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GASTROENTEROLOGY 2004;126:322–342

PECIAL REPORTS AND REVIEWS

nterohepatic Bile Salt Transporters in Normal Physiology andiver Disease

ERD A. KULLAK–UBLICK,*,‡ BRUNO STIEGER,* and PETER J. MEIER*Division of Clinical Pharmacology and Toxicology; and ‡Division of Gastroenterology and Hepatology, Department of Internal Medicine,
niversity Hospital, Zurich, Switzerland
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he vectorial transport of bile salts from blood into bile isssential for the generation of bile flow, solubilization ofholesterol in bile, and emulsification of lipids in the intes-ine. Major transport proteins involved in the enterohepaticirculation of bile salts include the hepatocellular bile saltxport pump (BSEP, ABCB11), the apical sodium-de-endent bile salt transporter (ASBT, SLC10A2) in cholan-iocytes and enterocytes, the sodium-dependent hepato-yte bile salt uptake system NTCP (SLC10A1), the organicnion transporting polypeptides OATP-C (SLC21A6),ATP8 (SLC21A8) and OATP-A (SLC21A3), and the mul-idrug resistance protein MRP3 (ABCC3). Synthesis andransport of bile salts are intricately linked processeshat undergo extensive feedback and feed-forward reg-lation by transcriptional and posttranscriptional mech-nisms. A key regulator of hepatocellular bile salt ho-eostasis is the bile acid receptor/farnesoid X receptorXR, which activates transcription of the BSEP andATP8 genes and of the small heterodimer partner 1SHP). SHP is a transcriptional repressor that mediatesile acid-induced repression of the bile salt uptake sys-ems rat Ntcp and human OATP-C. A nuclear receptorhat activates rodent Oatp2 (Slc21a5) and human MRP2ABCC2) is the pregnane X receptor/steroid X receptorXR/SXR. Intracellular trafficking and membrane inser-ion of bile salt transporters is regulated by lipid, protein,nd extracellular signal-related kinases in response tohysiologic stimuli such as cyclic adenosine monophos-hate or taurocholate. Finally, dysfunction of individualile salt transporters such as BSEP, on account of ge-etic mutations, steric inhibition, suppression of genexpression, or disturbed signaling, is an important causef cholestatic liver disease.

ile acid research experienced a renaissance with theproclamation of a “bile-ology” role for bile acids as

ey signaling molecules in the enterohepatic circula-ion.1 The traditional image of bile acids as solvents ofholesterol in bile and lipid emulsifiers in the intestineas been revolutionized in recent years. An intriguingeature of bile acid metabolism is the efficiency of their

ycling through the liver and the intestine, with onlyinimal losses of about 0.5 g/d into feces. This loss is

ompensated for by de novo synthesis in the liver, arocess that is subject to extensive feedback autoregula-ion by bile acids. The coordinated action of bile saltransport proteins maintains ongoing hepatic extractionrom portal blood, biliary excretion, and absorption ofile salts in the intestine. These processes are essential forile acid and lipid homeostasis, as evidenced by the widepectrum of phenotypic abnormalities attributable to theysfunction of individual transporter molecules. Thiseview first summarizes the transport systems involved inhe enterohepatic circulation of bile salts and then fo-usses on recent insights into the regulation and diseasetates associated with defects of bile salt transport. Forurther information, the reader is referred to severalomplementary reviews that have appeared recently.2–7

Physiology of the EnterohepaticCirculation of Bile AcidsBile Acid Synthesis

Bile acids are synthesized from cholesterol byither a neutral or classical pathway resulting in forma-ion of cholic acid or by an acidic or alternative pathway

Abbreviations used in this paper: ABC, adenosine triphosphate–inding cassette; ATP, adenosine triphosphate; BAR, bile acid recep-or; BARE, bile acid response element; BRIC, benign recurrent intra-epatic cholestasis; CAR, constitutive androstane receptor; CDCA,henodeoxycholic acid; CYP, cytochrome P450 enzyme; DCA, deoxy-holic acid; FXR, farnesoid X receptor; HCC, hepatocellular carcinoma;NF, hepatocyte nuclear factor; I-BABP, ileal bile acid binding protein;PS, lipopolysaccharide; LXR, liver-X receptor; MDR, multidrug resis-ance gene product; OATP, organic anion transporting polypeptide;I3K, phosphoinositide 3-kinase; PPAR, peroxisome proliferator-acti-ated receptor; PXR, pregnane X receptor; RAR, retinoic acid receptor;dx, radixin; RXR, retinoic X receptor; SHP, small heterodimer partner;REBP, sterol regulatory element-binding protein; UDCA, ursodeoxy-holic acid.

© 2004 by the American Gastroenterological Association0016-5085/04/$30.00

doi:10.1053/j.gastro.2003.06.005

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January 2004 BILE SALT TRANSPORTERS IN LIVER DISEASE 323

eading to the synthesis of chenodeoxycholic acidCDCA). In the classical pathway, the rate-limiting en-oplasmic reticulum enzyme cholesterol-7�-hydroxylasecytochrome P450 enzyme [CYP] 7A1) hydroxylatesholesterol at the 7� position and the resulting 7�-ydroxycholesterol is 12�-hydroxylated by microsomalterol-12�-hydroxylase (CYP8B1).8 A cascade of down-tream reactions ensues, including hydroxylation of theliphatic side chain at position 27 via mitochondrialYP27.9 In the alternative pathway, 7�-hydroxylation isreceded by the formation of several different oxysterols.lthough 25- and 27-hydroxycholesterol are subse-uently 7�-hydroxylated by CYP7B1 oxysterol-7�-hy-roxylase, the oxysterol 24-hydroxycholesterol is a sub-trate of CYP39A1.10,11 Oxysterols are also substrates ofYP7A112 and 7�-hydroxylation of oxysterols blocks

heir ability to inhibit sterol regulatory element-bindingrotein (SREBP).13 Thus, 7�-hydroxylation of oxysterolsan markedly influence lipid metabolism through in-reasing the expression of genes normally regulated byREBP, notably the low-density lipoprotein receptor.14

his also explains why variation in the expression level ofYP7A1 correlates with the expression of SREBP-regu-

ated genes.15 In humans, the classical bile acid syntheticathway is predominant, with only about 10% of bilecids being produced via the alternative pathway. Thisxplains why �50% of the bile acid pool consists ofholic acid and a further 20% of its metabolite deoxy-holic acid (DCA), whereas CDCA constitutes �30% ofhe total bile acid pool in humans.16

Hepatocellular Bile Salt Excretion

After their synthesis in hepatocytes, bile acids arexcreted as C24 amides conjugated with glycine or tau-ine. Amidation of bile acids lowers the pKa to �3 andmproves their solubility in the intestinal lumen. Athysiologic pH in bile fluid and in blood the amides areresent in anionic salt form and are thus called bile salts.he term “bile salts” is used to denote the predominant

ransport form of glycine or taurine conjugated bilecids, which are negatively charged at physiological pH.n contrast, the term “bile acids” denotes newly synthe-ized bile acid derivatives exhibiting a terminal acidicroup. Under normal conditions, conjugated bile saltsre excreted into bile via the bile salt export pumpBSEP, ABCB11), an adenosine triphosphate (ATP)-inding cassette (ABC) transporter localized in the can-licular or apical domain of the hepatocyte plasma mem-rane (Figure 1).3 In human liver, BSEP is a 160-ilodalton protein that exhibits a high affinity for bilealts when expressed at high levels in baculovirus-in-

ected Sf9 insect cells. The Michaelis constant (Km) for3H]-taurocholate ranges between 4.25–7.9 �mol/L, andhe rank order of the intrinsic clearance of bile salts isaurochenodeoxycholate � taurocholate � tauroursode-xycholate � glycocholate.17,18 BSEP belongs to the

igure 1. Bile salt transporters in human liver and intestine. At theasolateral hepatocyte membrane, the major uptake system for con-

ugated bile salts is the Na�-dependent bile salt transporter NTCPSLC10A1). Na�-independent bile salt uptake is mediated by theultispecific organic anion transporting polypeptides OATP-A

SLC21A3), OATP-C (SLC21A6), and to a minor extent OATP8SLC21A8). At the canalicular hepatocyte membrane, excretion ofonovalent bile salts is mediated by the bile salt export pump BSEP

ABCB11), whereas excretion of nonbile salt organic anions such asilirubin glucuronides, as well as of divalent sulfated or glucu-onidated bile salts, is mediated by the multidrug resistance associ-ted protein MRP2 (ABCC2). Biliary excretion of phospholipids andholesterol is mediated by MDR3 (ABCB4) and ABCG5/ABCG8, re-pectively. The FIC1 gene product, which may be involved in bile saltransport, is also expressed at the canalicular membrane of hepato-ytes. MRP3 (ABCC3) is a basolateral efflux system for anionic con-ugates that also mediates low-affinity transport of monovalent bilealts. MRP3 is expressed in hepatocytes, cholangiocytes, and entero-ytes. MRP4 (ABCC4) is a basolateral efflux system for anionic con-ugates including sulfated bile salts, and its mouse homolog (Mrp4) isnduced in the livers of Fxr-/- mice that exhibit elevated concentrationsf bile salts.226 In cholangiocytes and enterocytes, the apical Na�-ependent bile salt transporter ASBT (SLC10A2) mediates bile saltptake across the luminal membrane. Basolateral efflux into theeriductular capillary plexus occurs via organic anion exchange mech-nisms and via MRP3. Ductular bile is modified by chloride channelsuch as the cystic fibrosis transmembrane conductance regulatorCFTR) and the chloride/bicarbonate exchanger (anion exchanger 2,E2). Enterocytes also express MRP2, the cholesterol transporterBCG5/ABCG8, and the PXR-inducible xenobiotic efflux pump MDR1t their apical membrane. Open symbols denote Na�-cotransportNTCP, ASBT) or anion exchange mechanisms; grey symbols repre-ent ATP-binding cassette (ABC) transporters.

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324 KULLAK–UBLICK, STIEGER, AND MEIER GASTROENTEROLOGY Vol. 126, No. 1

ultidrug resistance (MDR) gene family of ABC trans-orters with orthologous gene products in a variety ofnimal species including rat, mouse, rabbit, pig, and theittle skate.19–25 In mammalian species, the canalicularile salt concentration is about 1000-fold higher thanhat in portal blood, underscoring the need for an activeTP-dependent efflux system at the canalicular pole of

he hepatocyte.In addition to excretion of monovalent bile salts via

SEP, the canalicular MRP 2 is capable of transportinglucuronidated and sulfated bile salts.26 MRP2 mediatesxport of multiple organic anions including conjugatedilirubin from hepatocytes into bile and its enterohepaticxpression is confined to the canalicular domain of hepa-ocytes (Figure 1) and the apical domain of enterocytes inhe proximal small intestine. MRP2 expression is regu-ated by diverse mechanisms, including membrane re-rieval and insertion, translation, and transcription.3 Inddition to transport of anionic conjugates, MRP2 trans-orts cancer chemotherapeutics, uricosurics, antibiotics,eukotrienes, glutathione, toxins, and heavy metals.27

RP2 is thus important clinically because it modulateshe pharmacokinetics of many drugs. Genetic studiesave identified various naturally occurring mutationshat are associated with a loss of MRP2 expression and/orunction.28 In cholestatic liver disease, MRP2 expressions decreased.29–31 A second canalicular efflux pump forulfated conjugates is human ABCG2. Potential physio-ogic substrates include estrone-3-sulfate and dehydro-piandrosterone sulfate.32

The excretion of amphipathic bile salts into bile driveshe canalicular excretion of phosphatidylcholine and freeholesterol as unilamellar vesicles, the relative lipid com-osition of which determines the risk for cholesterolallstone formation.33 In the presence of an excess of bilealts, mixed micelles are formed and stored in the gall-ladder. Recently, a heterodimeric ABC transporter thats encoded by 2 oppositely oriented, closely opposedenes termed ABCG5 and ABCG8 has been identified asn apical transporter involved in cholesterol excretionnto bile (Figure 1).34,35 Another ABC transporter in-olved in cholesterol transport is ABCA1, which medi-tes cellular cholesterol efflux from peripheral macro-hages but is also expressed at the basolateral surface ofepatocytes and Caco2 cells.36,37 Absence of ABCA1 inice with a null allele does not affect biliary cholesterol

xcretion.38 Mutations in the ABCA1 gene in man arehe cause of Tangier disease, a familial high-densityipoprotein deficiency.39–41 Expression of both ABCG5/BCG8 and ABCA1 is subject to feedforward regulationy cholesterol through oxysterol-dependent activation of

he liver-X receptor (LXR), the master regulator of he-atic cholesterol homeostasis.42,43

Cholangiocyte Bile Salt Transport

After excretion by hepatocytes into the bile cana-iculus, primary hepatic bile is modified during its pas-age through the biliary tree by organic anion and elec-rolyte transport proteins expressed in biliary epithelialells (Figure 1). Cholangiocytes are capable of Na�-ependent uptake of bile salts via the apical sodium-ependent bile salt transporter ASBT (SLC10A2), that isxpressed at the apical or luminal membrane.44 Absorp-ion of bile salts within the biliary tree is the first stepithin the so-called cholehepatic shunt pathway that

llows for intrahepatic cycling of bile salts through theeriductular capillary plexus. Considering that luminalree concentrations of bile salts are in the millimolarange, it is unlikely that uptake of bile salts by cholan-iocytes quantitatively affects biliary bile salt concentra-ions under normal conditions. The physiological role ofile salt uptake by cholangiocytes probably pertains tohe regulatory effect of bile salts on intracellular signal-ng mechanisms including cholangiocellular mucin andicarbonate secretion. After uptake across the luminalholangiocyte membrane, bile salts are effluxed across theasolateral cholangiocyte membrane into the periductu-ar capillary plexus via an anion exchange mechanism.45

n man, this anion exchanger could be the organic anionransporting polypeptide A (OATP-A, SLC21A3), whichas been shown to be expressed in human gallbladder-erived biliary epithelial cells.46 In addition to an anionxchange mechanism, a certain proportion of bile saltsppears to be secreted via MRP3 (ABCC3). MRP3 haseen localized at the basolateral membrane of cholangio-ytes and human gallbladder epithelial cells in immuno-uorescence studies (Figure 1).47,48 A third possible can-idate for bile salt extrusion across the basolateralembrane is an alternatively spliced form of rat Asbt,

alled t-Asbt.49 The t-Asbt is specifically localized to theasolateral domain of cholangiocytes and transport stud-es in Xenopus laevis oocytes showed that t-Asbt canunction as a bile salt efflux protein.49

Intestinal Absorption of Bile Salts

A critical step in bile acid homeostasis is theireabsorption in the intestinal lumen, which is a majoreterminant of the bile acid pool size and the activity ofhe bile acid synthesizing enzymes CYP7A1 andYP27.50 Reabsorption occurs primarily in the ileum,nd the first step involves uptake into ileal columnarpithelium via the apical sodium-dependent bile saltransporter ASBT (SLC10A2), which has also been called

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leal bile acid transporter or ileal sodium-dependent bilealt transporter.51,52 The human ASBT protein consists of48 amino acids and is encoded by a major 4.0-kbranscript that has been detected in the ileum, cecum,nd kidney by northern blot analysis.53 In rats, expres-ion of Asbt has been shown on the apical surface of ilealnterocytes, renal proximal tubular cells, and largeholangiocytes.44,54 Human ASBT transports conjugatednd unconjugated bile salts with a higher affinity forDCA and DCA than for taurocholate.53 The ASBT gene

s localized on chromosome 13q33.55 In addition toa�-dependent ASBT-mediated bile salt uptake, Na�-

ndependent uptake down the length of the small intes-ine in rats is mediated by Oatp3 (Slc21a7), which haseen shown to be expressed at the apical surface of jejunalnterocytes.56 Rat Oatp3 has the highest affinity forlycine-conjugated dihydroxy bile salts, which is ingreement with previous jejunal perfusion studies inuinea pigs showing that dihydroxy bile salt conjugatesre absorbed more rapidly than trihydroxy conjugates inhe jejunum.57

After uptake into the enterocyte, bile salts are shuttledo the basolateral domain for efflux into the portal cir-ulation. Intracellular transport is mediated by the 14ilodalton ileal bile acid-binding protein (I-BABP), thats cytoplasmatically attached to ASBT.58 The bile saltinding site of I-BABP has been characterized by usingcombination of photoaffinity labeling, mass spectrom-

try and nuclear magnetic resonance structure.59 Baso-ateral extrusion of bile salts is mediated by MRP3ABCC3), a member of the MRP subfamily of transport-rs that is expressed at the basolateral membrane of ilealnd colonic enterocytes,60 hepatocytes,61,62 cholangio-ytes,47,62 and in kidney.63 MRP3 substrates include therganic anions estradiol-17�-glucuronide,64,65 bilirubinlucuronide,66 the monovalent bile salts taurocholate andlycocholate,67,68 and divalent sulfated bile salts.67 Al-hough MRP3 represents a bile salt efflux system at theasolateral membrane of enterocytes, human MRP3 has aower affinity for conjugated bile salts than rat Mrp3,uggesting that the overall contribution of human MRP3o basolateral bile salt efflux might be small.68,69

Hepatocellular Uptake of Bile Salts

The final step in the enterohepatic circulation ofile salts is the extraction from portal blood plasma byepatocytes. This process is described in detail in severalecent review articles.2–4 Bile salts circulate in plasmaightly bound to albumin and lipoproteins such as high-ensity lipoprotein.5 More than 80% of conjugatedile salts undergo single-pass extraction by the liver,redominantly via the Na�-taurocholate cotransporting

olypeptide (NTCP, SLC10A1). NTCP belongs to theame transporter family as ASBT, and the human proteinonsists of 349 amino acids with a high affinity foraurocholate (Km �6 �mol/L).70 It has been localized tohe basolateral hepatocyte membrane in rat and humaniver (Figure 1).71,72 In contrast to ASBT, the substratepecificity of rat Ntcp is not limited strictly to bile saltsut also includes sulfated sex steroids, bromosulfophtha-ein, thyroid hormones, and the drug conjugate chloram-ucil-taurocholate.2,72–74 The predominant role of Ntcpn Na�-dependent hepatocellular bile salt uptake is ev-dent from several observations: (1) the Km values ofa�-dependent taurocholate uptake by hepatocytes are

imilar to Km values in Ntcp expressing transfected cellines2; (2) decreases of Ntcp expression in cultured pri-

ary rat hepatocytes and in regenerating liver are par-lleled by decreased Na�-dependent taurocholate up-ake3; and (3) Na�-dependent taurocholate uptake inenopus laevis oocytes injected with total rat liver mes-

enger RNA (mRNA) is decreased by 95% after inhibi-ion of Ntcp expression by antisense oligonucleotides.75

Next to the sodium-dependent uptake system NTCP,a�-independent hepatic uptake of bile salts is mediated

y the organic anion transporting polypeptides (Oatps/ATPs) (Figure 1). The functional properties of OATPs

re reviewed in detail elsewhere.2,76,77 Both rodent anduman hepatocytes express 3 members of the OATPuperfamily at appreciable levels. Rat liver membersomprise Oatp1 (Slc21a1), Oatp2 (Slc21a5), and Oatp4Slc21a10). Oatp1 and Oatp2 belong to the OATP1Aubfamily and Oatp4 to the OATP1B subfamily, accord-ng to the recently proposed new classification and no-

enclature scheme for OATPs (see Note added inroof).77 All 3 rat liver Oatps transport bile salts withm values for taurocholate uptake of 32–50 �mol/L foratp1, 35 �mol/L for Oatp2, and 9–27 �mol/L foratp4.2 Human hepatocytes predominantly expressATP-C (SLC21A6) and OATP8 (SLC21A8), whichelong to the OATP1B subfamily, OATP-B (SLC21A9,member of the OATP2B subfamily)77 and OATP-A,hich represents the only human member of theATP1A subfamily.77,78 Of these 4 human OATPs,

elevant bile salt transport could be shown for OATP-,79–82 the major Na�-independent bile salt uptake sys-

em of human liver, OATP-A78 and to a lesser extentATP8.82 The Km of OATP-C–mediated taurocholateptake is in the range of 14–34 �mol/L.79,80 A secondmportant function of OATP-C is uptake of unconju-ated bilirubin from sinusoidal blood plasma.83,84

ATP-A is a 60-kilodalton bile salt transporter with aroad substrate specificity for xenobiotics.76–78,85 Its ex-

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ression levels appear to vary interindividually and oc-asional high levels of expression in human liver haveeen reported.86 Of note, OATP-A expression levels arencreased in liver tissue of patients with cholestatic liverisease.87 In summary, OATP-C and OATP-A are the 2a�-independent bile salt uptake systems of human

epatocytes, whereas OATP8 mediates taurocholateransport to a lesser extent and OATP-B does not trans-ort bile salts at all.82

Regulation of Bile Salt Transport andImplications for Liver Disease

Virtually all bile salt transport systems mentionedarlier are not only constitutively expressed but alsoubject to extensive regulation, mainly at the level ofene transcription. These regulatory mechanisms repre-ent adaptive responses to (1) intracellular accumulationf bile salts and other amphipathic compounds that act asigands for nuclear receptors and (2) changes in thectivation of critical transcription factors in diseasetates. Through the coordinated transcriptional regula-ion of transport proteins at the plasma membrane andntracellular cytochrome P450 enzymes, the liver andntestine form a functional enterohepatic unit that canapidly metabolize and eliminate cholestatic bile saltsnd toxic xenobiotic compounds. Hence, bile salts canerve as model compounds for studying the effect ofenobiotics on gene expression. In addition to the tran-criptional cascades that regulate bile salt transporterxpression, several posttranscriptional mechanisms aremportant for the short-term adaptive requirements ofransport protein targeting and membrane insertion inesponse to substrate flux and induction of protein ki-ase–signaling pathways.

Transcriptional Regulation of BileSalt Transporters

The requirement for a regulatory network con-rolling the activity of bile salt uptake and efflux systemss evident from the fact that the intracellular accumula-ion of bile salts leads to cholestasis, hepatocyte apopto-is, and parenchymal damage.88 Bile salts can regulateot only uptake and efflux systems but also key bile acidynthetic enzymes through the action of critical tran-cription factors that bind to “bile acid responsive ele-ents” (BAREs) in the regulatory regions of the corre-

ponding genes. Bile salts, sterols, and fatty acids areatural ligands of nuclear hormone receptors expressed iniver and intestine (Table 1), and among these the bilecid receptor/farnesoid X receptor (BAR/FXR) has aredominant role in regulating bile acid synthesis andile salt transport. FXR (also called RIP14 and HRP1)

elongs to the NR1 family of nuclear receptors that alsoontains the pregnane X receptor (PXR), the peroxisomeroliferator-activated receptor � (PPAR�), and the LXR.XR and LXR are closely related and belong to theR1H subfamily. FXR is highly expressed in the liver

nd intestine but also in adrenal gland and kidney.89,90

he most effective activator of FXR is CDCA, with anffective concentration (EC50) of about 10–20 �mol/L inV-1 cells (green monkey kidney cells).91 The secondaryile acids lithocholic acid and DCA are less effective, andhe hydrophilic bile acid ursodeoxycholic acid (UDCA)as been reported to be inactive.90 Nuclear receptorsenerally bind to AG(G/T)TCA-like hexameric repeatotifs as heterodimers with the retinoid X receptor

RXR).Activation of bile salt transporter genes by the

AR/FXR. Bile salt transporter genes that are directly orndirectly regulated by FXR include BSEP, MRP2, rattcp, OATP-C and OATP8 in hepatocytes, and I-BABP

n the intestine.92–98 BSEP, MRP2, OATP8, and I-BABPave an inverted repeat element in the promoter regionhat directly binds the FXR/RXR heterodimer and isctivated by CDCA.92–94,97,98 Activation of the bile saltfflux pumps BSEP and MRP2 by bile acids is an im-ortant adaptive mechanism by which hydrophobic bilecids can promote their own efflux into bile. Interest-ngly, the cholestatic bile acid lithocholic acid inhibitshe BSEP promoter and strongly decreases BSEP expres-ion in primary human hepatocytes and HepG2 cellshrough antagonism of FXR activity.99 This could ben important mechanism of cholestasis induced by cho-estatic bile salts. The induction of OATP8, which isainly a xenobiotic and peptide uptake system,82,100–102

ay serve to maintain the hepatocellular clearance ofenobiotics in cholestatic situations when other basolat-ral uptake systems such as OATP-C are down-regu-ated.29,30 None of the Oatps in rodent liver has beenhown to be activated by FXR. However, rodent Oatp2s induced by lithocholic acid through a PXR-mediatedechanism.103–105

Repression of bile salt transporter genes throughndirect FXR pathways. Rat Ntcp, human OATP-C and

ouse Asbt are transcriptionally repressed by bile saltshrough FXR-mediated induction of the small het-rodimer partner (SHP)1. SHP is a nuclear receptorNR0B2) that negatively interacts with other nucleareceptors such as the liver receptor homolog (LRH)1 (alsoalled fetal transcription factor [FTF], NR5A2) andepatocyte nuclear factor (HNF) 4�, essential activatorsf the bile acid synthetic enzymes CYP7A1 andYP8B1.106–110 Denson et al.95,111 showed that the rat

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able 1. Nuclear Receptors Involved in the Regulation of Bile

Nuclear receptors Ligands Transp

regnane X receptor PXR Xenobioticsrifampin,RU486,steroids,phenobarbital,St. John’swort, statins,bile salts, bileacid precursors

1 MDR

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onstitutive androstanereceptor

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1 OATP

1 BSEP1 MRP

eroxisome proliferatoractivated receptor �

PPAR� fatty acidsfibratesDHEAS

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lucocorticoid receptor GR glucocorticoids 1 ASBT

etinoic acid receptor RAR� retinoids 1 Ntcp

mall heterodimer partner SHP-1 activated by FXR 2 Ntcp

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ure in HepG2 cells.111 This suppressive effect of IL-1�n RAR�:RXR� transactivation involves mitogen-acti-ated protein kinase pathways, specifically phosphoryla-ion of RXR by the c-Jun N-terminal kinase.112

Mouse but not rat Asbt promoter activity is repressedn Caco2 cells by CDCA.113 The mouse Asbt promoterinds and is activated by LRH1. SHP diminishes thectivity of the mouse Asbt promoter and partially offsetsts activation by LRH1. The bile acid response is medi-

t and Drug Transporters

roteins Functional consequences References

CB1) Increased efflux of xenobiotics fromhepatocytes and enterocytes

119,125

21a5) Increased hepatocellular uptake of organicanions and xenobiotics (e.g., digoxin)

104,105

CC2) Increased efflux of anionic conjugates andxenobiotics from hepatocytes andenterocytes

94

CC2) Increased apical efflux of anionicconjugates and xenobiotics fromhepatocytes and enterocytes

94

CC3) Increased basolateral efflux of anionicconjugates and xenobiotics fromhepatocytes, cholangiocytes, andenterocytes

127

Increased bile acid protein binding in ilealenterocytes

98

C21A8) Increased hepatocellular uptake of organicanions and xenobiotics (e.g., digoxin)

97

B11) Increased biliary excretion of bile salts 92,93CC2) Increased efflux of anionic conjugates and

xenobiotics from hepatocytes andenterocytes

94

b4) Increased phospholipid flippase at thecanalicular hepatocyte plasmamembrane

132

10A2) Increased Na�-dependent bile salt uptakein ileum

116

10A2) Increased Na�-dependent bile salt uptakein ileum

138

0a1) Increased Na�-dependent bile salt uptakeinto hepatocytes

111

0a1) Decreased Na�-dependent bile salt uptakeinto hepatocytes

95

Decreased Na�-independent bile saltuptake into hepatocytes

96

CC3) Increased basolateral efflux of anionicconjugates and xenobiotics fromhepatocytes, cholangiocytes andenterocytes

129

Increased cholesterol efflux frommacrophages, hepatocytes andenterocytes

37,227–229

CG8 Increased cholesterol secretion into bile 42,230

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ted by FXR and can be antagonized by overexpression ofdominant-negative FXR in Caco2 cells.113

The mechanisms leading to down-regulation of theATP-C gene in cholestasis have been shown to involveile acid mediated repression of HNF1�, the majorranscriptional activator of OATP-C.114 HNF1� RNAnd protein levels in cultured hepatoma cells are de-reased �50% by exposure to CDCA and the humanNF1� gene contains a classic BARE in the minimalromoter region.96 This BARE contains an HNF4�inding site and the central mechanism by which bilecids repress HNF1� is decreased activation by HNF4�.n addition, HNF4� nuclear binding activity is de-reased by CDCA and the human HNF4� gene promoters repressed by CDCA through a SHP-independentechanism.96 These findings indicate a further regula-

ory cascade by which bile acids regulate hepatic geneshrough suppression of the master transcription factorsNF1� and HNF4�. In man, this mechanism may even

redominate over the effects of bile acids on LRH1,specially because human CYP7A1 and ASBT—unlikeheir rodent counterparts—are not activated byRH1.106,107,113,115,116

Role of the PXR/SXR. The second nuclear recep-or, next to FXR, that regulates bile salt transporterenes is PXR, also known as SXR in humans.117–119

igands of PXR include rifampicin, RU486, St. John’sort extract, clotrimazol, lovastatin, phenobarbital, bile

alts, and bile acid precursors.93,120–123 PXR is the classicxenosensor,” which binds as a heterodimer with RXR to“xenobiotic response element” in the promoter of theuman CYP3A4 gene.117,124 PXR is expressed mainly inhe liver and intestine, and other target genes that arectivated by PXR include human MDR1,125 MRP2,94

nd rodent Oatp2.103,105 PXR can also act as a transcrip-ional repressor, as shown for mouse cholesterol-7�-hy-roxylase,103 although the exact mechanism of repressions unknown. Until today, MRP2 and rodent Oatp2 arehe only bile salt transporters shown to be regulated byXR. Reduced expression of Oatp2 in ethinylestradiol-

nduced cholestasis in rats is associated with reduceduclear-binding activity of PXR.126 The humanATP-A gene also contains a clustered PXR consensusite that binds PXR (Jung et al., unpublished data, June002), and it is conceivable that the induction ofATP-A expression observed in certain cholestatic pa-

ients is a PXR-related effect.87

Role of other transcription factors in the regula-ion of bile salt transporter genes. The role of nucleareceptors other than FXR and PXR in regulating bilealt transport is less well established (Table 1). The

asolateral bile salt transporter Mrp3, which is stronglynduced in obstructive cholestasis in rats and compen-ates for decreased Mrp2 expression,47 is induced byonstitutive androstane receptor (CAR).127,128 CAR is auclear receptor known to strongly activate CYP2B1xpression, and recent evidence suggests that bilirubinay induce translocation of CAR from the cytosol to the

ucleus, suggesting a regulatory role of CAR in choles-atic liver disease. In addition to regulation by CAR, theuman MRP3 gene possesses 2 alpha-1 fetoprotein tran-cription factor (FTF) binding sites that confer induc-bility by FTF.129 FTF mRNA levels are increased byDCA in human colonic cells. Whether hepatic MRP3xpression is also induced by bile salts through an FTF-ediated mechanism has not been studied.The rat Ntcp gene is induced by the pituitary hormone

rolactin, leading to increased Ntcp mRNA and anncreased Vmax for Na�-/taurocholate cotransport inepatocytes from postpartum rats.130 Prolactin binds tohe prolactin receptor and triggers a signaling cascadehat results in recruitment of Stat5, a member of theignal transducers and activators of transcription family.uclear translocation of phosphorylated liver Stat5 is

orrelated with suckling-induced increases in serum pro-actin levels. The Ntcp gene promoter is transactivated bytat5, which binds to interferon-�–activated sequence–ike elements in the promoter sequence.131

The apical sodium-dependent bile salt transporterSBT and the hepatocyte canalicular phospholipidippase Mdr2/MDR3 (ABCB4) are activated byPAR�.116,132 A key function of PPAR� is the regula-ion of genes involved in various steps of fatty acidetabolism133 and PPAR� ligands include fatty acids

nd fibrate drugs. A possible link between ileal bile saltbsorption via ASBT and hepatic fatty acid catabolismas long been postulated on the basis of the observationhat patients with type IV hypertriglyceridemia exhibitecreased intestinal bile salt absorption and reduced ilealxpression of ASBT mRNA and protein by 32% and3%, respectively.134,135 Although the cause-effect rela-ionship of this association remains to be established, thending that PPAR� regulates the human ASBT genepens the possibility that decreased activity of PPAR�known to be a highly polymorphic gene) could be theause of both hypertriglyceridemia and decreased ilealile salt absorption. Sequence polymorphisms of theSBT gene itself as a cause of decreased ASBT expression

n hypertriglyceridemia have been excluded.136 Anothermplication of ASBT induction by PPAR� concernsptake of bile salts by biliary epithelial cells expressingSBT. Fibrate drugs are ligands of PPAR� and an

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mportant adverse effect of fibrate treatment is the in-reased risk of cholesterol gallstone formation. Fibratesecrease biliary bile salt excretion and consequently in-rease biliary cholesterol saturation. Although 1 mecha-ism is the suppression of hepatic bile acid synthesis,137

ncreased expression of ASBT at the apical surface ofholangiocytes secondary to fibrate-induced PPAR� ac-ivation could increase cholangiocellular bile salt absorp-ion, thereby contributing to the increased biliary cho-esterol/bile salt ratio.

An important mechanism of induction of intestinala�-dependent bile salt transport is glucocorticoid re-

eptor (GR)-mediated transactivation of ASBT gene ex-ression. The GR is a nuclear steroid hormone receptorhat is activated by nanomolar concentrations of glu-ocorticoids. The human ASBT gene has been shown toe transactivated by the GR and its ligands dexametha-one and budesonide.138 This is of particular interestith respect to the fact that patients with Crohn’s diseaseave decreased ileal ASBT expression and consequentlyuffer from intestinal bile salt malabsorption.139,140 Thus,ne of the beneficial effects of glucocorticoid treatment inrohn’s disease could be the induction of ileal ASBTxpression, thereby ameliorating the chologenic compo-ent of diarrhea.

Posttranscriptional Regulation ofHepatocellular Bile Salt Transporters

Hepatocytes are exposed to variable loads of bilealts with especially high bile salt concentrations inostprandial portal blood plasma. These rapid changes inhe hepatocellular bile salt transport capacity cannot beccounted for by transcriptional regulation but requireosttranscriptional adaptive processes. Such posttran-criptional modifications include alterations in proteinargeting by recruitment of carriers from intracellularools, leading to an increased transporter density at thelasma membrane. Alternatively, functional activity ofhe transporters may be modulated by protein phosphor-lation/dephosphorylation or by protein-protein interac-ions.

Canalicular transporters. Canalicular ABC trans-orters such as rat Bsep, Mdr1, and Mdr2 have beenhown to be targeted to the canalicular membrane inesponse to adenosine 3,5-cyclic monophosphatecAMP) and taurocholate.141 Because pretreatment withycloheximide, an inhibitor of protein biosynthesis, doesot prevent this increase in canalicular transporter den-ity, recruitment of transporters must occur from anxisting intracellular pool rather than from enhancedranscription or translation. The “cAMP pool” probablyiffers from the “taurocholate pool.”142 Newly synthe-

ized Bsep, after being targeted through an endosomalompartment, accumulates in an intrahepatic cAMP poolnd later equilibrates with the taurocholate pool. Underasal conditions, the majority of Mdr1, Mdr2, and Bseproteins appear to reside in intrahepatic pools rather thann the canalicular membranes as indicated by an intra-epatic/canalicular transporter ratio of �6:1.141

Posttranscriptional regulation of canalicular ABCransporters is under the control of the lipid kinaseI3K.143,144 PI3K is required for recruitment and vesic-lar trafficking of ABC transporters from intracellularools to the canalicular membrane after stimulation byaurocholate.144 After administration of the PI3K inhib-tor wortmannin, taurocholate-induced bile salt secretions decreased by 50%. PI3K is also necessary for maximalTP-dependent transport of Mrp2, Mdr2, and Bsep (butot Mdr1) in the canalicular membrane, presumablyecause of specific interaction of PI3K lipid productsith these ABC transporters.141 Next to PI3K, proteininase C isoforms are also important for intracellularransporter trafficking to the canalicular membrane. Ac-ivation of protein kinase C by UDCA or by the phorbolster 12-myristate-13-acetate increases the density ofrp2 transporter molecules at the canalicular mem-

rane.145,146 Additional mechanisms of UDCA-inducednsertion of transporters into the canalicular membraneight involve p38 mitogen-activated protein kinase and

xtracellular signal-related kinases.147,148 Furthermore,ecruitment of intracellular Bsep to the canalicular mem-rane is also induced by hypo-osmolarity, whereas hy-erosmolarity causes cholestasis through retrieval of bothsep and Mrp2 into intracellular vesicles.149

Finally, canalicular localization of Mrp2 is dependentn the presence of radixin (Rdx), a member of thezrin-Rdx-moesin family of proteins that crosslink actinlaments and integral membrane proteins.150 Rdx isoncentrated in bile canalicular membranes, and its ab-ence in Rdx / mice leads to decreased Mrp2 densitys compared with other canalicular proteins such as-glycoproteins.150 In vitro binding studies show thatadixin associates directly with the carboxy-terminal cy-oplasmic domain of human MRP2.150 This is the firstemonstration of a protein-protein interaction with aanalicular transporter molecule that directly affects pro-ein targeting and function.

Basolateral transporters. Studies with isolatedat hepatocytes show an enhanced sodium-dependentptake of taurocholate after addition of cAMP.151 Thisncreased bile salt transport activity is paralleled by arotein synthesis-independent translocation of Ntcprom an endosomal compartment to the basolateral cell

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ole of hepatocytes.152 Immunoprecipitation experi-ents show that Ntcp is a serine/threonine phosphopro-

ein which, on addition of cAMP, is dephosphorylated byrotein phosphatase 2B in cultured rat hepatocytes.153,154

he state of phosphorylation of Ntcp determines itsistribution between the plasma membrane and the en-osomal compartment, whereby cAMP-mediated de-hosphorylation of Ntcp leads to an increased retentionf Ntcp in the plasma membrane.155 Insertion and re-rieval of Ntcp from the basolateral plasma membraneollows vesicular transport routes of hepatocytes. Thisrocess is controlled by the phosphoinositide 3-kinasePI3K)/protein kinase B pathway and relies on intacticrofilaments.156–158 In addition to Ntcp, Oatp1 activ-

ty has also been shown to be regulated by cAMP.reatment of hepatocytes by cAMP leads to a 75%

eduction in the uptake of the prototypic Oatp1 sub-trate bromosulfophthalein into hepatocytes.159 In con-rast to the regulation of Ntcp-mediated bile salt trans-ort, cAMP does not alter the carrier density of Oatp1,ut most probably reduces the Vmax of Oatp1 by in-ucing phosphorylation of the transporter in the plasmaembrane.160

The physiologic stimuli altering the phosphorylationtate of Ntcp and Oatp1, as well as the carrier density oftcp in the basolateral plasma membrane, have not been

lucidated. It was shown that addition of ATP4 toepatocytes significantly reduces the uptake of bromo-ulfophthalein into hepatocytes, implying a role of pu-inergic receptors.159 After a meal, hepatocytes are notnly challenged with a significant bile salt load but alsoy high portal venous concentrations of nutrients such aslucose and amino acids. The latter have been shown toctivate compensatory volume decrease mechanisms inepatocytes.161 Along these lines, it was shown that cellwelling of hepatocytes leads to a translocation of Ntcpnto the basolateral plasma membrane via the PI3Kathway.162

Disease States Associated WithDisturbances of Bile Salt TransportHereditary Defects of Bile Salt Transport

The role of bile salt transporters in the pathogen-sis of liver disease is a dynamic field of research thategan in the late 1950s when it was realized that certainorms of cholestasis have an increased prevalence in se-ected families, indicating a genetic basis.163–166 Mean-hile, distinct clinical entities have been defined that are

aused by mutations in transporter genes, notably in theamilial intrahepatic cholestasis 1 (FIC1, ATP8B1),SEP (ABCB11), and MDR3 (ABCB4) genes in the case

f progressive familial intrahepatic cholestasis (PFIC)ypes 1, 2, and 3, respectively.3,4,167,168 All 3 types ofFIC are inherited in an autosomal recessive fashion andan be distinguished on the basis of clinical, biochemical,nd histological features.

PFIC type 1 (PFIC1, Byler’s Disease). This disor-er is characterized by jaundice and severe pruritus in theresence of high serum concentrations of bile salts butormal gamma-glutamyltranspeptidase and cholesterol.iver histology shows cholestasis and progression to cir-hosis without ductular proliferation. PFIC1 is caused byutations of the coding sequence of the FIC1 gene

ATP8B1), which is located on chromosome 18q 21-2169 and which is expressed in liver and small intestine.n the liver, FIC1 has been localized to the canalicularembrane of hepatocytes and to cholangiocytes.170 FIC1

ncodes a P-type adenosine triphophatase putatively in-olved in the transport of phospholipids and possibly bilealts,171 although direct transport studies have not yeteen reported. When this function is defective, the trans-ort of bile salts is impaired in the liver as well as in thentestine resulting in cholestasis and chronic wateryiarrhea.172 Mutations in the FIC1 gene are also re-ponsible for benign recurrent intrahepatic cholestasisBRIC).171

PFIC type 2 (PFIC2, Byler Syndrome). This dis-rder presents with permanent jaundice from the onsetnd liver failure occurs more rapidly than in PFIC1. It isaused by mutations of the BSEP (ABCB11) gene, whichs located on chromosome 2q 24.173 Children with PFIC2o not express BSEP.174 When PFIC2-related BSEP mu-ations are introduced artificially into rat Bsep and ex-ressed in Madin–Darby canine kidney and Sf9 insectells, the G238V, E297G, G982R, R1153C, and1268Q mutations prevent the protein from trafficking

o the apical membrane, whereas the G238V mutanteems to be rapidly degraded by proteasomes.175

hereas mutation C336S affects neither Bsep transportctivity nor trafficking, mutations E297G, G982R,1153C, and R1268Q abolish taurocholate transport

ctivity. Impaired bile salt secretion results in accumu-ation of bile salts within hepatocytes followed by hep-tocellular injury, apoptosis, and/or necrosis. A clinicalyndrome with recurrent intrahepatic cholestasis butormal liver architecture in an adolescent patient haseen associated with compound heterozygosity for the297G and a novel R432T mutation,176 suggesting thatertain adult forms of cholestasis may also be caused bySEP mutations and reduced transport function.

PFIC type 3 (PFIC3, MDR3 deficiency). In con-rast to PFIC1 and PFIC2, this disorder exhibits high

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erum GGT and shows ductular proliferation and annflammatory infiltrate in the early stages, whichrogress to biliary cirrhosis. It is caused by homozygousutations of the MDR3 (ABCB4) gene.177 This results

n a lack of MDR3 P-glycoprotein, the phospholipidxport pump of the canalicular membrane, and in anbsence of phospholipids (phosphatidylcholine) fromile. Because biliary phospholipids form mixed micellesith bile salts and thereby protect the biliary epithelium

gainst toxic bile salt effects, their lack in the bile ofatients with MDR3 deficiency leads to toxic injury ofhe biliary epithelium. MDR3 deficiency can cause aide spectrum of clinical phenotypes ranging from neo-atal cholestasis to intrahepatic cholestasis of pregnancynd cirrhosis of adulthood.178

BRIC. Like PFIC1, this disorder is also caused bymutation in the FIC1 (ATP8B1) gene, although not allRIC phenotypes are attributable to FIC1 muta-

ions.176,179 It is characterized by recurrent bouts ofholestasis in the adult with intermittent symptom-freeeriods lasting from months to several years. In contrasto PFIC1, BRIC does not lead to progressive liver dam-ge. Serum bile salt concentrations are elevated prior tohe increase of other cholestatic markers. Fecal loss of bilealts due to intestinal malabsorption is increased in non-ymptomatic BRIC patients.180 As mentioned earlier,IC1 is also expressed in the small intestine and appearso be important for intestinal bile salt absorption.

Dubin–Johnson syndrome. The Dubin–Johnsonyndrome is an autosomal recessive disorder characterizedy conjugated hyperbilirubinemia, increased urinary ex-retion of coproporphyrin I, deposits of a black pigmentn centrolobular hepatocytes, and prolonged bromosul-ophthalein retention.181 In contrast to PFIC, hepaticunction is preserved. The syndrome is caused by thebsence of MRP2 protein from the canalicular hepatocyteembrane182 because of mutations of the MRP2 gene

ABCC2).181,183,184 The MRP2Delta(R,M) mutation,hich describes the deletion of Arg1392 and Met1393,

auses disturbed maturation and trafficking of the pro-ein from the endoplasmic reticulum to the Golgi com-lex and impaired sorting of the glycoprotein to thepical membrane.185 The MRP2 I1173F mutation,hich denotes the exchange of the hydrophobic amino

cid isoleucine 1173 with phenylalanine in a predictedxtracellular loop of MRP2, leads to retention of MRP2n the endoplasmic reticulum and degradation by pro-easomes in transfected HEK293 cells, and is associatedith a loss of ATP-dependent transport of leukotriene4.186 Absent MRP2 function may be compensated

or by increased expression of MRP3 at the basolateral

epatocyte membrane, as suggested by immunofluores-ence studies on liver sections from a Dubin–Johnsonatient.61

Polymorphisms of basolateral bile salt trans-orter genes. In contrast to the established pathogeneticignificance of gene mutations in the bile excretory sys-ems of the canalicular hepatocyte membrane, the role ofefects in basolateral bile salt uptake transporters is lessell defined. Mechanistically, reduced hepatocellular up-

ake of cholephilic compounds because of dysfunction ofbile salt uptake system is unlikely to cause hepatic

arenchymal damage, whereas it may influence the he-atic clearance of a drug that is normally extracted frominusoidal blood via the same transporter. Mutations inhe human NTCP (SLC10A1) gene that are associatedith defective Na�-dependent bile salt uptake have noteen described to date.187 However, the Na�-indepen-ent organic anion transporting polypeptides appear toe highly polymorphic, notably the 4 OATPs expressedn human liver (OATP-A [SLC21A3], OATP-BSLC21A9], OATP-C [SLC21A6], and OATP8SLC21A8]).76 The functional consequences of polymor-hisms in the OATP-C gene—the major Na�-indepen-ent bile salt uptake system—have been determined initro.188 The transport capacity for estrone-3-sulfate andstradiol-17�-glucuronide is severely compromised byeveral nonsynonymous polymorphisms affecting highlyonserved amino acids within the putative transmem-rane regions and extracellular loop 5, even though theseatients have no obvious clinical phenotype. TheLC21A6-L193R amino acid substitution is a naturallyccurring mutation resulting in defective protein matu-ation and absent in vitro transport function.189 Poly-orphisms have also been identified in the OATP-A andATP8 genes,76 although the functional and clinical

onsequences remain to be defined.

Acquired Defects of Bile Salt Transport

Bile salt transporters are vulnerable targets ofumerous cholestatic metabolites and signaling path-ays (Table 2). Syndromes in which direct inhibition of

unction or expression of bile salt transporters are a causef cholestasis include drug-induced cholestasis, sepsis-ssociated cholestasis, and intrahepatic cholestasis ofregnancy. The changes in transporter expression thatccur in extrahepatic cholestasis are secondary adaptivehanges that result from the impairment of bile flow andetention of biliary constituents within the hepatic pa-enchyma.

Drug-induced cholestasis. An important mecha-ism of drug-induced cholestasis is inhibition of the bilealt export pump with accumulation of bile salts in

T

B

H

P


able 2. Role of Bile Salt Transporters in the Pathogenesis of Liver Disease

SpeciesTransportprotein

Genesymbol Physiologic function Alterations in liver disease References

asolateral transport proteins

Rat Ntcp Slc10a1 Na�-dependent hepatocellularbile salt uptake

Decreased expression in rat models of cholestasisDecreased mRNA and protein levels during pregnancy,

associated with decreased nuclear binding of HNF1� andRAR�:RXR�

201,211,231232

Human NTCP SLC10A1 Na�-dependent hepatocellularbile salt uptake

Decreased mRNA and protein levels in human cholestaticliver disease

30,187

Decreased expression in HCC 72

Rat Oatp1 Slc21a1 Multispecific uptake oforganic anions andamphipathic compounds

Decreased expression in bile duct ligation and in ethinylestradiol induced cholestasis

211,233

Oatp2 Slc21a5 Multispecific uptake oforganic anions and ofcardiac glycosides(digoxin)

Decreased mRNA but not protein levels in carbontetrachloride induced liver injury

Decreased mRNA and protein levels in ethinylestradiol-induced cholestasis

234

126

Oatp4 Slc21a10 Multispecific uptake oforganic anions andamphipathic compounds

Decreased expression in bile duct ligation and sepsis 235

Human OATP-C SLC21A6 Hepatocellular uptake of bilesalts and other organicanions

Reduced mRNA in PSC and inflammatory cholestasisDecreased expression in HCC

29,30217

OATP8 SLC21A8 Hepatocellular uptake oforganic anions, peptides,and xenobiotics

Decreased expression in HCC because of increasedexpression of the transcriptional repressor HNF3�

218

Rat/human Mrp1/MRP1 ABCC1 Efflux of cytotoxic cations andnon–bile salt organicanions

Increased expression in hepatoma cells and sepsis 199,236

Rat/human Mrp3/MRP3 ABCC3 Efflux of organic anions, bilesalts, and anticanceragents

Increased expression in Eisai Hyperbilirubinemic Rats and inbile duct ligation

Increased expression in Dubin–Johnson syndrome andprimary biliary cirrhosis

23761

epatocyte canalicular transport proteins

Mouse/rat/human

Bsep/Bsep/BSEP

ABCB11 Canalicular efflux of bile salts Gene mutations and absence of the protein in patients withPFIC2, characterized by low GGT levels and reduced biliarybile acid excretion

174,238

Compound heterozygosity for the E297G/R432T mutations ina patient with recurrent intrahepatic cholestasis

176

Reduced mRNA and canalicular BSEP staining in humaninflammatory cholestasis

30

Cisinhibition by cholestatic drugs such as cyclosporine A 190

Transinhibition by the cholestatic estrogen metaboliteestradiol-17�-D-glucuronide

190,239

Increased expression in C57L/J gallstone-susceptible mice,despite reduced bile salt excretory capacity

220,240

Mouse/rat/human

Mdr2/Mdr2/MDR3

ABCB4 Biliary excretion ofphospholipids

Mdr2 / knockout mice exhibit an absence ofphospholipids in bile and develop progressive liver diseasewith portal inflammation, bile duct proliferation and fibrosis

241

PFIC3, characterized by high GGT levels and absentlipoprotein X in serum, is caused by mutations in theMDR3 gene (chromosome 7q21)

177

MDR3 mutations in PFIC3 are associated with intrahepaticcholestasis of pregnancy

242

Rat/human Mrp2/MRP2 ABCC2 Canalicular excretion oforganic anions

Decreased mRNA and protein levels in bile duct ligation andendotoxinemia

200,243

Decreased canalicular density of Mrp2 transporter moleculesin endotoxinemia, taurolithocholate cholestasis, and bileduct ligation

145,200,243

Mutations in the rat Mrp2 gene cause hereditary conjugatedhyperbilirubinemia

244

Mutations in the human MRP2 gene cause the Dubin–Johnsonsyndrome with absent protein expression

181,183

MRP2 function is inhibited by anabolic 17�-alkylated steroids 245,246

Decreased canalicular MRP2 staining in PBC andinflammatory cholestasis

30,31

Decreased mRNA levels in PSC 29

Human FIC1 ATP8B1 Putative aminophospholipidtranslocator

P-type ATPase, positional candidate in genetic linkageanalysis of PFIC1 (Byler’s disease) and BRIC

171

SC, primary sclerosing cholangitis; PBC, primary biliary cirrhosis.

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epatocytes and subsequent liver damage. Inhibition ofsep by drugs can be tested in transport assays usingsep-expressing membrane vesicles from Sf9 insect cells.yclosporine A, rifamycin SV, rifampicin, gliben-lamide, and the thiazolidinedione insulin sensitizerrug troglitazone cis-inhibit Bsep/BSEP-mediated3H]taurocholate transport, providing a potential mech-nism for intrahepatic cholestasis caused by thesegents.17,18,190,191 Cyclosporine A additionally leads toisturbances of Bsep localization at the canalicular mem-rane and to disruption of the pericanalicular F-actinytoskeleton, an effect that was not observed with themmunosuppressive agent tacrolimus (FK506).192 Thendothelin antagonist bosentan also inhibits Bsep func-ion while increasing Mrp2-dependent bilirubin excre-ion and bile salt-independent bile flow.193,194 The sub-trate specificity of Bsep has been suggested to extendot only to bile salts but also to vinblastine, calcein-cetoxymethyl ester, and the linear hexapeptideitekiren, which could cause cholestasis through inter-ction with the canalicular efflux of bile salts.21 Anotherotential Bsep substrate and inhibitor is the nonsteroidalnti-inflammatory agent sulindac, an established hepato-oxin. Sulindac appears to be secreted into the bileanaliculus in unconjugated form via a canalicular bilealt export system and is passively absorbed by the bileuct epithelium, thereby inducing a bicarbonate-richholeresis.195 Because of continuous cycling within theholehepatic shunt pathway, high local concentrations ofulindac could be reached within the hepatocyte thatause cholestasis by inhibition of canalicular bile saltfflux.

Sepsis-associated cholestasis. Cholestasis is aell-recogized complication in patients with sep-

is.196,197 Sepsis-associated cholestasis is more commonith gram-negative infections and is caused by the effectf bacterial endotoxins on hepatocellular transport sys-ems.2 In experimental sepsis secondary to lipopolysac-haride (LPS) administration, ATP-dependent uptake of�mol/L taurocholate in canalicular plasma membrane

esicles is reduced to 53% of controls without an appar-nt change in Km, suggesting a decrease in the numberf Bsep molecules in the canalicular membrane.197 Re-uced canalicular secretion of bile salts is caused by a2% reduction in Bsep mRNA levels198 and a concom-tant decrease in protein199 in endotoxin-treated rats. Thexpression of the canalicular organic anion transporterrp2 is decreased even more profoundly, amounting to

1% of controls in the LPS model.198 Furthermore, LPSas been shown to induce an early and selective but

eversible retrieval of Mrp2 from the canalicular mem-rane.200

Administration of either LPS, tumor necrosis factor � orL-1� to rodents causes a decrease in mRNA levels of theepatocellular bile salt uptake system Ntcp at the basolat-ral membrane.201,202 Administration of IL-6, a marker ofacteremia, reduces hepatocellular taurocholate uptakeithout affecting Ntcp mRNA levels.201,203 The down-

egulation of Ntcp in endotoxin-induced cholestasis canartly be explained by decreased binding activity of theuclear transcription factors HNF1� and RAR�:RXR� tohe Ntcp gene promoter.111,112,204 The effects of endotoxinn bile salt transport can be attenuated by pretreatment ofats with anti-tumor necrosis factor-� antibodies.205

Intrahepatic cholestasis of pregnancy. Intra-epatic cholestasis of pregnancy (ICP) is a syndromeharacterized by pruritus and biochemical cholestasis ofarying severity. It accounts for about 20% of pregnan-y-associated liver diseases that present with jaundice.he familial clustering of ICP, the high regional preva-

ence, the higher prevalence among mothers and sistersf patients with ICP, and the susceptibility to ethinylstradiol-induced cholestasis in subjects with a familyistory of ICP implicates one or several genetic traits inhe pathogenesis.206–208 The coexistence of ICP in PFIC3s associated with defects of the MDR3 (ABCB4) gene, asuggested by the identification of nonsense mutations inhe MDR3 gene of women with this diagnosis.178,209

ext to polymorphisms of the MDR3 and possibly theSEP genes, the role of cholestatic estrogen metabolitesppears to play a major role in the pathogenesis of ICP.hus, ATP-dependent canalicular taurocholate transport

s reduced by 63% after ethinyl estradiol treatment ofats,210 which is attributable to the 47% reduction ofsep protein levels after 5 days of ethinyl estradiol

reatment.198 Down-regulation of hepatocellular bile saltptake systems (Ntcp and Oatp1) has also been ob-erved.211 However, direct inhibition of Bsep transportunction by the cholestatic estrogen metabolite estradiol-7�-D-glucuronide may represent a major pathogeneticactor in estradiol-induced cholestasis.190,212

Expression of Bile Salt Transporters inHepatocellular Carcinoma

The pharmacological interest in expression of bilealt transporters in hepatocellular carcinomas (HCC) re-ates to the fact that cytostatic agents covalently linkedo bile salts could be shuttled into HCC tumor cellsrovided that the appropriate bile salt uptake systems arexpressed at adequate levels. Cytostatic drug conjugatesonsisting of a bile salt moiety such as glycocholate oraurocholate and a cytostatic agent such as cisplatin or

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hlorambucil have been evaluated with respect to liverrganotropism and uptake into liver tumors. Bamet-R2s a cisplatin-glycocholic acid conjugate that is taken upnto rat HCC after intravenous injection.213 A cisplatin-DCA conjugate called Bamet-UD2 was shown to ef-

ectively inhibit the growth of orthotopically implantedouse Hepa 1-6 hepatomas in the livers of nude miceith only low in vivo toxicity.214,215 Both Bamet-R2 andamet-UD2 are substrates of the bile salt uptake trans-orters of human liver, NTCP, OATP-A, and OATP-.216 Interestingly, they are also substrates of the organication transporters OCT1 and OCT2, that belong to theLC22A family of solute carriers.216 Next to the cispla-in-bile acid conjugates, a chlorambucil-taurocholateonjugate has also been shown to be a substrate of NTCPnd OATP-A.72

The usefulness of these drug targeting approacheslearly depends on the expression levels of bile saltptake systems in human HCC cells compared with theurrounding nontumor liver parenchyma. The level ofTCP mRNA in 5 surgically resected HCCs was re-

uced to 56% in northern blots compared with peritu-or nonmalignant liver tissue and immunofluorescent

nalysis indicated the presence of NTCP protein at theurface of HCC cells.72 The OATP-C mRNA was re-uced in 11 out of 20 HCCs by real-time polymerasehain reaction.217 Expression of OATP8, which exhibitsow bile salt transport activity,82 was decreased in 60% ofCCs on the mRNA and protein level compared with

urrounding nontumor liver tissue from the same pa-ients.218 This decrease was atrributable to an increase inNF3� expression in these HCCs because HNF3�

inds to and transcriptionally represses the OATP8ene.218 Expression of the organic anion and bile saltfflux systems MRP1, MRP2, and MRP3 has also beentudied in HCCs by immunostaining. Whereas no sur-ace expression could be shown for MRP1, plasma mem-rane staining of MRP2 and MRP3 was detectable in7% and 100% of HCCs examined, respectively.219

hether the novel cytostatic bile acid conjugate drugsre substrates of MRP2 and MRP3, which could com-romise intracellular accumulation of these agents, re-ains to be established.

Role of Bile Salt Transporters in CholesterolGallstone Disease

Although several bile salt transporter genes haveeen identified within susceptibility loci in the gall-tone-susceptible mouse, the exact contribution of eachuantitative trait locus to cholesterol gallstone formations still under investigation. Candidate “Lith” genes in-olved in bile salt transport include mouse Bsep (Abcb11),

rp2 (Abcc2), Fic1 (Atp8b1), Ntcp (Slc10a1), AsbtSlc10a2), and Oatp1 (Slc21a1).220 Alterations in the ex-ression or function of these transport proteins may causehanges in the composition of bile that contribute toholesterol gallstones. The Bsep gene is contained withinhe Lith1 quantitative trait locus, and the Lith1 locusonfers high cholesterol secretion rates in combinationith elevated bile salt and phospholipid secretion rates in

usceptible mice, a phenotype resembling that of obeseuman gallstone patients.221 The Lith1 locus is thusssociated with a gain of Bsep function and an increase inelative and absolute cholesterol secretion rates. Anotherene contained within the Lith1 locus is the oxysterol-ependent nuclear receptor LXR, that activates tran-cription of several cholesterol homeostatic genes includ-ng ABCA1 and ABCG5/ABCG8.222 This could alsoxplain the increase in pigment secretion that is associ-ted with the Lith1 locus. The role of the ileal bile saltransporter gene Asbt (Slc10a2) in cholesterol gallstoneormation could relate to the fact that decreased ASBTunction in man (e.g., in Crohn’s disease138) is associatedith increased colonic bile salt levels, which promoteilirubin absorption and increased bilirubin secretionnto bile.223 The exact contribution of the other bile saltransporter Lith genes to the pathogenesis of cholesterolallstones remains elusive at present because mutationsf the corresponding genes in man (e.g., MRP2 in theubin–Johnson syndrome or FIC1 in PFIC1 and BRIC)

re not associated with an increased incidence of chole-ithiasis. In contrast, the Lith gene Mdr2 (MDR3 in man,BCB4), which does not transport bile salts, appears tolay a role in certain forms of cholelithiasis in patientshat exhibit biochemical evidence of chronic cholestasis,ecurrence of symptoms after cholecystectomy, and aigh cholesterol/phospholipid ratio in bile.224 In 1 pa-ient, the consecutive onset of cholelithiasis in adoles-ence, followed by recurrent intrahepatic cholestasis ofregnancy, and finally biliary cirrhosis in adulthood haseen associated with a mutation at codon 535 in exon 14f the MDR3 gene.225

ConclusionsThe 2 clinical situations in which dysfunction of

ile salt transporters become most apparent are choles-atic liver disease and intestinal bile salt malabsorption.he former results from an “overflow” of bile salts in the

iver and systemic circulation caused by a disturbance ofepatocellular bile salt transport and excretion; the lattereads to an “underfilling” of the bile salt pool and con-equently decreased biliary bile salt excretion. The clin-cal manifestations of cholestasis are elevated serum

mmbdtcaubdnpthds

mtfcnsO(2PstaSSSOStc


arkers of cholestasis, pruritus, jaundice, intestinalalassimilation, and ultimately liver damage. Intestinal

ile salt malabsorption manifests itself as chologeniciarrhea and may contribute to gallstone formation. Be-ween these 2 extremes of defective bile salt transport, aomplex network of regulatory cascades governs thedaptive responses to physiological and exogenous stim-li such as postprandial increases in the portal venousile salt load, intake of xenobiotics, hormonal influencesuring pregnancy, and septicemia. Whereas the intesti-al bile salt transporter ASBT has become an establishedharmacological target for inhibition of bile salt absorp-ion in the treatment of lipid disorders, the usefulness ofepatocellular bile salt transporters for liver-directedrug targeting strategies awaits confirmation in humantudies.

Note added in proof. Following submission of thisanuscript, the new OATP classification and nomencla-

ure system, as indicated on page 325 of this review, wasormalized and accepted by the HUGO Gene Nomen-lature Committee (HGNC). According to this phyloge-etically based and species-independent classificationystem, all Oatps/OATPs are classified within theATP/SLCO superfamily of membrane transport systems

Hagenbuch B and Meier PJ, Pflugers Arch Eur J Physiol003, in press; Hediger M et al., Pflugers Arch Eur Jhysiol 2003, in press; http://www.pharmaconference.org/

lctable.asp). Concerning the Oatps/OATPs mentioned inhis review, the new protein names and the new gene symbolsre as follows: Oatp1a1/Slco1a1 (previously called Oatp1/lc21a1), Oatp1a4/Slco1a4 (Oatp2/Slc21a5), Oatp1b2/lco1b2 (Oatp4/Slc21a10), OATP1A2/SLCO1A2 (OATP-A/LC21A3) OATP1B1/SLCO1B1 (OATP-C/SLC21A6),ATP1B3/SLCO1B3 (OATP8/SLC21A8), OATP2B1/

LCO2B1 (OATP-B/SLC21A9). We suggest using onlyhe new nomenclature in future in order to avoid furtheronfusion.

References1. Chawla A, Saez E, Evans RM. “Don’t know much bile-ology.” Cell

2000;103:1–4.2. Kullak–Ublick GA, Stieger B, Hagenbuch B, Meier PJ. Hepatic

transport of bile salts. Semin Liver Dis 2000;20:273–292.3. Stieger B, Meier PJ. Bile salt transporters. Annu Rev Physiol

2002;64:635–661.4. Trauner M, Boyer JL. Bile salt transporters: molecular character-

ization, function and regulation. Physiol Rev 2003;83:633–671.5. Wolkoff AW, Cohen DE. Bile acid regulation of hepatic physiol-

ogy. I. Hepatocyte transport of bile acids. Am J Physiol Gastro-intest Liver Physiol 2003;284:G175–G179.

6. Chiang JYL. Bile acid regulation of hepatic physiology. III. Bileacids and nuclear receptors. Am J Physiol Gastrointest LiverPhysiol 2003;284:G349–G356.

7. Kullak-Ublick GA, Meier PJ. Mechanisms of cholestasis. ClinLiver Dis 2000;4:357–385.

8. Eggertsen G, Olin M, Andersson U, Ishida H, Kubota S, HellmanU, Okuda KI, Bjorkhem I. Molecular cloning and expression ofrabbit sterol 12alpha-hydroxylase. J Biol Chem 1996;271:32269–32275.

9. Cali JJ, Russell DW. Characterization of human sterol 27-hydrox-ylase. A mitochondrial cytochrome P-450 that catalyzes multipleoxidation reaction in bile acid biosynthesis. J Biol Chem 1991;266:7774–7778.

10. Schwarz M, Lund EG, Lathe R, Bjorkhem I, Russell DW. Identi-fication and characterization of a mouse oxysterol 7alpha-hy-droxylase cDNA. J Biol Chem 1997;272:23995–24001.

11. Li-Hawkins J, Lund EG, Bronson AD, Russell DW. Expressioncloning of an oxysterol 7alpha-hydroxylase selective for 24-hydroxycholesterol. J Biol Chem 2000;275:16543–16549.

12. Norlin M, Andersson U, Bjorkhem I, Wikvall K. Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A).J Biol Chem 2000;275:34046–34053.

13. Schroepfer GJ Jr. Oxysterols: modulators of cholesterol metab-olism and other processes. Physiol Rev 2000;80:361–554.

14. Dueland S, Trawick JD, Nenseter MS, MacPhee AA, Davis RA.Expression of 7 alpha-hydroxylase in non-hepatic cells results inliver phenotypic resistance of the low density lipoprotein recep-tor to cholesterol repression. J Biol Chem 1992;267:22695–22698.

15. Miyake JH, Doung XD, Strauss W, Moore GL, Castellani LW,Curtiss LK, Taylor JM, Davis RA. Increased production of apoli-poprotein B-containing lipoproteins in the absence of hyperlip-idemia in transgenic mice expressing cholesterol 7alpha-hydrox-ylase. J Biol Chem 2001;276:23304–23311.

16. Kullak-Ublick GA, Paumgartner G, Berr F. Long-term effects ofcholecystectomy on bile acid metabolism. Hepatology 1995;21:41–45.

17. Byrne JA, Strautnieks SS, Mieli-Vergani G, Higgins CF, Linton KJ,Thompson RJ. The human bile salt export pump: characteriza-tion of substrate specificity and identification of inhibitors. Gas-troenterology 2002;123:1649–1658.

18. Noe J, Stieger B, Meier PJ. Functional expression of the cana-licular bile salt export pump of human liver. Gastroenterology2002;123:1659–1666.

19. Gerloff T, Stieger B, Hagenbuch B, Madon J, Landmann L, RothJ, Hofmann AF, Meier PJ. The sister of P-glycoprotein representsthe canalicular bile salt export pump of mammalian liver. J BiolChem 1998;273:10046–10050.

20. Green RM, Hoda F, Ward KL. Molecular cloning and character-ization of the murine bile salt export pump. Gene 2000;241:117–123.

21. Lecureur V, Sun D, Hargrove P, Schuetz EG, Kim RB, Lan L-B,Schuetz JD. Cloning and expression of murine sister of P-glyco-protein reveals a more discriminating transporter than MDR1/P-glycoprotein. Mol Pharmacol 2000;57:24–35.

22. Noe J, Hagenbuch B, Meier PJ, St-Pierre MV. Characterization ofthe mouse bile salt export pump overexpressed in the baculo-virus system. Hepatology 2001;33:1223–1231.

23. Xu G, Pan LX, Li H, Forman BM, Erickson SK, Shefer S, BollineniJ, Batta AK, Christie J, Wang TH, Michel J, Yang S, Tsai R, Lai L,Shimada K, Tint GS, Salen G. Regulation of the farnesoid Xreceptor (FXR) by bile acid flux in rabbits. J Biol Chem 2002;277:50491–50496.

24. Childs S, Lin Yeh R, Georges E, Ling V. Identification of a sistergene to P-glycoprotein. Cancer Res 1995;55:2029–2034.

25. Ballatori N, Rebbeor JF, Connolly GC, Seward DJ, Lenth BE,Henson JH, Sundaram P, Boyer JL. Bile salt excretion in skateliver is mediated by a functional analog of Bsep/Spgp, the bilesalt export pump. Am J Physiol 2000;278:G57–G63.

26. Akita H, Suzuki H, Ito K, Kinoshita S, Sato N, Takikawa H,Sugiyama Y. Characterization of bile acid transport mediated by


multidrug resistance associated protein 2 and bile salt exportpump. Biochim Biophys Acta 2001;1511:7–16.

27. Gerk PM, Vore M. Regulation of expression of the multidrugresistance-associated protein 2 (MRP2) and its role in drugdisposition. J Pharmacol Exp Ther 2002;302:407–415.

28. Suzuki H, Sugiyama Y. Single nucleotide polymorphisms in mul-tidrug resistance associated protein 2 (MRP2/ABCC2): its im-pact on drug disposition. Adv Drug Deliv Rev 2002;54:1311–1331.

29. Oswald M, Kullak–Ublick GA, Paumgartner G, Beuers U. Expres-sion of hepatic transporters OATP-C and MRP2 in primary scle-rosing cholangitis. Liver 2001;21:247–253.

30. Zollner G, Fickert P, Zenz R, Fuchsbichler A, Stumptner C, Ken-ner L, Ferenci P, Stauber RE, Krejs GJ, Denk H, Zatloukal K,Trauner M. Hepatobiliary transporter expression in percutane-ous liver biopsies of patients with cholestatic liver diseases.Hepatology 2001;33:633–646.

31. Kullak-Ublick GA, Baretton GB, Oswald M, Renner EL, Paumgart-ner G, Beuers U. Expression of the hepatocyte canalicular mul-tidrug resistance protein (MRP2) in primary biliary cirrhosis.Hepatol Res 2002;23:78–82.

32. Suzuki M, Suzuki H, Sugimoto Y, Sugiyama Y. ABCG2 transportssulfated conjugates of steroids and xenobiotics. J Biol Chem2003;278:22644–22649.

33. Paigen B, Carey MC. Gallstones. In: King RA, Rotter JI, MotulskyAG, eds. The genetic basis of common diseases. Oxford Univer-sity Press, 2002:298–335.

34. Yu L, Li-Hawkins J, Hammer RE, Berge KE, Horton JD, Cohen JC,Hobbs HH. Overexpression of ABCG5 and ABCG8 promotesbiliary cholesterol secretion and reduces fractional absorptionof dietary cholesterol. J Clin Invest 2002;110:671–680.

35. Yu L, Hammer RE, Li-Hawkins J, von Bergmann K, Lutjohann D,Cohen JC, Hobbs HH. Disruption of Abcg5 and Abcg8 in micereveals their crucial role in biliary cholesterol secretion. ProcNatl Acad Sci U S A 2002;99:16237–16242.

36. Neufeld EB, Demosky SJ Jr, Stonik JA, Combs C, Remaley AT,Duverger N, Santamarina-Fojo S, Brewer HB Jr. The ABCA1transporter functions on the basolateral surface of hepatocytes.Biochem Biophys Res Commun 2002;297:974–979.

37. Ohama T, Hirano K, Zhang Z, Aoki R, Tsujii K, Nakagawa-ToyamaY, Tsukamoto K, Ikegami C, Matsuyama A, Ishigami M, Sakai N,Hiraoka H, Ueda K, Yamashita S, Matsuzawa Y. Dominant ex-pression of ATP-binding cassette transporter-1 on basolateralsurface of Caco-2 cells stimulated by LXR/RXR ligands. Bio-chem Biophys Res Commun 2002;296:625–630.

38. Groen AK, Bloks VW, Bandsma RH, Ottenhoff R, Chimini G,Kuipers F. Hepatobiliary cholesterol transport is not impaired inAbca1-null mice lacking HDL. J Clin Invest 2001;108:843–850.

39. Brooks-Wilson A, Marcil M, Clee SM, Zhang L-H, Roomp K, vanDam M, Yu L, Brewer C, Collins JA, Molhuizen HOF, Loubser O,Ouelette BFF, Fichter K, Ashbourne-Excoffon KJD, Sensen CW,Scherer S, Mott S, Denis M, Martindale D, Frohlich J, Morgan K,Koop B, Pimstone S, Kastelein JJP, Genest JJ, Hayden MR.Mutations in ABC1 in Tangier disease and familial high-densitylipoprotein deficiency. Nat Genet 1999;22:336–345.

40. Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Died-erich W, Drobnik W, Barlage S, Buchler C, Porsch-Ozcurumez M,Kaminski WE, Hahmann HW, Oette K, Rothe G, Aslanidis C,Lackner KJ, Schmitz G. The gene encoding ATP-binding cassettetransporter 1 is mutated in Tangier disease. Nat Genet 1999;22:347–351.

41. Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette J-C, DeleuzeJ-F, Brewer HB, Duverger N, Denefle P, Assmann G. Tangierdisease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat Genet 1999;22:352–355.

42. Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H, Man-gelsdorf DJ. Regulation of ATP-binding cassette sterol transport-

ers ABCG5 and ABCG8 by the liver X receptors � and �. J BiolChem 2002;277:18793–18800.

43. Francis GA, Annicotte JS, Auwerx J. Liver X receptors: XcretingXol to combat atherosclerosis. Trends Mol Med 2002;8:455–458.

44. Lazaridis KN, Pham L, Tietz P, Marinelli RA, deGroen PC, LevineS, Dawson PA, LaRusso NF. Rat cholangiocytes absorb bileacids at their apical domain via the ileal sodium-dependent bileacid transporter. J Clin Invest 1997;100:2714–2721.

45. Benedetti A, Di Sario A, Marucci L, Svegliati Baroni G, Schtein-gart CD, Ton Nu HT, Hofmann AF. Carrier-mediated transport ofconjugated bile acids across the basolateral membrane of bili-ary epithelial cells. Am J Physiol 1997;272:G1416–G1424.

46. Chignard N, Mergey M, Veissiere D, Parc R, Capeau J, Poupon R,Paul A, Housset C. Bile acid transport and regulating functionsin the human biliary epithelium. Hepatology 2001;33:496–503.

47. Soroka CJ, Lee JM, Azzaroli F, Boyer JL. Cellular localization andup-regulation of multidrug resistance-associated protein 3 inhepatocytes and cholangiocytes during obstructive cholestasisin rat liver. Hepatology 2001;33:783–791.

48. Rost D, Konig J, Weiss G, Klar E, Stremmel W, Keppler D.Expression and localization of the multidrug resistance proteinsMRP2 and MRP3 in human gallbladder epithelia. Gastroenter-ology 2001;121:1203–1208.

49. Lazaridis KN, Tietz P, Wu T, Kip S, Dawson PA, LaRusso NF.Alternative splicing of the rat sodium/bile acid transporterchanges its cellular localization and transport properties. ProcNatl Acad Sci USA 2000;97:11092–11097.

50. Xu G, Shneider BL, Shefer S, Nguyen LB, Batta AK, Tint GS,Arrese M, Thevananther S, Ma L, Stengelin S, Kramer W, Green-blatt D, Pcolinsky M, Salen G. Ileal bile acid transport regulatesbile acid pool, synthesis, and plasma cholesterol levels differ-ently in cholesterol-fed rats and rabbits. J Lipid Res 2000;41:298–304.

51. Kramer W, Girbig F, Glombik H, Corsiero D, Stengelin S, WeylandC. Identification of a ligand-binding site in the Na�/bile acidcotransporting protein from rabbit ileum. J Biol Chem 2001;276:36020–36027.

52. Wang W, Xue S, Ingles SA, Chen Q, Diep AT, Frankl HD, Stolz A,Haile RW. An association between genetic polymorphisms in theileal sodium-dependent bile acid transporter gene and the riskof colorectal adenomas. Cancer Epidemiol Biomarkers Prev2001;10:931–936.

53. Craddock AL, Love MW, Daniel RW, Kirby LC, Walters HC, WongMH, Dawson PA. Expression and transport properties of thehuman ileal and renal sodium-dependent bile acid transporter.Am J Physiol 1998;274:G157–G169.

54. Shneider BL, Dawson PA, Christie D-M, Hardikar W, Wong MH,Suchy FJ. Cloning and molecular characterization of the ontog-eny of a rat ileal sodium-dependent bile acid transporter. J ClinInvest 1995;95:745–754.

55. Oelkers P, Kirby LC, Heubi JE, Dawson PA. Primary bile acidmalabsorption caused by mutations in the ileal sodium-depen-dent bile acid transporter gene (SLC10A2). J Clin Invest 1997;99:1880–1887.

56. Walters HC, Craddock AL, Fusegawa H, Willingham MC, DawsonPA. Expression, transport properties, and chromosomal locationof organic anion transporter subtype 3. Am J Physiol 2000;279:G1188–G1200.

57. Amelsberg A, Schteingart CD, Ton-Nu HT, Hofmann AF. Carrier-mediated jejunal absorption of conjugated bile acids in theguinea pig. Gastroenterology 1996;110:1098–1106.

58. Gong YZ, Everett ET, Schwartz DA, Norris JS, Wilson FA. Molec-ular cloning, tissue distribution, and expression of a 14-kDa bileacid-binding protein from rat ileal cytosol. Proc Natl Acad SciU S A 1994;91:4741–4745.

59. Kramer W, Sauber K, Baringhaus KH, Kurz M, Stengelin S,


Lange G, Corsiero D, Girbig F, Konig W, Weyland C. Identificationof the bile acid-binding site of the ileal lipid-binding protein byphotoaffinity labeling, matrix-assisted laser desorption ioniza-tion-mass spectrometry, and NMR structure. J Biol Chem 2001;276:7291–7301.

60. Rost D, Mahner S, Sugiyama Y, Stremmel W. Expression andlocalization of the multidrug resistance-associated protein 3 inrat small and large intestine. Am J Physiol Gastrointest LiverPhysiol 2002;282:G720–G726.

61. Konig J, Rost D, Cui Y, Keppler D. Characterization of the humanmultidrug resistance protein isoform MRP3 localized to thebasolateral hepatocyte membrane. Hepatology 1999;29:1156–1163.

62. Kool M, van der Linden M, de Haas M, Scheffer GL, Vree JML,Smith AJ, Jansen G, Peters GJ, Ponne N, Scheper RJ, OudeElferink RPJ, Baas F, Borst P. MRP3, an organic anion trans-porter able to transport anti-cancer drugs. Proc Natl Acad SciUSA 1999;96:6914–6919.

63. Scheffer GL, Kool M, de Haas M, de Vree JM, Pijnenborg AC,Bosman DK, Oude Elferink RP, van der Valk P, Borst P, ScheperRJ. Tissue distribution and induction of human multidrug resis-tant protein 3. Lab Invest 2002;82:193–201.

64. Hirohashi T, Suzuki H, Sugiyama Y. Characterization of thetransport properties of cloned rat multidrug resistance-associ-ated protein 3 (MRP3). J Biol Chem 1999;274:15181–15185.

65. Zeng H, Liu G, Rea PA, Kruh GD. Transport of amphipathicanions by human multidrug resistance protein 3. Cancer Res2000;60:4779–4784.

66. Keppler D, Kamisako T, Leier I, Cui Y, Nies AT, Tsujii H, Konig J.Localization, substrate specificity, and drug resistance con-ferred by conjugate export pumps of the MRP family. Adv En-zyme Regul 2000;40:339–349.

67. Hirohashi T, Suzuki H, Takikawa H, Sugiyama Y. ATP-dependenttransport of bile salts by rat multidrug resistance-associatedprotein 3 (mrp3). J Biol Chem 2000;275:2905–2910.

68. Zelcer N, Saeki T, Bot I, Kuil A, Borst P. Transport of bile acidsin multidrug-resistance-protein 3-overexpressing cells co-trans-fected with the ileal Na�-dependent bile-acid transporter. Bio-chem J 2003;369:23–30.

69. Akita H, Suzuki H, Hirohashi T, Takikawa H, Sugiyama Y. Trans-port activity of human MRP3 expressed in Sf9 cells: compara-tive studies with rat MRP3. Pharm Res 2002;19:34–41.

70. Hagenbuch B, Meier PJ. Molecular cloning, chromosomal local-ization, and functional characterization of a human liver Na�/bile acid cotransporter. J Clin Invest 1994;93:1326–1331.

71. Stieger B, Hagenbuch B, Landmann L, Hoechli M, Schroeder A,Meier PJ. In situ localization of the hepatocytic Na�/tauro-cholate cotransporting polypeptide in rat liver. Gastroenterology1994;107:1781–1787.

72. Kullak-Ublick GA, Glasa J, Boeker C, Oswald M, Gruetzner U,Hagenbuch B, Stieger B, Meier PJ, Beuers U, Kramer W, WessG, Paumgartner G. Chlorambucil-taurocholate is transported bybile acid carriers expressed in human hepatocellular carcino-mas. Gastroenterology 1997;113:1295–1305.

73. Meier PJ, Eckhardt U, Schroeder A, Hagenbuch B, Stieger B.Substrate specificity of sinusoidal bile acid and organic anionuptake systems in rat and human liver. Hepatology 1997;26:1667–1677.

74. Kullak-Ublick GA, Ismair MG, Kubitz R, Schmitt M, Haussinger D,Stieger B, Hagenbuch B, Meier PJ, Beuers U, Paumgartner G.Stable expression and functional characterization of a Na�-taurocholate cotransporting green fluorescent protein in humanhepatoblastoma HepG2 cells. Cytotechnology 2000;34:1–9.

75. Hagenbuch B, Scharschmidt BF, Meier PJ. Effect of antisenseoligonucleotides on the expression of hepatocellular bile acidand organic anion uptake systems in Xenopus laevis oocytes.Biochem J 1996;316:901–904.

76. Tirona RG, Kim RB. Pharmacogenomics of organic anion-trans-porting polypeptides (OATP). Adv Drug Deliv Rev 2002;54:1343–1352.

77. Hagenbuch B, Meier PJ. The superfamily of organic anion trans-porting polypeptides. Biochim Biophys Acta 2003;1609:1–18.

78. Kullak-Ublick GA, Hagenbuch B, Stieger B, Schteingart CD, Hof-mann AF, Wolkoff AW, Meier PJ. Molecular and functional char-acterization of an organic anion transporting polypeptide clonedfrom human liver. Gastroenterology 1995;109:1274–1282.

79. Abe T, Kakyo M, Tokui T, Nakagomi R, Nishio T, Nakai D,Nomura H, Unno M, Suzuki M, Naitoh T, Matsuno S, Yawo H.Identification of a novel gene family encoding human liver-spe-cific organic anion transporter LST-1. J Biol Chem 1999;274:17159–17163.

80. Hsiang B, Zhu Y, Wang Z, Wu Y, Sasseville V, Yang W-P, Kirch-gessner TG. A novel human hepatic organic anion transportingpolypeptide (OATP2). J Biol Chem 1999;274:37161–37168.

81. Konig J, Cui Y, Nies AT, Keppler D. A novel human organic aniontransporting polypeptide localized to the basolateral hepatocytemembrane. Am J Physiol 2000;278:G156–G164.

82. Kullak-Ublick GA, Ismair MG, Stieger B, Landmann L, Huber R,Pizzagalli F, Fattinger K, Meier PJ, Hagenbuch B. Organic aniontransporting polypeptide B (OATP-B) and its functional compari-son with three other OATPs of human liver. Gastroenterology2001;120:525–533.

83. Cui Y, Konig J, Leier I, Buchholz U, Keppler D. Hepatic uptake ofbilirubin and its conjugates by the human organic anion trans-porter SLC21A6. J Biol Chem 2001;276:9626–9630.

84. Briz O, Serrano MA, Macias RI, Gonzalez-Gallego J, Marin JJ.Role of organic anion-transporting polypeptides, OATP-A, OATP-Cand OATP-8 in the human placenta-maternal liver tandem excre-tory pathway for foetal bilirubin. Biochem J 2003;371:897–905.

85. Bossuyt X, Mueller M, Meier PJ. Multispecific amphipathic sub-strate transport by an organic anion transporter of human liver.J Hepatol 1996;25:733–738.

86. Schiffer R, Neis M, Holler D, Rodriguez F, Geier A, Gartung C,Lammert F, Dreuw A, Zwadlo-Klarwasser G, Merk H, Jugert F,Baron JM. Active influx transport is mediated by members of theorganic anion transporting polypeptide family in human epider-mal keratinocytes. J Invest Dermatol 2003;120:285–291.

87. Kullak-Ublick GA, Beuers U, Fahney C, Hagenbuch B, Meier PJ,Paumgartner G. Identification and functional characterization ofthe promoter region of the human organic anion transportingpolypeptide gene. Hepatology 1997;26:991–997.

88. Faubion WA, Guicciardi ME, Miyoshi H, Bronk SF, Roberts PJ,Svingen PA, Kaufmann SH, Gores GJ. Toxic bile salts inducerodent hepatocyte apoptosis via direct activation of Fas. J ClinInvest 1999;103:137–145.

89. Forman BM, Goode E, Chen J, Oro AE, Bradley DJ, Perlmann T,Noonan DJ, Burka LT, McMorris T, Lamph WW, Evans RM,Weinberger C. Identification of a nuclear receptor that is acti-vated by farnesol metabolites. Cell 1995;81:687–693.

90. Chiang JYL. Bile acid regulation of gene expression: roles ofnuclear hormone receptors. Endocr Rev 2002;23:443–463.

91. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A,Hull MV, Lustig KD, Mangelsdorf DJ, Shan B. Identification of anuclear receptor for bile acids. Science 1999;284:1362–1365.

92. Ananthanarayanan M, Balasubramanian N, Makishima M, Man-gelsdorf DJ, Suchy FJ. Human bile salt export pump (BSEP)promoter is transactivated by the farnesoid X receptor/bile acidreceptor (FXR/BAR). J Biol Chem 2001;276:28857–28865.

93. Schuetz EG, Strom S, Yasuda K, Lecureur V, Assem M, BrimerC, Lamba J, Kim RB, Ramachandran V, Komoroski BJ, Venkat-aramanan R, Cai H, Sinal CJ, Gonzalez FJ, Schuetz JD. Disruptedbile acid homeostasis reveals an unexpected interaction amongnuclear hormone receptors, transporters, and cytochromeP450. J Biol Chem 2001;276:39411–39418.

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94. Kast HR, Goodwin B, Tarr PT, Jones SA, Anisfeld AM, Stoltz CM,Tontonoz P, Kliewer S, Willson TM, Edwards PA. Regulation ofmultidrug resistance-associated protein 2 (ABCC2) by the nu-clear receptors pregnane X receptor, farnesoid X-activated re-ceptor, and constitutive androstane receptor. J Biol Chem2002;277:2908–2915.

95. Denson LA, Sturm E, Echevarria W, Zimmerman TL, MakishimaM, Mangelsdorf DJ, Karpen SJ. The orphan nuclear receptor,shp, mediates bile acid-induced inhibition of the rat bile acidtransporter, ntcp. Gastroenterology 2001;121:140–147.

96. Jung D, Kullak-Ublick GA. Hepatocyte nuclear factor 1�: a keymediator of the effect of bile acids on gene expression. Hepa-tology 2003;37:622–631.

97. Jung D, Podvinec M, Meyer UA, Mangelsdorf DJ, Fried M, MeierPJ, Kullak-Ublick GA. Human organic anion transporting polypep-tide 8 promoter is transactivated by the farnesoid X receptor/bile acid receptor. Gastroenterology 2002;122:1954–1966.

98. Grober J, Zaghini I, Fujii H, Jones SA, Kliewer SA, Willson TM,Ono T, Besnard P. Identification of a bile acid-responsive ele-ment in the human ileal bile acid-binding protein gene. Involve-ment of the farnesoid X receptor/9-cis-retinoic acid receptorheterodimer. J Biol Chem 1999;274:29749–29754.

99. Yu J, Lo JL, Huang L, Zhao A, Metzger E, Adams A, Meinke PT,Wright SD, Cui J. Lithocholic acid decreases expression of bilesalt export pump through farnesoid X receptor antagonist activ-ity. J Biol Chem 2002;277:31441–31447.

00. Konig J, Cui Y, Nies AT, Keppler D. Localization and genomicorganization of a new hepatocellular organic anion transportingpolypeptide. J Biol Chem 2000;275:23161–23168.

01. Ismair MG, Stieger B, Cattori V, Hagenbuch B, Fried M, MeierPJ, Kullak-Ublick GA. Hepatic uptake of cholecystokinin octapep-tide by organic anion transporting polypeptides Oatp4 andOATP8 of rat and human liver. Gastroenterology 2001;121:1185–1190.

02. Ismair MG, Stanca C, Ha HR, Renner EL, Meier PJ, Kullak-UblickGA. Interactions of glycyrrhizin with organic anion transportingpolypeptides of rat and human liver. Hepatol Res 2003;26:343–347.

03. Staudinger JL, Goodwin B, Jones SA, Hawkins-Brown D, Mac-Kenzie KI, LaTour A, Liu Y, Klaassen CD, Brown KK, Reinhard J,Willson TM, Koller BH, Kliewer SA. The nuclear receptor PXR isa lithocholic acid sensor that protects against liver toxicity. ProcNatl Acad Sci U S A 2001;98:3369–3374.

04. Hagenbuch N, Reichel C, Stieger B, Cattori V, Fattinger KE,Landmann L, Meier PJ, Kullak-Ublick GA. Effect of phenobarbitalon the expression of bile salt and organic anion transporters ofrat liver. J Hepatol 2001;34:881–887.

05. Guo GL, Staudinger J, Ogura K, Klaassen CD. Induction of ratorganic anion transporting polypeptide 2 by pregnenolone-16�-carbonitrile is via interaction with pregnane X receptor. MolPharmacol 2002;61:832–839.

06. Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, MooreLB, Galardi C, Wilson JG, Lewis MC, Roth ME, Maloney PR,Willson TM, Kliewer SA. A regulatory cascade of the nuclearreceptors FXR, SHP-1, and LRH-1 represses bile acid biosynthe-sis. Mol Cell 2000;6:517–526.

07. Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J,Mangelsdorf DJ. Molecular basis for feedback regulation of bileacid synthesis by nuclear receptors. Mol Cell 2000;6:507–515.

08. del Castillo-Olivares A, Gil G. Suppression of sterol 12�-hydrox-ylase transcription by the short heterodimer partner: insightsinto the repression mechanism. Nucleic Acids Res 2001;29:4035–4042.

09. Zhang M, Chiang JYL. Transcriptional regulation of the humansterol 12�-hydroxylase gene (CYP8B1). J Biol Chem 2001;276:41690–41699.

10. Lee YK, Dell H, Dowhan DH, Hadzopoulou-Cladaras M, Moore

DD. The orphan nuclear receptor SHP inhibits hepatocyte nu-clear factor 4 and retinoid X receptor transactivation: two mech-anisms for repression. Mol Cell Biol 2000;20:187–195.

11. Denson LA, Auld KL, Schiek DS, McClure MH, Mangelsdorf DJ,Karpen SJ. Interleukin-1� suppresses retinoid transactivation oftwo hepatic transporter genes involved in bile formation. J BiolChem 2000;275:8835–8843.

12. Li D, Zimmerman TL, Thevananther S, Lee H-Y, Kurie JM, KarpenSJ. Interleukin-1�-mediated suppression of RXR: RAR transac-tivation of the Ntcp promoter is JNK-dependent. J Biol Chem2002;277:31416–31422.

13. Chen F, Ma L, Dawson PA, Sinal CJ, Sehayek E, Gonzalez FJ,Breslow J, Ananthanarayanan M, Shneider BL. Liver receptorhomologue-1 mediates species- and cell line-specific bile acid-dependent negative feedback regulation of the apical sodium-dependent bile acid transporter. J Biol Chem 2003;278:19909–19916.

14. Jung D, Hagenbuch B, Gresh L, Pontoglio M, Meier PJ, Kullak-Ublick GA. Characterization of the human OATP-C (SLC21A6)gene promoter and regulation of liver-specific OATP genes byhepatocyte nuclear factor 1�. J Biol Chem 2001;276:37206–37214.

15. Chen W, Owsley E, Yang Y, Stroup D, Chiang JY. Nuclear recep-tor-mediated repression of human cholesterol 7�-hydroxylasegene transcription by bile acids. J Lipid Res 2001;42:1402–1412.

16. Jung D, Fried M, Kullak-Ublick GA. Human apical sodium-depen-dent bile salt transporter gene (SLC10A2) is regulated by theperoxisome proliferator-activated receptor �. J Biol Chem 2002;277:30559–30566.

17. Blumberg B, Sabbagh W Jr, Juguilon H, Bolado J Jr, van MeterCM, Ong ES, Evans RM. SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev 1998;12:3195–3205.

18. Xie W, Barwick JL, Downes M, Blumberg B, Simon CM, NelsonMC, Neuschwander Tetri BA, Brunt EM, Guzelian PS, Evans RM.Humanized xenobiotic response in mice expressing nuclearreceptor SXR. Nature 2000;406:435–439.

19. Synold TW, Dussault I, Forman BM. The orphan nuclear receptorSXR coordinately regulates drug metabolism and efflux. NatMed 2001;7:584–590.

20. Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT,Kliewer SA. The human orphan nuclear receptor PXR is acti-vated by compounds that regulate CYP3A4 gene expression andcause drug interactions. J Clin Invest 1998;102:1016–1023.

21. Moore LB, Goodwin B, Jones SA, Wisely GB, Serabjit Singh CJ,Willson TM, Collins JL, Kliewer SA. St. John’s wort induceshepatic drug metabolism through activation of the pregnane Xreceptor. Proc Natl Acad Sci U S A 2000;97:7500–7502.

22. Moore LB, Parks DJ, Jones SA, Bledsoe RK, Consler TG, Stim-mel JB, Goodwin B, Liddle C, Blanchard SG, Willson TM, CollinsJL, Kliewer SA. Orphan nuclear receptors constitutive andro-stane receptor and pregnane X receptor share xenobiotic andsteroid ligands. J Biol Chem 2000;275:15122–15127.

23. Goodwin B, Gauthier KC, Umetani M, Watson MA, LochanskyMI, Collins JL, Leitersdorf E, Mangelsdorf DJ, Kliewer SA, RepaJJ. Identification of bile acid precursors as endogenous ligandsfor the nuclear xenobiotic pregnane X receptor. Proc Natl AcadSci U S A 2003;100:223–228.

24. Liddle C, Goodwin B. Regulation of hepatic drug metabolism:role of the nuclear receptors PXR and CAR. Semin Liver Dis2002;22:115–122.

25. Geick A, Eichelbaum M, Burk O. Nuclear receptor responseelements mediate induction of intestinal MDR1 by rifampin.J Biol Chem 2001;276:14581–14587.

26. Geier A, Dietrich CG, Gerloff T, Haendly J, Kullak-Ublick GA,Stieger B, Meier PJ, Matern S, Gartung C. Regulation of baso-lateral organic anion transporters in ethinylestradiol-induced

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1


cholestasis in the rat. Biochim Biophys Acta 2003;1609:87–94.

27. Cherrington NJ, Hartley DP, Li N, Johnson DR, Klaassen CD.Organ distribution of multidrug resistance proteins 1, 2, and 3(Mrp1, 2, and 3) mRNA and hepatic induction of Mrp3 byconstitutive androstane receptor activators in rats. J PharmacolExp Ther 2002;300:97–104.

28. Xiong H, Yoshinari K, Brouwer KL, Negishi M. Role of constitu-tive androstane receptor in the in vivo induction of Mrp3 andCYP2B1/2 by phenobarbital. Drug Metab Dispos 2002;30:918–923.

29. Inokuchi A, Hinoshita E, Iwamoto Y, Kohno K, Kuwano M, Uchi-umi T. Enhanced expression of the human multidrug resistanceprotein 3 by bile salt in human enterocytes. A transcriptionalcontrol of a plausible bile acid transporter. J Biol Chem 2001;276:46822–46829.

30. Ganguly TC, Liu Y, Hyde JF, Hagenbuch B, Meier PJ, Vore M.Prolactin increases hepatic Na�/taurocholate cotransport activ-ity and messenger RNA post partum. Biochem J 1994;303:33–36.

31. Ganguly TC, O’Brien ML, Karpen SJ, Hyde JF, Suchy FJ, Vore M.Regulation of the rat liver sodium-dependent bile acid cotrans-porter gene by prolactin. J Clin Invest 1997;99:2906–2914.

32. Kok T, Bloks VW, Wolters H, Havinga R, Jansen PL, Staels B,Kuipers F. Peroxisome proliferator-activated receptor alpha(PPARalpha)-mediated regulation of multidrug resistance 2(Mdr2) expression and function in mice. Biochem J 2003;369:539–547.

33. Desvergne B, Wahli W. Peroxisome proliferator-activated recep-tors: nuclear control of metabolism. Endocr Rev 1999;20:649–688.

34. Angelin B, Hershon KS, Brunzell JD. Bile acid metabolism inhereditary forms of hypertriglyceridemia: evidence for an in-creased synthesis rate in monogenic familial hypertriglyceride-mia. Proc Natl Acad Sci U S A 1987;84:5434–5438.

35. Duane WC, Hartich LA, Bartman AE, Ho SB. Diminished geneexpression of ileal apical sodium bile acid transporter explainsimpaired absorption of bile acid in patients with hypertriglycer-idemia. J Lipid Res 2000;41:1384–1389.

36. Love MW, Craddock AL, Angelin B, Brunzell JD, Duane WC,Dawson PA. Analysis of the ileal bile acid transporter gene,SLC10A2, in subjects with familial hypertriglyceridemia. Arterio-scler Thromb Vasc Biol 2001;21:2039–2045.

37. Post SM, Duez H, Gervois PP, Staels B, Kuipers F, Princen HM.Fibrates suppress bile acid synthesis via peroxisome prolifera-tor-activated receptor-alpha-mediated downregulation of choles-terol 7alpha-hydroxylase and sterol 27-hydroxylase expression.Arterioscler Thromb Vasc Biol 2001;21:1840–1845.

38. Jung D, Fantin AC, Scheurer U, Fried M, Kullak–Ublick GA.Human ileal bile acid transporter gene ASBT (SLC10A2) istransactivated by the glucocorticoid receptor. Gut 2004;53:(inpress).

39. Scheurlen C, Kruis W, Bull U, Stellaard F, Lang P, PaumgartnerG. Comparison of 75SeHCAT retention half-life and fecal con-tent of individual bile acids in patients with chronic diarrhealdisorders. Digestion 1986;35:102–108.

40. Davie RJ, Hosie KB, Grobler SP, Newbury Ecob RA, Keighley MR,Birch NJ. Ileal bile acid malabsorption in colonic Crohn’s dis-ease. Br J Surg 1994;81:289–290.

41. Kipp H, Arias IM. Trafficking of canalicular ABC transporters inhepatocytes. Annu Rev Physiol 2002;64:595–608.

42. Kipp H, Pichetshote N, Arias IM. Transporters on demand:intrahepatic pools of canalicular ATP binding cassette transport-ers in rat liver. J Biol Chem 2001;276:7218–7224.

43. Misra S, Ujhazy P, Varticovski L, Arias IM. Phosphoinositide3-kinase lipid products regulate ATP-dependent transport bysister of P-glycoprotein and multidrug resistance associated

protein 2 in bile canalicular membrane vesicles. Proc Natl AcadSci U S A 1999;96:5814–5819.

44. Misra S, Ujhazy P, Gatmaitan Z, Varticovski L, Arias IM. The roleof phosphoinositide 3-kinase in taurocholate-induced traffickingof ATP-dependent canalicular transporters in rat liver. J BiolChem 1998;273:26638–26644.

45. Beuers U, Bilzer M, Chittattu A, Kullak Ublick GA, Keppler D,Paumgartner G, Dombrowski F. Tauroursodeoxycholic acid in-serts the apical conjugate export pump, Mrp2, into canalicularmembranes and stimulates organic anion secretion by proteinkinase C-dependent mechanisms in cholestatic rat liver. Hepa-tology 2001;33:1206–1216.

46. Kubitz R, Huth C, Schmitt M, Horbach A, Kullak Ublick G,Haussinger D. Protein kinase C-dependent distribution of themultidrug resistance protein 2 from the canalicular to the ba-solateral membrane in human HepG2 cells. Hepatology 2001;3:340–350.

47. Kurz AK, Graf D, Schmitt M, vom Dahl S, Haussinger D. Taur-oursodesoxycholate-induced choleresis involves p38(MAPK) ac-tivation and translocation of the bile salt export pump in rats.Gastroenterology 2001;121:407–419.

48. Paumgartner G, Beuers U. Ursodeoxycholic acid in cholestaticliver disease: mechanisms of action and therapeutic use revis-ited. Hepatology 2002;36:525–531.

49. Schmitt M, Kubitz R, Lizun S, Wettstein M, Haussinger D.Regulation of the dynamic localization of the rat Bsep gene-encoded bile salt export pump by anisoosmolarity. Hepatology2001;33:509–518.

50. Kikuchi S, Hata M, Fukumoto K, Yamane Y, Matsui T, Tamura A,Yonemura S, Yamagishi H, Keppler D, Tsukita S. Radixin defi-ciency causes conjugated hyperbilirubinemia with loss of Mrp2from bile canalicular membranes. Nat Genet 2002;31:320–325.

51. Grune S, Engelking LR, Anwer MS. Role of intracellular calciumand protein kinases in the activation of hepatic Na�/tauro-cholate cotransport by cyclic AMP. J Biol Chem 1993;268:17734–17741.

52. Mukhopadhayay S, Ananthanarayanan M, Stieger B, Meier PJ,Suchy FJ, Anwer MS. cAMP increases liver Na�-taurocholatecotransport by translocating transporter to plasma membranes.Am J Physiol 1997;273:G842–G848.

53. Mukhopadhyay S, Ananthanarayanan M, Stieger B, Meier PJ,Suchy FJ, Anwer MS. Sodium taurocholate cotransportingpolypeptide is a serine, threonine phosphoprotein and is de-phosphorylated by cyclic adenosine monophosphate. Hepatol-ogy 1998;28:1629–1636.

54. Webster CR, Blanch C, Anwer MS. Role of PP2B in cAMP-induced dephosphorylation and translocation of NTCP. Am JPhysiol Gastrointest Liver Physiol 2002;283:G44–G50.

55. Mukhopadhyay S, Webster CR, Anwer MS. Role of protein phos-phatases in cyclic AMP-mediated stimulation of hepatic Na�/taurocholate cotransport. J Biol Chem 1998;273:30039–30045.

56. Webster CR, Anwer MS. Role of the PI3K/PKB signaling path-way in cAMP-mediated translocation of rat liver Ntcp. Am JPhysiol 1999;277:G1165–G1172.

57. Webster CR, Srinivasulu U, Ananthanarayanan M, Suchy FJ,Anwer MS. Protein kinase B/Akt mediates cAMP- and cell swell-ing-stimulated Na�/taurocholate cotransport and Ntcp translo-cation. J Biol Chem 2002;277:28578–28583.

58. Dranoff JA, McClure M, Burgstahler AD, Denson LA, CrawfordAR, Crawford JM, Karpen SJ, Nathanson MH. Short-term regu-lation of bile acid uptake by microfilament-dependent transloca-tion of rat ntcp to the plasma membrane. Hepatology 1999;30:223–229.

59. Campbell CG, Spray DC, Wolkoff AW. Extracellular ATP4- mod-

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1


ulates organic anion transport by rat hepatocytes. J Biol Chem1993;268:15399–15404.

60. Glavy JS, Wu SM, Wang PJ, Orr GA, Wolkoff AW. Down-regulationby extracellular ATP of rat hepatocyte organic anion transport ismediated by serine phosphorylation of oatp1. J Biol Chem2000;275:1479–1484.

61. Haussinger D. The role of cellular hydration in the regulation ofcell function. Biochem J 1996;313:697–710.

62. Webster CR, Blanch CJ, Phillips J, Anwer MS. Cell swelling-induced translocation of rat liver Na(�)/taurocholate cotrans-port polypeptide is mediated via the phosphoinositide 3-kinasesignaling pathway. J Biol Chem 2000;275:29754–29760.

63. Hsia DY, Boggs JD, Driscoll SG, Gellis SS. Prolonged obstruc-tive jaundice in infancy. V. The genetic components in neonatalhepatitis. Am J Dis Child 1958;485–491.

64. Tygstrup N. Intermittent possibly familial intrahepatic choles-tatic jaundice. Lancet 1960;1:1171–1172.

65. Clayton RJ, Iber FL, Ruebner BH, Mckusick VA. Byler disease.Fatal familial intrahepatic cholestasis in an Amish kindred.J Pediatr 1965;67:1025–1028.

66. Gray OP, Saunders RA. Familial intrahepatic cholestatic jaun-dice in infancy. Arch Dis Child 1966;41:320–328.

67. Kullak-Ublick GA, Beuers U, Paumgartner G. Hepatobiliary trans-port. J Hepatol 2000;32(Suppl 1):3–18.

68. Stanca C, Jung D, Meier PJ, Kullak-Ublick GA. Hepatocellulartransport proteins and their role in liver disease. World J Gas-troenterol 2001;7:157–169.

69. Carlton VE, Knisely AS, Freimer NB. Mapping of a locus forprogressive familial intrahepatic cholestasis (Byler disease) to18q21-q22, the benign recurrent intrahepatic cholestasis re-gion. Hum Mol Genet 1995;4:1049–1053.

70. Eppens EF, van Mil SW, de Vree JM, Mok KS, Juijn JA, OudeElferink RP, Berger R, Houwen RH, Klomp LW. FIC1, the proteinaffected in two forms of hereditary cholestasis, is localized inthe cholangiocyte and the canalicular membrane of the hepa-tocyte. J Hepatol 2001;35:436–443.

71. Bull LN, van Eijk MJ, Pawlikowska L, DeYoung JA, Juijn JA, LiaoM, Klomp LW, Lomri N, Berger R, Scharschmidt BF, Knisely AS,Houwen RH, Freimer NB. A gene encoding a P-type ATPasemutated in two forms of hereditary cholestasis. Nat Genet1998;18:219–224.

72. Bull LN, Carlton VE, Stricker NL, Baharloo S, DeYoung JA,Freimer NB, Magid MS, Kahn E, Markowitz J, DiCarlo FJ,McLoughlin L, Boyle JT, Dahms BB, Faught PR, Fitzgerald JF,Piccoli DA, Witzleben CL, O’Connell NC, Setchell KD, AgostiniRM Jr, Kocoshis SA, Reyes J, Knisely AS. Genetic and morpho-logical findings in progressive familial intrahepatic cholestasis(Byler disease [PFIC-1] and Byler syndrome): evidence for het-erogeneity. Hepatology 1997;26:155–164.

73. Strautnieks SS, Kagalwalla AF, Tanner MS, Knisely AS, Bull L,Freimer N, Kocoshis SA, Gardiner RM, Thompson RJ. Identifica-tion of a locus for progressive familial intrahepatic cholestasisPFIC2 on chromosome 2q24. Am J Hum Genet 1997;61:630–633.

74. Jansen PL, Strautnieks SS, Jacquemin E, Hadchouel M, SokalEM, Hooiveld GJ, Koning JH, De Jager Krikken A, Kuipers F,Stellaard F, Bijleveld CM, Gouw A, Van Goor H, Thompson RJ,Muller M. Hepatocanalicular bile salt export pump deficiency inpatients with progressive familial intrahepatic cholestasis. Gas-troenterology 1999;117:1370–1379.

75. Wang L, Soroka CJ, Boyer JL. The role of bile salt export pumpmutations in progressive familial intrahepatic cholestasis typeII. J Clin Invest 2002;110:965–972.

76. Kullak-Ublick GA, Kerb R, Mullhaupt B, Penger A, Brinkmann U,Stieger B, Meier PJ. A novel R432T mutation in the bile saltexport pump gene (BSEP; ABCB11) is associated with recurrent

intrahepatic cholestasis in an adolescent patient [abstract].Hepatology 2001;34:216A.

77. de Vree JM, Jacquemin E, Sturm E, Cresteil D, Bosma PJ, AtenJ, Deleuze JF, Desrochers M, Burdelski M, Bernard O, OudeElferink RP, Hadchouel M. Mutations in the MDR3 gene causeprogressive familial intrahepatic cholestasis. Proc Natl Acad SciU S A 1998;95:282–287.

78. Jacquemin E, De Vree JM, Cresteil D, Sokal EM, Sturm E,Dumont M, Scheffer GL, Paul M, Burdelski M, Bosma PJ, Ber-nard O, Hadchouel M, Elferink RP. The wide spectrum of multi-drug resistance 3 deficiency: from neonatal cholestasis to cir-rhosis of adulthood. Gastroenterology 2001;120:1448–1458.

79. Floreani A, Molaro M, Mottes M, Sangalli A, Baragiotta A, RodaA, Naccarato R, Clementi M. Autosomal dominant benign recur-rent intrahepatic cholestasis (BRIC) unlinked to 18q21 and2q24. Am J Med Genet 2000;95:450–453.

80. Bijleveld CM, Vonk RJ, Kuipers F, Havinga R, Boverhof R, Koop-man BJ, Wolthers BG, Fernandes J. Benign recurrent intrahe-patic cholestasis: altered bile acid metabolism. Gastroenterol-ogy 1989;97:427–432.

81. Wada M, Toh S, Taniguchi K, Nakamura T, Uchiumi T, Kohno K,Yoshida I, Kimura A, Sakisaka S, Adachi Y, Kuwano M. Muta-tions in the canalicular multispecific organic anion transporter(cMOAT) gene, a novel ABC transporter, in patients with hyper-bilirubinemia II/Dubin-Johnson syndrome. Hum Mol Genet1998;7:203–207.

82. Kartenbeck J, Leuschner U, Mayer R, Keppler D. Absence of thecanalicular isoform of the MRP gene-encoded conjugate exportpump from the hepatocytes in Dubin-Johnson syndrome. Hepa-tology 1996;23:1061–1066.

83. Paulusma CC, Kool M, Bosma PJ, Scheffer GL, Ter Borg F,Scheper RJ, Tytgat GNJ, Borst P, Baas F, Oude Elferink RPJ. Amutation in the human canalicular multispecific organic aniontransporter gene causes the Dubin-Johnson syndrome. Hepa-tology 1997;25:1539–1542.

84. Paulusma CC, Oude Elferink RP. The canalicular multispecificorganic anion transporter and conjugated hyperbilirubinemia inrat and man. J Mol Med 1997;75:420–428.

85. Keitel V, Kartenbeck J, Nies AT, Spring H, Brom M, Keppler D.Impaired protein maturation of the conjugate export pump mul-tidrug resistance protein 2 as a consequence of a deletionmutation in Dubin-Johnson syndrome. Hepatology 2000;32:1317–1328.

86. Keitel V, Nies AT, Brom M, Hummel Eisenbeiss J, Spring H,Keppler D. A common Dubin-Johnson syndrome mutation im-pairs protein maturation and transport activity of MRP2(ABCC2). Am J Physiol Gastrointest Liver Physiol 2003;284:G165–G174.

87. Shneider BL, Fox VL, Schwarz KB, Watson CL, Ananthanaray-anan M, Thevananther S, Christie DM, Hardikar W, SetchellKDR, Mieli-Vergani G, Suchy FJ, Mowat AP. Hepatic basolateralsodium-dependent bile acid transporter expression in two un-usual cases of hypercholanemia and in extrahepatic biliaryatresia. Hepatology 1997;25:1176–1183.

88. Tirona RG, Leake BF, Merino G, Kim RB. Polymorphisms inOATP-C: identification of multiple allelic variants associated withaltered transport activity among European- and African-Ameri-cans. J Biol Chem 2001;276:35669–35675.

89. Michalski C, Cui Y, Nies AT, Nuessler AK, Neuhaus P, ZangerUM, Klein K, Eichelbaum M, Keppler D, Konig J. A naturallyoccurring mutation in the SLC21A6 gene causing impairedmembrane localization of the hepatocyte uptake transporter.J Biol Chem 2002;277:43058–43063.

90. Stieger B, Fattinger K, Madon J, Kullak-Ublick GA, Meier PJ.Drug- and estrogen-induced cholestasis through inhibition of thehepatocellular bile salt export pump (Bsep) of rat liver. Gastro-enterology 2000;118:422–430.

1

1

1

1

1

1

1

1

1

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2


91. Funk C, Ponelle C, Scheuermann G, Pantze M. Cholestaticpotential of troglitazone as a possible factor contributing totroglitazone-induced hepatotoxicity: in vivo and in vitro interac-tion at the canalicular bile salt export pump (Bsep) in the rat.Mol Pharmacol 2001;59:627–635.

92. Roman ID, Fernandez-Moreno MD, Fueyo JA, Roma MG,Coleman R. Cyclosporin A induced internalization of the bile saltexport pump in isolated rat hepatocyte couplets. Toxicol Sci2003;71:276–281.

93. Fattinger K, Funk C, Pantze M, Weber C, Reichen J, Stieger B,Meier PJ. The endothelin antagonist bosentan inhibits the can-alicular bile salt export pump: a potential mechanism for he-patic adverse reactions. Clin Pharmacol Ther 2001;69:223–231.

94. Fouassier L, Kinnman N, Lefevre G, Lasnier E, Rey C, Poupon R,Elferink RP, Housset C. Contribution of mrp2 in alterations ofcanalicular bile formation by the endothelin antagonist bosen-tan. J Hepatol 2002;37:184–191.

95. Bolder U, Trang NV, Hagey LR, Schteingart CD, Ton-Nu HT, CerreC, Elferink RP, Hofmann AF. Sulindac is excreted into bile by acanalicular bile salt pump and undergoes a cholehepatic circu-lation in rats. Gastroenterology 1999;117:962–971.

96. Moseley RH. Sepsis-associated cholestasis. Gastroenterology1997;112:302–306.

97. Bolder U, Ton-Nu H-T, Schteingart CD, Frick E, Hofmann AF.Hepatocyte transport of bile acids and organic anions in endo-toxemic rats: impaired uptake and secretion. Gastroenterology1997;112:214–225.

98. Lee JM, Trauner M, Soroka CJ, Stieger B, Meier PJ, Boyer JL.Expression of the bile salt export pump is maintained afterchronic cholestasis in the rat. Gastroenterology 2000;118:163–172.

99. Vos TA, Hooiveld GJEJ, Koning H, Childs S, Meijer DKF,Moshage H, Jansen PLM, Muller M. Up-regulation of the multi-drug resistance genes, mrp1 and mdr1b, and down-regulationof the organic anion transporter, mrp2, and the bile salt trans-porter, spgp, in endotoxemic rat liver. Hepatology 1998;28:1637–1644.

00. Kubitz R, Wettstein M, Warskulat U, Haussinger D. Regulationof the multidrug resistance protein 2 in the rat liver by lipopoly-saccharide and dexamethasone. Gastroenterology 1999;116:401–410.

01. Green RM, Beier D, Gollan JL. Regulation of hepatocyte bile salttransporters by endotoxin and inflammatory cytokines in ro-dents. Gastroenterology 1996;111:193–198.

02. Kullak-Ublick GA. Regulation of organic anion and drug trans-porters of the sinusoidal membrane. J Hepatol 1999;31:563–573.

03. Green RM, Whiting JF, Rosenbluth AB, Beier D, Gollan JL. Inter-leukin-6 inhibits hepatocyte taurocholate uptake and sodium-potassium adenosinetriphosphatase activity. Am J Physiol1994;267:G1094–G1100.

04. Trauner M, Arrese M, Lee H, Boyer JL, Karpen SJ. Endotoxindownregulates rat hepatic ntcp gene expression via decreasedactivity of critical transcription factors. J Clin Invest 1998;101:2092–2100.

05. Whiting JF, Green RM, Rosenbluth AB, Gollan JL. Tumor necro-sis factor-� decreases hepatocyte bile salt uptake and medi-ates endotoxin-induced cholestasis. Hepatology 1995;22:1273–1278.

06. Leevy CB, Koneru B, Klein KM. Recurrent familial prolongedintrahepatic cholestasis of pregnancy associated with chronicliver disease [see comments]. Gastroenterology 1997;113:966–972.

07. Mella JG, Roschmann E, Glasinovic JC, Alvarado A, Scrivanti M,Volk BA. Exploring the genetic role of the HLA-DPB1 locus in

Chileans with intrahepatic cholestasis of pregnancy. J Hepatol1996;24:320–323.

08. Meng LJ, Reyes H, Axelson M, Palma J, Hernandez I, Ribalta J,Sjovall J. Progesterone metabolites and bile acids in serum ofpatients with intrahepatic cholestasis of pregnancy: effectof ursodeoxycholic acid therapy. Hepatology 1997;26:1573–1579.

09. Gendrot C, Bacq Y, Brechot M-C, Lansac J, Andres C. A secondheterozygous MDR3 nonsense mutation associated with intra-hepatic cholestasis of pregnancy [letter]. J Med Genet 2003;40:e32.

10. Bossard R, Stieger B, O’Neill B, Fricker G, Meier PJ. Ethinylestra-diol treatment induces multiple canalicular membrane transportalterations in rat liver. J Clin Invest 1993;91:2714–2720.

11. Simon FR, Fortune J, Iwahashi M, Gartung C, Wolkoff AW,Sutherland E. Ethinyl estradiol cholestasis involves alterationsin expression of liver sinusoidal transporters. Am J Physiol1996;271:G1043–G1052.

12. Vore M, Liu Y, Huang L. Cholestatic properties and hepatictransport of steroid glucuronides. Drug Metab Rev 1997;29:183–203.

13. Monte MJ, Dominguez S, Palomero MF, Macias RIR, Marin JJG.Further evidence of the usefulness of bile acids as moleculesfor shuttling cytostatic drugs toward liver tumors. J Hepatol1999;31:521–528.

14. Dominguez MF, Macias RI, Izco Basurko I, de La Fuente A,Pascual MJ, Criado JM, Monte MJ, Yajeya J, Marin JJ. Low in vivotoxicity of a novel cisplatin-ursodeoxycholic derivative (Bamet-UD2) with enhanced cytostatic activity versus liver tumors.J Pharmacol Exp Ther 2001;297:1106–1112.

15. Larena MG, Martinez Diez MC, Monte MJ, Dominguez MF, Pas-cual MJ, Marin JJ. Liver organotropism and biotransformation ofa novel platinum-ursodeoxycholate derivative, Bamet-UD2, withenhanced antitumour activity. J Drug Target 2001;9:185–200.

16. Briz O, Serrano MA, Rebollo N, Hagenbuch B, Meier PJ, KoepsellH, Marin JJ. Carriers involved in targeting the cytostatic bileacid-cisplatin derivatives cis-diammine-chloro-cholylglycinate-platinum(II) and cis-diammine-bisursodeoxycholate-platinum(II)toward liver cells. Mol Pharmacol 2002;61:853–860.

17. Kinoshita M, Miyata M. Underexpression of mRNA in humanhepatocellular carcinoma focusing on eight loci. Hepatology2002;36:433–438.

18. Vavricka SR, Jung D, Fried M, Grutzner U, Meier PJ, Kullak-UblickGA. The human organic anion transporting polypeptide 8(SLCO1B3) gene is transcriptionally repressed by hepatocytenuclear factor 3� in hepatocellular carcinoma. J Hepatol (inpress).

19. Nies AT, Konig J, Pfannschmidt M, Klar E, Hofmann WJ, KepplerD. Expression of the multidrug resistance proteins MRP2 andMRP3 in human hepatocellular carcinoma. Int J Cancer 2001;94:492–499.

20. Lammert F, Carey MC, Paigen B. Chromosomal organization ofcandidate genes involved in cholesterol gallstone formation: amurine gallstone map. Gastroenterology 2001;120:221–238.

21. Wang DQ, Lammert F, Paigen B, Carey MC. Phenotypic charac-terization of lith genes that determine susceptibility to choles-terol cholelithiasis in inbred mice. Pathophysiology of biliarylipid secretion. J Lipid Res 1999;40:2066–2079.

22. Carey MC, Paigen B. Epidemiology of the American Indians’burden and its likely genetic origins. Hepatology 2002;36:781–791.

23. Brink MA, Slors JF, Keulemans YC, Mok KS, de Waart DR, CareyMC, Groen AK, Tytgat GN. Enterohepatic cycling of bilirubin: aputative mechanism for pigment gallstone formation in ilealCrohn’s disease. Gastroenterology 1999;116:1420–1427.

24. Rosmorduc O, Hermelin B, Poupon R. MDR3 gene defect in

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adults with symptomatic intrahepatic and gallbladder choles-terol cholelithiasis. Gastroenterology 2001;120:1459–1467.

25. Lucena JF, Herrero JI, Quiroga J, Sangro B, Garcia-Foncillas J,Zabalegui N, Sola J, Herraiz M, Medina JF, Prieto J. A multidrugresistance 3 gene mutation causing cholelithiasis, cholestasisof pregnancy, and adulthood biliary cirrhosis. Gastroenterology2003;124:1037–1042.

26. Zelcer N, Reid G, Wielinga P, Kuil A, van Der Heijden I, SchuetzJD, Borst P. Steroid- and bile acid-conjugates are substrates ofhuman MRP4 (ABCC4). Biochem J 2003;371:361–367.

27. Repa JJ, Turley SD, Lobaccaro JA, Medina J, Li L, Lustig K, ShanB, Heyman RA, Dietschy JM, Mangelsdorf DJ. Regulation ofabsorption and ABC1-mediated efflux of cholesterol by RXRheterodimers. Science 2000;289:1524–1529.

28. Costet P, Luo Y, Wang N, Tall AR. Sterol-dependent transacti-vation of the ABC1 promoter by the liver X receptor/retinoid Xreceptor. J Biol Chem 2000;275:28240–28245.

29. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear recep-tors and lipid physiology: opening the X-files. Science 2001;294:1866–1870.

30. Fitzgerald ML, Moore KJ, Freeman MW. Nuclear hormone recep-tors and cholesterol trafficking: the orphans find a new home. JMol Med 2002;80:271–281.

31. Gartung C, Ananthanarayanan M, Rahman MA, Schuele S,Nundy S, Soroka CJ, Stolz A, Suchy FJ, Boyer JL. Down-regula-tion of expression and function of the rat liver Na�/bile acidcotransporter in extrahepatic cholestasis. Gastroenterology1996;110:199–209.

32. Arrese M, Trauner M, Ananthanarayanan M, Pizarro M, Solis N,Accatino L, Soroka C, Boyer JL, Karpen SJ, Miquel JF, Suchy FJ.Down-regulation of the Na�/taurocholate cotransportingpolypeptide during pregnancy in the rat. J Hepatol 2003;38:148–155.

33. Dumont M, Jacquemin E, D’Hont C, Descout C, Cresteil C,Haouzi D, Desrochers M, Stieger B, Hadchouel M, Erlinger S.Expression of the liver Na�-independent organic anion trans-porting polypeptide (oatp-1) in rats with bile duct ligation.J Hepatol 1997;27:1051–1056.

34. Geier A, Kim SK, Gerloff T, Dietrich CG, Lammert F, Karpen SJ,Stieger B, Meier PJ, Matern S, Gartung C. Hepatobiliary organicanion transporters are differentially regulated in acute toxic liverinjury induced by carbon tetrachloride. J Hepatol 2002;37:198–205.

35. Kakyo M, Unno M, Tokui T, Nakagomi R, Nishio T, Iwasashi H,Nakai D, Seki M, Suzuki M, Naitoh T, Matsuno S, Yawo H, AbeT. Molecular characterization and functional regulation of anovel rat liver-specific organic anion transporter rlst-1. Gastro-enterology 1999;117:770–775.

36. Roelofsen H, Vos TA, Schippers IJ, Kuipers F, Koning H,Moshage H, Jansen PLM, Muller M. Increased levels of the
multidrug resistance protein in lateral membranes of proliferat- f
ing hepatocyte-derived cells. Gastroenterology 1997;112:511–521.

37. Hirohashi T, Suzuki H, Ito K, Ogawa K, Kume K, Shimizu T,Sugiyama Y. Hepatic expression of multidrug resistance-asso-ciated protein-like proteins maintained in Eisai hyperbiliru-binemic rats. Mol Pharmacol 1998;53:1068–1075.

38. Strautnieks SS, Bull LN, Knisely AS, Kocoshis SA, Dahl N, ArnellH, Sokal E, Dahan K, Childs S, Ling V, Tanner MS, KagalwallaAF, Nemeth A, Pawlowska J, Baker A, Mieli Vergani G, FreimerNB, Gardiner RM, Thompson RJ. A gene encoding a liver-specificABC transporter is mutated in progressive familial intrahepaticcholestasis. Nat Genet 1998;20:233–238.

39. Huang L, Smit JW, Meijer DK, Vore M. Mrp2 is essential forestradiol-17beta(beta-D-glucuronide)-induced cholestasis inrats. Hepatology 2000;32:66–72.

40. Green RM, Ward K. Phenotypic characterization of ATP-depen-dent canalicular bile salt transport in C57L/J and AKR/J mice[abstract]. Hepatology 2000;32:390A.

41. Smit JJM, Schinkel AH, Oude Elferink RPJ, Groen AK, WagenaarE, van Deemter L, Mol CAAM, Ottenhoff R, van der Lugt NMT,van Roon MA, van der Valk MA, Offerhaus GJA, Berns AJM,Borst P. Homozygous disruption of the murine mdr2 P-glycopro-tein gene leads to a complete absence of phospholipid from bileand to liver disease. Cell 1993;75:451–462.

42. Jacquemin E, Cresteil D, Manouvrier S, Boute O, Hadchouel M.Heterozygous non-sense mutation of the MDR3 gene in familialintrahepatic cholestasis of pregnancy [letter]. Lancet 1999;353:210–211.

43. Trauner M, Arrese M, Soroka CJ, Ananthanarayanan M, KoeppelTA, Schlosser SF, Suchy FJ, Keppler D, Boyer JL. The rat cana-licular conjugate export pump (mrp2) is down-regulated in intra-hepatic and obstructive cholestasis. Gastroenterology 1997;113:255–264.

44. Paulusma CC, Bosma PJ, Zaman GJR, Bakker CTM, Otter M,Scheffer GL, Scheper RJ, Borst P, Oude Elferink RPJ. Congenitaljaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science 1996;271:1126–1128.

45. Descotes G, Osman H, Gallas JF, Penacchio E, Moreau C.Functional, biochemical, and morphological hepatobiliary ef-fects in rats chronically exposed to a steroidal antiandrogen.Toxicol Appl Pharmacol 1996;137:23–33.

46. Silva MO, Reddy KR, McDonald T, Jeffers LJ, Schiff ER. Danazol-induced cholestasis. Am J Gastroenterol 1989;84:426–428.

Received March 25, 2003. Accepted June 12, 2003.Address requests for reprints to: Gerd A. Kullak-Ublick, M.D., Depart-ent of Internal Medicine, University Hospital, CH-8091 Zurich, Swit-

erland. e-mail: [email protected]; fax: (41) 1-255-4503.Supported by grants 32-59155.99, 632-062773 and 31-64140.00

rom the Swiss National Science Foundation, Bern, Switzerland.

Enterohepatic bile salt transporters in normal physiology and liver disease

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