Notch-IGF1 signaling during liver regeneration drives biliary epithelial cell expansion and inhibits hepatocyte differentiation

In the adult liver, a population of facultative progenitor cells called biliary epithelial cells (BECs) proliferate and differentiate into cholangiocytes and hepatocytes after injury, thereby restoring liver function. In mammalian models of chronic liver injury, Notch signaling is essential for bile duct formation from these cells. However, the continual proliferation of BECs and differentiation of hepatocytes in these models have limited their use for determining whether Notch signaling is required for BECs to replenish hepatocytes after injury in the mammalian liver. Here, we used a temporally restricted model of hepatic repair in which large-scale hepatocyte injury and regeneration are initiated through the acute loss of Mdm2 in hepatocytes, resulting in the rapid, coordinated proliferation of BECs. We found that transient, early activation of Notch1- and Notch3-mediated signaling and entrance into the cell cycle preceded the phenotypic expansion of BECs into hepatocytes. Notch inhibition reduced BEC proliferation, which resulted in failure of BECs to differentiate into hepatocytes, indicating that Notch-dependent expansion of BECs is essential for hepatocyte regeneration. Notch signaling increased the abundance of the insulin-like growth factor 1 receptor (IGF1R) in BECs, and activating IGFR signaling increased BEC numbers but suppressed BEC differentiation into hepatocytes. These results suggest that different signaling mechanisms control BEC expansion and hepatocyte differentiation.

Downregulation of STAT3 enhanced chemokine expression and neutrophil recruitment in biliary atresia

Biliary atresia (BA) is an immune related disorder and STAT3 is a key signalling molecule in inflammation. This study was designed to clarify the function of STAT3 in BA. STAT3 expression was examined in patients and a mouse BA model in which STAT3 levels were further altered with a specific inhibitor or activator. Neutrophil accumulation and the levels of the neutrophil chemoattractants CXCL1 and IL-8 were determined. The effects of STAT3 inhibition on IL-8 expression were examined in human biliary epithelial cell cultures. Functional changes in liver STAT3+ neutrophils in the mouse model were analysed with 10x single cells RNAseq methods. Results showed STAT3 and p-STAT3 expression was reduced in BA liver tissue compared to control samples. Administration of a STAT3 inhibitor increased jaundice and mortality and reduced body weight in BA mice. In contrast, the STAT3 activator ameliorated BA symptoms. Extensive neutrophil accumulation together with CXCL1 upregulation, both of which were suppressed by an anti-CXCL1 antibody, were observed in the STAT3 inhibitor-treated group. Recombinant IL-8 administration increased disease severity in BA mice, and the STAT3 activator had the reverse effect. Inhibiting STAT3 increased apoptosis of human biliary epithelial cells together with upregulated IL-8 expression. RNAseq analysis revealed reduced the numbers of STAT3 expressing neutrophil in BA which was accompanied by marked enhanced interferon related anti-viral activities. In conclusion, STAT3 reduction, enhanced IL-8 and CXCL1 expression and promoted the accumulation of interferon responsive neutrophils resulting in biliary epithelial cell damage in BA.

Mathematical analysis of the effect of portal vein cells on biliary epithelial cell differentiation through the Delta-Notch signaling pathway

Objective: The Delta-Notch signaling pathway induces the differentiation of initially homogeneous progenitor cells in many biological contexts. A mathematical modeling of this signaling pathway on Drosophila wing vein differentiation suggested the importance of synthesis rate of components of this signaling pathway. The epithelial differentiation of bile ducts in the developing liver is unique in that portal vein smooth muscle cells express extremely high amount of Delta ligands and act as a disturbance. In the present study, the importance of synthesis rate of Delta ligands and Notch receptors is mathematically examined using the model for Drosophila wing vein differentiation. Results: The epithelial differentiation was dependent on the synthesis rate of Delta ligands and Notch receptors as far as examined. This result supports the importance of synthesis rate in Delta-Notch signaling pathway components in a disturbance-dependent context as well.

NOTCH-YAP1/TEAD-DNMT1 axis regulates hepatocyte reprogramming into intrahepatic cholangiocarcinoma

ABSTRACTIntrahepatic cholangiocarcinoma (ICC), a disease of poor prognosis, has increased in incidence. It is challenging to treat due to intra- and inter-tumoral heterogeneity, which in part is attributed to diverse cellular origin. Indeed, co-expression of AKT and NICD in hepatocytes (HCs) yielded ICC, with similarity to proliferative, Notch-activated, and stem cell-like subclasses of clinical ICC. NICD regulated SOX9 and YAP1 during ICC development. Yap1 deletion or TEAD inhibition impaired HC-to-biliary epithelial cell (BEC) reprogramming and ICC proliferation; Sox9 loss repressed tumor growth; and Yap1-Sox9 combined loss abolished ICC development in AKT-NICD model. DNMT1 was discovered as a novel downstream effector of YAP1-TEAD complex that directed HC-to-BEC/ICC fate-switch. DNMT1 loss prevented Notch-dependent HC-to-ICC development, and DNMT1 re-expression restored ICC development following TEAD repression. Coexpression of DNMT1 with AKT was sufficient to induce hepatic tumor development including ICC. Thus, we have identified a novel NOTCH-YAP1/TEAD-DNMT1 axis essential for HC-driven ICC development.SIGNIFICANCEWe evaluated the clinical relevance of hepatocyte-driven ICC model and revealed critical but distinct roles of YAP1 and SOX9 in AKT-NICD-driven hepatocyte-derived ICC. We also identified NOTCH-YAP1/TEAD-DNMT1 axis as a critical driver for hepatocyte-to-ICC reprogramming, which might have biological and therapeutic implications in ICC subsets.