Julie Venter

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.

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.

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.

H3 histamine receptor agonist inhibits biliary growth of BDL rats by downregulation of the cAMP-dependent PKA/ERK1/2/ELK-1 pathway

Histamine regulates many functions by binding to four histamine G-coupled receptor proteins (H1R, H2R, H3R and H4R). As H3R exerts their effects by coupling to Gαi/o proteins reducing adenosine 3′, 5′-monophosphate (cAMP) levels (a key player in the modulation of cholangiocyte hyperplasia/damage), we evaluated the role of H3R in the regulation of biliary growth. We posed the following questions: (1) Do cholangiocytes express H3R? (2) Does in vivo administration of (R)-(α)-(−)-methylhistamine dihydrobromide (RAMH) (H3R agonist), thioperamide maleate (H3R antagonist) or histamine, in the absence/presence of thioperamide maleate, to bile duct ligated (BDL) rats regulate cholangiocyte proliferation? and (3) Does RAMH inhibit cholangiocyte proliferation by downregulation of cAMP-dependent phosphorylation of protein kinase A (PKA)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ets-like gene-1 (Elk-1)? The expression of H3R was evaluated in liver sections by immunohistochemistry and immunofluorescence, and by real-time PCR in cholangiocyte RNA from normal and BDL rats. BDL rats (immediately after BDL) were treated daily with RAMH, thioperamide maleate or histamine in the absence/presence of thioperamide maleate for 1 week. Following in vivo treatment of BDL rats with RAMH for 1 week, and in vitro stimulation of BDL cholangiocytes with RAMH, we evaluated cholangiocyte proliferation, cAMP levels and PKA, ERK1/2 and Elk-1 phosphorylation. Cholangiocytes from normal and BDL rats express H3R. The expression of H3R mRNA increased in BDL compared to normal cholangiocytes. Histamine decreased cholangiocyte growth of BDL rats to a lower extent than that observed in BDL RAMH-treated rats; histamine-induced inhibition of cholangiocyte growth was partly blocked by thioperamide maleate. In BDL rats treated with thioperamide maleate, cholangiocyte hyperplasia was slightly higher than that of BDL rats. In vitro, RAMH inhibited the proliferation of BDL cholangiocytes. RAMH inhibition of cholangiocyte growth was associated with decreased cAMP levels and PKA/ERK1/2/Elk-1 phosphorylation. Downregulation of cAMP-dependent PKA/ERK1/2/Elk-1 phosphorylation (by activation of H3R) is important in the inhibition of cholangiocyte growth in liver diseases.

Serotonin metabolism is dysregulated in cholangiocarcinoma, which has implications for tumor growth.

Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. Symptoms are usually evident after blockage of the bile duct by the tumor, and at this late stage, they are relatively resistant to chemotherapy and radiation therapy. Therefore, it is imperative that alternative treatment options are explored. We present novel data indicating that the metabolism of serotonin is dysregulated in cholangiocarcinoma cell lines, compared with normal cholangiocytes, and tissue and bile from cholangiocarcinoma patients. Specifically, there was an increased expression of tryptophan hydroxylase 1 and a suppression of monoamine oxidase A expression (enzymes responsible for the synthesis and degradation of serotonin, respectively) in cholangiocarcinoma. This resulted in an increased secretion of serotonin from cholangiocarcinoma and increased serotonin in the bile from cholangiocarcinoma patients. Increased local serotonin release may have implications on cholangiocarcinoma cell growth. Serotonin administration increased cholangiocarcinoma cell growth in vitro, whereas inhibition of serotonin synthesis decreases tumor cell growth both in vitro and in vivo. The data presented here represent the first evidence that serotonin metabolism is dysregulated in cholangiocarcinoma and that modulation of serotonin synthesis may represent an alternative target for the development of therapeutic strategies.

Caffeic acid phenethyl ester decreases cholangiocarcinoma growth by inhibition of NF-κB and induction of apoptosis

Caffeic acid phenethyl ester (CAPE) inhibits the growth of tumor cells and is a known inhibitor of nuclear factor kappa beta (NF‐κB), which is constitutively active in cholangiocarcinoma (CCH) cells. We evaluated the effects of CAPE on CCH growth both in vitro and in vivo. Inhibition of NF‐κB DNA‐binding activity was confirmed in nuclear extracts treated with CAPE at 50, 40 and 20 μM. CAPE decreases the expression of NF‐κB1 (p50) and RelA (p65). CAPE decreased the growth of a number of CCH cells but not normal cholangiocytes. Cell cycle decrease was seen by a decrease in PCNA protein expression and the number of BrdU‐positive cells treated with CAPE at 20 μM compared to vehicle. Inhibition of growth and increased cell cycle arrest of Mz‐ChA‐1 cells by CAPE were coupled with increased apoptosis. Bax expression was increased, whereas Bcl‐2 was decreased in cells treated with CAPE compared to vehicle. In vivo studies were performed in BALB/c nude (nu/nu) mice implanted subcutaneously with Mz‐ChA‐1 cells and treated with daily IP injections of DMSO or CAPE (10 mg/kg body weight in DMSO) for 77 days. Tumor growth was decreased and tumor latency was increased 2‐fold in CAPE compared to vehicle‐treated nude mice. In tumor samples, decreased CCH growth by CAPE was coupled with increased apoptosis. CAPE both in vivo and in vitro decreases the growth of CCH cells by increasing apoptosis. These results demonstrate that CAPE might be an important therapeutic tool in the treatment of CCH.

Knockout of secretin receptor reduces large cholangiocyte hyperplasia in mice with extrahepatic cholestasis induced by bile duct ligation

During bile duct ligation (BDL), the growth of large cholangiocytes is regulated by the cyclic adenosine monophosphate (cAMP)/extracellular signal‐regulated kinase 1/2 (ERK1/2) pathway and is closely associated with increased secretin receptor (SR) expression. Although it has been suggested that SR modulates cholangiocyte growth, direct evidence for secretin‐dependent proliferation is lacking. SR wild‐type (WT) (SR+/+) or SR knockout (SR−/−) mice underwent sham surgery or BDL for 3 or 7 days. We evaluated SR expression, cholangiocyte proliferation, and apoptosis in liver sections and proliferating cell nuclear antigen (PCNA) protein expression and ERK1/2 phosphorylation in purified large cholangiocytes from WT and SR−/− BDL mice. Normal WT mice were treated with secretin (2.5 nmoles/kg/day by way of osmotic minipumps for 1 week), and biliary mass was evaluated. Small and large cholangiocytes were used to evaluate the in vitro effect of secretin (100 nM) on proliferation, protein kinase A (PKA) activity, and ERK1/2 phosphorylation. SR expression was also stably knocked down by short hairpin RNA, and basal and secretin‐stimulated cAMP levels (a functional index of biliary growth) and proliferation were determined. SR was expressed by large cholangiocytes. Knockout of SR significantly decreased large cholangiocyte growth induced by BDL, which was associated with enhanced apoptosis. PCNA expression and ERK1/2 phosphorylation were decreased in large cholangiocytes from SR−/− BDL compared with WT BDL mice. In vivo administration of secretin to normal WT mice increased ductal mass. In vitro, secretin increased proliferation, PKA activity, and ERK1/2 phosphorylation of large cholangiocytes that was blocked by PKA and mitogen‐activated protein kinase kinase inhibitors. Stable knockdown of SR expression reduced basal cholangiocyte proliferation. SR is an important trophic regulator sustaining biliary growth. Conclusion: The current study provides strong support for the potential use of secretin as a therapy for ductopenic liver diseases. HEPATOLOGY 2010

Leptin enhances cholangiocarcinoma cell growth

Cholangiocarcinoma is a strongly aggressive malignancy with a very poor prognosis. Effective therapeutic strategies are lacking because molecular mechanisms regulating cholangiocarcinoma cell growth are unknown. Furthermore, experimental in vivo animal models useful to study the pathophysiologic mechanisms of malignant cholangiocytes are lacking. Leptin, the hormone regulating caloric homeostasis, which is increased in obese patients, stimulates the growth of several cancers, such as hepatocellular carcinoma. The aim of this study was to define if leptin stimulates cholangiocarcinoma growth. We determined the expression of leptin receptors in normal and malignant human cholangiocytes. Effects on intrahepatic cholangiocarcinoma (HuH-28) cell proliferation, migration, and apoptosis of the in vitro exposure to leptin, together with the intracellular pathways, were then studied. Moreover, cholangiocarcinoma was experimentally induced in obese fa/fa Zucker rats, a genetically established animal species with faulty leptin receptors, and in their littermates by chronic feeding with thioacetamide, a potent carcinogen. After 24 weeks, the effect of leptin on cholangiocarcinoma development and growth was assessed. Normal and malignant human cholangiocytes express leptin receptors. Leptin increased the proliferation and the metastatic potential of cholangiocarcinoma cells in vitro through a signal transducers and activators of transcription 3-dependent activation of extracellular signal-regulated kinase 1/2. Leptin increased the growth and migration, and was antiapoptotic for cholangiocarcinoma cells. Moreover, the loss of leptin function reduced the development and the growth of cholangiocarcinoma. The experimental carcinogenesis model induced by thioacetamide administration is a valid and reproducible method to study cholangiocarcinoma pathobiology. Modulation of the leptin-mediated signal could be considered a valid tool for the prevention and treatment of cholangiocarcinoma.