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

尽管肝细胞的表型是固定的，但最近的证据表明，肝细胞可以在体外分化为胆管样细胞。以前，我们报道，培养的大鼠肝细胞经历树突状形态与胆管细胞角蛋白（CK）的表达，当他们第一次聚集和嵌入在I型胶原凝胶基质。在这里，使用类器官培养，我们表明，肝细胞可以形成真实的导管结构，蛋白质酪氨酸磷酸化和Notch信号可能参与了这一过程。培养3周以上。肝细胞形成由层粘连蛋白和基底膜包围的圆形管状结构。形态发生和胆管特异性CK 19的表达均通过增加蛋白酪氨酸磷酸化而增强，而通过抑制促分裂原活化蛋白激酶（MKK 1）或磷脂酰肌醇（PI）3-激酶而抑制。此外，Notch配体（Jagged 1. Jagged 2）和Notch 1，以及几个Notch靶标（Hes 2，HERP 2）。我们还通过电泳迁移率变动分析筛选了CK 19启动子内的蛋白结合位点，并确定了三个蛋白结合似乎通过培养肝细胞而增强的位点。我们的研究结果表明，成熟肝细胞的表型是可塑的，特定的蛋白酪氨酸磷酸化途径和Notch信号参与化生分化。

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.