Nox4 Plays a Role in TGF-β-Dependent Lens Epithelial to Mesenchymal Transition

PURPOSE. Transforming growth factor-beta induces an epithelial to mesenchymal transition (EMT) in the lens, presented as an aberrant growth and differentiation of lens epithelial cells. Studies in other models of EMT have shown that TGF-beta-driven EMT is dependent on the expression of the reactive oxygen species (ROS)-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase-4 (Nox4). We investigate the role of this enzyme in TGF-beta-induced lens EMT and determine whether it is required for this pathologic process. METHODS. Rat lens epithelial explants were used to investigate the role of Nox4 in TGF-beta-driven lens EMT. Nox1-4 expression and localization was determined by immunolabeling and/or RT-PCR. NADPH-oxidase-produced ROS were visualized microscopically using the fluorescent probe, dihydroethidium (DHE). VAS2870, a pan-NADPH oxidase inhibitor, was used to determine the specificity of Nox4 expression and its role in ROS production, and subsequently TGF-beta-driven EMT. RESULTS. We demonstrate, for the first time to our knowledge, in rat lens epithelial explants that TGF-beta treatment induces Nox4 (but not Nox1-3) expression and activity. Increased Nox4 expression was first detected at 6 to 8 hours following TGF-beta treatment and was maintained in explants up to 48 hours. At 8 hours after TGF-beta treatment, Nox4 was observed in cell nuclei, while at later stages in the EMT process (at 48 hours), Nox4 was predominately colocalized with alpha-smooth muscle actin. The inhibition of Nox4 expression and activity using VAS2870 inhibited EMT progression. CONCLUSIONS. Transforming growth factor-beta drives the expression of the ROS-producing enzyme Nox4 in rat lens epithelial cells and Nox4 inhibition can impede the EMT process.