G. Alpini

Opposing Actions of Endocannabinoids on Cholangiocarcinoma Growth RECRUITMENT OF Fas AND Fas LIGAND TO LIPID RAFTS

Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. Modulation of the endocannabinoid system is being targeted to develop possible therapeutic strategies for a number of cancers; therefore, we evaluated the effects of the two major endocannabinoids, anandamide and 2-arachidonylglycerol, on numerous cholangiocarcinoma cell lines. Although anandamide was antiproliferative and proapoptotic, 2-arachidonylglycerol stimulated cholangiocarcinoma cell growth. Specific inhibitors for each of the cannabinoid receptors did not prevent either of these effects nor did pretreatment with pertussis toxin, a Gi/o protein inhibitor, suggesting that anandamide and 2-arachidonylglycerol did not exert their diametric effects through any known cannabinoid receptor or through any other Gi/o protein-coupled receptor. Using the lipid raft disruptors methyl-β-cyclodextrin and filipin, we demonstrated that anandamide, but not 2-arachidonylglycerol, requires lipid raft-mediated events to inhibit cellular proliferation. Closer inspection of the lipid raft structures within the cell membrane revealed that although anandamide treatment had no observable effect 2-arachidonylglycerol treatment effectively dissipated the lipid raft structures and caused the lipid raft-associated proteins lyn and flotillin-1 to disperse into the surrounding membrane. In addition, anandamide, but not 2-arachidonylglycerol, induced an accumulation of ceramide, which was required for anandamide-induced suppression of cell growth. Finally we demonstrated that anandamide and ceramide treatment of cholangiocarcinoma cells recruited Fas and Fas ligand into the lipid rafts, subsequently activating death receptor pathways. These findings suggest that modulation of the endocannabinoid system may be a target for the development of possible therapeutic strategies for the treatment of this devastating cancer.

Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice

Aims: We have shown that large and small cholangiocytes, which reside primarily in large and small intrahepatic bile ducts, respectively, have different functions and responses to injuries. However, there are no systematic studies of the molecular differences between small and large cholangiocytes, which would explain cholangiocyte heterogeneity. To evaluate the differential gene expression between small and large cholangiocytes, microarray analysis was performed.

Morphological and Functional Features of Hepatic Cyst Epithelium in Autosomal Dominant Polycystic Kidney Disease

We evaluated the morphological and functional features of hepatic cyst epithelium in adult autosomal dominant polycystic kidney disease (ADPKD). In six ADPKD patients, we investigated the morphology of cyst epithelium apical surface by scanning electron microscopy and the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF1), IGF1 receptors (IGF1-R), growth hormone receptor, the proliferation marker proliferating cell nuclear antigen, and pAKT by immunohistochemistry and immunofluorescence. Proliferation of liver cyst-derived epithelial cells was evaluated by both MTS proliferation assay and [(3)H]thymidine incorporation into DNA. The hepatic cyst epithelium displayed heterogeneous features, being normal in small cysts (<1 cm), characterized by rare or shortened cilia in 1- to 3-cm cysts, and exhibiting the absence of both primary cilia and microvilli in large cysts (>3 cm). Cyst epithelium showed marked immunohistochemical expression of ER, growth hormone receptor, IGF1, IGF1-R, proliferating cell nuclear antigen, and pAKT. IGF1 was 10-fold more enriched in the hepatic cyst fluid than in serum. Serum-deprived liver cyst-derived epithelial cells proliferated when exposed to 17beta-estradiol and IGF1 and when exposed to human cyst fluid. ER or IGF1-R antagonists inhibited the proliferative effect of serum readmission, cyst fluid, 17beta-estradiol, and IGF1. Our findings could explain the role of estrogens in accelerating the progression of ADPKD and may suggest a potential benefit of therapeutic strategies based on estrogen antagonism.

Knockout of α -calcitonin gene-related peptide reduces cholangiocyte proliferation in bile duct ligated mice

The role of sensory innervation in the regulation of liver physiology and the pathogenesis of cholestatic liver disease are undefined. Biliary proliferation has been shown to be coordinately controlled by parasympathetic and sympathetic innervation of the liver. The aim of our study was to address the role of the sensory neuropeptide calcitonin gene-related peptide (α-CGRP) in the regulation of cholangiocyte proliferation during cholestasis induced by extrahepatic bile duct obstruction (BDL). Our study utilized a knockout (KO) mouse model, which lacks the sensory neuropeptide α-CGRP. Wild-type (WT) and α-CGRP KO mice were subjected to sham surgery or BDL for 3 and 7 days. In addition, immediately after BDL, WT and KO mice were administered the CGRP receptor antagonist (CGRP8–37) for 3 and 7 days by osmotic minipumps. Liver sections and isolated cholangiocytes were evaluated for proliferation markers. Isolated WT BDL (3 days) cholangiocytes were stimulated with α- and β-CGRP and evaluated for proliferation and cAMP-mediated signaling. Lack of α-CGRP inhibits cholangiocyte proliferation induced by BDL at both 3 and 7 days. BDL-induced cholangiocyte proliferation in WT mice was associated with increases of circulating α-CGRP levels. In vitro, α- and β-CGRP stimulated proliferation in purified BDL cholangiocytes, induced elevation of cAMP levels, and stimulated the activation of cAMP-dependent protein kinase A and cAMP response element binding protein DNA binding. In conclusion, sensory innervation of the liver and biliary expression of α-CGRP play an important role in the regulation of cholangiocyte proliferation during cholestasis.

Alfa and beta estrogen receptors and the biliary tree

This manuscript summarizes recent data showing that estrogens and their receptors play an important role in modulating cholangiocyte proliferation. We have recently demonstrated that rat cholangiocytes express both estrogen receptors (ER)-alpha and -beta subtypes, while hepatocytes only express ER-alpha. ER and especially the ER-beta subtype, are overexpressed in cholangiocytes proliferating after bile duct ligation (BDL) in the rat, in association with enlarged bile duct mass and with enhanced estradiol serum levels. Cholangiocyte proliferation, during BDL, is impaired by estrogen antagonists (tamoxifen, ICI 182,780) which furthermore, induce the overexpression of Fas antigen and activate apoptosis of proliferating cholangiocytes. 17beta-estradiol stimulates, in vitro cholangiocyte proliferation, and this effect is individually blocked by tamoxifen or ICI 182,780. Cholangiocyte proliferation during BDL was associated with an enhanced protein expression of phosphorylated extracellular regulated kinases (ERK)1/2 which is, in contrast, negatively modulated by tamoxifen in association with its antiproliferative effect. This indicates a major involvement of the ERK system in the estrogen modulation of cholangiocyte proliferation.

Regulators of Cholangiocyte Proliferation.

Cholangiocytes, a small population of cells within the normal liver, have been the focus of a significant amount of research over the past two decades because of their involvement in cholangiopathies such as primary sclerosing cholangitis and primary biliary cholangitis. This article summarizes landmark studies in the field of cholangiocyte physiology and aims to provide an updated review of biliary pathogenesis. The historical approach of rodent extrahepatic bile duct ligation and the relatively recent utilization of transgenic mice have led to significant discoveries in cholangiocyte pathophysiology. Cholangiocyte physiology is a complex system based on heterogeneity within the biliary tree and a number of signaling pathways that serve to regulate bile composition. Studies have expanded the list of neuropeptides, neurotransmitters, and hormones that have been shown to be key regulators of proliferation and biliary damage. The peptide histamine and hormones, such as melatonin and angiotensin, angiotensin, as well as numerous sex hormones, have been implicated in cholangiocyte proliferation during cholestasis. Numerous pathways promote cholangiocyte proliferation during cholestasis, and there is growing evidence to suggest that cholangiocyte proliferation may promote hepatic fibrosis. These pathways may represent significant therapeutic potential for a subset of cholestatic liver diseases that currently lack effective therapies.

Histamine regulation of biliary proliferation

Cholangiocytes, which line the biliary epithelium, are hormone-responsive and responsible for the modification of canalicular bile before reaching the duodenum. Cholangiocytes are the target cells in cholangiopathies such as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) that are characterized by cholangiocyte proliferation/loss [1]. The biliary epithelium is morphologically and functionally heterogeneous and is lined by small ( 15 lm in diameter) cholangiocytes lining small and large bile ducts, respectively [2]. Cholangiocyte responses are regulated by both Ca2+- and cAMP-dependent signaling [2–5].

Cholangiocarcinoma development: The resurgence of bile acids

C holangiocarcinoma (CCA) is a rare malignant cancer arising from cholangiocytes, the epithelial cells lining the biliary epithelium. Despite being overall a rare neoplasm (3% of all gastrointestinal tumors worldwide), CCA is the second most common primary hepatic neoplasm, after hepatocellular carcinoma (HCC). CCA still represents a challenge for patients and clinicians. While the incidence of the disease appears to have increased over the past 30 years, prognosis of untreated patients has remained substantially unchanged and dismal. Treatment options are limited: surgical resection of the tumor remains the sole curative approach, but it can be achieved only in a minority of patients. Liver transplantation with neoadjuvant chemoradiation is currently reserved only for a few patients affected by perihilar CCA who meet stringent inclusion criteria. In this complex scenario, a deeper understanding of the mechanisms leading to CCA formation is eagerly sought, since it holds the hope for the generation of new and effective therapeutic options to treat such a deadly disease. In the present issue of HEPATOLOGY, Liu et al. report that conjugated bile acids are able to sustain CCA growth and invasiveness through the activation of the sphingosine 1-phosphate receptor 2 (S1PR2), a new bile acid receptor identified recently. The authors demonstrate that S1PR2 is the predominant sphingosine 1-phosphate (S1P) receptor expressed in rat and human CCA cell lines. Importantly, human CCA tissues showed significantly higher S1PR2 levels as compared to nontumor samples obtained from the same patients. In line with what was previously shown for rat hepatocytes, conjugated bile acid stimulation of S1PR2 induced the activation of ERK1/2 and AKT. The increased nuclear p-ERK1/2 levels were blunted by the incubation with JTE-013, a S1PR2 selective antagonist. Moreover, by using two different in vitro models the authors prove that taurocholic acid (TCA) exerts proproliferative effects on rat and human cell lines that are blocked by treatment with JTE-013 or by shRNA knockdown of S1PR2. In fact, treatment with JTE-013 alone reduced CCA cell proliferation compared to baseline, suggesting stimulatory autocrine/ paracrine functions of the S1P secreted by the cells. Finally, Liu et al. show that TCA-mediated activation of S1PR2 is also involved in CCA cells invasiveness. Indeed, TCA stimulated cell migration both in the Matrigel invasion assay and in scratch assays, which were again inhibited by S1PR2 antagonism (Fig. 1). Despite that the majority of CCA are sporadic, several conditions associated with biliary inflammation

Mechanisms of cholangiocyte responses to injury

Cholangiocytes, epithelial cells that line the biliary epithelium, are the primary target cells for cholangiopathies including primary sclerosing cholangitis and primary biliary cholangitis. Quiescent cholangiocytes respond to biliary damage and acquire an activated neuroendocrine phenotype to maintain the homeostasis of the liver. The typical response of cholangiocytes is proliferation leading to bile duct hyperplasia, which is a characteristic of cholestatic liver diseases. Current studies have identified various signaling pathways that are associated with cholangiocyte proliferation/loss and liver fibrosis in cholangiopathies using human samples and rodent models. Although recent studies have demonstrated that extracellular vesicles and microRNAs could be mediators that regulate these messenger/receptor axes, further studies are required to confirm their roles. This review summarizes current studies of biliary response and cholangiocyte proliferation during cholestatic liver injury with particular emphasis on the secretin/secretin receptor axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Author Correction: The role of the secretin/secretin receptor axis in inflammatory cholangiocyte communication via extracellular vesicles

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.