Liver‐specific β‐catenin knockout (β‐Catenin‐LKO) mice have revealed an essential role of β‐catenin in metabolic zonation where it regulates pericentral gene expression and in initiating liver ...regeneration (LR) after partial hepatectomy (PH), by regulating expression of Cyclin‐D1. However, what regulates β‐catenin activity in these events remains an enigma. Here we investigate to what extent β‐catenin activation is Wnt‐signaling‐dependent and the potential cell source of Wnts. We studied liver‐specific Lrp5/6 KO (Lrp‐LKO) mice where Wnt‐signaling was abolished in hepatocytes while the β‐catenin gene remained intact. Intriguingly, like β‐catenin‐LKO mice, Lrp‐LKO exhibited a defect in metabolic zonation observed as a lack of glutamine synthetase (GS), Cyp1a2, and Cyp2e1. Lrp‐LKO also displayed a significant delay in initiation of LR due to the absence of β‐catenin‐TCF4 association and lack of Cyclin‐D1. To address the source of Wnt proteins in liver, we investigated conditional Wntless (Wls) KO mice, which lacked the ability to secrete Wnts from either liver epithelial cells (Wls‐LKO), or macrophages including Kupffer cells (Wls‐MKO), or endothelial cells (Wls‐EKO). While Wls‐EKO was embryonic lethal precluding further analysis in adult hepatic homeostasis and growth, Wls‐LKO and Wls‐MKO were viable but did not show any defect in hepatic zonation. Wls‐LKO showed normal initiation of LR; however, Wls‐MKO showed a significant but temporal deficit in LR that was associated with decreased β‐catenin‐TCF4 association and diminished Cyclin‐D1 expression. Conclusion: Wnt‐signaling is the major upstream effector of β‐catenin activity in pericentral hepatocytes and during LR. Hepatocytes, cholangiocytes, or macrophages are not the source of Wnts in regulating hepatic zonation. However, Kupffer cells are a major contributing source of Wnt secretion necessary for β‐catenin activation during LR. (Hepatology 2014;60:964–976)
Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to ...cholangiocytes to assist in the repair and vice versa, as shown by various fate‐tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up‐regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low‐density lipoprotein‐related protein 5/6, transgenic mice expressing stable β‐catenin, and in vitro studies, we show a role of Wnt signaling through β‐catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a β‐catenin‐independent manner. Conclusion: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both β‐catenin‐dependent and ‐independent manners. (Hepatology 2016;64:1652‐1666)
Abstract Among the adult organs, liver is unique for its ability to regenerate. A concerted signaling cascade enables optimum initiation of the regeneration process following insults brought about by ...surgery or a toxicant. Additionally, there exists a cellular redundancy, whereby a transiently amplifying progenitor population appears and expands to ensure regeneration, when differentiated cells of the liver are unable to proliferate in both experimental and clinical scenarios. One such pathway of relevance in these phenomena is Wnt/β-catenin signaling, which is activated relatively early during regeneration mostly through post-translational modifications. Once activated, β-catenin signaling drives the expression of target genes that are critical for cell cycle progression and contribute to initiation of the regeneration process. The role and regulation of Wnt/β-catenin signaling is now documented in rats, mice, zebrafish and patients. More recently, a regenerative advantage of the livers in β-catenin overexpressing mice was reported, as was also the case after exogenous Wnt-1 delivery to the liver paving the way for assessing means to stimulate the pathway for therapeutics in liver failure. β-Catenin is also pertinent in hepatic oval cell activation and differentiation. However, aberrant activation of the Wnt/β-catenin signaling is reported in a significant subset of hepatocellular cancers (HCC). While many mechanisms of such activation have been reported, the most functional means of aberrant and sustained activation is through mutations in the β-catenin gene or in AXIN1/2, which encodes for a scaffolding protein critical for β-catenin degradation. Intriguingly, in experimental models hepatic overexpression of normal or mutant β-catenin is insufficient for tumorigenesis. In fact β-catenin loss promoted chemical carcinogenesis in the liver due to alternate mechanisms. Since most HCC occur in the backdrop of chronic hepatic injury, where hepatic regeneration is necessary for maintenance of liver function, but at the same time serves as the basis of dysplastic changes, this Promethean attribute exhibits a Jekyll and Hyde behavior that makes distinguishing good regeneration from bad regeneration essential for targeting selective molecular pathways as personalized medicine becomes a norm in clinical practice. Could β-catenin signaling be one such pathway that may be redundant in regeneration and indispensible in HCC in a subset of cases?
Highlights • β-catenin localization in hepatocellular carcinoma tissue array is characterized. • Glutamine synthetase correlates with nuclear β-catenin localization in HCC. • Nuclear β-catenin in HCC ...correlates with decreased intratumoral fibrosis. • Mice expressing normal or mutant-β-catenin show equal fibrosis and HCC after TAA. • Lack of β-catenin in HCC correlates with absence of inflammation and fibrosis.
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While the pathology of biliary fibrosis is well described, the signaling pathways involved in the proliferation and activity of the cholangiocyte compartment during cholestatic liver ...injury are incompletely understood. β‐Catenin, the chief downstream effector of the Wnt signaling pathway, has been shown to play an important role during bile duct development but its role in adult bile duct homeostasis remains undetermined. Hepatocyte and cholangiocyte‐specific β‐catenin knockout (KO) when fed a diet containing 0.1% 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) for 1–2 weeks showed decreased atypical ductular reaction as compared to wildtype littermates (WT). KO and WT were next subjected to bile duct ligation (BDL), which led to an increase in bilirubin in both WT and KO mice after 14 days. However, levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, and gamma glutamyl transpeptidase (GGTP) were significantly lower in KO when compared to WT. This was associated with a dramatic decrease in ductular reaction and fibrosis in β‐catenin KO livers. Further analysis yielded a notable decrease in total hepatic bile acids (BA) in the KO, which was associated with an increased FXR/SHP2 activation. This led to reduced BA synthesis and increased excretion. Thus, loss of β‐ catenin limits cholestatic injury in multiple models by modulating BA biosynthesis. These findings support an important role of Wnt/β‐catenin signaling in bile duct homeostasis and repair. This study was funded b
y
NIH grant 1R01DK62277 to SPM.
Loss of β‐catenin is often associated with increased apoptosis in the liver. Knowing the role of TNF‐α in regulating cell death, we explored the susceptibility of hepatocyte‐specific β‐catenin ...knockout (KO) mice and their wildtype littermates (WT) to intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) or TNF‐α after D‐galactosamine (GalN) or actinomycin D (ActD) pre‐treatment. Paradoxically, KO mice are refractory to GalN/LPS, ActD/LPS and GalN/TNF‐α and show significantly lower morbidity than the WT animals. Analysis of liver enzymes, TUNEL staining and caspase activity confirmed the presence of massive hepatic injury in the WT but not in the KO. To determine the mechanism, we analyzed the anti‐apoptotic transcription factor NF‐κB. Levels of NF‐κB (p65), phosphoserine‐536‐p65 and downstream targets Fas and Traf‐1 were elevated in the randomly fed KO mice over WT. Interestingly, we found a decrease in hepatic p65 in KO fasted overnight, however fasted and re‐fed KO showed greater expression and activation of p65 than WT. Multiple mechanisms induce basal NF‐κB activation in KO. Higher expression of TLR4, an LPS receptor; increased CD45+ve inflammatory cells (cell source of TNF‐α and IL‐6); & loss of basal p65‐β‐catenin association that prevents NF‐κB nuclear translocation, all support baseline NF‐κB activation in KO. Thus, we observed a paradoxical protection from TNF‐α‐mediated apoptosis in β‐catenin KO animals owing to the preexisting priming of the liver through NF‐κB activation, which is due to multiple mechanisms. Therefore, β‐catenin through its interactions with NF‐κB may be an important component of balancing hepatocyte life and death and therefore pertinent in regulating liver homeostasis.
Suppression of β‐catenin signaling in liver is associated with increased apoptosis through unknown mechanisms. Loss of Met, a downstream target of β‐catenin, has been also been shown to induce ...apoptosis through dissociation of the Met‐Fas complex. We hypothesized that loss of β‐catenin in hepatocytes would exacerbate Fas‐mediated apoptosis. However, we demonstrate that β‐catenin loss paradoxically protects against Fas‐mediated apoptosis. Analysis of mRNA expression levels in wild‐type (WT) and β‐catenin knockout (KO) livers reveals a 9‐fold increase in HGF expression in KO, while baseline levels of Met protein are decreased in KO compared to WT. Expression of Fas protein is modestly increased in KO under unstimulated conditions. We also identify a novel interaction of Fas and β‐catenin in WT livers at baseline, which was dramatically lower in KO. KO and WT mice were next injected intravenously with Jo‐2 antibody (a Fas agonist) and monitored for morbidity. While 100% of WT mice are morbid or dead by 6 hours, 35% of the KO mice were alive and showed a marked reduction in liver injury by histology, serum biochemistry and TUNEL immunohistochemistry. Interestingly, a number of proteins known to be protective against Fas‐mediated apoptosis, such as c‐Jun, heat‐shock proteins and NF‐κB, are basally upregulated in KO livers as assessed by gene array. Thus despite increased HGF mRNA expression and decreased levels of Met protein, we report a decreased susceptibility of β‐catenin KO mice to Fas‐mediated cell death, which appears to be due to the presence of basal chronic injury and apoptosis, which primes the KO livers for protection through upregulation of multiple known anti‐apoptotic factors.
Because healthy, non‐diseased donor livers for orthotopic liver transplantation are scarce, the development of alternative sources of functioning hepatocytes as a bridge to transplantation is ...critical. However, the availability of viable hepatocytes in numbers large enough for use in cell therapy is currently limited. Hepatocytes overexpressing a mutated form of β‐catenin may have a homing, growth, and regenerative advantage over wild‐type (WT) cells, thus decreasing the numbers of cells needed for transplantation. Our laboratory has previously shown that transgenic (TG) hepatocytes overexpressing Ser45‐mutated‐β‐catenin proliferate faster in culture than WT hepatocytes and show increased viability after 120 hours. However, the replicative capacity of these TG hepatocytes is unchanged from that of WT, as they senesce at approximately the same number of passages as WT hepatocytes in long‐term culture. Addtionally, expression of telomerase is equivalent in both WT and TG livers, indicating that TG hepatocytes containing mutated β‐catenin do not proliferate indefinitely. Preliminary results suggest that transplanted (Tx) WT hepatocytes can contribute to the livers of hepatectomized (PHx) β‐catenin KO livers (a model of delayed regeneration), but do not contribute to the livers of WT mice. Furthermore, the reduction in AST and ALT seen in PHx/Tx mice at D30 suggests that these transplanted hepatocytes may help to ameliorate damage and improve function. Currently ongoing cell transplantation experiments in which hepatocytes overexpressing TG mutated β‐catenin are transplanted into KO animals will elucidate the role of β‐catenin in repopulation of damaged livers.
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