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Background & Aims: Primary biliary cholangitis (PBC) is a chronic cholangiopathy characterised by immuno-mediated injury of interlobular bile ducts leading to intrahepatic cholestasis and progressive liver fibrosis. PBC histology is characterised by portal inflammation, progressive fibrosis, ductopenia, and the appearance of the so-called ductular reaction. The aim of the present study was to investigate the pathogenetic relevance of ductular reaction in PBC. Methods: Liver biopsies were collected from naïve people with PBC (N = 87). Clinical–serological parameters were obtained at diagnosis and after 1 year of ursodeoxycholic acid (UDCA) treatment. Histological staging was performed on all slides according to multiple scoring systems and criteria for PBC. Liver samples were obtained from Mdr2−/− mice treated with or without UDCA. Samples were processed for histology, immunohistochemistry, and immunofluorescence. Results: Ductular reaction in people with PBC correlated with the disease stage and liver fibrosis, but not with disease activity; an extensive ductular reaction correlated with serum alkaline phosphatase levels at diagnosis, response to UDCA, and individuals’ estimated survival, independently from other histological parameters, including disease stage. In people with PBC, reactive ductules were associated with the establishment of junctions with bile canaliculi and with fibrogenetic cell activation. Consistently, in a mouse model of intrahepatic cholestasis, UDCA treatment was effective in reducing ductular reaction and fibrosis and increasing ductular–canalicular junctions. Conclusions: Extensive ductular reaction outlines a severe histologic phenotype in PBC and is associated with an inadequate therapy response and a worse estimated prognosis. Lay summary: In people affected by primary biliary cholangitis (PBC), the histological appearance of extensive ductular reaction identifies individuals at risk of progressive fibrosis. Ductular reaction at dia

Background and Aims: Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models. Approach and Results: While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2–/–) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2–/–- mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2–/–- mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2–/– mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2–/– mice, respectively. 5HT levels increase in Mdr2–/– mice and in PSC human patients compared to their controls and decrease in serum of Mdr2–/– mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2–/– mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls. Conclusions: Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.

Primary sclerosing cholangitis (PSC) is characterized by increased mast cell (MC) infiltration, biliary damage and hepatic fibrosis. Cholangiocytes secrete stem cell factor (SCF), which is a chemoattractant for c-kit expressed on MCs. We aimed to determine if blocking SCF inhibits MC migration, biliary damage and hepatic fibrosis. Methods: FVB/NJ and Mdr2−/− mice were treated with Mismatch or SCF Vivo-Morpholinos. We measured (i) SCF expression and secretion; (ii) hepatic damage; (iii) MC migration/activation and histamine signaling; (iv) ductular reaction and biliary senescence; and (v) hepatic fibrosis. In human PSC patients, SCF expression and secretion were measured. In vitro, cholangiocytes were evaluated for SCF expression and secretion. Biliary proliferation/senescence was measured in cholangiocytes pretreated with 0.1% BSA or the SCF inhibitor, ISK03. Cultured HSCs were stimulated with cholangiocyte supernatant and activation measured. MC migration was determined with cholangiocytes pretreated with BSA or ISK03 loaded into the bottom of Boyden chambers and MCs into top chamber. Results: Biliary SCF expression and SCF serum levels increase in human PSC. Cholangiocytes, but not hepatocytes, from SCF Mismatch Mdr2−/− mice have increased SCF expression and secretion. Inhibition of SCF in Mdr2−/− mice reduced (i) hepatic damage; (ii) MC migration; (iii) histamine and SCF serum levels; and (iv) ductular reaction/biliary senescence/hepatic fibrosis. In vitro, cholangiocytes express and secrete SCF. Blocking biliary SCF decreased MC migration, biliary proliferation/senescence, and HSC activation. Conclusion: Cholangiocytes secrete increased levels of SCF inducing MC migration, contributing to biliary damage/hepatic fibrosis. Targeting MC infiltration may be an option to ameliorate PSC progression.

Primary Biliary Cholangitis (PBC) is an uncommon, chronic, cholangiopathy of autoimmune origin and unknown etiology characterized by positive anti-mitochondrial autoantibodies (AMA), female preponderance and progression to cirrhosis if left untreated. The diagnosis is based on AMA- or PBC-specific anti-nuclear antibody (ANA)-positivity in the presence of a cholestatic biochemical profile, histologic confirmation being mandatory only in seronegative cases. First-line treatment is ursodeoxycholic acid (UDCA), which is effective in preventing disease progression in about two thirds of the patients. The only approved second-line treatment is obeticholic acid. This article summarizes the most relevant conclusions of a meeting held in Lugano, Switzerland, from September 23rd-25th 2018, gathering basic and clinical scientists with various background from around the world to discuss the latest advances in PBC research. The meeting was dedicated to Ian Mackay, pioneer in the field of autoimmune liver diseases. The role of liver histology needs to be reconsidered: liver pathology consistent with PBC in AMA-positive individuals without biochemical cholestasis is increasingly reported, raising the question as to whether biochemical cholestasis is a reliable disease marker for both clinical practice and trials. The urgent need for new biomarkers, including more accurate markers of cholestasis, was also widely discussed during the meeting. Moreover, new insights in interactions of bile acids with biliary epithelia in PBC provide solid evidence of a role for impaired epithelial protection against potentially toxic hydrophobic bile acids, raising the fundamental question as to whether this bile acid-induced epithelial damage is the cause or the consequence of the autoimmune attack to the biliary epithelium. Strategies are needed to identify difficult-to-treat patients at an early disease stage, when new therapeutic approaches targeting immunologic pathways, in addition to bile acid

Melatonin, a neuroendocrine hormone synthesized by the pineal gland and cholangiocytes, decreases biliary hyperplasia and liver fibrosis during cholestasis-induced biliary injury via melatonin-dependent autocrine signaling through increased biliary arylalkylamine N-acetyltransferase (AANAT) expression and melatonin secretion, downregulation of miR-200b and specific circadian clock genes. Melatonin synthesis is decreased by pinealectomy (PINX) or chronic exposure to light. We evaluated the effect of PINX or prolonged light exposure on melatonin-dependent modulation of biliary damage/ductular reaction/liver fibrosis. Studies were performed in male rats with/without BDL for 1 week with 12:12 h dark/light cycles, continuous light or after 1 week of PINX. The expression of AANAT and melatonin levels in serum and cholangiocyte supernatant were increased in BDL rats, while decreased in BDL rats following PINX or continuous light exposure. BDL-induced increase in serum chemistry, ductular reaction, liver fibrosis, inflammation, angiogenesis and ROS generation were significantly enhanced by PINX or light exposure. Concomitant with enhanced liver fibrosis, we observed increased biliary senescence and enhanced clock genes and miR-200b expression in total liver and cholangiocytes. In vitro, the expression of AANAT, clock genes and miR-200b was increased in PSC human cholangiocyte cell lines (hPSCL). The proliferation and activation of HHStecs (human hepatic stellate cell lines) were increased after stimulating with BDL cholangiocyte supernatant and further enhanced when stimulated with BDL rats following PINX or continuous light exposure cholangiocyte supernatant via intracellular ROS generation. Conclusion: Melatonin plays an important role in the protection of liver against cholestasis-induced damage and ductular reaction.

Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct–ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin-1 receptor (NK-1R). To identify whether SP regulates liver fibrosis during cholestasis, wild-type or NK-1R knockout (NK-1R–/–) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2–/–) mice treated with either an NK-1R antagonist (L-733,060) or saline were used. Additionally, wild-type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK-1R in both BDL and Mdr2–/–mice compared to wild-type mice. Expression of tachykinin precursor 1 and NK-1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK-1R decreased BDL-induced liver fibrosis, and treatment with L-733,060 resulted in decreased liver fibrosis in Mdr2–/–mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor-Î21 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK-1R–/–mice with BDL surgery or Mdr2–/–mice treated with L-733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L-733,060 inhibited SP-induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L-733,060 partially reversed the SP-induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP-induced increase of senescence-related gene expression in cultured cholangiocytes. Conclusion: Collectively, our results demonstrate the regulatory effects of the SP/NK-1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury. (Hepatology 2017

Melatonin therapy or prolonged exposure to complete darkness reduces biliary hyperplasia and liver fibrosis in bile-duct–ligated (BDL) rats; however, no information exists in primary sclerosing cholangitis (PSC). Thus, we aimed to determine the therapeutic effects of prolonged dark therapy or melatonin administration on hepatic fibrosis in the multidrug resistance gene 2–knockout (Mdr2-/-) mouse model of PSC. Melatonin levels, biliary mass, liver fibrosis, angiogenesis and miR-200b expression were evaluated in wild-type and Mdr2-/- mice exposed to darkness or melatonin treatment or in male patients with PSC and healthy controls. Mdr2-/- mice were also treated with miR-200b inhibitor or control before evaluating biliary mass, liver fibrosis, and angiogenesis. After overexpression of arylalkylamine N-acetyltransferase (AANAT; the enzyme regulating melatonin synthesis) or inhibition of miR-200b in cholangiocytes and hepatic stellate cells in vitro, we evaluated angiogenesis and fibrosis gene expression. After exposure to darkness or administration of melatonin, Mdr2-/- mice show elevated serum melatonin levels and inhibition of biliary mass, along with reduction of liver fibrosis and angiogenesis. MicroRNA PCR analysis demonstrated that miR-200b expression increased in Mdr2-/- mice and patients with PSC compared with controls and decreased in Mdr2-/- mice subjected to dark exposure or melatonin treatment. Inhibition of miR-200b in Mdr2-/- ablates biliary proliferation, liver fibrosis, and angiogenesis. In vitro, overexpression of AANAT or inhibition of miR-200b in cholangiocytes and hepatic stellate cells decreased the expression of miR-200b, angiogenesis, and fibrosis genes. Dark therapy or targeting melatonin/miR-200b axis may be important in the management of biliary damage and liver fibrosis in cholangiopathies including PSC.

Biliary-committed progenitor cells (small mouse cholangiocytes; SMCCs) from small bile ducts are more resistant to hepatobiliary injury than large mouse cholangiocytes (LGCCs) from large bile ducts. The definitive endoderm marker, forkhead box A2 (FoxA2), is the key transcriptional factor that regulates cell differentiation and tissue regeneration. Our aim was to characterize the translational role of FoxA2 during cholestatic liver injury. Messenger RNA expression in SMCCs and LGCCs was assessed by polymerase chain reaction (PCR) array analysis. Liver tissues and hepatic stellate cells (HSCs) from primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients were tested by real-time PCR for methylation, senescence, and fibrosis markers. Bile duct ligation (BDL) and multidrug resistance protein 2 (MDR2) knockout mice (MDR2-/-) were used as animal models of cholestatic liver injury with or without healthy transplanted large or small cholangiocytes. We demonstrated that FoxA2 was notably enhanced in murine liver progenitor cells and SMCCs and was silenced in human PSC and PBC liver tissues relative to respective controls that are correlated with the epigenetic methylation enzymes, DNA methyltransferase (DNMT) 1 and DNMT3B. Serum alanine aminotransferase and aspartate aminotransferase levels in nonobese diabetic/severe combined immunodeficiency mice engrafted with SMCCs post-BDL showed significant changes compared to vehicle-treated mice, along with improved liver fibrosis. Enhanced expression of FoxA2 was observed in BDL mouse liver after SMCC cell therapy. Furthermore, activation of fibrosis signaling pathways were observed in BDL/MDR2-/- mouse liver as well as in isolated HSCs by laser capture microdissection, and these signals were recovered along with reduced hepatic senescence and enhanced hepatic stellate cellular senescence after SMCC engraft. Conclusion: The definitive endoderm marker and the positive regulator of biliary development, FoxA

Fibroblast growth factor receptor 2 (FGFR2) might have an important role in the pathogenesis and biology of cholangiocarcinoma (CCA). We examined FGFR expression in CCA tumor specimens obtained from patients and CCA cell lines, and then determined the effects of the novel FGFR inhibitor, derazantinib (DZB; formally, ARQ 087), which is currently in clinical phase 2 trials for intrahepatic CCA. DZB inhibited the growth of CCA cell lines in a dose-dependent manner, and extracellular signal-regulated kinase 1/2 and AKT. It also activated apoptotic and cell growth arrest signaling. DZB reduced the in vitro invasiveness and the expression of key epithelial-mesenchymal transition genes. The in vitro data correlated with the expression of FGFRs in human CCA specimens by immunohistochemistry (FGFR1, 30% positive; and FGFR2, 65% positive) and the CCA cell lines assayed by Western blot analysis. These correlated in vitro studies suggest that FGFR may play an important role in the pathogenesis and biology of CCA. Our findings support the notion that FGFR inhibitors, like DZB, should be further evaluated at the clinical stage as targeted therapy for CCA treatment.

Background & Aims Proliferating cholangiocytes secrete and respond to neuroendocrine hormones, including secretin. We investigated whether secretin secreted by S cells and cholangiocytes stimulates biliary proliferation in mice. Methods Cholestasis was induced in secretin knockout (Sct-/-) and wild-type (control) mice by bile duct ligation (BDL). At days 3 and 7 after BDL, control and Sct-/- mice received tail-vein injections of morpholinos against microRNA 125b or let7a. One week later, liver tissues and cholangiocytes were collected. Immunohistochemical, immunoblot, luciferase reporter, and real-time polymerase chain reaction assays were performed. Intrahepatic bile duct mass (IBDM) and proliferation were measured. Secretin secretion was measured in conditioned media from cholangiocytes and S cells and in serum and bile. Results Secretin secretion was increased in supernatants from cholangiocytes and S cells and in serum and bile after BDL in control mice. BDL Sct-/- mice had lower IBDM, reduced proliferation, and reduced production of vascular endothelial growth factor (VEGF) A and nerve growth factor (NGF) compared with BDL control. BDL and control mice given morpholinos against microRNA 125b or let7a had increased IBDM. Livers of mice given morpholinos against microRNA 125b had increased expression of VEGFA, and those treated with morpholinos against microRNA let7a had increased expression of NGF. Secretin regulated VEGF and NGF expression that negatively correlated with microRNA 125b and let7a levels in liver tissue. Conclusions After liver injury, secretin produced by cholangiocytes and S cells reduces microRNA 125b and let7a levels, resulting in up-regulation of VEGF and NGF. Modulation of cholangiocyte expression of secretin could be a therapeutic approach for biliary diseases. © 2014 by the AGA Institute.

Cholangiocarcinoma (CCA) is a devastating biliary cancer. Melatonin is synthesized in the pineal gland and peripheral organs from serotonin by two enzymes, serotonin N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT). Cholangiocytes secrete neuroendocrine factors, including serotonin-regulating CCA growth by autocrine mechanisms. Melatonin exerts its effects by interaction with melatonin receptor type 1A/1B (MT1/MT2) receptors. We propose that 1) in CCA, there is decreased expression of AANAT and ASMT and secretion of melatonin, changes that stimulate CCA growth; and 2) in vitro overexpression of AANAT decreases CCA growth. We evaluated the 1) expression of AANAT, ASMT, melatonin, and MT1/MT2 in human nonmalignant and CCA lines and control and CCA biopsy samples; 2) melatonin levels in nonmalignant and CCA lines, and bile and serum from controls and patients with intrahepatic CCA; 3) effect of melatonin on the growth and expression of AANAT/ASMT and MT1/MT2 in CCA lines implanted into nude mice; and 4) effect of AANAT overexpression on the proliferation, apoptosis, and expression of MT1/MT2 in Mz-ChA-1 cells. The expression of AANAT, ASMT, and melatonin decreased, whereas MT1/MT2 expression increased in CCA lines and biopsy samples. Melatonin secretion decreased in the supernatant of CCA lines and bile of CCA patients. Melatonin decreased xenograft CCA tumor growth in nude mice by increased AANAT/ ASMT and melatonin, along with reduced MT1/MT2 expression. Overexpression of AANAT in Mz-ChA-1 cells inhibited proliferation and MT1/ MT2 expression and increased apoptosis. There is dysregulation of the AANAT/ASMT/melatonin ¡ melatonin receptor axis in CCA, which inhibited melatonin secretion and subsequently enhanced CCA growth. © 2011 the American Physiological Society

Cholangiocarcinoma (CCA) is a devastating liver tumour arising from malignant transformation of bile duct epithelial cells. Cancer stem cells (CSC) are a subset of tumour cells endowed with stem-like properties, which play a role in tumour initiation, recurrence and metastasis. In appropriate conditions, CSC form 3D spheres (SPH), which retain stem-like tumour-initiating features. Here, we found different expression of iron proteins indicating increased iron content, oxidative stress and higher expression of CSC markers in CCA-SPH compared to tumour cells growing as monolayers. Exposure to the iron chelator desferrioxamine decreased SPH forming efficiency and the expression of CSC markers and stem-like genes, whereas iron had an opposite effect. Microarray profiles in CCA samples (n = 104) showed decreased H ferritin, hepcidin and ferroportin expression in tumours respect to surrounding liver, whereas transferrin receptor was up-regulated. Moreover, we found a trend toward poorer outcome in CCA patients with elevated expression of ferritin and hepcidin, two major proteins of iron metabolism. These findings, which represent the first evidence of a role for iron in the stem cell compartment as a novel metabolic factor involved in CCA growth, may have implications for a better therapeutic approach.

Hepatic fibrosis occurs during the progression of primary sclerosing cholangitis (PSC) and is characterized by accumulation of extracellular matrix proteins. Proliferating cholangiocytes and activated hepatic stellate cells (HSCs) participate in the promotion of liver fibrosis during cholestasis. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone synthesized by hypothalamic neurons and the biliary epithelium and exerts its biological effects on cholangiocytes by interaction with the receptor subtype (GnRHR1) expressed by cholangiocytes and HSCs. Previously, we demonstrated that administration of GnRH to normal rats increased intrahepatic biliary mass (IBDM) and hepatic fibrosis. Also, miR-200b is associated with the progression of hepatic fibrosis; however, the role of the GnRH/GnRHR1/miR-200b axis in the development of hepatic fibrosis in PSC is unknown. Herein, using the mouse model of PSC (multidrug resistance gene 2 knockout), the hepatic knockdown of GnRH decreased IBDM and liver fibrosis. In vivo and in vitro administration of GnRH increased the expression of miR-200b and fibrosis markers. The GnRH/GnRHR1 axis and miR-200b were up-regulated in human PSC samples. Cetrorelix, a GnRHR1 antagonist, inhibited the expression of fibrotic genes in vitro and decreased IBDM and hepatic fibrosis in vivo. Inhibition of miR-200b decreased the expression of fibrosis genes in vitro in cholangiocyte and HSC lines. Targeting the GnRH/GnRHR1/miR-200b axis may be key for the management of hepatic fibrosis during the progression of PSC.

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

Background & Aims A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche. Methods CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14+ with CCA-sphere conditioned medium. Results CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p = 0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14+ macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163+ set was found in the tumor front of human CCA specimens (n = 23) and correlated with a high level of serum cancer antigen 19.9 (n = 17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n = 12). S

Cyclic adenosine monophosphate (cAMP) is generated by adenylyl cyclases (ACs), a group of enzymes with different tissue specificity and regulation. We hypothesized that AC isoforms are heterogeneously expressed along the biliary tree, are associated with specific secretory stimuli, and are differentially modulated in cholestasis. Small duct and large duct cholangiocytes were isolated from controls and from lipopolysaccharide-treated or alpha-naphthylisothiocyanate-treated rats. AC isoform expression was assessed via real-time polymerase chain reaction. Secretion and cAMP levels were measured in intrahepatic bile duct units after stimulation with secretin, forskolin, HCO(3) (-)/CO(2), cholinergic agonists, and beta-adrenergic agonists, with or without selected inhibitors or after silencing of AC8 or soluble adenylyl cyclase (sAC) with small interfering RNA. Gene expression of the Ca(2+)-insensitive isoforms (AC4, AC7) was higher in small duct cholangiocytes, whereas that of the Ca(2+)-inhibitable (AC5, AC6, AC9), the Ca(2+)/calmodulin-stimulated AC8, and the soluble sAC was higher in large duct cholangiocytes. Ca(2+)/calmodulin inhibitors and AC8 gene silencing inhibited choleresis and cAMP production stimulated by secretin and acetylcholine, but not by forskolin. Secretion stimulated by isoproterenol and calcineurin inibitors was cAMP-dependent and gamma-aminobutyric acid-inhibitable, consistent with activation of AC9. Cholangiocyte secretion stimulated by isohydric changes in [HCO(3) (-)](i) was cAMP-dependent and inhibited by sAC inhibitor and sAC gene silencing. Treatment with lipopolysaccharide or alpha-naphthylisothiocyanate increased expression of AC7 and sAC but decreased expression of the other ACs. Conclusion: These studies demonstrate a previously unrecognized role of ACs in biliary pathophysiology. In fact: (1) AC isoforms are differentially expressed in cholangiocyte subpopulations; (2) AC8, AC9, and sAC mediate cholangiocyte secretion in response to sec