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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 knockout 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 maybe one of the mechanisms contributing to the role of β-catenin in cell survival. [ABSTRACT FROM AUTHOR]

The Wnt/β-catenin pathway plays an important role in embryonic liver development, morphogenesis, and organogenesis. Here, we report on the activation of β-catenin during early postnatal liver growth. Modulation of β-catenin expression was studied in CD-1 mice livers over a time course of 0 to 30 postnatal days (PD) and 3 mo. Increases in total and active β-catenin were observed in developing livers from PD 5 to 20. A concomitant increase in the β-catenin-transcription factor (TCF) complex along with nuclear and cytoplasmic β-catenin was also evident, which coincided with ongoing hepatocyte proliferation by PCNA immunohistochemistry. This activation of β-catenin was multifactorial, including cyclical inhibition of glycogen synthase kinase-3β, suppression of casein kinase-IIαα, and a transient increase in β-catenin gene expression. Coprecipitation experiments revealed the formation of the β-catenin-cadherin complex at PD 5, whereas adequate β-catenin-c-Met complex at the hepatocyte membrane did not form until PD 20, which might be contributing to the free β-catenin pool during early postnatal growth. Furthermore, β-catenin liver-specific knockout mice exhibited smaller livers at PD 30, secondary to diminished hepatocyte proliferation. These data indicate that the activation of β-catenin is critical for early postnatal liver growth and development. [ABSTRACT FROM AUTHOR]

The wnt/beta-catenin pathway plays an important role in liver biology. Because of its diverse roles in liver, it is critical to identify novel targets, which might dictate such roles. Using ?-catenin conditional knockout mice (KO), we identified regucalcin as a novel target of this pathway. Regucalcin is a Ca2+ binding protein, which regulates Ca2+ transport from cytoplasm to nucleus in regenerating liver. mRNA expression of regucalcin was downregulated over 35-fold in KO as compared to wild-type controls (WT), which was confirmed by RT-PCR. An analysis of regucalcin protein expression by Western blotting showed the presence of regucalcin in WT at 1, 3 & 6 months, whereas it was decreased by 1 month and absent after 3 months in KO. Immunohistochemistry confirmed normal expression of regucalcin protein in nucleus and cytosol of WT hepatocytes, which was markedly diminished in KO. Also, beta-catenin overexpressing mice showed elevated levels of regucalcin as compared to the WT. An analysis of the promoter region of mouse regucalcin gene revealed at least 3 putative Tcf-binding sites. Finally, regucalcin protein expression mirrors that of beta-catenin nuclear translocation following 2/3 partial hepatectomy in the mouse. Also, higher regucalcin levels were observed in HepG2 (active-beta-catenin) as compared to Hep3B (normal beta-catenin) hepatoma cells, suggesting a possible link between beta-catenin and regucalcin in beta-catenin-active HCCs. In conclusion, regucalcin is regulated by beta-catenin in the liver, and may play a role in liver regeneration and tumorigenesis. [ABSTRACT FROM AUTHOR]

Hepatocellular cancer (HCC) is a disease of poor prognosis. Owing to the similarities between the processes of development and carcinogenesis, we utilized early developing livers to recognize differentially expressed genes and analyzed their expression in HCC. Livers from various mouse embryos (E11-E18) were utilized for gene and protein analysis. Patient HCC tissues (n=22), normal adjacent preneoplastic tissue and normal livers were examined for PDGFRα by Western and Real-Time PCR. The upstream effectors such as PDGF-A and PDGF-C were also examined. Finally, therapeutic effect of anti-PDGFRα antibody (MAb3G3; ImClone) was investigated on human hepatoma cells. A significantly higher expression of PDGFRct is observed during early liver development (p<0.001). 14/22 (>60%) of HCC samples showed a 1.5-7-fold increase in total PDGFRα levels as compared to controls (p<0.01). 17/21 additional samples revealed cytoplasmic PDGFRα and activated-PDGFRα (Tyr754) staining in tumors only. PDGFRα gene expression was increased in 3, PDGF-AA in 3 and PDGF-CC in 4 patients with elevated PDGFRα protein. Hep3B and HepG2 cells, which showed elevated and activated PDGFRα, when treated with MAb3G3, showed significant decrease in cell proliferation and survival (p<0.01). Thus around 70% of HCC patients had elevated PDGFRα levels in the tumor core, due to diverse mechanisms and its inhibition, might have therapeutic consequences in HCC. [ABSTRACT FROM AUTHOR]

β-Catenin is temporally expressed during early liver development. We utilized floxed β-catenin mice (lox-P sites flanking Ex2-6) & FoxA3-Cre transgenic mice to generate liver-specific β-catenin knockouts (ko). No viable offsprings were obtained with gestational lethality at around E17 stage. A significant decrease in β-catenin was observed in endodermal-derived hepatic epithelial cells in the liver after E12 by immunohistochemistry (IHC) and western blot (WB). There was a significant decrease in liver size (2-5 times) as compared to the control littermates beyond E14 stage. H&E showed decreased epithelial cells in the k.o. livers, which was confirmed by decreased numbers of E-cadherin, αFP and HNF4α-positive cells by FACS, IHC and WB. No defect in hematopoiesis was detected by the colony-forming cell unit assay. The epithelial cells from the k.o. livers failed to propagate in cultures. In addition, the hepatocytes remained undifferentiated based on histology and low glycogen, albumin & transferrin. The deficient hepatocyte compartment was due to decreased proliferation (PCNA) & survival (TUNEL). TEM studies showed epithelial cell blebbing reminiscent of oncotic death. Hepatocytes showed increased oxidative stress by MDA and 4-HNE assay secondary to decreased GSTs. Thus β-catenin is essential in normal liver development and is critical for hepatocyte maturation, expansion & survival, which are indispensable to survival. [ABSTRACT FROM AUTHOR]