Pirfenidone anti-fibrotic effects are partially mediated by the inhibition of MUC1 bioactivation

// Beatriz Ballester 1 , 2 , * , Javier Milara 1 , 2 , 3 , 4 , * and Julio Cortijo 1 , 2 , 5 1 Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain 2 CIBERES, Health Institute Carlos III, Valencia, Spain 3 Health Research Institute INCLIVA, Valencia, Spain 4 Pharmacy Unit, Clinic University Hospital, Valencia, Spain 5 Research and Teaching Unit, University General Hospital Consortium, Valencia, Spain * These authors contributed equally to this work Correspondence to: Beatriz Ballester, email: beaballester7@gmail.com Javier Milara, email: xmilara@hotmail.com Keywords: pirfenidone; idiopathic pulmonary fibrosis; MUC1; transforming growth factor beta-1; fibroblasts Received: December 11, 2019     Accepted: March 03, 2020     Published: April 14, 2020 ABSTRACT Pirfenidone is a pleiotropic molecule approved to treat idiopathic pulmonary fibrosis (IPF). Pirfenidone has demonstrated to downregulate transforming growth factor-β1 (TGF-β1) cellular effects. However, its anti-fibrotic mechanism remains unclear. Here, we aim to analyze the effects of pirfenidone on the TGF-β1 canonical and non-canonical pathways, as well as, on the most characteristic IPF cellular processes. Results observed in this work showed that TGF-β1-induced canonical SMAD3 and non-canonical ERK1/2 phosphorylations were not inhibited by pirfenidone in alveolar A549 and lung fibroblasts MRC5 cells. In contrast, pirfenidone inhibited TGF-β1-induced MUC1-CT Thr 41 (1224) and Tyr 46 (1229) phosphorylations, thus reducing the β-catenin activation. Additionally, immunoprecipitation and immunofluorescence studies in ATII cells and lung fibroblasts showed that pirfenidone inhibited the formation and nuclear translocation of the transcriptional fibrotic TGF-β1-induced phospho-SMAD3/MUC1-CT/active-β-catenin complex, and consequently the SMAD-binding element activation (SBE). This study provided also evidence of the inhibitory effect of pirfenidone on the TGF-β1-induced ATII to mesenchymal and fibroblast to myofibroblast transitions, fibroblast proliferation and ATII and fibroblast senescence. Therefore, it indicates that pirfenidone’s inhibitory effect on TGF-β1-induced fibrotic cellular processes is mediated by the inhibition of MUC1-CT phosphorylation, β-catenin activation, nuclear complex formation of phospho-SMAD3/MUC1-CT/active β-catenin and SBE activation, which may be of value to further develop anti-fibrotic IPF therapies.