Transplantation/Immunology
LPS-Induced Epithelial-Mesenchymal Transition of Intrahepatic Biliary Epithelial Cells

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Background

Recent studies have revealed that the epithelial-mesenchymal transition (EMT) of bile duct epithelial cells is engaged in hepatic fibrogenesis. However, the association between etiological factors of liver disease such as virus or bacterial infection and EMT remains to be investigated. The present study focuses on the inductive role of endotoxin, the main component of the cell wall's ectoblast of gram-negative bacteria, in the EMT of human intrahepatic biliary epithelial cells (HIBEpiCs).

Methods

The expressions of E-cadherin, S100A4, α-SMA, TGF-β1, and Smad2/3 in HIBEpiCs cultured with or without lipopolysaccharide LPS, were detected by real-time PCR and Western blotting. We blocked the expression of TGF-β1 using paclitaxel and knocked down Smad2/3 by siRNA to explore the role of TGF-β1/Smad2/3 pathway in the EMT of HIBEpiCs.

Results

Resting HIBEpiCs showed epithelioid cobblestone morphology, and underwent a phenotypic change to produce bipolar cells with a fibroblastic morphology when co-cultured with LPS. After LPS stimulation and the up-regulation of mRNA and protein expression of TGF-β1 and Smad2/Smad3, the mRNA and protein expression of mesenchymal markers (S100A and α-SMA) increased significantly. Paclitaxel inhibited the mRNA and protein expression of TGF-β1 in vitro. Knock-down of Smad2/3 by siRNA led to up-regulation of epithelial markers E-cadherin and down-regulation of S100A and α-SMA, indicating a reversal of the EMT.

Conclusions

LPS can induce the expression of TGF-ß1 and a subsequent EMT in HIBEpiCs, and the inhibition of TGF-ß1 or Smad 2/3 could reverse this EMT, suggesting that LPS may play a potential role in the EMT of HIBEpiCs.

Introduction

Repeated biliary infection is a characteristic feature of a range of chronic inflammatory liver diseases including hepatolithiasis (HL) and biliary stricture or obstruction. Biliary chronic inflammation plays an important role in liver fibrosis or even cirrhosis. The causative organism of biliary infection is often gram-negative bacteria, which could release endotoxin and induce inflammatory reaction. Studies have showed that when the IBECs are exposed to a high level of lipopolysaccharide during bacterial infections, IBECs often show cellular damage and pathologic proliferation. This response of IBECs plays a role in the progression of liver fibrogenesis or even cirrhosis [1].

Although the activation of hepatic stellate cells remains a central event in liver fibrosis, the role of epithelial-mesenchymal transition (EMT) of both hepatocytes and cholangiocytes in liver fibrosis is attracting great attentions. EMT has been implicated in a variety of biological processes such as fibrogenesis, embryonic development, and tumor progression. Recent clinical and animal studies have demonstrated that bile duct epithelial cells can undergo EMT, thereby contributing to hepatic fibrosis 2, 3, 4, 5. Consistent with these studies, our previous retrospective clinical study shown that we observed the stimulation of the TGF-β1/smad2/3 signaling pathway followed by the loss of epithelial markers and the acquirement of mesenchymal markers in bile duct epithelial cells from patients with primary hepatolithiasis.

Transforming growth factor beta1 (TGF-β1) is known to be the most potent inducer of EMT, and it initiates morphological transition of the cells from an epithelial to a fibroblastic appearance, accompanied by a loss of epithelial cell markers such as E-cadherin and a gain of mesenchymal cell markers such as vimentin, fibronectin, and N-cadherin 6, 7 However, how the TGF-β1 is induced in chronic liver diseases remains unclear. As infection is a common pathogenic process of chronic liver disease that mainly involves bile duct injury, and also participates in liver fibrosis, we hypothesized that biliary infection is related to the induction of TGF-β1-mediated EMT and subsequent fibrogenesis. Therefore, in this study, primary IBECs were cultured with LPS to investigate whether these can activate TGFβ/Smads signaling pathway and induce EMT.

Section snippets

Antibodies and Reagents

Antibodies and reagents included anti-TGF-β1, anti-E-cadherin, anti-S100A4, anti-p-Smad 2/3, and anti-α-SMA (DAKO, Glostrup, Denmark); Smad2/3 siRNA and control siRNA (Santa Cruz Technology, Santa Cruz, CA); paclitaxel and LPS (Sigma Chemical Co., St. Louis, MO, USA); Takara PrimeScript TMRT-PCR Kit (Takara Biotechnology Co., Dalian, China); RIMP1640 (Sigma Chemical Co.); and Trizol (Invitrogen, Carlsbad, CA, USA).

Cell Culture

Human intrahepatic Biliary Epithelial Cells (HIBEpiCs) were purchased from the

Effects of LPS on TGF-β1 mRNA Expression by HIBEpiCs

Twenty-four hours after the administration of LPS, marked TGF-β1 mRNA was detected. The expression of TGF-β1 mRNA reached the peak at 48 h, but the mRNA level decreased after 72 h. These results suggest that the expression of TGF-β1 was induced by LPS and reached a peak value at 48 h (Fig. 1 P < 0.01; ∗∗P < 0.05). Therefore, we detected the expression of proteins involved in the EMT process at 48 and 72 h in the following experiments.

LPS-induced expression of E-cadherin, S100A4, and α-SMA in HIBEpiCs

Resting HIBEpiCs showed epithelioid cobblestone morphology,

Discussion

In this study, stimulation with LPS causes a TGFβ/Smads-mediated EMT of HIBEpiCs. These results indicated that biliary infection played a potential role in inducing EMT of bile duct epithelial cells and subsequent peribiliary fibrosis. Biliary infection is an independent risk factor for liver injury and fibrosis. However, the exact molecular mechanism remains unclear. Bacteria, mainly gram-negative bacteria, retrograde into the biliary tract through cholangiopancreatic ampulla. LPS, endotoxin

Acknowledgments

The authors acknowledge support for this work by the Science and Technology Foundation of Guizhou Province (no. 2009GZ14798).

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2

These authors contributed equally to this work.

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