Thioredoxin Has a Growth Factor/Cytokine-Like Property that Generates Reactive Oxygen Species (ROS) and Stimulates Cell Growth in Human Lens Epithelial Cells (HLE B3)

Abstract

Purpose:: We have undertaken this study to investigate the mechanism/s by which extra cellular thioredoxin (Trx) stimulated the expression of several antioxidant enzymes in human lens epithelial cells observed previously (Yogorova et al., Exp Eye Res., 2006).

Methods:: Purified recombinant human Trx was used throughout the studies. Trx-stimulated cell proliferation was determined by manual cell counting. Trx-induced ROS generation was examined using dichlorofluorescein (DCFH) fluorescence by FACS analysis. NADPH oxidase activity was measured by superoxide anion production using lucigenin-amplified chemiluminescence in Trx treated and untreated HLE B3 cells. H2O2 was quantified using the method of Hildebrant et al.

Results:: Recombinant human Trx (5 µM) added to culture medium in the absence of fetal bovine serum stimulated the proliferation of HLE B3 cells over a 3 day period. Treating HLE B3 cells with 20 µM Trx resulted in increased DCF fluorescence over the untreated control cells, indicating Trx could stimulate intracellular ROS generation. Co-treatment of the cells with catalase (1 mg/ml) and Trx resulted in a significant decrease in ROS generation. Treatment of HLE B3 cells for 30 min with a NADPH oxidase inhibitor, diphenyleneiodonium chloride (10 µM), prior to Trx treatment resulted in ~ 50% decrease in DCF fluorescence. Cells treated with Trx (20 µM) showed two-fold increase in NADPH oxidase activity as compared to the untreated control.

Conclusions:: Based on above observations we propose that Trx possesses a novel growth factor/cytokine-like function in stimulating cell proliferation and specific antioxidant enzyme expressions via ROS-mediated redox signaling by activating membrane-bound NADPH oxidase enzyme.

Keywords: growth factors/growth factor receptors • signal transduction • proliferation