Methotrexate-Induced Epithelial-Mesenchymal Transition in the Alveolar Epithelial Cell Line A549

Methotrexate (MTX) therapy of certain cancers and rheumatoid arthritis often induces serious interstitial lung complications including pulmonary fibrosis. In this study, we investigated the epithelial-mesenchymal transition (EMT) induced by MTX and by transforming growth factor (TGF)-beta 1 in the human alveolar epithelial cell line A549 in order to develop new strategies for the prevention of EMT. First, we examined the effect of TGF-beta 1 and MTX on cell morphology and the expression of EMT-related mRNAs in A549 cells. Then, the effects of SB431542 (SB), a potent inhibitor of TGF-beta receptor kinase, and a neutralizing antibody for TGF-beta 1 on the phenotypic changes of A549 cells induced by TGF-beta 1 and MTX were examined. After incubation with TGF-beta 1 and MTX, the mRNA expression of epithelial markers such as cytokeratin 19 was reduced, while that of mesenchymal markers such as alpha-smooth muscle actin was increased. SB suppressed the development of morphological changes and partially rescued alterations in mRNA expression of EMT markers induced by MTX. In addition, the enhancement of SMAD2 phosphorylation by MTX was also prevented by SB. On the other hand, EMT-related changes induced by MTX were not affected by a neutralizing antibody for TGF-beta 1. We have demonstrated that phenotypic changes of A549 cells induced by MTX are partly mediated by a TGF-beta 1-related intracellular signaling pathway, although TGF-beta 1 itself is not directly involved in this process.