Benjamin Cieply

Wnt/β-Catenin Signaling Mediates Oval Cell Response in Rodents

Adult hepatic stem cells or oval cells are facultative stem cells in the liver that are activated during regeneration only during inhibition of innate hepatocyte proliferation. On the basis of its involvement in liver cancer, regeneration, and development, we investigated the role of the Wnt/β‐catenin pathway in oval cell response, which was initiated in male Fisher rats with 2‐acetylaminofluorine and two‐third partial hepatectomy (PHX). Extensive oval cell activation and proliferation were observed at 5 and 10 days post‐PHX, as indicated by hematoxylin‐eosin and proliferating cell nuclear antigen analysis. A noteworthy increase in total and active β‐catenin was observed at this time, which was localized to the oval cell cytoplasm and nuclei by immunohistochemistry and confirmed by double immunofluorescence. A concomitant increase in Wnt‐1 in hepatocytes along with increased expression of Frizzled‐2 in oval cells was observed. This paracrine mechanism coincided with a decrease in Wnt inhibitory factor‐1 and glycogen synthase kinase‐3β down‐regulation leading to β‐catenin stabilization. To strengthen its role, β‐catenin conditional knockout mice were treated with 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine to induce oval cell activation. A dramatic decrease in the A6‐positive oval cell numbers in the absence of β‐catenin demonstrated a critical role of β‐catenin in oval cell biology. Conclusion: The Wnt/β‐catenin pathway plays a key role in the normal activation and proliferation of adult hepatic stem cells. (HEPATOLOGY 2007.)

Unique phenotype of hepatocellular cancers with exon-3 mutations in beta-catenin gene†

Wnt/β‐catenin signaling plays an important role in liver development and regeneration. Its aberrant activation, however, is observed in a subset of primary hepatocellular cancers (HCCs). In the current study, we compare and contrast the tumor characteristics of HCC in the presence or absence of mutations in the β‐catenin gene (CTNNB1). Frozen HCCs (n = 32), including five fibrolamellar (FL) variants, and control livers (n = 3) from Health Sciences Tissue Bank and Department of Surgery at the University of Pittsburgh Medical Center, were examined for mutations in CTNNB1, protein levels of β‐catenin, tyrosine‐654‐phosphorylated‐β‐catenin (Y654‐β‐catenin), and glutamine synthetase (GS). Missense mutations in the exon‐3 of CTNNB1were identified in 9/32 HCCs. Total β‐catenin levels were higher than controls in most tumors; however, GS was exclusively increased in HCCs with mutations. Phenotypically, greater percentages of mutated HCCs showed macrovascular and microvascular invasion. Also, the tumor size was greater than double in mutated HCCs. High levels of total β‐catenin protein were observed in multinodular tumors independent of β‐catenin mutations. In addition, significant cases with mutations showed absence of cirrhosis. Finally, the highest levels of Y654‐β‐catenin were exclusively observed in fibrolamellar (FL)‐HCC cases. Conclusion: Thus, HCCs that harbor missense mutations in exon‐3 of CTNNB1 exhibit, histologically, a more aggressive phenotype. Also, CTNNB1 mutations might lead to HCC in the absence of cirrhosis. Finally, FL‐HCC cases display a unique up‐regulation of tyrosine‐phosphorylated‐β‐catenin, suggesting robust receptor tyrosine kinase signaling in this tumor type. (HEPATOLOGY 2009.)

Activation of Wnt/β-catenin pathway during hepatocyte growth factor–induced hepatomegaly in mice†

Hepatocyte growth factor (HGF) and β‐catenin both play a crucial role in stimulating hepatocyte proliferation, but whether these 2 pathways cooperate in inducing hepatocyte proliferation is unclear. We have previously reported that β‐catenin forms a complex with c‐Met (HGF receptor) that undergoes dissociation because of β‐catenin tyrosine phosphorylation on stimulation by HGF. It is also known that delivery of the human HGF gene cloned in a plasmid under a CMV promoter results in hepatomegaly in mice. In addition, recently characterized β‐catenin transgenic mice also showed hepatomegaly. The present study was based on the hypothesis that HGF‐induced hepatomegaly is mediated, at least in part, by activation of the Wnt/β‐catenin pathway. Here we report that delivery of the human HGF gene delivery in mice led to hepatomegaly via β‐catenin activation in the liver in 1‐ and 4‐week studies. The mechanisms of β‐catenin activation in the 1‐week study included loss of c‐Met–β‐catenin association as well as canonical β‐catenin activation, leading to its nuclear translocation. In the 4‐week study, β‐catenin activation was observed via canonical mechanisms, whereas the c‐Met–β‐catenin complex remained unchanged. In both studies there was an associated increase in the E‐cadherin–β‐catenin association at the membrane. In addition, we generated liver‐specific β‐catenin knockout mice, which demonstrated significantly smaller livers. HGF gene delivery failed to induce hepatomegaly in these β‐catenin conditionally null mice. In conclusion, β‐catenin‐ and HGF‐mediated signaling pathways cooperate in hepatocyte proliferation, which may be crucial in liver development, regeneration following partial hepatectomy, and pathogenesis of hepatocellular carcinoma. (HEPATOLOGY 2006;44:992–1002.)

Liver-Specific β-Catenin Knockout Mice Exhibit Defective Bile Acid and Cholesterol Homeostasis and Increased Susceptibility to Diet-Induced Steatohepatitis

Although the role of Wnt/beta-catenin signaling in liver growth and development is well established, its contribution in non-neoplastic hepatic pathologies has not been investigated. Here, we examine the role of beta-catenin in a murine model of diet-induced liver injury. Mice with hepatocyte-specific beta-catenin deletion (KO) and littermate controls were fed the steatogenic methionine and choline-deficient (MCD) diet or the corresponding control diet for 2 weeks and characterized for histological, biochemical, and molecular changes. KO mice developed significantly higher steatohepatitis and fibrosis on the MCD diet compared with wild-type mice. Both wild-type and KO livers accumulated triglyceride on the MCD diet but, unexpectedly, higher hepatic cholesterol levels were observed in KO livers on both control and MCD diets. Gene expression analysis showed that hepatic cholesterol accumulation in KO livers was not attributable to increased synthesis or uptake. KO mice had lower expression of bile acid synthetic enzymes but exhibited higher hepatic bile acid and serum bilirubin levels, suggesting defects in bile export. Therefore, loss of beta-catenin in the liver leads to defective cholesterol and bile acid metabolism in the liver and increased susceptibility to developing steatohepatitis in the face of metabolic stress.

β-Catenin Regulates Vitamin C Biosynthesis and Cell Survival in Murine Liver

Because the Wnt/β-catenin pathway plays multiple roles in liver pathobiology, it is critical to identify gene targets that mediate such diverse effects. Here we report a novel role of β-catenin in controlling ascorbic acid biosynthesis in murine liver through regulation of expression of regucalcin or senescence marker protein 30 and l-gulonolactone oxidase. Reverse transcription-PCR, Western blotting, and immunohistochemistry demonstrate decreased regucalcin expression in β-catenin-null livers and greater expression in β-catenin overexpressing transgenic livers, HepG2 hepatoma cells (contain constitutively active β-catenin), regenerating livers, and in hepatocellular cancer tissues that exhibit β-catenin activation. Interestingly, coprecipitation and immunofluorescence studies also demonstrate an association of β-catenin and regucalcin. Luciferase reporter and chromatin immunoprecipitation assays verified a functional TCF-4-binding site located between −163 and −157 (CTTTGCA) on the regucalcin promoter to be critical for regulation by β-catenin. Significantly lower serum ascorbate levels were observed in β-catenin knock-out mice secondary to decreased expression of regucalcin and also of l-gulonolactone oxidase, the penultimate and last (also rate-limiting) steps in the synthesis of ascorbic acid, respectively. These mice also show enhanced basal hepatocyte apoptosis. To test if ascorbate deficiency secondary to β-catenin loss and regucalcin decrease was contributing to apoptosis, β-catenin-null hepatocytes or regucalcin small interfering RNA-transfected HepG2 cells were cultured, which exhibited significant apoptosis that was alleviated by the addition of ascorbic acid. Thus, through regucalcin and l-gulonolactone oxidase expression, β-catenin regulates vitamin C biosynthesis in murine liver, which in turn may be one of the mechanisms contributing to the role of β-catenin in cell survival.