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Intrahepatic cholangiocarcinoma (ICC) is a devastating liver cancer with extremely high intra- and inter-tumoral molecular heterogeneity, partly due to its diverse cellular origins. We investigated clinical relevance and the molecular mechanisms underlying hepatocyte (HC)-driven ICC development.Expression of ICC driver genes in human diseased livers at risk for ICC development were examined. The sleeping beauty and hydrodynamic tail vein injection based Akt-NICD/YAP1 ICC model was used to investigate pathogenetic roles of SRY-box transcription factor 9 (SOX9) and yes-associated protein 1 (YAP1) in HC-driven ICC. We identified DNA methyltransferase 1 (DNMT1) as a YAP1 target, which was validated by loss- and gain-of-function studies, and its mechanism addressed by chromatin immunoprecipitation sequencing.Co-expression of AKT and Notch intracellular domain (NICD)/YAP1 in HC yielded ICC that represents 13% to 29% of clinical ICC. NICD independently regulates SOX9 and YAP1 and deletion of either, significantly delays ICC development. Yap1 or TEAD inhibition, but not Sox9 deletion, impairs HC-to-biliary epithelial cell (BEC) reprogramming. DNMT1 was discovered as a novel downstream effector of YAP1-TEAD complex that directs HC-to-BEC/ICC fate switch through the repression of HC-specific genes regulated by master regulators for HC differentiation, including hepatocyte nuclear factor 4 alpha, hepatocyte nuclear factor 1 alpha, and CCAAT/enhancer-binding protein alpha/beta. DNMT1 loss prevented NOTCH/YAP1-dependent HC-driven cholangiocarcinogenesis, and DNMT1 re-expression restored ICC development following TEAD repression. Co-expression of DNMT1 with AKT was sufficient to induce tumor development including ICC. DNMT1 was detected in a subset of HCs and dysplastic BECs in cholestatic human livers prone to ICC development.We identified a novel NOTCH-YAP1/TEAD-DNMT1 axis essential for HC-to-BEC/ICC conversion, which may be relevant in cholestasis-to-ICC pathogenesis in the clinic.

Yes-associated protein 1 (YAP1) is a transcriptional coactivator that activates transcriptional enhanced associate domain transcription factors upon inactivation of the Hippo signaling pathway, to regulate biological processes like proliferation, survival, and differentiation. YAP1 is most prominently expressed in biliary epithelial cells (BECs) in normal adult livers and during development. In the current review, we will discuss the multiple roles of YAP1 in the development and morphogenesis of bile ducts inside and outside the liver, as well as in orchestrating the cholangiocyte repair response to biliary injury. We will review how biliary repair can occur through the process of hepatocyte-to-BEC transdifferentiation and how YAP1 is pertinent to this process. We will also discuss the liver's capacity for metabolic reprogramming as an adaptive mechanism in extreme cholestasis, such as when intrahepatic bile ducts are absent due to YAP1 loss from hepatic progenitors. Finally, we will discuss the roles of YAP1 in the context of pediatric pathologies afflicting bile ducts, such as Alagille syndrome and biliary atresia. In conclusion, we will comprehensively discuss the spatiotemporal roles of YAP1 in biliary development and repair after biliary injury while describing key interactions with other well-known developmental pathways.

Metabolic heterogeneity or functional zonation is a key characteristic of the liver that allows different metabolic pathways to be spatially regulated within the hepatic system and together contribute to whole body homeostasis. These metabolic pathways are segregated along the portocentral axis of the liver lobule into three hepatic zones: periportal, intermediate or midzonal, and perivenous. The liver performs complementary or opposing metabolic functions within different hepatic zones while synergistic functions are regulated by overlapping zones, thereby maintaining the overall physiological stability. The Wnt/β-catenin signaling pathway is well known for its role in liver growth, development, and regeneration. In addition, the Wnt/β-catenin pathway plays a fundamental and dominant role in hepatic zonation and signals to orchestrate various functions of liver metabolism and pathophysiology. The β-catenin protein is the central player in the Wnt/β-catenin signaling cascade, and its activation is crucial for metabolic patterning of the liver. However, dysregulation of Wnt/β-catenin signaling is also implicated in different liver pathologies, including those associated with metabolic syndrome. β-Catenin is preferentially localized in the central region of the hepatic lobule surrounding the central vein and regulates multiple functions of this region. This review outlines the role of Wnt/β-catenin signaling pathway in controlling the different metabolic processes surrounding the central vein and its relation to liver homeostasis and dysfunction.

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of β-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of β-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-β signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of β-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

ANKS6 is a ciliary protein that localizes to the proximal compartment of the primary cilium, where it regulates signaling. Mutations in the ANKS6 gene cause multiorgan ciliopathies in humans, which include laterality defects of the visceral organs, renal cysts as part of nephronophthisis and congenital hepatic fibrosis (CHF) in the liver. Although CHF together with liver ductal plate malformations are common features of several human ciliopathy syndromes, including nephronophthisis-related ciliopathies, the mechanism by which mutations in ciliary genes lead to bile duct developmental abnormalities is not understood. Here, we generated a knockout mouse model of Anks6 and show that ANKS6 function is required for bile duct morphogenesis and cholangiocyte differentiation. The loss of Anks6 causes ciliary abnormalities, ductal plate remodeling defects and periportal fibrosis in the liver. Our expression studies and biochemical analyses show that biliary abnormalities in Anks6-deficient livers result from the dysregulation of YAP transcriptional activity in the bile duct-lining epithelial cells. Mechanistically, our studies suggest, that ANKS6 antagonizes Hippo signaling in the liver during bile duct development by binding to Hippo pathway effector proteins YAP1, TAZ and TEAD4 and promoting their transcriptional activity. Together, this study reveals a novel function for ANKS6 in regulating Hippo signaling during organogenesis and provides mechanistic insights into the regulatory network controlling bile duct differentiation and morphogenesis during liver development.

Cholangiopathies are chronic, progressive diseases of the biliary tree, and can be either acquired or genetic. The primary target is the cholangiocyte (CC), the cell type lining the bile duct that is responsible for bile modification and transport. Despite advances in our understanding and diagnosis of these diseases in recent years, there are no proven therapeutic treatments for the majority of the cholangiopathies, and liver transplantation is the only life-extending treatment option for patients with end-stage cholestatic liver disease. One potential therapeutic strategy is to facilitate endogenous repair of the biliary system, which may alleviate intrahepatic cholestasis caused by these diseases. During biliary injury, hepatocytes (HC) are known to alter their phenotype and acquire CC-like features, a process known as cellular reprogramming. This brief review discusses the potential ways in which reprogrammed HC may contribute to biliary repair, thereby restoring bile flow and reducing the severity of cholangiopathies. Some of these include modifying bile to reduce toxicity, serving as a source of de novo CC to repair the biliary epithelium, or creating new channels to facilitate bile flow.

Chronic cholestasis results from bile secretory defects or impaired bile flow with few effective medical therapies available. Thyroid hormone triiodothyronine and synthetic thyroid hormone receptor agonists, such as sobetirome (GC-1), are known to impact lipid and bile acid (BA) metabolism and induce hepatocyte proliferation downstream of Wnt/β-catenin signaling after surgical resection; however, these drugs have yet to be studied as potential therapeutics for cholestatic liver disease. Herein, GC-1 was administered to ATP binding cassette subfamily B member 4 (Abcb4(−/−); Mdr2(−/−)) knockout (KO) mice, a sclerosing cholangitis model. KO mice fed GC-1 diet for 2 and 4 weeks had decreased serum alkaline phosphatase but increased serum transaminases compared with KO alone. KO mice on GC-1 also had higher levels of total liver BA due to alterations in expression of BA detoxification, transport, and synthesis genes, with the net result being retention of BA in the hepatocytes. Interestingly, GC-1 does not induce hepatocyte proliferation or Wnt/β-catenin signaling in KO mice, likely a result of decreased thyroid hormone receptor β expression without Mdr2. Therefore, although GC-1 treatment induces a mild protection against biliary injury in the early stages of treatment, it comes at the expense of hepatocyte injury and is suboptimal because of lower expression of thyroid hormone receptor β. Thus, thyromimetics may have limited therapeutic benefits in treating cholestatic liver disease.

Congenital hepatic fibrosis (CHF) is a developmental liver disease that is caused by mutations in genes that encode for ciliary proteins and is characterized by bile duct dysplasia and portal fibrosis. Recent work has demonstrated that mutations in ANKS6 can cause CHF due to its role in bile duct development. Here, we report a novel ANKS6 mutation, which was identified in an infant presenting with neonatal jaundice due to underlying biliary abnormalities and liver fibrosis. Molecular analysis revealed that ANKS6 liver pathology is associated with the infiltration of inflammatory macrophages to the periportal fibrotic tissue and ductal epithelium. To further investigate the role of macrophages in CHF pathophysiology, we generated a novel liver-specific Anks6 knockout mouse model. The mutant mice develop biliary abnormalities and rapidly progressing periportal fibrosis reminiscent of human CHF. The development of portal fibrosis in Anks6 KO mice coincided with the accumulation of inflammatory monocytes and macrophages in the mutant liver. Gene expression and flow cytometric analysis demonstrated the preponderance of M1- over M2-like macrophages at the onset of fibrosis. A critical role for macrophages in promoting peribiliary fibrosis was demonstrated by depleting the macrophages with clodronate liposomes which effectively reduced inflammatory gene expression and fibrosis, and ameliorated tissue histology and biliary function in Anks6 KO livers. Together, this study demonstrates that macrophages play an important role in the initiation of liver fibrosis in ANKS6-deficient livers and their therapeutic elimination may provide an avenue to mitigate CHF in patients.

Expansion of biliary epithelial cells (BECs) during ductular reaction (DR) is observed in liver diseases including cystic fibrosis (CF), and associated with inflammation and fibrosis, albeit without complete understanding of underlying mechanism. Using two different genetic mouse knockouts of β-catenin, one with β-catenin loss is hepatocytes and BECs (KO1), and another with loss in only hepatocytes (KO2), we demonstrate disparate long-term repair after an initial injury by 2-week choline-deficient ethionine-supplemented diet. KO2 show gradual liver repopulation with BEC-derived β-catenin-positive hepatocytes and resolution of injury. KO1 showed persistent loss of β-catenin, NF-κB activation in BECs, progressive DR and fibrosis, reminiscent of CF histology. We identify interactions of β-catenin, NFκB, and CF transmembranous conductance regulator (CFTR) in BECs. Loss of CFTR or β-catenin led to NF-κB activation, DR, and inflammation. Thus, we report a novel β-catenin-NFκB-CFTR interactome in BECs, and its disruption may contribute to hepatic pathology of CF., eLife digest The liver has an incredible capacity to repair itself or ‘regenerate’ – that is, it has the ability to replace damaged tissue with new tissue. In order to do this, the organ relies on hepatocytes (the cells that form the liver) and bile duct cells (the cells that form the biliary ducts) dividing and transforming into each other to repair and replace damaged tissue, in case the insult is dire. During long-lasting or chronic liver injury, bile duct cells undergo a process called ‘ductular reaction’, which causes the cells to multiply and produce proteins that stimulate inflammation, and can lead to liver scarring (fibrosis). Ductular reaction is a hallmark of severe liver disease, and different diseases exhibit ductular reactions with distinct features. For example, in cystic fibrosis, a unique type of ductular reaction occurs at late stages, accompanied by both inflammation and fibrosis. Despite the role that ductular reaction plays in liver disease, it is not well understood how it works at the molecular level. Hu et al. set out to investigate how a protein called β-catenin – which can cause many types of cells to proliferate – is involved in ductular reaction. They used three types of mice for their experiments: wild-type mice, which were not genetically modified; and two strains of genetically modified mice. One of these mutant mice did not produce β-catenin in biliary duct cells, while the other lacked β-catenin both in biliary duct cells and in hepatocytes. After a short liver injury – which Hu et al. caused by feeding the mice a specific diet – the wild-type mice were able to regenerate and repair the liver without exhibiting any ductular reaction. The mutant mice that lacked β-catenin in hepatocytes showed a temporary ductular reaction, and ultimately repaired their livers by turning bile duct cells into hepatocytes. On the other hand, the mutant mice lacking β-catenin in both hepatocytes and bile duct cells displayed sustained ductular reactions, inflammation and fibrosis, which looked like that seen in patients with liver disease associated to cystic fibrosis. Further probing showed that β-catenin interacts with a protein called CTFR, which is involved in cystic fibrosis. When bile duct cells lack either of these proteins, another protein called NF-B gets activated, which causes the ductular reaction, leading to inflammation and fibrosis. The findings of Hu et al. shed light on the role of β-catenin in ductular reaction. Further, the results show a previously unknown interaction between β-catenin, CTFR and NF-B, which could lead to better treatments for cystic fibrosis in the future.

Primary sclerosing cholangitis (PSC) is a rare, chronic, cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts. We have previously demonstrated the importance of Wnt/β-catenin signaling in mouse models of PSC. In this study, we wished to determine the clinical relevance of β-catenin localization in patient samples. In livers explanted from patients diagnosed with PSC, the majority (12/16; 75%) lacked β-catenin protein expression. Biopsies from patients post-transplant were classified as recurrent or non-recurrent based on pathology reports and then scored for β-catenin activation as a function of immunohistochemical localization. Despite lack of statistical significance, patients with recurrent primary disease (n = 11) had a greater percentage of samples with nuclear, transcriptionally active β-catenin (average 58.8%) than those with no recurrence (n = 10; 40.53%), while non-recurrence is correlated with β-catenin staining at the cell surface (average 52.63% for non-recurrent vs. 27.34% for recurrent), as determined by three different methods of analysis. β-catenin score and years-to-endpoint are both strongly associated with recurrence status (p = 0.017 and p = 0.00063, respectively). Finally, there was significant association between higher β-catenin score and increased alkaline phosphatase, a marker of biliary injury and disease progression. Thus, β-catenin expression and activation changes during the progression of PSC, and its localization may be a useful prognostic tool for predicting recurrence of this disease.

We previously identified an up‐regulation of specific Wnt proteins in the cholangiocyte compartment during cholestatic liver injury and found that mice lacking Wnt secretion from hepatocytes and cholangiocytes showed fewer proliferating cholangiocytes and high mortality in response to a 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) diet, a murine model of primary sclerosing cholangitis. In vitro studies demonstrated that Wnt7b, one of the Wnts up‐regulated during cholestasis, induces proliferation of cholangiocytes in an autocrine manner and increases secretion of proinflammatory cytokines. We hypothesized that loss of Wnt7b may exacerbate some of the complications of cholangiopathies by decreasing the ability of bile ducts to induce repair. Wnt7b‐flox mice were bred with Krt19‐cre mice to deplete Wnt7b expression in only cholangiocytes (CC) or with albumin‐Cre mice to delete Wnt7b expression in both hepatocytes and cholangiocytes (HC + CC). These mice were placed on a DDC diet for 1 month then killed for evaluation. Contrary to our expectations, we found that mice lacking Wnt7b from CC and HC + CC compartments had improved biliary injury, decreased cellular senescence, and lesser bile acid accumulation after DDC exposure compared to controls, along with decreased expression of inflammatory cytokines. Although Wnt7b knockout (KO) resulted in fewer proliferating cholangiocytes, CC and HC + CC KO mice on a DDC diet also had more hepatocytes expressing cholangiocyte markers compared to wild‐type mice on a DDC diet, indicating that Wnt7b suppression promotes hepatocyte reprogramming. Conclusion: Wnt7b induces a proproliferative proinflammatory program in cholangiocytes, and its loss is compensated for by conversion of hepatocytes to a biliary phenotype during cholestatic injury., Wnt7b has previously been shown to be upregulated in mouse models of cholestatic liver injury. Here, we delete Wnt7b from either cholangiocytes alone, or from both hepatocytes and cholangiocytes, and find that cholangiocyte proliferation is impaired after 5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) treatment. The decreased regeneration of the biliary compartment is compensated for by conversion of hepatocytes to a biliary phenotype, resulting in decreased biliary injury.

Intrahepatic cholangiocarcinoma (ICC), a disease of poor prognosis, has increased in incidence. It is challenging to treat due to intra- and inter-tumoral heterogeneity, which in part is attributed to diverse cellular origin. Indeed, co-expression of AKT and NICD in hepatocytes (HCs) yielded ICC, with similarity to proliferative, Notch-activated, and stem cell-like subclasses of clinical ICC. NICD regulated SOX9 and YAP1 during ICC development. Yap1 deletion or TEAD inhibition impaired HC-to-biliary epithelial cell (BEC) reprogramming and ICC proliferation; Sox9 loss repressed tumor growth; and Yap1-Sox9 combined loss abolished ICC development in AKT-NICD model. DNMT1 was discovered as a novel downstream effector of YAP1-TEAD complex that directed HC-to-BEC/ICC fate-switch. DNMT1 loss prevented Notch-dependent HC-to-ICC development, and DNMT1 re-expression restored ICC development following TEAD repression. Coexpression of DNMT1 with AKT was sufficient to induce hepatic tumor development including ICC. Thus, we have identified a novel NOTCH-YAP1/TEAD-DNMT1 axis essential for HC-driven ICC development.SIGNIFICANCEWe evaluated the clinical relevance of hepatocyte-driven ICC model and revealed critical but distinct roles of YAP1 and SOX9 in AKT-NICD-driven hepatocyte-derived ICC. We also identified NOTCH-YAP1/TEAD-DNMT1 axis as a critical driver for hepatocyte-to-ICC reprogramming, which might have biological and therapeutic implications in ICC subsets.

SummaryYAP1 regulates cell plasticity during liver injury, regeneration and cancer, but its role in liver development is unknown. YAP1 activity was detected in biliary cells and in cells at the hepato-biliary bifurcation in single-cell RNA-sequencing analysis of developing livers. Hepatoblast deletion ofYap1led to no impairment in Notch-driven SOX9+ ductal plate formation, but prevented the formation of the abutting second layer of SOX9+ ductal cells, blocking the formation of a patent intrahepatic biliary tree. Intriguingly, the mice survived for 8 months with severe cholestatic injury and without any hepatocyte-to-biliary transdifferentiation. Ductular reaction in the perihilar region suggested extrahepatic biliary proliferation likely seeking the missing intrahepatic biliary network. Long-term survival of these mice occurred through hepatocyte adaptation via reduced metabolic and synthetic function including altered bile acid metabolism and transport. Overall, we show YAP1 as a key regulator of bile duct development while highlighting a profound adaptive capability of hepatocytes.

β-Catenin, the downstream effector of the Wnt signaling, plays important roles in hepatic development, regeneration, and tumorigenesis. However, its role at hepatocyte adherens junctions (AJ) is relatively poorly understood, chiefly due to spontaneous compensation by γ-catenin. We simultaneously ablated β- and γ-catenin expression in mouse liver by interbreeding β-catenin-γ-catenin double-floxed mice and Alb-Cre transgenic mice. Double knockout mice show failure to thrive, impaired hepatocyte differentiation, cholemia, ductular reaction, progressive cholestasis, inflammation, fibrosis, and tumorigenesis, which was associated with deregulation of tight junctions (TJ) and bile acid transporters, leading to early morbidity and mortality, a phenotype reminiscent of progressive familial intrahepatic cholestasis (PFIC). To address the mechanism, we specifically and temporally eliminated both catenins from hepatocytes using adeno-associated virus 8 carrying Cre-recombinase under the thyroid-binding globulin promoter (AAV8-TBG-Cre). This led to a time-dependent breach of the blood-biliary barrier associated with sequential disruption of AJ and TJ verified by ultrastructural imaging and intravital microscopy, which revealed unique paracellular leaks around individual hepatocytes, allowing mixing of blood and bile and leakage of blood from one sinusoid to another. Molecular analysis identified sequential losses of E-cadherin, occludin, claudin-3, and claudin-5 due to enhanced proteasomal degradation, and of claudin-2, a β-catenin transcriptional target, which was also validated in vitro. Conclusion We report partially redundant function of catenins at AJ in regulating TJ and contributing to the blood-biliary barrier. Furthermore, concomitant hepatic loss of β- and γ-catenin disrupts structural and functional integrity of AJ and TJ via transcriptional and posttranslational mechanisms. Mice with dual catenin loss develop progressive intrahepatic cholestasis, providing a unique model to study diseases such as PFIC. (Hepatology 2018;67:2320-2337).

Cholestatic liver diseases result from impaired bile flow and are characterized by inflammation, atypical ductular proliferation (ADP), and fibrosis. The Wnt/β-catenin pathway plays a role in bile duct development, yet its role in cholestatic injury remains indeterminate. Liver-specific β-catenin knockout (KO) mice and wild-type (WT) littermates were subjected to cholestatic injury via bile duct ligation or short-term exposure to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Intriguingly, KO exhibit a dramatic protection from liver injury, fibrosis, and ADP, which coincided with significantly decreased total hepatic bile acids (BA). This led to the discovery of a novel role for β-catenin in regulating BA synthesis and transport through regulation of farnesoid X receptor (FXR) activation. We show that β-catenin functions as both an inhibitor of nuclear translocation and as a nuclear co-repressor through formation of a physical complex with FXR. Loss of β-catenin expedited FXR nuclear localization and FXR/RXRα association, culminating in small heterodimer protein (SHP) promoter occupancy and activation in response to BA or FXR agonist. Conversely, accumulation of β-catenin sequesters FXR, thus inhibiting its activation. Finally, exogenous suppression of β-catenin expression during cholestatic injury reduces β-catenin/FXR complex, activates FXR to decrease total BA and alleviates hepatic injury. Conclusion: We have identified a novel FXR/β-catenin interaction whose modulation via β-catenin suppression promotes FXR activation and decreases hepatic BA, which may provide unique therapeutic opportunities in cholestatic liver diseases. This article is protected by copyright. All rights reserved.

An important role for β-catenin in regulating p65 (a subunit of NF-κB) during acute liver injury has recently been elucidated through use of conditional β-catenin knockout mice, which show protection from apoptosis through increased activation of p65. Thus, we hypothesized that the p65/β-catenin complex may play a role in regulating processes such as cell proliferation during liver regeneration. We show through in vitro and in vivo studies that the p65/β-catenin complex is regulated through the TNF-α pathway and not through Wnt signaling. However, this complex is unchanged after partial hepatectomy (PH), despite increased p65 and β-catenin nuclear translocation as well as cyclin D1 activation. We demonstrate through both in vitro silencing experiments and chromatin immunoprecipitation after PH that β-catenin, and not p65, regulates cyclin D1 expression. Conversely, using reporter mice we show p65 is activated exclusively in the nonparenchymal (NPC) compartment during liver regeneration. Furthermore, stimulation of macrophages by TNF-α induces activation of NF-κB and subsequent secretion of Wnts essential for β-catenin activation in hepatocytes. Thus, we show that β-catenin and p65 are activated in separate cellular compartments during liver regeneration, with p65 activity in NPCs contributing to the activation of hepatocyte β-catenin, cyclin D1 expression, and subsequent proliferation.

BACKGROUND AND AIMS Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.

SUMMARY Yes-associated protein 1 (YAP1) regulates cell plasticity during liver injury, regeneration, and cancer, but its role in liver development is unknown. We detect YAP1 activity in biliary cells and in cells at the hepatobiliary bifur-cation in single-cell RNA sequencing analysis of developing livers. Deletion of Yap1 in hepatoblasts does not impair Notch-driven SOX9+ ductal plate formation but does prevent the formation of the abutting second layer of SOX9+ ductal cells, blocking the formation of a patent intrahepatic biliary tree. Intriguingly, these mice survive for 8 months with severe cholestatic injury and without hepatocyte-to-biliary transdifferentiation. Ductular reaction in the perihilar region suggests extrahepatic biliary proliferation, likely seeking the missing intrahepatic biliary network. Long-term survival of these mice occurs through hepatocyte adaptation via reduced metabolic and synthetic function, including altered bile acid metabolism and transport. Overall, we show YAP1 as a key regulator of bile duct development while highlighting a profound adaptive capability of hepatocytes., Graphical Abstract, In brief Molina et al. demonstrate that loss of Yap1 in developing livers leads to absence of intrahepatic bile ducts, preventing bile secretion from liver to small intestines. Yap1 knockout mice are small and jaundiced and exhibit adaptation via altering bile acid transport and metabolism, but they eventually die around 8 months of age.

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)

Background and aims The Wnt/β-catenin signaling pathway has a well-described role in liver pathobiology. Its suppression was recently shown to decrease bile acid (BA) synthesis, thus preventing the development of cholestatic liver injury and fibrosis after bile duct ligation (BDL). Approach and results To generalize these observations, we suppressed β-catenin in Mdr2 knockout (KO) mice, which develop sclerosing cholangitis due to regurgitation of BA from leaky ducts. When β-catenin was knocked down (KD) in KO for 2 weeks, hepatic and biliary injury were exacerbated in comparison to KO given placebo, as shown by serum biochemistry, ductular reaction, inflammation, and fibrosis. Simultaneously, KO/KD livers displayed increased oxidative stress and senescence and an impaired regenerative response. Although the total liver BA levels were similar between KO/KD and KO, there was significant dysregulation of BA transporters and BA detoxification/synthesis enzymes in KO/KD compared with KO alone. Multiphoton intravital microscopy revealed a mixing of blood and bile in the sinusoids, and validated the presence of increased serum BA in KO/KD mice. Although hepatocyte junctions were intact, KO/KD livers had significant canalicular defects, which resulted from loss of hepatocyte polarity. Thus, in contrast to the protective effect of β-catenin KD in BDL model, β-catenin KD in Mdr2 KO aggravated rather than alleviated injury by interfering with expression of BA transporters, hepatocyte polarity, canalicular structure, and the regenerative response. Conclusions The resulting imbalance between ongoing injury and restitution led to worsening of the Mdr2 KO phenotype, suggesting caution in targeting β-catenin globally for all cholestatic conditions.

Background & Aims Porphyrias result from anomalies of heme biosynthetic enzymes and can lead to cirrhosis and hepatocellular cancer. In mice, these diseases can be modeled by administration of a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes accumulation of porphyrin intermediates, resulting in hepatobiliary injury. Wnt/β-catenin signaling has been shown to be a modulatable target in models of biliary injury; thus, we investigated its role in DDC-driven injury. Methods β-Catenin (Ctnnb1) knockout (KO) mice, Wnt co-receptor KO mice, and littermate controls were fed a DDC diet for 2 weeks. β-Catenin was exogenously inhibited in hepatocytes by administering β-catenin dicer-substrate RNA (DsiRNA), conjugated to a lipid nanoparticle, to mice after DDC diet and then weekly for 4 weeks. In all experiments, serum and livers were collected; livers were analyzed by histology, western blotting, and real-time PCR. Porphyrin was measured by fluorescence, quantification of polarized light images, and liquid chromatography-mass spectrometry. Results DDC-fed mice lacking β-catenin or Wnt signaling had decreased liver injury compared to controls. Exogenous mice that underwent β-catenin suppression by DsiRNA during DDC feeding also showed less injury compared to control mice receiving lipid nanoparticles. Control livers contained extensive porphyrin deposits which were largely absent in mice lacking β-catenin signaling. Notably, we identified a network of key heme biosynthesis enzymes that are suppressed in the absence of β-catenin, preventing accumulation of toxic protoporphyrins. Additionally, mice lacking β-catenin exhibited fewer protein aggregates, improved proteasomal activity, and reduced induction of autophagy, all contributing to protection from injury. Conclusions β-Catenin inhibition, through its pleiotropic effects on metabolism, cell stress, and autophagy, represents a novel therapeutic approach for patients with porphyria. Lay summary Porphyrias are disorders resulting from abnormalities in the steps that lead to heme production, which cause build-up of toxic by-products called porphyrins. Liver is commonly either a source or a target of excess porphyrins, and complications can range from minor abnormalities to liver failure. In this report, we inhibited Wnt/β-catenin signaling in an experimental model of porphyria, which resulted in decreased liver injury. Targeting β-catenin affected multiple components of the heme biosynthesis pathway, thus preventing build-up of porphyrin intermediates. Our study suggests that drugs inhibiting β-catenin activity could reduce the amount of porphyrin accumulation and help alleviate symptoms in patients with porphyria.

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.

Activation of Wnt/β-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless ( Wls -LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls -LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased β-catenin–T-cell factor 4 interaction and cyclin-D1 expression. Wls -LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in β-catenin–T-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of β-catenin signaling and hepatocyte proliferation, both in vitro and in vivo . We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses β-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process.

Background & Aims Liver fibrosis, hepatocellular necrosis, inflammation, and proliferation of liver progenitor cells are features of chronic liver injury. Mouse models have been used to study the end-stage pathophysiology of chronic liver injury. However, little is known about differences in the mechanisms of liver injury among different mouse models because of our inability to visualize the progression of liver injury in vivo in mice. We developed a method to visualize bile transport and blood-bile barrier (BBlB) integrity in live mice. Methods C57BL/6 mice were fed a choline-deficient, ethionine-supplemented (CDE) diet or a diet containing 0.1% 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) for up to 4 weeks to induce chronic liver injury. We used quantitative liver intravital microscopy (qLIM) for real-time assessment of bile transport and BBlB integrity in the intact livers of the live mice fed the CDE, DDC, or chow (control) diets. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, real-time polymerase chain reaction, and immunoblots. Results Mice with liver injury induced by a CDE or a DDC diet had breaches in the BBlB and impaired bile secretion, observed by qLIM compared with control mice. Impaired bile secretion was associated with reduced expression of several tight-junction proteins (claudins 3, 5, and 7) and bile transporters (NTCP, OATP1, BSEP, ABCG5, and ABCG8). A prolonged (2-week) CDE, but not DDC, diet led to re-expression of tight junction proteins and bile transporters, concomitant with the reestablishment of BBlB integrity and bile secretion. Conclusions We used qLIM to study chronic liver injury, induced by a choline-deficient or DDC diet, in mice. Progression of chronic liver injury was accompanied by loss of bile transporters and tight junction proteins.

The Wnt/β-catenin pathway is critical for liver development, differentiation, metabolism, and homeostasis. Aberrations in this pathway have also been identified in many pathophysiological conditions, such as nonalcoholic steatohepatitis, fibrosis, and hepatobiliary neoplasias. Recent research has uncovered a role for Wnt/β-catenin signaling in regulating cell survival or cell death after acute or chronic liver injury as well. In this chapter, we will provide background on this signaling pathway as well as various mechanisms involved in apoptosis and survival in both experimental and clinical models of liver injury. We will then discuss the role of Wnt/β-catenin signaling in the context of various types of liver injury, focusing on two types of regulation: direct (through transcription of target genes and physical interaction with survival pathway components) and indirect (through regulation of processes such as oxidative stress and inflammation during liver injury). Finally, we will discuss the therapeutic implications of modulating this pathway to impact cell survival during liver injury.

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 lack of glutamine synthetase (GS), Cyp1a2 and Cyp2e1. Lrp-LKO also displayed a significant delay in initiation of LR due to 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 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.

β-Catenin signaling is implicated in hepatocellular carcinoma (HCC), although its role in inflammation, fibrosis, and proliferation is unclear. Commercially available HCC tissue microarray (TMA) of 89 cases was assessed for β-catenin, one of its transcriptional targets glutamine synthetase (GS), proliferation (PCNA), inflammation (CD45), and fibrosis (Sirius Red). HCC cells transfected with wild-type (WT) or mutant-β-catenin were evaluated for β-catenin-T cell factor transactivation by TOPFlash reporter activity and expression of certain targets. Hepatocyte-specific-serine-45-mutated β-catenin transgenic mice (TG) and controls (Con) were used to study thioacetamide (TAA)-induced hepatic fibrosis and tumorigenesis. Sustained β-catenin activation was only observed in mutant-, not WT-β-catenin transfected HCC cells. Aberrant intratumoral β-catenin stabilization was evident in 33% cases with 9% showing predominant nuclear with some cytoplasmic (N/C) localization and 24% displaying predominant cytoplasmic with occasional nuclear (C/N) localization. N/C β-catenin was associated with reduced fibrosis (p = 0.017) and tumor-wide GS staining (p < 0.001) while C/N correlated with increased intratumoral inflammation (p = 0.064) and proliferation (p = 0.029). A small subset of HCC patients (15.5%) lacked β-catenin staining and exhibited low inflammation and fibrosis (p < 0.05). TG and Con mice exposed to TAA showed comparable development of fibrosis and progression to cirrhosis and HCC. Taken together the data suggests a complex relationship of β-catenin, inflammation, fibrosis and HCC. GS staining is highly sensitive in identifying HCC with nuclear β-catenin, which may in turn represent β-catenin mutations, and does so with high negative predictive value. Also, β-catenin mutations and cirrhosis do not appear to cooperate in HCC pathogenesis in mice and men.

Aberrant platelet-derived growth factor receptor-α (PDGFRα) signaling is evident in a subset of hepatocellular cancers (HCCs). However, its role and regulation in hepatic physiology remains elusive. In the current study, we examined PDGFRα signaling in liver development and regeneration. We identified notable PDGFRα activation in hepatic morphogenesis that, when interrupted by PDGFRα-blocking antibody, led to decreased hepatoblast proliferation and survival in embryonic liver cultures. We also identified temporal PDGFRα overexpression, which is regulated by epidermal growth factor (EGF) and tumor necrosis factor α, and its activation at 3 to 24 hours after partial hepatectomy. Through generation of hepatocyte-specific PDGFRA knockout (KO) mice that lack an overt phenotype, we show that absent PDGFRα compromises extracelluar signal-regulated kinases and AKT activation 3 hours after partial hepatectomy, which, however, is alleviated by temporal compensatory increases in the EGF receptor (EGFR) and the hepatocyte growth factor receptor (Met) expression and activation along with rebound activation of extracellular signal-regulated kinases and AKT at 24 hours. These untimely increases in EGFR and Met allow for normal hepatocyte proliferation at 48 hours in KO, which, however, are aberrantly prolonged up to 72 hours. Intriguingly, such compensation also was visible in primary KO hepatocyte cultures but not in HCC cells after siRNA-mediated PDGFRα knockdown. Thus, temporal activation of PDGFRα in liver development is important in hepatic morphogenesis. In liver regeneration, despite increased signaling, PDGFRα is dispensable owing to EGFR and Met compensation, which is unique to normal hepatocytes but not HCC cells.

Wnt/β-catenin signaling plays an important role in hepatic homeostasis, especially in liver development, regeneration, and cancer, and loss of β-catenin signaling is often associated with increased apoptosis. To elucidate how β-catenin may be regulating hepatocyte survival, we investigated the susceptibility of β-catenin conditional knockout (KO) mice and their wild-type (WT) littermates to Fas and tumor necrosis factor-α (TNF-α), two common pathways of hepatocyte apoptosis. While comparable detrimental effects from Fas activation were observed in WT and KO, a paradoxical survival benefit was observed in KO mice challenged with D-galactosamine/lipopolysaccharide. KO mice showed significantly lower morbidity and liver injury due to early, robust, and protracted activation of NF-κB in the absence of β-catenin. Enhanced NF-κB activation in KO mice was associated with increased basal inflammation and Toll-like receptor 4 expression and lack of the p65/β-catenin complex in hepatocytes. The p65/β-catenin complex in WT livers underwent temporal dissociation allowing for NF-κB activation to regulate hepatocyte survival following TNF-α-induced hepatic injury. Decrease of total β-catenin protein but not its inactivation induced p65 activity, whereas β-catenin stabilization either chemically or due to mutations repressed it in hepatomas in a dose-dependent manner, whereas β-catenin stabilization repressed it either chemically or due to mutations. Conclusion: The p65/β-catenin complex in hepatocytes undergoes dynamic changes during TNF-α–induced hepatic injury and plays a critical role in NF-κB activation and cell survival. Modulation of β-catenin levels is a unique mode of regulating NF-κB activity and thus may present novel opportunities in devising therapeutics in specific hepatic injuries. (HEPATOLOGY 2013)