Effect of thioltransferase (glutaredoxin) deletion on cellular sensitivity to oxidative stress and cell proliferation in lens epithelial cells of thioltransferase knockout mouse

Stefan Löfgren, M. Rohan Fernando, Kui Yi Xing, Yin Wang, Charles A. Kuszynski, Ye Shih Ho, Marjorie F. Lou

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30 Scopus citations

Abstract

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 H2O 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 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.

Original languageEnglish (US)
Pages (from-to)4497-4505
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume49
Issue number10
DOIs
StatePublished - Oct 1 2008

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ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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