Epimorphin Regulates Bile Duct Formation via Effects on Mitosis Orientation in Rat Liver Epithelial Stem-Like Cells

Abstract

Understanding how hepatic precursor cells can generate differentiated bile ducts is crucial for studies on epithelial morphogenesis and for development of cell therapies for hepatobiliary diseases. Epimorphin (EPM) is a key morphogen for duct morphogenesis in various epithelial organs. The role of EPM in bile duct formation (DF) from hepatic precursor cells, however, is not known. To address this issue, we used WB-F344 rat epithelial stem-like cells as model for bile duct formation. A micropattern and a uniaxial static stretch device was used to investigate the effects of EPM and stress fiber bundles on the mitosis orientation (MO) of WB cells. Immunohistochemistry of liver tissue sections demonstrated high EPM expression around bile ducts in vivo. In vitro, recombinant EPM selectively induced DF through upregulation of CK19 expression and suppression of HNF3α and HNF6, with no effects on other hepatocytic genes investigated. Our data provide evidence that EPM guides MO of WB-F344 cells via effects on stress fiber bundles and focal adhesion assembly, as supported by blockade EPM, β1 integrin, and F-actin assembly. These blockers can also inhibit EPM-induced DF. These results demonstrate a new biophysical action of EPM in bile duct formation, during which determination of MO plays a crucial role.

Role of epimorphin in bile duct formation of rat liver epithelial stem‐like cells: Involvement of small G protein RhoA and C/EBPβ

“…We supposed that some phosphorylation of FAK for limited adhesion is independent of RhoA, since similar phosphorylation of FAK at a basal level was observed between WB RhoA-DN cells in contact with PT67 EPM cells, and normal WB-F344 cells in contact with PT67 cells (Clark et al, 1998). This presumption is also supported by our previous results that EPM promoted the assembly of focal adhesions during biliary differentiation of WB-F344 cells (Zhou et al, 2010). Moreover, we found RhoA dependence of the phosphorylation of ERK, which was confirmed as a downstream molecular of FAK signaling pathway in WB-F344 cells.…”

“…Cells were treated as we previously described (Zhang et al, 2008;Zhou et al, 2010). Briefly, permeabilized cells were incubated with RhoA, CK19, GGT primary antibodies (1:100, Santa Cruz Biochemistry).…”

“…1A) (Zhou et al, 2010). Here, NIH 3T3-cell-derived PT67 packaging cell line (PT67) was transfected with plasmids containing expression constructs for mouse EPM (Fig.…”

Effects of stress fiber contractility on uniaxial stretch guiding mitosis orientation and stress fiber alignment

“…It has been reported that intracellular cues and extracellular microenvironments are involved in the spindle positioning process. Independent research groups have demonstrated that stress fibers (SFs, comprised of F-actin and myosin) and integrin-mediated cell adhesion to the extracellular matrix (ECM) are essential to the MO determination (Fernandez-Minan et al, 2007;Toyoshima and Nishida, 2007;Woolner et al, 2008;Zhou et al, 2010). In consideration of these findings and the fact that mechanical forces induce stress fiber orientation (SFO; Takemasa et al, 1997;Wang, 2000;Kaunas et al, 2006), we hypothesized that the mechanical microenvironment may guide MO via its effect on SFO.…”

A polymorphism in the integrin αV subunit gene affects the progression of primary biliary cirrhosis in Japanese patients

“…Epimorphin is one of the ligands of the integrin aVb1 receptor located on the cellular membrane [31] and is expressed in hepatic stellate cells and myofibroblasts during liver injury in mice [32,33]. After binding to its receptor, epimorphin signals regulate normal bile duct formation by mediating mitosis orientation [34]. Because integrin aV, coded by ITGAV, is a component of the integrin aVb1 receptor, the minor G allele at rs1448427 SNP in ITGAV in PBC patients may affect the expression of integrin aVb1 receptor and its affinity to epimorphin, leading to diminution of the function of integrin aV, as well as the diminution of epimorphin-integrin aVb1 receptor signaling.…”