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

Beatriz Ballester,Javier Milara,Julio Cortijo

doi:10.18632/oncotarget.27526

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 Thr41 (1224) and Tyr46 (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.

Highlights

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by extensive accumulation of abnormal extracellular matrix (ECM) in the lung and variable progression among patients, leading to death 3-5 years after diagnosis [1]
The non-canonical ERK1/2 phosphorylation pathway was increased in A549 and MRC5 cells by transforming growth factor-β1 (TGF-β1) action, to SMAD3 phosphorylation, pirfenidone did not prevent ERK1/2 phosphorylation in both types of cells (Figure 1A, 1B)
We found that mucin 1 (MUC1)-cytoplasmic tail (CT) was increased in lung tissue from idiopathic pulmonary fibrosis (IPF) patients, but not in the lungs of healthy subjects, and located mostly in hyperplasic alveolar type II epithelial cells (ATII) cells and fibroblasts in fibrotic areas

Summary

Introduction

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by extensive accumulation of abnormal extracellular matrix (ECM) in the lung and variable progression among patients, leading to death 3-5 years after diagnosis [1]. Invasive myofibroblasts in IPF lungs have multiple origins [4] including lung resident fibroblasts or mesenchymal/myofibroblast transformations of alveolar type II epithelial cells (ATII) [5]. Both type of cells, fibroblasts and ATII cells, acquire senescent identities, which are able to promote lung fibrosis [6, 7]. Transforming growth factor (TGF)-β1 is probably the most potent pro-fibrotic factor [8] This cytokine is known to promote fibroblasts differentiation and ECM production [9], leading to pulmonary fibrosis mainly through the SMAD-dependent canonical pathway [10]. Aberrant activity of the developmental Wnt/β-catenin signaling pathway has emerged recently as a fundamental concept in fibrogenesis [11, 12]

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