Role of Nrf2 in Methotrexate-Induced Epithelial-Mesenchymal Transition in Alveolar A549 Cells.

Methotrexate (MTX) is known to induce serious lung diseases, such as pulmonary fibrosis. Although we demonstrated that MTX is associated with epithelial-mesenchymal transition (EMT), the underlying mechanism remains unclear. Nuclear factor erythroid 2-related factor 2 (Nrf2), an oxidative stress response regulator, is related to EMT induction. In the present study, we examined the association of Nrf2 with the MTX-induced EMT in the alveolar epithelial cell line A549. MTX treatment decreased the mRNA expression of heme oxidase-1 (HO-1), a target of Nrf2, which was inhibited by co-treatment with diethyl maleate (DEM), an Nrf2 activator. Additionally, the MTX-induced increase in reactive oxygen species (ROS) production was significantly suppressed by DEM. Furthermore, DEM decreased mRNA/protein expression levels of α-smooth muscle actin (SMA), a representative EMT marker, which were upregulated by MTX. Nuclear expression and localization of Nrf2 were suppressed by MTX treatment, which led to a decrease in Nrf2 activity. Finally, in Nrf2 knockdown cells, the MTX-induced enhancement of α-SMA mRNA/protein expression was not observed, indicating that downregulation of Nrf2 may play a critical role in the MTX-induced EMT in A549 cells. These results suggest that Nrf2-regulated transcriptional activity would be associated with the MTX-induced EMT induction.