Phenotypic plasticity of mature hepatocytes: investigation of the mechanisms for aberrant cytokeratin 19 expression in hepatocytes

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Despite widely-accepted notion that phenotype of hepatocytes is fixed, recent evidence has suggested that they can differentiate into bile duct-like cells in vitro. Previously we reported that cultured rat hepatocytes underwent dendritic morphogenesis with expression of bile duct cytokeratins (CK), when they were first aggregated and embedded within a type l collagen gel matrix. Here, using the organoid cultures, we show that hepatocytes can form real ductular structures and that protein tyrosine phosphorylation and Notch signaling may be involved in the process. After culture for more than three weeks. hepatocytes formed round ductular structures surrounded by laminin and basement membranes. Both the morphogenesis and bile duct-specific CK19 expression were enhanced by increased protein tyrosine phosphorylation, while suppressed by inhibition of mitogen-activated protein kinase kinase (MKK1) or phosphatidyl inositol (Pl) 3-kinase. Moreover, there was an increase in the expression of Notch ligands (Jagged1. Jagged2) and Notch1, as well as several Notch targets (Hes2, HERP2) during culture. We also screened protein-binding sites within the CK19 promoter by electrophoresis mobility shift assay, and identified three sites where protein binding appeared to be enhanced by cultule of hepatocytes. Our results indicate that the phenotype of mature hepatocytes is plastic and that specific protein tyrosine phosphorylation pathways and Notch signaling are involved in the metaplastic differentiation.