Regulation of thioltransferase expression in human lens epithelial cells

PURPOSE. To study how the expression of thioltransferase (TTase), a critica l thiol repair and dethiolating enzyme, is regulated in human lens epitheli al cells under oxidative stress. Also to examine whether depleting the prim ary 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 mil lion) and gradually weaned from serum in the medium before exposing to 0.1 mM H2O, 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 activ ity and for reverse transcription-polymerase chain reaction (RT-PCR) or Nor thern hybridization analysis to quantify TTase mRNA. The effect of GSH depl etion on TTase mRNA expression was examined by treating the cells with buth ionine S,R-sulfoximine (BSO); I-chloro, 2,4-dinitrobenzene (CDNB); or 1,3-b is (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, CDN B, and BCNU resulted in a minimum change in TTase expression. It is notewor thy that when cells depleted of GSH were subjected to oxidative stress, TTa se expression was also found to be strongly upregulated. CONCLUSIONS. These observations suggest that the upregulation of TTase expr ession in the lens epithelial cells could be an adaptive response of the ce lls to combat oxidative stress to restore the vital functions of the lens p roteins and enzymes. Such regulation is independent of cellular GSH concent ration.