β‐catenin Overexpressing Hepatocytes Reduce Bile Stasis by Contributing to Cholangiocyte‐Like Phenotype in Murine Model of Intrahepatic Cholestasis

Study Objective To investigate the role of β‐catenin overexpression in hepatocytes (HC) in hepatocyte‐to‐biliary reprogramming during intrahepatic cholestasis. Hypothesis We previously reported that mice transgenic for Ser‐45 mutant β‐catenin (TG) show increased hepatocyte‐to‐biliary reprogramming after administration of the porphyrinogenic biliary toxin 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) diet for 1 month. However, whether these cholangiocyte‐like hepatocytes fully convert into cholangiocytes (CC) or maintain an intermediate phenotype, remains unclear. We hypothesized that TG livers will have more HC‐derived CC than wild type and contribute to functional de novo biliary branches that will alleviate disease severity. Methods Age matched WT control (Con) and TG, both containing ROSA26‐stop flox/flox , were injected with AAV8‐TBG‐Cre to permanently label hepatocytes with EYFP and then fed 0.1% DDC diet for different time points ‐ 30d, 60d, 90d, 120d, 150d. Before sacrifice, bile flow analysis was measured for 1h to obtain flow rate and bile was collected. Liver histology and serum biochemistry were analyzed for fibrosis and parameters of injury. Porphyrin measurement was assessed in the liver tissues. Relative gene expression for bile transporter genes and immunofluorescence staining were performed to determine the expression of biliary markers. Results TG mice showed significantly increased bile flow rate as compared to Con mice after 150d of DDC diet. Liver histology and porphyrin measurement analysis showed significantly less porphyrin accumulation in TG as compared to the Con. Analysis of bile homeostasis and transport genes demonstrated comparable levels of expression in TG and Con mice suggesting a possible mechanism by which HC contribute to bile duct formation and thus, increase bile flow rate observed in TG. HC‐derived ducts positive for both EYFP and Sox9 were observed more frequently in TG mice compared to Con. Nevertheless, serum biochemistry did not show any differences in hepatic and biliary injury in these groups. Conclusions Mice with β‐catenin overexpressing HC showed significantly improved bile flow rate and reduced porphyrin accumulation in response to the biliary toxin DDC. Using HC fate‐tracing, we observed increased biliary cell markers in TG hepatocytes that is suggestive of the mechanism of enhanced bile flow. Further studies would be focused on assessing the functional comparison of these HC‐derived CC to native CC and evaluate if β‐catenin overexpression mediates similar biliary reprogramming in response to short‐term DDC diet.