Export pumps for glutathione S-conjugates

Free Radical Biology & Medicine, Vol. 27, Nos. 9/10, pp. 985–991, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter

PII S0891-5849(99)00171-9

Forum EXPORT PUMPS FOR GLUTATHIONE S-CONJUGATES DIETRICH KEPPLER Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany

Abstract—The release of glutathione S– conjugates from cells is an ATP-dependent process mediated by integral membrane glycoproteins belonging to the recently discovered multidrug-resistance protein (MRP) family. Many lipophilic compounds conjugated with glutathione, glucuronate, or sulfate are substrates for export pumps of the MRP family. In humans six MRP isoforms encoded by different genes have been cloned. Orthologs of MRP have been identified in many species including yeast, plants, and nematodes. Human MRP1 and MRP2 are currently best characterized with respect to substrate specificity by measurements of ATP-dependent transport into inside-out membrane vesicles. High-affinity substrates include the glutathione S-conjugate leukotriene C4, S-(2,4dinitrophenyl)glutathione, bilirubin glucuronosides, and 17␤-glucuronosyl estradiol. In addition, glutathione disulfide is transported by MRP1 and MRP2. Reduced glutathione may be released from cells in a process directly or indirectly mediated by members of the MRP family. Proteins of the MRP family are indispensable for transport of glutathione S-conjugates and glutathione disulfide into the extracellular space and play, therefore, a decisive role in detoxification and defense against oxidative stress. © 1999 Elsevier Science Inc. Keywords—ATP-binding cassette (ABC) transporters, ATP-dependent transport, Conjugate export pumps, DubinJohnson syndrome, GSSG transport, Leukotriene C4 transport, MRP1, MRP2, MRP3, MRP family of transporters, Multidrug resistance proteins, Oxidative stress, Free radicals

INTRODUCTION

mastocytoma cells [10]. The identification of the membrane proteins mediating the ATP-dependent transport of glutathione S-conjugates was a consequence of the molecular characterization of the 190kDa glycoprotein mediating the transport of the endogenous glutathione S-conjugate leukotriene C4 [11]. This protein turned out to be the murine ortholog of the multidrug resistance protein [12] that had been cloned in 1992 by Cole et al.[13] from multidrug resistant human lung cancer cells and identified as an integral membrane glycoprotein belonging to the superfamily of ATP-binding cassette (ABC) transporters. This multidrug-resistance protein (abbreviated MRP or MRP1; symbol ABCC1) is very different from MDR1 P-glycoprotein which shares only 15 % amino acid identity [14]. MRP1 mediates the ATPdependent transport of many glutathione S-conjugates, in addition to glucuronosides and sulfoconjugates [15– 19]. MRP1 has been the first member of the growing family of conjugate export pumps cloned from mammals, yeast, plants, and nematodes [20 –23]. For many of these export pumps a role has been established in glutathione-dependent drug resistance and detoxification [14,20,22,24,25]. Moreover, because of the MRP-

The conjugation of many lipophilic substances with glutathione precedes their transport across the plasma membrane into the extracellular space [1,2]. The transport of glutathione S-conjugates, including glutathione disulfide, has been recognized as a primary-active, ATP-dependent process that was characterized in inside-out plasma membrane vesicles from many cell types including erythrocytes [3,4], hepatocytes [5–7], heart [8,9], and Address correspondence to: Dr. Dietrich Keppler, Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany; Tel: ⫹49 (6221) 42-2400; Fax: ⫹49 (6221) 42-2402; E-Mail: [email protected]. Dietrich Keppler studied medicine at the universities in Munich and Freiburg, Germany. He joined the Biochemistry Department of Freiburg University in 1966 and became a Professor of Biochemistry in 1975. In 1987 he became Head of the Division of Tumor Biochemistry of the Deutsches Krebsforschungszentrum and Professor of Tumor Biochemistry at the University of Heidelberg. His research interests have included molecular mechanisms of liver injuries, particularly galactosamine-induced hepatitis, pyrimidine nucleotide biosynthesis and metabolism, and the metabolism and transport of leukotrienes. Studies on the transport of the endogenous glutathione S-conjugate leukotriene C4 led to the molecular identification and cloning of membrane proteins mediating the ATP-dependent transport of lipophilic compounds conjugated with glutathione, glucuronate, or sulfate. 985

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mediated export of glutathione disulfide from cells [26], MRP family members play a role in the defense against oxidative stress. MRP family of ATP-dependent conjugate export pumps Elucidation of the sequence [13] and function [15–20] of human MRP1 pointed to the presence of related proteins in other tissues, such as liver [27–29] and kidney [30,31]. The apical isoform MRP2, with an amino acid identity of 49% with MRP1, has been localized to the hepatocyte canalicular membrane [28,29] and to the apical membrane of proximal tubules in the kidney [30,31]. Human MRP3, with an amino acid identity of 58% with MRP1, has been localized to the basolateral hepatocyte membrane [32,33]. Human MRP4, MRP5, and MRP6 [21,34] have only been partially characterized [21,35]. The yeast (Saccharomyces cerevisiae) cadmium factor 1 (YCF1) gene encodes another member of the MRP family that functions as an ATP-dependent vacuolar glutathione S-conjugate pump [36]. The amino acid identity of yeast YCF1 with human MRP1 is 40% [14] and 41% with human MRP2 [20]. Several MRP-related genes encoding ATP-dependent glutathione S-conjugate export pumps have been cloned more recently from the plant Arabidopsis thaliana [22]. In the soil nematode Caenorhabditis elegans, 4 MRP-related transporters were identified that contribute to detoxification of cadmium and arsenite, possibly by pumping glutathione S-conjugates of these heavy metals [37]. The increasing number of members of the MRP family in different organisms is evident from the comparison of dendrograms published in recent years [23]. The MRP family members that have been functionally characterized so far share the property of ATP-dependent export pumps for anionic conjugates and lipophilic anions. Many of these transport proteins enable the sequestration and terminal excretion of conjugates formed in so-called phase II reactions of detoxification. Substrate specificity of the ATP-dependent conjugate export pumps encoded by the genes MRP1, MRP2, and MRP3 The functional characterization of recombinant human MRP1 [16 –19,26,38,39], MRP2 [25,40], and MRP3 [33,41] indicates that these proteins are conjugate export pumps with overlapping substrate specificities but with marked kinetic differences. For human MRP1, a ranking of substrates according to the Vmax/Km ratios is as follows [38]: LTC4 ⬎ LTD4 ⬎ S-(2,4-dinitrophenyl)glutathione ⬎ 17␤-glucuronosyl estradiol ⬎ monoglucuronosyl bilirubin ⬎ 3␣-sulfatolithocholyl taurine ⬎ GSSG.

This ranking is similar for human MRP2. However, the Km value of MRP2 for LTC4 and 17␤-glucuronosyl estradiol is 10-fold and 4.8-fold higher, respectively, than for MRP1 [25]. The glutathione S-conjugate of aflatoxin B1 was identified as another high-affinity substrate for MRP1 [39]. Interestingly, this potent carcinogen (unconjugated aflatoxin B1) is also transported by MRP1 in an ATP-dependent manner when GSH (5 mM) is added to allow for cotransport, possibly after formation of a labile complex [39]. It should be noted that both MRP1 [23] and MRP2 [42] are also transporting unconjugated amphiphilic anions, such as the amphiphilic penta-anion Fluo-3 with Km values of 12 and 3.7 ␮M, respectively [42]. Cloning of rat MRP3 and determination of its substrate specificity in inside-out membrane vesicles from transfected cells indicated that it is preferentially transporting glucuronosides, such as 17␤-glucuronosyl estradiol, but that glutathione S-conjugates are relatively poor substrates [41]. These data suggest that the substrate specificities of MRP1 and MRP2 differ less than MRP1 and MRP3 or MRP2 and MRP3, although, based on the overall amino acid identity, MRP1, and MRP2 differ much more than MRP1 and MRP3 [32,33]. A more detailed comparison of substrate specificities will be possible when the transport function of recombinant human MRP3 has been analyzed in membrane vesicles. Role of MRP isoforms in the release of GSH from cells The transport proteins involved in the release of GSH from cells may include members of the MRP family [19,24,43– 46]. This is indicated by MRP1-dependent cotransport of GSH with natural product toxins such as aflatoxin and vincristine [19,39,43,44] and by the ATPdependent low-affinity transport of GSH by a yeast ortholog of MRP, the transporter YCF1 [46]. Furthermore, MRP2-dependent release of GSH into the extracellular space of MRP2-transfected cells and into bile of rats with varying levels of MRP2 in the hepatocyte canalicular membrane has been demonstrated [45]. The latter studies suggest that MRP2 functions as a low-affinity export pump for the release of GSH across apical membrane domains. Since hepatocellular GSH concentrations are high (5–10 mM), MRP2 might serve an important physiological function in the maintenance of GSH-dependent bile flow and in hepatic GSH turnover [45]. However, it is not possible at present to discriminate between a MRP2-mediated cotransport of GSH together with an endogenous yet unidentified cosubstrate and a transport of GSH alone. Interestingly, GSH export catalyzed by MRP3 was not detectable [33]. Additional transport proteins involved in the release of GSH from hepatocytes have been described both in

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Fig. 1. Transport and detoxification of endogenous substances, drugs, and carcinogens. Conjugates with glutathione, glucuronate, or sulfate leave the cell by ATP-dependent transport mediated by a member of the MRP family. Conjugates are retained in the cell in the absence of MRP-mediated export [29,50].

the canalicular [47] and in the sinusoidal [48] membrane. The organic anion transporter OATP1 from rat liver was shown to release GSH across the sinusoidal membrane into the blood by exchange with hydrophobic glutathione S-conjugates such as leukotriene C4 [48]. This countertransport by OATP1 provides not only a molecular basis for GSH release but also for the apparently unidirectional uptake of leukotriene C4 into hepatocytes, because of the high intracellular and the relatively low extracellular GSH concentration [48]. Function of MRP family members in detoxification and defense against oxidative stress The sequence of uptake of endogenous and xenobiotic lipophilic substances into cells followed by oxidation, conjugation with glutathione or alternative anionic groups, and ATP-dependent export by a member of the MRP family is of vital importance in detoxification and cellular homeostasis (Fig. 1). These different steps or phases in detoxification may also be designated phase 0, phase 1, phase 2, and phase 3 [49]. Studies in mutant animals which lack MRP2 in their hepatocyte canalicular

membrane [29] demonstrate that glutathione S-conjugates cannot be released into bile [50] and excretion across the basolateral membrane can serve as an alternative pathway [32]. The redundancy with regard to the occurrence of several MRP family members in most cells [21] seems to prevent intracellular accumulation of conjugates. The ATP-dependent MRP-mediated export of conjugates represents an indispensable terminal step in detoxification (Fig. 1). This conclusion is in line with the synergistic effect of an overexpression of both, a glutathione S-transferase (GST P1-1 or GST A1-1) and MRP1, leading to high-level resistance to the cytotoxic action of several drugs [51,52]. Furthermore, overexpression of recombinant MRP2 confers resistance of human and canine cells to several cytotoxic agents including cisplatin [25]. A number of cell types and tissues release GSSG upon external addition of hydroperoxides or other conditions inducing oxidative stress, indicating that the GSSG reductase pathways may be insufficient [53–55]. Identification of the proteins releasing GSSG from cells as MRP1 and MRP2 [26] suggests that MRP family members play an essential role in the control of the intracel-

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Fig. 2. MRP-mediated export of glutathione disulfide. GSSG is a substrate for MRP1 or MRP2 [26]. Under conditions of oxidative stress, the reduction of GSSG by glutathione reductase may become rate-limiting, thus leading to an increase in the export of GSSG [53–55]. Filled arrows indicate enhanced formation of hydroperoxides; open arrows indicate supply of metabolites counteracting oxidative stress.

lular GSSG level and that MRP-mediated GSSG export may serve as a mechanism to compensate oxidative stress when GSSG reductase becomes rate-limiting (Fig. 2). It is consistent with this view that oxidative stress enhances the expression of MRP1 in cultured cells [56]. Mutants deficient in MRP1 or MRP2 Mice lacking the MRP1 protein have been generated by embryonic stem cell technology [57]. These animals are deficient in the release of LTC4 from leukotrienegenerating cells and lack ATP-dependent transport of glutathione S-conjugates across the erythrocyte plasma membrane [57]. These findings provide excellent support and confirmation of the function of MRP1 identified previously [15–19]. MRP2 deficiency is the basis of the Dubin-Johnson syndrome in humans [58] and several mutations in the coding sequence of the MRP2 gene have been identified which cause the absence of the MRP2 protein from the hepatocyte canalicular membrane [59,60]. The lack of functional MRP2 protein, therefore, leads to the characteristic increase of anionic conjugates, including bilirubin glucuronosides, in blood serum. Upregulation of MRP3 in the basolateral hepatocyte membrane mediates the efflux of the conjugates from hepatocytes into blood [32].

Mutant rat strains lacking the MRP2 protein because of point mutations in the coding sequence have been identified [28,61] and studied extensively [7,27,50,62]. These MRP2-deficient mutants not only lack ATP-dependent transport of glutathione S-conjugates across the hepatocyte canalicular membrane [7,27,29] but are also deficient in the hepatobiliary elimination of many anionic conjugates including LTC4 [50,62]. The mutant rats GY/TR⫺ [62] and EHBR [61] have provided much, although indirect, information on the substrate specificity of rat MRP2 [62]. Regulation of conjugate export pumps of the MRP family Regulation of MRP isoforms has been observed both on the level of localization and membrane insertion of the transport protein [63– 65] and on the level of transcriptional regulation [56,66 – 68]. For MRP1, transcriptional suppression by wild-type p53, possibly by diminishing the effect of the powerful transcription activator Sp1, has been demonstrated [67]. Therefore, a loss of wild-type p53, as it occurs in many tumors, may contribute to an upregulation of the MRP1 gene and to MRP1-mediated multidrug resistance [67]. It will be of interest to elucidate in further detail the regulatory sequences of the MRP1 gene involved in this process [67],

Glutathione S-conjugate pumps

as well as the regulatory sequences mediating the upregulation of MRP1 expression during oxidative stress [56]. A manifold upregulation of MRP3 expression has been described in rat liver in obstructive jaundice induced by bile duct ligation and in the MRP2-deficient mutant rat EHBR [68]. A similar upregulation may occur in the liver of patients with Dubin-Johnson syndrome [32]. At present, the signal for this upregulation of MRP3 has not been identified. Transcriptional upregulation of rat MRP2 gene expression by 2-acetylaminofluorene and cisplatin has been characterized by detailed studies on the 5⬘-flanking sequence of the MRP2 gene [66]. These toxic agents also lead to enhanced amounts of MRP2 protein in the hepatocyte canalicular membrane of the rat. For the rat MRP2 protein in hepatocytes, regulation by endocytic retrieval and exocytic insertion plays a significant role [63– 65]. The MRP2-mediated conjugate transport in hepatocytes is regulated by cAMP-stimulated sorting of MRP2 to the apical membrane [65]. Furthermore, osmosensitive reversible localization of MRP2 has been established both in hepatocyte couplets and in the isolated perfused rat liver where hyperosmotic exposure retrieved MRP2 to intracellular vesicles [64]. Endocytic retrieval of rat MRP2 together with a downregulation of MRP2 gene expression is also observed in endotoxin-induced cholestasis and after bile duct ligation [63]. The endotoxin-induced decrease of MRP2 in the hepatocyte canalicular membrane explains the wellknown decrease in the excretion of MRP2 substrates into bile in endotoxemia. A more detailed understanding of the regulation of glutathione S-conjugate transport by expression of MRP genes and by regulation of protein sorting to the plasma membrane represents a challenge for research in the near future. Acknowledgements — Research in the author’s laboratory has been supported by grants from the Deutsche Forschungsgemeinschaft through SFB 352 and SFB 601, by the Tumorzentrum HeidelbergMannheim, and by the Forschungsschwerpunkt Transplantation Heidelberg. Contributions to the work by members of the Division of Tumor Biochemistry, both past and present, are gratefully acknowledged.

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ABBREVIATIONS

ABCC—symbol for ATP-binding cassette transporter of subgroup C CYPs— cytochromes P450 EHBR—Eisai hyperbilirubinemic mutant rat GSH—reduced glutathione GSSG— oxidized glutathione or glutathione disulfide GY/TR⫺—Groningen yellow/transport-deficient mutant rat LTC4—leukotriene C4 MRP—multidrug resistance protein or multidrug resistance-associated protein MRP2—multidrug resistance protein 2 or apical multidrug resistance protein, also termed canalicular isoform of MRP (cMRP), or canalicular multispecific organic anion transporter (cMOAT) PAPS—3⬘-phosphoadenylylsulfate UDPGlcUA—UDPglucuronate