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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 & Aims: Biliary tree, liver, and pancreas share a common embryological origin. We previously demonstrated the presence of stem/progenitor cells of endodermal origin in the adult human extrahepatic biliary tree. This study evaluated the human foetal biliary trees as sources of stem/progenitor cells of multiple endodermal-derived mature fates., Methods: Human foetal intrahepatic and extrahepatic biliary tree tissues and isolated cells were tested for cytoplasmic and surface markers of stem cells and committed progenitors, as well as endodermal transcription factors requisite for a liver versus pancreatic fate. In vitro and in vivo experiments were conducted to evaluate the potential mature fates of differentiation., Results: Foetal biliary tree cells proliferated clonogenically for more than 1 month on plastic in a serum-free Kubota medium. After culture expansion, cells exhibited multipotency and could be restricted to certain lineages under defined microenvironments, including hepatocytes, cholangiocytes, and pancreatic islet cells. Transplantation of foetal biliary tree cells into the livers of immunodeficient mice resulted in effective engraftment and differentiation into mature hepatocytes and cholangiocytes., Conclusions: Foetal biliary trees contain multipotent stem/progenitor cells comparable with those in adults. These cells can be easily expanded and induced in vitro to differentiate into liver and pancreatic mature fates, and engrafted and differentiated into mature cells when transplanted in vivo. These findings further characterise the development of these stem/progenitor cell populations from foetuses to adults, which are thought to contribute to liver and pancreas organogenesis throughout life., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Unlabelled: Cl(-) /HCO3- anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR-506) - predicted as candidate to target AE2 mRNA - for the decreased expression of AE2 in PBC. Real-time quantitative polymerase chain reaction showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is up-regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in three-dimensional (3D)-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3'untranslated region (3'UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR-506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity., Conclusion: miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3'UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Aims: The aims of this study were to detect the infection rates of DNA viruses in liver tissue of biliary atresia and to investigate the effect of perinatal infection of cytomegalovirus in biliary atresia., Methods: A total of 85 liver biopsies (taken during Kasai portoenterostomy) were tested by fluorescence quantitative polymerase chain reaction for DNA viruses (herpes simplex virus [HSV], Epstein-Barr virus [EBV], varicella zoster virus [VZV], cytomegalovirus [HCMV], and adenovirus). Immunocytochemical detection of CMV-pp65 antigenemia assay was used to detect the presence of viral protein in liver samples. Human intrahepatic biliary epithelial cells was infected by the laboratory strain AD169 of HCMV in vitro., Results: Virus DNA was found in the biopsies (51/85 HCMV, 5/85 ADV, 3/85 EBV). The biopsies of 2 patients were tested positive for 2 viruses simultaneously. They include one case of HCMV in combination with ADV and one case of ASV in combination with EBV. CMV-pp65 antigenemia were distributed in hepatocyte, vascular endothelial cell, and biliary duct endothelial cell. The cytopathic effect and apoptosis were observed after HCMVAD169 infected human intrahepatic biliary epithelial cells at 6 days., Conclusion: Human intrahepatic biliary epithelial cell is the target cell of HCMV. The etiology of biliary atresia is probably multifactorial. The perinatal infection of HCMV is one of the important etiologies for biliary atresia in China.

Stem/progenitors have been identified intrahepatically in the canals of Hering and extrahepatically in glands of the biliary tree. Glands of the biliary tree (peribiliary glands) are tubulo-alveolar glands with mucinous and serous acini, located deep within intrahepatic and extrahepatic bile ducts. We have shown that biliary tree stem/progenitors (BTSCs) are multipotent, giving rise in vitro and in vivo to hepatocytes, cholangiocytes or pancreatic islets. Cells with the phenotype of BTSCs are located at the bottom of the peribiliary glands near the fibromuscular layer. They are phenotypically heterogeneous, expressing transcription factors as well as surface and cytoplasmic markers for stem/progenitors of liver (e.g. SOX9/17), pancreas (e.g. PDX1) and endoderm (e.g. SOX17, EpCAM, NCAM, CXCR4, Lgr5, OCT4) but not for mature markers (e.g. albumin, secretin receptor or insulin). Subpopulations co-expressing liver and pancreatic markers (e.g. PDX1(+)/SOX17(+)) are EpCAM(+/-), and are assumed to be the most primitive of the BTSC subpopulations. Their descendants undergo a maturational lineage process from the interior to the surface of ducts and vary in the mature cells generated: pancreatic cells in hepatopancreatic ducts, liver cells in large intrahepatic bile ducts, and bile duct cells along most of the biliary tree. We hypothesize that there is ongoing organogenesis throughout life, with BTSCs giving rise to hepatic stem cells in the canals of Hering and to committed progenitors within the pancreas. The BTSCs are likely to be central to normal tissue turnover and injury repair and to be key elements in the pathophysiology of liver, pancreas and biliary tree diseases, including oncogenesis., (© 2011 The Authors. Journal of Anatomy © 2011 Anatomical Society.)

The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Primary biliary cirrhosis (PBC) is histologically characterized by chronic nonsuppurative destructive cholangitis (CNSDC) associated with destruction of small bile ducts. The pathogenesis of PBC, predominance of female, or the reason why biliary duct is selectively involved, however, remains unknown. Infectious or non-infectious noxious insults such as xenobiotic chemicals may precipitate in the individual having a genetic background of PBC. Activation of innate immune response seems to be a key event in early PBC, leading to the autoimmune injury of small intrahepatic bile duct. Biliary epithelial cells possess an innate immune system consisting of the Toll-like receptor (TLR) family and recognize pathogen-associated molecular patterns (PAMPs). In PBC, dysregulated biliary innate immunity, namely hyper-responsiveness to PAMPs, is associated with the pathogenesis of cholangiopathy. Moreover, the targeted biliary epithelial cells (BEC) may play an active role in the perpetuation of autoimmunity by attracting immune cells via chemokine secretion. Biliary innate immune responses induce the production of two chemokines, CX3CL1 (fractalkine) and several Th1 shift chemokines, causing the migration of inflammatory cells including NK cells. TLR 4 ligand-stimulated NK cells destroy autologous BECs in the presence of interferon alpha (IFN-α) synthesized by TLR 3 ligand-stimulated monocytes. These findings give new insights in the pathogenesis of this mysterious disease, PBC.

Unlabelled: Human cholangiocytes are continuously exposed to millimolar levels of hydrophobic bile salt monomers. We recently hypothesized that an apical biliary HCO3- umbrella might prevent the protonation of biliary glycine-conjugated bile salts and uncontrolled cell entry of the corresponding bile acids, and that defects in this biliary HCO3- umbrella might predispose to chronic cholangiopathies. Here, we tested in vitro whether human cholangiocyte integrity in the presence of millimolar bile salt monomers is dependent on (1) pH, (2) adequate expression of the key HCO3- exporter, anion exchanger 2 (AE2), and (3) an intact cholangiocyte glycocalyx. To address these questions, human immortalized cholangiocytes and cholangiocarcinoma cells were exposed to chenodeoxycholate and its glycine/taurine conjugates at different pH levels. Bile acid uptake was determined radiochemically. Cell viability and apoptosis were measured enzymatically. AE2 was knocked down by lentiviral short hairpin RNA. A cholangiocyte glycocalyx was identified by electron microscopy, was enzymatically desialylated, and sialylation was quantified by flow cytometry. We found that bile acid uptake and toxicity in human immortalized cholangiocytes and cholangiocarcinoma cell lines in vitro were pH and AE2 dependent, with the highest rates at low pH and when AE2 expression was defective. An apical glycocalyx was identified on cholangiocytes in vitro by electron microscopic techniques. Desialylation of this protective layer increased cholangiocellular vulnerability in a pH-dependent manner., Conclusion: A biliary HCO3- umbrella protects human cholangiocytes against damage by bile acid monomers. An intact glycocalyx and adequate AE2 expression are crucial in this process. Defects of the biliary HCO3- umbrella may lead to the development of chronic cholangiopathies., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Unlabelled: Functional pluripotent characteristics have been observed in specific subpopulations of hepatic cells that express some of the known cholangiocyte markers. Although evidence indicates that specific cytokines, granulocyte macrophage colony-stimulating factors (GM-CSFs), and stem cell factors (SCFs) may be candidate treatments for liver injury, the role of these cytokines in intrahepatic biliary epithelium remodeling is unknown. Thus, our aim was to characterize the specific cytokines that regulate the remodeling potentials of cholangiocytes after 70% partial hepatectomy (PH). The expression of the cytokines and their downstream signaling molecules was studied in rats after 70% PH by immunoblotting and in small and large murine cholangiocyte cultures (SMCCs and LMCCs) by immunocytochemistry and real-time polymerase chain reaction (PCR). There was a significant, stable increase in SCF and GM-CSF levels until 7 days after PH. Real-time PCR analysis revealed significant increases of key remodeling molecules, such as S100 calcium-binding protein A4 (S100A4) and miR-181b, after SCF plus GM-CSF administration in SMCCs. SMCCs produced significant amounts of soluble and bound SCFs and GM-CSFs in response to transforming growth factor-beta (TGF-β). When SMCCs were incubated with TGF-β plus anti-SCF+GM-CSF antibodies, there was a significant decrease in S100A4 expression. Furthermore, treatment of SMCCs with SCF+GM-CSF significantly increased matrix metalloproteinases (MMP-2 and MMP-9) messenger RNA as well as miR-181b expression, along with a reduction of metalloproteinase inhibitor 3. Levels of MMP-2, MMP-9, and miR-181b were also up-regulated in rat liver and isolated cholangiocytes after PH., Conclusion: Our data suggest that altered expression of SCF+GM-CSF after PH can contribute to biliary remodeling (e.g., post-transplantation) by functional deregulation of the activity of key signaling intermediates involved in cell expansion and multipotent differentiation., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Unlabelled: Notch signaling and hepatocyte nuclear factor-6 (HNF-6) are two genetic factors known to affect lineage commitment in the bipotential hepatoblast progenitor cell (BHPC) population. A genetic interaction involving Notch signaling and HNF-6 in mice has been inferred through separate experiments showing that both affect BHPC specification and bile duct morphogenesis. To define the genetic interaction between HNF-6 and Notch signaling in an in vivo mouse model, we examined the effects of BHPC-specific loss of HNF-6 alone and within the background of BHPC-specific loss of recombination signal binding protein immunoglobulin kappa J (RBP-J), the common DNA-binding partner of all Notch receptors. Isolated loss of HNF-6 in this mouse model fails to demonstrate a phenotypic variance in bile duct development compared to control. However, when HNF-6 loss is combined with RBP-J loss, a phenotype consisting of cholestasis, hepatic necrosis, and fibrosis is observed that is more severe than the phenotype seen with Notch signaling loss alone. This phenotype is associated with significant intrahepatic biliary system abnormalities, including an early decrease in biliary epithelial cells, evolving to ductular proliferation and a decrease in the density of communicating peripheral bile duct branches. In this in vivo model, simultaneous loss of both HNF-6 and RBP-J results in down-regulation of both HNF-1β and Sox9 (sex determining region Y-related HMG box transcription factor 9)., Conclusion: HNF-6 and Notch signaling interact in vivo to control expression of downstream mediators essential to the normal development of the intrahepatic biliary system. This study provides a model to investigate genetic interactions of factors important to intrahepatic bile duct development and their effect on cholestatic liver disease phenotypes., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Cholangitis arising from biliary infection dominates the prognosis in Caroli's disease. To clarify the influences of bacterial infection on the biliary cystogenesis, in vivo and in vitro studies were performed using the polycystic kidney (PCK) rat as an animal model of Caroli's disease. Cholangitis became a frequent histological finding in aged PCK rats, and neovascularization around the bile ducts also increased in aged PCK rats. Immunohistochemistry revealed that expression of vascular endothelial growth factor (VEGF) was increased in PCK rat biliary epithelium. In vitro, PCK cholangiocytes overexpressed VEGF, and the supernatant of cultured PCK cholangiocytes significantly increased the proliferative activity, migration, and tube formation of cultured rat vascular endothelial cells. Stimulation with lipopolysaccharide (LPS) further induced VEGF expression in PCK cholangiocytes, which might be mediated by signaling pathways involving phosphatidylinositol 3-kinase (PI3K)-Akt and c-Jun N-terminal kinase (JNK). Both LPS and VEGF increased cell proliferative activity in PCK cholangiocytes, and siRNA against VEGF significantly reduced LPS-induced cell proliferation. Thus, LPS-induced overexpression of VEGF in the biliary epithelium may lead to hypervascularity around the bile ducts; concurrently, LPS and VEGF act as cell proliferation factors for cholangiocytes. Biliary infection may thus exacerbate biliary cystogenesis in PCK rats., (Copyright © 2011. Published by Elsevier Inc.)

Unlabelled: The identification of resident stem cells in the mouse gallbladder is, to date, unexplored. In addition, the relationship between adult gallbladder stem cells and intrahepatic bile duct (IHBD) cells is not well understood. The aim of this study was to isolate stem cells from an adult mouse gallbladder and determine whether they were unique, compared to IHBD cells. By limiting dilution analyses and index sorts, we found that an EpCAM(+) CD49f(hi) epithelial cell subpopulation from primary gallbladder is enriched in colony-forming cells, compared to EpCAM(+) CD49f(lo) cells. EpCAM(+) CD49f(hi) cells expressed cluster of differentiation (CD)29, CD133, and stem cell antigen-1, but were negative for lineage markers CD31, CD45, and F4/80. Using a novel feeder cell-culture system, we observed long-term (>passage 20) and clonal expansion of the EpCAM(+) CD49f(hi) cells in vitro. In a matrigel differentiation assay, EpCAM(+) CD49f(+) cells expanding in vitro underwent organotypic morphogenesis forming ductular structures and cysts. These structures are similar to, and recapitulate a transport function of, primary gallbladder. EpCAM(+) CD49f(+) cells also engraft into the subcutaneous space of recipient mice. We compared primary gallbladder and IHBD cells by flow cytometry and found phenotypic differences in the expression of CD49f, CD49e, CD81, CD26, CD54, and CD166. In addition, oligonucleotide microarrays showed that the expanded EpCAM(+) CD49f(+) gallbladder cells and IHBD cells exhibit differences related to lipid and drug metabolism. Notable genes that were different are cytochrome P450, glutathione S-transferase, Indian hedgehog, and solute carrier family genes., Conclusion: We have isolated an epithelial cell population from primary mouse gallbladder with stem cell characteristics and found it to be unique, compared to IHBD cells., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Background: During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic tissue. The aim of this study was to develop protocols for three-dimensional visualization of protein expression, hepatic portal structures and human hepatic cholangiocyte tubulogenesis., Results: Protocols were developed to digitally visualize portal vessel branching and protein expression of hepatic cell lineage and extracellular matrix deposition markers in three dimensions. Samples from human prenatal livers ranging from 7 weeks + 2 days to 15½ weeks post conception as well as adult normal and acetaminophen intoxicated liver were used. The markers included cytokeratins (CK) 7 and 19, the epithelial cell adhesion molecule (EpCAM), hepatocyte paraffin 1 (HepPar1), sex determining region Y (SRY)-box 9 (SOX9), laminin, nestin, and aquaporin 1 (AQP1).Digital three-dimensional reconstructions using CK19 as a single marker protein disclosed a fine network of CK19 positive cells in the biliary tree in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic biliary tree forms through several developmental stages involving an initial transition of primitive hepatocytes into cholangiocytes shaping the ductal plate followed by a process of maturation and remodeling where the intrahepatic biliary tree develops through an asymmetrical form of cholangiocyte tubulogenesis., Conclusions: The developed protocols provide a novel and sophisticated three-dimensional visualization of vessels and protein expression in human liver during development and disease.

ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. Dynamic, multiscale, live cell imaging (confocal and total internal reflection fluorescence microscopy and a luminescence detection system with a high sensitivity charge-coupled device camera) was utilized to detect vesicular ATP release from cell populations, single cells, and the submembrane space of a single cell. In response to increases in cell volume, BECs release ATP, which was dependent on intact microtubules and vesicular trafficking pathways. ATP release occurred as stochastic point source bursts of luminescence consistent with exocytic events. Parallel studies identified ATP-enriched vesicles ranging in size from 0.4 to 1 μm that underwent fusion and release in response to increases in cell volume in a protein kinase C-dependent manner. Present in all models, SLC17A9 contributed to ATP vesicle formation and regulated ATP release. The findings are consistent with the existence of an SLC17A9-dependent ATP-enriched vesicular pool in biliary epithelium that undergoes regulated exocytosis to initiate purinergic signaling.

Objective: To investigate the role of IL-6/STAT3 pathway in the proliferation of cholangiocytes after liver transplantation and determine whether or not rapamycin (RPM) depresses the regeneration of cholangiocytes by blocking the activation of STAT3., Methods: Rats were randomized into OLT-1 h and OLT-12 h groups (supplied livers preserved for 1 or 12 h), anti-sIL-6R group (rats of the OLT-12 h group injected intravenously with 16.7 µg/kg anti-rat sIL-6R antibody at 1 hour pre-operation and daily post-operation), RPM group (rats of the OLT-12 h group injected intraperitoneally with 0.05 mg/kg RPM for 3 days pre-operation and daily post-operation) and sham group (transverse laparotomy and closure without liver manipulation). At 1, 3, 7, 14 d post-operation, the IL-6 concentration in liver homogenate and cholangiocytes proliferation were detected by ELISA (enzyme linked immunosorbent assay) and histochemistry respectively. The expressions of IL-6 mRNA, phosphorylated-STAT3 and cyclin D1 protein in cholangiocytes were determined by real-time RT-PCR (reverse transcription-polymerase chain reaction) or Western blot. The DNA binding activity of STAT3 was determined by electrophoretic mobility shift assay. The serum concentrations of ALP (alkaline phosphatase) and GGT (γ-glutamyltransferase) were also measured., Results: The minimal expressions of IL-6, p-STAT3, cyclin D1 and DNA binding activity of STAT3 were detected in OLT-1h group. And a slight increase of IOD (integral optical density) ratio (38 ± 10 and 22 ± 7) indicated a mild cholangiocytes proliferation. The concentrations of GGT were (69 ± 6) U/L, (34 ± 4) U/L and ALP (86 ± 9) U/L, (45 ± 3) U/L. The expression of IL-6 in liver homogenate were (273 ± 20) ng/g, (159 ± 18) ng/g and 0.40 ± 0.04, 0.23 ± 0.04 in cholangiocytes. The expressions of P-STAT3 were 0.420 ± 0.023 and 0.230 ± 0.040 in cholangiocytes and cyclin D1 0.580 ± 0.023 and 0.420 ± 0.015 respectively. Cholangiocytes responded to extended cold preservation with severe bile duct injures and marked increases in IL-6 secretion, p-STAT3 and cyclin D1 protein expression and DNA binding activity of STAT3, followed by compensatory cholangiocytes regeneration. Meanwhile biochemical index and morphology indicated that bile duct injury recovered at 14 d post-operation. The IOD ratios were 38 ± 10 and 22 ± 7 respectively. The expressions of IL-6 were (659 ± 28) and (446 ± 23) ng/g in liver homogenate and 0.73 ± 0.06 and 0.54 ± 0.04 in cholangiocytes. The expression of P-STAT3 were 0.72 ± 0.04 and 0.58 ± 0.06 in cholangiocytes and cyclin D1 0.88 ± 0.04 and 0.74 ± 0.07 respectively. However, anti-sIL-6R inhibited the cholangiocytes proliferation and reduced the expressions of IL-6, STAT3 and cyclin D1. The DNA binding activity of STAT3 with cellular injury and the increases of serum ALP or GGT were also abrogated by the administration of anti-sIL-6R. With similar results, the RPM treatment had insignificant effects on the expression of IL-6., Conclusion: The IL-6/STAT3 pathway initiates the cholangiocytes regeneration after liver transplantation so as to accelerate the biliary recovery. However RPM represses the cholangiocytes regeneration by inhibiting the STAT3 activation. It may have a negative effect on the healing and recovery of bile ducts in grafts with extended cold preservation.

Purpose of Review: The aim of this brief review is to provide an up-to-date view of the role played by α-smooth muscle actin-positive cancer-associated fibroblastic cells in promoting intrahepatic cholangiocarcinoma progression., Recent Findings: An increase in α-smooth muscle actin-positive cancer-associated fibroblastic cells in the stroma of intrahepatic cholangiocarcinoma has recently been demonstrated to accelerate cholangiocarcinoma progression. However, our understanding of the evolving cellular and molecular interactions between these stromal cells and cholangiocarcinoma cells in relation to promoting intrahepatic cholangiocarcinoma progression is only just beginning to be elucidated. Imbalances in multifactorial growth factor/cytokine signaling, activation of Hedgehog-GLI signaling and of proteases involved in extracellular matrix remodeling, and matricellular protein-protein and protein-cholangiocarcinoma cell interactions, as well as hypoxia, all appear to factor into the complex and dynamic interactive mechanisms through which cancer-associated fibroblastic cells crosstalk with cholangiocarcinoma cells to promote intrahepatic cholangiocarcinoma progression. Novel three-dimensional organotypic co-culture models are being developed to facilitate relevant studies of cancer-associated fibroblastic cell/cholangiocarcinoma cell interactions that may more accurately mimic physiologically pertinent features of the tumor., Summary: Increasing our understanding of critical interactive pathways by which cancer-associated fibroblastic cells crosstalk with cholangiocarcinoma cells to promote tumor progression can lead to the development of novel multitargeting strategies for intrahepatic cholangiocarcinoma therapy.

Specific accumulation and cell cycle arrest were observed in human cholangiocarcinoma cells by hybrid liposomes composed of 90 mol% L-alpha-dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylene(21)dodecyl ether (C(12)(EO)(21)) without affecting normal cholangiocytes., (Copyright 2010 Elsevier Ltd. All rights reserved.)

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.)

Background: Research in biliary atresia has been hindered by lack of a suitable animal model. Lampreys are primitive vertebrates with distinct larval and adult life cycle stages. During metamorphosis the biliary system of the larval lamprey disappears. Lamprey metamorphosis has been proposed as a model for biliary atresia. We have begun to explore cellular events during lamprey metamorphosis by assessing for cholangiocyte apoptosis., Materials and Methods: Sea lamprey larvae were housed under controlled environmental conditions. Premetamorphic larvae were induced to undergo metamorphosis by exposure to 0.01% KClO(4). Animals were photographed weekly, and the stage of metamorphosis was assigned based upon external features. Livers were harvested and processed for routine histology and immunohistochemistry. DNA fragmentation was detected using deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays and cholangiocytes were identified with antibodies to cytokeratin-19. Percent TUNEL+ cholangiocytes at different stages of metamorphosis was determined., Results: The percentage of TUNEL+ cholangiocytes was 10% in premetamorphic (stage 0) lamprey (n = 6), 51% at stage 1 (n = 6), 40% at stage 2 (n = 5), 18% at stage 3 (n = 5), and 9% stage 4 (n = 4). Routine hemotoxylin and eosin stained paraffin-embedded tissue sections revealed frequent apoptotic bodies at stages 3 and 4 of metamorphosis without histologic evidence of necrosis., Conclusions: DNA fragmentation is identified at the earliest stages of metamorphosis during induced metamorphosis in lampreys. Additional studies are necessary to validate this potentially valuable animal model., (Copyright 2010 Elsevier Inc. All rights reserved.)

Objective: To investigate whether lipopolysaccharide (LPS) stimulates cholangiocyte proliferation via the IL-6/STAT3 pathway in vivo., Methods: Rats were randomized into three groups: LPS group (injected intravenously with LPS 2.5 mg/kg), anti-IL-6 group (injected intravenously with anti-IL-6 0.5 mg/kg 1hr after LPS injection), and control group. At 6, 12, 24, 48 and 72 h after LPS injection, LPS concentration in plasma was detected by kinetic turbidimetric limulus test. IL-6 concentrations in liver homogenate was determinded by ELISA, cholangiocyte proliferation was checked by immunohistochemistry, expression of IL-6 mRNA was quantified by real-time RT-PCR, the level of phophorylated-STAT3 (P-STAT3) protein was analyzed by western blotting., Results: Cholangiocytes responded to LPS by a marked increase in cell proliferation, IL-6 secretion and P-STAT3 expression. Anti-IL-6 neutralizing antibody inhibited LPS-induced cholangiocytes proliferation, and decreased levels of IL-6 and p-STAT3., Conclusions: LPS promotes cholangiocyte proliferation through the IL-6/STAT3 pathway.

Purpose: The aim of this study was to evaluate the effect of inflammation on NAD(P)H: quinone oxidoreductase-1 (NQO1), the xenobiotic metabolizing and antioxidant enzyme protecting cells against electrophiles and reactive oxygen species in biliary cancer (cholangiocarcinoma) cells., Methods: Human cholangiocarcinoma cell line, KKU-OCA17 and HeLa Chang liver cells were treated with inflammatory cytokine combinations (interferon-gamma, interleukin-1beta and tumor necrosis factor-alpha) for 48 h before NQO1 activity was assayed. Oxidant status was examined by assays of formation of nitric oxide (NO) and superoxide, and glutathione (GSH) levels. Expression of NQO1 was assessed by a reverse transcription and polymerase chain reaction. Effects of S-nitroso-glutathione (GSNO) were examined if the effects of inflammatory cytokines could be mimicked by nitric oxide donor., Results: NQO1 activity in KKU-OCA17 and HeLa Chang liver cells was suppressed by cytokine combination. Cytokines induced formation of NO and suppression of redox ratios of GSH and glutathione disulfide (GSSG). GSNO produced the similar effects as cytokines on KKU-OCA17, in contrast, GSNO induced increase of NQO1 activity in HeLa Chang liver cells. The treatment of cytokines or GSNO suppressed expression of NQO1 in KKU-OCA17 and HeLa Chang liver cells., Conclusions: Inflammatory cytokines induced oxidative stress and this is associated with suppression of NQO1, whereas may contribute to differential susceptibility of biliary epithelial cells to chemical-induced cytotoxicity and carcinogenesis.

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

Transdifferentiation of cells from a patient's own liver into pancreatic beta-cells could be useful for beta-cell replacement. We hypothesized that intrahepatic biliary epithelial cells (IHBECs) could become a new source of insulin-producing cells. IHBECs isolated from adult mice were expanded using our novel culture method termed, collagen-embedded floating culture method (CEFCM). With CEFCM, IHBECs formed three-dimensional ductal cysts and rapidly expanded their number by about 15-fold within 2 weeks. Over 90% of cells were positive for cytokeratin 7 and 19. At day 14, IHBECs were transfected with adenoviral (Ad)- pancreas duodenum homeobox 1 (Pdx-1), NeuroD or Pdx-1/VP16. After 7 additional days in serum- and insulin-free differentiation medium (DM), cell phenotypes were determined by RT-PCR, immunostaining and ELISA for insulin. In DM control IHBECs started to express some endocrine progenitor genes (Neurog3, NeuroD, Nkx6.1, and Pdx-1) but lacked insulin gene (Ins) mRNA. Transduced expression of PDX-1, NEUROD or PDX-1/VP16 led to expression of not only INS but also GLUT2 and prohormone convertase 1 and 2. About 3% of 4000 cells counted in PDX-1/VP16 transduced cultures stained strongly for C-peptide suggesting that a subpopulation may have the capacity for differentiation. Transduced cells released insulin (Ad-PDX-1 0.08+/-0.05, Ad-NEUROD 0.33+/-0.09, Ad-PDX-1/VP16 0.37+/-0.14 ng/1x10(5) cells after 48 h in culture). IHBECs can be markedly expanded, and then with molecular manipulation a subpopulation of these cells can differentiate towards a beta-cell phenotype. This approach may lead to a new source of beta-cells that can be used for transplantation in diabetes.

Background: Cholangiocytes are exposed to endotoxins (lipopolysaccharide, LPS) in a variety of biliary inflammations. It is known that LPS enhances the release of interleukin (IL)-6, a potent cholangiocyte mitogen. However, the role of LPS in cholangiocyte proliferation in vivo is unknown. Aims To investigate whether LPS stimulates cholangiocyte proliferation in vivo via the IL-6/STAT3 pathway., Methods: Rats were randomized into four groups: the LPS group (injected intravenously with LPS 2.5 mg/kg), anti-IL-6 group (injected intravenously with anti-IL-6 0.5 mg/kg 1 h after LPS injection), RPM group (treated with RPM 0.4 mg/kg intraperitoneally 30 min before LPS injection), and control group. At 6, 12, 24, 48, and 72 h after LPS injection, LPS in plasma was detected by kinetic turbidimetric limulus test. IL-6 concentrations in liver homogenate and cholangiocyte proliferation were determined by ELISA or immunohistochemistry, respectively. Expression of IL-6 mRNA and phosphorylated-STAT3 (P-STAT3) protein in cholangiocytes was analyzed by real-time RT-PCR and western blotting., Results: Cholangiocytes responded to LPS by a marked increase in cell proliferation, IL-6 secretion, and P-STAT3 expression. Anti-IL-6 neutralizing antibody inhibited LPS-induced proliferation of cholangiocytes and decreased levels of IL-6 and STAT3. Furthermore, after being treated with RPM, STAT3 activation was also depressed, which resulted a decreased proliferation of cholangiocytes., Conclusions: LPS promotes cholangiocyte proliferation through the IL-6/STAT3 pathway, while RPM shows a depressive effect in this pathway.

There has been a rapid growth in our understanding of the molecular bases of primary biliary cirrhosis (PBC). These efforts were initiated when the immunodominant mitochondrial autoantigen was cloned and sequenced. Using the recombinant cloned antigen as a tool, research has focused on the effector mechanisms of disease and the uniqueness of the primary target tissue, the intrahepatic bile ducts. Most recently, there have been experimental data suggesting that innate immunity changes may be critical to the initiation and perpetuation of the autoimmune injury, as in the case of the enhanced response of monocytes and memory B cells to infectious stimulation and environmental mimics. These observations are important as they help fill in the many gaps which remain on the most difficult subject of autoimmunity, etiology. Indeed, based on the available data, several experimental models of PBC have been developed. These models illustrate and suggest that PBC can be initiated by several mechanisms, all of which lead to loss of tolerance to the mitochondrial antigens. However, once this adaptive response develops, it appears that much of the subsequent pathology is exacerbated by innate responses. We suggest that future therapeutic efforts in PBC will depend heavily on understanding the nature of this innate immune responses and methodology to blunt their cytotoxicity.

The human intrahepatic biliary epithelium is composed of a morphologically heterogeneous population of epithelial cells. During liver cirrhosis, new biliary ductular structures develop at the portal margins that express markers of immaturity such as CD56 and Bcl-2. These markers are also expressed transiently on immature biliary duct precursors during embryological development; thus their reappearance during cirrhosis suggests a recapitulation of ontogenesis during some liver conditions. Here we describe methods, based on the differential expression of membrane markers, for separating immature biliary epithelial cells from those associated with mature ducts. We also describe two- and three-dimensional culture models for the maintenance of mature and immature populations in vitro. Both populations readily establish colonies in monolayer culture but only cells from mature ducts can be maintained in medium-term culture as serially proliferating, passageable cultures; immature cells deteriorate and detach within 2-3 weeks of isolation. In three-dimensional collagen gel culture, both mature and immature populations form duct-like structures with clearly definable lumena that persist for up to 6 weeks.

Epithelial-mesenchymal transitions (EMTs) play an important role in tissue construction during embryogenesis, and evidence suggests that this process may also help to remodel some adult tissues after injury. Activation of the hedgehog (Hh) signaling pathway regulates EMT during development. This pathway is also induced by chronic biliary injury, a condition in which EMT has been suggested to have a role. We evaluated the hypothesis that Hh signaling promotes EMT in adult bile ductular cells (cholangiocytes). In liver sections from patients with chronic biliary injury and in primary cholangiocytes isolated from rats that had undergone bile duct ligation (BDL), an experimental model of biliary fibrosis, EMT was localized to cholangiocytes with Hh pathway activity. Relief of ductal obstruction in BDL rats reduced Hh pathway activity, EMT, and biliary fibrosis. In mouse cholangiocytes, coculture with myofibroblastic hepatic stellate cells, a source of soluble Hh ligands, promoted EMT and cell migration. Addition of Hh-neutralizing antibodies to cocultures blocked these effects. Finally, we found that EMT responses to BDL were enhanced in patched-deficient mice, which display excessive activation of the Hh pathway. Together, these data suggest that activation of Hh signaling promotes EMT and contributes to the evolution of biliary fibrosis during chronic cholestasis.

We report a 50-year-old male patient with primary liver carcinoma exhibiting dual hepatocellular and biliary epithelial differentiations associated with citrin deficiency (asymptomatic adult-onset type II citrullinemia, CTLN2). Although so far 14 CTLN2 patients with hepatocellular carcinoma have been reported, this report describes a unique case of liver carcinoma showing the features of both hepatocellular and cholangiocellular carcinoma. In addition to the clinical data of the 14 patients reported previously, the findings in our patient suggest that the citrin deficiency might be one of the key disorders causing hepatocellular carcinoma especially at younger ages and can also play an important role in hepatocarcinogenesis of the hepatic progenitor cells, which have the bipotential to differentiate into both hepatocytes and cholangiocytes.

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.

This study reports on morphological features of hepatic portal tracts in the liver of a rhesus monkey. The light microscope shows that the number of each type of principal component comprising a portal tract varies but that there are usually one to five lymphatics, one bile ductule, one bile duct, one arteriolar and one arterial branch of the hepatic artery, and one hepatic portal vein. Bile ductules, in cross section, have 6-10 cells (mostly low pyramidal, but with a few cuboidal) bordering the lumen, an outside diameter of from about 20 to 25 microm, and a luminal diameter of from 2 to 10 microm. Bile ducts, in cross section, have more than 10 cells (about equal numbers of low pyramidal and cuboidal) bordering the lumen, an outside diameter greater than 25 microm and a luminal diameter of greater than 10 microm. The term "pyramidal" has not previously been applied to the cells of the ductules and ducts. The monkey tracts show several cytological features previously undescribed, viz., abortive cilia and basal bodies in the duct cells, abortive cilia in the ductule cells, and an occasional aggregation of ribosomes in arterial endothelial cells. They also show a major histological feature previously mentioned but not illustrated, viz., bundles of nerve processes which exhibit a preferential location, i.e., proximity to the arterioles and arteries.

The epithelial cells lining intrahepatic bile ducts (i.e., cholangiocytes), like many cell types in the body, have primary cilia extending from the apical plasma membrane into the bile ductal lumen. Cholangiocyte cilia express proteins such as polycystin-1, polycystin-2, fibrocystin, TRPV4, P2Y12, AC6, that account for ciliary mechano-, osmo-, and chemo-sensory functions; when these processes are disturbed by mutations in genes encoding ciliary-associated proteins, liver diseases (i.e., cholangiociliopathies) result. The cholangiociliopathies include but are not limited to cystic and fibrotic liver diseases associated with mutations in genes encoding polycystin-1, polycystin-2, and fibrocystin. In this review, we discuss the functions of cholangiocyte primary cilia, their role in the cholangiociliopathies, and potential therapeutic approaches., (Copyright (c) 2008 Wiley-Liss, Inc.)

Cellular senescence is reportedly involved in cholangiopathy in primary biliary cirrhosis and oxidative stress is proposed as a pathogenetic factor in biliary epithelial cells (BECs). This study investigated the involvement of proinflammatory cytokines (IFN-beta, IFN-gamma and TNF-alpha) and ataxia telangiectasia-mutated (ATM)/p53/ p21(WAF1/Cip1) pathway with respect to oxidative stress in cellular senescence of BECs. H(2)O(2) treatment (oxidative stress) induced phosphorylation (activation) of ATM and p53 and also p21(WAF1/Cip1) expression in BECs. Treatment with inflammatory cytokines generated reactive oxygen species (ROS) in cultured BECs followed by activation of the ATM/p53/p21(WAF1/Cip1) pathway and the induction of cellular senescence. Pre-treatment with ATM inhibitor (2-aminopurine) and antioxidant (N-acetylcysteine) significantly blocked the cellular senescence of BECs induced by oxidative stress or inflammatory cytokines. In conclusion, proinflammatory cytokines induce ROS generation and activate the ATM/p53/p21(WAF1/Cip1) pathway, followed by biliary epithelial senescence. This senescent process may be involved in the development of destructive cholangiopathy in humans.

Background/aims: Biliary epithelial cells (BECs) possess negative regulatory mechanisms of Toll-like receptor (TLR)-based tolerance to bacteria (e.g. endotoxin tolerance). Viral infections of the Reoviridae genus with a dsRNA genome are suspected to be part of the aetiology of biliary atresia (BA), but the negative biliary mechanisms remain unexplored., Methods: Cultured human intrahepatic BECs (HIBECs) pretreated with polyinosinic-polycytidylic acid [poly(I:C)] (a synthetic analogue of viral dsRNA) for 24 h were exposed to poly(I:C) in fresh medium. The activation of nuclear factor-kappaB (NF-kappaB) and the expression of myxovirus resistance protein A (MxA) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNAs were evaluated. Moreover, after the pretreatment, the transition of these molecules was examined in poly(I:C)-free conditions., Results: Treatment with poly(I:C) significantly upregulated NF-kappaB activity in fresh HIBECs, and pretreatment failed to show tolerance to poly(I:C). The production of MxA and TRAIL was also preserved. Moreover, upregulation in the pretreated HIBECs was well preserved in poly(I:C)-free medium for at least 72 h., Conclusions: BECs fail to show tolerance to poly(I:C), and once innate immunity is activated it is sustained in poly(I:C)-free conditions, suggesting that the initiation of the immune response to dsRNA in HIBECs and its presence after the clearance of virus are closely associated with the progression of BA.

Unlabelled: Previous studies from our laboratory have demonstrated that hepatocytes can transdifferentiate into biliary epithelium (BE) both in vivo and in vitro; however, the mechanisms are unclear. The current study was designed to investigate the mechanisms of hepatocyte transdifferentiation in vitro. Rat hepatocytes were cultured in roller bottles to obtain hepatocyte organoid cultures, which were stimulated with various growth factors (GFs) including hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), stem cell factor (SCF), macrophage-stimulating protein (MSP), fibroblast growth factor-a (FGF-a), fibroblast growth factor-b (FGF-b), and fibroblast growth factor-8b (FGF-8b). Only the cultures treated with HGF, EGF, and their combination exhibited formation of hepatocyte-derived biliary epithelium (BE) despite the presence and activation of all the pertinent cognate membrane receptors of the rest of the GFs. Microarray analysis of the organoid cultures identified specific up-regulation of approximately 500 target genes induced by HGF and EGF, including members of the extracellular matrix (ECM) protein family, Wnt/beta-catenin pathway, transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) pathway, and CXC (cysteine-any amino acid-cysteine) chemokines. To investigate the downstream signaling involved in hepatocyte to biliary epithelial cell (BEC) transdifferentiation, we investigated expression and activities of mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)] as well as serine/threonine kinase AKT. The analysis indicated that AKT phosphorylation was particularly increased in cultures treated with HGF, EGF, and their combination. Whereas phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely inhibited biliary epithelium formation, AKT inhibitor could only moderately reduce formation of BE in the organoid cultures treated with HGF+EGF. Most of the HGF+EGF target genes were altered by LY294002., Conclusion: Taken together, these data indicate that hepatocyte to BE transdifferentiation is regulated by HGF and EGF receptors and that PI3 kinase-mediated signaling independent of AKT is a crucial component of the transdifferentiation process.

Cholangiocytes, epithelial cells lining the biliary tree, have primary cilia extending from their apical membrane into the ductal lumen. Although important in disease, cilia also play a vital role in normal cellular functions. We reported that cholangiocyte cilia are sensory organelles responding to mechanical stimuli (i.e., luminal fluid flow) by alterations in intracellular Ca(2+) and cAMP. Because cholangiocyte cilia are also ideally positioned to detect changes in composition and tonicity of bile, we hypothesized that cilia also function as osmosensors. TRPV4, a Ca(2+)-permeable ion channel, has been implicated in signal transduction of osmotic stimuli. Using purified rat cholangiocytes and perfused intrahepatic bile duct units (IBDUs), we found that TRPV4 is expressed on cholangiocyte cilia, and that hypotonicity induces an increase in intracellular Ca(2+) in a TRPV4-, ciliary-, and extracellular calcium-dependent manner. The osmosensation of luminal tonicity by ciliary TRPV4 induces bicarbonate secretion, the main determinant of ductal bile formation, by a mechanism involving apical ATP release. Furthermore, the activation of TRPV4 in vivo, by its specific agonist, 4alphaPDD, induces an increase in bile flow as well as ATP release and bicarbonate secretion. Our results suggest that cholangiocyte primary cilia play an important role in ductal bile formation by acting as osmosensors.

Background & Aims: Bicarbonate secretion is a primary function of cholangiocytes. Either adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca(2+) can mediate bicarbonate secretion, but these are thought to act through separate pathways. We examined the role of the inositol 1,4,5-trisphosphate receptor (InsP3R) in mediating bicarbonate secretion because this is the only intracellular Ca(2+) release channel in cholangiocytes., Methods: Intrahepatic bile duct units (IBDUs) were microdissected from rat liver then luminal pH was examined by confocal microscopy during IBDU microperfusion. Cyclic AMP was increased using forskolin or secretin, and Ca(2+) was increased using acetylcholine (ACh) or adenosine triphosphate (ATP). Apyrase was used to hydrolyze extracellular ATP, and suramin was used to block apical P2Y ATP receptors. In selected experiments, IBDUs were pretreated with short interfering RNA (siRNA) to silence expression of specific InsP3R isoforms., Results: Both cAMP and Ca(2+) agonists increased luminal pH. The effect of ACh on luminal pH was reduced by siRNA for basolateral (types I and II) but not apical (type III) InsP3R isoforms. The effect of forskolin on luminal pH was reduced by a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and by siRNA for the type III InsP3R. Luminal apyrase or suramin blocked the effects of forskolin but not ACh on luminal pH., Conclusions: Cyclic AMP-induced ductular bicarbonate secretion depends on an autocrine signaling pathway that involves CFTR, apical release of ATP, stimulation of apical nucleotide receptors, and then activation of apical, type III InsP3Rs. The primary role of CFTR in bile duct secretion may be to regulate secretion of ATP rather than to secrete chloride and/or bicarbonate.

Calcium (Ca2+) pathways are important in cell volume regulation in many cells, but its role in volume regulatory processes in cholangiocytes is unclear. Thus, we have investigated the role of Ca2+ in regulatory volume decrease (RVD) in cholangiocytes using freshly isolated bile duct cell clusters (BDCCs) from normal mouse. No significant increase in [Ca2+]i was observed during RVD, while ionomycin and ATP showed significant increases. Confocal imaging also showed no significant changes in the levels or distributions of intracellular Ca2+ during RVD. Cell volume study by quantitative videomicroscopy indicated that removal and chelation of extracellular Ca2+ by ethylene glycol-bis (beta-aminoethyl ether)-N,N,N-tetraacetic acid (EGTA) or administration of nifedipine did not affect RVD but verapamil significantly inhibited the RVD. Moreover, Ca2+ agonists or inhibitors of Ca2+ release from intracellular stores had no significant effect on RVD. However, 1,2-bis (2-aminophenoxy) ethane-N,N,N'N'-tetraacetic acid-AM (BAPTA-AM) showed significant decreases in [Ca2+]i and significantly inhibited RVD, which was reversed with coadministration of valinomycin, suggesting that BAPTA-AM-induced inhibition is due to potassium conductance or other cellular processes requiring permissive [Ca2+](i. These findings indicate that an increase in [Ca2+]i or extracellular Ca2+ is not required for RVD but Ca2+ has a permissive role in RVD of mouse cholangiocytes.

Xanthogranulomatous cholecystitis (XGC) is characterized by the infiltration of numerous foamy macrophages. Bacterial infection is thought to be involved in the pathogenesis of XGC. Using XGC and cultured murine biliary epithelial cells (BEC), the participation of E. coli and the role of the scavenger receptor class A (SCARA), as well as chemokine(C-X-C motif) ligand 16 (CXCL16) and its receptor chemokine(C-X-C motif) receptor 6 (CXCR6), were examined in the pathogenesis of XGC. E. coli components and genes were detected in XGC on immunohistochemistry and polymerase chain reaction (PCR), respectively. SCARA-recognizing E. coli was found in foamy macrophages aggregated in xanthogranulomatous lesions. CXCL16, which functions as a membrane-bound molecule and soluble chemokine to induce adhesion and migration of CXCR6(+) cells, was detected on gallbladder epithelia, and CXCR6(+)/CD8(+) T cells and CXCR6(+)/CD68(+) macrophages were also accumulated. In cultured BEC, CXCL16 mRNA and secreted soluble CXCL16 were constantly detected and upregulated by treatment with E. coli and lipopolysaccharide through Toll-like receptor 4. These suggest that SCARA in macrophages is involved in the phagocytosis of E. coli followed by foamy changes and that bacterial infection causes the upregulation of CXCL16 in gallbladder epithelia, leading to the chemoattraction of macrophages via CXCL16-CXCR6 interaction and formation of the characteristic histology of XGC.

Background: Chronic colitis-harboring TCRalpha(- / - ) x AIM(- / - ) mice showed PBC-like bile duct damage in the liver. Bacterial infection is one of the candidates for the pathogenesis of PBC. We demonstrated that the bacterial cell wall component lipotheicoic acid (LTA) was detected at sites of inflammation around damaged bile ducts in PBC patients. The aim of this study was to investigate the pathophysiology of the liver and other organs in TCRalpha(- / - ) x AIM(- / - ) mice., Methods: Thirteen female TCRalpha(- / - ) x AIM(- / - ) mice were sacrificed at 24 weeks of age. The liver, stomach, small intestine, colon, pancreas, kidney and spleen were studied for pathological examination. Using anti-LTA antibody as the primary antibody, an immunohistochemical study was carried out., Results: In the liver, LTA was mainly detected in the portal area with inflammation, and some of the cytoplasm of hepatocytes. Inflammations were also observed in the stomach, intestine, pancreas and kidney. Throughout the gastrointestinal tract, from the stomach to the colon, LTA was detected in the epithelium at sites of inflammation. Furthermore, LTA was detected around both pancreatic ducts with inflammation and distal renal tubules with inflammation., Conclusions: The development of inflammations in the liver as well as extensive organs, strongly suggests a close relationship between bile duct damage and systemic multifocal epithelial inflammations, perhaps involving bacterial LTA, in TCRalpha(- / - ) x AIM(- / - ) mice.

The mechanism of transformation of biliary epithelium leading to intestinal metaplasia, which is significantly involved in biliary diseases, remains unclear. CDX2, an intestine-specific transcription factor, is thought to regulate intestinal mucin MUC2 (mucus core protein) expression. We took advantage of polycystic kidney (PCK) rats as a model of chronic suppurative cholangitis with intestinal metaplasia and of cultured biliary epithelial cells (BECs) from PCK rats to clarify the causal relation between bacterial components such as pathogen-associated molecular patterns (PAMPs) and the development of intestinal metaplasia of bile ducts. Histological, immunohistochemical, and in situ hybridization studies were conducted in PCK rat livers. In cultured BECs, CDX2 and MUC2 were expressed following treatment with PAMPs and inhibitors (anti-Toll-like receptor (TLR)2/TLR4 antibody, nuclear factor-kappaB (NF-kappaB) inhibitor MG132). Chronic suppurative cholangitis with intestinal metaplasia developed as the PCK rats aged, and intestinal metaplasia and aberrant CDX2 and MUC2 expression developed in parallel. Intraluminal bacteria and the expression of TLR2 and TLR4 in BECs were demonstrated in the bile ducts, showing chronic suppurative cholangitis. In cultured BECs, treatment with PAMPs induced upregulation of CDX2 and MUC2 expression, and this effect was abolished by pretreatment with anti-TLR2 and anti-TLR4 antibody and MG132. A knockdown of CDX2 by CDX2 small interfering RNA inhibited MUC2 expression in cultured BECs induced by PAMPs, and transfection of CDX2 expression vector induced MUC2 expression. In conclusion, bacterial components may induce upregulation of the CDX2 expression followed by MUC2 expression via TLR and the NF-kappaB system in cultured BECs, and could be related to the development of intestinal metaplasia of the bile ducts.

Unlabelled: The Notch signaling pathway is involved in determination of cell fate and control of cell proliferation in multiple organ systems. Jag1 encodes a ligand in the Notch pathway and has been identified as the disease-causing gene for the developmental disorder Alagille syndrome. Evidence from the study of human disease and mouse models has implicated Jag1 as having an important role in the development of bile ducts. We have derived a conditional knockout allele (Jag1(loxP)) to study the role of Jag1 and Notch signaling in liver and bile duct development. We crossed Jag1(loxP) mice with a transgenic line carrying Cre recombinase under the control of the albumin promoter and alpha-fetoprotein enhancer to ablate Jag1 in hepatoblasts. The liver-specific Jag1 conditional knockout mice showed normal bile duct development. To further decrease Notch pathway function, we crossed the Jag1 conditional knockout mice with mice carrying the hypomorphic Notch2 allele, and bile duct anatomy remained normal. When Jag1 conditional mice were crossed with mice carrying the Jag1 null allele, the adult progeny exhibited striking bile duct proliferation., Conclusion: These results indicate that Notch signaling in the liver is sensitive to Jag1 gene dosage and suggest a role for the Notch pathway in postnatal growth and morphogenesis of bile ducts.

Methionine aminopeptidases (MetAP) are proteases which remove the N-terminal methionine from newly synthesized proteins. Associations of MetAP2 with tumor progression of different cancers have been repeatedly reported. We aim to determine if MetAP2 is expressed in cholangiocarcinomas (CCA) and investigate to see if it would be a useful therapeutic target. We evaluated MetAP2 expression by immunohistochemistry in 82 patients of intrahepatic CCA. MetAP2 was expressed in bile ducts to various degrees. It was occasionally expressed with weak staining in normal bile duct epithelium but was strikingly over-expressed in dysplastic bile duct epithelia, primary and metastatic CCA tissues (p < 0.001). The increased expression of MetAP2 in proliferating bile duct was evident. All metastatic tumors had stronger expression of MetAP2 than the corresponding primary tumors. Fumagillin, a MetAP2 specific inhibitor, significantly inhibited cell proliferation in dose dependent manner and the degree of growth inhibition was dependent on the amount of cellular enzyme. The present study highlights the involvement of MetAP2 in an early event of carcinogenesis of CCA. The findings represent the first description of increased MetAP2 expression in CCA. The inhibition of enzyme activity using MetAP2 inhibitors may be a potential strategy for long-term control of tumor development and progression in CCA patients.

Mucin secreted by mucosal epithelial cells plays a role in the protection of the mucosal surface and also is involved in pathological processes. So far, MUC1-4, 5AC, 5B, 6-8, 11-13 and 15-17 genes coding the backbone mucin core protein have been identified in humans. Their diverse physiological distribution and pathological alterations have been reported. Trefoil factor family (TFF) peptides are mucin-associated molecules co-expressed with MUC mucins and involved in the maintenance of mucosal barrier and the biological behavior of epithelial and carcinoma cells. Intrahepatic biliary system is a route linking the bile canaliculi and the extrahepatic bile duct for the excretion of bile synthesized by hepatocytes. Biliary epithelial cells line in the intrahepatic biliary system, secreting mucin and other molecules involved in the maintenance and regulation of the system. In this review, the latest information regarding properties, expression profiles and regulation of MUC mucins and TFF peptides in the intrahepatic biliary system is summarized. In particular, we focus on the expression profiles and their significance of MUC mucins in developmental and normal livers, various hepatobiliary diseases and intrahepatic cholangiocarcinoma.

Cryptosporidium parvum attaches to intestinal and biliary epithelial cells via specific molecules on host-cell surface membranes including Gal/GalNAc-associated glycoproteins. Subsequent cellular entry of this parasite depends on host-cell membrane alterations to form a parasitophorous vacuole via activation of phosphatidylinositol 3-kinase (PI-3K)/Cdc42-associated actin remodelling. How C. parvum hijacks these host-cell processes to facilitate its infection of target epithelia is unclear. Using specific probes to known components of sphingolipid-enriched membrane microdomains (SEMs), we detected aggregation of host-cell SEM components at infection sites during C. parvum infection of cultured human biliary epithelial cells (i.e. cholangiocytes). Activation and membrane translocation of acid-sphingomyelinase (ASM), an enzyme involved in SEM membrane aggregation, were also observed in infected cells. Pharmacological disruption of SEMs and knockdown of ASM via a specific small interfering RNA (siRNA) significantly decreased C. parvum attachment (by approximately 84%) and cellular invasion (by approximately 88%). Importantly, knockdown of ASM and disruption of SEMs significantly blocked C. parvum-induced accumulation of Gal/GalNAc-associated glycoproteins at infection sites by approximately 90%. Disruption of SEMs and knockdown of ASM also significantly blocked C. parvum-induced activation of host-cell PI-3K and subsequent accumulation of Cdc42 and actin by up to 75%. Our results suggest an important role of SEMs for C. parvum attachment to and entry of host cells, likely via clustering of membrane-binding molecules and facilitating of C. parvum-induced actin remodelling at infection sites through activation of the PI-3K/Cdc42 signalling pathway.

Purpose and Experimental Design: To identify novel molecular targets for the treatment of intrahepatic cholangiocarcinoma (ICC), the second most common type of primary hepatobiliary cancer, we earlier analyzed genome-wide expression profiles of genes in 25 ICCs. Among the genes whose expression levels were commonly elevated in the tumors, we identified a novel gene termed RASGEF1A that encodes a putative Ras guanine nucleotide exchange factor domain-containing protein., Results: We showed in this article that RASGEF1A protein has a guanine nucleotide exchange activity to K-RAS, H-RAS, and N-RAS proteins in vitro. Consistently, exogenous RASGEF1A expression increased the activity of Ras. In addition, suppression of RASGEF1A by small interfering RNA retarded the growth of cholangiocarcinoma cells. Interestingly, COS7 cells expressing exogenous RASGEF1A showed enhanced cellular motility in Transwell and wound-healing assays., Conclusions: These data suggest that elevated expression of RASGEF1A may play an essential role for proliferation and progression of ICC. Our data indicate that RASGEF1A may be a promising therapeutic target for the majority of ICCs.

In the PCK rat, a rodent model of Autosomal Recessive Polycystic Kidney Disease (ARPKD), a spontaneous splicing mutation of Pkhd1 initiates hepatic cyst development. Cystic cholangiocytes possess short and malformed cilia that do not express fibrocystin, the Pkhd1 protein. During the disease course, cysts continue to grow; however, the mechanisms underlying cyst progression are unclear due in part to the lack of suitable cell lines to study cystogenesis. Here, we describe the development of a PCK-derived cholangiocyte cell line (PCK-CCL). Normal rat cholangiocytes (NRCs) were used as a control. The PCK-CCL maintained a cholangiocyte phenotype as assessed by the expression of the CK-19, CK-7 and GGT. PCK-CCL grown on collagen formed a polarized monolayer with well-developed junctional complexes, and distinct apical and basolateral membranes. Compared to NRCs, cilia in the PCK-CCL were short and malformed and did not express fibrocystin. The PCK-CCL exhibited a higher rate of proliferation (P<0.05) with a doubling time approximately half that of NRCs. By RT-PCR analysis of exons 33-37, an approximately 800 bp product of Pkhd1 was amplified in NRCs. In contrast and as expected, in the PCK-CCL, the Pkhd1 amplicon was smaller ( approximately 630 bp) reflecting the IVS35-2A --> T mutation. PCK-CCL and NRCs seeded in 3-D cultures formed cystic structures; however, the PCK cysts expanded progressively up to day 21 while cysts formed by NRCs remained the same size after day 9. In summary, we have developed a cholangiocyte cell line from the PCK rat that retains properties of the cholangiocytes lining hepatic cysts in vivo. The cells have been grown continuously for approximately 18 month and 45 passages without crisis or senescence. The morphology and growth characteristics of the PCK-CCL are consistent with those seen in vivo in the PCK rat, suggesting that this cell line will be useful in dissecting the mechanisms of hepatic cyst formation.

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.