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Background & Aims: Cholangiopathies are chronic liver diseases in which damaged cholangiocytes trigger a proinflammatory and profibrotic reaction. The nuclear vitamin D receptor (VDR) is highly expressed in cholangiocytes and exerts immune-regulatory functions in these cells. In the present study, we examined the protective function of VDR and other vitamin D signaling pathways in chronic cholangiopathy and cholangiocytes., Methods: Vdr was invalidated in Abcb4 knockout mice, a widely used animal model of chronic cholangiopathy. The impact of vitamin D signaling on cholangiopathy features was examined in vivo and in cholangiocytes (primary and cell lines)., Results: Cholangiopathy features (i.e, cholestasis, ductular reaction and fibrosis) were aggravated in Vdr;Abcb4 double knockout mice compared to the Abcb4 simple knockout, and associated with an overexpression of proinflammatory factors. The proinflammatory phenotype of cholangiocytes was also exacerbated following VDR silencing in vitro. The expression of proinflammatory factors and the severity of cholangiopathy were reduced in the double knockout mice treated with the vitamin D analog calcipotriol or with vitamin D. In vitro, the inflammatory response to TNFα was significantly reduced by calcipotriol in biliary cells silenced for VDR, and this effect was abolished by co-silencing the plasma membrane receptor of vitamin D, protein disulfide-isomerase A3 (PDIA3)., Conclusions: Our results demonstrate an anti-inflammatory role of VDR signaling in cholangiocytes and cholangiopathy. They also provide evidence for PDIA3-mediated anti-inflammatory effects of vitamin D and vitamin D analog in these settings., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nuclear receptors (NR), the largest family of transcription factors, control many physiological and pathological processes. To gain insight into hepatic NR and their potential as therapeutic targets in cholestatis, we determined their expression in individual cell types of the mouse liver in normal and cholestatic conditions. Hepatocytes, cholangiocytes, hepatic stellate cells (HSC), sinusoidal endothelial cells (SEC) and Kupffer cells (KC) were isolated from the liver of mice with acute or chronic cholestasis (i.e. bile duct-ligated or Abcb4 -/- mice, respectively) and healthy controls. The expression of 43 out of the 49 NR was evidenced by RT-qPCR in one or several liver cell types. Expression of four NR was restricted to non-parenchymal liver cells. In normal conditions, NR were expressed at higher levels in individual cell types when compared to total liver. Half of the NR expressed in the liver had maximal expression in non-parenchymal cells. After bile duct ligation, NR mRNA changes occurred mostly in non-parenchymal cells and mainly consisted in down-regulations. In Abcb4 -/- mice, NR mRNA changes were equally frequent in hepatocytes and non-parenchymal cells. Essentially down-regulations were found in hepatocytes, HSC and cholangiocytes, as opposed to up-regulations in SEC and KC. While undetectable in total liver, Vdr expression was up-regulated in all non-parenchymal cells in Abcb4 -/- mice. In conclusion, non-parenchymal liver cells are a major site of NR expression. During cholestasis, NR expression is markedly altered mainly by down-regulations, suggesting major changes in metabolic activity. Thus, non-parenchymal cells are important new targets to consider in NR-directed therapies., (Copyright © 2017. Published by Elsevier B.V.)

The gallbladder stores and concentrates bile between meals. Gallbladder motor function is regulated by bile acids via the membrane bile acid receptor, TGR5, and by neurohormonal signals linked to digestion, for example, cholecystokinin and FGF15/19 intestinal hormones, which trigger gallbladder emptying and refilling, respectively. The cycle of gallbladder filling and emptying controls the flow of bile into the intestine and thereby the enterohepatic circulation of bile acids. The gallbladder also largely contributes to the regulation of bile composition by unique absorptive and secretory capacities. The gallbladder epithelium secretes bicarbonate and mucins, which both provide cytoprotection against bile acids. The reversal of fluid transport from absorption to secretion occurs together with bicarbonate secretion after feeding, predominantly in response to an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent pathway triggered by neurohormonal factors, such as vasoactive intestinal peptide. Mucin secretion in the gallbladder is stimulated predominantly by calcium-dependent pathways that are activated by ATP present in bile, and bile acids. The gallbladder epithelium has the capacity to absorb cholesterol and provides a cholecystohepatic shunt pathway for bile acids. Changes in gallbladder motor function not only can contribute to gallstone disease, but also subserve protective functions in multiple pathological settings through the sequestration of bile acids and changes in the bile acid composition. Cholecystectomy increases the enterohepatic recirculation rates of bile acids leading to metabolic effects and an increased risk of nonalcoholic fatty liver disease, cirrhosis, and small-intestine carcinoid, independently of cholelithiasis. Among subjects with gallstones, cholecystectomy remains a priority in those at risk of gallbladder cancer, while others could benefit from gallbladder-preserving strategies. © 2016 American Physiological Society. Compr Physiol 6:1549-1577, 2016., (Copyright © 2016 John Wiley & Sons, Inc.)

E-cadherin is a cell-to-cell adhesion molecule involved in epithelial cell behavior, tissue formation and cancer suppression. In the liver, E-cadherin is expressed by hepatocytes and biliary epithelial cells. However, the exact role of E-cadherin in hepatic pathophysiology remains largely unknown. Recently, specific loss of E-cadherin in liver epithelial cells has been shown to favor periportal fibrosis, periportal inflammation and liver cancer progression, suggesting that E-cadherin is a central liver protector., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Background: Nuclear receptors (NRs) form a family of 48 members. NRs control hepatic processes such as bile acid homeostasis, lipid metabolism and mechanisms involved in fibrosis and inflammation. Due to their central role in the regulation of hepatoprotective mechanisms, NRs are promising therapeutic targets in cholestatic disorders., Key Messages: NRs can be classified into five different physiological clusters. NRs from the 'bile acids and xenobiotic metabolism' and from the 'lipid metabolism and energy homeostasis' clusters are strongly expressed in the liver. Furthermore, NRs from these clusters, such as farnesoid X receptor α (FXRα), pregnane X receptor (PXR) and peroxisome proliferator-activated receptors (PPARs), have been associated with the pathogenesis and the progression of cholestasis. The latter observation is also true for vitamin D receptor (VDR), which is barely detectable in the whole liver, but has been linked to cholestatic diseases. Involvement of VDR in cholestasis is ascribed to a strong expression in nonparenchymal liver cells, such as biliary epithelial cells, Kupffer cells and hepatic stellate cells. Likewise, NRs from other physiological clusters with low hepatic expression, such as estrogen receptor α (ERα) or reverse-Erb α/β (REV-ERB α/β), may also control pathophysiological processes in cholestasis., Conclusions: In this review, we will describe the impact of individual NRs on cholestasis. We will then discuss the potential role of these transcription factors as therapeutic targets., (2015 S. Karger AG, Basel.)

Unlabelled: The ABCB4 transporter mediates phosphatidylcholine (PC) secretion at the canalicular membrane of hepatocytes and its genetic defects cause biliary diseases. Whereas ABCB4 shares high sequence identity with the multidrug transporter, ABCB1, its N-terminal domain is poorly conserved, leading us to hypothesize a functional specificity of this domain. A database of ABCB4 genotyping in a large series of patients was screened for variations altering residues of the N-terminal domain. Identified variants were then expressed in cell models to investigate their biological consequences. Two missense variations, T34M and R47G, were identified in patients with low-phospholipid-associated cholelithiasis or intrahepatic cholestasis of pregnancy. The T34M and R47G mutated proteins showed no or minor defect, respectively, in maturation and targeting to the apical membrane, in polarized Madin-Darby Canine Kidney and HepG2 cells, whereas their stability was similar to that of wild-type (WT) ABCB4. By contrast, the PC secretion activity of both mutants was markedly decreased. In silico analysis indicated that the identified variants were likely to affect ABCB4 phosphorylation. Mass spectrometry analyses confirmed that the N-terminal domain of WT ABCB4 could undergo phosphorylation in vitro and revealed that the T34M and R47G mutations impaired such phosphorylation. ABCB4-mediated PC secretion was also increased by pharmacological activation of protein kinases A or C and decreased by inhibition of these kinases. Furthermore, secretion activity of the T34M and R47G mutants was less responsive than that of WT ABCB4 to protein kinase modulators., Conclusion: We identified disease-associated variants of ABCB4 involved in the phosphorylation of its N-terminal domain and leading to decreased PC secretion. Our results also indicate that ABCB4 activity is regulated by phosphorylation, in particular, of N-terminal residues., (© 2014 by the American Association for the Study of Liver Diseases.)

Unlabelled: Alterations in apical junctional complexes (AJCs) have been reported in genetic or acquired biliary diseases. The vitamin D nuclear receptor (VDR), predominantly expressed in biliary epithelial cells in the liver, has been shown to regulate AJCs. The aim of our study was thus to investigate the role of VDR in the maintenance of bile duct integrity in mice challenged with biliary-type liver injury. Vdr(-/-) mice subjected to bile duct ligation (BDL) displayed increased liver damage compared to wildtype BDL mice. Adaptation to cholestasis, ascertained by expression of genes involved in bile acid metabolism and tissue repair, was limited in Vdr(-/-) BDL mice. Furthermore, evaluation of Vdr(-/-) BDL mouse liver tissue sections indicated altered E-cadherin staining associated with increased bile duct rupture. Total liver protein analysis revealed that a truncated form of E-cadherin was present in higher amounts in Vdr(-/-) mice subjected to BDL compared to wildtype BDL mice. Truncated E-cadherin was also associated with loss of cell adhesion in biliary epithelial cells silenced for VDR. In these cells, E-cadherin cleavage occurred together with calpain 1 activation and was prevented by the silencing of calpain 1. Furthermore, VDR deficiency led to the activation of the epidermal growth factor receptor (EGFR) pathway, while EGFR activation by EGF induced both calpain 1 activation and E-cadherin cleavage in these cells. Finally, truncation of E-cadherin was blunted when EGFR signaling was inhibited in VDR-silenced cells., Conclusion: Biliary-type liver injury is exacerbated in Vdr(-/-) mice by limited adaptive response and increased bile duct rupture. These results indicate that loss of VDR restricts the adaptation to cholestasis and diminishes bile duct integrity in the setting of biliary-type liver injury., (Copyright © 2013 The Authors. HEPATOLOGY published by Wiley on behalf of the American Association for the Study of Liver Diseases.)

Background & Aims: Patients with cystic fibrosis (CF) have poorly defined defects in biliary function. We evaluated the effects of cystic fibrosis transmembrane conductance regulator (CFTR) deficiency on the enterohepatic disposition of bile acids (BAs)., Methods: Bile secretion and BA homeostasis were investigated in Cftr(tm1Unc) (Cftr-/-) and CftrΔF508 (ΔF508) mice., Results: Cftr-/- and ΔF508 mice did not grow to normal size, but did not have liver abnormalities. The gallbladders of Cftr-/- mice were enlarged and had defects in emptying, based on (99m)technetium-mebrofenin scintigraphy or post-prandial variations in gallbladder volume; gallbladder contraction in response to cholecystokinin-8 was normal. Cftr-/- mice had abnormal gallbladder bile and duodenal acidity, and overexpressed the vasoactive intestinal peptide-a myorelaxant factor for the gallbladder. The BA pool was larger in Cftr-/- than wild-type mice, although there were no differences in fecal loss of BAs. Amounts of secondary BAs in portal blood, liver, and bile of Cftr-/- mice were much lower than normal. Expression of genes that are induced by BAs, including fibroblast growth factor-15 and BA transporters, was lower in the ileum but higher in the gallbladders of Cftr-/- mice, compared with wild-type mice, whereas enzymes that synthesize BA were down-regulated in livers of Cftr-/- mice. This indicates that BAs underwent a cholecystohepatic shunt, which was confirmed using cholyl-(Ne-NBD)-lysine as a tracer. In Cftr-/- mice, cholecystectomy reversed most changes in gene expression and partially restored circulating levels of secondary BAs. The ΔF508 mice overexpressed vasoactive intestinal peptide and had defects in gallbladder emptying and in levels of secondary BAs, but these features were less severe than in Cftr-/- mice., Conclusions: Cftr-/- and CftrΔF508 mice have defects in gallbladder emptying that disrupt enterohepatic circulation of BAs. These defects create a shunt pathway that restricts the amount of toxic secondary BAs that enter the liver., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Vitamin D through the vitamin D nuclear receptor (VDR) plays a key role in mineral ion homeostasis. The liver is central in vitamin D synthesis, however the direct involvement of the vitamin D-VDR axis on the liver remains to be evaluated. In this review, we will describe vitamin D metabolism and the mechanisms of homeostatic control. We will also address the associations between the vitamin D-VDR axis and pathological liver entities, such as non-alcoholic fatty liver disease, autoimmune liver disease, viral hepatitis and liver cancer. The link between liver diseases and the vitamin D-VDR axis will be discussed in light of evidences arising from in vitro and in vivo studies. Finally, we will consider the therapeutic potential of the vitamin D-VDR axis in liver diseases., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

The biliary epithelium is organized as a single layer of biliary epithelial cells lining the biliary tree. Biliary epithelial cells have three major biological functions: protection, secretion and proliferation. These functions are all controlled by nuclear receptors. The biliary tree conveys bile, a complex fluid containing toxics such as endotoxins, from the liver to the duodenum. Active protection against endotoxins can be elicited by the vitamin D receptor or the farnesoid X receptor (FXR), thus avoiding constant inflammation of the biliary epithelium. Anti-inflammatory activities may be triggered by PPAR-α and -γ, which are also able to inhibit the deleterious effect of bacterial products. Secretion, a major function of biliary epithelial cells, is mainly regulated by circulating factors. Luminal factors, such as bile salts, may also control fluid secretion by classical intracellular pathways, membrane receptors or nuclear receptors. FXR or the glucocorticoid receptor have indeed been shown to increase the expression of genes encoding membrane-bound proteins that participate in biliary epithelial cell secretion. Biliary epithelial cells are quiescent cells that are able to proliferate in pathophysiological settings. Inhibition of estrogen receptor signaling decreases pathological biliary epithelial cell proliferation. Furthermore, progesterone, through the progesterone receptor, increases biliary epithelial cells proliferation. Taken together these observations suggest that nuclear receptors are involved in the control of biliary epithelial cell biology. A better delineation of the specific biliary epithelial cell functions controlled by nuclear receptors may shed light on potential therapeutic molecular targets of cholangiopathies., (Copyright © 2011 S. Karger AG, Basel.)

Loss of NM23-H1 expression correlates with the degree of metastasis and with unfavorable clinical prognosis in several types of human carcinoma. However, the mechanistic basis for the metastasis suppressor function of NM23-H1 is obscure. We silenced NM23-H1 expression in human hepatoma and colon carcinoma cells and methodologically investigated effects on cell-cell adhesion, migration, invasion, and signaling linked to cancer progression. NM23-H1 silencing disrupted cell-cell adhesion mediated by E-cadherin, resulting in β-catenin nuclear translocation and T-cell factor/lymphoid-enhancing factor-1 transactivation. Further, NM23-H1 silencing promoted cellular scattering, motility, and extracellular matrix invasion by promoting invadopodia formation and upregulating several matrix metalloproteinases (MMP), including membrane type 1 MMP. In contrast, silencing the related NM23-H2 gene was ineffective at promoting invasion. NM23-H1 silencing activated proinvasive signaling pathways involving Rac1, mitogen-activated protein kinases, phosphatidylinositol 3-kinase (PI3K)/Akt, and src kinase. Conversely, NM23-H1 was dispensable for cancer cell proliferation in vitro and liver regeneration in NM23-M1 null mice, instead inducing cellular resistance to chemotherapeutic drugs in vitro. Analysis of NM23-H1 expression in clinical specimens revealed high expression in premalignant lesions (liver cirrhosis and colon adenoma) and the central body of primary liver or colon tumors, but downregulation at the invasive front of tumors. Our findings reveal that NM23-H1 is critical for control of cell-cell adhesion and cell migration at early stages of the invasive program in epithelial cancers, orchestrating a barrier against conversion of in situ carcinoma into invasive malignancy., (© 2010 AACR.)

Backgrounds & Aims: Under normal conditions, the biliary tract is a microbial-free environment. The absence of microorganisms has been attributed to various defense mechanisms that include the physicochemical and signaling actions of bile salts. Here, we hypothesized that bile salts may stimulate the expression of a major antimicrobial peptide, cathelicidin, through nuclear receptors in the biliary epithelium., Methods: The expression of cathelicidin was analyzed in human liver samples by immunostaining and reverse-transcription quantitative polymerase chain reaction. The regulation of cathelicidin expression by the endogenous bile salt, chenodeoxycholic acid, and by the therapeutic bile salt, ursodeoxycholic acid (UDCA), was assessed in human biliary epithelial cells in which endogenous nuclear receptor expression was blunted by siRNA or dominant-negative strategies., Results: In the human liver, biliary epithelial cells show intense immunoreactivity for cathelicidin and for the vitamin D receptor. In cultured biliary epithelial cells, chenodeoxycholic acid and UDCA induce cathelicidin expression through 2 different nuclear receptors: the farnesoid X receptor and the vitamin D receptor, respectively. Importantly, vitamin D further increases the induction of cathelicidin expression by both bile salts. In a prototypical inflammatory biliary disease (ie, primary biliary cirrhosis), we document that hepatic expressions of the vitamin D receptor and of cathelicidin significantly increased with UDCA therapy., Conclusions: Our results indicate that bile salts may contribute to biliary tract sterility by controlling epithelial cell innate immunity. They further suggest that in inflammatory biliary diseases, which involve bacterial factors, a strategy systematically combining UDCA with vitamin D would increase therapeutic efficacy.

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) anchors and regulates apical membrane proteins in epithelia. EBP50 is inducible by estrogen and may affect cell proliferation, although this latter function remains unclear. The goal of this study was to determine whether EBP50 was implicated in the ductular reaction that occurs in liver disease. EBP50 expression was examined in normal human liver, in human cholangiopathies (ie, cystic fibrosis, primary biliary cirrhosis, and primary sclerosing cholangitis), and in rats subjected to bile-duct ligation. The regulation of EBP50 by estrogens and its impact on proliferation were assessed in both bile duct-ligated rats and Mz-Cha-1 human biliary epithelial cells. Analyses of cell isolates and immunohistochemical studies showed that in normal human liver, EBP50 is expressed in the canalicular membranes of hepatocytes and, together with ezrin and cystic fibrosis transmembrane conductance regulator, in the apical domains of cholangiocytes. In both human cholangiopathies and bile duct-ligated rats, EBP50 was redistributed to the cytoplasmic and nuclear compartments. EBP50 underwent a transient increase in rat cholangiocytes after bile-duct ligation, whereas such expression was down-regulated in ovariectomized rats. In addition, in Mz-Cha-1 cells, EBP50 underwent up-regulation and intracellular redistribution in response to 17beta-estradiol, whereas its proliferation was inhibited by siRNA-mediated EBP50 knockdown. These results indicate that both the expression and distribution of EBP50 are regulated by estrogens and contribute to the proliferative response in biliary epithelial cells.

Background & Aims: In the past decade, there has been a rising incidence of hepatocellular carcinoma (HCC) and a progressive increase in HCC-related mortality in the United States and Western Europe. The poor survival of patients with HCC is largely related to the lack of reliable tools for early diagnosis., Methods: We have applied proteomics tools to the comparative analysis of protein profiles between HCC and adjacent nontumor tissues as a means for discovering novel molecular markers., Results: Forty-seven protein spots that showed reproducible variation were identified by mass spectrometry, corresponding to 23 distinct genes. A positive correlation between transcript and protein level variations was observed for only 7 out of the 23 genes. Proteolytic cleavage accounted for the discrepancies between messenger RNA and protein level changes for 7 genes including calreticulin, PDIA3, PDI, and GRP78. We detected a fragment of each of these 4 endoplasmic reticulum proteins in the culture supernatant of the PLC-PRF5 hepatoma cell line, suggesting that their cleavage leads to release of selected cleaved products in the extracellular compartment. We also detected calreticulin and PDIA3 cleavage products in sera of patients with HCC. A statistically highly significant difference in calreticulin and PDIA3 fragment serum levels between patients with HCC and healthy individuals was observed. Amounts of calreticulin and PDIA3 fragments were also significantly different between patients with HCC and at-risk patients (patients with chronic hepatitis or cirrhosis)., Conclusions: Specific isoforms in general and cleavage products in particular should therefore be further evaluated as new markers for HCC.

Recent reports in patients with PFIC1 have indicated that a gene defect in ATP8B1 could cause deregulations in bile salt transporters through decreased expression and/or activity of FXR. This study aimed to: (1) define ATP8B1 expression in human hepatobiliary cell types, and (2) determine whether ATP8B1 defect affects gene expressions related to bile secretion in these cells. ATP8B1 expression was detected by RT-PCR in hepatocytes and cholangiocytes isolated from normal human liver and gallbladder. ATP8B1 mRNA levels were 20- and 200-fold higher in bile duct and gallbladder epithelial cells, respectively, than in hepatocytes. RT-PCR analyses of the liver from two patients with PFIC1, one with PFIC2, one with biliary atresia, showed that, compared to normal liver, hepatic expressions of FXR, SHP, CYP7A1, ASBT were decreased at least by 90% in all cholestatic disorders. In contrast, NTCP transcripts were less decreased (by < or = 30% vs. 97%) in PFIC1 as compared with other cholestatic disorders, while BSEP transcripts, in agreement with BSEP immunohistochemical signals, were normal or less decreased (by 50% vs. 97%). CFTR hepatic expression was decreased (by 80%), exclusively in PFIC1, while bile duct mass was not reduced, as ascertained by cytokeratin-19 immunolabeling. In Mz-ChA-2 human biliary epithelial cells, a significant decrease in CFTR expression was associated with ATP8B1 invalidation by siRNA. In conclusion, cholangiocytes are a major site ofATP8B1 hepatobiliary expression. A defect of ATP8B1 along with CFTR downregulation can impair the contribution of these cells to bile secretion, and potentially explain the extrahepatic cystic fibrosis-like manifestations that occur in PFIC1.

Vasoactive intestinal peptide receptor-1 (VPAC1) is the high-affinity receptor of vasoactive intestinal peptide (VIP), a major regulator of bile secretion. To better define the level at which VPAC1 stimulates bile secretion, we examined its expression in the different cell types participating in bile formation (i.e., hepatocytes, bile duct, and gallbladder epithelial cells). Because VPAC1 expression was previously shown to be regulated by nuclear receptors, we tested the hypothesis that it may be regulated by the farnesoid X receptor (FXR). Quantitative RT-PCR and immunoblot analyses of cell isolates indicated that VPAC1 is expressed in all three cell types lining the human biliary tree, with predominant expression in the gallbladder. In primary cultures of human gallbladder epithelial cells, VIP induced cAMP production and chloride secretion. Analysis of the VPAC1 gene revealed the presence of potential FXR response element sequences, and both FXR and RXRalpha expressions were detected in gallbladder epithelial cells. In these cells, the FXR pharmacological agonist GW4064 upregulated VPAC1 expression in a dose-dependent manner, and this effect was antagonized by the RXRalpha ligand, 9-cis retinoic acid. Chenodeoxycholate activated endogenous FXR in gallbladder epithelial cells, as ascertained by electromobility shift assay and upregulation of the FXR target gene, small heterodimer partner. Chenodeoxycholate also provoked an increase in VPAC1 mRNA and protein content in these cells. In conclusion, FXR agonists may increase gallbladder fluid secretion through transcriptional activation of VPAC1, which may contribute to the regulation of bile secretion by bile salts and to a protective effect of FXR pharmacological agonists in gallstone disease.

Biliary function is essential for intestinal absorption of fat, homeostasis of cholesterol and elimination of diverse metabolic end-products. Bile is elaborated in hepatocyte canaliculi and modified by cholangiocytes through both secretion and absorption processes. The main determinant of bile formation is an osmotic filtration process resulting from active transport of bile acids and other osmotic solutes. Most of the membrane transporters ensuring bile formation have now been identified. The expression of these membrane transporters is regulated in particular through transcriptional mechanisms under the control of nuclear receptors activated by ligands, such as bile acids, which act as endogenous steroids synthesized from cholesterol in hepatocytes. Monogenic cholestatic diseases illustrate the key role of membrane transporters in biliary function. Bile acids are potent modulators of transporters and thus trigger an adaptative response to cholestasis. The extent of this adaptative response could explain the compelling phenotypic variability of cholestatic diseases in childhood and adults. The firstline medical treatment is currently ursodeoxycholic acid. In case of failure of this medical treatment, liver transplantation is required. Recent progress in the molecular pathogenesis of bile formation and cholestatic liver diseases is expected to provide the design for drugs targeted to the molecular abnormalities responsible of cholestatic diseases.

Fluid and ion secretion in the gallbladder is mainly triggered by the intracellular second messenger cAMP. We examined the action of bile salts on the cAMP-dependent pathway in the gallbladder epithelium. Primary cultures of human gallbladder epithelial cells were exposed to agonists of the cAMP pathway and/or to bile salts. Taurochenodeoxycholate and tauroursodeoxycholate increased forskolin-induced cAMP accumulation to a similar extent, without affecting cAMP basal levels. This potentiating effect was abrogated after PKC inhibition, whereas both taurochenodeoxycholate and tauroursodeoxycholate induced PKC-alpha and -delta translocation to cell membranes. Consistent with a PKC-mediated stimulation of cAMP production, the expression of six adenylyl cyclase isoforms, including PKC-regulated isoforms 5 and 7, was identified in human gallbladder epithelial cells. cAMP-dependent chloride secretion induced by isoproterenol, a beta-adrenergic agonist, was significantly increased by taurochenodeoxycholate and by tauroursodeoxycholate. In conclusion, endogenous and therapeutic bile salts via PKC regulation of adenylyl cyclase activity potentiate cAMP production in the human gallbladder epithelium. Through this action, bile salts may increase fluid secretion in the gallbladder after feeding.

Hepatic resection in cirrhotic patients is associated with impaired liver regeneration and poor clinical outcome. Because experimental cirrhosis is associated with hepatic cell hypoxia, we herein investigated whether hypoxia might alter the mechanisms of liver regeneration in the cirrhotic liver. Cirrhosis was induced by diethylnitrosamine in rats. Immunohistochemistry was performed to assess hepatocellular hypoxia and proliferation 24 hours after a two-thirds partial hepatectomy (PH) in cirrhotic and control rats. Cultured hepatocytes and myofibroblastic hepatic stellate cells were submitted to hypoxia using anaerobic jars. Hepatocyte growth factor (HGF) and c-Met expressions were determined by reverse transcriptase-polymerase chain reaction, Northern blot, and Western blot. In control rats, hypoxia was restricted to perivenular hepatocytes, and PH induced a marked increase in hepatocyte proliferation and in liver HGF expression, whereas c-Met expression remained unchanged. In cirrhotic rats, hypoxia was detected virtually in all of the hepatocytes, and PH induced no significant change in hepatocyte proliferation and in liver HGF expression, whereas c-Met expression was decreased as compared to normal livers. In vitro, the expression of HGF in myofibroblastic hepatic stellate cells and of c-Met in hepatocytes underwent a dramatic decrease under hypoxia. Our results suggest that hepatocellular hypoxia causes inhibition of HGF (and of c-Met)-mediated proliferation and thereby might contribute to liver regeneration failure in cirrhotic liver.