Function And Mechanism Of Epimorphin In Normal Development And Anormogenesis Of Liver Cell

The liver is a extreme heterogeneous organ in human body. Hepatic parenchymal cells are surrounded and sustained at least seven kinds of stroma cells (including hepatic stellate cells, Kuppfer cells, sinusoidal endothelial cells, etc.) and extracellular components secreted by these cells, which composed hepatic parenchymal cells'microenvironment. These microenvironment components mainly contain soluble growth factors, insoluble extracellular matrix (ECM), and intercellular interaction. In normal liver development, hepatic parenchymal cells are regulated by their microenvironment in an accurate order and perform correct physiological function; however, once the microenvironment was broken down, these parenchymal cells will develop toward anormogenesis which leads to inflammation, fibrosis and tumorigenesis. The academic dissertation mainly investigated the function and mechanism of epimorphin, an important mesenchymal associated protein, in normal development and anormogenesis of liver cell.Part I. Molecular mechanism of epimorphin (EPM) regulating liver stem cell differentiation into bile duct formationHepatic parenchymal cells contains two epithelial cells parenchymal hepatocytes and bile duct epithelial cells. A small number of hepatic progenitors or hepatic stem cells which possess the potential to differentiate into both parenchymal hepatocytes and bile duct epithelial cells, are believed to remain undifferentiated throughout development and reside even in the adult liver, although their precise characteristics and physiological significance have not been clearly documented. It is important to understand the mechanism involved in stem cell fate determination in unique local microenvironments, which have effects on their proliferation, differentiation and morphogenesis. It is now known that ECM components in the hepatic mesenchyme are themselves able to induce hepatic stem cell differentiation, among which there is a membrane protein called epimorhpin (EPM) specificly secreted by hepatic stellate cells (HSC). Several pieces of accumulating evidence confirmed that important morphogenic roles for EPM in many epithelial organs including mammary glands, pancreas, lung, gallbladder, hair follicles, skin, intestine, etc. Some reports suggested EPM expression is enhanced in the late stage of liver regeneration and involved in the differentiation of hepatic stem-like cells in vitro. Our previous study shown that i-EPM has the ability to guide mitosis orientation (MO) determination of the WB cells along the tangential direction of cell-EPM contact surface via mediating focal adhesion assembly and F-actin bundles alignment, which may be vital to bile duct-like formation of the WB cells. But intracellular molecular signaling pathway of the bile duct formation of hepatic stem cells induced by EPM remains largely unknown. Therefore, our first aim was to present some of the molecular mechanisms by which EPM mediates bile duct formation.First, we established a better biliary differentiation model by co-culture of EPM-overexpressed mesenchymal cells (PT67EPM) with WB cells. In the co-culture system, we showed that EPM could promote WB cells differentiation into bile duct-like structures. RT-PCR and Western blot analysis showed an elevation of biliary differentiation markers, including a modest elevation of Yp, aquaporin-1, Cx43, and a strong elevation of, CK19 and GGT (IV). It is worth noting that elevated CK19 and GGT proteins by immunofluorescence assay showed were limited to be expressed on elongated WB cells but not the control cells. Taken together, these results confirmed that EPM was able to induce differentiation of WB cells into duct formation. The direct cause of cell morphological chang is variation of cystoskeleton arrangement. To confirm this conclusion, we used immunofluorescence assay and the Laser-Confocal Microscopy (CLSM) images showed that EPM could induce the F-actin rearrangement of WB cells. At the same time, we also found a small G protein closely related to cell morphologic change and cystoskeleton RhoA (total RhoA and GTP-bound RhoA), was elevated involved in bile duct formation of EPM-induced WB cells. Moreover, the signaling pathway of EPM was analyzed by Western blot. Also, a dominant negative (DN) RhoA-WB cell line (WBRhoA-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 cells through effects on RhoA and C/EBPβ, implicating a dual aspect of this morphoregulator in bile duct epithelial morphogenesis.PartⅡ. Function and mechanism of EPM in invasion and metastasis of human hepatocellular carcinomaRecently, many studies have highlighted the importance of cross-talk between tumor cells and their microenvironment and the contribution of stroma cells to tumor progression. It has been demonstrated that various stroma cell types are recruited to neoplasms, where they are activated, and substantially promote the proliferation, invasiveness and metastatic potential of cancer cells. HSC belong to one of the most important stroma cell types in ECM of hepatocellular carcinoma (HCC). In the tumor environment, HSC undergo the transition from quiescent to activated state and affect cancer cell proliferation and invasiveness. Activated HSCs can secrete a variety of protein molecules in a paracrine manner, such as growth factors, signaling molecules and soluble mediators. Among them, EPM, on which we pay much more attention, is also present. We then presumed that EPM, a epithelium morphogenesis factor, probably participates in regulation of hepatocarcinogenesis, while FAK-ERK pathway is very important to tumorigenesis. There are only two independent reports about EPM in tumor: transgenic mice expressing the extracellular form of EPM in mammary epithelial cells develop alveolar hyperplasias and have a high incidence of mammary neoplasia; the lack of EPM inhibits chronic inflammation-associated colon carcinogenesis in mice. Although EPM is known to be involved in liver development and regeneration, its function in liver cancer has not been characterized. Therefore, our second aim was to carry out the function and mechanism of EPM in hepatocarcinogenesis.To investigate in more detail the biological function of EPM in hepatocellular tumor, we established two ectopic EPM-expressing HCC cell lines named 97H-pIEPM and 97L-pIEPM. We performed MTT cell proliferation and colony formation assays and xenograft experiments to examine whether EPM affects cell proliferation. The results indicated that EPM didn't affect cancer cell proliferation in vitro or tumor growth in vivo to a significant extent. Then, we used Matrigel invasion assay and orthotopical liver implantation for intrahepatic metastasis assay, and all the data in vitro and in vivo supported an important role for EPM in hepatocellular tumor invasion and metastasis. To determine how EPM facilitates HCC cell invasion, the expression of matrix metalloproteinase family (MMPs), proteolytic enzymes involved in tumor invasion, were analyzed by RT-PCR. The results showed that only MMP-9 was dramatically western blot results. To examine the mechanism by which EPM activates MMP-9 expression, two important kinases FAK and ERK, known to mediate intergrin signaling, were analyzed. We found that phosphorylation of FAK and ERK1/2 was up-regulated in EPM transfected HCC cells. Block both of FAK and ERK1/2 in EPM-expressing cells significantly inhibited MMP-9 expression and reduced invasive ability of the HCC cells. These observations demonstrate that EPM activates FAK and ERK signaling pathway to facilitate MMP-9 expression and stimulate cancer cell invasion.To summarize, our researches demonstrated that EPM regulated rat liver epithelial stem-like cells differentiation to bile duct via activation of RhoA-FAK-ERK-C/EBPβsignaling pathway for the first time and provided the first evidence suggesting that EPM secreting from HSCs could promote HCC cell invasion and metastasis via FAK-ERK-MMP-9 pathway. Our researches not only supply viewpoint that the ECM provides biochemical and mechanical contextual information conveyed in liver normal development and liver epithelium morphogenesis, but also propose a new theory that interruption of the EPM-FAK-ERK-MMP-9 pathway may be a useful therapeutic approach for controlling HCC metastasis.