Genetic cholestasis has been dissected through genetic investigation. The major PFIC genes are now described. ATP8B1 encodes FIC1, ABCB11 encodes BSEP, ABCB4 encodes MDR3, TJP2 encodes TJP2, NR1H4 encodes FXR, and MYO5B encodes MYO5B. The full spectra of phenotypes associated with mutations in each gene are discussed, along with our understanding of the disease mechanisms. Differences in treatment response and targets for future treatment are emerging.

We screened DNAs from 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in nine genes encoding components of ATP-binding cassette subfamily B (ABCB/MDR/TAP) by directly sequencing the entire applicable genomic regions except for repetitive elements. This approach identified 297 SNPs and 29 insertion/deletion polymorphisms among the nine genes. Of the 297 SNPs, 50 were identified in the ABCB1 gene, 14 in TAP], 35 in TAP2, 48 in ABCB4, 13 in ABCB7, 21 in ABCB8, 21 in ABCB9, 13 in ABCB10, and 82 in ABCB11. Thirteen were located in 5' flanking regions, 237 in introns, 37 in exons, and 10 in 3' flanking regions. These variants may contribute to investigations of possible correlations between genotypes and disease-susceptibility phenotypes or responsiveness to drug therapy.

Intrahepatic cholestasis, or impairment of bile flow, is an important manifestation of inherited and acquired liver disease. In recent years, human genetic and molecular studies have identified several genes, the disruption of which results in cholestasis. ATP8B1 (FIC1), ABCB11 (BSEP), and ABCB4 (MDR3) are disrupted in forms of progressive familial intrahepatic cholestasis (PFIC) and related disorders. Mutations in BAAT, TJP2 (ZO-2), and EPHX1 have been identified in patients with hypercholanemia. A CLDN1 mutation was recently reported in patients with ichthyosis, leukocyte vacuoles, alopecia and sclerosing cholangitis (ILVASC), and North American Indian childhood cirrhosis (NAIC) is associated with a missense mutation in CIRH1A. Alagille syndrome patients carry mutations in JAG1, and mutations in VPS33B have been identified in patients with arthrogryposis, renal dysfunction and cholestasis syndrome (ARC). Identification of these genes, and characterization of the proteins they encode, is enhancing our understanding of the biology of the enterohepatic circulation in health and disease.

Direct-acting antiviral drugs (DAAs) are currently replacing antiviral therapy for Hepatitis C infection. Treatment related side effects are even worse and the emergence of resistant viruses must be avoided because of the direct-antiviral action. Altogether it remains a challenge to take treatment decisions in a clinical setting with cost restrictions. Genetic host factors are hereby essential to implement an individualized treatment concept. In recent years results on different genetic variants have been published with a strong association with therapy response, fibrosis and treatment-related side effects. Polymorphisms of the IL28B gene were identified as accurate predictors for therapy response and spontaneous clearance of HCV infection and are already used for diagnostic decisions. For RBV-induced side effects, such as hemolytic anemia, associations to genetic variants of inosine triphosphatase (ITPA) were described and different SLC28 transporters for RBV-uptake have been successfully analyzed. Fibrosis progression has been associated with variants of Vitamin D receptor (VDR) and ABCB11 (bile salt export pump). Cirrhotic patients especially have a high treatment risk and low therapy response, so that personalized antiviral treatment is mandatory. This review focuses on different host genetic variants in the pathogenesis of Hepatitis C at the beginning of a new area of treatment.

Drug-induced cholestasis (DIC) is poorly understood and its preclinical prediction is mainly limited to assessing the compound's potential to inhibit the bile salt export pump (BSEP). Here, we evaluated two 3D spheroid models, one from primary human hepatocytes (PHH) and one from HepaRG cells, for the detection of compounds with cholestatic liability. By repeatedly co-exposing both models to a set of compounds with different mechanisms of hepatotoxicity and a non-toxic concentrated bile acid (BA) mixture for 8 days we observed a selective synergistic toxicity of compounds known to cause cholestatic or mixed cholestatic/hepatocellular toxicity and the BA mixture compared to exposure to the compounds alone, a phenomenon that was more pronounced after extending the exposure time to 14 days. In contrast, no such synergism was observed after both 8 and 14 days of exposure to the BA mixture for compounds that cause non-cholestatic hepatotoxicity. Mechanisms behind the toxicity of the cholestatic compound chlorpromazine were accurately detected in both spheroid models, including intracellular BA accumulation, inhibition of ABCB11 expression and disruption of the F-actin cytoskeleton. Furthermore, the observed synergistic toxicity of chlorpromazine and BA was associated with increased oxidative stress and modulation of death receptor signalling. Combined, our results demonstrate that the hepatic spheroid models presented here can be used to detect and study compounds with cholestatic liability.

OBJECTIVE

To determine if specific mutations were present in Asian patients with progressive familial intrahepatic cholestasis (PFIC) type 2 caused by defects in bile salt export pump (BSEP), encoded by ABCB11.

METHODS

A combination of denaturing high-performance liquid chromatography (DHPLC) and direct sequencing was used to screen ABCB11 mutations in 18 Taiwanese patients with low gamma-glutamyltransferase PFIC or benign recurrent intrahepatic cholestasis (BRIC). Polymorphisms were also analyzed in patients with PFIC (n = 21), neonatal cholestasis (n = 23), and control subjects (n = 88).

RESULTS

Seven mutations in 4 of 16 patients with PFIC from different families were detected by DHPLC, including M183V, V284L, R303K, R487H, W493X, G1004D, and 1145delC. G1004D was found in a patient with BRIC. L827I was found in another patient with neonatal cholestasis. Absent or defective BSEP staining was found in the liver of patients with mutations. Polymorphisms V444A and A865V, with an allele frequencies 75.6% and 0.6%, respectively, were found in our population. No differences were found between patients with cholestasis and control subjects.

CONCLUSIONS

One-fourth of Taiwanese patients with PFIC/BRIC had compound heterozygous or single heterozygous ABCB11 mutations without hot spots. All of the mutations were different from those detected in Western countries.

Born at 27 weeks gestation, a child of consanguineous parents of Pakistani origin required prolonged parenteral nutrition. She developed jaundice, with extensive fibrosis and architectural distortion at liver biopsy; jaundice resolved with supportive care. Serum γ-glutamyl transpeptidase values were within normal ranges. The bile acids in her plasma and urine were >85% unconjugated (non-amidated). Two genes encoding bile-acid amidation enzymes were sequenced. No mutations were found in BAAT, encoding bile acid-CoA : aminoacid N-acyl transferase. The patient was homozygous for the missense mutation c.1012C>> T in SLC27A5, predicted to alter a highly conserved amino-acid residue (p.H338Y) in bile acid-CoA ligase (BACL). She also was homozygous for the missense mutation c.1772A>> G in ABCB11, predicted to alter a highly conserved amino-acid residue (p.N591S) in bile salt export pump (BSEP). BACL is essential for reconjugation of bile acids deconjugated by gut bacteria, and BSEP is essential for hepatocyte-canaliculus export of conjugated bile acids. A female sibling born at term had the same bile-acid phenotype and SLC27A5 genotype, without clinical liver disease. She was heterozygous for the c.1772A>> G ABCB11 mutation. This is the first report of a mutation in SLC27A5. The amidation defect may have contributed to cholestatic liver disease in the setting of prematurity, parenteral nutrition, and homozygosity for an ABCB11 mutation.

BACKGROUND

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific disorder, which is characterized by raised serum bile acid level and potential adverse fetal outcome. Farnesoid X receptor (FXR), also known as a bile acid receptor, was found to be expressed in placenta with low level. Whether activation of FXR by specific agonists could regulate the pathogenesis of ICP is still unclear.

METHODS

A model of maternal cholestasis was induced by administration of 17α-ethynylestradiol (E2) in pregnant mice for 6 days. We explored the regulatory effect of WAY-362450 (W450), a highly selective and potent FXR agonist on placenta.

RESULTS

In this study, we demonstrated that administration of E2 increased bile acid levels in mouse serum, liver and amniotic fluid. Bile acid levels were significantly decreased after W450 treatment. W450 protected against the impairment of placentas induced by E2, including severe intracellular edema and apoptosis of trophoblasts. Moreover, W450 significantly induced the expressions of FXR target bile acid transport gene ATP-binding cassette, sub-family B (MDR/TAP), member 11 (Abcb11;Bsep) in placenta. W450 could also attenuate placental oxidative stress and increase the expressions of antioxidant enzymes Prdx1 and Prdx3.

CONCLUSIONS

In conclusion, our data demonstrated that FXR agonist W450 modulated bile acid balance and protected against placental oxidative stress. Thus, our results support that potent FXR agonists might represent promising drugs for the treatment of ICP.

In the present study MRP2/ABCC2 and BSEP/ABCB11 expression were investigated in sandwich cultured (SC) human and rat hepatocytes exposed to the proinflammatory cytokines. The investigation was also done in lipopolysaccharide (LPS)-treated rats. In SC human hepatocytes, both absolute protein and mRNA levels of MRP2/ABCC2 were significantly down-regulated by TNF-α, IL-6, or IL-1β. In contrast to mRNA decrease, which was observed for BSEP/ABCB11, the protein amount was significantly increased by IL-6 or IL-1β. A discrepancy between the change in BSEP/ABCB11 mRNA and protein levels was encountered in SC human hepatocytes treated with proinflammatory cytokines. In SC rat hepatocytes, Mrp2/Abcc2 mRNA was down-regulated by TNF-α and IL-6, whereas the protein level was decreased by all three cytokines. Down-regulations of both Bsep/Abcb11 mRNA and protein levels were found in SC rat hepatocytes exposed to TNF-α or IL-1β. Administration of LPS triggered the release of the proinflammatory cytokines and caused the decrease of Mrp2/Abcc2 and Bsep/Abcb11 protein in liver at 24 h post-treatment; however, the Mrp2 and Bsep protein levels rebounded at 48 h post-LPS treatment. In total, our results indicate that proinflammatory cytokines regulate the expression of MRP2/Mrp2 and BSEP/Bsep and for the first time demonstrate the differential effects on BSEP/Bsep expression between SC human and rat hepatocytes. Furthermore, the agreement between transporter regulation in vitro in SC rat hepatocytes and in vivo in LPS-treated rats during the acute response phase demonstrates the utility of in vitro SC hepatocyte models for predicting in vivo effects.

We describe a PFIC2 patient with a good response to ursodeoxycholic acid for 9 years. We found two novel ABCB11 gene mutations in the patient, i.e. I498T and 2098delA. The correlation of the patient's genotypes with the clinical course supports the existence of a phenotypic continuum between BRIC2 and PFIC2.

Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5>abcg2>abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2',7'-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11 activities, in accordance with low abcg2 and abcb11 transcript levels. Our data indicate that transporter expression and activity patterns in the different trout cell lines are irrespective of the tissue of origin, but are determined by factors of cell cultivation.

OBJECTIVE

PFIC2 is caused by mutations in ABCB11 encoding BSEP. In most cases affected children need liver transplantation that is thought to be curative. We report on two patients who developed recurrent normal GGT cholestasis mimicking primary BSEP disease, after liver transplantation.

METHODS

PFIC2 diagnosis was made in infancy in both patients on absence of canalicular BSEP immunodetection and on ABCB11 mutation identification. Liver transplantation was performed at age 9 (patient 1) and 2.8 (patient 2) years without major complications. Cholestasis with normal GGT developed 17 and 4.8years after liver transplantation, in patient 1 and patient 2, respectively, during an immunosuppression reduction period.

RESULTS

Liver biopsies showed canalicular cholestasis, giant hepatocytes, and slight lobular fibrosis, without evidence of rejection or biliary complications. An increase in immunosuppression resulted in cholestasis resolution in only one patient. Both patients developed atrial fibrillation, and one melanonychia. The newborn of patient 1 developed transient neonatal normal GGT cholestasis. Immunofluorescence staining of normal human liver sections with patient's sera, collected at the time of cholestasis, and using an anti-human IgG antibody to detect serum antibodies, showed reactivity to a canalicular epitope, likely to be BSEP. Indeed, Western blot analysis showed that patient 2 serum recognized rat Bsep.

CONCLUSIONS

Allo-immune mediated BSEP dysfunction may occur after liver transplantation in PFIC2 patients leading to a PFIC2 like phenotype. Extrahepatic features and/or offspring transient neonatal cholestasis of possible immune mediated mechanisms, may be associated. Increasing the immunosuppressive regimen might be an effective therapy.

OBJECTIVE

Our aims were to identify and functionally characterize coding region nonsynonymous single nucleotide polymorphisms in the hepatic efflux transporter, bile salt export pump (BSEP; ABCB11), and to assess interindividual variability in BSEP expression.

METHODS

We identified 24 single nucleotide polymorphisms, including nine nonsynonymous variants, in ABCB11 from genomic DNA of approximately 250 ethnically diverse healthy individuals using denaturing high-performance liquid chromatography analysis and DNA sequencing. Wild type and variant BSEP were generated and functionally characterized for taurocholate transport activity in vitro in HeLa cells using a recombinant vaccinia-based method. BSEP expression was assessed by real-time mRNA analysis, western blot analysis, and immunofluorescence confocal microscopy.

RESULTS

For the most part, polymorphisms were rare and ethnic-dependent. In vitro functional studies revealed several rare variants, including 616A>G, 1674G>C, 1772A>G, and 3556G>A, to be associated with significantly impaired taurocholate transport activity while the 890A>G variant trended towards impaired function but was not statistically significant. The 3556G>A variant was associated with reduced cell surface to total protein expression compared with wild-type BSEP. Expression of BSEP by mRNA and protein analysis was determined from a bank of human liver samples. Wide interindividual variability was noted in both mRNA (19-fold) and protein (31-fold) expression levels. The common variant 1331T>C was associated with significantly reduced hepatic BSEP mRNA levels.

CONCLUSIONS

Accordingly, our study indicates there are functionally relevant polymorphisms in ABCB11 which may be of potential relevance in the predisposition to acquired liver disorders such as drug-induced cholestasis.

Genetic susceptibility in the causation of gallbladder diseases was recognized as early as 1937. A major gallstone susceptibility locus (Lith1) was identified in 1995 by quantitative trait locus mapping in mice. Two attractive positional and functional candidate genes in LXRA and ABCB11 are located in this interval. ABCB11 is associated with progressive familial cholestasis. This study was undertaken to investigate LXRA and ABCB11 as candidate genes for gallstone disease in humans. Eight hundred and ten patients who underwent cholecystectomy for symptomatic gallstone disease (median age of onset, 50 years) were compared with 718 sex-matched control individuals. Control individuals were sonographically free of gallstones. Haplotype tagging and all known coding single nucleotide polymorphisms (SNPs) were genotyped for ABCB11 (n=29) and LXRA (n=10). The investigated high-risk patient sample provides a power of greater than 80% for the detection of odds ratios down to 1.55. No evidence of association of the two genes in the single point tagging markers, coding variants or in the sliding window haplotype analysis was detected (all nominal single-point P values>or=.08). In conclusion, in the investigated German sample, no evidence of association of ABCB11 and LXRA to gallstone susceptibility was detected. The gallstone trait is not allelic to progressive familial cholestasis at the ABCB11 locus. Systematic fine mapping of the Lith1 region is required to identify the causative genetic variants for gallstone in mice and humans.

The nuclear receptors liver X receptor (LXR) alpha and farnesoid X receptor (FXR) are positive and negative regulators of cholesterol 7alpha-hydroxylase (CYP7A1) transcription, respectively. To clarify their roles in the regulation of CYP7A1 in mice, we investigated mRNA expression of their target genes in the livers of C57BL/6 mice fed the following five diets for 2 weeks: a standard diet, cholic acid, cholesterol, cholesterol+high fat, or an atherogenic diet (cholic acid+cholesterol+high fat). The mRNA level of ATP-binding cassette transporter (ABC) A1 gene, one of LXRalpha target genes, significantly increased on the diets containing cholic acid and/or cholesterol+high fat, but not on the diet containing cholesterol alone. On the other hand, the mRNA levels of the FXR target genes ABCB11, ABCC2, and short heterodimer partner increased only on the diet containing cholic acid with or without cholesterol+high fat. Surprisingly, cholesterol alone or cholesterol+high fat did not affect CYP7A1 mRNA level, whereas cholic acid with or without cholesterol+high fat greatly reduced the level. Thus, in the atherogenic diet-fed mice, cholic acid component is needed for the FXR activation, and FXR dominantly regulates CYP7A1 transcription.

Long-Evans Cinnamon (LEC) rats exhibit a genetic defect in Atp7b gene, which is homologous to the human Wilson's disease gene, resulting in an inability to mobilize copper from the liver. This study was undertaken to gain insight into the relationship between liver copper accumulation and plasma lipid profile, circulating lipoprotein composition, hepatic sterol metabolism and biliary lipid secretion rates in 12-week-old LEC rats compared to control Long-Evans rats. Concomitant with hepatic copper deposition, LEC rats displayed increased content of triglycerides (TGs), free cholesterol (FC) and cholesteryl ester (CE) in the liver. Hepatic concentrations of malondialdehyde (MDA), an index of lipid peroxidation were also significantly elevated in LEC rats (50%). This steatosis was associated with aberrant microsomal apolipoprotein (apo) B-100 and microsomal triglyceride transfer protein (MTP) content, hypotriglyceridemia, hypocholesterolemia and abnormalities in both circulating lipoprotein composition and size. Atypical hepatobiliary sterol metabolism was established by the assessment of the activity of key intracellular enzymes for cholesterol homeostasis, which demonstrated, with respect to controls, a 40% reduction in 3-hydroxy-3-methylglutaryl coenzyme A reductase, a 30% reduction in cholesterol 7alpha-hydroxylase, and a 54% reduction in acyl CoA:cholesterol acyltransferase. During a 6-h biliary drainage, a decline in the bile acid output was recorded and might be linked to the low protein expression of the bile salt export pump (BSEP or ABCB11). Our data emphasize the crucial role of copper balance in hepatic sterol homeostasis and lipoprotein metabolism in LEC rats. Additional studies are needed to delineate the mechanisms of these disorders.

The bile salt export pump (BSEP, ABCB11) is the primary transporter of bile acids from the hepatocyte to the biliary system. This rate-limiting step in bile formation is essential to the formation of bile salt dependent bile flow, the enterohepatic circulation of bile acids, and the digestion of dietary fats. Mutations in BSEP are associated with cholestatic diseases such as progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), drug-induced cholestasis, and intrahepatic cholestasis of pregnancy. Development of clinical therapies for these conditions necessitates a clear understanding of the cell biology of biosynthesis, trafficking, and transcriptional and translational regulation of BSEP. This chapter will focus on the molecular and cell biological aspects of this critical hepatic membrane transporter.

Cholestasis is characterised by impaired bile secretion and accumulation of bile salts in the organism. Hereditary cholestasis is a heterogeneous group of rare autosomal recessive liver disorders, which are characterised by intrahepatic cholestasis, pruritus, and jaundice and caused by defects in genes related to the secretion and transport of bile salts and lipids. Phenotypic manifestation is highly variable, ranging from progressive familial intrahepatic cholestasis (PFIC)-with onset in early infancy and progression to end-stage liver disease-to a milder intermittent mostly nonprogressive form known as benign recurrent intrahepatic cholestasis (BRIC). Cases have been reported of initially benign episodic cholestasis that subsequently transitions to a persistent progressive form of the disease. Therefore, BRIC and PFIC seem to represent two extremes of a continuous spectrum of phenotypes that comprise one disease. Thus far, five representatives of PFIC (named PFIC1-5) caused by pathogenic mutations present in both alleles of ATP8B1, ABCB11, ABCB4, TJP2, and NR1H4 have been described. In addition to familial intrahepatic cholestasis, partial defects in ATP8B1, ABCB11, and ABCB4 predispose patients to drug-induced cholestasis and intrahepatic cholestasis in pregnancy. This review summarises the current knowledge of the clinical manifestations, genetics, and molecular mechanisms of these diseases and briefly outlines the therapeutic options, both conservative and invasive, with an outlook for future personalised therapeutic strategies.

Riociguat is a new soluble guanylate cyclase stimulator under development for pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension. So far, the interaction potential of riociguat with other drugs is nearly unknown. Therefore, we assessed in vitro the potency of riociguat to inhibit important drug metabolising enzymes (cytochrome P450 (CYP) 3A4, CYP2C19, and CYP2D6) and drug transporters (P-glycoprotein (P-gp/ABCB1), breast cancer resistance protein (BCRP/ABCG2), and organic anion transporting polypeptides (OATP) 1B1 and 1B3). In addition we evaluated its substrate characteristics for P-gp, BCRP, and the multidrug resistance-associated protein 1 (MRP1/ABCC1). We also assessed riociguat's inducing properties on important drug metabolising enzymes and transporters and investigated its ability to activate the pregnane-X-receptor (PXR). Riociguat was identified as a weak to moderate inhibitor of P-gp (f2-value: 11.7 ± 4.8 μM), BCRP (IC50 = 46.2 ± 20.3 μM), OATP1B1 (IC50 = 34.1 ± 3.15 μM), OATP1B3 (IC50 = 50.3 ± 7.5 μM), CYP2D6 (IC50 = 12.4 ± 0.74 μM), and CYP2C19 (IC50 = 46.1 ± 7.14 μM). Furthermore, it induced mRNA expression of BCRP/ABCG2 (3-fold at 20 μM) and to a lesser extent of CYP3A4 (2.3-fold at 20 μM), UGT1A4, and ABCB11. The only weak inducing properties were confirmed by weak activation of PXR. Considering its systemic concentrations its interaction potential as a perpetrator drug seems to be low. In contrast, our data suggest that riociguat is a P-gp substrate and might therefore act as a victim drug when co-administered with strong P-gp inductors or inhibitors.

OBJECTIVE

The bile acid-activated farnesoid X receptor (FXR) is a nuclear receptor regulating bile acid, glucose and cholesterol homeostasis. Obeticholic acid (OCA), a promising drug for the treatment of non-alcoholic steatohepatitis (NASH) and type 2 diabetes, activates FXR. Mouse studies demonstrated that FXR activation by OCA alters hepatic expression of many genes. However, no data are available on the effects of OCA in the human liver. Here we generated gene expression profiles in human precision cut liver slices (hPCLS) after treatment with OCA.

METHODS

hPCLS were incubated with OCA for 24 h. Wild-type or FXR(-/-) mice received OCA or vehicle by oral gavage for 7 days.

RESULTS

Transcriptomic analysis showed that well-known FXR target genes, including NR0B2 (SHP), ABCB11 (BSEP), SLC51A (OSTα) and SLC51B (OSTβ), and ABCB4 (MDR3) are regulated by OCA in hPCLS. Ingenuity pathway analysis confirmed that 'FXR/RXR activation' is the most significantly changed pathway upon OCA treatment. Comparison of gene expression profiles in hPCLS and mouse livers identified 18 common potential FXR targets. ChIP-sequencing in mouse liver confirmed FXR binding to IR1 sequences of Akap13, Cgnl1, Dyrk3, Pdia5, Ppp1r3b and Tbx6.

CONCLUSIONS

Our study shows that hPCLS respond to OCA treatment by upregulating well-known FXR target genes, demonstrating its suitability to study FXR-mediated gene regulation. We identified six novel bona-fide FXR target genes in both mouse and human liver. Finally, we discuss a possible explanation for changes in high or low density lipoprotein observed in NASH and primary biliary cholangitis patients treated with OCA based on the genomic expression profile in hPCLS.

Retinoic acid (RA) and bile acids share common roles in regulating lipid homeostasis and insulin sensitivity. In addition, the receptor for RA (retinoid x receptor) is a permissive partner of the receptor for bile acids, farnesoid x receptor (FXR/NR1H4). Thus, RA can activate the FXR-mediated pathway as well. The current study was designed to understand the effect of all-trans RA on bile acid homeostasis. Mice were fed an all-trans RA-supplemented diet and the expression of 46 genes that participate in regulating bile acid homeostasis was studied. The data showed that all-trans RA has a profound effect in regulating genes involved in synthesis and transport of bile acids. All-trans RA treatment reduced the gene expression levels of Cyp7a1, Cyp8b1, and Akr1d1, which are involved in bile acid synthesis. All-trans RA also decreased the hepatic mRNA levels of Lrh-1 (Nr5a2) and Hnf4α (Nr2a1), which positively regulate the gene expression of Cyp7a1 and Cyp8b1. Moreover, all-trans RA induced the gene expression levels of negative regulators of bile acid synthesis including hepatic Fgfr4, Fxr, and Shp (Nr0b2) as well as ileal Fgf15. All-trans RA also decreased the expression of Abcb11 and Slc51b, which have a role in bile acid transport. Consistently, all-trans RA reduced hepatic bile acid levels and the ratio of CA/CDCA, as demonstrated by liquid chromatography-mass spectrometry. The data suggest that all-trans RA-induced SHP may contribute to the inhibition of CYP7A1 and CYP8B1, which in turn reduces bile acid synthesis and affects lipid absorption in the gastrointestinal tract.

Farnesoid X receptor (FXR) is a bile acid-sensing nuclear receptor that controls bile acid homeostasis. It has been suggested that downregulation of FXR contributes to the pathogenesis of an inherited disorder of bile secretion caused by mutations in ATP8B1. We have investigated the relationship between ATP8B1 knockdown and FXR downregulation in the human hepatoblastoma cell line HepG2. Transfection of HepG2 cells with ATP8B1 small interfering RNA (siRNA) duplexes led to a 60% reduction in the endogenous levels of ATP8B1 mRNA and protein and a concomitant decrease in FXR mRNA and protein content, as well as in FXR phosphorylation. This decrease was accompanied by a marked reduction in mRNA levels of a subset of FXR targets, such as bile salt export pump (ABCB11), small heterodimer partner, and uridine 5'-diphosphate-glucuronosyltransferase. ATP8B1 inhibition specifically targeted FXR since mRNA expression of other prominent nuclear receptors, such as pregnane X receptor and constitutive androstane receptor, or liver-enriched transcription factors, such as hepatocyte nuclear factor 1alpha (HNF-1alpha) and HNF-4alpha, was not altered. The expression of other key genes involved in bile acid synthesis, detoxification, and transport also remained unchanged upon ATP8B1 knockdown. Supporting the specificity of the effect, siRNA-mediated silencing of ABCB11, whose defect is associated with another inherited disorder of bile secretion, did not affect FXR expression. Treatment with the synthetic FXR agonist GW4064 was able to partially neutralize ATP8B1 siRNA-mediated FXR downregulation and fully counteract inhibition of FXR target genes. Collectively these findings indicate that ATP8B1 knockdown specifically downregulates FXR, and this action can be circumvented by treatment with FXR agonists.

BACKGROUND

Cholangiocarcinoma (CC) is increasing in incidence, but its pathogenesis remains poorly understood. Chronic inflammation of the bile duct and cholestasis are major risk factors, but most cases in the West are sporadic. Genetic polymorphisms in biliary transporter proteins have been implicated in benign biliary disease and, in the case of progressive familial cholestasis, have been associated with childhood onset of CC. In the current study, five biologically plausible candidate genes were investigated: ABCB11 (BSEP), ABCB4 (MDR3), ABCC2 (MRP2), ATP8B1 (FIC1) and NR1H4 (FXR).

METHODS

DNA was collected from 172 Caucasian individuals with confirmed CC. A control cohort of healthy Caucasians was formed. Seventy-three SNPs were selected using the HapMap database to capture genetic variation around the five candidate loci. Genotyping was undertaken with a competitive PCR-based system. Confirmation of Hardy-Weinberg equilibrium and Cochran-Armitage trend testing were performed using PLINK. Haplotype frequencies were compared using haplo.stats.

RESULTS

All 73 SNPs were in Hardy-Weinberg equilibrium. Four SNPs in ABCB11 were associated with altered susceptibility to CC, including the V444A polymorphism, but these associations did not retain statistical significance after Bonferroni correction for multiple testing. Haplotype analysis of the genotyped SNPs in ATP8B1 identified significant differences in frequencies between cases and controls (global p value of 0.005).

CONCLUSIONS

Haplotypes in ATP8B1 demonstrated a significant difference between CC and control groups. There was a trend towards significant association of V444A with CC. Given the biological plausibility of polymorphisms in ABCB11 and ATP8B1 as risk modifiers for CC, further study in a validation cohort is required.