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A S L D A b st ra ct s increase in circulating Galanin in the serum. Galanin immunoreactivity was observed in both cholangiocytes and hepatocytes, whereas GalR1 was found predominantly in cholangiocytes. Systemic treatment of rats with M617 increased both CK-19 expression and IBDM in both sham and BDL-treated animals. In vitro, treatment of the mouse cholangiocyte cell line with M617 increased ERK1/2 and RSK-1 activity. There was a concomitant increase in cholangiocyte proliferation after M617 treatment that could be blocked by pretreatment with inhibitors for ERK1/2 and RSK-1. Conclusions: Data presented here demonstrate a direct stimulatory role for Galanin on cholangiocyte proliferation under physiological (sham) and pathological (BDL) conditions via a mechanism involving the activation of ERK1/2 and subsequent phosphorylation of RSK-1. Targeting Galanin or GalR1 may prove a useful strategy to regulate biliary mass during cholestatic liver injury.

A S L D A b st ra ct s increase in circulating Galanin in the serum. Galanin immunoreactivity was observed in both cholangiocytes and hepatocytes, whereas GalR1 was found predominantly in cholangiocytes. Systemic treatment of rats with M617 increased both CK-19 expression and IBDM in both sham and BDL-treated animals. In vitro, treatment of the mouse cholangiocyte cell line with M617 increased ERK1/2 and RSK-1 activity. There was a concomitant increase in cholangiocyte proliferation after M617 treatment that could be blocked by pretreatment with inhibitors for ERK1/2 and RSK-1. Conclusions: Data presented here demonstrate a direct stimulatory role for Galanin on cholangiocyte proliferation under physiological (sham) and pathological (BDL) conditions via a mechanism involving the activation of ERK1/2 and subsequent phosphorylation of RSK-1. Targeting Galanin or GalR1 may prove a useful strategy to regulate biliary mass during cholestatic liver injury.

Background: The intestinal mucus layer protects the epithelium against noxious agents and pathogenic bacteria present in the gastrointestinal tract. It is composed of mucins, predominantly mucin-2 (Muc2), secreted by goblet cells of the intestine. Experimental alcoholic liver disease is dependent on the translocation of bacterial products across the intestinal barrier into the systemic circulation, which induces an inflammatory response in the liver and contributes to steatohepatitis. The aim of our study was to investigate the impact of the intestinal mucus layer and in particular Muc2 on alcoholic liver disease. Methods and Results: We used the Tsukamoto-French mouse model which involves continuous intragastric feeding of isocaloric diet (n=4-5) or alcohol for 1 week in Muc2-/(n=9) and wildtype (WT) mice (n=10). Muc2 was abundantly expressed in the small and large intestine of WT mice, but undetectable in the liver. The intestinal mucus layer was considerably thinner in Muc2 deficient mice as shown by PAS staining. Alcohol feeding did not result in a compensatory upregulation of other intestinal mucins in Muc2-/mice. After alcohol feeding mice deficient in Muc2 were protected from steatohepatitis as evidenced by significantly lower ALT levels and hepatic triglyceride concentrations. In addition, hepatic oxidative stress was significantly reduced in Muc2-/mice following intragastric alcohol feeding as shown by immunohistochemistry for 4-hydroxynonenal (4-HNE) and by TBARS assay. There was no significant difference in plasma alcohol levels or hepatic alcohol metabolizing enzymes alcohol dehydrogenase 1 (ADH1) and cytochrome p450 enzyme 2E1 (Cyp2E1) in alcohol fed Muc2-/as compared to WT mice. Most notably, Muc2-/mice had significantly lower systemic plasma LPS levels after alcohol feeding. In contrast to WT mice, Muc2-/mice did not exhibit intestinal bacterial overgrowth, but a higher amount of intestinal probiotic Lactobacillus after alcohol administration as shown by qPCR for 16S rRNA and Lactobacillus spp. The antimicrobial proteins Reg3b and Reg3g were found to be expressed at significantly higher levels in the proximal small intestine in isocaloric diet and alcohol fed Muc2-/mice relative to WT mice as assessed by qPCR and Western blotting. As Reg3b and Reg3g are bactericidal c-type lectins, an increase in their expression might contribute to the observed reduction of the bacterial burden and suppression of bacterial overgrowth in the intestine. Conclusion: Intestinal mucin-2 deficiency protects from alcoholic steatohepatitis. We suggest a pathway that involves higher expression of enteric antimicrobial molecules which suppresses alcohol-associated intestinal bacterial overgrowth. Subsequently, lower amounts of bacterial products such as endotoxin translocate into the systemic circulation and cause less alcoholic liver disease.

Background: The intestinal mucus layer protects the epithelium against noxious agents and pathogenic bacteria present in the gastrointestinal tract. It is composed of mucins, predominantly mucin-2 (Muc2), secreted by goblet cells of the intestine. Experimental alcoholic liver disease is dependent on the translocation of bacterial products across the intestinal barrier into the systemic circulation, which induces an inflammatory response in the liver and contributes to steatohepatitis. The aim of our study was to investigate the impact of the intestinal mucus layer and in particular Muc2 on alcoholic liver disease. Methods and Results: We used the Tsukamoto-French mouse model which involves continuous intragastric feeding of isocaloric diet (n=4-5) or alcohol for 1 week in Muc2-/(n=9) and wildtype (WT) mice (n=10). Muc2 was abundantly expressed in the small and large intestine of WT mice, but undetectable in the liver. The intestinal mucus layer was considerably thinner in Muc2 deficient mice as shown by PAS staining. Alcohol feeding did not result in a compensatory upregulation of other intestinal mucins in Muc2-/mice. After alcohol feeding mice deficient in Muc2 were protected from steatohepatitis as evidenced by significantly lower ALT levels and hepatic triglyceride concentrations. In addition, hepatic oxidative stress was significantly reduced in Muc2-/mice following intragastric alcohol feeding as shown by immunohistochemistry for 4-hydroxynonenal (4-HNE) and by TBARS assay. There was no significant difference in plasma alcohol levels or hepatic alcohol metabolizing enzymes alcohol dehydrogenase 1 (ADH1) and cytochrome p450 enzyme 2E1 (Cyp2E1) in alcohol fed Muc2-/as compared to WT mice. Most notably, Muc2-/mice had significantly lower systemic plasma LPS levels after alcohol feeding. In contrast to WT mice, Muc2-/mice did not exhibit intestinal bacterial overgrowth, but a higher amount of intestinal probiotic Lactobacillus after alcohol administration as shown by qPCR for 16S rRNA and Lactobacillus spp. The antimicrobial proteins Reg3b and Reg3g were found to be expressed at significantly higher levels in the proximal small intestine in isocaloric diet and alcohol fed Muc2-/mice relative to WT mice as assessed by qPCR and Western blotting. As Reg3b and Reg3g are bactericidal c-type lectins, an increase in their expression might contribute to the observed reduction of the bacterial burden and suppression of bacterial overgrowth in the intestine. Conclusion: Intestinal mucin-2 deficiency protects from alcoholic steatohepatitis. We suggest a pathway that involves higher expression of enteric antimicrobial molecules which suppresses alcohol-associated intestinal bacterial overgrowth. Subsequently, lower amounts of bacterial products such as endotoxin translocate into the systemic circulation and cause less alcoholic liver disease.

Cholangiocarcinoma (CCA) originates from the neoplastic transformation of cholangiocytes, which are the epithelial cells that line the bile ducts. CCAs are neuroendocrine tumors and secrete and respond to neuropeptides with alterations in cell proliferation via autocrine pathways. For example, the expression/secretion of neuroendocrine factors such as NPY and serotonin are dysregulated in cholangiocarcinoma, which exert local control on tumor cell proliferation. The neuropeptide substance P (SP) stimulates normal biliary hyperplasia by binding to the neurokinin-1 receptor (NK-1R) stimulating ERK1/2 activity. Neutral endopeptidase (NEP) is a metalloprotease that degrades a number of secreted neuropeptides including SP. The AIM of our study was to evaluate the role of NEP in the autocrine regulation of CCA growth by SP. METHODS: The intra- and extra-hepatic CCA lines, Mz-ChA-1, SG231, CCLP, HuCCT-1, HuH-28, and TFK-1, and non-malignant biliary line, H-69, were utilized. We evaluated NK1R, tachykinin 1 (TAC1, gene encoding SP) and NEP expression by qPCR, FACS and immunofluorescence in cell lines and by immunohistochemistry in commercially available CCA tissue arrays. The secretion of SP in all lines was measured by EIA. The dose-dependent effect of SP (10−6 to 10−11 M for 48 hrs) on cell growth and SP-induced (10−7 M) cell growth in the presence/absence of PD98059 (MEK1 inhibitor) was evaluated by MTS proliferation assay. The effect of SP on phosphorylation of ERK1/2 was determined by Western blot in the presence/absence of PD98059. To determine the role of NEP in the regulation of CCA growth, NEP was overexpressed in Mz-ChA-1 cells before evaluating: (i) NEP expression by qPCR and SP secretion by EIA kits; (ii) proliferation by MTS assay; and (iii) expression of Cyclin D1, PCNA and VEGF-A/C (factors regulating CCA growth). RESULTS: All CCA cell lines and human CCA tissue array samples were positive for NK-1R and TAC1. The CCA lines secreted higher levels of SP compared to H69. Correspondingly, NEP expression levels were significantly reduced in CCA lines compared to nonmalignant H69 cells and in human CCA tissue arrays compared to normal bile ducts. SP increased CCA growth (∼60% at 10−7 M), which was dependent upon ERK1/2 activity. Overexpression of NEP (∼80%, the SP degrading enzyme) in Mz-ChA-1 cells decreased SP secretion and significantly decreased cell growth, which was associated with decreased expression of Cyclin D1, PCNA and VEGF-A/C compared to control transfected Mz-ChA-1. CONCLUSIONS: SP is mitogenic for CCA regulating growth in an autocrine fashion that is associated with decreased NEP expression. Targeting the NEP/SP/NK1R axis may represent a novel therapeutic approach for cholangiocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3023. doi:1538-7445.AM2012-3023

correlated these data with the simultaneous presence of hepatitis B and C (HBV and HCV) infection.Methods: We quantified methylation levels at 19 CpG loci (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, p16, APC, CDH1, RUNX3, RIZ1, SFRP2, MINT31, COX2, MINT1, CACNA1G, RASSF2, MINT2, Reprimo, DCC) using combined bisulfite restriction analysis (COBRA). In total, 81 advanced tumors (well-differentiated HCCs >2 cm in size or moderately-poorly differentiated HCC), 12 early stage tumors (dysplastic nodules or well-differentiated HCC 2 cm size showing denser methylation compared with early tumors at these CpG loci. Additionally, HCV-related tumors tended to carry higher methylation levels at Group3 loci compared with HBV-related and virus-negative tumor, in both early and advanced tumors. Conclusions: Aberrant methylation is a frequent event in the liver and sequentially progresses from non-cancerous liver to early stage cancers and finally to advanced HCC. The cumulative differences in methylation levels among various liver tissues (precancerous livers, early and advanced tumors) suggest that aberrant methylation is an early event in HCC that progresses with the advancing stage. Lastly, simultaneous HCV infection may accelerate the carcinogenetic process in HCC.

lower levels of lAb than those isolated from normal animals (LDC Tx: 25±8%; SDC Tx: 83±5%; LDC Wt: 74±8%; SDC Wt: 80±3%). When LDC and SDC isolated from Tx mice were used as stimulatory cells in a 72 hr MLC they show an impaired T-cell stimulatory activity as respect to DC isolated from Wt animals (see figure). Conclusions: our data show that in the early phase of hepatocarcinogenesis there is a systemic immundepression since both resident LDC and SDC isolated from tx mice have a weak T-cell stimulator activity, as compared to Wt animals. However the poor T-cell stimulator activity of LDC and SDC was regardless of lAb expression. This study was supported by MIUR grant no. 2003069954.

The objectives of this review are to outline the recent findings related to the morphological heterogeneity of the biliary epithelium and the heterogeneous pathophysiological responses of different sized bile ducts to liver gastrointestinal hormones and peptides and liver injury/toxins with changes in apoptotic, proliferative and secretory activities. The knowledge of biliary function is rapidly increasing because of the recognition that biliary epithelial cells (cholangiocytes) are the targets of human cholangiopathies, which are characterized by proliferation/damage of bile ducts within a small range of sizes. The unique anatomy, morphology, innervation and vascularization of the biliary epithelium are consistent with function of cholangiocytes within different regions of the biliary tree. The in vivo models [e.g., bile duct ligation (BDL), partial hepatectomy, feeding of bile acids, carbon tetrachloride (CCl4) or alpha-naphthylisothiocyanate (ANIT)] and the in vivo experimental tools [e.g., freshly isolated small and large cholangiocytes or intrahepatic bile duct units (IBDU) and primary cultures of small and large murine cholangiocytes] have allowed us to demonstrate the morphological and functional heterogeneity of the intrahepatic biliary epithelium. These models demonstrated the differential secretory activities and the heterogeneous apoptotic and proliferative responses of different sized ducts. Similar to animal models of cholangiocyte proliferation/injury restricted to specific sized ducts, in human liver diseases bile duct damage predominates specific sized bile ducts. Future studies related to the functional heterogeneity of the intrahepatic biliary epithelium may disclose new pathophysiological treatments for patients with cholangiopathies.

Menin (MEN1) is a tumor-suppressor protein in neuroendocrine tissue. Therefore, we tested the novel hypothesis that menin regulates cholangiocarcinoma proliferation. Menin and miR-24 expression levels were measured in the following intrahepatic and extrahepatic cholangiocarcinoma (CCA) cell lines, Mz-ChA-1, TFK-1, SG231, CCLP, HuCCT-1, and HuH-28, as well as the nonmalignant human intrahepatic biliary line, H69. miR-24 miRNA and menin protein levels were manipulated invitro in Mz-ChA-1 cell lines. Markers of proliferation and angiogenesis (Ki-67, vascular endothelial growth factors A/C, vascular endothelial growth factor receptors 2/3, angiopoietin 1/2, and angiopoietin receptors 1/2) were evaluated. Mz-ChA-1 cells were injected into the flanks of nude mice and treated with miR-24 inhibitor or inhibitor scramble. Menin expression was decreased in advanced CCA specimens, whereas miR-24 expression was increased in CCA. Menin overexpression decreased proliferation, angiogenesis, migration, and invasion. Inhibition of miR-24 increased menin protein expression while decreasing proliferation, angiogenesis, migration, and invasion. miR-24 was shown to negatively regulate menin expression by luciferase assay. Tumor burden and expression of proliferative and angiogenic markers was decreased in the miR-24 inhibited tumor group compared to controls. Interestingly, treated tumors were more fibrotic than the control group. miR-24–dependent expression of menin may be important in the regulation of nonmalignant and CCA proliferation and may be an additional therapeutic tool for managing CCA progression.

Background Prolactin promotes proliferation of several cells. Prolactin receptor exists as two isoforms: long and short, which activate different transduction pathways including the Ca2+-dependent PKC-signaling. No information exists on the role of prolactin in the regulation of the growth of female cholangiocytes. The rationale for using cholangiocytes from female rats is based on the fact that women are preferentially affected by specific cholangiopathies including primary biliary cirrhosis. We propose to evaluate the role and mechanisms of action by which prolactin regulates the growth of female cholangiocytes. Results Normal cholangiocytes express both isoforms (long and short) of prolactin receptors, whose expression increased following BDL. The administration of prolactin to normal female rats increased cholangiocyte proliferation. In purified normal female cholangiocytes, prolactin stimulated cholangiocyte proliferation, which was associated with increased [Ca2+]i levels and PKCβ-I phosphorylation but decreased PKCα phosphorylation. Administration of an anti-prolactin antibody to BDL female rats decreased cholangiocyte proliferation. Normal female cholangiocytes express and secrete prolactin, which was increased in BDL rats. The data show that prolactin stimulates normal cholangiocyte growth by an autocrine mechanism involving phosphorylation of PKCβ-I and dephosphorylation of PKCα. Conclusion We suggest that in female rats: (i) prolactin has a trophic effect on the growth of normal cholangiocytes by phosphorylation of PKCβ-I and dephosphorylation of PKCα; and (iii) cholangiocytes express and secrete prolactin, which by an autocrine mechanism participate in regulation of cholangiocyte proliferation. Prolactin may be an important therapeutic approach for the management of cholangiopathies affecting female patients.