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Effect of Thioltransferase (Glutaredoxin) Deletion on Cellular Sensitivity to Oxidative Stress and Cell Proliferation in Lens Epithelial Cells of Thioltransferase Knockout Mouse

¹From the Department of Veterinary and Biomedical Sciences and the ²Center for Redox Biology, University of Nebraska-Lincoln, Lincoln, Nebraska; the ⁵Departments of Pathology and Microbiology and ⁷Ophthalmology, University of Nebraska Medical Center, Omaha, Nebraska; and the ⁶Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan.

PURPOSE. To examine the physiological function of the thioltransferase (TTase)/glutathione (GSH) system in the lens using TTase knockout mouse (TTase^–/–) lens epithelial cells (LECs) as a model.

METHODS. Primary LEC cultures were obtained from wild-type (TTase^+/+) and TTase^–/– mice. Characterization and validation of the cells were determined by immunoblotting for TTase and -crystallin proteins and by immunohistochemistry for glutathionylated proteins. Cell proliferation was examined by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and BrdU analysis, and cell apoptosis after H₂O₂ stress was assessed by fluorescence-activated cell sorter analysis. Reloading of TTase protein into the TTase^–/– cells was achieved with reagent.

RESULTS. Primary LEC cultures obtained from wild-type (TTase^+/+) and TTase^–/– mice were characterized and found to contain lens-specific -crystallin protein. Western blot analysis confirmed the absence of TTase protein in the TTase^–/– cells and its presence in the wild-type cells. TTase^–/– LECs had significantly lower levels of glutathione (GSH) and protein thiols with extensive elevation of glutathionylated proteins, and they exhibited less resistance to oxidative stress than did TTase^+/+ cells. These cells were less viable and more apoptotic, and they had a reduced ability to remove H₂O₂ after challenge with low levels of H₂O₂. Reloading of purified TTase into the TTase^–/– cells restored the antioxidant function in TTase^–/– cells to a near normal state.

CONCLUSIONS. These findings confirm the importance of TTase in regulating redox homeostasis and suggest a new physiological function in controlling cell proliferation in the lens epithelial cells.

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				Y. Wang and M. F. Lou The Regulation of NADPH Oxidase and Its Association with Cell Proliferation in Human Lens Epithelial Cells Invest. Ophthalmol. Vis. Sci., May 1, 2009; 50(5): 2291 - 2300. [Abstract] [Full Text] [PDF]