The bile salt export pump (BSEP) in health and disease

Clinics and Research in Hepatology and Gastroenterology (2012) 36, 536—553

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MINI REVIEW

The bile salt export pump (BSEP) in health and disease Ralf Kubitz ∗, Carola Dröge , Jan Stindt , Katrin Weissenberger , Dieter Häussinger Medical faculty of the Heinrich-Heine-University of Düsseldorf, Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital, Düsseldorf, Germany Available online 12 July 2012

Summary The bile salt export pump (BSEP) is the major transporter for the secretion of bile acids from hepatocytes into bile in humans. Mutations of BSEP are associated with cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 2 (PFIC-2), benign recurrent intrahepatic cholestasis type 2 (BRIC-2) and genetic polymorphisms are linked to intrahepatic cholestasis of pregnancy (ICP) and drug-induced liver injury (DILI). Detailed analysis of these diseases has considerably increased our knowledge about physiology and pathophysiology of bile secretion in humans. This review focuses on expression, localization, and function, short- and long-term regulation of BSEP as well as diseases association and treatment options for BSEP-associated diseases. © 2012 Elsevier Masson SAS. All rights reserved.

Introduction A major function of the liver is the transport of bile salts from blood into bile. Bile salt secretion is required for the removal of cholesterol, which is insoluble in water. Within bile cholesterol is incorporated into mixed micelles, which contain large amounts of bile acids (BA) and phospholipids [1]. 12—20 g of bile acids have to be excreted into bile in order to eliminate 500 mg of cholesterol per day. Therefore, effective transport systems have evolved to ensure bile acid excretion. These include the bile salt export pump (BSEP, gene symbol ABCB11) at the

∗

Corresponding author. Tel.: +49 211 8119648. E-mail address: [email protected] (R. Kubitz).

canalicular (apical) membrane of hepatocytes as well as the sodium-taurocholate cotransporting polypeptide (NTCP) [2] and organic anion transporting polypeptides (OATPs) [3], which mediate uptake of bile salts from blood into hepatocytes. Secretion of bile acids from hepatocytes back into blood may be achieved by the organic solute transporter OST␣/OST␤ [4] and the multidrug resistance associated protein 4 (MRP4) [5,6]. At the canalicular membrane, the multidrug resistance protein 3 (MDR3, gene symbol ABCB4) flops phosphatidylcholine into bile, and forms a functional unit together with the heterodimeric cholesterol transporter ABCG5/8 and BSEP, because their substrates (phosphatidylcholine, cholesterol, bile acids) together constitute mixed micelles (Fig. 1). Canalicular secretion is regarded as the rate-controlling step of the vectorial transport of bile acids through hepatocytes [7—9]. Due to the crucial localization of BSEP and

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The bile salt export pump (BSEP) in health and disease

PFIC-1 BRIC-1 PFIC-2 BRIC-2 ICP/ DILI DubinJohnson-S.

FIC1

537

ATP8B1

ATP7B

M. Wilson

BSEP

MDR3

PFIC-3 LPAC ICP

ABCB11

MRP2 ABCC2

ABCB4

SitoABCG5/8 sterolemia

Figure 1 Canalicular transporter proteins and associated liver diseases. Model of a hepatocyte couplet with transporter proteins at the canalicular membrane. ATP8B1 (FIC1) is a putative aminophospholipid flippase, mutations are associated with progressive familial intrahepatic cholestasis type 1 (PFIC-1) and benign recurrent intrahepatic cholestasis type 1 (BRIC-1). Its activity probably is a precondition for proper bile salt export pump (BSEP) function. Mutations of BSEP (ABCB11) are related to PFIC-2, BRIC-2 and drug-induced liver injury (DILI). The multidrug resistance protein 3 (MDR3/ABCB4) acts as a floppase for phosphatidylcholine. PFIC-3, LPAC (low phospholipid associated cholelithiasis syndrome) and ICP (intrahepatic cholestasis of pregnancy) are related to mutations of MDR3. Mutations of the heterodimeric cholesterol transporter ABCG5/8 are related to sitosterolemia (characterized by increased serum levels of plant sterols and premature atherosclerosis). The substrates of BSEP (bile acids), MDR3 (phospholipids) and ABCG5/8 (cholesterol) form mixed micelles in bile. Mutations of the bilirubin transporter multidrug resistance associated protein 2 (MRP2/ABCC2) are linked to the Dubin-Johnson-Syndrome, characterized by conjugated hyperbilirubinemia. Morbus Wilson (copper storage disease) is caused by mutations of the copper transporter ATP7B.

the multiple effects of bile acids, alteration of BSEP may directly or indirectly affect processes such as liver regeneration [10], glucose homeostasis [11], energy expenditure [12], atherosclerosis [13], or carcinogenesis [14]. Elevated bile acid concentrations may be toxic due to their detergent properties [15] and hydrophobic bile acids may activate proapoptotic pathways [16]. All these aspects may explain why BSEP plays a central role in many liver diseases.

Expression, localization and function of bile salt export pump (BSEP) The bile salt export pump BSEP (ABCB11) belongs to the MDR/TAP subfamily of ATP binding cassette transporter (ABC transporter) together with P-glycoprotein (P-gp/or MDR1, gene: ABCB1) and MDR3 (ABCB4). ABCB11 is localized on the long arm of chromosome 2 (2q24) in humans [17]. BSEP consists of 27 coding exons following the untranslated first exon. The protein includes 1.321 amino acids with a molecular mass of approximately 160 kDa (Fig. 2). On a molecular level, BSEP is composed of 12 transmembrane helices and two large nucleotide binding domains. BSEP is responsible for the bile salt-dependent bile flow. It mainly transports monovalent bile acids including taurin and glycine conjugates of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) as well as the secondary bile acid deoxycholic acid (DCA) [18]. In addition, it secretes ursodeoxycholic acid (UDCA) and its conjugates into bile. Km-values of human BSEP for taurocholate (TC), taurochenodeoxycholate (TCDC),

glycocheno-deoxycholate (GCDC) and glycocholate (GC) are 6.2 ␮M, 6.6 ␮M, 7.5 ␮M and 21.7 ␮M, respectively, as determined in vesicles from transfected HEK293 cells. Calculated intrinsic clearance values (Vmax/Km) resulted in a rank order of TCDC > GCDC > TC > GC [19]. Because CDCA and its conjugates are potentially more toxic for hepatocytes in vivo [20] and in vitro [16,20] a ‘‘better’’ elimination of chenodeoxycholate as compared to cholate derivatives appears favourable. BSEP is exclusively expressed in hepatocytes and is mainly localized at the canalicular membrane. It is not entirely clear, if BSEP reaches the canalicular membrane directly from the Golgi apparatus to the canalicular membrane [21] or indirectly via the basolateral membrane [22]. The latter possibility is favoured by the observation that anti-BSEP antibodies, which may develop in children with PFIC-2 and liver transplantation, selectively reach the canaliculi within less than 30 minutes [23] in the intact organ, suggesting binding of the antibodies by BSEP at the basolateral membrane followed by transcytosis. Targeting of BSEP from the Golgi to the canalicular membrane is stimulated by the p38MAP kinase and protein kinase C [24], which are (together with extracellular-signalregulated kinases) otherwise involved in bile acid induced exocytosis of transporter bearing vesicles [25,26]. Internalisation of BSEP from the canalicular membrane is regulated by HCLS1-associated protein X-1 (Hax1). Depletion of Hax1 leads to an increase of BSEP at the apical membrane in MDCK cells. Likewise, a dominant negative form of cortactin (lacking the C-terminal SH3 domain), an interacting partner of Hax1, increases apical BSEP in MDCK cells [27]. Cortactin

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R. Kubitz et al. membrane asymmetry. This may be essential for resistance of the canalicular membrane towards detergent effects of bile acids [34] and was suggested to be directly required for BSEP activity [34,35]. Another possible mechanism of interaction between FIC1 and BSEP involves FXR (see below for details concerning FXR) and the atypical protein kinase C zeta (PKC␨), which has a prominent canalicular localization [36]. It was shown that inhibition of PKC␨ by a pseudosubstrate or its downregulation by siRNA reduced the nuclear localization of FXR [37], which eventually reduces BSEP expression. The interplay between FIC1 and BSEP on different levels (membrane asymmetry/transporter activity or signalling/transporter expression) explains the development of cholestasis in patients with FIC1 mutations similar to patients with BSEP mutations. However, apart from its role in cholestasis FIC1 deficiency causes several symptoms such as diarrhoea or hearing loss, which are not observed in BSEP disease and which are explained by the extrahepatic expression of FIC1 [38,39]. If in turn reduced BSEP activity influences the proposed FIC1-PKC␨-FXR cascade is not known yet. Furthermore, Bsep function depends on membrane cholesterol content [40,41]. Interference with cholesterol synthesis by pravastatin, a HMG-CoA reductase inhibitor, not only decreases serum cholesterol levels but also ameliorates cholestasis in bile duct-ligated rats along with an increase in Bsep expression [42].

Long-term regulation of bile salt export pump (BSEP) Figure 2 Molecular model of bile salt export pump (BSEP). A model of BSEP was constructed according to [23] using the crystal structure of the multidrug transporter Sav1866 from S. aureus (Protein Data Bank entry: 2HYD) as a template. The putative transmembrane helices (TMH) of the N-terminal half of the BSEP are shown in green, the TMH of the C-terminal half are shown in blue. They were calculated by the transmembrane helices prediction tool from http://www.enzim.hu/hmmtop [229]. The extracellular loops (representing the probable binding sites for anti-BSEP-autoantibodies, see text) of the N-terminal half are given in magenta. Within the nucleotide binding domains the Walker A motifs are shown in red, the Walker B motif in green and the ABC transporter signature in yellow.

phosphorylation is induced by cell shrinkage via a p47phox and Fyn-dependent pathway, which results in endocytosis of Bsep and Mrp2 and subsequent cholestasis [28]. A tyrosinebased motif at the C-terminus was recently shown to be essential for dynamin- and clathrin-dependent endocytosis of BSEP [29] involving the AP2 adaptor complex [30]. Cortactin is known to interact with dynamin [31] and this interaction may thus represent a link between external stimuli and transporter endocytosis. The ‘‘familial intrahepatic cholestasis 1’’ protein 1 (FIC1, ATP8B1) belongs to the large family of P-type ATPase ion pumps with ubiquitous expression [32]. The exact function of FIC1 and especially its downstream signalling is still under debate. FIC1 probably acts as a flippase, which transports phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet of cell membranes [33] thereby maintaining plasma

Long-term regulation of BSEP is complex and has to fulfil many demands. It occurs in large parts on the level of transcription. Major regulators of BSEP expression are the farnesoid X receptor (FXR), the liver receptor homolog 1 (Lrh1) and the nuclear factor erythroid 2-related factor 2 (Nrf2) (Fig. 3). The farnesoid X receptor (FXR/Fxr for the human and rodent orthologues) together with its obligate partner RXR␣ directly transactivates the human [43,44] and rat BSEP/Bsep promoter [45] after bile acid binding to FXR/Fxr. In Fxr knockout mice, basal expression of mouse Bsep (mBsep) is diminished and inducibility of Bsep expression by cholic acid feeding is almost completely abolished [46]. Chenodeoxycholate (CDCA), the predominant bile acid in humans, is the most potent natural agonist for FXR [47,48]. CDCA facilitates the recruitment of the coactivator complex ASCOM (including the coactivator NCOA6 and the H3K4 lysine methyltransferase MLL3) leading to methylation of histones within the mBsep promoter region, a process shown to be essential for Fxr-dependent Bsep expression [49]. Bile acids activate the BSEP promoter via FXR in the rank order of CDCA > DCA > CA > UDCA with a ratio of 25:20:18:8 in vitro, similar to the ratios found in HepG2 cells (∼50:17:5:1) [50], whereas lithocholic acid (LCA) was identified as a ‘‘natural’’ antagonist [51]. The steroid receptor coactivator-2 (SRC-2; also known as nuclear receptor coactivator-2 [NCOA2]) was also shown to cooperatively interact with BSEP promoter stimulation by FXR [52]. SRC-2 is activated via phosphorylation by the AMP activated protein kinase (AMPK), which is regarded as an energy depletion sensing kinase. SRC-2 possesses an intrinsic

The bile salt export pump (BSEP) in health and disease

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Figure 3 Regulation of the bile salt export pump (BSEP) promoter. Bile acids (BA) are major regulators of BSEP expression through activation of the farnesoid X receptor (FXR). The activating complex ASCOM (see text for details) is recruited by BA/FXR for the methylation (Me) of histones within the BSEP promoter. The nuclear factor erythroid 2-related factor 2 (NRF2) is activated by oxidative stress (e.g. by certain ‘‘toxic’’ BA), binds to a Maf recognition element (MARE) and transactivates the BSEP promoter. The steroid receptor coactivator-2 (SRC2) is activated by the liver kinase B1 (LKB1) and the ‘‘energy sensor kinase’’ AMP activated protein kinase (AMPK) and activates gene expression via acetylation (Ac) of histones. Liver receptor homolog 1 (LRH1) transactivates BSEP and CYP7A1, the rate-controlling enzyme of BA synthesis. Increased BSEP transcription eventually increases BSEP protein expression and removal of intracellular BA. (FXRE: FXR response element; LRHRE: LRH response element; NTCP: Na+ -taurocholate cotransporting polypeptide; ROS: reactive oxygen species; RXR: Retinoid X receptor; the arrows indicate stimulation/activation; dotted lines represent BA flux.)

histone acetyltransferase activity and is recruited to promoter sites of genes. Acetylation of histones by SRC-2 in vicinity to the promoter sites increases the accessibility of the related genes for transcription. It was suggested that the AMPK-SRC2/FXR-BSEP cascade eventually would enhance fat absorption from the intestine via increased BSEP-dependent bile salt secretion [52], representing another link between energy and bile acid homeostasis [53]. The importance of SRC-2 was highlighted by the observation that BSEP mRNA and protein expression was significantly reduced in hepatocytes-specific SRC-2 knockout mice, whereas deletion of SRC-1 or SRC-3 has no effect [52]. Interaction of SRC-2 with several other nuclear receptors including the retinoid X receptor [54,55] or the peroxisome proliferator-activated receptor gamma [56] has been demonstrated. The liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), acts upstream of the AMPK and in addition activates other kinases from the AMPK-related kinase family. LKB1 regulates cell polarity and, via AMPK, cellular energy homeostasis. Interestingly, liver-specific knockout of LKB1 led to defective bile duct formation, impaired bile acid clearance and a marked retention of mBsep in intracellular pools along with reduced NtcpmRNA and protein expression [57]. In Fxr wildtype mice mBsep expression is upregulated or at least maintained after bile duct ligation (BDL). In Fxr knockout mice (Fxr−/− ) mBsep is almost undetectable with or without BDL. Absent mBsep expression leads to reduced pressure within the biliary tree and absence of bile infarcts after BDL [58] suggesting a beneficial effect of Fxr

repression in BDL. In addition, a stronger upregulation of Mrp4 and Mrp3 expression was observed in Fxr−/− mice [58], which otherwise is repressed by Fxr [59]. Upregulation of MRP4 probably represents an overflow mechanism, which protects hepatocytes from increased bile acids concentrations. In line with this, an almost 10-fold induction of MRP4 was observed in humans with progressive familial intrahepatic cholestasis [60]. Whether stimulation or inhibition of Fxr is more beneficial may depend on the species but also on the type of cholestasis. In rats with obstructive cholestasis, stimulation of Fxr by the Fxr agonist GW4064 has a protective effect [61]. In HepG2 cells stigmasterol acetate, a phytosterol component of intravenous nutrition, suppresses FXR-target genes including BSEP, FGF-19, OST␣/␤, and SHP. It was suggested that stigmasterol is directly involved in total parenteral nutrition-associated cholestasis due to its FXR-antagonistic action [62]. In mice treated with alphanaphthylisothiocyanate (ANIT) liver injury was more evident in Fxr−/− mice as compared to wildtype or pregnane X receptor knockout mice (Pxr−/− ), in line with a protective role of Fxr in ANIT-induced intrahepatic cholestasis [63]. The liver receptor homolog 1 (Lrh1, NR5A2) is a key regulator of cholesterol 7 alpha-hydroxylase (CYP7A1), a rate-controlling enzyme for BA synthesis from cholesterol. Lrh1 is another regulator of Bsep and in hepatocyte-specific Lrh1 knockout mice a reduced Bsep expression was observed [64] along with reduced cholic acid synthesis [65]. Likewise, the nuclear factor erythroid 2-related factor 2 (Nrf2) positively regulates Bsep [66] and in addition

540 Mrp2, Mrp3 and Mrp4 [67,68]. Nrf2 is a sensor for oxidative stress [69,70], which may be relevant for counteracting reactive oxygen species (ROS) induced by toxic bile acids [71]. Nrf2 binds to the Bsep promoter approx. two hundred base pairs in front of the transcription start site to a musculo-aponeurotic fibrosacroma (Maf) recognition element (MARE) [66]. A single application of perfluorodecanoic acid, a known agonist of peroxisome proliferator-activated receptor ␣ (PPAR␣) induces Mrp3 up to four-fold and Mrp4 more than 30-fold in a Nrf2-dependent manner [72], underscoring the hepatoprotective role of Nrf2. It was even suggested that some beneficial effects of UDCA are mediated by Nrf2 [68]. In the regenerating liver after partial hepatectomy, Bsep expression remains unaffected [73], whereas the sinusoidal uptake transporter Ntcp is downregulated by up to 90% [74] along with a sustained upregulation of the bile salt exporter Mrp4 [75]. Likewise, Oatp1 and 2 (rat) are reduced at the mRNA and protein level by 50 to 60% [73], preventing uncontrolled uptake of potentially toxic substrates into hepatocytes. Interestingly, despite reduced net uptake of bile acids (leading to a 10-fold increase in serum bile salts) bile flow and bile acid excretion into bile per gram body weight remains unaltered [76] or even increase after partial hepatectomy [77]. Although increased bile acid concentrations are generally considered to be toxic, elevated bile acid levels and downstream signalling are required for proper liver regeneration [10].

Short-term regulation of bile salt export pump (BSEP) Rapid adaptation of the activity of canalicular transporter proteins including Bsep may be achieved by rapid vesicular insertion and retrieval into and from the canalicular membrane, which represents a major mechanism for short-term regulation of bile formation [8,78]. The number of transporter molecules and consequently the maximum transport rate Vmax may be adjusted within minutes according to actual needs. This mechanism is involved when changes in hepatocellular hydration [79], hormones [80], bile salts [26,81,82] or oxidative stress [83] occur. Cell volume changes are of central importance, irrespective if they are induced by anisotonic exposure, amino acid uptake, insulin, or ethanol [84,85]. Increments in cell volume increase bile salt secretion in a microtubules-dependent manner [86] and involve rapid insertion of intracellular stored Bsep into the canalicular membrane as shown by immunohistochemistry [79]. Similarly, the bilirubin transporter Mrp2 is regulated by osmolarity [87,88], lipopolysaccharide [89], or oxidative stress [90], however, following hyperosmotic exposure, Bsep and Mrp2 are retrieved into different intracellular vesicular compartments [79]. Only 15% of retrieved vesicles contained both, Bsep and Mrp2, which may be due to different retrieval mechanisms for these transporters. Interestingly, within the canalicular membrane, Bsep is found in the same bile salt resistant lipid microdomains like Mrp2 or transporters such as AbcG5 [91] and it is not yet known how endocytosis of Bsep independent of Mrp2 is achieved. In line with transporter specific mechanisms of regulation, Bsep expression was unchanged in radixin knock out mice

R. Kubitz et al. (a cytoskeletal protein of the ezrin-radixin-moesin family), whereas canalicular Mrp2 protein expression was lacking despite normal Mrp2-mRNA levels [92]. Taurocholate induces hepatocyte swelling, therefore swelling-induced Bsep insertion may acts as feed forward regulation of canalicular secretion [85]. Such a response would also accelerate enterohepatic circulation of bile acids after ingestion of a meal, which in turn triggers nutrient-driven hepatocyte swelling by the concentrative uptake of amino acids into hepatocytes. In addition to endo-/exocytosis Bsep may be regulated by covalent modifications. Coexpression of mBsep and mouse PKC alpha in Sf9 insect cells led to phosphorylation of mBsep [93]. Furthermore, short-chain ubiquitination of BSEP has been demonstrated, which may initiate degradation of BSEP [94]. Signalling events in the above mentioned processes are complex but have been unravelled in large parts. For example, hepatocyte swelling is sensed by integrins (␣5 ß1 ), consecutively triggering activation of focal adhesion kinase, c-Src and mitogen activated protein kinases Erks and p38MAPK [26,95—97]. On the other hand, hyperosmotic Bsep (and Mrp2) retrieval involves endosomal osmosensing with activation of acidic sphingomyelinase, followed by ceramide formation, protein kinase C␨ activation, p47phox phosphorylation and NADPH oxidase activation. The resulting oxidative stress signal induces an activation of the Src family kinase Fyn, which then triggers Bsep retrieval probably via cortactin phosphorylation [28]. Tauroursodeoxycholate (TUDC) stimulates bile acid secretion in part via the same pathways as cell swelling (for detailed review see [98]). In addition, TUDC [99] or taurolithocholic acid [100] activate different PKC isoforms. In the intact organ, activation of Ca2+ -dependent PKC isoforms (cPKC) by phorbolesters or thymeleatoxin, a selective agonist for cPKCs, induces cholestasis [101] along with retrieval of Bsep. cPKCs together with phosphoinositide 3-kinase (PI3K) are also involved in mediating the cholestatic effect of estradiol 17␤-D-glucuronide (E[2]17G) [102,103]. Evidence was presented that Ca2+ via type II inositol 1,4,5-trisphosphate receptor (InsP3R2) is necessary for maintenance of rat Bsep activity, as shown in sandwich cultures of hepatocytes [104].

Pharmacology of bile salt export pump (BSEP) Although BSEP almost exclusively transports bile salts, endogenous substances as well as many drugs can act as BSEP inhibitors [105,106]. A correlation between potency of BSEP inhibition by different pharmaceuticals and risk of cholestatic drug-induced liver injury (DILI) was observed, which was attributed in part to the physicochemical properties of the compound such as molecular weight, nonpolar surface area and the partition coefficient [107]. Compounds known to inhibit BSEP were found to be hepatocytotoxic in a sandwich culture model, when bile acids such as GCDC were added together with these compounds [108], supporting the concept that intracellular accumulation of bile acids represent an important mechanism of drug-induced liver injury [109]. It has been proposed that the potential of BSEP inhibition should be considered in the course of drug development

The bile salt export pump (BSEP) in health and disease just as properties such as reactive metabolite formation, cytotoxicity and mitochondrial toxicity [110]. Z-Guggulsterone (a plant steroid found in the resin of Commiphora wightii) acts as a FXR antagonist. It was shown that it induces BSEP as well as expression of cholesteryl ester hydrolase (CES1), thereby affecting cholesterol metabolism. Z-Guggulsterone is metabolized by CYP3A4 and its metabolites even more increase BSEP but not CES1. These differential effects on BSEP and CES1 expression may explain why different dosages of Z-Guggulsterone either increase or decrease cholesterol levels [111]. Finally, scoparone, a compound from a Chinese herb, potentiated BSEP expression by increasing the effect of CDCA on the human BSEP promoter [112].

Bile salt export pump (BSEP)-related liver diseases Several transporter proteins are localized within the canalicular membrane, which are associated with (monogenetic) liver diseases including progressive familial intrahepatic cholestasis (PFIC) type 1 to 3, benign recurrent intrahepatic cholestasis (BRIC) type 1 and 2, low phospholipid associated cholelithiasis (LPAC), intrahepatic cholestasis of pregnancy (ICP), Wilson’s disease, Sitosterolemia and the Dubin-Johnson-Syndrome. In addition to inherited diseases BSEP has been recognized to be involved in acquired forms of cholestasis such as drug-induced liver injury (DILI) (Fig. 1).

Progressive familial intrahepatic cholestasis type 2 (PFIC-2) The term ‘‘progressive familial intrahepatic cholestasis’’ (PFIC) was used in order to distinguish cholestasis-induced end stage liver diseases in childhood, which could not be attributed to neonatal or paediatric cholestasis such as biliary atresia, Alagille syndrome or Aagenaes syndrome [113,114]. These criteria were for example consistent with Byler’s disease, a severe form of cholestasis with low gamma-glutamyltranspeptidase (␥GT) levels, which was first observed in an Amish family descending from Jacob and Nancy Byler [115]. Mutations of FIC1 were shown to be responsible for Byler’s disease [116—118], which was termed PFIC-1, accordingly. A second, FIC1-independent low ␥GTcholestasis, formerly termed ‘‘Byler-like syndrome’’ [119] could be linked to mutations of ABCB11/BSEP and was termed PFIC-2 [17,116]. In contrast to PFIC-1 and -2, PFIC3 (due to mutations of ABCB4/MDR3) is characterized by increased ␥GT levels. Commonly, normal ␥GT levels in PFIC1/-2 are ascribed to the low levels of (free) bile salts in bile, and consequently less damage of cholangiocytes [120]. PFIC-2 patients usually present within the first 6 months of life. Jaundice, itching, and growth failure are typical findings in PFIC-2, but some children may present with severe haemorrhage due to vitamin K deficiency. Pruritus usually is more severe in PFIC-2 (and PFIC-1) as compared to PFIC-3 [121]. All PFIC-patients commonly have significantly lower serum cholesterol levels as compared to other forms of paediatric cholestasis [122]. PFIC-2 patients have a higher risk to develop hepatocellular carcinoma [123—125] or cholangiocarcinoma (CC)

541 [126]. A role of BSEP for the development of CC was also suggested on the basis of an association to the BSEP polymorphism p.V444A in a study of 172 CC-patients [127]. The occurrence of pancreatic adenocarcinoma was reported in a patient with PFIC-2, who was treated by biliary diversion [128]. Reduced or even absent expression of canalicular BSEP is a typical feature of PFIC-2 and is of diagnostic value [60,126,129—131], whereas light and electron microscopy findings are variable and non-specific in PFIC-2 [131]. Absent BSEP expression is self-evident for premature stop-codons or frame shift-mutations. However, BSEP deficiency in the context of missense mutations is more difficult to explain. Possible causes include: firstly, direct interference with splicing efficiency as shown for several PFIC-2-relevant missense mutations [132]. Single nucleotide exchanges may reside within so-called exonic splicing enhancers (ESE). ESEs are hexanucleotides that are involved in the guidance of the splicing machinery to splice sites. By the use of an in vitro minigene approach, interference with splicing was shown for c.1445A > G (p.D482G), c.1757C > T (p.T586I), c.3432C > A (p.S1144R), c.3458G > A (p.R1153H), c.3460T > C (p.S1154P), c.3691C > T (p.R1231 W) and c.3692G > A (p.R1231Q) [132]. If these mutations affect splicing in vivo as in vitro needs to be confirmed; secondly, up to 30% of proteins are degraded by the ER-associated degradation (ERAD) pathway [133]. Induction of protein misfolding by missense mutations and consecutive elimination by ERAD may be another reason for decreased BSEP expression. Interestingly, the very common polymorphism p.V444A of BSEP (allele frequency > 50%), which may occur isoallelic to mutations, strongly increases ERAD, as shown in expression studies of cloned BSEP [23]; thirdly, a decrease in half-life may shorten residency of BSEP at the canalicular membrane as shown for the two common PFIC-2 mutations p.E297G and p.D482G in Madin-Darby canine kidney (MDCK) cells [134]. Ubiquitination may induce endocytosis and thereby can initiate degradation of membrane proteins [135,136]. A higher proportion of short-chain ubiquitination was observed in BSEPE297G and BSEPD482G as compared to wildtype BSEP suggesting that ubiquitin dependent degradation is involved in the reduction of transporter half live [94].

Benign recurrent intrahepatic cholestasis type 2 (BRIC-2) Milder forms of BSEP-associated cholestatic liver diseases have been recognized recently and were termed ‘‘benign recurrent intrahepatic cholestasis type 2’’ (BRIC-2) [137]. The clinical presentation of BRIC-2 is similar to BRIC-1 [138], which is caused by ‘‘mild’’ mutations of FIC1 [117,139]. A higher incidence of gall stones was observed in BRIC-2 as compared to BRIC-1 patients [137], a clinical finding, which may guide the diagnostic approach in patients with recurrent, self-limiting cholestasis and low ␥GT levels. Mutations such as p.E186G, p.E297G, p.R432T, p.A570T, p.T923P, p.A926P, p.G1004D, p.R1050C and p.R1128H have been described in BRIC-2 patients [137,140—142] (Table 1). While some BRIC-2 patients have only one (heterozygous) PFIC-2 mutation, others have mutations on both alleles (compound heterozygous or homozygous). There might also

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Table 1

Genetic variants of the bile salt export pump (bile salt export pump [BSEP]/ABCB11). PFIC M1V G19R L50S M62K C68Y C107R I112T W114R Y157C A167T A167V I182K M183T M183V G188W M217R R223C S226L G238V T242I A257G V284L E297G R303G R303K Q312H R313S G327E W330R

C336S W342G A382G R387H A390P G410D L413W I420T D440E G455E K461E T463I Q466K R470Q Y472C V481E D482G R487H R487P N490D I498T G499E I512T N515T R517H F540L I541L I541T F548Y

D549V G556R G562D A570T L581F A588V S593R I627T E636G R698C S699P E709K G758R G766R Y818F R832C R832H T859R A865V Q869P G877R S901R R948C N979D G982R G1004D T1029K G1032R A1044P

L1055P C1083Y A1110E S1114R G1116E G1116F G1116R S1120N R1128C S1144R R1153C R1153H S1154P N1173D T1210P N1211D V1212F R1231Q R1231W L1242I D1243G R1268Q A1283V G1292V G1298R

Nonsense mutations (premature stop-codons)

S25X E96X W330X Y354X R415X R470X

Y472X W493X R520X I528X R575X Q702X

Y772X Q791X R928X Y1041X R1057X Q1058X

R1090X V1147X Q1215X R1235X E1302X

ICP

Other liver diseases

Genetic variants without disease association

E135K E137K E186G L198P E297G G374S A390P R432T V444A I498T A570T T586I G648V T655I T923P A926P R948C G1004D R1050C G1116R R1128H L1197G R1231Q

E137K L198P E297G R415Q V444A D482G N591S T655I

T87R M123T S194P L198P G260D E297K V444A T510T T655I D676Y P710P L827I G855R E1186K

V43I S56L Q121K R128H I206V V284A G295C G295R G295S R299K R303K L339V H423R V444A V444D V444G A459V I468I R487L Q546K Q558H E592Q V597M R616G T619A M677L M677V R696Q R698H

S701P L712L A865D A865G S874P I939M R958Q F959C F959V T965S F971L F971Y L1006F N1009H K1145N I1183T

R. Kubitz et al.

Missense mutations

BRIC/NFC

PFIC

BRIC/NFC

Splice site mutations

76 + 3G > T 77-1G > C 99-1G > T 150 + 3A > C 390-1G > A 611 + 1G > A

908 + 1delG 908 + 1G > T 908 + 1G > A 1435-13 -8del 2012-8T > G 2178 + 1G > A

2178 + 1G > T 2179-2A > G 2343 + 1G > T 2343 + 2T > C 2611-2A > T R1001R

3057-2A > G 3213 + 1delG 3213 + 4A > G 3213 + 5G > A

Deletions/insertions/frame shifts

Q101Dfs8X T127Hfs6X N199Ifs14X L232Cfs9X R303Sfs17X V368Rfs27X

L380Wfs18X A382 A388del P456Pfs24X H484Rfs5X I528Sfs21X I610Qfs45X

G648Vfs5X K700Sfs12X T919del K930Efs92X K930Efs79X K969 K972del

Q1058Hfs38X I1061Vfs34X L1165del A1192Efs50X T1256Tfs40X

Synonymous variants without disease association

R33R D36D R52R D58D

F90F I134I S136S V195V

L232L Y269Y Q312Q G319G E395E

I416I G418G F427F A535A

ICP

Other liver diseases

Genetic variants without disease association Q159Q Q361Q

F959Hfs1X F959Gfs48X

G557G V597V A804A G817G

I876I G937G Y981Y G1004G

A1028A K1070K T1086T A1110A

The bile salt export pump (BSEP) in health and disease

Table 1 (Continued)

K1145K

The overview shows ≈ 290 known variants of BSEP on the protein level, except splice site mutations, which are shown on cDNA level. The taxonomy and denomination of genetic variations follows the guidelines of the Human Variation Nomenclature Society (http://www.hgvs.org/mutnomen/). The variants are clustered according to the genetic type of mutation/polymorphism and to their connection with diseases (PFIC: progressive familial intrahepatic cholestasis; BRIC: benign recurrent intrahepatic cholestasis; NFC: non-fibrosing cholestasis; ICP: intrahepatic cholestasis of pregnancy). Some mutations and synonymous variants are not (yet) associated with diseases. For a number of mutations/variants the formal proof for a causal relationship to a distinct phenotype is pending. The variants are collected from different databases (http://www.lovd.nl/2.0/; http://www.ncbi.nlm.nih.gov/SNP/; http://pharmacogenetics.ucsf.edu/; http://abcmutations.hegelab.org/; http://www.hgmd.org/) and from literature [116,123,126,129,131,132,137,141—143,152,159,162,165,195,211—228]. The variants printed in bold have been detected in patients analysed in Düsseldorf and are not yet implemented in databases.

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Figure 4 Membrane expression of wildtype bile salt export pump (BSEP) and BSEP-G374S in HEK293 cells. (A) Wildtype BSEP-EYFP (BSEP-wt) and (B) BSEP-G374S-EYFP (green) were transfected in HEK293 cells. Endogenous Na+ -K+ -ATPase was stained in red and colocalization between BSEP and Na+ -K+ -ATPase was measured by fluorescence densitometry in single cells. While expression of Na+ -K+ -ATPase was not significantly different (C), total (D) and plasma membrane expression (E) of BSEP was significantly higher (*) in cells transfected with mutant BSEP-G374S as compared to wildtype BSEP. Although G374S is associated with a severe cholestatic phenotype, there is no obvious trafficking defect.

be a transition between BRIC-2 and PFIC-2 as shown for a patient compound heterozygous for p.I498T and c.2098delA [143] or siblings with the homozygous mutation p.G374S. In the latter patients, development of liver cirrhosis was delayed and occurred after childhood. Amino acid 374 is part of the putative sixth transmembrane helix and lies in vicinity to the channel pore, which may explain the severely reduced transport capacity of BSEPG374S (Stindt, Häussinger, Kubitz, unpublished). While an inverse correlation between the amount of BSEP at the plasma membrane and the severity of phenotypes has been described for other mutations [144] p.G374S showed an increased membrane expression of BSEP (as compared to wildtype BSEP) despite the severity of the phenotype (Weissenberger, Häussinger, Kubitz, unpublished) (Fig. 4).

Intrahepatic cholestasis of pregnancy and bile salt export pump (BSEP) Pruritus is the principal symptom of intrahepatic cholestasis of pregnancy (ICP), whereas jaundice occurs in only 10% of patients [145]. It most often starts during the third trimester of pregnancy [146]. ICP has a distinct geographical prevalence and affects 0,1% of pregnancies in Canada and more than 25% of Araucanians in Chile [147]. Increased

serum bile acid concentrations are almost diagnostic for ICP. Bile acid levels above 40 ␮mol/l are predictive for ICP-related complications [147] such as preterm birth or asphyxia [145,147,148]. Most ICP-patients experience spontaneous recovery within 2 weeks after delivery. In order to exclude that transaminases are elevated independent of ICP, follow up until complete normalization of liver enzymes should be assured [149]. A few ‘‘common’’ BSEP mutations (including p.E297G, p.D482G and p.N591S) have been detected in ICP-patients in heterozygous form and account for about 1.4% (7/491) of ICP-patients [150]. Canalicular expression of BSEP with the ‘‘typical’’ ICP mutation p.N591S was higher than that of the BRIC-2 (p.A570 T, p.R1050 C) or PFIC-2 mutations (p.D482G, p.E297G) [144]. Recently, a neonate has been described with homozygousity for p.N591S and, in addition, a homozygous mutation (p.H338Y) in the SLC27A5 gene. SLC27A5 encodes the bile acyl-CoA synthetase, an enzyme responsible for re-conjugation of de-conjugated BA (as part of the enterohepatic circulation) [151]. This child presented with liver fibrosis within the first year of life, while a sibling was free of symptoms although it had the same homozygous SLC27A5 mutation, while it was heterozygous for p.N591S [151], demonstrating a ‘‘dose effect’’ of N591S. Apart from rare mutations, the common BSEP polymorphism p.V444A (c.1331T > C, rs2287622, valine to alanine at

The bile salt export pump (BSEP) in health and disease position 444) has been linked to ICP. The C-allele is more frequent in ICP-patients as compared to healthy pregnant women [150,152,153]. Contraceptive-induced cholestasis (CIC) is similar to ICP and presents with pruritus, and may be more strongly associated with p.V444A [153]. Estrogens are glucuronidated and consecutively secreted into bile by the multidrug resistance related protein 2 (Mrp2). Within bile glucuronidated estrogens transinhibit Bsep [154]. In addition, estrogens induce vesicular retrieval of Bsep in rat liver [80] and 17␣-ethinylestradiol was shown to downregulate Bsep expression in mice [155]. Apart from estrogens metabolites also sulphated progesterone metabolites are increased in ICP [156,157] and may add to BSEP inhibition [158].

Anti-bile salt export pump (BSEP) antibodies and cholestasis When medical or surgical treatment (see below) of PFIC2 patients fails, liver transplantation is often required. It has been observed, that some of these patients suffer from otherwise unexplained recurrent cholestasis similar to preceding PFIC-2. In some of these patients de novo formation of anti-BSEP antibodies could be demonstrated [23,159,160]. Due to our experience (five cases), these antibodies always bind to an extracellular loop of the N-terminal half of BSEP (Fig. 2). It is speculated that the complete absence of BSEP expression (e.g. in the presence of Stopor frame shift-mutations) in the native liver is responsible for the failure to develop auto-tolerance towards BSEP, and therefore BSEP is recognized as a foreign antigen after transplantation. Treatment of children with anti-BSEP antibodies includes a change of immune-suppressants (e.g. from a calcineurin inhibitor to sirolimus [161]), plasmapheresis or immune-adsorption [23]. B-cell depletion by rituximab (directed against the CD20 epitope) represents another treatment option.

Drug-induced liver injury The common BSEP polymorphism p.V444A (c.1331T > C) is not only associated to ICP (see above), but predisposes for the development of drug-induced liver injury (DILI). Allele frequency of the C-allele in patients with DILI was 76% as compared to 59% in controls corresponding to an Odds ratio of 3 [162]. The common explanation for the relation between p.V444A and ICP or DILI is a decreased expression of BSEP in the presence of the polymorphism as shown in a small cohort [132,163], whereas no differences in the transport kinetics of the two variants (valine or alanine at position 444 of BSEP) were observed [162]. Morgan et al. determined IC50 values from 200 model compounds in a vesicle based assay and could identify 38 compounds, which inhibited BSEP-mediated TC transport with IC50 values below 25 ␮M [164]. Ogimura et al. investigated potential BSEP-related toxicity of 26 compounds in an in vitro cell model. Drugs such as Ritonavir, Cyclosporin A, Simvastatin and other became toxic in the presence, but not in the absence of bile acids. Apart from some substances (e.g. verapamil) there was a good correlation between the

545 results of the two studies, suggesting that certain drugs cause cholestasis through direct inhibition of BSEP function.

Other liver diseases in relation to bile salt export pump (BSEP) There is no association between BSEP polymorphisms/mutations and primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC) [165,166]. However, BSEP expression may be severely reduced in MDR3dependent ICP when p.V444A is present [167]. Furthermore, p.V444A may influence development of liver fibrosis in patients with chronic hepatitis C infection. The C-allele (corresponding to alanine) was associated with more severe liver fibrosis [168]. In a single cohort of 352 patients the C-allele (heterozygous or homozygous) was associated to a lower end of treatment response (ETR) and sustained virological response (SVR) [169]. The polymorphism p.V444A has a less stringent impact on HCV treatment as compared to IL-28B polymorphisms [170—173] and further studies are needed [174] to more precisely define the role of BSEP in interferon-based therapy regimens of HCV. There is evidence for an association between BSEP and cholangiocarcinoma (CC). In a recent study a higher prevalence of p.V444A was observed in CC-patients [127]. Restricted BSEP expression due to the genetic polymorphism p.V444A or due to BSEP mutations may result in altered biliary bile salt concentrations, which in turn may have an impact of cholangiocyte proliferation or apoptosis [175]. In the course of liver surgery, when a Pringle manoeuvre is carried out (interruption of blood flow through the hepatic artery and portal vein) ischemia-reperfusion injury may develop. Higher presurgical heat shock protein 70 (Hsp70) and lower Bsep mRNA levels have been associated with lower incidence of postoperative jaundice and a protective role of Hsp70 has been discussed [176]. In critically ill patients from the intensive care unit, elevation of serum bile acids levels were observed and were correlated with a reversal of BA transport due to decreased BSEP and increased basolateral MRP3 expression [177]. Finally, sepsis-associated cholestasis is regularly observed in critically ill patients. It is mediated by lipopolysaccharides (LPS) that trigger the downregulation of Bsep and Mrp2 [89,178,179] involving cytokines such as IL-6, IL-1␣ or TNF␣ [180—182]. In human liver, LPSdependent downregulation of BSEP may largely occur on a posttranscriptional level [183]. LPS-induced endocytosis of Bsep occurs in a zonal pattern with the strongest internalization in periportal hepatocytes [182]. Bsep and the bilirubin transporter Mrp2 may be endocytosed in different compartments [184]. Coadministration of dexamethasone along with LPS prevented cholestasis [89] as did feeding of rats with taurine [185]. Likewise, applying heat shock to livers prior to LPS treatment results in earlier recovery of transporter mRNA. Under these conditions physical interaction between heat shock protein 70 and Bsep was observed, which may protect Bsep from degradation [186].

546

Treatment of bile salt export pump (BSEP)-associated liver diseases In patients with PFIC-2 surgical and medical treatment options intent to prevent growth retardation and complication of chronic liver diseases [187]. Supportive treatment includes supplementation of fat-soluble vitamin K and D in order to prevent haemorrhage and rickets. Administration of medium chain triglycerides, which are absorbed independent of bile acids [187,188] may circumvent malabsorption. Partial biliary external diversion (PEBD) [189—191] aims to bypass the terminal ileum in order to remove bile acids from the enterohepatic circulation. PEBD improves clinical symptoms, growth, histology and liver function tests [191] and a success rate (drop of serum bile acid levels below 10 ␮mol/l, normalization of transaminases) of about 75% can be expected [190]. A laparoscopic approach of PEBD is possible and less invasive [192,193]. Standard treatment of PFIC includes ursodeoxycholic acid (UDCA) in a dose of 20 to 30 mg/kg/day [194], which was shown to be effective in PFIC-2 [39,169,195], PFIC-3 [149,196,197] or PFIC-1 patients [198], although failure of UDCA has also been reported [199]. The underlying genetic variants determine the responsiveness to UDCA therapy; in general treatment is more likely to be effective in patients with missense than with premature stop-codon mutations [196]. UDCA replaces toxic bile salts [200] and may amount to 40% of total serum bile salt concentration. In addition, UDCA may induce choleresis by vesicular insertion of Bsep [26,82] and may counteract bile acid induced apoptosis [201]. UDCA was shown to increase the expression of BSEP in human liver [202] and to restore rat Bsep expression in estrogen-induced cholestasis [203]. Furthermore, although UDCA is less active as an FXR agonist as compared to CDCA or CA, it may effectively induce FXR protein expression and thereby could influence BSEP expression [50]. BRIC patients do not seem to benefit from phenobarbital or cholestyramin [138], two compounds usually used in cholestatic patients [204]. Although characterized by spontaneous resolution of cholestasis, cholestatic episodes in BRIC may be disabling especially due to severe pruritus. Treatment with liver support systems has been shown to be effective and may relieve itching for months [205—207]. Rifampicin has been reported to be effective for treating pruritus in PFIC-patients [208]. Rifampicin not only induces expression of drug metabolising enzymes but also increases BSEP and MRP4 expression [209] along with a reduction of NTCP, altogether lowering intra-hepatocytic bile salt concentrations. It has been suggested that induction of MRP4/Mrp4 acts as an overflow mechanism in order to prevent bile acid overload of hepatocytes [7,60,202,210]. Chaperons may gain a role in the treatment of inherited diseases such as PFIC. At least, 4-phenylbutyrate, which is used for the treatment of urea cycle defects and which may act as a chemical chaperon, was shown to increase membrane expression of BSEP [134].

Concluding remarks The bile salt export pump is crucial for canalicular secretion of bile acids. Mutations of BSEP are associated from

R. Kubitz et al. moderate to severe forms of cholestasis including progressive familial intrahepatic cholestasis (PFIC) type 2, benign recurrent cholestasis (BRIC) type 2 and intrahepatic cholestasis of pregnancy (ICP). Detailed analyses of mutations and genetic variants in humans have promoted our knowledge about bile formation and will in future open new therapeutic strategies for (cholestatic) liver diseases.

Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.

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