Tumor necrosis factor-α promotes bile ductular transdifferentiation of mature rat hepatocytes in vitro.

We previously showed that mature hepatocytes could transdifferentiate into bile ductular cells when placed in a collagen-rich microenvironment. To explore the mechanism of transdifferentiation, we examined whether inflammatory cytokines affected the phenotype of hepatocytes in a three-dimensional culture system. Spheroidal aggregates of rat hepatocytes were embedded within a type I collagen gel matrix and cultured in the presence of various cytokines. In the control, hepatocytes gradually lost expression of albumin, tyrosine aminotransferase, and hepatocyte nuclear factor (HNF)-4α, while aberrantly expressed bile ductular markers, including cytokeratin 19 (CK 19) and spermatogenic immunoglobulin superfamily (SgIGSF). Among the cytokines examined, tumor necrosis factor (TNF)-α inhibited expression of albumin and HNF-4α, both at mRNA and protein levels. After culturing for 2 weeks with TNF-α, hepatocytic spheroids were transformed into extensively branching tubular structures composed of CK 19- and SgIGSF-positive small cuboidal cells. These cells responded to secretin with an increase in secretion and expressed functional bile duct markers. TNF-α also induced the phosphorylation of Jun N-terminal kinase (JNK) and c-Jun, and the morphogenesis was inhibited by SP600125, a specific JNK inhibitor. Furthermore, in chronic rat liver injury induced by CCl(4) , ductular reaction in the centrilobular area demonstrated strong nuclear staining of phosphorylated c-Jun. Our results demonstrate that TNF-α promotes the ductular transdifferentiation of hepatocytes and suggest a role of TNF-α in the pathogenesis of ductular reaction.

Recovery of mature hepatocytic phenotype following bile ductular transdifferentiation of rat hepatocytes in vitro.

We previously demonstrated that mature rat hepatocytes transdifferentiate to bile ductular cells when cultured in a three-dimensional collagen-rich matrix. Here, we show that the phenotype of transdifferentiated hepatocytes can be reversed by modulating culture conditions. Spheroidal aggregates of hepatocytes were cultured within a collagen gel matrix in the presence of serum and tumor necrosis factor-α. Spheroids transformed into ductular structures composed of small cuboidal cells, lost the expression of hepatocytic markers, whereas aberrantly expressed bile ductular markers. The transdifferentiated cells were then retrieved from the gels, plated on surfaces coated with a basement membrane-like material, and cultured in serum-free media. Cells spontaneously formed spheroidal aggregates and recovered hepatocytic phenotype. Dexamethasone (Dex), which suppressed the phosphorylation of ERK and Jun N-terminal kinase, facilitated the recovery, and the combination with interleukin-6 or oncostatin M resulted in the recovery of hepatocyte nuclear factor 4 α protein expression and the typical hepatocytic morphology, and a decrease in the expression of bile ductular markers. A cDNA microarray analysis revealed that the hepatocyte-specific mRNA expression profile was recovered in these cells. Our results demonstrate that hepatocytes are able to recover their phenotypes following bile ductular transdifferentiation, suggesting that hepatocytic and bile ductular phenotypes may be mutually reversible.