ERK/GSK3β/Snail signaling mediates radiation-induced alveolar epithelial-to-mesenchymal transition

Radiotherapy is one of the major treatment regimes for thoracic malignancies, but can lead to severe lung complications including pneumonitis and fibrosis. Recent studies suggest that epithelial-to-mesenchymal transition (EMT) plays an important role in tissue injury leading to organ fibrosis. To investigate whether radiation can induce EMT in lung epithelial cells and also to understand the potential mechanism(s) associated with this change, rat alveolar type II lung epithelial RLE-6TN cells were irradiated with 8 Gy of Cs-137 gamma-rays. Western blot and immunofluorescence analyses revealed a time-dependent decrease in E-cadherin with a concomitant increase in alpha-smooth muscle actin (alpha-SMA) and vimentin after radiation, suggesting that the epithelial cells acquired a mesenchymal-like morphology. Protein levels and nuclear translocation of Snail, the key inducer of EMT, were significantly elevated in the irradiated cells. Radiation also induced a time-dependent inactivation of glycogen synthase kinase-3 beta (GSK3 beta), an endogenous inhibitor of Snail. A marked increase in phosphorylation of ERK1/2, but not JINK or p38, was observed in irradiated RLE-6TN cells. Silencing ERK1/2 using siRNAs and the MEK/ERK inhibitor U0126 attenuated the radiation-induced phosphorylation of GSK3 beta and altered the protein levels of Snail, alpha-SMA, and E-cadherin in RLE-6TN cells. Preincubating RLE-6TN cells with N-acetylcysteine, an antioxidant, abolished the radiation-induced phosphorylation of ERK and altered protein levels of Snail, E-cadherin, and alpha-SMA. These findings reveal, for the first time, that radiation-induced EMT in alveolar type II epithelial cells is mediated by the ERK/GSK3 beta/Snail pathway. (C) 2011 Elsevier Inc. All rights reserved.