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SEMINARS IN LIVER DISEASE—VOL. 20, NO. 3, 2000 Hepatic Secretion of Conjugated Drugs and Endogenous Substances DIETRICH KEPPLER, M.D. and JÖRG KÖNIG, Ph.D. ABSTRACT Conjugate export pumps of the multidrug resistance protein (MRP) family mediate the ATPdependent secretion of anionic conjugates across the canalicular and the basolateral hepatocyte membrane into bile and sinusoidal blood, respectively. Xenobiotic and endogenous lipophilic substances may be conjugated with glutathione, glucuronate, sulfate, or other negatively charged groups and thus become substrates for export pumps of the MRP family. The apical isoform, MRP2 (gene symbol ABCC2), has been localized to the apical membrane of several polarized epithelia and particularly to the canalicular membrane of hepatocytes. Absence of functionally active MRP2 glycoprotein from this membrane domain prevents the secretion of many anionic conjugates into bile. Prototypic endogenous substrates of high affinity for recombinant human MRP2 include bisglucuronosyl bilirubin, monoglucuronosyl bilirubin, and the glutathione S-conjugate leukotriene C4. Several mutations in the human MRP2 gene have been identified that lead to the absence of MRP2 from the canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome. MRP2-mediated conjugate export represents a decisive final step in the detoxification of drugs, toxins, and endogenous substances. The basolateral isoform, MRP3 (gene symbol ABCC3), is upregulated in MRP2 deficiency and in extrahepatic cholestasis. MRP3 mediates the ATP-dependent transport of anionic conjugates, particularly of glucuronides and sulfoconjugates, across the basolateral hepatocyte membrane into sinusoidal blood. The inverse regulation of MRP3 and MRP2 expression under many conditions is consistent with their distinct localization and with a compensatory role of MRP3 in the hepatic secretion of anionic conjugates during impaired transport into bile. KEY WORDS: conjugate export pumps, Dubin-Johnson syndrome, MRP transporters MRP2, MRP3 Hepatocytes actively convert exogenous and endogenous substances into anionic conjugates with glutathione, glucuronate, sulfate, or other negatively charged moieties. This conjugation of lipophilic sub- stances precedes their transport into the extracellular space. Under physiologic conditions, these conjugates are secreted across the canalicular membrane into bile by an ATP-dependent conjugate export pump.1–6 The Objectives Upon completion of this article, the reader should be able to 1) summarize the role of the conjugate export pumps of the multidrug resistance protein (MRP) family in the hepatic handling of endogenous and xenobiotic substances, 2) summarize the prototypic substrates of the apical conjugate export pump MRP2, 3) summarize the molecular basis of Dubin-Johnson syndrome, and 4) discuss the compensatory role of the basolateral conjugate export pump MRP3 in cholestasis and Dubin-Johnson syndrome. Accreditation The Indiana University School of Medicine is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. Credit The Indiana University School of Medicine designates this educational activity for a maximum of 1.0 hours credit toward the AMA Physicians Recognition Award in category one. Each physician should claim only those hours of credit that he/she actually spent in the educational activity. Disclosure Statements have been obtained regarding the authors’ relationships with financial supporters of this activity. There is no apparent conflict of interest related to the context of participation of the authors of this article. From the Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany. Reprint requests: Dr. D. Keppler, Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany. E-mail: [email protected] Copyright © 2000 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel.: +1(212) 584-4662. 0272-8087,p;2000,20,03,265,272,ftx,en;sld00073x 265 266 SEMINARS IN LIVER DISEASE—VOL. 20, NO. 3, 2000 molecular identification and cloning of this canalicular conjugate export pump (MRP2)7–9 was a consequence of the discovery that the multidrug resistance protein 1 (MRP1) transports similar substrates, including glutathione S-conjugates, glucuronides, and sulfoconjugates.10–13 Mutant rat strains deficient in the hepatobiliary secretion of conjugates and additional organic anions14,15 were shown to lack the multidrug resistance protein 2 (MRP2) in the canalicular membrane8,9 due to mutations in the rat MRP2 gene, leading to premature termination codons.8,16 Observations in these mutant rats prove the concept that anionic conjugates of many lipophilic substances cannot exit across the plasma membrane into bile in the absence of the ATPdependent export pump MRP2. The lack of this protein prevents the secretion into bile of substances such as the glutathione S-conjugate leukotriene C415 and N-acetyl leukotriene E4,17 both high-affinity substrates for MRP2.3,18 Evidently, there is no alternative transport protein in the canalicular membrane for the secretion of these conjugates. It is important to note, however, that many conjugates formed in the hepatocytes can be transported into the sinusoidal space, particularly under pathophysiologic conditions associated with an impaired function of MRP2.17,19 The recent localization of another member of the MRP family, MRP3, to the basolateral membrane of hepatocytes20,21 suggests that basolateral isoforms of the MRP family can support the secretion of conjugates from hepatocytes into blood. In this article we consider the role of MRP family members in the hepatocellular secretion of anionic substances formed in the conjugation phase (phase II) of detoxification of drugs and endogenous substances. Moreover, we review consequences of mutations in the MRP2 gene leading to the Dubin-Johnson syndrome in humans. CONJUGATE EXPORT PUMPS OF THE MRP FAMILY Members of the MRP family have been identified in a variety of different organisms, including yeast, nematodes, plants, and mammals. The major function of this ATP-binding cassette (ABC) transporters is the export of anionic conjugates from cells. In humans, the MRP family currently comprises six characterized members, known as MRP1 (symbol ABCC1); MRP2 (ABCC2), also known as canalicular MRP or canalicular multispecific organic anion transporter; MRP3 (ABCC3); MRP4 (ABCC4); MRP5 (ABCC5); and MRP6 (ABCC6). The deduced amino acid numbers range from 1,325 amino acids for MRP4 to 1,545 amino acids for MRP2 (Table 1). The length of the proteins and the membrane topology discriminates the MRPs from the MDR Pglycoproteins. In contrast to the typical six plus six transmembrane segment topology described for members of the MDR family,22,23 four of the six current members of the MRP family exhibit an additional aminoterminal membrane-spanning domain represented by an extension of approximately 200 amino acids.24 In addition to computational methods, the topology of MRP1 and MRP2 has been studied by mutational analyses, limited proteolysis experiments,25 and epitope insertion studies.26–28 Both transporters are predicted to consist of a P-glycoprotein-like core structure with two ATP-binding domains and two transmembrane regions, in addition to a third transmembrane region located TABLE 1. Members of the MRP Family with Amino Acid Identities Relative to MRP2 Identity (%) Accession Number 1531 1545 1527 1325 1437 1503 48 100 46 36 36 38 NM_004996 X96395 Y17151 AF071202 AF104942 AF076622 MRP2 1564 82 Z49144 Rat MRP2 MRP3 MRP6 1541 1523 1502 78 46 36 X96393 U73038 AB010466 Mouse MRP1 MRP2 MRP5 1528 1543 1436 48 77 36 AF022908 AF227274 AB019003 Species Protein Symbol Chromosome Human MRP1 MRP2 MRP3 MRP4 MRP5 MRP6 ABCC1 ABCC2 ABCC3 ABCC4 ABCC5 ABCC6 16p13.1 10q24 17q21.3 13q32 3q27 16p13.1 Rabbit ABCC, ABC subfamily C. Amino Acids 267 HEPATIC SECRETION—KEPPLER, KÖNIG amino-proximal in front of the core sequence. A remarkable topologic feature of MRP1 and MRP2 represents their amino-terminus, which was predicted to be extracellular. This was first described for MRP2 on the basis of topology prediction programs9 and subsequently experimentally established both for MRP1 by epitope insertion studies29 and for MRP2 by direct immunofluorescence microscopy.18 MRP1 and MRP2 are the best characterized members of the MRP family, and both share a similar substrate specificity.30 MRP1, the founding member of the family, was cloned from a drug-selected human lung cancer cell line.31 The MRP1 gene spans approximately 200 kilo-base pairs (kbp) and is located on chromosome 16p13.12-13; the gene contains 31 exons.32 The human MRP2 protein consists of 1,545 amino acids and is encoded by a gene located on chromosome 10q23q24.9,18,19,33–36 The MRP2 gene spans approximately 45 kbp and contains 32 exons ranging in size from 56 to 255 bp.37 Each nucleotide binding domain of the MRP2 gene is encoded by three exons. The comparison of the genomic organization of MRP237,38 with the genomic organization of the MRP1 gene32 displays remarkable similarities indicated by the size and number of exons and by 21 identical splice sites when viewed on the amino acid level.37 Recently, the genomic organization of the MRP3 gene became accessible as a genomic cosmid clone (GenBank accession AC004590). The MRP3 gene contains 31 exons. Interestingly, MRP1, MRP2, and MRP3 have 21 identical splice sites when viewed on an amino acid alignment. Despite the fact that these three MRP isoforms share only a relatively low degree of amino acid identity, a close evolutionary relationship of these transporters is indicated by their similar genomic organization. The identification of MRP3, MRP4, MRP5, and MRP6 was mainly based on the analysis of the expressed sequence tag database39 followed by the cloning of partial cDNA sequences40 and subsequently of the complete cDNA of the respective transporter. MRP1–5 are encoded by genes located on different chromosomes (Table 1). Among the more recently identified MRP isoforms (MRP3–5), MRP3 is best characterized with respect to its tissue-specific expression, its transport function, and its localization.20,21,41–43 Unlike the substrates for MRP1 and MRP2, glutathione S-conjugates are poor substrates for MRP3.42 On the other hand, other glucuronosyl conjugates, including 17-glucuronosyl estradiol, are substrates for MRP1, MRP2, and MRP3. In contrast to MRP1 and MRP2 from rat and human, rat MRP3 is able to transport sulfated and nonsulfated bile salts.42 Moreover, MRP3 is also able to confer drug resistance against several epipodophyllotoxins and methotrexate.21 The tissue distribution of MRP3 exhibits similarities with the tissue distribution of MRP2, and both proteins are expressed in liver and colon.20,21,30 APICAL CONJUGATE EXPORT PUMP MRP2 MRP2 has been the second member of the MRP family to be cloned, localized, and functionally characterized.7–9,16,18,19,30,33–36,44–49 Up to now, it is the only conjugate export pump detected in the apical (canalicular) membrane domain of hepatocytes. MRP2 has also been localized to the apical membrane of kidney proximal tubules,47,48 of intestinal epithelia, and various polarized cells in culture.49–52 It should be noted that other known members of the human MRP family have been localized to the basolateral domain of polarized cells.30 The localization of MRP2 is dynamic as shown by the endocytic retrieval and exocytic insertion of the protein in rat hepatocytes.53–57 Endocytic retrieval followed by downregulation of MRP2 gene expression is also observed in rat hepatocytes after endotoxin-induced cholestasis56,57 and after duct ligation.55 The endotoxininduced decrease of MRP2 in the canalicular membrane explains the well-known impairment of the excretion of MRP2 substrates into bile in endotoxemia. This is exemplified by the early and potent inhibition of cysteinyl leukotriene secretion across the canalicular membrane into rat bile after endotoxin administration.58,59 The substrate specificity of MRP2 has been elucidated in a stepwise fashion, starting from hepatobiliary elimination studies in MRP2-deficient mutant rats,6,14,15,60 leading to measurements of ATP-dependent transport using inside-out-oriented canalicular membrane vesicles derived from normal and MRP2-deficient mutant rat liver,2–7,9 and finally by transport measurements using recombinant MRP2 from human and other species.18,30,36,46 Based on the work with recombinant human MRP2 studied in membrane vesicles, the prototypic high-affinity substrates include monoglucuronosyl bilirubin, bisglucuronosyl bilirubin, and the endogenous glutathione S-conjugate leukotriene C4 (Table 2). A large number of glutathione S-conjugates, glucuronides, and sulfoconjugates of drugs and other xenobiotics are also substrates for MRP2, although the affinity for most drug conjugates is much lower than for the prototypic TABLE 2. Selected Substrates for the Apical Conjugate Export Pump MRP2 (Human Recombinant) Substrate Monoglucuronosyl bilirubin Bisglucuronosyl bilirubin Leukotriene C4 S-glutathionyl 2,4-dinitrobenzene 17-Glucuronosyl estradiol para-Aminohippurate Km Value (µM) 0.7 0.9 1.0 6.5 7.2 880 Reference Kamisako et al., 199944 Kamisako et al., 199944 Cui et al., 199918 Evers et al., 199836 Cui et al., 199918 Leier et al., 200062 268 SEMINARS IN LIVER DISEASE—VOL. 20, NO. 3, 2000 endogenous substrates.61 In addition, a number of nonconjugate amphiphilic anions, such as the lipophilic pentaanion Fluo349,52 and the monoanionic paraaminohippurate,62 are MRP2 substrates. Because MRP2 shares only 48% identical amino acids with MRP1 (Table 1), it cannot be anticipated that known inhibitors for MRP163 will also interfere with MRP2-mediated transport. Inhibitors acting on MRP2, although less potently than on MRP1,11 include the quinoline derivative MK5719 and cyclosporin A.52 Selective inhibitors for MRP2 may be of interest to overcome MRP2-mediated drug resistance18,36 and to enhance the intestinal absorption of drugs that are MRP2 substrates and are otherwise transported back into the intestinal lumen. MEMBERS OF THE MRP FAMILY LOCALIZED TO THE BASOLATERAL MEMBRANE The founding member of the MRP family, MRP1,31 has been localized to the basolateral membrane of polarized pig kidney cells after transfection with human MRP1 cDNA.64 In hepatocytes, however, the expression of MRP1 mRNA is extremely low31 and the immunoreactive proteins detected at the basolateral hepatocyte membrane by use of antibodies, raised against human MRP1,7,19 could only be fully identified after the more recent cloning of the isoforms MRP320,21 and MRP6.30,65–67 MRP3 and MRP6 have been localized to the basolateral membrane of human hepatocytes and other polarized epithelial cells.20,21,30,65 MRP6 is constitutively and highly expressed in hepatocytes and kidney,66 but its substrate specificity has not yet been elucidated. MRP3 is low under normal conditions in hepatocytes and upregulated in cholestatic liver disease.20,21,41–43 Under many conditions, MRP3 is regulated inversely when compared with MRP2.68 It is of in- terest that MRP3 has also been localized to the basolateral membrane of cholangiocytes21 and intestinal epithelia where it may contribute to the enterohepatic circulation of bile salts.42,43,69 MRP2 DEFICIENCY IN DUBIN-JOHNSON SYNDROME Some mutations in the MRP2 gene are associated with the absence of the MRP2 protein from the hepatocyte canalicular membrane.34,37 Several different mutations in the MRP2 gene have been discovered in humans34,37,38,70 and rats.8,16 The Dubin-Johnson syndrome in humans is an autosomal recessively inherited disorder characterized by conjugated hyperbilirubinemia and pigment deposition in the liver.71–73 The deficient transport of monoglucuronosyl bilirubin and bisglucuronosyl bilirubin and other anionic conjugates from hepatocytes into bile is caused by the absence or the functional impairment of the MRP2 protein in the canalicular membrane.19,34,37,74 So far, however, mutations in the MRP2 gene leading to a functionally deficient protein inserted into the hepatocyte canalicular membrane have not been identified. Furthermore, we have neither detected truncated MRP2 protein in the hepatocytes from a DubinJohnson syndrome patient with a stop codon in exon 23 nor in a patient with a 6-nucleotide deletion in exon 30.37 Current knowledge on the sites of mutations in the coding sequence and in splice sites of the MRP2 gene in Dubin-Johnson syndrome together with the exon–intron organization of the human MRP2 gene are depicted in Figure 1. Determination of the exon–intron organization of the gene has been a prerequisite for the elucidation of mutations underlying Dubin-Johnson syndrome.37,38 The currently known mutations in the MRP2 gene are scattered preferentially over the 3-proximal half of the mRNA including the exons encoding both nucleotidebinding domains (Fig. 1). FIG. 1. Mutations in the MRP2 gene leading to Dubin-Johnson syndrome. The genomic organization of the human MRP2 gene is characterized by 32 exons and a size of the total gene of about 45 kbp.37 The exon–intron boundaries (GenBank accession AJ132244), the number of exons, and both ATP-binding domains are indicated. Arrows indicate the sites of mutations currently identified. F1–F4 correspond to mutations identified in Fukuoka,38,70 S1 indicates a mutation identified in Saga,75 H1 and H2 correspond to mutations studied in Heidelberg,37 A1 is an identical mutation as H1 and was first described in Amsterdam,34 and T1 designates the mutation present in a large group of Iranian Jews analyzed in Tel Aviv.76 HEPATIC SECRETION—KEPPLER, KÖNIG Established mutations in patients with DubinJohnson syndrome include a nonsense mutation leading to a premature termination codon,34,37 a missense mutation affecting the first nucleotide-binding domain,38,70 a deletion mutation leading to the loss of two amino acids in the second nucleotide-binding domain,37 splice junction mutations leading to exon deletions and premature termination codons,38,70,75 and a mutation causing an isoleucin to phenylalanine exchange in position 1173.76 Furthermore, mutations were identified in two wellcharacterized hyperbilirubinemic rat strains, which have been considered as animal models of the human Dubin-Johnson syndrome, the GY/TR mutant rat8 and the Eisai hyperbilirubinemic rat.16 These mutations introduce premature termination codons at codon 401 and 855 in GY/TR and Eisai hyperbilirubinemic rat mutant rats, respectively. In both mutant livers, however, no truncated proteins were detected,9 and the MRP2 269 mRNA was below detectability as analyzed by Northern blotting.8,9,16 The introduction of premature termination codons may lead to a decrease in the level of mRNA by a mechanism termed “nonsense mediated decay.”77 In the case of a stop codon 5 of the last splice site, this is recognized during translation, and the mRNA is subjected to decay.77 It is likely that the absence of the MRP2 protein from the hepatocyte canalicular membrane in certain cases of Dubin-Johnson syndrome19,37,74 is also a consequence of the rapid degradation of the mutated mRNA. Other mutations in the MRP2 gene may lead to a reduced stability of the protein, may affect the interaction of the MRP2 protein with chaperone proteins, may influence trafficking and apical localization of the protein, or may lead to an apically localized but functionally deficient MRP2 protein. These alternatives should be considered and possibly investigated for each of the mutations in the MRP2 gene, unless mutations FIG. 2. Localization and function of the conjugate export pumps MRP2 and MRP3 in normal and MRP2-deficient hepatocytes. (Top) The uptake of various substances across the basolateral membrane, followed by conjugation, and MRP2-mediated export across the apical (canalicular) membrane is shown. (Bottom) Situation in extrahepatic cholestasis or MRP2 deficiency with the compensatory function of MRP3 mediating export of conjugates across the basolateral membrane into sinusoidal blood. 270 leading to a premature termination codon allow for a simpler interpretation of the consequences. SEMINARS IN LIVER DISEASE—VOL. 20, NO. 3, 2000 plifying one of several conditions under which MRP3 and MRP2 are inversely regulated in liver and hepatocyte-derived cells.68 INTERACTION OF THE APICAL AND BASOLATERAL CONJUGATE EXPORT PUMPS: MRP2 AND MRP3 The release on anionic conjugates from hepatocytes into sinusoidal blood under conditions of extrahepatic cholestasis or MRP2 deficiency can be mediated by the basolateral export pump MRP3 (Fig. 2). This is indicated by the basolateral localization of MRP3 in human and rat hepatocytes20,21 and by the substrate preference of MRP3 for anionic conjugates formed inside hepatocytes both under normal conditions and during cholestasis or MRP2 deficiency.42,78,79 Substrates for MRP3 include glucuronosyl bilirubin,78 17-glucuronosyl estradiol,42,79 and sulfated bile salts such as sulfatolithocholyl taurine and sulfatochenodeoxycholyl taurine,42,79 whereas glutathione conjugates are relatively poor substrates for MRP3 when compared with MRP1 and MRP2.79 The pronounced expression of MRP3 in the basolateral membrane of hepatocytes from patients with Dubin-Johnson syndrome20 serves as a compensatory transport pathway for conjugates that cannot be secreted into bile and must therefore be released into blood followed by renal excretion. The conjugated hyperbilirubinemia in Dubin-Johnson syndrome suggests that the release of bilirubin glucuronides from hepatocytes via MRP3 is more rapid than renal excretion. The compensatory role of MRP3 in MRP2 deficiency and in extrahepatic cholestasis is also consistent with the upregulation of MRP3 in rat liver after bile duct ligation and in rats with hereditary MRP2 deficiency.80 The noninvasive assessment of hepatobiliary and renal secretion of anionic conjugates in rats under such conditions has indicated a prolonged time period for intracellular storage and metabolism and an approximately threefold extension of hepatic excretion half-times followed by the complete renal elimination of substances that are eliminated under normal conditions via MRP2 into bile.17 The molecular mechanisms leading to the upregulation of MRP3 have not been sufficiently elucidated.42,68,79,80,81 Characterization of the 5-flanking region of human MRP3 demonstrates that MRP3 is under the control of a TATA-less promoter and that Sp1 binding sites may be involved in the regulation of its expression.81 Evidence has not been obtained supporting a direct action of bilirubin as an inducer.81 The basal promoter activity of human MRP3 is only 4 % of that measured for MRP268; however, MRP3 promoter activity, mRNA, and protein are markedly increased after disruption of microtubules by nocadazol.68 This treatment leads, on the other hand, to a downregulation of promoter activity, mRNA, and protein of MRP2, exem- ABBREVIATIONS ABC ABCC kbp MRP ATP-binding cassette ATP-binding cassette transporter subfamily C kilo-base pair multidrug resistance protein REFERENCES 1. 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