Ashley Cast

Intrahepatic Bile Duct Regeneration in Mice Does Not Require Hnf6 or Notch Signaling through Rbpj

The potential for intrahepatic bile duct (IHBD) regeneration in patients with bile duct insufficiency diseases is poorly understood. Notch signaling and Hnf6 have each been shown to be important for the morphogenesis of IHBDs in mice. One congenital pediatric liver disease characterized by reduced numbers of IHBDs, Alagille syndrome, is associated with mutations in Notch signaling components. Therefore, we investigated whether liver cell plasticity could contribute to IHBD regeneration in mice with disruptions in Notch signaling and Hnf6. We studied a mouse model of bile duct insufficiency with liver epithelial cell-specific deficiencies in Hnf6 and Rbpj, a mediator of canonical Notch signaling. Albumin-Cre Hnf6(flox/flox)Rbpj(flox/flox) mice initially developed no peripheral bile ducts. The evolving postnatal liver phenotype was analyzed using IHBD resin casting, immunostaining, and serum chemistry. With age, Albumin-Cre Hnf6(flox/flox)Rbpj(flox/flox) mice mounted a ductular reaction extending through the hepatic tissue and then regenerated communicating peripheral IHBD branches. Rbpj and Hnf6 were determined to remain absent from biliary epithelial cells constituting the ductular reaction and the regenerated peripheral IHBDs. We report the expression of Sox9, a marker of biliary epithelial cells, in cells expressing hepatocyte markers. Tissue analysis indicates that reactive ductules did not arise directly from preexisting hilar IHBDs. We conclude that liver cell plasticity is competent for regeneration of IHBDs independent of Notch signaling via Rbpj and Hnf6.

MCTP2 is a dosage-sensitive gene required for cardiac outflow tract development.

Coarctation of the aorta (CoA) and hypoplastic left heart syndrome (HLHS) have been reported in rare individuals with large terminal deletions of chromosome 15q26. However, no single gene important for left ventricular outflow tract (LVOT) development has been identified in this region. Using array-comparative genomic hybridization, we identified two half-siblings with CoA with a 2.2 Mb deletion on 15q26.2, inherited from their mother, who was mosaic for this deletion. This interval contains an evolutionary conserved, protein-coding gene, MCTP2 (multiple C2-domains with two transmembrane regions 2). Using gene-specific array screening in 146 individuals with non-syndromic LVOT obstructive defects, another individual with HLHS and CoA was found to have a de novo 41 kb intragenic duplication within MCTP2, predicted to result in premature truncation, p.F697X. Alteration of Mctp2 gene expression in Xenopus laevis embryos by morpholino knockdown and mRNA overexpression resulted in the failure of proper OT development, confirming the functional importance of this dosage-sensitive gene for cardiogenesis. Our results identify MCTP2 as a novel genetic cause of CoA and related cardiac malformations.

An essential and highly conserved role for Zic3 in left-right patterning, gastrulation and convergent extension morphogenesis

Mutations in ZIC3 result in X-linked heterotaxy in humans, a syndrome consisting of left-right (L-R) patterning defects, midline abnormalities, and cardiac malformations. Similarly, loss of function of Zic3 in mouse results in abnormal L-R patterning and cardiac development. However, Zic3 null mice also exhibit defects in gastrulation, neural tube closure, and axial patterning, suggesting the hypothesis that Zic3 is necessary for proper convergent extension (C-E) morphogenesis. To further investigate the role of Zic3 in early embryonic development, we utilized two model systems, Xenopus laevis and zebrafish, and performed loss of function analysis using antisense morpholino-mediated gene knockdown. Both Xenopus and zebrafish demonstrated significant impairment of C-E in Zic3 morphants. L-R patterning was also disrupted, indicating that the role of Zic3 in L-R axis development is conserved across species. Correlation of L-R patterning and C-E defects in Xenopus suggests that early C-E defects may underlie L-R patterning defects at later stages, since Zic3 morphants with moderate to severe C-E defects exhibited an increase in laterality defects. Taken together, these results demonstrate a functional conservation of Zic3 in L-R patterning and uncover a previously unrecognized role for Zic3 in C-E morphogenesis during early vertebrate development.

Epithelial VEGF signaling is required in the mouse liver for proper sinusoid endothelial cell identity and hepatocyte zonation in vivo

Vascular endothelial growth factor (VEGF) is crucial for vascular development in several organs. However, the specific contribution of epithelial-VEGF signaling in the liver has not been tested. We used a mouse model to specifically delete Vegf from the liver epithelial lineages during midgestational development and assessed the cell identities and architectures of epithelial and endothelial tissues. We find that without epithelial-derived VEGF, the zonal endothelial and hepatocyte cell identities are altered. We also find decreased portal vein and hepatic artery branching coincident with an increase in hepatic hypoxia postnatally. Together, these data indicate that VEGF secreted from the hepatic epithelium is required for normal differentiation of cells and establishment of three-dimensional vascular branching and zonal architectures in both epithelial and endothelial hepatic tissues.

Activation of CDK4 Triggers Development of Non-alcoholic Fatty Liver Disease

The development of non-alcoholic fatty liver disease (NAFLD) is a multiple step process. Here, we show that activation of cdk4 triggers the development of NAFLD. We found that cdk4 protein levels are elevated in mouse models of NAFLD and in patients with fatty livers. This increase leads to C/EBPα phosphorylation on Ser193 and formation of C/EBPα-p300 complexes, resulting in hepatic steatosis, fibrosis, and hepatocellular carcinoma (HCC). The disruption of this pathway in cdk4-resistant C/EBPα-S193A mice dramatically reduces development of high-fat diet (HFD)-mediated NAFLD. In addition, inhibition of cdk4 by flavopiridol or PD-0332991 significantly reduces development of hepatic steatosis, the first step of NAFLD. Thus, this study reveals that activation of cdk4 triggers NAFLD and that inhibitors of cdk4 may be used for the prevention/treatment of NAFLD.

De novo formation of the biliary system by TGFβ-mediated hepatocyte transdifferentiation

Transdifferentiation is a complete and stable change in cell identity that serves as an alternative to stem-cell-mediated organ regeneration. In adult mammals, findings of transdifferentiation have been limited to the replenishment of cells lost from preexisting structures, in the presence of a fully developed scaffold and niche1. Here we show that transdifferentiation of hepatocytes in the mouse liver can build a structure that failed to form in development—the biliary system in a mouse model that mimics the hepatic phenotype of human Alagille syndrome (ALGS)2. In these mice, hepatocytes convert into mature cholangiocytes and form bile ducts that are effective in draining bile and persist after the cholestatic liver injury is reversed, consistent with transdifferentiation. These findings redefine hepatocyte plasticity, which appeared to be limited to metaplasia, that is, incomplete and transient biliary differentiation as an adaptation to cell injury, based on previous studies in mice with a fully developed biliary system3–6. In contrast to bile duct development7–9, we show that de novo bile duct formation by hepatocyte transdifferentiation is independent of NOTCH signalling. We identify TGFβ signalling as the driver of this compensatory mechanism and show that it is active in some patients with ALGS. Furthermore, we show that TGFβ signalling can be targeted to enhance the formation of the biliary system from hepatocytes, and that the transdifferentiation-inducing signals and remodelling capacity of the bile-duct-deficient liver can be harnessed with transplanted hepatocytes. Our results define the regenerative potential of mammalian transdifferentiation and reveal opportunities for the treatment of ALGS and other cholestatic liver diseases.In a mouse model of a human cholestatic liver disease caused by impaired NOTCH signalling, hepatocytes transdifferentiate into cholangiocytes and form a therapeutically effective biliary system, driven by TGFβ signalling.

RNA binding protein CUGBP1 inhibits liver cancer in a phosphorylation dependent manner

ABSTRACT Despite intensive investigations, mechanisms of liver cancer are not known. Here, we identified an important step of liver cancer, which is the neutralization of tumor suppressor activities of an RNA binding protein, CUGBP1. The translational activity of CUGBP1 is activated by dephosphorylation at Ser302. We generated CUGBP1-S302A knock-in mice and found that the reduction of translational activity of CUGBP1 causes development of a fatty liver phenotype in young S302A mice. Examination of liver cancer in diethylnitrosamine (DEN)-treated CUGBP1-S302A mice showed these mice develop much more severe liver cancer that is associated with elimination of the mutant CUGBP1. Searching for mechanisms of this elimination, we found that the oncoprotein gankyrin (Gank) preferentially binds to and triggers degradation of dephosphorylated CUGBP1 (de-ph-S302-CUGBP1) or S302A mutant CUGBP1. To test the role of Gank in degradation of CUGBP1, we generated mice with liver-specific deletion of Gank. In these mice, the tumor suppressor isoform of CUGBP1 is protected from Gank-mediated degradation. Consistent with reduction of CUGBP1 in animal models, CUGBP1 is reduced in patients with pediatric liver cancer. Thus, this work presents evidence that de-ph-S302-CUGBP1 is a tumor suppressor protein and that the Gank-UPS-mediated reduction of CUGBP1 is a key event in the development of liver cancer.

Vascular patterning sets the stage for macro and micro hepatic architecture

Background The liver is a complex organ with a variety of tissue components that require a precise architecture for optimal function of metabolic and detoxification processes. As a result of the delicate orchestration required between the various hepatic tissues, it is not surprising that impairment of hepatic function can be caused by a variety of factors leading to chronic liver disease. Results Despite the growing rate of chronic liver disease, there are currently few effective treatment options besides orthotopic liver transplantation. Better therapeutic options reside in the potential for genetic and cellular therapies that promote progenitor cell activation aiding de novo epithelial and vascular regeneration, cell replacement, or population of bioartificial hepatic devices. In order to explore this area of new therapeutic potential, it is crucial to understand the factors that promote hepatic function through regulating cell identities and tissue architecture. Conclusions In this commentary, we review the signals regulating liver cell fates during development and regeneration and highlight the importance of patterning the hepatic vascular systems to set the groundwork for the macro and micro hepatic architecture of the epithelium. Developmental Dynamics 244:497–506, 2015.

PARP1 activation increases expression of modified tumor suppressors and pathways underlying development of aggressive hepatoblastoma

Hepatoblastoma (HBL) is a pediatric liver cancer that affects children under the age of three. Reduction of tumor suppressor proteins (TSPs) is commonly seen in liver cancer. However, in our studies we find that aggressive, chemo-resistant HBLs exhibit an elevation of TSPs. HBL patients with a classic phenotype have reduced TSP levels, but patients with aggressive HBL express elevated TSPs that undergo posttranslational modifications, eliminating their tumor suppression activities. Here we identify unique aggressive liver cancer domains (ALCDs) that are activated in aggressive HBL by PARP1-mediated chromatin remodeling leading to elevation of modified TSPs and activation of additional cancer pathways: WNT signaling and β-catenin. Inhibition of PARP1 blocks activation of ALCDs and normalizes expression of corresponding genes, therefore reducing cell proliferation. Our studies reveal PARP1 activation as a mechanism for the development of aggressive HBL, further suggesting FDA-approved PARP1 inhibitors might be used for treatment of patients with aggressive HBL.Leila Valanejad et al. report increased expression of modified tumor suppressor proteins (TSPs) with loss of tumor suppressor activity in aggressive, chemotherapy-resistant hepatoblastoma. They find that TSP upregulation occurs via PARP1-mediated chromatin remodeling, leading to activation of multiple cancer-associated pathways.

Gankyrin Promotes Tumor-Suppressor Protein Degradation to Drive Hepatocyte Proliferation

Background & Aims Uncontrolled liver proliferation is a key characteristic of liver cancer; however, the mechanisms by which this occurs are not well understood. Elucidation of these mechanisms is necessary for the development of better therapy. The oncogene Gankyrin (Gank) is overexpressed in both hepatocellular carcinoma and hepatoblastoma. The aim of this work was to determine the role of Gank in liver proliferation and elucidate the mechanism by which Gank promotes liver proliferation. Methods We generated Gank liver-specific knock-out (GLKO) mice and examined liver biology and proliferation after surgical resection and liver injury. Results Global profiling of gene expression in GLKO mice showed significant changes in pathways involved in liver cancer and proliferation. Investigations of liver proliferation after partial hepatectomy and CCl4 treatment showed that GLKO mice have dramatically inhibited proliferation of hepatocytes at early stages after surgery and injury. In control LoxP mice, liver proliferation was characterized by Gank-mediated reduction of tumor-suppressor proteins (TSPs). The failure of GLKO hepatocytes to proliferate is associated with a lack of down-regulation of these proteins. Surprisingly, we found that hepatic progenitor cells of GLKO mice start proliferation at later stages and restore the original size of the liver at 14 days after partial hepatectomy. To examine the proliferative activities of Gank in cancer cells, we used a small molecule, cjoc42, to inhibit interactions of Gank with the 26S proteasome. These studies showed that Gank triggers degradation of TSPs and that cjoc42-mediated inhibition of Gank increases levels of TSPs and inhibits proliferation of cancer cells. Conclusions These studies show that Gank promotes hepatocyte proliferation by elimination of TSPs. This work provides background for the development of Gank-mediated therapy for the treatment of liver cancer. RNA sequencing data can be accessed in the NCBI Gene Expression Omnibus: GSE104395.