Regulation of thioltransferase expression in human lens epithelial cells.
OBJECTIVE
To study how the expression of thioltransferase (TTase), a critical thiol repair and dethiolating enzyme, is regulated in human lens epithelial cells under oxidative stress. Also to examine whether depleting the primary cellular antioxidant glutathione (GSH) in these cells has any influence on TTase expression under the same conditions.


METHODS
Human lens epithelial cells (B3) were grown to confluence (1.6 million) and gradually weaned from serum in the medium before exposing to 0.1 mM H2O2 for 2 hours. Cells were removed at the time intervals of 0, 5, 10, 15, 30, 60, and 120 minutes for protein measurements of GSH and TTase activity and for reverse transcription-polymerase chain reaction (RT-PCR) or Northern hybridization analysis to quantify TTase mRNA. The effect of GSH depletion on TTase mRNA expression was examined by treating the cells with buthionine S,R-sulfoximine (BSO); 1-chloro, 2,4-dinitrobenzene (CDNB); or 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). Lens epithelial cells, depleted of cellular GSH by treatment with BCNU, were subjected to oxidative stress to examine the effect on TTase activity and mRNA level.


RESULTS
A transient increase was detected in TTase mRNA after 5 minutes of H2O2 treatment. The upregulation reached a maximum of 80% above the normal level by 10 minutes and gradually decreased as the oxidant was detoxified by the cells. Manipulation of cellular GSH level by treatment with BSO, CDNB, and BCNU resulted in a minimum change in TTase expression. It is noteworthy that when cells depleted of GSH were subjected to oxidative stress, TTase expression was also found to be strongly upregulated.


CONCLUSIONS
These observations suggest that the upregulation of TTase expression in the lens epithelial cells could be an adaptive response of the cells to combat oxidative stress to restore the vital functions of the lens proteins and enzymes. Such regulation is independent of cellular GSH concentration.
Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln 68583-0905, USA.
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N
Raghavachari
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Krysan
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K
Xing
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MF
Lou
D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002330:Carmustine / Q000494:pharmacology; D002478:Cells, Cultured; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D054477:Glutaredoxins; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007908:Lens, Crystalline / Q000187:drug effects / Q000201:enzymology*; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D011490:Protein Disulfide Reductase (Glutathione); D012333:RNA, Messenger / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
D004137:Dinitrochlorobenzene; D054477:Glutaredoxins; D012333:RNA, Messenger; D019328:Buthionine Sulfoximine; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D005978:Glutathione; D002330:Carmustine