Loss of Anks6 leads to YAP deficiency and liver abnormalities.

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.

Dysregulated Bile Transporters and Impaired Tight Junctions During Chronic Liver Injury in Mice.

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.

Dual catenin loss in murine liver causes tight junctional deregulation and progressive intrahepatic cholestasis.

ß-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 ß- and γ-catenin expression in mouse liver by interbreeding ß-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 ß-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 ß- 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).

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. ß-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 ß-catenin. Enhanced association of ß-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 ß-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.

WNT5A inhibits hepatocyte proliferation and concludes ß-catenin signaling in liver regeneration.

Activation of Wnt/ß-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 ß-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 ß-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 ß-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses ß-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process.

Gliotoxin-induced changes in rat liver regeneration after partial hepatectomy.

BACKGROUND: Hepatic non-parenchymal cells (NPCs), encompassing hepatic stellate cells (HSCs), macrophages and endothelial cells, synthesize new hepatocyte growth factor (HGF) during liver regeneration (LR), and also play an important function in matrix production at the end of regeneration. AIMS: The aim of this study was to determine whether ablating NPCs either during hepatocyte proliferation or during matrix resynthesis will have any effect on LR. METHODS: Rats were injected with either gliotoxin (which induces NPC apoptosis) or vehicle control at various stages during partial hepatectomy (PH). NPCs and hepatocytes were also treated in vitro with gliotoxin. RESULTS: Proliferating cells were abundant in control livers 24 h after PH, while in gliotoxin-treated rats, mitosis was absent, apoptotic NPCs were apparent and HGF was decreased. In vitro studies demonstrated a > 50% decrease in cell viability in NPC cultures, while hepatocyte viability and proliferation were unaffected. Chronic elimination of NPCs over a period of 5 days after PH led to increased desmin-positive HSCs and fewer alpha smooth muscle actin-expressing HSCs. Finally, there was continued proliferation of hepatocytes and decreased collagen I and TGF-ß when HSCs, the matrix-producing NPCs, were ablated during later stages of LR. CONCLUSIONS: Ablation of NPCs at early time points after PH interferes with liver regeneration, while their ablation at late stages causes impairment in the termination of LR, demonstrating a time-dependent regulatory role of NPCs in the regenerative process.

Role and regulation of PDGFRα signaling in liver development and regeneration.

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.

Accelerated liver regeneration and hepatocarcinogenesis in mice overexpressing serine-45 mutant beta-catenin.

UNLABELLED: The Wnt/beta-catenin pathway is implicated in the pathogenesis of hepatocellular cancer (HCC). We developed a transgenic mouse (TG) in the FVB strain that overexpresses Ser45-mutated-beta-catenin in hepatocytes to study the effects on liver regeneration and cancer. In the two independent TG lines adult mice show elevated beta-catenin at hepatocyte membrane with no increase in the Wnt pathway targets cyclin-D1 or glutamine synthetase. However, TG hepatocytes upon culture exhibit a 2-fold increase in thymidine incorporation at day 5 (D5) when compared to hepatocytes from wildtype FVB mice (WT). When subjected to partial hepatectomy (PH), dramatic increases in the number of hepatocytes in S-phase are evident in TG at 40 and WT at 72 hours. Coincident with the earlier onset of proliferation, we observed nuclear translocation of beta-catenin along with an increase in total and nuclear cyclin-D1 protein at 40 hours in TG livers. To test if stimulation of beta-catenin induces regeneration, we used hydrodynamic delivery of Wnt-1 naked DNA to control mice, which prompted an increase in Wnt-1, beta-catenin, and known targets, glutamine synthetase (GS) and cyclin-D1, along with a concomitant increase in cell proliferation. beta-Catenin-overexpressing TG mice, when followed up to 12 months, showed no signs of spontaneous tumorigenesis. However, intraperitoneal delivery of diethylnitrosamine (DEN), a known carcinogen, induced HCC at 6 months in TG mice only. Tumors in TG livers showed up-regulation of beta-catenin, cyclin-D1, and unique genetic aberrations, whereas other canonical targets were unremarkable. CONCLUSION: beta-Catenin overexpression offers growth advantage during liver regeneration. Also, whereas no spontaneous HCC is evident, beta-catenin overexpression makes TG mice susceptible to DEN-induced HCC.

Beta-catenin regulates vitamin C biosynthesis and cell survival in murine liver.

Because the Wnt/beta-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 beta-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 beta-catenin-null livers and greater expression in beta-catenin overexpressing transgenic livers, HepG2 hepatoma cells (contain constitutively active beta-catenin), regenerating livers, and in hepatocellular cancer tissues that exhibit beta-catenin activation. Interestingly, coprecipitation and immunofluorescence studies also demonstrate an association of beta-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 beta-catenin. Significantly lower serum ascorbate levels were observed in beta-catenin knock-out 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 beta-catenin loss and regucalcin decrease was contributing to apoptosis, beta-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, beta-catenin regulates vitamin C biosynthesis in murine liver, which in turn may be one of the mechanisms contributing to the role of beta-catenin in cell survival.