Effect of thioltransferase (glutaredoxin) deletion on cellular sensitivity to oxidative stress and cell proliferation in lens epithelial cells of thioltransferase knockout mouse.
OBJECTIVE
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 alpha-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 alpha-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.
Löfgren
Stefan
S
Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0905, USA.
Fernando
M Rohan
MR
Xing
Kui-Yi
KY
Wang
Yin
Y
Kuszynski
Charles A
CA
Ho
Ye-Shih
YS
Lou
Marjorie F
MF
D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D003600:Cytosol; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D054477:Glutaredoxins / Q000494:pharmacology / Q000502:physiology*; D005978:Glutathione / Q000502:physiology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000633:toxicity; D007908:Lens, Crystalline / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018384:Oxidative Stress; D011994:Recombinant Proteins / Q000494:pharmacology; D038202:alpha-Crystallin A Chain / Q000378:metabolism
D054477:Glutaredoxins; D011994:Recombinant Proteins; D038202:alpha-Crystallin A Chain; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005978:Glutathione