Roles Of RhoA In Liver Precursor Cell Differentiation

Oval cells are the commonly accepted liver precursor cells which have the ability to differentiate into hepatocytes and biliary cells in vitro. WB-F344 cells derived from Fischer male rat liver are the commonly used in vitro model. When cultured on Matrigel, liver precursor epithelium WB-F344 cells could be induced to differentiate into biliary cells and formed islets. Cells that linked the islets were strenched along the islets. Biliay markers such as Yp, Aquaporin and CK19 were found upregulated. Matrigel is an artificial membrane containing mesenchymal components which, in vivo, contribute to the emergence of bile ducts in close contact with the portal mesenchyme. Epimorphin (EPM, also named syntaxin 2), a mesenchymal cell surface-associated molecule, is highly conserved in mammalian. EPM can selevtively initiates two key processes of tubulogenesis: branching morphogenesis and luminal morphogenesis depending upon its polar/apolar presentation. Our previous researches found that EPM could affect biliary commitment of WB-F344 cells and the stress fibre alignment in those cells. Furthermore, RhoA expression was upregulated in both Matrigel and EPM induced biliary differentiation. RhoA was first described as the membrane transformation regulators, and numerous studies have shown that RhoA functions as regulator of cytokinesis in proliferation, and as molecular switches in stem cell commitment and differentiation. Roles of RhoA indicate that it may take part in the Matrigel/EPM induced biliary lineage commitment and the following morphogenesis. This thesis research was up to investigate the roles of Matrigel, EPM and RhoA in biliary commitment of WB-F344 cells.At first WB-F344 cells were seeded on Matrigel to investigate cell morphological changes, biliary gene and RhoA expression. Meanwhile EPM was transfected into PT67 cells via Lipofectamine 2000 and the stable EPM expressing cell line was screened. WB-F344 cells were then cocultured with EPM-PT67/PT67 cells which were seeded as feeder layers, EPM-PT67/PT67 cells were pretreated with mitomycin before coculture. WB-F344 cocultured with EPM-PT67 cells were found forming bile duct like structures. Then RhoA positive and negative mutant(constitutively active, CA; dominant negative, DN) expressing vectors were constructed and stably transfected into WB-F344 cells via repsectively pfu point mutation and lentivirus transfection system. All the experiments were executed with control WB-F344 cells which was transfected with a lentirus only contains GFP. To investigate the function of EPM, RhoA CA, RhoA DN and control WB-F344 cells were cocultured with mitomycin pretreated EPM-PT67/PT67 feeder layers. To investigate the role of RhoA-ROCK in RhoA CA induced biliary commitment, ROCK inhibitor Y27632 and confocal observation were used. Meanwhile, according to previous studies the effectors such as FAK and ERK which were key factors in EPM signal pathway were investigated by western blotting.WB-F344 cells did change cell shape and formed bile duct like structures when seeded on Matrigel or cocultured with EPM-PT67. Biliary genes such as Yp, Aquaporin, CK19 and CX43 was upregulated according to the RT-PCR results. Meanwhile RhoA was upregulated as well. RhoA gene was successfully point mutated and highly transfected into WB-F344 cells by lentivirus transfection system, both at gene and protein level. Although RhoA didn't affect the cell cycle and cell growth of WB-F344 cells, it did change cell morphologies. RhoA DN WB cells were large and branchy, flat and mutually indistiguishable. On the contrary, RhoA CA WB cells were round, had a high nuclear/cytoplasmic ratio and the clear cell-cell boudary. RhoA CA WB cells showed classical ultrastructures of bile ductular cells. Cells attached closely to adjacent cells through intracellular tight junctional complexes, and adjacent cells also formed well-defined narrow lumina. Cells had small mitochondria, the nucleus of cells were oval, and the nucleous was more diffuse. While control oval cells had more abundant rough surfaced endoplasmic reticulum (rER), big mitochondria and glycogen particles which were only present in control cells. The nucleus was round and the nucleolus was large and contained a distinct nucleoclonema. Accompanying upregulation of biliary lineage markers and morphological changes, cells with ectopic active RhoA expression were found to form bile-duct like structures even without Matrigel/EPM treatment, while those with negative RhoA didn't form structures even induced by EPM. Further reserches showed that Y27632 eliminated RhoA CA/EPM induced bile duct structure formation. F-actin cytoplasmic staining also suggested that RhoA-ROCK signal pathway was involved in the biliary commitment of WB cells, lots of F-actin bundles were well aligned along the strenched cell direction in RhoA CA WB cells formed bile duct structures. Finally according to wesern blotting results RhoA was found phosphorylateing focal adhesion kinase (FAK) and MAPK 42/44. Then the signal pathways in which both EPM and RhoA function were EPM→αⅴβ1 integrin→RhoA→FAK→ERK1/2→MMP3/CEBPβ→Differetiation and EPM→αⅴβ1 integrin→RhoA→ROCK→Stress Fibre Formation→Morphogenesis.In a word, we successfully set up the Matrigel in vitro culture and EPM-PT67 coculture systems in which WB-F344 cells were found differentiating into biliary cells and RhoA was found playing an essential role in these process. Besides, our results suggested that RhoA play an obligatory role in EPM induced WB-F344 cell differentiation and further luminal morphogenesis, loss of RhoA function could eliminated EPM induced biliary lineage commitment. We also proposed the possible signal pathways in which RhoA and EPM function. Our research provided quite convenient models to study the hepatic differentiation in vitro, could faciliate the mechanism studies in hepatic differentiation and had broad prospects for clinical trials.