Troglitazone Attenuates TGF-β1-Induced EMT in Alveolar Epithelial Cells via a PPARγ-Independent Mechanism

Beiyun Zhou,Mihaela Ivan,Lucas Demaio,Jiao Luo,Manda Sai Krishnaveni,Yixin Liu,Stephen T Buckley,Kwang-Jin Kim,Vipul Patel,Carsten Ehrhardt,Zea Borok,Edward D Crandall,Saverio Bellusci

doi:10.1371/journal.pone.0038827

Abstract

Peroxisome proliferator activated receptor γ (PPARγ) agonists are effective antifibrotic agents in a number of tissues. Effects of these agents on epithelial-mesenchymal transition (EMT) of primary alveolar epithelial cells (AEC) and potential mechanisms underlying effects on EMT have not been well delineated. We examined effects of troglitazone, a synthetic PPARγ agonist, on transforming growth factor (TGF)-β1-induced EMT in primary rat AEC and an alveolar epithelial type II (AT2) cell line (RLE-6TN). TGF-β1 (2.5 ng/mL) induced EMT in both cell types, as evidenced by acquisition of spindle-like morphology, increased expression of the mesenchymal marker α-smooth muscle actin (α-SMA) and downregulation of the tight junctional protein zonula occludens-1 (ZO-1). Concurrent treatment with troglitazone (or rosiglitazone), ameliorated effects of TGF-β1. Furthermore, following stimulation with TGF-β1 for 6 days, troglitazone reversed EMT-related morphological changes and restored both epithelial and mesenchymal markers to control levels. Treatment with GW9662 (an irreversible PPARγ antagonist), or overexpression of a PPARγ dominant negative construct, failed to inhibit these effects of troglitazone in AEC. Troglitazone not only attenuated TGF-β1-induced phosphorylation of Akt and glycogen synthase kinase (GSK)-3β, but also inhibited nuclear translocation of β-catenin, phosphorylation of Smad2 and Smad3 and upregulation of the EMT-associated transcription factor SNAI1. These results demonstrate inhibitory actions of troglitazone on TGF-β1-induced EMT in AEC via a PPARγ-independent mechanism likely through inhibition of β-catenin-dependent signaling downstream of TGF-β1, supporting a role for interactions between TGF-β and Wnt/β-catenin signaling pathways in EMT.

Highlights

Idiopathic pulmonary fibrosis (IPF) is a progressive disorder of unknown etiology characterized by accumulation of fibroblasts/ myofibroblasts and marked deposition of extracellular matrix components [1]
We examined the effects of troglitazone, a synthetic peroxisome proliferator-activated receptor gamma (PPARc) ligand, on transforming growth factor (TGF)-b1-mediated Epithelial-mesenchymal transition (EMT) in both primary alveolar epithelial cells (AEC) and a non-transformed rat lung epithelial cell line, RLE-6TN [20]
Following treatment with TGF-b1 for 12 days, primary AEC exhibited a marked alteration in cell morphology, changing from the characteristic organized ‘cobblestone’ appearance of differentiated epithelial cell monolayers to a disorganized elongated fibroblast-like phenotype (Figure 1A)

Summary

Introduction

Idiopathic pulmonary fibrosis (IPF) is a progressive disorder of unknown etiology characterized by accumulation of fibroblasts/ myofibroblasts and marked deposition of extracellular matrix components [1]. We examined the effects of troglitazone, a synthetic PPARc ligand, on TGF-b1-mediated EMT in both primary AEC and a non-transformed rat lung epithelial cell line, RLE-6TN [20].

Results

Conclusion