Role of β-catenin in the adult liver
Authors: Frank J. Gonzalez
Journal: Hepatology (2006)

With its involvement in the Wnt signal transduction pathway and in tumorigenesis, β-catenin is an intensely studied protein.1 In tissues and quiescent cells, β-catenin is found localized in the cytosol and associated with the inner plasma membrane where it is tethered to cadherin and α-catenin forming a link to the cytoskeleton (Fig. 1). In this context, β-catenin is thought to play a role in cell-cell communication and adhesion, and possibly cell morphology. In the cytosol, β-catenin is bound to a number of proteins including adenomatous polyposis coli (APC) and the axins and, in non-stimulated cells, undergoes continuous turnover by proteolytic degradation that is stimulated by phosphorylation at specific serine and threonine residues. Localization and degradation of β-catenin are controlled by phosphorylation, most notably by casein kinase 1 and glycogen synthase kinase-3β (GSK-3β). Following activation of the Wnt pathway through binding of Wnt ligands to frizzled receptors on the cell surface, a protein, disheveled, inhibits GSK-3β leading to lower rates of phosphorylation and degradation of β-catenin, which results in its accumulation in the cytosol and nucleus. In the nucleus, β-catenin serves to co-activate a family of Lef/Tcf transcription factors that stimulate transcription of target genes including those encoding cyclin D and c-myc that promote cell proliferation. This influence on cell proliferation is the molecular basis for the role of β-catenin in tumorigenesis, in particular solid tumors of the breast, colon, liver, lung, gastric, prostate and skin.2 In colon carcinogenesis, β-catenin is the terminal signaling molecule associated with familial adenomatous polyposis that is due to germline mutations in the APC gene.3 A mouse model of this disorder also exists.4 Uncontrolled cell proliferation and associated tumorigenesis are activated by not only mutations in APC, but also mutations in the β-catenin protein that result in constitutive activation and nuclear accumulation of the protein. Schematic of the Wnt signal transduction pathway. The pathway is described in the text. Axin, axis inhibition; Dsh, dishevelled; CK1, casein kinase 1; LRP, lipoprotein-receptor related protein; PP2A, protein phosphatase 2A; β-TrCP, beta-transducin repeat-containing protein. Figure derived from ScienceSlides, Chapel Hill, NC. APC, adenomatous polyposis coli. β-catenin has a role in mammalian development as revealed by β-catenin-null mice that are embryonic lethal at day 7 post coitum due to a gastrulation defect with specific deficits in ectoderm and mesoderm formation.5 β-Catenin also functions in development of organ systems. During the course of liver development, there is a change in the nuclear accumulation of the protein with a notable decrease in the nuclear/cytoplasmic ratio that would be consistent with hepatocyte proliferation during liver growth.6 Antisense knock-down of β-catenin in day 10 embryonic cultures impairs liver development in vitro.7 In keeping with its known role in activating cell proliferation, β-catenin undergoes cellular stabilization and nuclear translocation during the course of liver regeneration after partial hepatectomy.8 In contrast, during matrigel-induced differentiation of primary hepatocytes in culture, β-catenin is stabilized but there is a concomitant decrease in its nuclear accumulation and an association with the hepatocyte growth factor receptor.9 In most tissues of mature mice including the colon, β-catenin is in the cytosol in its transcriptionally inactive (phosphorylated) state. In the non-dividing adult liver, the protein is expressed but is localized largely at the inner plasma membrane with little cytoplasmic and no detectable nuclear staining.6 Thus, in quiescent liver, β-catenin is probably undergoing the usual Ser/Thr phosphorylation and proteolytic turnover, and upon signaling for proliferation, the protein accumulates and translocates to the nucleus. In order to determine the role of β-catenin in liver, a conditional knockout mouse was generated and described in the current issue of HEPATOLOGY.10 The Cre/loxP system was used with the Cre transgene driven by the albumin promoter. In adult mice with liver-specific disruption of the β-catenin gene (Ctnnb1), β-catenin protein expression was abolished in hepatocytes; due to the cell specificity of the albumin promoter, disruption of Ctnnb1 would only be expected in hepatocytes and not in non-parenchymal cell such as Kupffer and Ito cells. In addition, the promoter is not significantly expressed until after birth with maximal expression occurring at around 2 weeks of age. Thus, Cre recombinase activity and disruption of Ctnnb1 would not be expected until the mice are at least two to three weeks old. While the liver weight of Ctnnb1-null mice was reduced by 20% in mature animals, the overall architecture of the liver was not significantly different from wild-type controls. This indicates that β-catenin has little influence on cell-cell interaction and cell architecture that are important in liver physiology. Of interest, no marked physiological deficiencies were reported suggesting that liver function was not significantly altered. These data suggest that loss of β-catenin in late development has only a minor impact on formation of the liver other that the decrease in mass of the organ. Most interestingly, there was little difference in cell proliferation and apoptosis between control and β-catenin liver-null mice suggesting that the lower liver weight is likely due to a lower rate of hepatocyte proliferation during later stages of development.10 Interestingly, transgenic mice overexpressing β-catenin have a 15% to 20% increase in liver size that is accompanied by an increase in cell proliferation that is evident even at age 3 months, long after liver growth and development is complete when mice reach puberty.11 Directed gene expression analysis revealed an alteration in a small number of genes including that encoding glutamine synthetase (Glu1) and a couple of cytochrome P450s, CYP1A2 (Cyp1a2) and CYP2E1 (Cyp2e1), that were downregulated in the absence of β-catenin.10 These changes in gene expression were found to influence blood ammonia levels in high protein fed mice and decreased sensitivity to the hepatotoxic effects of the commonly used analgesic drug acetaminophen. Indeed, in agreement with this observation, mice lacking expression of CYP1A2 and CYP2E1 are resistant to acetaminophen-induced hepatotoxicity.12 The lack of expression of Glu1 was consistent with earlier studies where Glu1 was identified as a potential target gene of β-catenin by transgenic over-expression of β-catenin in mice.13 Activation of the Wnt pathway by co-culturing epithelial cells with fetal hepatocytes and adenovirus-forced expression of β-catenin also implicated β-catenin in control of the Glu1.14 However, direct activation of the gene through a Lef/Tcf binding site has not been demonstrated thus leaving open a direct role for β-catenin in regulation of Glu1. In agreement with the current report, correlative studies examining the association between β-catenin, glutamine synthase and cytochromes P450s suggested that the Wnt pathway influences the latter enzymes.15 Mouse tumors having high glutamine synthase and activated β-catenin expressed elevated hepatic levels of CYP2B, CYP2C and CYP2E1 as compared to tumors with low glutamine synthase and β-catenin. Reporter gene assays revealed that β-catenin could markedly augment the expression of the rat CYP2B1 promoter15; analysis of the CYP2E1 gene promoter was not done. In the current study, Sekine et al. report that putative Lef/Tcf binding sites were found in upstream regions of the glutamine synthase gene (Glu1), and the Cyp1a2 and Cyp2e1 genes.10 However, experiments were not done to determine whether any of these sites were functionally relevant. It is not uncommon to have sites that share a consensus binding site for a transcription factors that are functionally irrelevant in a specific cellular context but may become functional in other cell types or under various stimuli. For example, the gene encoding phosphoenolpyruvate carboxykinase (PEPCK), expressed in liver and other tissues, has upstream cis-elements for several transcription factors and is under complex, tissue-specific and hormone-dependent control in keeping with its critical role in gluconeogenesis.16 While little information exists on the mechanism of regulation of Glu1, earlier studies using in vitro transcription17 and analysis of HNF1α-null mice18 revealed that the Cyp2e1 gene is controlled by the liver-enriched transcription factor HNF1α. Expression of the gene encoding CYP1A2 is also controlled by HNF1α as well as other transcription factors.19-21 The null mice studies suggest that liver-enriched transcription factors predominately control P450 genes in vivo. Thus, lack of β-catenin might have a direct or indirect role in HNF1α signaling on these genes as opposed to influencing Lef/Tcf transcription of Cyp1a2 and Cyp2e1. The authors have done a directed but limited analysis of transcription factor expression in the liver of the β-catenin liver-null mice and found no difference in expression of liver-enriched transcription factors. This result is not surprising since disruption of hepatocyte-enriched transcription factors usually results in debilitating phenotypes. For example, HNF1α-null mice are dwarf and diabetic.22 While global analysis of gene expression by microarray was not reported, what is most surprising from the current study is the small number of genes that have altered expression in liver of β-catenin liver-null mice. In addition to Glu1, Cyp1a2 and Cyp2e1, expression of the glutamine transporter, ornithine aminotransferase, ammonia transporter, and RhBG genes are also virtually extinguished in these mice.10 However, to my knowledge, scant information is available on the mechanisms regulating these latter genes; none have been functionally characterized for transcription factor binding sites. In addition, no common thread functionally links these genes, unlike those of other bonafide Lef/Tcf target genes controlling cell proliferation. Expression of these known Lef/Tcf target genes such as those encoding cyclin D1, c-myc, urokinase, and EGF receptor were not analyzed in the β-catenin liver-null mice but it is unlikely that they are affected since they would only be expressed in proliferating cells. While the presence of low levels of β-catenin in the nuclei of adult liver, not detectable by immunohistochemistry, cannot be excluded, the lack of β-catenin in the nucleus of hepatocytes suggests that Glu1, Cyp1a2 and Cyp2e1 are not directly activated by Lef/Tcf and β-catenin. Perhaps, absence of β-catenin alters selective signal transduction pathways that affect the phosphorylation status of hepatocyte enriched transcription factors or their coactivators. This could lead to altered HNF1α signaling in the absence of β-catenin and lower expression of Cyp1a2 and Cyp2e1. More studies are required to determine the role of β-catenin in control of gene expression in the adult liver. Many studies remain that could exploit the conditional null mouse system to examine the role of β-catenin in liver development and physiology. Since the β-catenin liver-null mice were produced using an albumin Cre transgene that is activated between 1 and 2 weeks after birth, the role of β-catenin in liver during early embryogenesis could not be assessed. Perhaps use of another promoter such as that controlling α-fetoprotein that is expressed in early embryos will be of value in the study of earlier stages of development. It would also be of interest to determine the effect of temporal disruption of β-catenin expression in mature mice using the tamoxifen-activated Cre under control of the albumin promoter.23 In particular, if the function of β-catenin is indirect and not due to Lef/Tcf, this strategy could address whether the lower expression of the putative β-catenin target genes is the result of changes in chromatin architecture that occur during the later stages of liver development in the absence of β-catenin. Altered chromatin structure or methylation status could result in downregulation of the putative target genes described above. While the role of β-catenin in modulating gene expression in the adult liver requires additional studies, the data from Sekine et al., would suggest that this factor has only a minor role in liver physiology in the absence of dietary, toxicologic or metabolic stress.10 The β-catenin liver-null mice will be an important model to determine the role of β-catenin in liver regeneration, cirrhosis and hepatocarcinogenesis. Based on earlier studies, these processes should be attenuated since they are dependent on the ability of hepatocytes to proliferate. It would also be of interest to subject the β-catenin liver-null mice to other dietary stresses such as high fat or ethanol feeding where the liver is forced to work overtime. Perhaps under these conditions, additional functions for β-catenin in the hepatocyte would emerge. In any case, this study shows the value of using conditional gene disruption to determine the function of important intracellular signaling molecules.