is one of the causative factors in the progression and etiology of age-related cataract. The Peroxiredoxin 6 (Prdx6), a savior for cells from internal or external environmental stresses, plays a role in cellular signaling by detoxifying reactive oxygen species (ROS) and thereby controlling gene regulation. Using targeted inactivation of the Prdx6 gene, here we show that Prdx6-deficient lens epithelial cells (LECs) are more vulnerable to UV-triggered cell death, a major cause of skin disorders including cataractogenesis, and these cells display abnormal protein profiles. PRDX6-depleted LECs showed phenotypic changes and formed lentoid body, a characteristic of terminal cell differentiation and epithelial mesenchymal transition (EMT). Prdx6^-/- LECs exposed to UV-B showed higher ROS expression, and were prone to apoptosis compared to wild-type LECs, underscoring protective role for Prdx6. The comparative proteomic analysis using fluorescence-based difference gel electrophoresis along with Mass spectrometry and database searching revealed a total number of 13 proteins that were differentially expressed in Prdx6^-/- cells. Six proteins were upregulated, where as expression of 7 proteins were decreased compared with Prdx6^+/+ LECs. Among the cytoskeleton associated proteins which were highly expressed in Prdx6-deficient LECs was tropomyosin (Tm) 2β. Protein blot and real-time PCR validated dramatic increase of Tm2β and Tm1 expression in these cells. Importantly, Prdx6^+/+ LECs showed similar pattern of Tm2β protein expression following TGFβ or H₂O₂ treatment. An extrinsic supply of PRDX6 could restore the Tm2β expression, demonstrating that PRDX6 may attenuate adverse signaling in cells and thereby maintains cellular homeostasis. By exploring redox-proteomic (Prdx6^-/-) and characterization and identification of the abnormally expressed proteins, and their attenuation by PRDX6 delivery should provide a base to develop a novel therapeutic interventions to postponing ROS-mediated abnormal signaling deleterious to cells or tissues.

peroxiredoxin; oxidative stress; proteomics; lens epithelial cells