Role of epimorphin in bile duct formation of rat liver epithelial stem-like cells: Involvement of small G protein RhoA and C/EBPβ
Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing, China. Journal of Cellular Physiology
(Impact Factor: 3.84).
11/2011; 226(11):2807-16. DOI: 10.1002/jcp.22625
Epimorphin/syntaxin 2 is a high conserved and very abundant protein involved in epithelial morphogenesis in various organs. We have shown recently that epimorphin (EPM), a protein exclusively expressed on the surface of hepatic stellate cells and myofibroblasts of the liver, induces bile duct formation of hepatic stem-like cells (WB-F344 cells) in a putative biophysical way. Therefore, the aim of this study was to present some of the molecular mechanisms by which EPM mediates bile duct formation. We established a biliary differentiation model by co-culture of EPM-overexpressed mesenchymal cells (PT67(EPM)) with WB-F344 cells. Here, we showed that EPM could promote WB-F344 cells differentiation into bile duct-like structures. Biliary differentiation markers were also elevated by EPM including Yp, Cx43, aquaporin-1, CK19, and gamma glutamyl transpeptidase (GGT). Moreover, the signaling pathway of EPM was analyzed by focal adhesion kinase (FAK), extracellular regulated kinase 1/2 (ERK1/2), and RhoA Western blot. Also, a dominant negative (DN) RhoA-WB-F344 cell line (WB(RhoA-DN)) was constructed. We found that the levels of phosphorylation (p) of FAK and ERK1/2 were up-regulated by EPM. Most importantly, we also showed that RhoA is necessary for EPM-induced activation of FAK and ERK1/2 and bile duct formation. In addition, a dual luciferase-reporter assay and CHIP assay was performed to reveal that EPM regulates GGT IV and GGT V expression differentially, possibly mediated by C/EBPβ. Taken together, these data demonstrated that EPM regulates bile duct formation of WB-F344 cells through effects on RhoA and C/EBPβ, implicating a dual aspect of this morphoregulator in bile duct epithelial morphogenesis.
Available from: James Pritchett
- "In the adult liver, EPIM is expressed in the connective tissue surrounding blood vessels, along the sinusoidal lining where HSCs reside ,  and in mesenchyme surrounding the bile duct where it is thought to play a role in duct formation , . In vivo, there is a reduction in EPIM expression following liver injury and HSC activation . "
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ABSTRACT: Background and Aims
Liver fibrosis is a major cause of morbidity and mortality. It is characterised by excessive extracellular matrix (ECM) deposition from activated hepatic stellate cells (HSCs). Although potentially reversible, treatment remains limited. Understanding how ECM influences the pathogenesis of the disease may provide insight into novel therapeutic targets for the disease. The extracellular protein Epimorphin (EPIM) has been implicated in tissue repair mechanisms in several tissues, partially, through its ability to manipulate proteases. In this study, we have identified that EPIM modulates the ECM environment produced by activated hepatic stellate cells (HSCs), in part, through down-regulation of pro-fibrotic Sex-determining region Y-box 9 (SOX9).
Influence of EPIM on ECM was investigated in cultured primary rat HSCs. Activated HSCs were treated with recombinant EPIM or SOX9 siRNA. Core fibrotic factors were evaluated by immunoblotting, qPCR and chromatin immunoprecipitation (ChIP).
During HSC activation EPIM became significantly decreased in contrast to pro-fibrotic markers SOX9, Collagen type 1 (COL1), and α- Smooth muscle actin (α-SMA). Treatment of activated HSCs with recombinant EPIM caused a reduction in α-SMA, SOX9, COL1 and Osteopontin (OPN), while increasing expression of the collagenase matrix metalloproteinase 13 (MMP13). Sox9 abrogation in activated HSCs increased EPIM and MMP13 expression.
These data provide evidence for EPIM and SOX9 functioning by mutual negative feedback to regulate attributes of the quiescent or activated state of HSCs. Further understanding of EPIM's role may lead to opportunities to modulate SOX9 as a therapeutic avenue for liver fibrosis.
Available from: Wen Long Yue
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ABSTRACT: The high incidence rate of hepatocellular carcinoma (HCC) is mainly the result of frequent metastasis and tumor recurrence. Unfortunately, the underlying molecular mechanisms driving HCC metastasis are still not fully understood. It has been demonstrated that tumor stroma cells contribute to primary tumor growth and metastasis. Within the HCC environment, activated hepatic stellate cells (HSCs) can release a number of molecules and enhance cancer cell proliferation and invasiveness in a paracrine manner. Here, for the first time, we demonstrate that epimorphin (EPM; also called syntaxin-2), an extracellular protein, is strongly elevated in activated HSCs within tumor stroma. We show that knockdown of EPM expression in HSCs substantially abolishes their effects on cancer cell invasion and metastasis. Ectopic expression of EPM in HCC cancer cells enhances their invasiveness; we demonstrate that the cells expressing EPM have markedly increased metastasis potential. Furthermore, EPM-mediated invasion and metastasis of cancer cells is found to require up-regulation of matrix metalloproteinase-9 (MMP-9) through the activation of focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) axis. CONCLUSION: Our results show that EPM, secreted by activated HSCs within HCC stroma, promotes invasion and metastasis of cancer cells by activating MMP-9 expression through the FAK-ERK pathway.
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ABSTRACT: An essential function of the liver is the formation and secretion of bile, a complex aqueous solution of organic and inorganic compounds essential as route for the elimination of body cholesterol as unesterified cholesterol or as bile acids. In bile, a considerable amount of otherwise insoluble cholesterol is solubilized by carriers including two other classes of lipids, namely phospholipid and bile acids. Formation of bile and generation of bile flow are driven by the active secretion of bile acids, lipids and electrolytes into the canalicular and bile duct lumens followed by the parallel movement of water. Thus, water has to cross rapidly into and out of the cell interior driven by osmotic forces. Bile as a fluid, results from complicated interplay of hepatocyte and cholangiocyte uptake and secretion, concentration, by involving a number of transporters of lipids, anions, cations, and water. The discovery of the aquaporin water channels, has clarified the mechanisms by which water, the major component of bile (more than 95%), moves across the hepatobiliary epithelia. This review is focusing on novel acquisitions in liver membrane lipidic and water transport and functional participation of aquaporin water channels in multiple aspects of hepatobiliary fluid balance. Involvement of aquaporins in a series of clinically relevant hepatobiliary disorders are also discussed.
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