Involvement of epithelial-mesenchymal transition in methotrexate-induced pulmonary fibrosis

Epithelial-mesenchymal transition (EMT) plays a pivotal event in the development of pulmonary fibrosis. We have previously reported that methotrexate (MTX)-induced alveolar epithelial cell injury followed by pulmonary fibrosis as a result of the recruitment and proliferation of myofibroblasts. However, there is no data concerning whether EMT occurs in MTX-induced pulmonary fibrosis. In the present study, therefore, we investigated the expression of EMT markers such as E-cadherin, alpha-SMA, and vimentin by immunofluorescence analysis in mouse lung tissues after administration of MTX. We found that vimentin and alpha-SMA-positive cells of the MTX-induced pulmonary fibrosis were increased; on the other hand, E-cadherin was decreased, indicating that epithelial cells act as the main source of mesenchymal expansion. These results exhibited the down-regulation of E-cadherin expression and the up-regulation of alpha-smooth muscle actin (alpha-SMA) in primary mouse alveolar epithelial cells (MAECs) and A549 cell lines. Additionally, MTX-induced A549 cells exhibited an EMT-like phenotype accompanied by the elevation of the expression of interleukin-6 (IL-6) and transforming growth factor (TGF)-beta 1, as well as an enhancement of migration. All of these findings suggest that MTX-induced pulmonary fibrosis occurs via EMT.