Hepatocyte ß-catenin loss is compensated by Insulin-mTORC1 activation to promote liver regeneration.

BACKGROUND AND AIMS: Liver regeneration (LR) following partial hepatectomy (PH) occurs via activation of various signaling pathways. Disruption of a single pathway can be compensated by activation of another pathway to continue LR. The Wnt-ß-catenin pathway is activated early during LR and conditional hepatocyte loss of ß-catenin delays LR. Here, we study mechanism of LR in the absence of hepatocyte-ß-catenin. APPROACH AND RESULTS: Eight-week-old hepatocyte-specific Ctnnb1 knockout mice (ß-catenin ΔHC ) were subjected to PH. These animals exhibited decreased hepatocyte proliferation at 40-120 h and decreased cumulative 14-day BrdU labeling of <40%, but all mice survived, suggesting compensation. Insulin-mediated mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) activation was uniquely identified in the ß-catenin ΔHC mice at 72-96 h after PH. Deletion of hepatocyte regulatory-associated protein of mTOR (Raptor), a critical mTORC1 partner, in the ß-catenin ΔHC mice led to progressive hepatic injury and mortality by 30 dys. PH on early stage nonmorbid Raptor ΔHC -ß-catenin ΔHC mice led to lethality by 12 h. Raptor ΔHC mice showed progressive hepatic injury and spontaneous LR with ß-catenin activation but died by 40 days. PH on early stage nonmorbid Raptor ΔHC mice was lethal by 48 h. Temporal inhibition of insulin receptor and mTORC1 in ß-catenin ΔHC or controls after PH was achieved by administration of linsitinib at 48 h or rapamycin at 60 h post-PH and completely prevented LR leading to lethality by 12-14 days. CONCLUSIONS: Insulin-mTORC1 activation compensates for ß-catenin loss to enable LR after PH. mTORC1 signaling in hepatocytes itself is critical to both homeostasis and LR and is only partially compensated by ß-catenin activation. Dual inhibition of ß-catenin and mTOR may have notable untoward hepatotoxic side effects.

NOTCH-YAP1/TEAD-DNMT1 Axis Drives Hepatocyte Reprogramming Into Intrahepatic Cholangiocarcinoma.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Dual ß-Catenin and γ-Catenin Loss in Hepatocytes Impacts Their Polarity through Altered Transforming Growth Factor-ß and Hepatocyte Nuclear Factor 4α Signaling.

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 ß-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 ß-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-ß 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 ß-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by ß-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

Nuclear factor erythroid 2-related factor 2 and ß-Catenin Coactivation in Hepatocellular Cancer: Biological and Therapeutic Implications.

BACKGROUND AND AIMS: HCC remains a major unmet clinical need. Although activating catenin beta-1 (CTNNB1) mutations are observed in prominent subsets of HCC cases, these by themselves are insufficient for hepatocarcinogenesis. Coexpression of mutant CTNNB1 with clinically relevant co-occurrence has yielded HCCs. Here, we identify cooperation between ß-catenin and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling in HCC. APPROACH AND RESULTS: Public HCC data sets were assessed for concomitant presence of CTNNB1 mutations and either mutations in nuclear factor erythroid-2-related factor-2 (NFE2L2) or Kelch like-ECH-associated protein 1 (KEAP1), or Nrf2 activation by gene signature. HCC development in mice and similarity to human HCC subsets was assessed following coexpression of T41A-CTNNB1 with either wild-type (WT)-, G31A-, or T80K-NFE2L2. Based on mammalian target of rapamycin complex 1 activation in CTNNB1-mutated HCCs, response of preclinical HCC to mammalian target of rapamycin (mTOR) inhibitor was investigated. Overall, 9% of HCC cases showed concomitant CTNNB1 mutations and Nrf2 activation, subsets of which were attributable to mutations in NFE2L2/KEAP1. Coexpression of mutated CTNNB1 with mutant NFE2L2, but not WT-NFE2L2, led to HCC development and mortality by 12-14 weeks. These HCCs were positive for ß-catenin targets, like glutamine synthetase and cyclin-D1, and Nrf2 targets, like NAD(P)H quinone dehydrogenase 1 and peroxiredoxin 1. RNA-sequencing and pathway analysis showed high concordance of preclinical HCC to human HCC subset showing activation of unique (iron homeostasis and glioblastoma multiforme signaling) and expected (glutamine metabolism) pathways. NFE2L2-CTNNB1 HCC mice were treated with mTOR inhibitor everolimus (5-mg/kg diet ad libitum), which led to >50% decrease in tumor burden. CONCLUSIONS: Coactivation of ß-catenin and Nrf2 is evident in 9% of all human HCCs. Coexpression of mutant NFE2L2 and mutant CTNNB1 led to clinically relevant HCC development in mice, which responded to mTOR inhibitors. Thus, this model has both biological and therapeutic implications.

Hepatic Stellate Cell-Specific Platelet-Derived Growth Factor Receptor-α Loss Reduces Fibrosis and Promotes Repair after Hepatocellular Injury.

Platelet-derived growth factor receptor (PDGFR)-α plays roles in cell survival, proliferation, and differentiation; however, its function in chronic liver injury sequelae, such as fibrosis, is unknown. Hepatic stellate cells (HSCs), the primary mediators of fibrosis, undergo activation, which entails differentiation to myofibroblasts, proliferation, migration, and collagen deposition, partially in response to PDGFs. To examine the role of PDGFR-α in HSCs, Lrat-Cre recombinase and Pdgfra-floxed mice were bred to generate Lrat-CrePdgfra-/- (knockout) animals, which were subjected to chronic liver injury through carbon tetrachloride treatment, bile duct ligation, and 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Although no major difference was observed after other types of liver injury, PDGFR-α loss in HSCs led to a significant albeit transient reduction in fibrosis after carbon tetrachloride injury, associated with increased HSC death and reduced migration. There was continued alleviation of hepatocellular injury in knockout mice despite ongoing carbon tetrachloride insult, associated with increased numbers of CD68 and F480 macrophages and increased clearance of damaged hepatocytes. Altogether our findings support a profibrotic role of PDGFR-α in HSCs during chronic liver injury in vivo via regulation of HSC survival and migration and affect the immune microenvironment, especially macrophages in clearing dying hepatocytes. Thus, our study provides a preclinical foundation for the future testing of therapeutic PDGFR-α inhibition in hepatic fibrosis, especially in combination with other therapies.

Hdac1 Regulates Differentiation of Bipotent Liver Progenitor Cells During Regeneration via Sox9b and Cdk8.

BACKGROUND & AIMS: Upon liver injury in which hepatocyte proliferation is compromised, liver progenitor cells (LPCs), derived from biliary epithelial cells (BECs), differentiate into hepatocytes. Little is known about the mechanisms of LPC differentiation. We used zebrafish and mouse models of liver injury to study the mechanisms. METHODS: We used transgenic zebrafish, Tg(fabp10a:CFP-NTR), to study the effects of compounds that alter epigenetic factors on BEC-mediated liver regeneration. We analyzed zebrafish with disruptions of the histone deacetylase 1 gene (hdac1) or exposed to MS-275 (an inhibitor of Hdac1, Hdac2, and Hdac3). We also analyzed zebrafish with mutations in sox9b, fbxw7, kdm1a, and notch3. Zebrafish larvae were collected and analyzed by whole-mount immunostaining and in situ hybridization; their liver tissues were collected for quantitative reverse transcription polymerase chain reaction. We studied mice in which hepatocyte-specific deletion of ß-catenin (Ctnnb1flox/flox mice injected with Adeno-associated virus serotype 8 [AAV8]-TBG-Cre) induces differentiation of LPCs into hepatocytes after a choline-deficient, ethionine-supplemented (CDE) diet. Liver tissues were collected and analyzed by immunohistochemistry and immunoblots. We performed immunohistochemical analyses of liver tissues from patients with compensated or decompensated cirrhosis or acute on chronic liver failure (n = 15). RESULTS: Loss of Hdac1 activity in zebrafish blocked differentiation of LPCs into hepatocytes by increasing levels of sox9b mRNA and reduced differentiation of LPCs into BECs by increasing levels of cdk8 mRNA, which encodes a negative regulator gene of Notch signaling. We identified Notch3 as the receptor that regulates differentiation of LPCs into BECs. Loss of activity of Kdm1a, a lysine demethylase that forms repressive complexes with Hdac1, produced the same defects in differentiation of LPCs into hepatocytes and BECs as observed in zebrafish with loss of Hdac1 activity. Administration of MS-275 to mice with hepatocyte-specific loss of ß-catenin impaired differentiation of LPCs into hepatocytes after the CDE diet. HDAC1 was expressed in reactive ducts and hepatocyte buds of liver tissues from patients with cirrhosis. CONCLUSIONS: Hdac1 regulates differentiation of LPCs into hepatocytes via Sox9b and differentiation of LPCs into BECs via Cdk8, Fbxw7, and Notch3 in zebrafish with severe hepatocyte loss. HDAC1 activity was also required for differentiation of LPCs into hepatocytes in mice with liver injury after the CDE diet. These pathways might be manipulated to induce LPC differentiation for treatment of patients with advanced liver diseases.

Loss of Wnt Secretion by Macrophages Promotes Hepatobiliary Injury after Administration of 3,5-Diethoxycarbonyl-1, 4-Dihydrocollidine Diet.

Exposure of mice to a diet containing 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) induces porphyrin accumulation, cholestasis, immune response, and hepatobiliary damage mimicking hepatic porphyria and sclerosing cholangitis. Although ß-catenin signaling promotes hepatocyte proliferation, and macrophages are a source of Wnts, the role of macrophage-derived Wnts in modulating hepatobiliary injury/repair remains unresolved. We investigated the effect of macrophage-specific deletion of Wntless, a cargo protein critical for cellular Wnt secretion, by feeding macrophage-Wntless-knockout (Mac-KO) and wild-type littermates a DDC diet for 14 days. DDC exposure induced Wnt11 up-regulation in macrophages. Mac-KO mice on DDC showed increased serum alkaline phosphatase, aspartate aminotransferase, direct bilirubin, and histologic evidence of more cell death, inflammation, and ductular reaction. There was impaired hepatocyte proliferation evidenced by Ki-67 immunostaining, which was associated with decreased hepatocyte ß-catenin activation and cyclin-D1 in Mac-KO. Mac-KO also showed increased CD45, F4/80, and neutrophil infiltration after DDC diet, along with increased expression of several proinflammatory cytokines and chemokines. Gene expression analyses of bone marrow-derived macrophages from Mac-KO mice and F4/80+ macrophages isolated from DDC-fed Mac-KO livers showed proinflammatory M1 polarization. In conclusion, this study shows that a lack of macrophage Wnt secretion leads to more DDC-induced hepatic injury due to impaired hepatocyte proliferation and increased M1 macrophages, which promotes immune-mediated cell injury.

ß-Catenin and Yes-Associated Protein 1 Cooperate in Hepatoblastoma Pathogenesis.

Hepatoblastoma (HB), the most common pediatric primary liver neoplasm, shows nuclear localization of ß-catenin and yes-associated protein 1 (YAP1) in almost 80% of the cases. Co-expression of constitutively active S127A-YAP1 and ΔN90 deletion-mutant ß-catenin (YAP1-ΔN90-ß-catenin) causes HB in mice. Because heterogeneity in downstream signaling is being identified owing to mutational differences even in the ß-catenin gene alone, we investigated if co-expression of point mutants of ß-catenin (S33Y or S45Y) with S127A-YAP1 led to similar tumors as YAP1-ΔN90-ß-catenin. Co-expression of S33Y/S45Y-ß-catenin and S127A-YAP1 led to activation of Yap and Wnt signaling and development of HB, with 100% mortality by 13 to 14 weeks. Co-expression with YAP1-S45Y/S33Y-ß-catenin of the dominant-negative T-cell factor 4 or dominant-negative transcriptional enhanced associate domain 2, the respective surrogate transcription factors, prevented HB development. Although histologically similar, HB in YAP1-S45Y/S33Y-ß-catenin, unlike YAP1-ΔN90-ß-catenin HB, was glutamine synthetase (GS) positive. However, both ΔN90-ß-catenin and point-mutant ß-catenin comparably induced GS-luciferase reporter in vitro. Finally, using a previously reported 16-gene signature, it was shown that YAP1-ΔN90-ß-catenin HB tumors exhibited genetic similarities with more proliferative, less differentiated, GS-negative HB patient tumors, whereas YAP1-S33Y/S45Y-ß-catenin HB exhibited heterogeneity and clustered with both well-differentiated GS-positive and proliferative GS-negative patient tumors. Thus, we demonstrate that ß-catenin point mutants can also collaborate with YAP1 in HB development, albeit with a distinct molecular profile from the deletion mutant, which may have implications in both biology and therapy.

Hepatocyte-Specific ß-Catenin Deletion During Severe Liver Injury Provokes Cholangiocytes to Differentiate Into Hepatocytes.

Liver regeneration after injury is normally mediated by proliferation of hepatocytes, although recent studies have suggested biliary epithelial cells (BECs) can differentiate into hepatocytes during severe liver injury when hepatocyte proliferation is impaired. We investigated the effect of hepatocyte-specific ß-catenin deletion in recovery from severe liver injury and BEC-to-hepatocyte differentiation. To induce liver injury, we administered choline-deficient, ethionine-supplemented (CDE) diet to three different mouse models, the first being mice with deletion of ß-catenin in both BECs and hepatocytes (Albumin-Cre; Ctnnb1flox/flox mice). In our second model, we performed hepatocyte lineage tracing by injecting Ctnnb1flox/flox ; Rosa-stopflox/flox -EYFP mice with the adeno-associated virus serotype 8 encoding Cre recombinase under the control of the thyroid binding globulin promoter, a virus that infects only hepatocytes. Finally, we performed BEC lineage tracing via Krt19-CreERT ; Rosa-stopflox/flox -tdTomato mice. To observe BEC-to-hepatocyte differentiation, mice were allowed to recover on normal diet following CDE diet-induced liver injury. Livers were collected from all mice and analyzed by quantitative real-time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. We show that mice with lack of ß-catenin in hepatocytes placed on the CDE diet develop severe liver injury with impaired hepatocyte proliferation, creating a stimulus for BECs to differentiate into hepatocytes. In particular, we use both hepatocyte and BEC lineage tracing to show that BECs differentiate into hepatocytes, which go on to repopulate the liver during long-term recovery. Conclusion: ß-catenin is important for liver regeneration after CDE diet-induced liver injury, and BEC-derived hepatocytes can permanently incorporate into the liver parenchyma to mediate liver regeneration.

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.

Bromodomain and Extraterminal (BET) Proteins Regulate Hepatocyte Proliferation in Hepatocyte-Driven Liver Regeneration.

Bromodomain and extraterminal (BET) proteins recruit key components of basic transcriptional machinery to promote gene expression. Aberrant expression and mutations in BET genes have been identified in many malignancies. Small molecule inhibitors of BET proteins such as JQ1 have shown efficacy in preclinical cancer models, including affecting growth of hepatocellular carcinoma. BET proteins also regulate cell proliferation in nontumor settings. We recently showed that BET proteins regulate cholangiocyte-driven liver regeneration. Here, we studied the role of BET proteins in hepatocyte-driven liver regeneration in partial hepatectomy (PHx) and acetaminophen-induced liver injury models in mice and zebrafish. JQ1 was injected 2 or 16 hours after PHx in mice to determine effect on hepatic injury, regeneration, and signaling. Mice treated with JQ1 after PHx displayed increased liver injury and a near-complete inhibition of hepatocyte proliferation. Levels of Ccnd1 mRNA and Cyclin D1 protein were reduced in animals injected with JQ1 16 hours after PHx and were even further reduced in animals injected with JQ1 2 hours after PHx. JQ1-treated zebrafish larvae after acetaminophen-induced injury also displayed notably impaired hepatocyte proliferation. In both models, Wnt signaling was prominently suppressed by JQ1. Our results show that BET proteins regulate hepatocyte proliferation-driven liver regeneration, and Wnt signaling is particularly sensitive to BET protein inhibition.

Hepatocyte-Derived Lipocalin 2 Is a Potential Serum Biomarker Reflecting Tumor Burden in Hepatoblastoma.

Hepatoblastoma (HB) is the most common pediatric liver malignant tumor. Previously, we reported co-activation of ß-catenin and Yes-associated protein-1 (YAP1) in 80% of HB. Hepatic co-expression of active ß-catenin and YAP1 via sleeping beauty transposon/transposase and hydrodynamic tail vein injection led to HB development in mice. Here, we identify lipocalin 2 (Lcn2) as a target of ß-catenin and YAP1 in HB and show that serum Lcn2 values positively correlated with tumor burden. Lcn2 was strongly expressed in HB tumor cells in our mouse model. A tissue array of 62 HB cases showed highest LCN2 expression in embryonal and lowest in fetal, blastemal, and small cell undifferentiated forms of HB. Knockdown of LCN2 in HB cells had no effect on cell proliferation but reduced NF-κB reporter activity. Next, liver-specific Lcn2 knockout (KO) mice were generated. No difference in tumor burden was observed between Lcn2 KO mice and wild-type littermate controls after sleeping beauty transposon/transposase and hydrodynamic tail vein injection delivery of active YAP1 and ß-catenin, although Lcn2 KO mice with HB lacked any serum Lcn2 elevation, demonstrating that transformed hepatocytes are the source of serum Lcn2. More blastemal areas and inflammation were observed within HB in Lcn2 KO compared with wild-type tumors. In conclusion, Lcn2 expressed in hepatocytes appears to be dispensable for the pathogenesis of HB. However, transformed hepatocytes secrete serum Lcn2, making Lcn2 a valuable biomarker for HB.

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).

Cyclooxygenase-2 deficiency impairs muscle-derived stem cell-mediated bone regeneration via cellular autonomous and non-autonomous mechanisms.

This study investigated the role of cyclooxygenase-2 (COX-2) expression by donor and host cells in muscle-derived stem cell (MDSC)-mediated bone regeneration utilizing a critical size calvarial defect model. We found that BMP4/green fluorescent protein (GFP)-transduced MDSCs formed significantly less bone in COX-2 knock-out (Cox-2KO) than in COX-2 wild-type (WT) mice. BMP4/GFP-transduced Cox-2KO MDSCs also formed significantly less bone than transduced WT MDSCs when transplanted into calvarial defects created in CD-1 nude mice. The impaired bone regeneration in the Cox-2KO MDSCBMP4/GFP group is associated with downregulation of BMP4-pSMAD1/5 signaling, decreased osteogenic differentiation and lowered proliferation capacity after transplantation, compared with WT MDSCBMP4/GFP cells. The Cox-2KO MDSCBMP4/GFP group demonstrated a reduction in cell survival and direct osteogenic differentiation in vitro These effects were mediated in part by the downregulation of Igf1 and Igf2. In addition, the Cox-2KO MDSCBMP4/GFP cells recruited fewer macrophages than the WT MDSC/BMP4/GFP cells in the early phase after injury. We concluded that the bone regeneration capacity of Cox-2KO MDSCs was impaired because of a reduction in cell proliferation and survival capacities, reduction in osteogenic differentiation and a decrease in the ability of the cells to recruit host cells to the injury site.

Thyroid Hormone Receptor-ß Agonist GC-1 Inhibits Met-ß-Catenin-Driven Hepatocellular Cancer.

The thyromimetic agent GC-1 induces hepatocyte proliferation via Wnt/ß-catenin signaling and may promote regeneration in both acute and chronic liver insufficiencies. However, ß-catenin activation due to mutations in CTNNB1 is seen in a subset of hepatocellular carcinomas (HCC). Thus, it is critical to address any effect of GC-1 on HCC growth and development before its use can be advocated to stimulate regeneration in chronic liver diseases. In this study, we first examined the effect of GC-1 on ß-catenin-T cell factor 4 activity in HCC cell lines harboring wild-type or mutated-CTNNB1. Next, we assessed the effect of GC-1 on HCC in FVB mice generated by hydrodynamic tail vein injection of hMet-S45Y-ß-catenin, using the sleeping beauty transposon-transposase. Four weeks following injection, mice were fed 5 mg/kg GC-1 or basal diet for 10 or 21 days. GC-1 treatment showed no effect on ß-catenin-T cell factor 4 activity in HCC cells, irrespective of CTNNB1 mutations. Treatment with GC-1 for 10 or 21 days led to a significant reduction in tumor burden, associated with decreased tumor cell proliferation and dramatic decreases in phospho-(p-)Met (Y1234/1235), p-extracellular signal-related kinase, and p-STAT3 without affecting ß-catenin and its downstream targets. GC-1 exerts a notable antitumoral effect on hMet-S45Y-ß-catenin HCC by inactivating Met signaling. GC-1 does not promote ß-catenin activation in HCC. Thus, GC-1 may be safe for use in inducing regeneration during chronic hepatic insufficiency.

Targeting ß-catenin in hepatocellular cancers induced by coexpression of mutant ß-catenin and K-Ras in mice.

Recently, we have shown that coexpression of hMet and mutant-ß-catenin using sleeping beauty transposon/transposase leads to hepatocellular carcinoma (HCC) in mice that corresponds to around 10% of human HCC. In the current study, we investigate whether Ras activation, which can occur downstream of Met signaling, is sufficient to cause HCC in association with mutant-ß-catenin. We also tested therapeutic efficacy of targeting ß-catenin in an HCC model. We show that mutant-K-Ras (G12D), which leads to Ras activation, cooperates with ß-catenin mutants (S33Y, S45Y) to yield HCC in mice. Affymetrix microarray showed > 90% similarity in gene expression in mutant-K-Ras-ß-catenin and Met-ß-catenin HCC. K-Ras-ß-catenin tumors showed up-regulation of ß-catenin targets like glutamine synthetase (GS), leukocyte cell-derived chemotaxin 2, Regucalcin, and Cyclin-D1 and of K-Ras effectors, including phosphorylated extracellular signal-regulated kinase, phosphorylated protein kinase B, phosphorylated mammalian target of rapamycin, phosphorylated eukaryotic translation initiation factor 4E, phosphorylated 4E-binding protein 1, and p-S6 ribosomal protein. Inclusion of dominant-negative transcription factor 4 at the time of K-Ras-ß-catenin injection prevented HCC and downstream ß-catenin and Ras signaling. To address whether targeting ß-catenin has any benefit postestablishment of HCC, we administered K-Ras-ß-catenin mice with EnCore lipid nanoparticles (LNP) loaded with a Dicer substrate small interfering RNA targeting catenin beta 1 (CTNNB1; CTNNB1-LNP), scrambled sequence (Scr-LNP), or phosphate-buffered saline for multiple cycles. A significant decrease in tumor burden was evident in the CTNNB1-LNP group versus all controls, which was associated with dramatic decreases in ß-catenin targets and some K-Ras effectors, leading to reduced tumor cell proliferation and viability. Intriguingly, in relatively few mice, non-GS-positive tumors, which were evident as a small subset of overall tumor burden, were not affected by ß-catenin suppression. CONCLUSION: Ras activation downstream of c-Met is sufficient to induce clinically relevant HCC in cooperation with mutant ß-catenin. ß-catenin suppression by a clinically relevant modality is effective in treatment of ß-catenin-positive, GS-positive HCCs. (Hepatology 2017;65:1581-1599).

Hepatocyte Wnts Are Dispensable During Diethylnitrosamine and Carbon Tetrachloride-Induced Injury and Hepatocellular Cancer.

Activation of the Wnt/ß-catenin signaling is reported in large subsets of hepatocellular carcinoma (HCC). Upregulation of Wnt genes is one contributing mechanism. In the current study, we sought to address the role of hepatocyte-derived Wnts in a model of hepatic injury, fibrosis, and carcinogenesis. We subjected hepatocyte-specific Wntless knockout mice (HP-KO), unable to secrete Wnts from hepatocytes, and littermate controls (HP-CON) to diethylnitrosamine and carbon tetrachloride (DEN/CCl4) and harvested at 3, 5, and 6 months for histological and molecular analysis. Analysis at 5 months displayed increased hepatic expression of several Wnts and upregulation of some, but not all, ß-catenin targets, without mutations in Ctnnb1. At 5 months, HP-CON and HP-KO had comparable tumor burden and injury; however, HP-KO uniquely showed small CK19+ foci within tumors. At 6 months, both groups were moribund with comparable tumor burden and CK19 positivity. While HCC histology was indistinguishable between the groups, HP-KO exhibited increased active ß-catenin and decreased c-Myc, Brd4, E-cadherin, and others. Hepatic injury, inflammation, and fibrosis were also indistinguishable at 3 months between both groups. Thus, lack of Wnt secretion from hepatocytes did not affect overall injury, fibrosis, or HCC burden, although there were protein expression differences in the tumors occurring in the two groups.

Mice lacking liver-specific ß-catenin develop steatohepatitis and fibrosis after iron overload.

BACKGROUND & AIMS: Iron overload disorders such as hereditary hemochromatosis and iron loading anemias are a common cause of morbidity from liver diseases and increase risk of hepatic fibrosis and hepatocellular carcinoma (HCC). Treatment options for iron-induced damage are limited, partly because there is lack of animal models of human disease. Therefore, we investigated the effect of iron overload in liver-specific ß-catenin knockout mice (KO), which are susceptible to injury, fibrosis and tumorigenesis following chemical carcinogen exposure. METHODS: Iron overload diet was administered to KO and littermate control (CON) mice for various times. To ameliorate an oxidant-mediated component of tissue injury, N-Acetyl-L-(+)-cysteine (NAC) was added to drinking water of mice on iron overload diet. RESULTS: KO on iron diet (KO +Fe) exhibited remarkable inflammation, followed by steatosis, oxidative stress, fibrosis, regenerating nodules and occurrence of occasional HCC. Increased injury in KO +Fe was associated with activated protein kinase B (AKT), ERK, and NF-κB, along with reappearance of ß-catenin and target gene Cyp2e1, which promoted lipid peroxidation and hepatic damage. Addition of NAC to drinking water protected KO +Fe from hepatic steatosis, injury and fibrosis, and prevented activation of AKT, ERK, NF-κB and reappearance of ß-catenin. CONCLUSIONS: The absence of hepatic ß-catenin predisposes mice to hepatic injury and fibrosis following iron overload, which was reminiscent of hemochromatosis and associated with enhanced steatohepatitis and fibrosis. Disease progression was notably alleviated by antioxidant therapy, which supports its chemopreventive role in the management of chronic iron overload disorders. LAY SUMMARY: Lack of animal models for iron overload disorders makes it hard to study the disease process for improving therapies. Feeding high iron diet to mice that lack the ß-catenin gene in liver cells led to increased inflammation followed by fat accumulation, cell death and wound healing that mimicked human disease. Administration of an antioxidant prevented hepatic injury in this model.

Rapid depletion of muscle progenitor cells in dystrophic mdx/utrophin-/- mice.

Duchenne muscular dystrophy (DMD) patients lack dystrophin from birth; however, muscle weakness becomes apparent only at 3-5 years of age, which happens to coincide with the depletion of the muscle progenitor cell (MPC) pools. Indeed, MPCs isolated from older DMD patients demonstrate impairments in myogenic potential. To determine whether the progression of muscular dystrophy is a consequence of the decline in functional MPCs, we investigated two animal models of DMD: (i) dystrophin-deficient mdx mice, the most commonly utilized model of DMD, which has a relatively mild dystrophic phenotype and (ii) dystrophin/utrophin double knock-out (dKO) mice, which display a similar histopathologic phenotype to DMD patients. In contrast to age-matched mdx mice, we observed that both the number and regeneration potential of dKO MPCs rapidly declines during disease progression. This occurred in MPCs at both early and late stages of myogenic commitment. In fact, early MPCs isolated from 6-week-old dKO mice have reductions in proliferation, resistance to oxidative stress and multilineage differentiation capacities compared with age-matched mdx MPCs. This effect may potentially be mediated by fibroblast growth factor overexpression and/or a reduction in telomerase activity. Our results demonstrate that the rapid disease progression in the dKO model is associated, at least in part, with MPC depletion. Therefore, alleviating MPC depletion could represent an approach to delay the onset of the histopathologies associated with DMD patients.

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.