Purpose: To molecularly clone the human lens thioredoxin (TXN) gene, characterize the recombinant protein (rTrx1) and study the regulation expression of thioredoxin (Trx1) in human lens epithelial cells under oxidative stress.
Methods: The human TXN gene was cloned from a human lens cDNA library. Trx1 activity was measured by insulin reduction assay. For study of the upregulation of Trx1, 1.6 million human lens epithelial cells (HLE B3) were exposed to H(2)O(2) (0.1 mM) for 0, 5, 10, 15, 20, and 30 minutes. The cells were lysed with lysis buffer and used for Trx1 activity assay, Western blot analysis, and real-time PCR.
Results: The sequence of the human lens TXN gene was identical with that of other human tissues. Recombinant Trx1 was sensitive to iodoacetic acid but showed strong resistance to oxidation (0.1 mM H(2)O(2)) at its approximate physiological protein level. Western blot analysis and assay of Trx activity revealed that Trx1 was expressed in HLE B3 cells and localized in epithelial, cortical, and nuclear regions of human and porcine lenses. In vivo oxidative stress of HLE B3 cells resulted in a 35% upregulation of the level of Trx1 protein after 10 minutes of H(2)O(2) treatment. Real-time PCR analysis showed an increase of approximately 50% in the level of Trx1 mRNA under the same oxidative stress conditions.
Conclusions: The upregulation of Trx1 in HLE B3 cells under oxidative stress and the presence of Trx1 in the lens suggest that the thioredoxin system may be an effective defense system that protects the lens against oxidative stress.