Oxidative Stress in TGF-β-induced lens EMT: it’s Nox what you think!

Abstract

Transforming Growth Factor-β (TGFβ) can induce an epithelial to mesenchymal transition (EMT) in lens that results in fibrotic cataract. It has previously shown that the reactive oxygen species (ROS) producing enzyme, NADPH oxidase 4 (Nox4), is upregulated in response to TGFβ-signalling. To better elucidate the role(s) of Nox4 in lens EMT, the present study characterised TGFβ-induced EMT and cataract formation using both in vitro and in vivo models, including Nox4 deletion strategies. We demonstrated that TGF-β can induce the upregulation of Nox4 in lens epithelial cells with a concomitant increase in reactive oxygen species (ROS) at early stages of in vitro culture. Pharmacological inhibition of Nox4 revealed that the presence of ROS at these early timepoints was due to Nox4 expression. When cultured over 5 days, pharmacological inhibition of Nox4 in lens cells delayed the progression of EMT by attenuating the formation of both TGF-β-induced capsular wrinkling and myofibroblasts, evidenced by the absence of alpha-smooth muscle actin (αSMA). One of the most notable findings of the present study was when mice overexpressing TGF-β specifically in the lens (that have anterior subcapsular cataracts) were crossed to Nox-4 deficient mice, and the lenses of their resultant progeny remaining transparent up to postnatal day 30. Despite this transparency, histology and immunolabelling of these same lenses revealed the presence of anterior subcapsular plaques; however, devoid of the typical EMT markers. Labelling of Nox4-deficient lens epithelial explants, and also the transgenic mouse lens sections revealed elevated pSmad2/3- and pERK1/2-signalling. Further qPCR analysis of these tissues revealed compensatory upregulation of Nox2 in mice deficient for Nox4. Moreover, TGFβ-treatment induced elevated but delayed ROS-labelling in Nox4-deficient lens epithelial explants. These results indicate that in mice at least, Nox4 plays a role in the development of TGFßinduced lens EMT leading to cataract, potentially by modulating pSmad2/3- and pERK1/2-signalling. In the absence of Nox4, there are other compensatory producers/sources of ROS, such as Nox2 and mitochondrial ROS. Taken together, these findings provide a better understanding of TGFß-induced lens EMT, as well as a platform allowing us to delineate putative Nox4 interactions with Smad2/3 and/or ERK1/2, as well as other signaling intermediates.