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The Hippo signaling pathway and the Notch signaling pathway are evolutionary conserved signaling cascades that have important roles in embryonic development of many organs. In murine liver, disruption of either pathway impairs intrahepatic bile duct development. Recent studies suggested that the Notch signaling receptor Notch2 is a direct transcriptional target of the Hippo signaling pathway effector YAP, and the Notch signaling is a major mediator of the Hippo signaling in maintaining biliary cell characteristics in adult mice. However, it remains to be determined whether the Hippo signaling pathway functions through the Notch signaling in intrahepatic bile duct development. We found that loss of the Hippo signaling pathway tumor suppressor Nf2 resulted in increased expression levels of the Notch signaling pathway receptor Notch2 in cholangiocytes but not in hepatocytes. When knocking down Notch2 on the background of Nf2 deficiency in mouse livers, the excessive bile duct development induced by Nf2 deficiency was suppressed by heterozygous and homozygous deletion of Notch2 in a dose-dependent manner. These results implicated that Notch signaling is one of the downstream effectors of the Hippo signaling pathway in regulating intrahepatic bile duct development.

Cholestasis is a condition that leads to chronic hepatobiliary inflammation, fibrosis, and eventually cirrhosis. Many microRNAs (miRs) are known to have a role in fibrosis progression; however, the role of miR-21 during cholestasis remains unknown. Therefore, the aim of this study was to elucidate the role of miR-21 during cholestasis-induced biliary hyperplasia and hepatic fibrosis. Wild-type (WT) and miR-21 -/- mice underwent Sham or bile duct ligation (BDL) for 1 week, before evaluating liver histology, biliary proliferation, hepatic stellate cell (HSC) activation, fibrotic response, and small mothers against decapentaplegic 7 (Smad-7) expression. In vitro, immortalized murine biliary cell lines (IMCLs) and human hepatic stellate cell line (hHSC) were treated with either miR-21 inhibitor or control before analyzing proliferation, apoptosis, and fibrotic responses. In vivo, the levels of miR-21 were increased in total liver and cholangiocytes after BDL, and loss of miR-21 decreased the amount of BDL-induced biliary proliferation and intrahepatic biliary mass. In addition, loss of miR-21 decreased BDL-induced HSC activation, collagen deposition, and expression of the fibrotic markers transforming growth factor-β1 and α-smooth muscle actin. In vitro, IMCL and hHSCs treated with miR-21 inhibitor displayed decreased proliferation and expression of fibrotic markers and enhanced apoptosis when compared with control treated cells. Furthermore, mice lacking miR-21 show increased Smad-7 expression, which may be driving the decrease in biliary hyperplasia and hepatic fibrosis. During cholestatic injury, miR-21 is increased and leads to increased biliary proliferation and hepatic fibrosis. Local modulation of miR-21 may be a therapeutic option for patients with cholestasis., Competing Interests: The authors have no conflicts of interest to declare.

The neurohypophysial hormone arginine vasopressin (AVP) acts by three distinct receptor subtypes: V1a, V1b, and V2. In the liver, AVP is involved in ureogenesis, glycogenolysis, neoglucogenesis and regeneration. No data exist about the presence of AVP in the biliary epithelium. Cholangiocytes are the target cells in a number of animal models of cholestasis, including bile duct ligation (BDL), and in several human pathologies, such as polycystic liver disease characterized by the presence of cysts that bud from the biliary epithelium. In vivo, liver fragments from normal and BDL mice and rats as well as liver samples from normal and ADPKD patients were collected to evaluate: (i) intrahepatic bile duct mass by immunohistochemistry for cytokeratin-19; and (ii) expression of V1a, V1b and V2 by immunohistochemistry, immunofluorescence and real-time PCR. In vitro, small and large mouse cholangiocytes, H69 (non-malignant human cholangiocytes) and LCDE (human cholangiocytes from the cystic epithelium) were stimulated with vasopressin in the absence/presence of AVP antagonists such as OPC-31260 and Tolvaptan, before assessing cellular growth by MTT assay and cAMP levels. Cholangiocytes express V2 receptor that was upregulated following BDL and in ADPKD liver samples. Administration of AVP increased proliferation and cAMP levels of small cholangiocytes and LCDE cells. We found no effect in the proliferation of large mouse cholangiocytes and H69 cells. Increases were blocked by preincubation with the AVP antagonists. These results showed that AVP and its receptors may be important in the modulation of the proliferation rate of the biliary epithelium.

Liver transplantation and cholangiocarcinoma induce biliary dysfunction following ischemia reperfusion (IR). The function of the intrahepatic biliary tree is regulated by both autocrine and paracrine factors. The aim of the study was to demonstrate that IR-induced damage of cholangiocytes is associated with altered expression of biliary angiogenic factors. Normal and bile duct ligation rats underwent 24-h sham or hepatic reperfusion after 30 min of transient occlusion of the hepatic artery (HAIR) or portal vein (PVIR) before collecting liver blocks and cholangiocyte RNA or protein. We evaluated liver histology, biliary apoptosis, proliferation and expression of VEGF-A/C, VEGFR-2/3, Ang-1/2, and Tie-1/2 in liver sections and isolated small and large cholangiocytes. Normal rat intrahepatic cholangiocyte cultures (NRICC) were maintained under standard conditions in normoxic or under a hypoxic atmosphere for 4 h and then transferred to normal conditions for selected times. Subsequently, we measured changes in biliary proliferation and apoptosis and the expression of VEGF-A/C and VEGFR-2/3. In vivo, HAIR (but not PVIR) induced damage of large bile ducts and decreased proliferation and secretin-stimulated cAMP levels. HAIR-induced damage of large bile ducts was associated with increased expression of VEGF-A/C, VEGFR-2/3, Ang-1/2, and Tie-1/2. In vitro, under hypoxic conditions, there was increased apoptosis and reduced proliferation of NRICC concomitant with enhanced expression of VEGF-A/C and VEGFR-2/3. The functional damage of large bile ducts by HAIR and hypoxia is associated with increased expression of angiogenic factors in small cholangiocytes, presumably due to a compensatory mechanism in response to biliary damage., (Copyright © 2015 the American Physiological Society.)

During cholestatic liver disease, there is dysregulation in the balance between biliary growth and loss in bile duct-ligated (BDL) rats modulated by neuroendocrine peptides via autocrine/paracrine pathways. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone that modulates reproductive function and proliferation in many cell types. We evaluated the autocrine role of GnRH in the regulation of cholangiocyte proliferation. The expression of GnRH receptors was assessed in a normal mouse cholangiocyte cell line (NMC), sham, and BDL rats. The effect of GnRH administration was evaluated in normal rats and in NMC. GnRH-induced biliary proliferation was evaluated by changes in intrahepatic bile duct mass and the expression of proliferation and function markers. The expression and secretion of GnRH in NMC and isolated cholangiocytes was assessed. GnRH receptor subtypes GnRHR1 and GnRHR2 were expressed in cholangiocytes. Treatment with GnRH increased intrahepatic bile duct mass as well as proliferation and function markers in cholangiocytes. Transient knockdown and pharmacologic inhibition of GnRHR1 in NMC decreased proliferation. BDL cholangiocytes had increased expression of GnRH compared with normal rats, accompanied by increased GnRH secretion. In vivo and in vitro knockdown of GnRH decreased intrahepatic bile duct mass/cholangiocyte proliferation and fibrosis. GnRH secreted by cholangiocytes promotes biliary proliferation via an autocrine pathway. Disruption of GnRH/GnRHR signaling may be important for the management of cholestatic liver diseases., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by selective destruction of intrahepatic cholangiocytes. Mechanisms underlying the development and progression of the disease are still controversial and largely undefined. Evidence suggests that PBC results from an articulated immunologic response against an immunodominant mitochondrial autoantigen, the E2 component of the pyruvate dehydrogenase complex (PDC-E2); characteristics of the disease are also the presence of disease-specific antimitochondrial autoantibodies (AMAs) and autoreactive CD4 and CD8 T cells. Recent evidence suggests that cholangiocytes show specific immunobiological features that are responsible for the selective targeting of those cells by the immune system. The immune reaction in PBC selectively targets small sized, intrahepatic bile ducts; although a specific reason for that has not been defined yet, it has been established that the biliary epithelium displays a unique heterogeneity, for which the physiological and pathophysiological features of small and large cholangiocytes significantly differ. In this review article, the authors provide a critical overview of the current evidence on the role of cholangiocytes in the immune-mediated destruction of the biliary tree that characterizes PBC., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Substance P (SP) promotes cholangiocyte growth during cholestasis by activating its receptor, NK1R. SP is a proteolytic product of tachykinin (Tac1) and is deactivated by membrane metalloendopeptidase (MME). This study aimed to evaluate the functional role of SP in the regulation of cholangiocarcinoma (CCA) growth. NK1R, Tac1, and MME expression and SP secretion were assessed in human CCA cells and nonmalignant cholangiocytes. The proliferative effects of SP (in the absence/presence of the NK1R inhibitor, L-733,060) and of L-733,060 were evaluated. In vivo, the effect of L-733,060 treatment or MME overexpression on tumor growth was evaluated by using a xenograft model of CCA in nu/nu nude mice. The expression of Tac1, MME, NK1R, PCNA, CK-19, and VEGF-A was analyzed in the resulting tumors. Human CCA cell lines had increased expression of Tac1 and NK1R, along with reduced levels of MME compared with nonmalignant cholangiocytes, resulting in a subsequent increase in SP secretion. SP treatment increased CCA cell proliferation in vitro, which was blocked by L-733,060. Treatment with L-733,060 alone inhibited CCA proliferation in vitro and in vivo. Xenograft tumors derived from MME-overexpressed human Mz-ChA-1 CCA cells had a slower growth rate than those derived from control cells. Expression of PCNA, CK-19, and VEGF-A decreased, whereas MME expression increased in the xenograft tumors treated with L-733,060 or MME-overexpressed xenograft tumors compared with controls. The study suggests that SP secreted by CCA promotes CCA growth via autocrine pathway. Blockade of SP secretion and NK1R signaling may be important for the management of CCA.

Neuropeptide Y (NPY) exerts its functions through six subtypes of receptors (Y₁-Y₆). Biliary homeostasis is regulated by several factors through autocrine/paracrine signaling. NPY inhibits cholangiocarcinoma growth; however, no information exists regarding the autocrine/paracrine role of NPY on biliary hyperplasia during cholestasis. The aims of this study were to determine: 1) the expression of NPY and Y₁-Y₅ in cholangiocytes and 2) the paracrine/autocrine effects of NPY on cholangiocyte proliferation. Normal or bile duct ligation (BDL) rats were treated with NPY, neutralizing anti-NPY antibody, or vehicle for 7 days. NPY and NPY receptor (NPYR) expression was assessed in liver sections and isolated cholangiocytes. NPY secretion was assessed in serum and bile from normal and BDL rats, as well as supernatants from normal and BDL cholangiocytes and normal rat cholangiocyte cell line [intrahepatic normal cholangiocyte culture (NRICC)]. We evaluated intrahepatic bile ductal mass (IBDM) in liver sections and proliferation in cholangiocytes. With the use of NRICC, the effects of NPY or anti-NPY antibody on cholangiocyte proliferation were determined. The expression of NPY and all NPYR were increased after BDL. NPY levels were lower in serum and cholangiocyte supernatant from BDL compared with normal rats. NPY secretion from NRICC was detected at both the basolateral and apical domains. Chronic NPY treatment decreased proliferating cellular nuclear antigen (PCNA) expression and IBDM in BDL rats. Administration of anti-NPY antibody to BDL rats increased cholangiocyte proliferation and IBDM. NPY treatment of NRICC decreased PCNA expression and increased the cell cycle arrest, whereas treatment with anti-NPY antibody increased proliferation. Therapies targeting NPY-mediated signaling may prove beneficial for the treatment of cholangiopathies.

Unlabelled: Large, but not small, cholangiocytes (1) secrete bicarbonate by interaction with secretin receptors (SRs) through activation of cystic fibrosis transmembrane regulator (CFTR), Cl(-) /HCO3 (-) (apex) anion exchanger 2 (Cl(-) /HCO3 (-) AE2), and adenylyl cyclase (AC)8 (proteins regulating large biliary functions) and (2) proliferate in response to bile duct ligation (BDL) by activation of cyclic adenosine monophosphate (cAMP) signaling. Small, mitotically dormant cholangiocytes are activated during damage of large cholangiocytes by activation of D-myo-inositol 1,4,5-trisphosphate/Ca(2+) /calmodulin-dependent protein kinase (CaMK) I. gamma-Aminobutyric acid (GABA) affects cell functions by modulation of Ca(2+) -dependent signaling and AC. We hypothesized that GABA induces the differentiation of small into large cholangiocytes by the activation of Ca(2+) /CaMK I-dependent AC8. In vivo, BDL mice were treated with GABA in the absence or presence of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) or N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7) before evaluating apoptosis and intrahepatic bile ductal mass (IBDM) of small and large cholangiocytes. In vitro, control- or CaMK I-silenced small cholangiocytes were treated with GABA for 3 days before evaluating apoptosis, proliferation, ultrastructural features, and the expression of CFTR, Cl(-) /HCO3 (-) AE2, AC8, and secretin-stimulated cAMP levels. In vivo administration of GABA induces the apoptosis of large, but not small, cholangiocytes and decreases large IBDM, but increased de novo small IBDM. GABA stimulation of small IBDM was blocked by BAPTA/AM and W7. Subsequent to GABA in vitro treatment, small cholangiocytes de novo proliferate and acquire ultrastructural and functional phenotypes of large cholangiocytes and respond to secretin. GABA-induced changes were prevented by BAPTA/AM, W7, and stable knockdown of the CaMK I gene., Conclusion: GABA damages large, but not small, cholangiocytes that differentiate into large cholangiocytes. The differentiation of small into large cholangiocytes may be important in the replenishment of the biliary epithelium during damage of large, senescent cholangiocytes., (Copyright © 2013 American Association for the Study of Liver Diseases.)

Unlabelled: Secretin stimulates ductal secretion by interacting with secretin receptor (SR) activating cyclic adenosine 3',5'-monophosphate/cystic fibrosis transmembrane conductance regulator/chloride bicarbonate anion exchanger 2 (cAMP⇒CFTR⇒Cl(-) /HCO 3- AE2) signaling that is elevated by biliary hyperplasia. Cholangiocytes secrete several neuroendocrine factors regulating biliary functions by autocrine mechanisms. Melatonin inhibits biliary growth and secretin-stimulated choleresis in cholestatic bile-duct-ligated (BDL) rats by interaction with melatonin type 1 (MT1) receptor through down-regulation of cAMP-dependent signaling. No data exist regarding the role of melatonin synthesized locally by cholangiocytes in the autocrine regulation of biliary growth and function. In this study, we evaluated the (1) expression of arylalkylamine N-acetyltransferase (AANAT; the rate-limiting enzyme for melatonin synthesis from serotonin) in cholangiocytes and (2) effect of local modulation of biliary AANAT expression on the autocrine proliferative/secretory responses of cholangiocytes. In the liver, cholangiocytes (and, to a lesser extent, BDL hepatocytes) expressed AANAT. AANAT expression and melatonin secretion (1) increased in BDL, compared to normal rats and BDL rats treated with melatonin, and (2) decreased in normal and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. The decrease in AANAT expression, and subsequent lower melatonin secretion by cholangiocytes, was associated with increased biliary proliferation and increased SR, CFTR, and Cl(-) /HCO 3- AE2 expression. Overexpression of AANAT in cholangiocyte cell lines decreased the basal proliferative rate and expression of SR, CFTR, and Cl(-) /HCO 3- AE2 and ablated secretin-stimulated biliary secretion in these cells., Conclusion: Local modulation of melatonin synthesis may be important for management of the balance between biliary proliferation/damage that is typical of cholangiopathies. (HEPATOLOGY 2013)., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Background: In several tumours the endogenous activity of histidine decarboxylase (HDC), the enzyme stimulating histamine synthesis, sustains the autocrine trophic effect of histamine on cancer progression. Cholangiocarcinoma is a biliary cancer with limited treatment options. Histamine interacts with four G-protein coupled receptors, H1-H4 histamine receptors (HRs)., Objective: To determine the effects of histamine stimulation and inhibition of histamine synthesis (by modulation of HDC) on cholangiocarcinoma growth., Methods: In vitro studies were performed using multiple human cholangiocarcinoma lines. The expression levels of the histamine synthetic machinery and HRs were evaluated along with the effects of histamine stimulation and inhibition on cholangiocarcinoma proliferation. A xenograft tumour model was used to measure tumour volume after treatment with histamine or inhibition of histamine synthesis by manipulation of HDC. Vascular endothelial growth factor (VEGF) expression was measured in cholangiocarcinoma cells concomitant with the evaluation of the expression of CD31 in endothelial cells in the tumour microenvironment., Results: Cholangiocarcinoma cells display (1) enhanced HDC and decreased monoamine oxidase B expression resulting in increased histamine secretion; and (2) increased expression of H1-H4 HRs. Inhibition of HDC and antagonising H1HR decreased histamine secretion in Mz-ChA-1 cells. Long-term treatment with histamine increased proliferation and VEGF expression in cholangiocarcinoma that was blocked by HDC inhibitor and the H1HR antagonist. In nude mice, histamine increased tumour growth (up to 25%) and VEGF expression whereas inhibition of histamine synthesis (by reduction of HDC) ablated the autocrine stimulation of histamine on tumour growth (~80%) and VEGF expression. No changes in angiogenesis (evaluated by changes in CD31 immunoreactivity) were detected in the in vivo treatment groups., Conclusion: The novel concept that an autocrine loop (consisting of enhanced histamine synthesis by HDC) sustains cholangiocarcinoma growth is proposed. Drug targeting of HDC may be important for treatment of patients with cholangiocarcinoma.

No information exists on the role of neuropeptide Y (NPY) in cholangiocarcinoma growth. Therefore, we evaluated the expression and secretion of NPY and its subsequent effects on cholangiocarcinoma growth and invasion. Cholangiocarcinoma cell lines and nonmalignant cholangiocytes were used to assess NPY mRNA expression and protein secretion. NPY expression was assessed by immunohistochemistry in human liver biopsies. Cell proliferation and migration were evaluated in vitro by MTS assays and matrigel invasion chambers, respectively, after treatment with NPY or a neutralizing NPY antibody. The effect of NPY or NPY depletion on tumor growth was assessed in vivo after treatment with NPY or the neutralizing NPY antibody in a xenograft model of cholangiocarcinoma. NPY secretion was upregulated in cholangiocarcinoma compared with normal cholangiocytes. Administration of exogenous NPY decreased proliferation and cell invasion in all cholangiocarcinoma cell lines studied and reduced tumor cell growth in vivo. In vitro, the effects of NPY on proliferation were blocked by specific inhibitors for NPY receptor Y2, but not Y1 or Y5, and were associated with an increase in intracellular d-myo-inositol 1,4,5-trisphosphate and PKCα activation. Blocking of NPY activity using a neutralizing antibody promoted cholangiocarcinoma growth in vitro and in vivo and increased the invasiveness of cholangiocarcinoma in vitro. Increased NPY immunoreactivity in human tumor tissue occurred predominantly in the center of the tumor, with less expression toward the invasion front of the tumor. We demonstrated that NPY expression is upregulated in cholangiocarcinoma, which exerts local control on tumor cell proliferation and invasion. Modulation of NPY secretion may be important for the management of cholangiocarcinoma.

Cholangiocarcinomas arise after the neoplastic transformation of the cholangiocytes that line the intra- and extrahepatic biliary epithelium. Symptoms usually do not present until late in the course of the disease, at which time they are relatively resistant to chemotherapeutic agents and as such are difficult to treat and display a poor prognosis. Because of the relative rarity of this disease, the overall volume of research into the molecular pathophysiology associated with this disease is small compared with other more prevalent tumors. However, the incidence of this devastating cancer is on the rise and renewed efforts to understand the pathogenesis of cholangiocarcinoma is needed to design novel therapeutic strategies to combat this disease. This review summarizes the recent advances into our knowledge and understanding of cholangiocarcinoma and highlights potential novel therapeutic strategies that may prove useful to treat this deadly disease.

Background: Polycystin-1 and -2 (PC-1 and PC-2) are critical components of primary cilia, which act as mechanosensors and drive cell response to injury. PC-1 activation involves the cleavage/processing of PC-1 cytoplasmic tail, driven by regulated intramembrane proteolysis or ubiquitine/proteasome, translocation in the nucleus and activation of transcription factors. Mutations of PC-1 or PC-2 occur in polycystic liver where cholangiocyte proliferation is enhanced., Aim: We evaluated the involvement of PC-1 and PC-2 in modulating cholangiocyte proliferation., Methods: We investigated rat cholangiocytes induced to proliferate by 17beta-oestradiol. Proliferation was evaluated by PCNA immunoblotting or [(3)H]-thymidine incorporation into DNA. PC-1 silencing was performed by siRNA, while inhibition of regulated intramembrane proteolysis or proteasome by gamma-secretase inhibitor, leupeptin or MG115., Results: Cholangiocyte proliferation was associated with decreased PC-1 and PC-2 expression, which was inversely correlated with enhanced PCNA. The selective silencing of PC-1 induced activation of cholangiocyte proliferation in association with decreased PC-1 expression. Two different regulated intramembrane proteolysis inhibitors, gamma-secretase-inhibitor and leupeptin, and the proteasome inhibitor, MG115, abolished the 17beta-oestradiol proliferative effect., Conclusions: PC-1 and PC-2 play a major role as modulators of cholangiocyte proliferation suggesting that primary cilia may act as sensors of cell injury driving, when activated, a proliferative cholangiocyte response to trigger the reparative processes., (Copyright 2009 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Histamine regulates functions via four receptors (HRH1, HRH2, HRH3, and HRH4). The d-myo-inositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/protein kinase C (PKC)/mitogen-activated protein kinase pathway regulates cholangiocarcinoma growth. We evaluated the role of HRH3 in the regulation of cholangiocarcinoma growth. Expression of HRH3 in intrahepatic and extrahepatic cell lines, normal cholangiocytes, and human tissue arrays was measured. In Mz-ChA-1 cells stimulated with (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH), we measured (a) cell growth, (b) IP(3) and cyclic AMP levels, and (c) phosphorylation of PKC and mitogen-activated protein kinase isoforms. Localization of PKCalpha was visualized by immunofluorescence in cell smears and immunoblotting for PKCalpha in cytosol and membrane fractions. Following knockdown of PKCalpha, Mz-ChA-1 cells were stimulated with RAMH before evaluating cell growth and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. In vivo experiments were done in BALB/c nude mice. Mice were treated with saline or RAMH for 44 days and tumor volume was measured. Tumors were excised and evaluated for proliferation, apoptosis, and expression of PKCalpha, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF receptor 2, and VEGF receptor 3. HRH3 expression was found in all cells. RAMH inhibited the growth of cholangiocarcinoma cells. RAMH increased IP(3) levels and PKCalpha phosphorylation and decreased ERK1/2 phosphorylation. RAMH induced a shift in the localization of PKCalpha expression from the cytosolic domain into the membrane region of Mz-ChA-1 cells. Silencing of PKCalpha prevented RAMH inhibition of Mz-ChA-1 cell growth and ablated RAMH effects on ERK1/2 phosphorylation. In vivo, RAMH decreased tumor growth and expression of VEGF and its receptors; PKCalpha expression was increased. RAMH inhibits cholangiocarcinoma growth by PKCalpha-dependent ERK1/2 dephosphorylation. Modulation of PKCalpha by histamine receptors may be important in regulating cholangiocarcinoma growth.

Caffeic acid phenethyl ester (CAPE) inhibits the growth of tumor cells and is a known inhibitor of nuclear factor kappa beta (NF-kappaB), 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-kappaB DNA-binding activity was confirmed in nuclear extracts treated with CAPE at 50, 40 and 20 microM. CAPE decreases the expression of NF-kappaB1 (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 microM 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.

Background: Endothelins (ET-1, ET-2, ET-3) are peptides with vasoactive properties interacting with ET(A) and ET(B) receptors. ET-1 inhibits secretin-stimulated ductal secretion (hallmark of cholangiocyte growth) of cholestatic rats by interaction with ET receptors., Aim: The aims of the studies were to evaluate (i) the effect of ET-1 on cholangiocarcinoma growth in Mz-ChA-1 cells and nude mice and (ii) whether ET-1 regulation of cholangiocarcinoma growth is associated with changes in the expression of vascular endothelial growth factor-A (VEGF-A), VEGF-C, VEGF receptor-2 (VEGFR-2) and VEGFR-3., Methods: We determined the expression of ET(A) and ET(B) receptors on normal and malignant (Mz-ChA-1) cholangiocytes and human cholangiocarcinoma tissue and the effect of ET-1 on the proliferation and expression of VEGF-A, VEGF-C (regulators of tumour angiogenesis) and its receptors, VEGFR-2 and VEGFR-3, in Mz-ChA-1 cells. In vivo, Mz-ChA-1 cells were injected into the flanks of athymic mice and injections of ET-1 or saline into the tumours were performed daily. The effect of ET-1 on tumour size, cell proliferation, apoptosis, collagen quantity and the expression of VEGF-A and VEGF-C and VEGFR-2 and VEGFR-3 were measured after 73 days., Results: Higher expression of ET(A) and ET(B) was observed in malignant compared with normal cholangiocytes. ET-1 inhibited proliferation and VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression of Mz-ChA-1 cells. Chronic ET-1 treatment decreased tumour volume, tumour cell proliferation and VEGF-A and VEGF-C expression but increased apoptosis and collagen tissue deposition compared with controls., Conclusions: Modulation of VEGF-A and VEGF-C (by ET-1) may be important for managing cholangiocarcinoma growth.

Background: Estrogens may induce the proliferation of neoplastic cells by activating neo-angiogenesis., Aim: To evaluate the effect of estrogens on the expression of vascular endothelial growth factor (VEGF) and related receptors (VEGF-R) in human cholangiocarcinoma and the role played by VEGF in mediating the proliferative effects of estrogens., Methods: Seven biopsies of intra-hepatic cholangiocarcinoma and the HuH-28 cell lines were investigated. Cell proliferation was measured by both PCNA Western blot and MTS proliferation assay., Results: By immunohistochemistry, biopsies of human cholangiocarcinoma stained positively for VEGF-A and VEGF-C and related receptors. HuH-28 cells expressed VEGF-A, -C, and VEGFR-1, -2, -3 and, their protein level was enhanced by 17beta-estradiol in association with the stimulation of cell proliferation. 17beta-Estradiol-stimulated proliferation of HuH-28 cells was blocked by 70% by VEGF-TRAP, a receptor-based VEGF inhibitor. 17beta-Estradiol induced the secretion of VEGF in the supernatant of HuH-28 cells. The stimulatory effect of 17beta-estradiol on the protein expression of VEGF-A, VEGF-C and VEGFR-1, -2, -3 was blocked by antagonists of ER (Ici182,780) or insulin-like growth factor 1-receptor (alphaIR3)., Conclusions: With the limitations of experiments performed in a cell line, our study indicates that VEGF plays a major role in mediating the proliferative effects of estrogens on human cholangiocarcinoma.

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.

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.

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.

Cholangiocarcinoma is a rare cancer originating from the neoplastic transformation of the epithelial cells (i.e. cholangiocytes) that line the biliary tract. The prognosis for patients with cholangiocarcinoma is grim due to lack of viable treatment options. The increase in world-wide incidence and mortality from cholangiocarcinoma highlights the importance of understanding the intracellular mechanisms that trigger the neoplastic transformation of cholangiocytes and the growth of biliary cancers. The purpose of the following review is to address what has been learned over the past decade concerning the molecular basis of cholangiocarcinogenesis. The material presented is divided into two sections: (1) mechanisms regulating neoplastic transformation of cholangiocytes; and (2) factors regulating cholangiocarcinoma growth. An understanding of the growth regulatory mechanisms of cholangiocarcinoma will lead to the identification of therapeutic targets for this devastating cancer.

Cholangiocarcinoma continues to be a challenging disease to treat. Systemic therapy is used in unresectable disease, disease progression after surgery, and in the palliative setting. Unfortunately, results of multiple phase II trials have rarely yielded positive results. As data on the molecular carcinogenesis of cholangiocarcinoma is developing, we are more able to understand the disease process and can use this understanding to create unique targeted therapies. We reviewed the role of c-Met/hepatocyte growth factor (HGF) in the development of cholangiocarcinoma. Furthermore, we explored the use of the c-Met guided cascade as a target to treat cholangiocarcinoma. We reviewed the current use and options for future development of c-Met agents to treat this disease.

Secretin stimulates ductal secretion by activation of cAMP --> PKA --> CFTR --> Cl(-)/HCO(3)(-) exchanger in cholangiocytes. We evaluated the expression of alpha(2A)-, alpha(2B)-, and alpha(2C)-adrenergic receptors in cholangiocytes and the effects of the selective alpha(2)-adrenergic agonist UK 14,304, on basal and secretin-stimulated ductal secretion. In normal rats, we evaluated the effect of UK 14,304 on bile and bicarbonate secretion. In bile duct-ligated (BDL) rats, we evaluated the effect of UK 14,304 on basal and secretin-stimulated 1) bile and bicarbonate secretion; 2) duct secretion in intrahepatic bile duct units (IBDU) in the absence or presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)/H(+) exchanger isoform NHE3; and 3) cAMP levels, PKA activity, Cl(-) efflux, and Cl(-)/HCO(3)(-) exchanger activity in purified cholangiocytes. alpha(2)-Adrenergic receptors were expressed by all cholangiocytes in normal and BDL liver sections. UK 14,304 did not change bile and bicarbonate secretion of normal rats. In BDL rats, UK 14,304 inhibited secretin-stimulated 1) bile and bicarbonate secretion, 2) expansion of IBDU luminal spaces, and 3) cAMP levels, PKA activity, Cl(-) efflux, and Cl(-)/HCO(3)(-) exchanger activity in cholangiocytes. There was decreased lumen size after removal of secretin in IBDU pretreated with UK 14,304. In IBDU pretreated with EIPA, there was no significant decrease in luminal space after removal of secretin in either the absence or presence of UK 14,304. The inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3. alpha(2)-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases.

Biogenic amines, such as serotonin, histamine, dopamine, and the catecholamines epinephrine and norepinephrine, regulate a multitude of cellular responses. A great deal of effort has been invested into understanding the effects of these molecules and their corresponding receptor systems on cholangiocyte secretion, apoptosis, and growth. This review summarizes the results of these efforts and highlights the importance of these regulatory molecules on the physiology and pathophysiology of cholangiocytes.

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 Galpha(i/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)-(alpha)-(-)-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.

In the last 15 years, the intrahepatic biliary tree has become the object of extensive studies, which highlighted the extraordinary biologic properties of cholangiocytes involved in bile formation, proliferation, injury repair, fibrosis, angiogenesis, and regulation of blood flow. Proliferation is a "typical" property of cholangiocytes and is key as a mechanism of repair responsible for maintaining the integrity of the biliary tree. Cholangiocyte proliferation occurs virtually in all pathologic conditions of liver injury where it is associated with inflammation, regeneration, and repair, thus conditioning the evolution of liver damage. Interestingly, proliferating cholangiocytes acquire the phenotype of neuroendocrine cells, and secrete different cytokines, growth factors, neuropeptides, and hormones, which represent potential mechanisms for cross talk with other liver cells. Many studies suggest the generation of a neuroendocrine compartment in the injured liver, mostly constituted by cells with cholangiocyte features, which functionally conditions the progression of liver disease. These insights on cholangiocyte pathophysiology will provide new potential strategies for the management of chronic liver diseases. The purpose of this review is to summarize the recent findings on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte biology.

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.

The scientific framework concerning estrogen effects on different tissues has expanded enormously during the last decades, when estrogen receptor (ER) subtypes were identified. Estrogens are not only essential for the female reproductive system, but they also control fundamental functions in other tissues including the cardiovascular system, bone, brain and liver. Recently, estrogens have been shown to target the biliary tree, where they modulate the proliferative and secretory activities of cholangiocytes, the epithelial cells lining bile ducts. By acting on both estrogen receptors (ER-alpha) and (ER-beta) subtypes, and by activating either genomic or non-genomic pathways, estrogens play a key role in the complex loop of growth factors and cytokines, which modulates the proliferative response of cholangiocytes to damage. Specifically, estrogens activate intracellular signalling cascades [ERK(1/2) (extracellular regulated kinases (1/2), PI3- kinase/AKT (phosphatidylinositol-3' kinase/AKT)] typical of growth factors such as insulin like growth factor (IGF1), nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), thus potentiating their action. In addition, estrogens stimulate the secretion of different growth factors in proliferating cholangiocytes. This review specifically deals with the recent advances related to the role and mechanisms by which estrogens modulate cholangiocyte functions in normal and pathological conditions.

Background/aims: We evaluated the role and mechanisms by which the GH/IGF1 axis modulates cholangiocyte proliferation., Methods: GH-receptors (GH-R), IGF1, IGFBP3 (binding protein 3), IGF1-R and receptor substrates (IRS) were evaluated in cholangiocytes of normal or bile duct-ligated (BDL) rat livers. The effects of GH and IGF1 on proliferation of normal quiescent cholangiocytes and the transduction pathways involved were investigated., Results: IGF1, GH-R, IGF1-R, IRS-1/2 were expressed in normal cholangiocytes and overexpressed in cholangiocytes proliferating after BDL which also secrete IGF1 in a higher amount than normal cells. IGFBP3, which may counter-regulate IGF1 effects, was decreased in BDL cholangiocytes. IGF1 promoted cholangiocyte proliferation in association with overexpression of p-IGF1R, IRS1, IRS-2, p-ERK1/2 and p-AKT. GH induced IGF1 expression and release in isolated cholangiocytes, and reproduced the effects of IGF1 but GH effects were abolished by IGF1-R blocking antibody, suggesting IGF1 as a mediator of GH. Finally, IGF1 and 17beta-estradiol reciprocally potentiated their proliferative effects on cholangiocytes, and by interacting at both receptor and post-receptor levels., Conclusions: Cholangiocytes respond to GH with production and release of IGF1 that modulates cell proliferation by transduction pathways involving IGF1-R, IRS1/2 and both ERK and PI3-kinase pathways. The biliary epithelium is a target of GH/IGF1 liver axis.

Background: The peribiliary plexus (PBP) plays a fundamental role in supporting the functions of the biliary epithelium. After common bile duct ligation (BDL) progressive PBP proliferation is demonstrated. We have, recently, demonstrated that the biliary epithelium express Vascular Endothelial Growth Factor (VEGF), both subtype -A and -B and VEGF receptors. Taking in consideration the wide extension of PBP during BDL, aim of our study is to investigate the role of VEGF in stimulating angiogenesis and also in the modulation of epithelial cells proliferation., Material and Methods: Experimental studies were performed by evaluating the effects of: a) endogenous VEGF neutralization by chronic administration of anti VEGF-C antibody on cholangiocyte proliferation in BDL rats and; b) the hepatic artery ligation (HAL) immediately after BDL followed by treatment (7 days) with a recombinant of VEGF-A (administered through IP implanted minipumps) on cholangiocyte proliferative activities., Results: Both administration of antiVEGF-C antibody and HAL decreases cholangiocyte proliferation. The decrease of cholangiocyte proliferation was associated with depressed VEGF-A protein expression. The administration of rVEGF-A to BDL, hepatic artery ligated rats prevented the decrease of cholangiocyte proliferation and VEGF-A expression as compared to BDL control rats., Conclusion: These data suggest that VEGF-C modulates the proliferative activities of cholangiocytes in experimental cholestasis and that circulating factors (i.e., VEGF) in the blood supply of the intra-hepatic biliary epithelium, play an important role in the balance between cholangiocyte proliferation/loss.

Background & Aims: We evaluated the expression of neurotrophins in rat cholangiocytes and the role and mechanisms by which nerve growth factor (NGF) modulates cholangiocyte proliferation., Methods: The expression of neurotrophins and their receptors was investigated by immunohistochemistry in liver sections and reverse-transcription polymerase chain reaction and immunoblots in isolated cholangiocytes. In vitro, the effect of NGF on cholangiocyte proliferation and signal transduction was investigated by immunoblotting for proliferating cell nuclear antigen, phosphorylated AKT (p-AKT), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated c-jun-N-terminal kinase, and phosphorylated p38. In vivo, rats that had undergone bile duct ligation (BDL) were treated with an anti-NGF antibody to immunoneutralize NGF and bile duct mass, proliferation, apoptosis, and inflammation were investigated by immunohistochemistry., Results: NGF and its TrkA receptor were expressed by normal rat cholangiocytes and up-regulated following BDL. Cholangiocytes secrete NGF, and secretion is increased in proliferating BDL cholangiocytes. In vitro, NGF stimulated cholangiocyte proliferation, which was associated with enhanced p-AKT and p-ERK1/2 expression. NGF proliferation in vitro was partially blocked by the MEK inhibitor (UO126) and completely ablated by the phosphatidylinositol 3-kinase inhibitor (wortmannin). In vitro, NGF and estrogens have an additive effect on cholangiocyte proliferation by acting on phosphorylated TrkA and p-ERK1/2. In vivo, immunoneutralization of NGF decreased bile duct mass in BDL rats, which was associated with depressed proliferation and enhanced apoptosis and with increased portal inflammation., Conclusions: Cholangiocytes secrete NGF and express NGF receptors. NGF induces cholangiocyte proliferation by activating the ERK and, predominantly, the phosphatidylinositol 3-kinase pathway and exerts an additive effect in combination with estrogens on proliferation.

Tauroursodeoxychate (TUDCA) is used for the treatment of cholangiopathies including primary sclerosing cholangitis, which is considered the primary risk factor for cholangiocarcinoma. The effect of TUDCA on cholangiocarcinoma growth is unknown. We evaluated the role of TUDCA in the regulation of growth of the cholangiocarcinoma cell line Mz-ChA-1. TUDCA inhibited the growth of Mz-ChA-1 cells in concentration- and time-dependent manners. TUDCA inhibition of cholangiocarcinoma growth was blocked by BAPTA-AM, an intracellular Ca(2+) concentration ([Ca(2+)](i)) chelator, and H7, a PKC-alpha inhibitor. TUDCA increased [Ca(2+)](i) and membrane translocation of the Ca(2+)-dependent PKC-alpha in Mz-ChA-1 cells. TUDCA inhibited the activity of MAPK, and this inhibitory effect of TUDCA was abrogated by BAPTA-AM and H7. TUDCA did not alter the activity of Raf-1 and B-Raf and the phosphorylation of MAPK p38 and JNK/stress-activated protein kinase. TUDCA inhibits Mz-ChA-1 growth through a signal-transduction pathway involving MAPK p42/44 and PKC-alpha but independent from Raf proteins and MAPK p38 and JNK/stress-activated protein kinases. TUDCA may be important for the treatment of cholangiocarcinoma.

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., Methods: Primary cultures of small and large cholangiocytes were isolated from normal mice (BALB/c), and immortalized by the introduction of the SV40 large T antigen gene. After cloning, small and large cholangiocyte cell lines were established. Their characteristic features were confirmed by electron microscopy (EM) and measurement of transepithelial electrical resistance (TER), and secretin-stimulated cAMP levels. Isolated total RNAs were hybridized with microarrays (Atlas Glass Array Mouse 1.0 and 3.8), which detects 4850 cDNA expressions. After hybridization, the fluorescent signals were scanned by a GenePix fluorescent scanner and analyzed using ArrayGauge software., Results: EM, TER and secretin-stimulated cAMP synthesis are consistent with the concept that small and large immortalized cholangiocytes originate from small and large ducts, respectively. When a cut-off value at the expression signal difference of 3.0 times was employed, 230 cDNAs among 4850 cDNAs (4.74%) were differentially expressed between small and large cholangiocytes. Of these 230 cDNAs, aquaporin 8, IL-2 receptor beta chain and caspase 9 were more strongly expressed by large cholangiocytes., Conclusions: Microarray successfully displayed characteristic differential cDNA expression between small and large cholangiocytes. This technique provides molecular information, which further supports our hypothesis that small and large bile ducts have different functions.

We sought to develop a cholangiocyte cell culture system that has preservation of receptors, transporters, and channels involved in secretin-induced secretion. Isolated bile duct fragments, obtained by enzyme perfusion of normal rat liver, were seeded on collagen and maintained in culture up to 18 wk. Cholangiocyte purity was assessed by staining for gamma-glutamyl transpeptidase (gamma-GT) and cytokeratin-19 (CK-19). We determined gene expression for secretin receptor (SR), cystic fibrosis transmembrane conductance regulator, Cl(-)/HCO(3)(-) exchanger, secretin-stimulated cAMP synthesis, Cl(-)/HCO(3) exchanger activity, secretin-stimulated Cl(-) efflux, and apical membrane-directed secretion in polarized cells grown on tissue culture inserts. Cultured cholangiocytes were all gamma-GT and CK-19 positive. The cells expressed SR and Cl(-)/HCO(3)(-) exchanger, and secretin-stimulated cAMP synthesis, Cl(-)/HCO(3)(-) exchanger activity, and Cl(-) efflux were similar to freshly isolated cholangiocytes. Forskolin (10(-4) M) induced fluid accumulation in the apical chamber of tissue culture inserts. In conclusion, we have developed a novel cholangiocyte line that has persistent HCO(3)(-), Cl(-), and fluid transport functions. This cell system should be useful to investigators who study cholangiocyte secretion.

The aim of this study was to determine whether taurocholate prevents vagotomy-induced cholangiocyte apoptosis. After bile duct ligation (BDL) + vagotomy, rats were fed taurocholate for 1 wk in the absence or presence of wortmannin. Caspase involvement was evaluated by measurement of caspase 8, 9, and 3 activities. Proliferation was determined by morphometry and PCNA immunoblots. Changes in phosphatidylinositol 3-kinase (PI3-kinase) activity were estimated by the expression of the phosphorylated Akt protein. Apically located Na(+)-dependent bile acid transporter (ABAT) expression and activity were evaluated by immunoblots and [(3)H]taurocholate uptake, respectively. Cholangiocyte apoptosis increased, whereas proliferation decreased in BDL + vagotomy rats. Taurocholate feeding prevented vagotomy effects on cholangiocyte functions, which were abolished by wortmannin. ABAT expression and activity as well as phosphorylated Akt protein expression were reduced by vagotomy but restored by taurocholate. The activities of caspase 8, 9, and 3 increased in BDL + vagotomy rats but were restored by taurocholate. The protective effect of taurocholate was associated with maintenance of ABAT activity, downregulation of caspase 8, 9, and 3, and activation of PI3-kinase. Bile acids are important in modulating cholangiocyte proliferation in denervated livers.

The objective of this article is to summarize the findings related to the notion that cholangiocytes, within small and large intrahepatic ducts, are heterogeneous regarding (1) morphology; (2) secretion in response to hormones and peptides and to bile acids; and (3) proliferation in response to injury or toxins, including bile duct ligation (BDL), acute carbon tetrachloride (CCl 4 ) administration, chronic feeding of bile salts (i.e., taurocholate [TC] or taurolithocholate [TLC]) or alpha-naphthylisothiocyanate (ANIT). After an overview of the morphology of the biliary epithelium, we provide a summary of cholangiocyte function, the in vivo models, and the in vitro experimental tools (i.e., small and large cholangiocytes or small and large intrahepatic bile duct units [IBDU]), which allowed us to demonstrate cholangiocyte heterogeneity. After a discussion on the receptors, transporters, and channels that are heterogeneously expressed by cholangiocytes, we discuss the different-sized ducts that differentially respond to injury and toxins. Finally, we review the human diseases that selectively target specific-sized ducts.

The aim of this study was to explore the intracellular signaling pathways involved in the stimulatory effects of estrogens on cholangiocyte proliferation. We investigated the tyrosine kinase-receptor pathway by evaluating the protein expression of total and phosphorylated mitogen-activated protein kinase (MAPK) isoform p44/p42 (e.g., extracellular signal-regulated kinase [ERK]1/2), the steroid-receptor coactivator Src and Shc (Src-homology/collagen protein). The study was performed in 3-week-old bile duct-ligated (BDL) rats, BDL rats treated with the antiestrogens, tamoxifen or Ici 182,780, and normal control rats. Proliferation was also evaluated in normal purified cholangiocytes treated with 17 beta estradiol in the presence or absence of tamoxifen, Ici 182,780, ERK, or Src inhibitors. After bile duct ligation, cholangiocyte proliferation was associated with a marked immunohistochemical nuclear positivity for phosphorylated (p)-ERK1/2, which was inhibited by in vivo treatment with tamoxifen or Ici 182,780. Protein expression of total and p-ERK1/2, and Shc in cholangiocytes isolated from BDL rats was markedly increased compared with controls and was inhibited by in vivo treatment with antiestrogens. In vitro, 17 beta estradiol-induced proliferation of isolated normal cholangiocyte was associated with increased (P <.01) protein expression of p-ERK1/2, Src, and Shc. Specific inhibitors of ER (Ici 182,780), ERK (U0125), and Src (PP2) inhibited in vitro 17 beta estradiol-induced cholangiocyte proliferation. In conclusion, this study showed that estrogens induced cholangiocyte proliferation by activating the Src/Shc/ERK pathway. This might suggest that pharmacologic modulation of ER, ERK, and/or Src could be proposed for the treatment of human pathology characterized by dysregulation of cholangiocyte proliferation.

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., (Copyright 2002 Elsevier Science Ireland Ltd.)

Growth factor signaling, mediated by the mitogen-activated protein kinase (MAPK) cascade, induces cell mitosis. Adenosine 3',5'-monophosphate (cAMP) may inhibit or stimulate mitosis (depending on the cell type) through the activation of MAPK and Raf proteins. Among Raf proteins, Raf-1 and B-Raf differentially regulate mitosis. Our aims were to evaluate the role and mechanisms of action of the alpha(2)-adrenergic agonist UK14,304 in the regulation of growth of the human cholangiocarcinoma cell line Mz-ChA-1. Immunocytochemistry and immunoblotting for alpha(2A)-, alpha(2B)-, or alpha(2C)-adrenergic receptor subtypes showed positive reaction in Mz-ChA-1 cells. We found that physiological concentrations of UK14,304 increased cAMP levels and inhibited proliferation and MAPK activity in Mz-ChA-1 cells. Mz-ChA-1 cells expressed Raf-1 and B-Raf. Epidermal growth factor (EGF) immediately and transiently stimulated Raf-1 activity, whereas B-Raf activity was increased with prolonged EGF stimulation. EGF-stimulated Raf-1 and B-Raf activities were both inhibited by UK14,304. UK14,304 did not affect Ras activity. In Mz-ChA-1 cells, alpha(2)-adrenoreceptor stimulation causes up-regulation of cAMP, which inhibits EGF-induced MAPK activity through an acute increase of Raf-1 and sustained activation of B-Raf. In conclusion, because alpha(2)-AR inhibition of growth occurred downstream of Ras, adrenergic stimulation or other stimulants of cAMP may overcome the Ras mutations and offer a new therapeutic approach for patients with cholangiocarcinoma.

Background & Aims: We investigated the expression of glucocorticoid receptors (GcRs) in the intrahepatic biliary epithelium and the role of corticosteroids in the regulation of cholangiocyte secretion., Methods: GcR was studied by immunohistochemistry, reverse-transcription polymerase chain reaction, and Western blots. The effects of dexamethasone and budesonide on biliary bicarbonate excretion and H+/HCO3- transport processes were investigated in bile fistula rats, isolated intrahepatic bile duct units (IBDUs), and purified cholangiocytes., Results: GcRs were expressed by rat cholangiocytes. Although acute administration of corticosteroids showed no effect, treatment for 2 days with dexamethasone or budesonide increased (P < 0.05) biliary bicarbonate concentration and secretion, which were blocked by the specific GcR antagonist, RU-486. IBDUs isolated from rats treated with dexamethasone or budesonide showed an increased (P < 0.05) activity of the Na+/H+ exchanger (NHE1 isoform) and Cl-/HCO3- exchanger (AE2 member), which was blocked by RU-486. Protein expression of NHE1 and AE2 and messenger RNA for NH1 but not AE2 were increased (P < 0.05) in isolated cholangiocytes by dexamethasone treatment., Conclusions: The intrahepatic biliary epithelium expresses GcR and responds to corticosteroids by increasing bicarbonate excretion in bile. This is caused by corticosteroid-induced enhanced activities and protein expression of transport processes driving bicarbonate excretion in the biliary epithelium.

The objective of this review article is to discuss the role of secretin and its receptor in the regulation of the secretory activity of intrahepatic bile duct epithelial cells (i.e., cholangiocytes). After a brief overview of cholangiocyte functions, we provide an historical background for the role of secretin and its receptor in the regulation of ductal secretion. We review the newly developed experimental in vivo and in vitro tools, which lead to understanding of the mechanisms of secretin regulation of cholangiocyte functions. After a description of the intracellular mechanisms by which secretin stimulates ductal secretion, we discuss the heterogeneous responses of different-sized intrahepatic bile ducts to gastrointestinal hormones. Furthermore, we outline the role of a number of cooperative factors (e.g., nerves, alkaline phosphatase, gastrointestinal hormones, neuropeptides, and bile acids) in the regulation of secretin-stimulated ductal secretion. Finally, we discuss other factors that may also play an important role in the regulation of secretin-stimulated ductal secretion.

Intrahepatic bile duct epithelial cells (i.e., cholangiocytes) are the target cells of chronic cholestatic liver diseases (i.e., cholangiopathies), which makes these cells of great interest to clinical hepatologists. This review will focus on "typical" cholangiocyte proliferation, whereas "atypical" (extension of cholangiocyte proliferation into parenchyma), and premalignant "oval" cell proliferation are reviewed elsewhere. The bile duct ligated (BDL) rat model, where most of the known mechanisms of cholangiocyte proliferation have been illustrated, was the first and remains the prototype animal model for "typical" cholangiocyte proliferation. Following a short overview of cholangiocyte functions, we briefly discuss the: (i) in vivo models [i.e., BDL (Fig. 1 and 4), chronic alpha-naphthylisothiocyanate (ANIT) or bile acid feeding (Fig. 2), acute carbon tetrachloride (CCl4) feeding and partial hepatectomy; and (ii) in vitro experimental tools [e.g., purified cholangiocytes and isolated intrahepatic bile duct units (IBDU)] that are key to the understanding of the mechanisms of "typical" cholangiocyte growth. In the second part of the review, we discuss a number of potential factors or conditions [e.g., gastrointestinal hormones, nerves, estrogens, blood supply, and growth factors] as well as the intracellular mechanisms [e.g., adenosine 3',5'-monophosphate (cAMP), and protein kinase C (PKC)] that may regulate "typical" cholangiocyte hyperplasia.

Background & Aims: We investigated the expression of estrogen receptor (ER) alpha and beta subtypes in cholangiocytes of normal and bile duct-ligated (BDL) rats and evaluated the role and mechanisms of estrogens in the modulation of cholangiocyte proliferation., Methods: ER-alpha and ER-beta were analyzed by immunohistochemistry, reverse-transcription polymerase chain reaction, and Western blotting in normal and BDL rats. The effects of the ER antagonists tamoxifen and ICI 182,780 on cholangiocyte proliferation were evaluated., Results: Cholangiocytes expressed both ER-alpha and ER-beta subtypes, whereas hepatocytes expressed only ER-alpha. In association with a marked cholangiocyte proliferation and with enhanced estradiol serum levels, the immunoreactivity for ER-alpha involved a 3-fold higher percentage of cholangiocytes in 3-week BDL than in normal rats; immunoreactivity for ER-beta showed a 30-fold increase. Western blot analysis showed that during BDL, the total amount of ER-beta in cholangiocytes was markedly increased (5-fold), whereas that of ER-alpha decreased slightly (-25%). Treatment with tamoxifen or ICI 182,780 of 3-week BDL rats inhibited cholangiocyte proliferation and induced overexpression of Fas antigen and apoptosis in cholangiocytes. In vitro, 17 beta estradiol stimulated proliferation of cholangiocyte, an effect blocked to the same extent by tamoxifen or ICI 182,780., Conclusions: This study suggests that estrogens and their receptors play a role in the modulation of cholangiocyte proliferation.

Bile duct damage and/or loss is limited to a range of duct sizes in cholangiopathies. We tested the hypothesis that CCl4 damages 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. CCl4 induces selective apoptosis of large ducts associated with loss of large cholangiocyte proliferation and secretion.

The aim of this study was to develop a model of selective duct damage restricted to hormone-responsive segments corresponding to the ducts damaged in primary biliary cirrhosis (PBC). Carbon tetrachloride (CCl4) was fed by gavage to rats, and 2, 7, 14, and 28 days later, small and large cholangiocytes were isolated. Apoptosis was determined in situ by morphology and in purified cholangiocytes by assessment of nuclear fragmentation by 4, 6-diamidino-2-phenylindole (DAPI) staining. Cholangiocyte proliferation was evaluated in situ by morphometry of liver sections stained for cytokeratin-19 (CK-19) and by proliferating cellular nuclear antigen (PCNA) staining in liver sections and in purified cholangiocytes by PCNA gene expression. Ductal secretion was assessed by measurement of secretin receptor (SR) gene expression and secretin-induced cyclic adenosine 3',5'-monophosphate (cAMP) synthesis and secretin-induced choleresis. Two days after CCl4 administration, there was an increased number of small ducts, but a reduction of large ducts. Apoptosis, observed only in large ducts, was associated with decreased DNA synthesis and ductal secretion. Conversely, small cholangiocytes expressed de novo the SR gene and secretin-stimulated cAMP synthesis 2 days after CCl4 treatment. Proliferation of large cholangiocytes was delayed until 7 days, which was associated with a transient increase in ductal secretion in vivo. CCl4 effects on cholangiocytes were reversed by day 28. CCl4 treatment causes a decrease in large duct mass as a result of a higher rate of apoptosis and absence of initial proliferation in large cholangiocytes. These processes were concomitant with a decrease of ductal secretion in large cholangiocytes. Small cholangiocytes appear resistant to CCl4-induced apoptosis, and proliferate and transiently compensate for loss of proliferative and secretory activity of large cholangiocytes.

Background & Aims: We have shown that taurocholate (TC) and taurolithocholate (TLC) interact in vitro with normal cholangiocytes, increasing DNA synthesis, secretin receptor (SR) gene expression, and adenosine 3',5'-cyclic monophosphate (cAMP) synthesis. To further extend these in vitro studies, we tested the hypothesis that bile acids (BAs) directly stimulate cholangiocyte proliferation and secretion in vivo., Methods: After feeding with TC or TLC (1% for 1-4 weeks), we assessed the following in vivo: (1) ductal proliferation by both morphometry and immunohistochemistry for proliferating cell nuclear antigen (PCNA) and measurement of [3H]thymidine incorporation; and (2) the effect of secretin on bile secretion and bicarbonate secretion in vivo. Genetic expression of H3-histone and SR and intracellular cAMP levels were measured in isolated cholangiocytes., Results: After BA feeding, there was an increased number of PCNA-positive cholangiocytes and an increased number of ducts compared with control rats. [3H]Thymidine incorporation, absent in control cholangiocytes, was increased in cholangiocytes from BA-fed rats. In BA-fed rats, there was increased SR gene expression (approximately 2.5-fold) and secretin-induced cAMP levels (approximately 3.0-fold) in cholangiocytes, which was associated with de novo secretin-stimulated bile flow and bicarbonate secretion., Conclusions: These data indicate that elevated BA levels stimulate ductal secretion and cholangiocyte proliferation.

We studied the expression of endothelin-1 (ET-1) receptors (ETA and ETB) and the effects of ET-1 on cholangiocyte secretion. The effects of ET-1 on cholangiocyte secretion were assessed in normal and bile duct-ligated (BDL) rats by measuring 1) basal and secretin-induced choleresis in vivo, 2) secretin receptor gene expression and cAMP levels in small and large cholangiocytes, and 3) luminal expansion in response to secretin in intrahepatic bile duct units (IBDU). ETA and ETB receptors were expressed by small and large cholangiocytes. ET-1 had no effect on basal bile flow or bicarbonate secretion in normal or BDL rats but decreased secretin-induced bicarbonate-rich choleresis in BDL rats. ET-1 decreased secretin receptor gene expression and secretin-stimulated cAMP synthesis in large cholangiocytes and secretin-induced luminal expansion in IBDU from normal or BDL rats. The inhibitory effects of ET-1 on secretin-induced cAMP synthesis and luminal duct expansion were blocked by specific inhibitors of the ETA (BQ-610) receptor. ET-1 inhibits secretin-induced ductal secretion by decreasing secretin receptor and cAMP synthesis, two important determinants of ductal secretion.