Effect of thioltransferase (glutaredoxin) deletion on cellular sensitivity to oxidative stress and cell proliferation in lens epithelial cells of thioltransferase knockout mouse.
Invest Ophthalmol Vis Sci
; 49(10): 4497-505, 2008 Oct.
Article
em En
| MEDLINE
| ID: mdl-18586881
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 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Estresse Oxidativo
/
Proliferação de Células
/
Glutarredoxinas
/
Cristalino
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Invest Ophthalmol Vis Sci
Ano de publicação:
2008
Tipo de documento:
Article
País de afiliação:
Estados Unidos