Role of Thioredoxin in the Response of Human Lens Epithelial Cells to Oxidative Stress

Abstract

Abstract: : Purpose: The present study was designed to examine the role of thioredoxin (TRx1) in protecting the human lens epithelial cells (HLE B3) from damage during the early stage of oxidative stress. Methods: Full-length human TRx1 gene was cloned from HLE B3 cells by RT-PCR. The TRx1 cDNA was inserted into the pCR3.1 eukaryotic expression vector. Overexpression of TRx1 gene in HLE B3 cells was achieved by stable transfection. Human Trx1 was overexpressed in E. coli BL-21 and purified to homogeneity. TRx activity was measured by insulin reduction assay. Enzyme assays for gluthatione peroxidase (GPx), glyceraldehyde-3-phosphate dehydrogenase (G-3PD) and thioltransferase (TTase) were measured by monitoring the oxidation of NADPH spectrophotometrically. HLE B3 cells were deprived of serum by incubating with 2% fetal bovine serum overnight and then in serum-free medium for 30 min before subjecting to a bolus of H₂O₂ (0.1 mM) for 15 min. The cells were disrupted by sonication in 2mM EDTA or lysed on plate with lysis buffer and used for enzyme activity assays and western blot analysis. Results: HLE B3 cells stably transfected with human TRx1 showed a increase of 1.2-fold in TRx activity compare to control. Further, TRx1-transfected cells showed a 20% increase activity and expression TTase protein. Exposing human GPx (commercial) to 0.1 mM H₂O₂ in vitro resulted in 50% loss of enzyme activity. Trx1 could be reactivate the H₂O₂-inactivated GPx activity when a high non-physiological level of TRx1 (50 µM) was used in vitro. However, when the cells were subjected to oxidative stress, nearly 50% of the GPx activity was inactivated in normal cells, but only 25% of GPx was inactivated in TRx1-transfected cells. Recombinant Trx1 also partially restored the G-3PD activity in the extract of cells pretreated by 0.1 mM H₂O₂. Conclusions: These results suggests a new physiological function of TRx1 as electron donor to repair the key enzyme GPx and that both repair enzyme systems, the NADPH-dependent thioredoxin/thioredoxin reductase and the GSH-dependent thioltransferase, contribute synergistically in protecting cells against oxidative stress.

Keywords: antioxidants • gene/expression • oxidation/oxidative or free radical damage