β‐Catenin, the downstream effector of the Wnt signaling, plays important roles in hepatic development, regeneration, and tumorigenesis. However, its role at hepatocyte adherens junctions (AJ) is relatively poorly understood, chiefly due to spontaneous compensation by γ‐catenin. We simultaneously ablated β‐ and γ‐catenin expression in mouse liver by interbreeding β‐catenin–γ‐catenin double‐floxed mice and Alb‐Cre transgenic mice. Double knockout mice show failure to thrive, impaired hepatocyte differentiation, cholemia, ductular reaction, progressive cholestasis, inflammation, fibrosis, and tumorigenesis, which was associated with deregulation of tight junctions (TJ) and bile acid transporters, leading to early morbidity and mortality, a phenotype reminiscent of progressive familial intrahepatic cholestasis (PFIC). To address the mechanism, we specifically and temporally eliminated both catenins from hepatocytes using adeno‐associated virus 8 carrying Cre‐recombinase under the thyroid‐binding globulin promoter (AAV8‐TBG‐Cre). This led to a time‐dependent breach of the blood–biliary barrier associated with sequential disruption of AJ and TJ verified by ultrastructural imaging and intravital microscopy, which revealed unique paracellular leaks around individual hepatocytes, allowing mixing of blood and bile and leakage of blood from one sinusoid to another. Molecular analysis identified sequential losses of E‐cadherin, occludin, claudin‐3, and claudin‐5 due to enhanced proteasomal degradation, and of claudin‐2, a β‐catenin transcriptional target, which was also validated in vitro. Conclusion: We report partially redundant function of catenins at AJ in regulating TJ and contributing to the blood–biliary barrier. Furthermore, concomitant hepatic loss of β‐ and γ‐catenin disrupts structural and functional integrity of AJ and TJ via transcriptional and posttranslational mechanisms. Mice with dual catenin loss develop progressive intrahepatic cholestasis, providing a unique model to study diseases such as PFIC. (H epatology 2018;67:2320‐2337).
Hepatocyte growth factor (HGF) and β-catenin have both been implicated in the pathogenesis of hepatocellular carcinoma (HCC). Human HGF (pCMV-HGF) gene delivery induces hepatomegaly in mice as is also evident in the β-catenin transgenic mice. The present study was aimed at examining any role of β-catenin in HGF-induced hepatomegaly. Here we report that human HGF gene delivery in a 1 week study in mice leads to hepatomegaly secondary to increased hepatocyte proliferation, which is accompanied by β-catenin activation in the liver. The primary mechanism of β-catenin activation is the loss of c-Met-β-catenin association at the hepatocyte membrane leading to nuclear translocation of β-catenin. In a four-week study, albeit β-catenin activation was observed along with significant hepatomegaly, c-Met-β-catenin complex remained unchanged. In addition, increased E-cadherin-β-catenin association was evident as well. HGF gene delivery failed to induce hepatomegaly in the β-catenin liver conditional knockout mice. These data demonstrate that importance of β-catenin in HGF-induced hepatocyte proliferation in vivo, thus highlighting the cooperation of HGF and β-catenin pathways in liver growth. (Supported by RSG-03-141-01-CNE and NIH - 1RO1DK62277 to SPSM).
Autosomal recessive polycystic kidney disease (ARPKD) is a monogenic disease characterized by development of hepatorenal cysts, pericystic fibrosis, and inflammation. Previous studies show that mast cell (MC) mediators such as histamine induce proliferation of cholangiocytes. We observed robust MC accumulation around liver cysts, but not kidney cysts, in polycystic kidney (PCK) rats (an animal model of ARPKD). Therefore, we hypothesized that MCs contribute to hepatic cyst growth in ARPKD. To test this hypothesis, we treated PCK rats with 1 of 2 different MC stabilizers, cromolyn sodium (CS) or ketotifen, or saline. The CS treatment decreased MC degranulation in the liver and reduced serum tryptase (an MC granule component). Interestingly, we observed an increase in liver to body weight ratio after CS treatment paralleled by a significant increase in individual cyst size. Hepatic fibrosis was not affected by CS treatment. The CS treatment increased hepatic cyst wall epithelial cell (CWEC) proliferation and decreased cell death. Ketotifen treatment also increased hepatic cyst size. In vitro, CS treatment did not affect proliferation of isolated hepatic CWECs from PCK rats. In contrast, CS decreased kidney to body weight ratio paralleled by a significant decrease in individual cyst size. The percentage of kidney to body weight ratio was strongly correlated with serum renin (an MC granule component). Ketotifen did not affect kidney cyst growth. Collectively, these data suggest that CS affects hepatic and renal cyst growth differently in PCK rats. Moreover, CS may be beneficial to renal cystic disease but may exacerbate hepatic cyst growth in ARPKD.
Abstract Rats selectively bred for the high intrinsic aerobic capacity runner (HCR) or low aerobic capacity runner (LCR) show pronounced differences in susceptibility for high‐fat/high sucrose (HFHS) diet‐induced hepatic steatosis and insulin resistance, replicating the protective effect of high aerobic capacity in humans. We have previously shown multiple systemic differences in energy and substrate metabolism that impacts steatosis between HCR and LCR rats. This study aimed to investigate hepatic‐specific mechanisms of action via changes in gene transcription. Livers of HCR rats had a greater number of genes that significantly changed in response to 3‐day HFHS compared with LCR rats (171 vs. 75 genes: >1.5‐fold, p < 0.05). HCR and LCR rats displayed numerous baseline differences in gene expression while on a low‐fat control diet (CON). A 3‐day HFHS diet resulted in greater expression of genes involved in the conversion of excess acetyl‐CoA to cholesterol and bile acid (BA) synthesis compared with the CON diet in HCR, but not LCR rats. These results were associated with higher fecal BA loss and lower serum BA concentrations in HCR rats. Exercise studies in rats and mice also revealed higher hepatic expression of cholesterol and BA synthesis genes. Overall, these results suggest that high aerobic capacity and exercise are associated with upregulated BA synthesis paired with greater fecal excretion of cholesterol and BA, an effect that may play a role in protection against hepatic steatosis in rodents.
HNF4α, the master regulator of hepatocyte differentiation, has been shown to inhibit hepatocyte proliferation via yet to be identified mechanisms. We investigated the mechanisms of HNF4α‐induced inhibition of hepatocyte proliferation using two novel HNF4α knockdown mouse models. Hepatocyte specific deletion of HNF4α resulted in increased hepatocyte proliferation. Comparative global gene expression analysis revealed a set of genes commonly regulated in both HNF4α deletion models. The majority of the down‐regulated genes are known HNF4α target genes involved in hepatic differentiation. Interestingly, many up‐regulated genes were associated with cell proliferation and cancer. HNF4α knockdown mice treated with the known hepatic carcinogen diethylnitrosamine developed a 7‐fold increase in number of tumors, as compared to controls. Furthermore, in vitro over expression of HNF4α in mouse HCC cells resulted in a decrease in cell proliferation along with a decrease in promitogenic gene expression. Taken together these data indicate that HNF4α inhibits hepatocyte proliferation by repression of specific pro‐mitogenic genes.
