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 language | English (US) |
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Pages (from-to) | 4497-4505 |
Number of pages | 9 |
Journal | Investigative Ophthalmology and Visual Science |
Volume | 49 |
Issue number | 10 |
DOIs | |
State | Published - Oct 2008 |
Externally published | Yes |
ASJC Scopus subject areas
- Ophthalmology
- Sensory Systems
- Cellular and Molecular Neuroscience