The primary transporter responsible for bile salt secretion is the bile salt export pump (BSEP, ABCB11), a member of the ATP-binding cassette (ABC) superfamily, which is located at the bile canalicular apical domain of hepatocytes. In humans, BSEP deficiency results in several different genetic forms of cholestasis, which include progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), as well as other acquired forms of cholestasis such as drug-induced cholestasis (DIC) and intrahepatic cholestasis of pregnancy (ICP). Because bile salts play a pivotal role in a wide range of physiologic and pathophysiologic processes, regulation of BSEP expression has been a subject of intense research. The authors briefly describe the molecular characteristics of BSEP and then summarize what is known about its role in the pathogenesis of genetic and acquired cholestatic disorders, emphasizing experimental observations from animal models and cell culture in vitro systems.
Cholestasis; progressive familial intrahepatic cholestasis; bile salt export pump (BSEP) mutations; polymorphisms; ATP-binding cassette (ABC) transporters; trafficking; recycling; ubiquitination
Progressive familial intrahepatic cholestasis, type 2 (PFIC2), characterized by cholestasis in infancy that may progress to cirrhosis, is caused by mutation in ABCB11, which encodes bile salt export pump (BSEP). We correlated histopathologic, immunohistochemical, and ultrastructural features in PFIC2 with specific mutations and clinical course. Twelve patients with clinical PFIC2 and ABCB11 mutations were identified, and 22 liver biopsy and explant specimens were assessed. All had hepatocellular cholestasis; most had canalicular bile plugs. At least 1 specimen from every patient had centrizonal/sinusoidal fibrosis, often with periportal fibrosis. Neonatal hepatitis-like features (inflammation, giant cells, necrosis) varied. In 2 of the 5 patients with paired specimens obtained > 6 months apart, lobular and portal fibrosis worsened. Transmission electron microscopy (EM) in all 9 patients studied showed canalicular dilatation, microvilli loss, abnormal mitochondrial internal structure, and varying intra-canalicular accumulation of finely granular bile. Canalicular staining for BSEP was absent in 10 patients and present in 2 patients, 1 of whom had intermittent symptoms. ABCB11 sequencing of all patients identified 6 novel and 10 previously described mutations, with nonsense, missense, and/or noncoding mutations in the 10 patients without immunohistochemically demonstrable BSEP. Missense and/or noncoding mutations were identified in the 2 patients with demonstrable BSEP, whose clinical course was more indolent. Mutations ending ABCB11 transcription appear linked, through hepatocellular necrosis and fibrosis, to worse outcome. In conclusion, light microscopy and electron microscopy findings in clinical PFIC2 can support diagnosis, but are variable and nonspecific. Therefore, no correlation between specific mutations and histopathology is yet possible.
PFIC2; progressive familial intrahepatic cholestasis; bile salt export pump; BSEP; ABCB11; neonatal hepatitis
PFIC II is a subtype of progressive familial intrahepatic cholestasis (PFIC) that is associated with mutations in the ABCB11 gene encoding the bile salt export pump (BSEP). However it is not known how these mutations cause this disease. To evaluate these mechanisms, we introduced seven PFIC II–associated missense mutations into rat Bsep and assessed their effects on Bsep membrane localization and transport function in MDCK and Sf9 cells, respectively. Five mutations, G238V, E297G, G982R, R1153C, and R1268Q, prevented the protein from trafficking to the apical membrane, and E297G, G982R, R1153C, and R1268Q also abolished taurocholate transport activity, possibly by causing Bsep to misfold. Mutation C336S affected neither Bsep transport activity nor the apical trafficking of Bsep, suggesting that this mutation alone may not cause this disease. D482G did not affect the apical expression but partially decreased the transport activity of Bsep. Mutant G238V was rapidly degraded in both MDCK and Sf9 cells, and proteasome inhibitor resulted in intracellular accumulation of this and other mutants, suggesting proteasome-mediated degradation plays an important role in expression of these PFIC II mutants. Our studies highlight the heterogeneous nature of PFIC II mutations and illustrate the significance of these mutations in the function and expression of Bsep.
Progressive familial intrahepatic cholestasis (PFIC) type 2 is caused by mutations in ABCB11, which encodes bile salt export pump (BSEP). We report a Thai female infant who presented with progressive cholestatic jaundice since 1 mo of age, with normal serum γ-glutamyltransferase. Immunohistochemical staining of the liver did not demonstrate BSEP along the canaliculi, while multidrug resistance protein 3 was expressed adequately. Novel mutations in ABCB11, a four-nucleotide deletion in exon 3, c.90_93delGAAA, and a single-nucleotide insertion in exon 5, c.249_250insT, were identified, with confirmation in her parents. These mutations were predicted to lead to synthesis of truncated forms of BSEP. Immunostaining and mutation analysis thus established the diagnosis of PFIC type 2.
ABCB11; Bile salt export pump; Progressive familial intrahepatic cholestasis
Partial External Biliary Diversion (PEBD) is a surgical intervention to treat children with Progressive Familial Intrahepatic Cholestasis (PFIC) and Alagille syndrome (AGS). PEBD can reduce disease progression, and examining the alterations in biliary lipid composition may be a prognostic factor for outcome.
Biliary lipid composition and the clinical course of AGS and PFIC patients were examined before and after PEBD.
Pre-PEBD bile from AGS patients had greater chenodeoxycholic/cholic acid (CDCA/CA), bile salt, cholesterol and phospholipid concentrations than PFIC patients. AGS patients, and PFIC patients with familial intrahepatic cholestasis 1 (FIC1) genotype, responded better to PEBD than PFIC patients with bile salt export protein (BSEP) genotype. After successful PEBD, AGS patients have higher biliary lipid concentrations than PFIC patients and PEBD also increases biliary phospholipid concentrations in FIC1 patients.
Both AGS and FIC1 patients can benefit from PEBD, and preserved biliary phospholipid concentrations may be associated with better outcomes post-PEBD.
The bile salt export pump (Bsep) represents the major bile salt transport system at the canalicular membrane of hepatocytes. When examined in model cell lines, genetic mutations in the BSEP gene impair its targeting and transport function, contributing to the pathogenesis of PFIC II. PFIC II mutations are known to lead to a deficiency of BSEP in human hepatocytes, suggesting that PFIC II mutants are unstable and degraded in the cell. To investigate this further, we have characterized the impact of several PFIC II mutations on the processing and stability of rat Bsep. G238V, D482G, G982R, R1153C and R1286Q all retain Bsep to the endoplasmic reticulum (ER) to different extents. Except for R1153C, the PFIC II mutants are degraded with varying half-lives. G238V and D482G are partially misfolded and can be stabilized by low temperature and glycerol. The proteasome provides the major degradation pathway for the PFIC II mutants, while the lysosome also contributes to the degradation of D482G. The PFIC II mutants appear to be more heavily ubiquitinated compared with the wild-type (wt) Bsep, and their ubiquitination is increased by the proteasome inhibitors. Overexpression of several E3 ubiquitin ligases, which are involved in ER-associated degradation (ERAD), lead to the decrease of both mutant and wt Bsep. Gene knockdown studies revealed that the ERAD E3s Rma1 and TEB4 contribute to the degradation of G238V, while HRD1 contributes to the degradation of a mutant lacking the lumenal glycosylation domain (ΔGly). Furthermore we present evidence that G982R weakly associates with various components of the ER quality control system. These data together demonstrate that the PFIC II mutants and ΔGly are degraded by the ERAD pathway.
The liver specific bile salt export pump (BSEP) is crucial for bile-acid dependent bile flow at the apical membrane. BSEP, a member of the family of structurally related ATP-Binding Cassette (ABC) proteins, is composed of 12 transmembrane segments (TMS) and 2 large cytoplasmic nucleotide binding domains (NBD). The regulation of trafficking of BSEP to and from the cell surface is not well understood, but is believed to play an important role in cholestatic liver diseases such as primary familial intrahepatic cholestasis type 2 (PFIC2). To address this issue, BSEP endocytosis was studied by immunofluorescence and a cell surface ELISA endocytosis reporter system using a chimera of the interleukin 2 receptor α (previously referred to as Tac) and the C-terminal tail of BSEP (TacCterm). An autonomous endocytosis motif in the carboxyl cytoplasmic terminus of BSEP was identified. We define this endocytic motif by site-directed mutagenesis as a canonical tyrosine-based motif 1310YYKLV1314 (Yxx∅). When expressed in HEK293T cells TacCterm is constitutively internalized via a dynamin- and clathrin-dependent pathway. Mutation of the Y1310Y1311 amino acids in TacCterm and in full length human BSEP blocks the internalization. Subsequent sequence analysis reveals this motif to be highly conserved between the closely related ABCB subfamily members that mediate ATP-dependent transport of broad substrate specificity.
Our results indicate constitutive internalization of BSEP is clathrin-mediated and dependent on the tyrosine-based endocytic motif at the C-terminal end of BSEP.
bile acid transporter; trafficking motif
Bile salts, cholesterol and phosphatidylcholine are secreted across the canalicular membrane of hepatocytes into bile by ATP-binding cassette (ABC) transporters. Secretion of bile salts by ABCB11 is essential for bile flow and for absorption of lipids and fat-soluble vitamins. ABCG5 and ABCG8 eliminate excess cholesterol and sterols from the body by secreting them into bile. There are two mechanisms to protect the canalicular membrane from solubilization by bile salts; ABCB4 secretes phosphatidylcholine into bile to form mixed micelles with bile salts, and ATP8B1 maintains the canalicular membrane in a liquid-ordered state. Three different forms of progressive familial intrahepatic cholestasis (PFIC) disorders, PFIC1, PFIC2 and PFIC3, are caused by mutations in ATP8B1, ABCB11 and ABCB4, respectively. Sitosterolemia is caused by mutations in ABCG5 and ABCG8. This article reviews the physiological roles of these canalicular transporters, and the pathophysiological processes and clinical features associated with their mutations.
ATP-binding cassette transporter; bile; canalicular membrane; cholestasis; cholesterol; P4 ATPase; phospholipids
Progressive familial intrahepatic cholestasis type 1 is a rare disease that is characterized by low serum γ-glutamyltransferase levels due to mutation in ATP8B1. We present a 23-year-old male who experienced persistent marked pruritus for eighteen years and recurrent jaundice for thirteen years, in addition to cholestasis that eventually became fatal. Genetic sequencing studies of the entire coding (exon) sequences of ATP8B1 and ABCB11 uncovered a novel heterozygous missense 3035G>T mutation (S1012I) and a synonymous 696T>C mutation in ATP8B1. The patient’s progression was associated with not only impaired familial intrahepatic cholestasis 1 (FIC1) function but also impaired bile salt export pump expression due to the impaired FIC1 function. Our findings show that patients with intermittent cholestasis can develop progressive liver disease even after several decades and require regular follow up.
ATP8B1; Bile salt export pump; Novel mutation; Progressive familial intrahepatic cholestasis type 1; Intermittent cholestasis
Progressive familial intrahepatic cholestasis (PFIC) refers to heterogeneous group of autosomal recessive disorders of childhood that disrupt bile formation and present with cholestasis of hepatocellular origin. The exact prevalence remains unknown, but the estimated incidence varies between 1/50,000 and 1/100,000 births.
Three types of PFIC have been identified and related to mutations in hepatocellular transport system genes involved in bile formation. PFIC1 and PFIC2 usually appear in the first months of life, whereas onset of PFIC3 may also occur later in infancy, in childhood or even during young adulthood. Main clinical manifestations include cholestasis, pruritus and jaundice. PFIC patients usually develop fibrosis and end-stage liver disease before adulthood. Serum gamma-glutamyltransferase (GGT) activity is normal in PFIC1 and PFIC2 patients, but is elevated in PFIC3 patients. Both PFIC1 and PFIC2 are caused by impaired bile salt secretion due respectively to defects in ATP8B1 encoding the FIC1 protein, and in ABCB11 encoding the bile salt export pump protein (BSEP). Defects in ABCB4, encoding the multi-drug resistant 3 protein (MDR3), impair biliary phospholipid secretion resulting in PFIC3.
Diagnosis is based on clinical manifestations, liver ultrasonography, cholangiography and liver histology, as well as on specific tests for excluding other causes of childhood cholestasis. MDR3 and BSEP liver immunostaining, and analysis of biliary lipid composition should help to select PFIC candidates in whom genotyping could be proposed to confirm the diagnosis. Antenatal diagnosis can be proposed for affected families in which a mutation has been identified. Ursodeoxycholic acid (UDCA) therapy should be initiated in all patients to prevent liver damage. In some PFIC1 or PFIC2 patients, biliary diversion can also relieve pruritus and slow disease progression. However, most PFIC patients are ultimately candidates for liver transplantation. Monitoring of hepatocellular carcinoma, especially in PFIC2 patients, should be offered from the first year of life. Hepatocyte transplantation, gene therapy or specific targeted pharmacotherapy may represent alternative treatments in the future.
Background and Aims
Inherited syndromes of intrahepatic cholestasis commonly result from mutations in the genes SERPINA1 (α1-antitrypsin deficiency), JAG1 (Alagille syndrome), ATP8B1 (Progressive Familial Intrahepatic Cholestasis type 1/PFIC1), ABCB11 (PFIC2), and ABCB4 (PFIC3). However, the large gene sizes and lack of mutational hotspots make it difficult to survey for disease-causing mutations in clinical practice. Here, we aimed to develop a technological tool that reads out the nucleotide sequence of these genes rapidly and accurately.
25-mer nucleotide probes were designed to identify each base for all exons, 10 bases of intronic sequence bordering exons, 280–500 bases upstream from the first exon for each gene, and 350 bases of the second intron of the JAG1 gene, and tiled using the Affymetrix resequencing platform. We then developed high-fidelity PCRs to produce amplicons using 1 ml of blood from each subject; amplicons were hybridized to the chip, and nucleotide calls were validated by standard capillary sequencing methods.
Hybridization of amplicons with the chip produced a high nucleotide sequence readout for all five genes in a single assay, with an automated call rate of 93.5% (range: 90.3–95.7%). The accuracy of nucleotide calls was 99.99% when compared with capillary sequencing. Testing the chip on subjects with cholestatic syndromes identified disease-causing mutations in SERPINA1, JAG1, ATP8B1, ABCB11 or ABCB4.
The resequencing chip efficiently reads SERPINA1, JAG1, ATP8B1, ABCB11 and ABCB4 with a high call rate and accuracy in one assay, and identifies disease-causing mutations.
The wide clinical spectrum of the ABCB4 gene (ATP-binding cassette subfamily B member 4) deficiency syndromes in humans includes low phospholipid-associated cholelithiasis (LPAC), intrahepatic cholestasis of pregnancy (ICP), oral contraceptives-induced cholestasis (CIC), and progressive familial intrahepatic cholestasis type 3 (PFIC3). No ABCB4 mutations are found in a significant proportion of patients with these syndromes. In the present study, 102 unrelated adult patients with LPAC (43 patients) or CIC/ICP (59 patients) were screened for ABCB4 mutations using DNA sequencing. Heterozygous ABCB4 point or short insertion/deletion mutations were found in 37% (16/43) of the LPAC patients and in 27% (16/59) of the ICP/CIC patients. High-resolution gene dosage methodologies were used in the 70 negative patients. Here, we describe for the first time ABCB4 partial or complete heterozygous deletions in 7% (3/43) of the LPAC patients, and in 2% (1/59) of the ICP/CIC patients. Our observations urge to systematically test patients with LPAC, ICP/CIC, and also children with PFIC3 for the presence of ABCB4 deletions using molecular tools allowing detection of gross rearrangements. In clinical practice, a comprehensive ABCB4 alteration-screening algorithm will permit the use of ABCB4 genotyping to confirm the diagnosis of LPAC or ICP/CIC, and allow familial testing. An early diagnosis of these biliary diseases may be beneficial because of the preventive effect of ursodeoxycholic acid on biliary complications. Further comparative studies of patients with well-characterized genotypes (including deletions) and phenotypes will help determine whether ABCB4 mutation types influence clinical outcomes.
ABCB4; deletion; biliary disease; LPAC; MDR3
Background & Aims
Progressive Familial Intrahepatic Cholestasis 1 (PFIC1) results from mutations in ATP8B1 (also known as FIC1), a putative aminophospholipid flippase. However conflicting hypotheses have been proposed for the pathogenesis of PFIC1. The aim of this study was to determine whether ATP8B1-deficiency produces cholestasis by altering the activity of the nuclear receptor FXR or by impairing the structure of the canalicular membrane
ATP8B1/Atp8b1 was knocked down in human and rat hepatocytes, and Caco2 cells using adenoviral and oligonucleotide siRNAs.
ATP8B1 mRNA and protein expression was greatly reduced in human and rat hepatocytes and Caco2 cells. In contrast, FXR expression and several FXR dependent membrane transporters (BSEP, MRP2) were unchanged at mRNA or protein levels in ATP8B1-deficient cells, whereas Mrp3 and Mrp4 were up-regulated in rat hepatocytes. FXR activity remained intact in these cells as evidenced by 6-ECDCA mediated induction of SHP, BSEP and MDR3/Mdr2. Fluorescent substrate excretion assays indicate that Bsep function was significantly reduced in Atp8b1-deficient rat hepatocytes although Bsep remained localized to the canalicular membrane. Exposure to the hydrophobic bile acid, CDCA resulted in focal areas of canalicular membrane disruption by electron microscopy and luminal accumulation of NBD-phosphatidylserine consistent with Atp8b1’s function as an aminophospholipid flippase.
ATP8B1- deficiency predisposes to cholestasis by favoring bile acid-induced injury in the canalicular membrane, but does not directly affect FXR expression, which may occur in PFIC1 as a secondary phenomenon associated to bile acid accumulation.
Prior loss-of-function analyses revealed that ATP8B1 (FIC1) post-translationally activated the Farnesoid X-Receptor (FXR).
Mechanisms underlying this regulation are elaborated upon by these gain-of-function studies in UPS cells, which lack endogenous FIC1 expression. FXR function was assayed in response to wild type and mutated FIC1 expression constructs using a human bile salt export pump (BSEP) promoter and a variety of cellular localization techniques.
FIC1 overexpression led to enhanced phosphorylation and nuclear localization of FXR that was associated with FXR-dependent activation of the BSEP promoter. The FIC1 effect was lost after mutation of the FXR response element in the BSEP promoter. Despite similar levels of FIC1 protein expression, Byler-disease FIC1 mutants did not activate BSEP, while benign recurrent intrahepatic cholestasis mutants partially activated BSEP. The FIC1 effect was dependent upon the presence of the FXR ligand, chenodeoxycholic acid. The FIC1 effect on FXR phosphorylation and nuclear localization and its effects on BSEP promoter activity could be blocked with protein kinase C (PKC) ζ inhibitors (pseudosubstrate or siRNA silencing). Recombinant PKCζ directly phosphorylated immunoprecipitated FXR. Mutation of threonine 442 of FXR to alanine yielded a dominant negative protein, while the phosphomimetic conversion to glutamate resulted in FXR with enhanced activity and nuclear localization. Inhibition of PKCζ in Caco-2 cells resulted in activation of the human apical sodium dependent bile acid transporter promoter.
These results demonstrate that FIC1 signals to FXR via PKCζ. FIC1-related liver disease is likely related to downstream effects of FXR on bile acid homeostasis. BRIC emanates from a partially functional FIC1 protein. Phosphorylation of FXR is an important mechanism for regulating its activity.
nuclear receptor; cholestasis; liver; ileum; bile acid
The exact molecular mechanism(s) of the disease that results from defects in the ATPase Class I Type 8B Member 1 gene remains controversial. Prior investigations of human ileum and in intestinal and ovarian cell lines have suggested that Familial Intrahepatic Cholestasis 1 (FIC1) activates the Farnesoid X-Receptor (FXR) via a pathway involving Protein Kinase C ζ (PKCζ). Translational investigations of human liver from individuals with FIC1 disease have been confounded by secondary affects of progressive cholestatic liver disease and limited numbers of samples for analysis. These studies, performed in primarily derived human hepatocytes, circumvent this issue. The canalicular bile salt export pump (BSEP) served as a downstream target of FXR. siRNA mediated silencing of FIC1 in human hepatocytes led to a reduction in both human BSEP promoter activity and BSEP protein expression, which correlated with a reduction in FXR expression and redistribution of its localization from the nucleus to the cytoplasm. These changes in BSEP expression could be reproduced by altering the expression of PKCζ; with a positive correlation of PKCζ activity and BSEP expression. Overall, these findings support the hypothesis that FIC1 enhances FXR signaling via a PKCζ dependent signaling pathway.
Progressive familial intrahepatic cholestasis (PFIC) occurs in many communities and races. A form of PFIC in five children from two consanguineous marriages in an Irish kindred is described. In addition, a review of clinical information from the records of three deceased members of the kindred strongly implies that they also suffered from PFIC. The children had a history of neonatal diarrhoea, sepsis, and intermittent jaundice that ultimately became permanent. They suffered intractable pruritus and growth retardation. Despite evidence of severe cholestasis, serum gamma-glutamyl transferase and cholesterol were normal in these children. Sweat sodium concentration were raised in three children. Liver histology showed severe intrahepatic cholestasis and hepatocellular injury. Urinary bile acid analysis revealed a non-specific pattern consistent with chronic cholestasis. These children suffer from a form of PFIC remarkably similar to that occurring in members of the Byler kindred.
The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.
BSEP disease results from mutations in ABCB11, which encodes the bile salt export pump (BSEP). BSEP disease is associated with an increased risk of hepatobiliary cancer.
A 36 year old woman with BSEP disease developed pancreatic adenocarcinoma at age 36. She had been treated with a biliary diversion at age 18. A 1.7 × 1.3 cm mass was detected in the pancreas on abdominal CT scan. A 2 cm mass lesion was found at the neck and proximal body of the pancreas. Pathology demonstrated a grade 2-3 adenocarcinoma with invasion into the peripancreatic fat.
Clinicians should be aware of the possibility of pancreatic adenocarcinoma in patients with BSEP disease.
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).
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.
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).
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.
Cholangiocarcinoma; Genetics; ABCB11; ABCB4; ABCC2; ATP8B1; NR1H4
The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into the bile canaliculus of hepatocytes. Its localization in the apical canalicular membrane is physiologically regulated by the demand to secrete biliary components. To gain insight into how such localization is regulated, we studied the intracellular trafficking of BSEP tagged with yellow fluorescent protein (YFP) in polarized WIF-B9 cells. Confocal imaging revealed that BSEP-YFP was localized at the canalicular membrane and in tubulo-vesicular structures either adjacent to the microtubule-organizing center or widely distributed in the cytoplasm. In the latter two locations, BSEP-YFP colocalized with rab11, an endosomal marker. Selective photobleaching experiments revealed that single BSEP-YFP molecules resided in canalicular membranes only transiently before exchanging with intracellular BSEP-YFP pools. Such exchange was inhibited by microtubule and actin inhibitors and was unaffected by brefeldin A, dibutyryl cyclic AMP, taurocholate, or PI 3-kinase inhibitors. Intracellular carriers enriched in BSEP-YFP elongated and dissociated as tubular elements from a globular structure adjacent to the microtubule-organizing center. They displayed oscillatory movement toward either canalicular or basolateral membranes, but only fused with the canalicular membrane. The pathway between canalicular and intracellular membranes that BSEP constitutively cycles within could serve to regulate apical pools of BSEP as well as other apical membrane transporters.
Background and aims: The aim of this study was to investigate the genetic aetiology of intrahepatic cholestasis of pregnancy (ICP) and the impact of known cholestasis genes (BSEP, FIC1, and MDR3) on the development of this disease.
Patients and methods: Sixty nine Finnish ICP patients were prospectively interviewed for a family history of ICP, and clinical features were compared in patients with familial ICP (patients with a positive family history, n=11) and sporadic patients (patients with no known family history of ICP, n=58). For molecular genetic analysis, 16 individuals from two independently ascertained Finnish ICP families were genotyped for the flanking markers for BSEP, FIC1, and MDR3.
Results: The pedigree structures in 16% (11/69) of patients suggested dominant inheritance. Patients with familial ICP had higher serum aminotransferase levels and a higher recurrence risk (92% v 40%). Both segregation of haplotypes and multipoint linkage analysis excluded BSEP, FIC1, and MDR3 genes in the studied pedigrees. Additionally, the MDR3 gene, previously shown to harbour mutations in ICP patients, was negative for mutations when sequenced in four affected individuals from the two families.
Conclusions: These results support the hypothesis that the aetiology of ICP is heterogeneous and that ICP is due to a genetic predisposition in a proportion of patients. The results of molecular genetic analysis further suggest that the previously identified three cholestasis genes are not likely to be implicated in these Finnish ICP families with dominant inheritance.
intrahepatic cholestasis of pregnancy; obstetric cholestasis; linkage analysis
The bile salt export pump (BSEP, ABCB11) is the major determinant of bile salt dependent bile secretion and its deficiency leads to cholestatic liver injury. BSEP/Bsep gene expression is regulated by the nuclear farnesoid X receptor (FXR). However, BSEP expression is retained in the liver of the Fxr−/− mice although reduced, indicating that there may be additional transcriptional factors that regulate its expression. The NF-E2-related factor-2 (Nrf2) plays a major role in response to oxidative stress by binding to antioxidant-responsive elements (ARE) that regulate many hepatic Phase I and II enzymes as well as hepatic efflux transporters. Computer software analysis of human BSEP reveals two Maf recognition elements (MAREs) from the sequence in the proximal promoter region where Nrf2 may bind. In this study we examine if Nrf2 plays a role in human BSEP expression and whether this might be mediated by the MAREs. Oltipraz, a potent activator of Nrf2, increased BSEP mRNA expression by ~ 7-fold in HepG2 cells and protein by ~ 70% in human hepatocytes. siRNAs lowered NRF2 expression in HepG2 cells and prevented the up-regulation of BSEP by oltipraz. Human BSEP promoter activity was stimulated by Nrf2 in a dose-dependent manner in luciferase reporter assays. Mutations of the predicted MARE1, but not MARE2, abolished this Nrf2 transcriptional activation. ChIP assays also demonstrated that Nrf2 specifically bound to MARE1, but not MARE2 regions in the BSEP promoter in HepG2 cells. Electrophoretic mobility shift assays further demonstrated direct binding of MARE1 in the BSEP promoter.
Nrf2 is a positive transcriptional regulator of human BSEP expression. Pharmacological activation of Nrf2 may be beneficial for cholestatic liver injury.
ATP-binding cassette (ABC) transporters; antioxidant-responsive element (ARE); oltipraz; bile secretion; gene regulation
Background: Intrahepatic cholestasis of pregnancy (ICP) affects approximately 0.7% of pregnancies in the UK and is associated with prematurity, fetal distress, and intrauterine death. Homozygous mutations in the ATP8B1 gene cause cholestasis with a normal serum gamma-glutamyl transpeptidase (γ-GT), and have been reported in two forms of cholestasis: progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis (BRIC).
Aims: To establish whether mutations in ATP8B1 are associated with ICP in British cases
Patients: Sixteen well phenotyped women with ICP without raised γ-GT were selected for sequence analysis. Subsequently, 182 patients and 120 controls were examined for the presence of the variants detected.
Methods: All coding exons were sequenced in 16 cases. Eight ICP cases, including two women carrying a mutation, were investigated using in vivo hepatic 31P magnetic resonance spectroscopy (MRS)
Results: Two heterozygous ATP8B1 transitions (208G>A and 2599C>T) that resulted in amino acid substitutions were identified; 208G>A was identified in three cases. MRS revealed an increased phosphodiester signal (Mann-Whitney U test, p = 0.03) and a decreased phosphomonoester/phosphodiester ratio (p = 0.04) in ICP cases compared with controls.
Conclusions: We were able to demonstrate ATP8B1 mutations in ICP. MRS studies suggest that susceptibility to ICP is associated with a relative rise in biliary phospholipid. These data also suggest that MRS may be used for non-invasive assessment of the liver and biliary constituents in cholestasis.
bile; liver; magnetic resonance spectroscopy; intrahepatic cholestasis of pregnancy
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.
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.
For the most part, polymorphisms were rare and ethnicity 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 – 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.
Accordingly, our study indicates that 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.
ABCB11; bile acid; bile salt export pump; cholestasis; pharmacogenetics; polymorphism; transporter
Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency.
We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains.
Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease.