Effect of thioltransferase (glutaredoxin) deletion on cellular sensitivity to oxidative stress and cell proliferation in lens epithelial cells of thioltransferase knockout mouse
| Autor: | Kui Yi Xing, Charles A. Kuszynski, Yin Wang, Marjorie F. Lou, M. Rohan Fernando, Ye-Shih Ho, Stefan Löfgren |
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| Rok vydání: | 2008 |
| Předmět: |
Cell Survival
Blotting Western Apoptosis Biology medicine.disease_cause alpha-Crystallin A Chain chemistry.chemical_compound Mice Cytosol Western blot Glutaredoxin Lens Crystalline medicine Animals Cells Cultured Glutaredoxins Cell Proliferation Mice Knockout medicine.diagnostic_test Cell growth Glyceraldehyde-3-Phosphate Dehydrogenases Epithelial Cells Glutathione Hydrogen Peroxide Blotting Northern Flow Cytometry Molecular biology Recombinant Proteins Blot Mice Inbred C57BL Oxidative Stress chemistry Oxidative stress |
| Zdroj: | Investigative ophthalmologyvisual science. 49(10) |
| ISSN: | 1552-5783 |
| Popis: | 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 α-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 H 2 O 2 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 α-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 H 2 O 2 after challenge with low levels of H 2 O 2 . 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. |
| Databáze: | OpenAIRE |
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