Epithelial-to-mesenchymal transition and fibroblast differentiation in idiopathic pulmonary fibrosis

Abstract

The aim of the present study is to explore the molecular mechanisms of epithelial-to-mesenchymal transition and fibroblast differentiation during the pathogenesis of idiopathic pulmonary fibrosis.

We identified 6 up-regulated and 3 down-regulated miRNAs in a human lung epithelial cell EMT model using miRNA microarray and real-time PCR. Overexpression of one of these up-regulated miRNAs, miR-424, increased the expression of ?-smooth muscle actin, an indicator of myofibroblast differentiation, but had no effects on the epithelial or mesenchymal cell markers. miR-424 enhanced the activity of the TGF-beta signaling pathway, as demonstrated by a luciferase reporter assay. Further experiments showed that miR-424 decreased the protein expression of Smurf2, a negative regulator of TGF-beta signaling, indicating that miR-424 exerts a forward regulatory loop in the TGF-beta signaling pathway. Our results suggest that miR-424 regulates the myofibroblast differentiation during EMT by potentiating the TGF-beta signaling pathway, likely through Smurf2.

Furthermore, we investigated the role of EZH2 in the differentiation of fibroblasts into myofibroblasts and underlying mechanisms. We found that EZH2 was up-regulated in the lungs of patients with IPF and mice with bleomycin-induced lung fibrosis. The up-regulation of EZH2 occurred in myofibroblasts. The inhibition of EZH2 by its inhibitor DZNep or shRNA reduced TGFbeta1-induced differentiation of human lung fibroblasts into myofibroblasts as demonstrated by the expression of myofibroblast marker alpha-smooth muscle actin, fibronectin and COL4A1 as well as contractility. DZNep inhibited Smad2/3 nuclear translocation without affecting Smad2/3 phosphorylation. Co-immunoprecipitation revealed a direct interaction between EZH2 and Smad2/3. DZNep treatment attenuated bleomycin-induced pulmonary fibrosis in mice. We conclude that EZH2 induces the differentiation of fibroblasts to myofibroblasts by binding and retaining Smad2/3 in the nucleus.