Carola M. Morell

Protein kinase A-dependent pSer(675) -β-catenin, a novel signaling defect in a mouse model of congenital hepatic fibrosis.

Genetically determined loss of fibrocystin function causes congenital hepatic fibrosis (CHF), Caroli disease (CD), and autosomal recessive polycystic kidney disease (ARPKD). Cystic dysplasia of the intrahepatic bile ducts and progressive portal fibrosis characterize liver pathology in CHF/CD. At a cellular level, several functional morphological and signaling changes have been reported including increased levels of 3′‐5′‐cyclic adenosine monophosphate (cAMP). In this study we addressed the relationships between increased cAMP and β‐catenin. In cholangiocytes isolated and cultured from Pkhd1del4/del4 mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser675‐phosphorylation of β‐catenin, its nuclear localization, and its transcriptional activity (western blot and TOP flash assay, respectively) along with a down‐regulation of E‐cadherin expression (immunocytochemistry and western blot); these changes were inhibited by the PKA blocker, PKI (1 μM). The Rho‐GTPase, Rac‐1, was also significantly activated by cAMP in Pkhd1del4/del4 cholangiocytes. Rac‐1 inhibition blocked cAMP‐dependent nuclear translocation and transcriptional activity of pSer675‐β‐catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1del4/del4 and was inhibited by: (1) PKI, (2) silencing β‐catenin (siRNA), and (3) the Rac‐1 inhibitor NSC 23766. Conclusion: These data show that in fibrocystin‐defective cholangiocytes, cAMP/PKA signaling stimulates pSer675‐phosphorylation of β‐catenin and Rac‐1 activity. In the presence of activated Rac‐1, pSer675‐β‐catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1del4/del4 cholangiocytes. β‐Catenin‐dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target. (Hepatology 2013;58:1713–1723)

hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α1-antitrypsin deficiency.

Graphical abstract

Notch signaling and progenitor/ductular reaction in steatohepatitis

Background and objective Persistent hepatic progenitor cells (HPC) activation resulting in ductular reaction (DR) is responsible for pathologic liver repair in cholangiopathies. Also, HPC/DR expansion correlates with fibrosis in several chronic liver diseases, including steatohepatitis. Increasing evidence indicates Notch signaling as a key regulator of HPC/DR response in biliary and more in general liver injuries. Therefore, we aimed to investigate the role of Notch during HPC/DR activation in a mouse model of steatohepatitis. Methods Steatohepatitis was generated using methionine-choline deficient (MCD) diet. For hepatocyte lineage tracing, R26R-YFP mice were infected with AAV8-TBG-Cre. Results MCD diet promoted a strong HPC/DR response that progressively diffused in the lobule, and correlated with increased fibrosis and TGF-β1 expression. Notch signaling was unchanged in laser-capture microdissected HPC/DR, whereas Notch receptors were down regulated in hepatocytes. However, in-vivo lineage tracing experiments identified discrete hepatocytes showing Notch-1 activation and expressing (the Notch-dependent) Sox9. Stimulation of AML-12 hepatocyte-cell line with immobilized Jag1 induced Sox9 and down-regulated albumin and BSEP expression. TGF-β1 treatment in primary hepatic stellate cells (HSC) induced Jag1 expression. In MCD diet-fed mice, αSMA-positive HSC were localized around Sox9 expressing hepatocytes, suggesting that Notch activation in hepatocytes was promoted by TGF-β1 stimulated HSC. In-vivo Notch inhibition reduced HPC response and fibrosis progression. Conclusion Our data suggest that Notch signaling is an important regulator of DR and that in steatohepatitis, hepatocytes exposed to Jag1-positive HSC, contribute to pathologic DR by undergoing Notch-mediated differentiation towards an HPC-like phenotype. Given the roles of Notch in fibrosis and liver cancer, these data suggest mesenchymal expression of Jag1 as an alternative therapeutic target.

Protein kinase a-dependent pSer675-β-catenin, a novel signaling defect in a mouse model of congenital hepatic fibrosis: Hepatology, Vol. 00, No. X, 2013

Genetically determined loss of fibrocystin function causes congenital hepatic fibrosis (CHF), Caroli disease (CD), and autosomal recessive polycystic kidney disease (ARPKD). Cystic dysplasia of the intrahepatic bile ducts and progressive portal fibrosis characterize liver pathology in CHF/CD. At a cellular level, several functional morphological and signaling changes have been reported including increased levels of 3′‐5′‐cyclic adenosine monophosphate (cAMP). In this study we addressed the relationships between increased cAMP and β‐catenin. In cholangiocytes isolated and cultured from Pkhd1del4/del4 mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser675‐phosphorylation of β‐catenin, its nuclear localization, and its transcriptional activity (western blot and TOP flash assay, respectively) along with a down‐regulation of E‐cadherin expression (immunocytochemistry and western blot); these changes were inhibited by the PKA blocker, PKI (1 μM). The Rho‐GTPase, Rac‐1, was also significantly activated by cAMP in Pkhd1del4/del4 cholangiocytes. Rac‐1 inhibition blocked cAMP‐dependent nuclear translocation and transcriptional activity of pSer675‐β‐catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1del4/del4 and was inhibited by: (1) PKI, (2) silencing β‐catenin (siRNA), and (3) the Rac‐1 inhibitor NSC 23766. Conclusion: These data show that in fibrocystin‐defective cholangiocytes, cAMP/PKA signaling stimulates pSer675‐phosphorylation of β‐catenin and Rac‐1 activity. In the presence of activated Rac‐1, pSer675‐β‐catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1del4/del4 cholangiocytes. β‐Catenin‐dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target. (Hepatology 2013;58:1713–1723)

31 MONOCYTE/MACROPHAGE PERIBILIARY RECRUITMENT BY PKHD1-DEFECTIVE CHOLANGIOCYTES INDUCES EXPRESSION OF αvβ6 INTEGRIN ON BILIARY CYSTS VIA TGF-β1 AND TNF-α IN CONGENITAL HEPATIC FIBROSIS

implementation of the current HBV-DNA screening assay, as well as a look-back of blood recipients. Methods: Sequence analysis revealed identical HBV sequences in donor and recipient. Back-up samples testing negative by NAT minipool at previous donations were individually tested by more sensitive assays (Roche Individual NAT, and home-made real-time PCR of S region). When available, pre and post-transfusion samples of recipients were tested for HBV markers. Sequence analyses were conducted by ABIPrism 3100. Results: 28 back-up samples (1–3 per patient) from 13 OBI carriers (2 anti-HBc only, 2 anti-HBs only, 8 anti HBs/antiHBc, 1 without any marker) were available. Overall, 62% tested positive with at least one of the two assays. Of them, 42% were positive by Individual NAT, and 58% by home-made protocol. Six donors gave at least one unit to 4 recipients, for whom both preand post-transfusion samples were available. One recipient acquired infection, as indicated by seroconversion and ALT elevation, two had pre-existing immunity to HBV, and one remained seronegative. Conclusions: A substantial proportion of donations containing HBVDNA are not identified by current NAT minipool assays. These donations can cause acute infections in recipients. Current approaches are highly ineffective, and alternative strategies based on more sensitive protocols or anti-HBc testing should be developed.

OC.10.4: PKA-DEPENDENT P-SER-675β-CATENIN PHOSPHORYLATION INCREASES CHOLANGIOCYTE MOTILITY IN A MOUSE MODEL OF FIBROPOLYCYSTIC LIVER DISEASES

the combination group and 29% in the Sorafenib alone group. The cumulative survival rates were 60%, 35% and 26% at 1-2-3 years, respectively in the combination group while, in the Sorafenib group alone were 37% and 0% at 1 and 2 years respectively (p<0.001). Conclusions: The combination between RFA of both HCC and PVTT plus Sorafenib significantly increases 3-year survival compared to the Sorafenib alone. Our data suggest that RFA of both HCC and PVTT represent a significant factor for increased survival.