Indoleamine 2,3-dioxygenase 1 limits hepatic inflammatory cells recruitment and promotes bile duct ligation-induced liver fibrosis.

Liver fibrosis is a course of chronic liver dysfunction, can develop into cirrhosis and hepatocellular carcinoma. Inflammatory insult owing to pathogenic factors plays a crucial role in the pathogenesis of liver fibrosis. Indoleamine 2,3-dioxygenase 1 (IDO1) can affect the infiltration of immune cells in many pathology processes of diseases, but its role in liver fibrosis has not been elucidated completely. Here, the markedly elevated protein IDO1 in livers was identified, and dendritic cells (DCs) immune-phenotypes were significantly altered after BDL challenge. A distinct hepatic population of CD11c+DCs was decreased and presented an immature immune-phenotype, reflected by lower expression levels of co-stimulatory molecules (CD40, MHCII). Frequencies of CD11c+CD80+, CD11c+CD86+, CD11c+MHCII+, and CD11c+CD40+ cells in splenic leukocytes were reduced significantly. Notably, IDO1 overexpression inhibited hepatic, splenic CD11c+DCs maturation, mature DCs-mediated T-cell proliferation and worsened liver fibrosis, whereas above pathological phenomena were reversed in IDO1-/- mice. Our data demonstrate that IDO1 affects the process of immune cells recruitment via inhibiting DCs maturation and subsequent T cells proliferation, resulting in the promotion of hepatic fibrosis. Thus, amelioration of immune responses in hepatic and splenic microenvironment by targeting IDO1 might be essential for the therapeutic effects on liver fibrosis.

Changes in transporters and metabolizing enzymes in the livers of rats with bile duct ligation.

PURPOSE: Bile duct ligation (BDL) in experimental animals is widely used as an animal model of liver cholestasis and fibrosis. The transcriptional process and plasma membrane localization of transporters are regulated by nuclear receptors and scaffold proteins, respectively. However, the detailed changes of these factors in the livers of BDL rats remain unclear. To clarify the effects of BDL on the levels of transporters and metabolizing enzymes, nuclear receptors, and scaffold proteins, we investigated changes in mRNA and protein levels of livers from BDL rats. METHODS: Membrane proteins and microsomes were prepared from rats with BDL. The mRNA levels of transporters and nuclear receptors in livers of control and BDL rats were examined by real-time reverse transcription polymerase chain reaction. The protein levels of transporters, metabolizing enzymes and scaffold proteins in membrane proteins and microsomes were determined by liquid chromatography-tandem mass spectrometry-based targeted proteomics. RESULTS: Mdr1a mRNA was significantly decreased at 1 and 2 weeks of BDL. The mRNA levels of MRP2 were significantly decreased. The mRNA levels of nuclear receptors were significantly decreased in livers of 1-week BDL rats. The protein levels of P-gp were significantly increased by BDL. Regarding scaffold proteins, the protein levels of ezrin, moesin and EBP50 were significantly decreased at 2 weeks of BDL. The protein levels of radixin were significantly increased at 1 week of BDL. In 1-week BDL rats, the protein levels of metabolizing enzymes such as CYP and UGT were significantly decreased. CONCLUSIONS: This study reports the comprehensive changes of transporters, metabolizing enzymes, nuclear receptors, and ezrin/radixin/moesin proteins in the livers of BDL rats. The expression levels of nuclear receptors and radixin that regulate the transcription and localization of CYP and/or transporters were decreased by BDL.

Transient c-Src Suppression During Endodermal Commitment of Human Induced Pluripotent Stem Cells Results in Abnormal Profibrotic Cholangiocyte-Like Cells.

Directed differentiation of human induced pluripotent stem cells (iPSCs) toward hepatobiliary lineages has been increasingly used as models of human liver development/diseases. As protein kinases are important components of signaling pathways regulating cell fate changes, we sought to define the key molecular mediators regulating human liver development using inhibitors targeting tyrosine kinases during hepatic differentiation of human iPSCs. A library of tyrosine kinase inhibitors was used for initial screening during the multistage differentiation of human iPSCs to hepatic lineage. Among the 80 kinase inhibitors tested, only Src inhibitors suppressed endoderm formation while none had significant effect on later stages of hepatic differentiation. Transient inhibition of c-Src during endodermal induction of human iPSCs reduced endodermal commitment and expression of endodermal markers, including SOX17 and FOXA2, in a dose-dependent manner. Interestingly, the transiently treated cells later developed into profibrogenic cholangiocyte-like cells expressing both cholangiocyte markers, such as CK7 and CK19, and fibrosis markers, including Collagen1 and smooth muscle actin. Further analysis of these cells revealed colocalized expression of collagen and yes-associated protein (YAP; a marker associated with bile duct proliferation/fibrosis) and an increased production of interleukin-6 and tumor necrosis factor-α. Moreover, treatment with verteporfin, a YAP inhibitor, significantly reduced expression of fibrosis markers. In summary, these results suggest that c-Src has a critical role in cell fate determination during endodermal commitment of human iPSCs, and its alteration in early liver development in human may lead to increased production of abnormal YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis. Stem Cells 2019;37:306-317.

Hdac1 Regulates Differentiation of Bipotent Liver Progenitor Cells During Regeneration via Sox9b and Cdk8.

BACKGROUND & AIMS: Upon liver injury in which hepatocyte proliferation is compromised, liver progenitor cells (LPCs), derived from biliary epithelial cells (BECs), differentiate into hepatocytes. Little is known about the mechanisms of LPC differentiation. We used zebrafish and mouse models of liver injury to study the mechanisms. METHODS: We used transgenic zebrafish, Tg(fabp10a:CFP-NTR), to study the effects of compounds that alter epigenetic factors on BEC-mediated liver regeneration. We analyzed zebrafish with disruptions of the histone deacetylase 1 gene (hdac1) or exposed to MS-275 (an inhibitor of Hdac1, Hdac2, and Hdac3). We also analyzed zebrafish with mutations in sox9b, fbxw7, kdm1a, and notch3. Zebrafish larvae were collected and analyzed by whole-mount immunostaining and in situ hybridization; their liver tissues were collected for quantitative reverse transcription polymerase chain reaction. We studied mice in which hepatocyte-specific deletion of ß-catenin (Ctnnb1flox/flox mice injected with Adeno-associated virus serotype 8 [AAV8]-TBG-Cre) induces differentiation of LPCs into hepatocytes after a choline-deficient, ethionine-supplemented (CDE) diet. Liver tissues were collected and analyzed by immunohistochemistry and immunoblots. We performed immunohistochemical analyses of liver tissues from patients with compensated or decompensated cirrhosis or acute on chronic liver failure (n = 15). RESULTS: Loss of Hdac1 activity in zebrafish blocked differentiation of LPCs into hepatocytes by increasing levels of sox9b mRNA and reduced differentiation of LPCs into BECs by increasing levels of cdk8 mRNA, which encodes a negative regulator gene of Notch signaling. We identified Notch3 as the receptor that regulates differentiation of LPCs into BECs. Loss of activity of Kdm1a, a lysine demethylase that forms repressive complexes with Hdac1, produced the same defects in differentiation of LPCs into hepatocytes and BECs as observed in zebrafish with loss of Hdac1 activity. Administration of MS-275 to mice with hepatocyte-specific loss of ß-catenin impaired differentiation of LPCs into hepatocytes after the CDE diet. HDAC1 was expressed in reactive ducts and hepatocyte buds of liver tissues from patients with cirrhosis. CONCLUSIONS: Hdac1 regulates differentiation of LPCs into hepatocytes via Sox9b and differentiation of LPCs into BECs via Cdk8, Fbxw7, and Notch3 in zebrafish with severe hepatocyte loss. HDAC1 activity was also required for differentiation of LPCs into hepatocytes in mice with liver injury after the CDE diet. These pathways might be manipulated to induce LPC differentiation for treatment of patients with advanced liver diseases.

Strong carcinogenic stress response induction of preneoplastic cells positive for GST-P in the rat liver: Physiological mechanism for initiation.

AIMS: To identify experimental conditions that induce preneoplastic cells positive for glutathione S-transferase P-form (GST-P) in the rat liver by new approaches, and analysis of the mechanism of cancer initiation based on the findings. MAIN METHODS: The experimental protocols employed to induce GST-P+ preneoplastic cells in rat liver were as follows. Protocol 1: adult rats were fed basal diet containing 2-acetylaminofluorene (AAF, 0.02% by wt) and high concentrations of N-acetyl-l-cysteine (0.5%) over 10 weeks. Protocol 2: rats were subjected to partial hepatectomy (2/3PH), followed by an AAF (0.04%) diet for two more weeks. Vibratome-prepared liver sections were then immunostained for GST-P. KEY FINDINGS: GST-P was inducible in the rat liver in response to the strong carcinogenic stress by AAF in the two experimental protocols. When examined immunocytochemically with vibratome sections, the biliary tracts of hepatocytes, GST-P+ single hepatocytes and foci were heavily positive for the marker enzyme in addition to ordinary cytosolic staining of preneoplastic cell populations. The biliary tracts of hepatocytes were severely injured, and the excretory portions of GST-P+ single hepatocytes were significantly injured. SIGNIFICANCE: The cytotoxic action of AAF that give rise to the GST-P+ single hepatocytes was suggested to be an injury to the excretory pump(s) and the duct of hepatocytes. A new physiological mechanism was hypothesized for the induction of preneoplastic cell populations in the rat liver instead of a genetic mechanism.

Exposure of Mice to 1,2-Dichloropropane Induces CYP450-Dependent Proliferation and Apoptosis of Cholangiocytes.

1,2-Dichloropropane (1,2-DCP) has been used as a paint remover in the industry. The International Agency for Research on Cancer reclassified this compound recently to group 1 (carcinogenic to humans) based on epidemiological studies of cholangiocarcinoma among offset-color proof-printing workers exposed to 1,2-DCP in Japan. Two-year rodent carcinogenicity bioassays demonstrated that 1,2-DCP induced tumors in liver and lung, but not in bile duct. The present study was designed to assess the toxic effects of 1,2-DCP on proliferation and apoptosis in mice bile duct and the role of cytochrome P450 (CYP450) in any such effect. Male C57BL/6JJcl mice were cotreated or untreated with 1-aminobenzotriazole (1-ABT), a CYP450 inhibitor, and exposed to inhalation of 1,2-DCP at 0, 50, or 250 ppm alone, or at 0, 50, 250, or 1250 ppm 8 h/day for 4 weeks. Exposure to 1,2-DCP increased proliferation and apoptosis of cholangiocytes and induced severe hepatic damage, but had no effect on the lungs. Cotreatment with 1-ABT abrogated the effects of 1,2-DCP on proliferation and apoptosis of cholangiocytes. The results revealed that 1,2-DCP induces proliferation and apoptosis of cholangiocytes and that this effect is mediated through CYP450.

Secretion of Thioredoxin Peroxidase Protein of Cat Liver Fluke Opisthorchis felineus during Modeling of Experimental Opisthorchiasis.

Mechanisms of thioredoxin peroxidase secretion by Opisthorchis felineus were studied in vivo and in vitro. Specific antibodies were obtained and used for western blotting and immunohistochemical detection in Syrian hamster model of opisthorchiasis. Secreted thioredoxin peroxidase protein was accumulated in the worm incubation medium under conditions of oxidative stress and in bile duct cells of hamsters with chronic opisthorchiasis.

Human Hepatic HepaRG Cells Maintain an Organotypic Phenotype with High Intrinsic CYP450 Activity/Metabolism and Significantly Outperform Standard HepG2/C3A Cells for Pharmaceutical and Therapeutic Applications.

Conventional in vitro human hepatic models for drug testing are based on the use of standard cell lines derived from hepatomas or primary human hepatocytes (PHHs). Limited availability, interdonor functional variability and early phenotypic alterations in PHHs restrict their use, whilst standard cell lines such as HepG2 lack a substantial and variable set of liver-specific functions such as CYP450 activity. Alternatives include the HepG2-derivative C3A cells selected as a more differentiated and metabolically active hepatic phenotype. Human HepaRG cells are an alternative organotypic co-culture model of hepatocytes and cholangiocytes reported to maintain in vivo-like liver-specific functions, including intact Phase I-III drug metabolism. In this study, we compared C3A and human HepaRG cells using phenotypic profiling, CYP450 activity and drug metabolism parameters to assess their value as hepatic models for pre-clinical drug testing or therapeutics. Compared with C3As, HepaRG co-cultures exhibit a more organotypic phenotype, including evidence of hepatic polarity with the strong expression of CYP3A4, the major isoform involved in the metabolism of over 60% of marketed drugs. Significantly greater CYP450 activity and expression of CYP1A2, CYP2E1 and CYP3A4 genes in HepaRG cells (comparable with that of human liver tissue) was demonstrated. Moreover, HepaRG cells also preferentially expressed the hepatic integrin α5 ß1 - an important modulator of cell behaviour including growth and survival, differentiation and polarity. Drug metabolite profiling of phenacetin (CYP1A2) and testosterone (CYP3A4) using LC-MS/MS and HPLC, respectively, revealed that HepaRGs had more intact (Phase I-II) metabolism profile. Thus, HepaRG cells significantly outperform C3A cells for the potential pharmaceutical and therapeutic applications.

The cystic fibrosis transmembrane conductance regulator controls biliary epithelial inflammation and permeability by regulating Src tyrosine kinase activity.

In the liver, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) regulates bile secretion and other functions at the apical membrane of biliary epithelial cells (i.e., cholangiocytes). CF-related liver disease is a major cause of death in patients with CF. CFTR dysfunction affects innate immune pathways, generating a para-inflammatory status in the liver and other epithelia. This study investigates the mechanisms linking CFTR to toll-like receptor 4 activity. We found that CFTR is associated with a multiprotein complex at the apical membrane of normal mouse cholangiocytes, with proteins that negatively control Rous sarcoma oncogene cellular homolog (Src) activity. In CFTR-defective cholangiocytes, Src tyrosine kinase self-activates and phosphorylates toll-like receptor 4, resulting in activation of nuclear factor kappa-light-chain-enhancer of activated B cells and increased proinflammatory cytokine production in response to endotoxins. This Src/nuclear factor kappa-light-chain-enhancer of activated B cells-dependent inflammatory process attracts inflammatory cells but also generates changes in the apical junctional complex and loss of epithelial barrier function. Inhibition of Src decreased the inflammatory response of CF cholangiocytes to lipopolysaccharide, rescued the junctional defect in vitro, and significantly attenuated endotoxin-induced biliary damage and inflammation in vivo (Cftr knockout mice). CONCLUSION: These findings reveal a novel function of CFTR as a regulator of toll-like receptor 4 responses and cell polarity in biliary epithelial cells; this mechanism is pathogenetic, as shown by the protective effects of Src inhibition in vivo, and may be a novel therapeutic target in CF-related liver disease and other inflammatory cholangiopathies. (Hepatology 2016;64:2118-2134).

PKCα regulates TMEM16A-mediated Cl⁻ secretion in human biliary cells.

TMEM16A is a newly identified Ca(2+)-activated Cl(-) channel in biliary epithelial cells (BECs) that is important in biliary secretion. While extracellular ATP stimulates TMEM16A via binding P2 receptors and increasing intracellular Ca(2+) concentration ([Ca(2+)]i), the regulatory pathways have not been elucidated. Protein kinase C (PKC) contributes to ATP-mediated secretion in BECs, although its potential role in TMEM16A regulation is unknown. To determine whether PKCα regulates the TMEM16A-dependent membrane Cl(-) transport in BECs, studies were performed in human biliary Mz-cha-1 cells. Addition of extracellular ATP induced a rapid translocation of PKCα from the cytosol to the plasma membrane and activation of whole cell Ca(2+)-activated Cl(-) currents. Currents demonstrated outward rectification and reversal at 0 mV (properties consistent with TMEM16A) and were inhibited by either molecular (siRNA) or pharmacologic (PMA or Gö6976) inhibition of PKCα. Intracellular dialysis with recombinant PKCα activated Cl(-) currents with biophysical properties identical to TMEM16A in control cells but not in cells after transfection with TMEM16A siRNA. In conclusion, our studies demonstrate that PKCα is coupled to ATP-stimulated TMEM16A activation in BECs. Targeting this ATP-Ca(2+)-PKCα signaling pathway may represent a therapeutic strategy to increase biliary secretion and promote bile formation.

Knockout of histidine decarboxylase decreases bile duct ligation-induced biliary hyperplasia via downregulation of the histidine decarboxylase/VEGF axis through PKA-ERK1/2 signaling.

Histidine is converted to histamine by histidine decarboxylase (HDC). We have shown that cholangiocytes 1) express HDC, 2) secrete histamine, and 3) proliferate after histamine treatment via ERK1/2 signaling. In bile duct-ligated (BDL) rodents, there is enhanced biliary hyperplasia, HDC expression, and histamine secretion. This studied aimed to demonstrate that knockdown of HDC inhibits biliary proliferation via downregulation of PKA/ERK1/2 signaling. HDC(-/-) mice and matching wild-type (WT) were subjected to sham or BDL. After 1 wk, serum, liver blocks, and cholangiocytes were collected. Immunohistochemistry was performed for 1) hematoxylin and eosin, 2) intrahepatic bile duct mass (IBDM) by cytokeratin-19, and 3) HDC biliary expression. We measured serum and cholangiocyte histamine levels by enzyme immunoassay. In total liver or cholangiocytes, we studied: 1) HDC and VEGF/HIF-1α expression and 2) PCNA and PKA/ERK1/2 protein expression. In vitro, cholangiocytes were stably transfected with shRNA-HDC plasmids (or control). After transfection we evaluated pPKA, pERK1/2, and cholangiocyte proliferation by immunoblots and MTT assay. In BDL HDC(-/-) mice, there was decreased IBDM, PCNA, VEGF, and HDC expression compared with BDL WT mice. Histamine levels were decreased in BDL HDC(-/-). BDL HDC(-/-) livers were void of necrosis and inflammation compared with BDL WT. PKA/ERK1/2 protein expression (increased in WT BDL) was lower in BDL HDC(-/-) cholangiocytes. In vitro, knockdown of HDC decreased proliferation and protein expression of PKA/ERK1/2 compared with control. In conclusion, loss of HDC decreases BDL-induced biliary mass and VEGF/HIF-1α expression via PKA/ERK1/2 signaling. Our data suggest that HDC is a key regulator of biliary proliferation.

Infiltration of inflammatory cells expressing mitochondrial proteins around bile ducts and in biliary epithelial layer may be involved in the pathogenesis in primary biliary cirrhosis.

AIMS: Serum antimitochondrial antibodies are characteristic in most patients with primary biliary cirrhosis (PBC); however, the significance of antimitochondrial antibodies in the pathogenesis of PBC remains unclear. We examined the extent and types of mitochondrial protein-expressing inflammatory cells and its association with deregulated autophagy of mitochondria in biliary epithelial lesions in PBC. METHODS: We examined the expression of pyruvate dehydrogenase complex-E2 component and a mitochondrial protein cytochrome c oxidase, subunit I in inflammatory cells in livers taken from patients with PBC (n=35) and control livers (n=64) including primary sclerosing cholangitis. Mitochondrial protein-expressing inflammatory cells were characterised by double immunofluorescence with surface markers. RESULTS: Infiltration of mitochondrial protein-expressing inflammatory cells was mainly observed in portal tracts and in the biliary epithelial layer and around the damaged small bile ducts in PBC. The extent of infiltration in portal tracts was significantly higher in PBC and early stage of chronic viral hepatitis than normal livers. The extent of infiltration around bile ducts and in biliary epithelial layer was significantly higher in early stage of PBC than control livers. Mitochondrial protein-expressing inflammatory cells included (1) CD68 and/or myeloperoxidase -positive monocytes/macrophages and (2) CD79a, CD38, CD138, IgM-positive and/or IgG-positive plasma cells. Colocalised expression of pyruvate dehydrogenase complex-E2 component and autophagy marker light chain 3ß was observed in the inflammatory cells. CONCLUSIONS: Mitochondrial protein-expressing inflammatory cells infiltrating around the damaged bile ducts and in biliary epithelial layers may be closely associated with the pathogenesis of bile duct lesion in PBC.

A novel keratin18 promoter that drives reporter gene expression in the intrahepatic and extrahepatic biliary system allows isolation of cell-type specific transcripts from zebrafish liver.

Heritable and acquired biliary disorders are an important cause of acute and chronic human liver disease. Biliary development and physiology have been studied extensively in rodent models and more recently, zebrafish have been used to uncover pathogenic mechanisms and potential therapies for these conditions. Here we report development of novel transgenic lines labeling the intrahepatic and extrahepatic biliary system of zebrafish larvae that can be used for lineage tracing and isolation of biliary-specific RNAs from mixed populations of liver cells. We show that GFP expression driven by a 4.4 kilobase promoter fragment from the zebrafish keratin18 (krt18) gene allows visualization of all components of the developing biliary system as early as 3 days post-fertilization. In addition, expression of a ribosomal fusion protein (EGFP-Rpl10a) in krt18:TRAP transgenic fish allows for enrichment of translated biliary cell mRNAs via translating ribosome affinity purification (TRAP). Future studies utilizing these reagents will enhance our understanding of the morphologic and molecular processes involved in biliary development and disease.

Inhibition of metalloprotease hyperactivity in cystic cholangiocytes halts the development of polycystic liver diseases.

OBJECTIVE: Polycystic liver diseases (PCLDs) are genetic disorders characterised by progressive bile duct dilatation and/or cyst development. Their pathogenesis is a consequence of hyperproliferation, hypersecretion and microRNA alterations in cholangiocytes. Here we evaluate the role of matrix metalloproteases (MMPs) in the hepatic cystogenesis of PCLDs. DESIGN: Metalloprotease activity was measured by microfluorimetric assays in normal and polycystic cholangiocyte cultures from humans and rats, and gene expression by real time quantitative PCR. The role of cytokines, oestrogens and growth factors present in the cystic fluid of PCLD patients was evaluated for MMP activity. The MMP inhibitor marimastat was examined for cystic expansion in vitro and in polycystic kidney (PCK) rats. RESULTS: Polycystic human and rat cholangiocytes displayed increased MMP activity, which was associated with increased mRNA levels of different MMPs. Interleukin (IL)-6 and IL-8, and 17ß-oestradiol, all stimulated MMP activity in human cholangiocytes. The presence of antibodies against IL-6 and/or IL-8 receptor/s inhibited baseline MMP hyperactivity of polycystic human cholangiocytes but had no effect on normal human cholangiocytes. MMP-3 was overexpressed in cystic cholangiocytes from PCLD human and PCK rat livers by immunohistochemistry. Marimastat reduced MMP hyperactivity of polycystic human and rat cholangiocytes and blocked the cystic expansion of PCK cholangiocytes cultured in three-dimensions. Chronic treatment of 8-week-old PCK rats with marimastat inhibited hepatic cystogenesis and fibrosis. CONCLUSIONS: PCLDs are associated with cholangiocyte MMP hyperactivity resulting from autocrine/paracrine stimulation by IL-6 and IL-8. Inhibition of this MMP hyperactivity with marimastat decreased hepatic cystogenesis in vitro and in an animal model of PCLD, offering a potential therapeutic tool.

Carcinogenic Potential of Biliary Epithelium of Congenital Choledochal Cyst Model in Rats: A Special Reference to HDAC Expression.

BACKGROUND/AIMS: Pancreaticobiliary maljunction (PBM) is well known to have carcinogenic potential. The goal of this study was to clarify the carcinogenic potential of the epithelium of choledochal cyst particularly by paying attention of HDAC1 and COX-2 expressions. METHODOLOGY: Six-week male Lewis rats were divided into two groups; 1) single dose of dibutyltin dichloride (8 mg/kg) was administered intravenously (DBTC group), 2) saline administration was done with the same manner (control group). Morphological changes of biliary tract and expressions of carcinogenesis-related genes, Ki67, HDAC1 and cyclooxygenase-2 (COX-2) were examined immunohistochemically after administration. RESULTS: In the DBTC group, the bile duct had been gradually dilated on day 3 after administration and the biliary epithelium of dilated bile duct was papillary proliferated on day 7. Ki67 and COX-2 expressions gradually increased and reached the highest value of 6.0% and 3.6% on day 14, respectively. HDAC1 expression increased at the early postoperative period prior to other oncogene, and reached the highest level of 15% on day 7. CONCLUSIONS: These findings suggested that HDAC1 played an important role in carcinogenesis of PBM through the regulation of COX-2.

PGE(2) induces MUC2 and MUC5AC expression in human intrahepatic biliary epithelial cells via EP4/p38MAPK activation.

BACKGROUND: MUC2 and MUC5AC overproduction is considered to be associated with hepatolithiasis and related to inflammation. However, mechanisms underlying MUC upregulation under inflammatory stimulation in human intrahepatic biliary epithelial cells (HIBECs) are not completely understood. MATERIAL AND METHODS: Expression of MUC2 and MUC5AC mRNA in HIBECs was detected by real-time PCR. Expression of COX-2, EP4, and phosphorylated ERK, JNK and p38MAPK protein was detected by Western blot. Concentrations of PGE2, IL-1ß and TNF-α in cell culture supernatants were measured using the Quantikine Elisa kit. RESULTS: COX-2 expression as well as PGE2 production in HIBECs was upregulated significantly by LPS, which was completely blocked by either TLR4 antagonist or NFκB inhibitor. Selective COX-2 inhibitor suppressed LPS-induced MUC2 and MUC5AC mRNA expression remarkably. Exogenous PGE2 increased MUC2 and MUC5AC mRNA expression in a dosage-dependent manner independent of IL-1ß and TNF-α. PGE2 receptor EP4 agonist elevated MUC2 and MUC5AC expression, whereas EP4 antagonist had the opposite effect. Expression of phosphorylated p38MAPK was upregulated by exogenous PGE2, and p38MAPK inhibitor reduced MUC2 and MUC5AC expression in HIBECs. In addition, it was found that levels of PGE2, MUC2 and MUC5AC in bile samples from the hepatic ducts affected by intrahepatic stones were significantly higher than those from the unaffected hepatic ducts of patients with hepatolithiasis. CONCLUSIONS: Our findings indicate that PGE2 induces MUC2 and MUC5AC expression in HIBECs via EP4-p38MAPK signaling.

Protective effect of gadolinium chloride on early warm ischemia/reperfusion injury in rat bile duct during liver transplantation.

BACKGROUND: Activation of Kupffer cell (KC) is acknowledged as a key event in the initiation and perpetuation of bile duct warm ischemia/reperfusion injury. The inhibitory effect of gadolinium chloride (GdCl(3)) on KC activation shows potential as a protective intervention in liver injury, but there is less research with regard to bile duct injury. METHODS: Sixty-five male Sprague-Dawley rats (200-250 g) were randomly divided into three experimental groups: a sham group (n = 15), a control group (n = 25), and a GdCl(3) group (n = 25). Specimen was collected at 0.5, 2, 6, 12 and 24 h after operation. Alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin (TBIL) of serum were measured. Tumor necrosis factor-α (TNF-α), Capase-3 activity and soluble Fas (sFas) were detected. The pathologic changes of bile duct were observed. Immunochemistry for bile duct Fas was performed. Apoptosis of bile duct cells was evaluated by the terminal UDP nick end labeling assay. RESULTS: GdCl(3) significantly decreased the levels of ALT, ALP and TBIL at 2, 6, 12, and 24 h, and increased serum sFas at 2, 6 and 12 h (P<0.05). TNF-α was lower in the GdCl(3) group than in the control group at 2, 6, 12 and 24 h (P<0.05). Preadministration of GdCl(3) significantly reduced the Caspase-3 activity and bile duct cell apoptosis at 2, 6, 12 and 24 h. After operation for 2, 6 and 12 h, the expression of Fas protein was lower in the GdCl(3) group than in the control group (P<0.05). CONCLUSIONS: GdCl(3) plays an important role in suppressing bile duct cell apoptosis, including decreasing ALT, ALP, TBIL and TNF-α; suppressing Fas-FasL-Caspase signal transduction during transplantation.

Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.

Although large cholangiocytes exert their functions by activation of cyclic adenosine 3',5'-monophosphate (cAMP), Ca(2+)-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1-H4HRs. H1HR acts by Gαq activating IP(3)/Ca(2+), whereas H2HR activates Gα(s) stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1-H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1-H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP(3) and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1-H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP(3) levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts.

Epidermal growth factor protects the apical junctional complexes from hydrogen peroxide in bile duct epithelium.

The tight junctions of bile duct epithelium form a barrier between the toxic bile and liver parenchyma. Disruption of tight junctions appears to have a crucial role in the pathogenesis of various liver diseases. In this study, we investigated the disruptive effect of hydrogen peroxide and the protective effect of epidermal growth factor (EGF) on the tight junctions and adherens junctions in the bile duct epithelium. Oxidative stress in NRC-1 and Mz-ChA-1 cell monolayers was induced by administration of hydrogen peroxide. Barrier function was evaluated by measuring electrical resistance and inulin permeability. Integrity of tight junctions, adherens junctions and the actin cytoskeleton was determined by imunofluorescence microscopy. Role of signaling molecules was determined by evaluating the effect of specific inhibitors. Hydrogen peroxide caused a rapid disruption of tight junctions and adherens junctions leading to barrier dysfunction without altering the cell viability. Hydrogen peroxide rapidly increased the levels of p-MLC (myosin light chain) and c-Src(pY418). ML-7 and PP2 (MLCK and Src kinase inhibitors) attenuated hydrogen peroxide-induced barrier dysfunction, tight junction disruption and reorganization of actin cytoskeleton. Pretreatment of cell monolayers with EGF ameliorated hydrogen peroxide-induced tight junction disruption and barrier dysfunction. The protective effect of EGF was abrogated by ET-18-OCH(3) and the Ro-32-0432 (PLCγ and PKC inhibitors). Hydrogen peroxide increased tyrosine phosphorylation of ZO-1, claudin-3, E-cadherin and ß-catenin, and pretreatment of cells with EGF attenuated tyrosine phosphorylation of these proteins. These results demonstrate that hydrogen peroxide disrupts tight junctions, adherens junctions and the actin cytoskeleton by an MLCK and Src kinase-dependent mechanism in the bile duct epithelium. EGF prevents hydrogen peroxide-induced tight junction disruption by a PLCγ and PKC-dependent mechanism.