title: Effect of thioltransferase (glutaredoxin) deletion on cellular sensitivity to oxidative stress and cell proliferation in lens epithelial cells of thioltransferase knockout mouse.
abstract: 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.
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0905, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Löfgren
authors.forename: M Rohan
authors.initials: MR
authors.lastname: Fernando
authors.forename: Kui-Yi
authors.initials: KY
authors.lastname: Xing
authors.forename: Yin
authors.initials: Y
authors.lastname: Wang
authors.forename: Charles A
authors.initials: CA
authors.lastname: Kuszynski
authors.forename: Ye-Shih
authors.initials: YS
authors.lastname: Ho
authors.forename: Marjorie F
authors.initials: MF
authors.lastname: Lou
mesh_terms: 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
chemical_list: D054477:Glutaredoxins; D011994:Recombinant Proteins; D038202:alpha-Crystallin A Chain; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005978:Glutathione