N-acetylcysteine inhibits alveolar epithelial-mesenchymal transition.

The ability of transforming growth factor-beta1 (TGF-beta1) to induce epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AEC) in vitro and in vivo, together with the demonstration of EMT in biopsies of idiopathic pulmonary fibrosis (IPF) patients, suggests a role for TGF-beta1-induced EMT in disease pathogenesis. We investigated the effects of N-acetylcysteine (NAC) on TGF-beta1-induced EMT in a rat epithelial cell line (RLE-6TN) and in primary rat alveolar epithelial cells (AEC). RLE-6TN cells exposed to TGF-beta1 for 5 days underwent EMT as evidenced by acquisition of a fibroblast-like morphology, downregulation of the epithelial-specific protein zonula occludens-1, and induction of the mesenchymal-specific proteins alpha-smooth muscle actin (alpha-SMA) and vimentin. These changes were inhibited by NAC, which also prevented Smad3 phosphorylation. Similarly, primary alveolar epithelial type II cells exposed to TGF-beta1 also underwent EMT that was prevented by NAC. TGF-beta1 decreased cellular GSH levels by 50-80%, whereas NAC restored them to approximately 150% of those found in TGF-beta1-treated cells. Treatment with glutathione monoethyl ester similarly prevented an increase in mesenchymal marker expression. Consistent with its role as an antioxidant and cellular redox stabilizer, NAC dramatically reduced intracellular reactive oxygen species production in the presence of TGF-beta1. Finally, inhibition of intracellular ROS generation during TGF-beta1 treatment prevented alveolar EMT, but treatment with H2O2 alone did not induce EMT. We conclude that NAC prevents EMT in AEC in vitro, at least in part through replenishment of intracellular GSH stores and limitation of TGF-beta1-induced intracellular ROS generation. We speculate that beneficial effects of NAC on pulmonary function in IPF may be mediated by inhibitory effects on alveolar EMT.