Col1A1 Production and Apoptotic Resistance in TGF-β1-Induced Epithelial-to-Mesenchymal Transition-Like Phenotype of 603B Cells

Jun Liu,Alex N Eischeid,Xian-Ming Chen,Srinivasa M Srinivasula

doi:10.1371/journal.pone.0051371

Abstract

Recent studies have suggested that proliferating cholangiocytes have an important role in the induction of fibrosis, either directly via epithelial-to-mesenchymal transition (EMT), or indirectly via activation of other liver cell types. Transforming growth factor beta 1 (TGF-β1), a critical fibrotic cytokine for hepatic fibrosis, is a potent EMT inducer. This study aimed to clarify the potential contributions of TGF-β1-induced EMT-like cholangiocyte phenotype to collagen production and cell survival of cholangiocytes in vitro. Mouse cholangiocytes (603B cells) were treated with TGF-β1 and EMT-like phenotype alterations were monitored by morphological changes and expression of EMT-associated genes. Alterations in Col1A1 gene, Col1A1-associated miR-29s, and pro-apoptotic genes were measured in TGF-β1-treated 603B cells. Snail1 knockdown was achieved using shRNA to evaluate the contribution of EMT-associated changes to Col1A1 production and cell survival. We found TGF-β1 treatment induced partial EMT-like phenotype transition in 603B cells in a Snail1-dependent manner. TGF-β1 also stimulated collagen α1(I) expression in 603B cells. However, this induction was not parallel to the EMT-like alterations and independent of Snail1 or miR-29 expression. Cells undergoing EMT-like changes showed a modest down-regulation of multiple pro-apoptotic genes and displayed resistance to TNF-α-induced apoptosis. TGF-β1-induced apoptosis resistance was attenuated in Snail1 knockdown 603B cells. TGF-β1-induced Col1A1 production seems to be independent of EMT-like transition and miR-29 expression. Nevertheless, TGF-β1-induced EMT may contribute to the increased survival capacity of cholangiocytes via modulating the expression of pro-apoptotic genes.

Highlights

Liver fibrosis results from the accumulation of interstitial or ‘‘scar’’ extracellular matrix (ECM) after either acute or chronic liver injury
We found that after 2 days of Transforming growth factor beta 1 (TGF-b1) treatment, E-cadherin mRNA levels were steadily decreased in 603B cells as compared with the controls (p,0.05, Fig. 1B)
Whether cholangiocytes can transdifferentiate into myofibroblast-like cells via epithelial-to-mesenchymal transition (EMT) to promote liver fibrosis development is still under debate [34]

Summary

Introduction

Liver fibrosis results from the accumulation of interstitial or ‘‘scar’’ extracellular matrix (ECM) after either acute or chronic liver injury. Cholangiocytes, epithelial cells lining the biliary tree, become aberrantly accumulated in the portal region and form clustered bile ducts during hepatic fibrosis [5]. Such reaction reflects a dysregulated balance between cell growth and cell death, referred as ‘‘ductular reaction’’ [6]. EMT describes the molecular reprogramming and phenotypic changes involved in the conversion of polarized immotile epithelial cells to motile mesenchymal cells [10] This process allows the remodeling of tissues during embryonic development and is recently implicated in tumor progression and development of fibrosis [11,12]. Cholangiocytes with mesenchymal features may contribute to the generation of fibroblast-like cells to facilitate collagen production and promote the ‘‘ductular reaction’’ at the portal region during hepatic fibrosis [13,14]

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