Shannon Glaser

Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation

We previously introduced the concept that intrahepatic bile duct epithelial cells, or cholangiocytes, are functionally heterogeneous. This concept is based on the observation that secretin receptor (SR) gene expression and secretin-induced cAMP synthesis are present in cholangiocytes derived from large (> 15 microns in diameter) but not small (< 15 microns in diameter) bile ducts. In work reported here, we tested the hypothesis that cholangiocytes are heterogeneous with regard to proliferative capacity. We assessed cholangiocyte proliferation in vivo by measurement of [3H]thymidine incorporation and in vitro by both [3H]thymidine incorporation and H3 histone gene expression in small (fraction 1) and large (fraction 2) cholangiocytes isolated from rats after bile duct ligation (BDL). In the two cholangiocyte subpopulations, we also studied basal somatostatin receptor (SSTR2) gene expression as well as the effects of somatostatin on 1) SR gene expression and secretin-induced cAMP synthesis and 2) [3H]thymidine incorporation and H3 histone gene expression. In normal rat liver, cholangiocytes, unlike hepatocytes, were mitotically dormant; after BDL, incorporation of [3H]thymidine markedly increased in cholangiocytes but not hepatocytes. When subpopulations of cholangiocytes were isolated after BDL, DNA synthesis assessed by both techniques was limited to large cholangiocytes, as was SSTR2 steady-state gene expression. In vitro, somatostatin inhibited SR gene expression and secretin-induced cAMP synthesis only in large cholangiocytes. Moreover, compared with no hormone, somatostatin inhibited DNA synthesis solely in large cholangiocytes. These results support the concept of the heterogeneity of cholangiocytes along the biliary tree, extend this concept to cholangiocyte proliferative activity, and imply that the proliferative compartment of cholangiocytes after BDL is located principally in the cholangiocytes lining large (> 15 microns) bile ducts.We previously introduced the concept that intrahepatic bile duct epithelial cells, or cholangiocytes, are functionally heterogeneous. This concept is based on the observation that secretin receptor (SR) gene expression and secretin-induced cAMP synthesis are present in cholangiocytes derived from large (>15 μm in diameter) but not small (<15 μm in diameter) bile ducts. In work reported here, we tested the hypothesis that cholangiocytes are heterogeneous with regard to proliferative capacity. We assessed cholangiocyte proliferation in vivo by measurement of [3H]thymidine incorporation and in vitro by both [3H]thymidine incorporation and H3 histone gene expression in small ( fraction 1) and large ( fraction 2) cholangiocytes isolated from rats after bile duct ligation (BDL). In the two cholangiocyte subpopulations, we also studied basal somatostatin receptor (SSTR2) gene expression as well as the effects of somatostatin on 1) SR gene expression and secretin-induced cAMP synthesis and 2) [3H]thymidine incorporation and H3 histone gene expression. In normal rat liver, cholangiocytes, unlike hepatocytes, were mitotically dormant; after BDL, incorporation of [3H]thymidine markedly increased in cholangiocytes but not hepatocytes. When subpopulations of cholangiocytes were isolated after BDL, DNA synthesis assessed by both techniques was limited to large cholangiocytes, as was SSTR2 steady-state gene expression. In vitro, somatostatin inhibited SR gene expression and secretin-induced cAMP synthesis only in large cholangiocytes. Moreover, compared with no hormone, somatostatin inhibited DNA synthesis solely in large cholangiocytes. These results support the concept of the heterogeneity of cholangiocytes along the biliary tree, extend this concept to cholangiocyte proliferative activity, and imply that the proliferative compartment of cholangiocytes after BDL is located principally in the cholangiocytes lining large (>15 μm) bile ducts.

Cholangiocyte proliferation and liver fibrosis

Cholangiocyte proliferation is triggered during extrahepatic bile duct obstruction induced by bile duct ligation, which is a common in vivo model used for the study of cholangiocyte proliferation and liver fibrosis. The proliferative response of cholangiocytes during cholestasis is regulated by the complex interaction of several factors, including gastrointestinal hormones, neuroendocrine hormones and autocrine or paracrine signalling mechanisms. Activation of biliary proliferation (ductular reaction) is thought to have a key role in the initiation and progression of liver fibrosis. The first part of this review provides an overview of the primary functions of cholangiocytes in terms of secretin-stimulated bicarbonate secretion--a functional index of cholangiocyte growth. In the second section, we explore the important regulators, both inhibitory and stimulatory, that regulate the cholangiocyte proliferative response during cholestasis. We discuss the role of proliferating cholangiocytes in the induction of fibrosis either directly via epithelial mesenchymal transition or indirectly via the activation of other liver cell types. The possibility of targeting cholangiocyte proliferation as potential therapy for reducing and/or preventing liver fibrosis, and future avenues for research into how cholangiocytes participate in the process of liver fibrogenesis are described.

Small mouse cholangiocytes proliferate in response to H1 histamine receptor stimulation by activation of the IP3/CaMK I/CREB pathway

Cholangiopathies are characterized by the heterogeneous proliferation of different-sized cholangiocytes. Large cholangiocytes proliferate by a cAMP-dependent mechanism. The function of small cholangiocytes may depend on the activation of inositol trisphosphate (IP(3))/Ca(2+)-dependent signaling pathways; however, data supporting this speculation are lacking. Four histamine receptors exist (HRH1, HRH2, HRH3, and HRH4). In several cells: 1) activation of HRH1 increases intracellular Ca(2+) concentration levels; and 2) increased [Ca(2+)](i) levels are coupled with calmodulin-dependent stimulation of calmodulin-dependent protein kinase (CaMK) and activation of cAMP-response element binding protein (CREB). HRH1 agonists modulate small cholangiocyte proliferation by activation of IP(3)/Ca(2+)-dependent CaMK/CREB. We evaluated HRH1 expression in cholangiocytes. Small and large cholangiocytes were stimulated with histamine trifluoromethyl toluidide (HTMT dimaleate; HRH1 agonist) for 24-48 h with/without terfenadine, BAPTA/AM, or W7 before measuring proliferation. Expression of CaMK I, II, and IV was evaluated in small and large cholangiocytes. We measured IP(3), Ca(2+) and cAMP levels, phosphorylation of CaMK I, and activation of CREB (in the absence/presence of W7) in small cholangiocytes treated with HTMT dimaleate. CaMK I knockdown was performed in small cholangiocytes stimulated with HTMT dimaleate before measurement of proliferation and CREB activity. Small and large cholangiocytes express HRH1, CaMK I, and CaMK II. Small (but not large) cholangiocytes proliferate in response to HTMT dimaleate and are blocked by terfenadine (HRH1 antagonist), BAPTA/AM, and W7. In small cholangiocytes, HTMT dimaleate increased IP(3)/Ca(2+) levels, CaMK I phosphorylation, and CREB activity. Gene knockdown of CaMK I ablated the effects of HTMT dimaleate on small cholangiocyte proliferation and CREB activation. The IP(3)/Ca(2+)/CaMK I/CREB pathway is important in the regulation of small cholangiocyte function.

Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver

Bile duct damage and/or loss is limited to a range of duct sizes in cholangiopathies. We tested the hypothesis that CCl4damages only large ducts. CCl4 or mineral oil was given to bile duct-ligated (BDL) rats, and 1, 2, and 7 days later small and large cholangiocytes were purified and evaluated for apoptosis, proliferation, and secretion. In situ, we measured apoptosis by morphometric and TUNEL analysis and the number of small and large ducts by morphometry. Two days after CCl4 administration, we found an increased number of small ducts and reduced number of large ducts. In vitro apoptosis was observed only in large cholangiocytes, and this was accompanied by loss of proliferation and secretion in large cholangiocytes and loss of choleretic effect of secretin. Small cholangiocytes de novo express the secretin receptor gene and secretin-induced cAMP response. Consistent with damage of large ducts, we detected cytochrome P-4502E1 (which CCl4 converts to its radicals) only in large cholangiocytes. CCl4induces selective apoptosis of large ducts associated with loss of large cholangiocyte proliferation and secretion.

Functional analysis of microRNAs in human hepatocellular cancer stem cells

MicroRNAs are endogenous small non‐coding RNAs that regulate gene expression and cancer development. A rare population of hepatocellular cancer stem cells (HSCs) holds the extensive proliferative and self‐renewal potential necessary to form a liver tumour. We postulated that specific transcriptional factors might regulate the expression of microRNAs and subsequently modulate the expression of gene products involved in phenotypic characteristics of HSCs. We evaluated the expression of microRNA in human HSCs by microarray profiling, and defined the target genes and functional effects of two groups of microRNA regulated by IL‐6 and transcriptional factor Twist. A subset of highly chemoresistant and invasive HSCs was screened with aberrant expressions of cytokine IL‐6 and Twist. We demonstrated that conserved let‐7 and miR‐181 family members were up‐regulated in HSCs by global microarray‐based microRNA profiling followed by validation with real‐time polymerase chain reaction. Importantly, inhibition of let‐7 increases the chemosensitivity of HSCs to sorafenib and doxorubicin whereas silencing of miR‐181 led to a reduction in HSCs motility and invasion. Knocking down IL‐6 and Twist in HSCs significantly reduced let‐7 and miR‐181 expression and subsequently inhibited chemoresistance and cell invasion. We showed that let‐7 directly targets SOCS‐1 and caspase‐3, whereas miR‐181 directly targets RASSF1A, TIMP3 as well as nemo‐like kinase (NLK). In conclusion, alterations of IL‐6‐ and Twist‐regulated microRNA expression in HSCs play a part in tumour spreading and responsiveness to chemotherapy. Our results define a novel regulatory mechanism of let‐7/miR‐181s suggesting that let‐7 and miR‐181 may be molecular targets for eradication of hepatocellular malignancies.

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.

Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium

Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2–4 μm2, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl−/HCO3− AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3′,5′-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl−/HCO3− exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree.

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.

Biliary repair and carcinogenesis are mediated by IL-33–dependent cholangiocyte proliferation

Injury to the biliary epithelium triggers inflammation and fibrosis, which can result in severe liver diseases and may progress to malignancy. Development of a type 1 immune response has been linked to biliary injury pathogenesis; however, a subset of patients with biliary atresia, the most common childhood cholangiopathy, exhibit increased levels of Th2-promoting cytokines. The relationship among different inflammatory drivers, epithelial repair, and carcinogenesis remains unclear. Here, we determined that the Th2-activating cytokine IL-33 is elevated in biliary atresia patient serum and in the livers and bile ducts of mice with experimental biliary atresia. Administration of IL-33 to WT mice markedly increased cholangiocyte proliferation and promoted sustained cell growth, resulting in dramatic and rapid enlargement of extrahepatic bile ducts. The IL-33-dependent proliferative response was mediated by an increase in the number of type 2 innate lymphoid cells (ILC2s), which released high levels of IL-13 that in turn promoted cholangiocyte hyperplasia. Induction of the IL-33/ILC2/IL-13 circuit in a murine biliary injury model promoted epithelial repair; however, induction of this circuit in mice with constitutive activation of AKT and YAP in bile ducts induced cholangiocarcinoma with liver metastases. These findings reveal that IL-33 mediates epithelial proliferation and suggest that activation of IL-33/ILC2/IL-13 may improve biliary repair and disruption of the circuit may block progression of carcinogenesis.

γ-Aminobutyric Acid Inhibits Cholangiocarcinoma Growth by Cyclic AMP–Dependent Regulation of the Protein Kinase A/Extracellular Signal-Regulated Kinase 1/2 Pathway

We studied the effect of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), in the regulation of cholangiocarcinoma growth. We determined the in vitro effect of GABA on the proliferation of the cholangiocarcinoma cell lines (Mz-ChA-1, HuH-28, and TFK-1) and evaluated the intracellular pathways involved. The effect of GABA on migration of Mz-ChA-1 cells was also evaluated. In vivo, Mz-ChA-1 cells were s.c. injected in athymic mice, and the effects of GABA on tumor size, tumor cell proliferation, apoptosis, collagen quantity, and the expression of vascular endothelial growth factor-A (VEGF-A) and VEGF-C (cancer growth regulators) were measured after 82 days. GABA decreased in vitro cholangiocarcinoma growth in a time-dependent and dose-dependent manner, by both cyclic AMP/protein kinase A- and D-myo-inositol-1,4,5-thriphosphate/Ca(2+)-dependent pathways, leading to down-regulation of extracellular signal-regulated kinase 1/2 phosphorylation. Blocking of GABA(A), GABA(B), and GABA(C) receptors prevented GABA inhibition of cholangiocarcinoma proliferation. GABA inhibited Mz-ChA-1 cell migration and, in vivo, significantly decreased tumor volume, tumor cell proliferation, and VEGF-A/C expression whereas increasing apoptosis compared with controls. An increase in collagen was evident in GABA-treated tumors. GABA decreases biliary cancer proliferation and reduces the metastatic potential of cholangiocarcinoma. GABA may represent a therapeutic agent for patients affected by malignancies of the biliary tract.