Beta-catenin in liver: A matter of life and death

Beta-catenin plays multiple roles in liver health and disease through regulation of proliferation, differentiation and metabolism. Elucidating the molecular basis of how beta-catenin regulates these diverse functions and others is the subject of this dissertation. While beta-catenin signaling undergoes temporal activation and its loss dampens liver regeneration (LR), the impact of stimulating this pathway remains unknown. We utilized transgenic (TG) mice expressing Ser45 mutated beta-catenin in hepatocytes to show a growth advantage both in vitro and during LR through cyclin-D1 regulation. Additionally, hydrodynamic delivery of Wnt-1 gene delivery induced beta-catenin activation and hepatocyte proliferation during LR. Regucalcin or senescence marker protein-30 (SMP30) was identified as a beta-catenin target in the liver through the use of hepatocyte-specific beta-catenin conditional knockout (KO) mice. SMP30 is a critical enzyme for the synthesis of ascorbic acid in murine hepatocytes, and its loss led to lower serum ascorbate levels in KO. KO hepatocytes displayed massive apoptosis in culture, which was blocked by addition of ascorbate to culture media. Additionally, apoptosis in HepG2 cells due to regucalcin knockdown was rescued by anti-oxidants. Thus, one mechanism of how beta-catenin regulates hepatocyte redox state and survival is through the control of regucalcin expression. KO livers displayed a basal increase in number of apoptotic hepatocytes. We explored the susceptibility of KO and wildtype (WT) controls to activation of the TNF-alpha mediated apoptotic pathway. Paradoxically, KO mice are refractory to D-galactosamine (GalN)/LPS, Actinomycin D (ActD)/LPS and GalN/TNF-alpha treatments showing lower morbidity than WT. NF-kappaB, a major pro-survival factor and its transcriptional targets were increased in KO basally and after injury due to lack of beta-catenin-p65 association, presence of increased basal inflammation and oxidative stress and increased TLR4 expression in KO livers. Additionally, p65 activation occurred earlier in KO than WT after LPS stimulation. Thus, paradoxical protection from TNF-alpha-mediated apoptosis in KOs occurs owing to pre-existing NF-kappaB activation that 'primes' the liver for protection against exogenous insult. Thus, we have identified beta-catenin as a pleiotropic factor regulating cell proliferation, cellular redox state and cell survival through specific genetic targets and protein-protein interactions. These findings have broad implications in acute and chronic hepatic diseases

Conditional Genetic Elimination of Hepatocyte Growth Factor in Mice Compromises Liver Regeneration after Partial Hepatectomy

Hepatocyte growth factor (HGF) has been shown to be indispensable for liver regeneration because it serves as a main mitogenic stimulus driving hepatocytes toward proliferation. We hypothesized that ablating HGF in adult mice would have a negative effect on the ability of hepatocytes to regenerate. Deletion of the HGF gene was achieved by inducing systemic recombination in mice lacking exon 5 of HGF and carrying the Mx1-cre or Cre-ERT transgene. Analysis of liver genomic DNA from animals 10 days after treatment showed that a majority (70-80%) of alleles underwent cre-induced genetic recombination. Intriguingly, however, analysis by RT-PCR showed the continued presence of both unrecombined and recombined forms of HGF mRNA after treatment. Separation of liver cell populations into hepatocytes and non-parenchymal cells showed equal recombination of genomic HGF in both cell types. The presence of the unrecombined form of HGF mRNA persisted in the liver in significant amounts even after partial hepatectomy (PH), which correlated with insignificant changes in HGF protein and hepatocyte proliferation. The amount of HGF produced by stellate cells in culture was indirectly proportional to the concentration of HGF, suggesting that a decrease in HGF may induce de novo synthesis of HGF from cells with residual unrecombined alleles. Carbon tetrachloride (CCl4)-induced regeneration resulted in a substantial decrease in preexisting HGF mRNA and protein, and subsequent PH led to a delayed regenerative response. Thus, HGF mRNA persists in the liver even after genetic recombination affecting most cells; however, PH subsequent to CCl4 treatment is associated with a decrease in both HGF mRNA and protein and results in compromised liver regeneration, validating an important role of this mitogen in hepatic growth. © 2013 Nejak-Bowen et al

Inverse correlation between the concentration of HGF in culture and production of HGF by HSCs.

<p>(<b>A</b>) Real-time PCR for HGF mRNA shows a dose-dependent decrease in HGF production by HSC-T6 cells in response to increasing concentrations of HGF in culture (*P<0.05; **P<0.01). (<b>B</b>) HGF protein production by HSC-T6 cells decreases in response to increased HGF, as measured by WB. Actin represents loading control. (<b>C</b>) Densitometry analysis on representative WB shown in (B).</p

Genotype and number of mice used in experiments.

<p>Genotype and number of mice used in experiments.</p

Liver regeneration stimulated by CCl4 depletes HGF mRNA and protein in HGF KO mice.

<p>(<b>A</b>) CCl4 treatment following genomic recombination further decreases full-length HGF mRNA, as assessed by real-time PCR. (<b>B</b>) WB shows decreased HGF expression in livers of HGF KO mice treated with CCl4 in combination with p(I):p(C), as compared to controls or those treated with p(I):p(C) only. Ponceau represents loading control.</p

Persistence of unrecombined HGF mRNA and protein in the livers of HGF^{ex.5 flox}; Cre^+/− mice after genomic recombination.

<p>(<b>A</b>) Schematic of the targeting strategy for conditional inactivation of the gene for HGF (top). Cre-mediated excision of the floxed HGF allele (middle) leads to the generation of a recombined allele (bottom) lacking exon 5. (<b>B</b>) Successful genomic deletion of HGF exon 5 after induction of recombination, as shown by PCR. Top - HGF^{ex.5 flox};Cre-ER^T mice; bottom - HGF^{ex.5 flox};Mx1-cre mice. (<b>C</b>) RT-PCR shows the presence of both recombined and unrecombined HGF mRNA in KO livers. (<b>D</b>) WB for HGF in control and HGF KO livers shows no differences after recombination. Ponceau represents loading control.</p

Recombination occurs in all hepatic cell populations, including those that produce HGF.

<p>(<b>A</b>) Separation of hepatic cell populations from HGF^{ex.5 flox};Mx1-cre mice into hepatocytes and NPCs shows recombination in both after p(I):p(C) treatment as compared to controls. (<b>B</b>) RT-PCR shows persistence of unrecombined HGF mRNA in both hepatocytes and NPCs. (<b>C</b>) Real-time PCR for full-length HGF mRNA shows a decrease in HGF in the NPC fraction after p(I):p(C) treatment. (<b>D</b>) WB for HGF in hepatocytes and NPCs shows that the amount of HGF is unchanged in KOs compared to controls, and is found mainly in the NPC fraction. Ponceau represents loading control.</p

Inhibiting Wnt Signaling Reduces Cholestatic Injury by Disrupting the Inflammatory Axis

Background & Aims: β-Catenin, the effector molecule of the Wnt signaling pathway, has been shown to play a crucial role in bile acid homeostasis through direct inhibition of farnesoid X receptor (FXR), which has pleiotropic effects on bile acid homeostasis. We hypothesize that simultaneous suppression of β-catenin signaling and activation of FXR in a mouse model of cholestasis will reduce injury and biliary fibrosis through inhibition of bile acid synthesis. Methods: To induce cholestasis, we performed bile duct ligation (BDL) on wild-type male mice. Eight hours after surgery, mice received FXR agonists obeticholic acid, tropifexor, or GW-4064 or Wnt inhibitor Wnt-C59. Severity of cholestatic liver disease and expression of target genes were evaluated after either 5 days or 12 days of treatment. Results: We found that although the FXR agonists worsened BDL-induced injury and necrosis after 5 days, Wnt-C59 did not. After 12 days of BDL, Wnt-C59 treatment, but not GW-4064 treatment, reduced both the number of infarcts and the number of inflammatory cells in liver. RNA sequencing analysis of whole livers revealed a notable suppression of nuclear factor kappa B signaling when Wnt signaling is inhibited. We then analyzed transcriptomic data to identify a cholangiocyte-specific signature in our model and demonstrated that Wnt-C59–treated livers were enriched for genes expressed in quiescent cholangiocytes, whereas genes expressed in activated cholangiocytes were enriched in BDL alone. A similar decrease in biliary injury and inflammation occurred in Mdr2 KO mice treated with Wnt-C59. Conclusions: Inhibiting Wnt signaling suppresses cholangiocyte activation and disrupts the nuclear factor kappa B–dependent inflammatory axis, reducing cholestatic-induced injury

Mice with Hepatic Loss of the Desmosomal Protein γ-Catenin Are Prone to Cholestatic Injury and Chemical Carcinogenesis

γ-Catenin, an important component of desmosomes, may also participate in Wnt signaling. Herein, we dissect the role of γ-catenin in liver by generating conditional γ-catenin knockout (KO) mice and assessing their phenotype after bile duct ligation (BDL) and diethylnitrosamine-induced chemical carcinogenesis. At baseline, KO and wild-type littermates showed comparable serum biochemistry, liver histology, and global gene expression. β-Catenin protein was modestly increased without any change in Wnt signaling. Desmosomes were maintained in KO, and despite no noticeable changes in gene expression, differential detergent fractionation revealed quantitative and qualitative changes in desmosomal cadherins, plaque proteins, and β-catenin. Enhanced association of β-catenin to desmoglein-2 and plakophilin-3 was observed in KO. When subjected to BDL, wild-type littermates showed specific changes in desmosomal protein expression. In KO, BDL deteriorated baseline compensatory changes, which manifested as enhanced injury and fibrosis. KO also showed enhanced tumorigenesis to diethylnitrosamine treatment because of Wnt activation, as also verified in vitro. γ-Catenin overexpression in hepatoma cells increased its binding to T-cell factor 4 at the expense of β-catenin-T-cell factor 4 association, induced unique target genes, affected Wnt targets, and reduced cell proliferation and viability. Thus, γ-catenin loss in liver is basally well tolerated. However, after insults like BDL, these compensations at desmosomes fail, and KO show enhanced injury. Also, γ-catenin negatively regulates tumor growth by affecting Wnt signaling