Transport of Bile Acids, Sulfated Steroids, Estradiol 17- -D- Glucuronide, and Leukotriene C4 by Human Multidrug Resistance Protein 8 (ABCC11)

We previously determined that expression of human multidrug resistance protein (MRP) 8, a recently described member of the MRP family of ATP-binding cassette transporters, enhances cellular extrusion of cyclic nucleotides and confers resistance to nucleotide analogs (J Biol Chem 278:29509–29514, 2003). However, the in vitro transport characteristics of the pump have not been determined. In this study, the substrate selectivity and biochemical activity of MRP8 is investigated using membrane vesicles prepared from LLC-PK1 cells transfected with MRP8 expression vector. Expression of MRP8 is shown to stimulate the ATP-dependent uptake of a range of physiological and synthetic lipophilic anions, including the glutathione S-conjugates leukotriene C4 and dinitrophenyl S-glutathione, steroid sulfates such as dehydroepiandrosterone 3-sulfate (DHEAS) and estrone 3-sulfate, glucuronides such as estradiol 17-Dglucuronide (E217 G), the monoanionic bile acids glycocholate and taurocholate, and methotrexate. In addition, MRP8 is competent in the in vitro transport of cAMP and cGMP, in accord with the results of our previously reported cellular studies. DHEAS, E217 G, and methotrexate were transported with Km and Vmax values of 13.0 0.8 M and 34.9 9.5 pmol/mg/min, 62.9 12 M and 62.0 5.2 pmol/mg/min, and 957 28 M and 317 17 pmol/mg/min, respectively. Based upon the stimulatory action of DHEAS on uptake of E217 G, the attenuation of this effect at high DHEAS concentrations and the lack of reciprocal promotion of DHEAS uptake by E217 G, a model involving nonreciprocal constructive interactions between some transport substrates is invoked. These results suggest that MRP8 participates in physiological processes involving bile acids, conjugated steroids, and cyclic nucleotides and indicate that the pump has complex interactions with its substrates. Investigations of members of the MRP family of ATPbinding cassette transporters have revealed a group of energy-dependent efflux pumps that are able to confer resistance to anticancer agents and transport a striking range of structurally diverse amphipathic anions (Kruh and Belinsky, 2003; Haimeur et al., 2004). Despite their conformity with respect to the transport of lipophilic anions, there are differences in the substrate ranges and functions of these pumps. MRP1 is a ubiquitous efflux pump for glutathione and glucuronate conjugates and plays a specific role in the extrusion of leukotriene C4 (LTC4) from mast cells (Leier et al., 1994; Jedlitschky et al., 1996; Loe et al., 1996). The substrate range of MRP2 is similar to that of MRP1, but the former pump, by virtue of its expression in canalicular (apical) membranes of hepatocytes, is responsible for the extrusion of glutathione, bilirubin glucuronide, and a variety of pharmaceutical agents into the bile (Ito et al., 1998; Cui et al., 1999; This work was supported in part by National Institutes of Health Grants CA73728 (to G.D.K.) and CA06927 and by an appropriation from the Commonwealth of Pennsylvania. Z.-S.C. is a recipient of a W. J. Avery Fellowship from the Fox Chase Cancer Center. Y.G. received support from Training Grant CA075266. 1 Current address: College of Pharmacy and Allied Health Professions, St. John’s University, New York, NY 11439. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.007138. ABBREVIATIONS: MRP, multidrug resistance protein (MRP1-MRP8, gene symbols ABCC1–ABCC6, ABCC10, and ABCC11; MOAT-B, MOAT-C, MOAT-D, and MOAT-E are alternative names for MRP4, MRP5, MRP3, and MRP6, respectively); LTC4, leukotriene C4; GC, glycocholate; TC, taurocholate; MTX, methotrexate; DHEAS, dehydroepiandrosterone 3-sulfate; DNP-SG, S-(2,4-dinitrophenyl)glutathione; E13S, estrone 3-sulfate; E217 G, 17 -estradiol 17-( -D-glucuronide); DHEA, dehydroepiandrosterone; E1, estrone; PGE1, prostaglandin E1; PGE2, prostaglandin E2; DHEAG, dehydroepiandrosterone 3-glucuronide; E13 G, estrone 3-( -D-glucuronide); E2, 17 -estradiol; E23 G, 17 -estradiol 3-( -D-glucuronide); E23 G17S, 17 -estradiol 3-( -D-glucuronide) 17-sulfate; E23S, 17 -estradiol 3-sulfate; E23S17 G, 17 -estradiol 3-sulfate 17-( -D-glucuronide); E3, estriol; E33 G, estriol 3-( -D-glucuronide); E33S, estriol 3-sulfate; E316 G, estriol 16-( -D-glucuronide); HEK, human embryonic kidney; PBS, phosphate-buffered saline; MK571, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid. 0026-895X/05/6702-545–557$20.00 MOLECULAR PHARMACOLOGY Vol. 67, No. 2 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 7138/1192670 Mol Pharmacol 67:545–557, 2005 Printed in U.S.A. 545 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from Kawabe et al., 1999; Gerk and Vore, 2002). MRP3, in addition to being able to transport glutathione and glucuronate conjugates, is also able to mediate the transport of the monoanionic bile constituents glycocholate (GC) and taurocholate (TC) (Hirohashi et al., 1999, 2000; Zeng et al., 2000). MRP4 and MRP5, which are structurally distinct from MRP1, MRP2, and MRP3, in that MRP4 and MRP5 possess two membrane spanning domains, whereas the latter proteins have three, have somewhat distinct substrate ranges (Belinsky et al., 1998). These two pumps are able to transport cAMP and cGMP (Jedlitschky et al., 2000; Chen et al., 2001; van Aubel et al., 2002; Wielinga et al., 2003), whereas cyclic nucleotides are not known to be transport substrates of the larger members of the family. Whereas MRP4 and MRP5 have capabilities that are not possessed by MRP1, MRP2, and MRP3, in certain respects the substrate range of at least MRP4 overlaps with those of the latter pumps. As is the case for MRP1, MRP2, and MRP3, MRP4 is able to mediate the transport of glutathione and glucuronate conjugates, folates, and methotrexate (MTX) (Chen et al., 2001, 2002). In addition, like MRP1, MRP4 is able to transport certain sulfated steroids, such as dehydroepiandrosterone 3-sulfate (DHEAS) and estrone 3-sulfate (E13S) (Qian et al., 2001; Zelcer et al., 2003b), and like MRP3, MRP4 has the facility for transporting monoanionic bile acids (Rius et al., 2003). Reports on MRP6 and MRP7, both of which possess three membrane spanning domains, indicate that MRP6 is able to transport glutathione conjugates and certain amphipathic cyclopentapeptides, and MRP7 has the facility for transporting glucuronate and possibly glutathione conjugates (Madon et al., 2000; Belinsky et al., 2002; Ilias et al., 2002; Chen et al., 2003a). Accumulating evidence indicates that certain MRPs have cooperative interactions with some of their substrates that may be consequent to the presence of multiple binding sites. The potential for MRPs to be activated in this manner was first raised in the context of the plant MRP AtMRP2, for which mutual stimulation by S-(2,4dinitrophenyl)glutathione (DNP-SG) and E217 G was described in a detailed study in which it was concluded that this activity was attributable to a mechanism other than cotransport from a common substrate binding site (Liu et al., 2001). More recent studies on mammalian MRPs provided additional support for cooperative interactions. MRP2-mediated transport of E217 G and bile acids was reported to exhibit mutual stimulation, and E217 G transport was also subject to stimulation by a range of compounds, including sulfanitran and sulfinpyrazone (Bodo et al., 2003; Zelcer et al., 2003a). In addition, MRP3-mediated transport of E217 G was reported to be stimulated by the sulfate conjugates of E3040, a benzothiazole derivative, and ethinylestradiol, a synthetic estrogen (Akita et al., 2002; Chu et al., 2004). Furthermore, in several instances, the agents that stimulated transport by MRP2 and MRP3 were not themselves susceptible to transport. Although the preponderance of evidence for how glutathione stimulates transport of uncharged lipophiles such as vincristine supports a working model involving cotransport from a common bipartite binding site composed of separate interacting surfaces for lipophilic and anionic moieties (for review, see Kruh and Belinsky, 2003), it has more recently been appreciated that glutathione can also stimulate transport of lipophilic anions, such as E13S and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol-glucuronide in the case of MRP1 and GC in the case of MRP4 (Leslie et al., 2001; Rius et al., 2003). This suggests the possibility that glutathione may have an additional binding site in MRP1 and MRP4 that is topologically distinct from the transport substrate binding pocket. MRP8 is a recently identified MRP family member whose structure resembles MRP4 and MRP5 with respect to possessing only two membrane spanning domains (Bera et al., 2001; Tammur et al., 2001; Yabuuchi et al., 2001). We previously determined that ectopic expression of MRP8 in LLC-PK1 cells enhances cellular efflux of cyclic nucleotides and confers resistance to certain anticancer and antiviral nucleotide analogs (Guo et al., 2003). However, the substrate range of the pump has not been determined and its potential physiological functions are largely unknown. Here, we investigate the substrate selectivity and biochemical activity of MRP8 using membrane vesicles prepared from LLC-PK1 cells transfected with MRP8 expression vector. It is shown that MRP8 is not only able to catalyze the ATP-energized transport of cyclic nucleotides but also that it is able to mediate the transport of a range of lipophilic anions, including the glutathione conjugate LTC4, sulfated steroids such as DHEAS and E13S, glucuronides such as 17 -estradiol 17-( -D-glucuronide) (E217 G), the bile constituents GC and TC, and monoglutamates such as MTX. Based upon the stimulatory action of DHEAS on uptake of E217 G, the attenuation of this effect at high DHEAS concentrations and the lack of reciprocal promotion of DHEAS uptake by E217 G, a model involving nonreciprocal constructive interactions between some transport substrates is invoked. These results suggest that MRP8 is involved in physiological processes involving bile acids, conjugated steroids, and cyclic nucleotides and indicate that the pump has complex interactions with its substrates. Materials and Methods Materials. [H]E217 G (45.0 Ci/mmol), [ H]dehydroepiandrosterone (DHEA; 74.0 Ci/mmol), [H]DHEAS (74.0 Ci/mmol), [H]LTC4 (130 Ci/mmol), [ H]estrone (E1; 65 Ci/mmol), [H]E13S (46 Ci/mmol), [H]TC (2.0 Ci/mmol), and [C]chenodeoxycholic acid (0.049 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). [H]cGMP (6.8 Ci/mmol), [H]cAMP (17 Ci/mmol), [H]MTX (23 Ci/mmol), [H]MTX-Glu2 (15 Ci/mmol), [H]MTX-Glu3 (17 Ci/mmol), and [H]folic acid (42 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA). [C]GC (0.056 Ci/mmol), [H]prostaglandin E1 (PGE1; 48 Ci/ mmol), and [H]prostaglandin E2 (PGE2; 187 Ci/mmol) were purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). [H]Cholic acid (25 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Creatine kinase, creatine phosphate, ATP, AMP, 1-chloro-2,4dinitrobenzene, unlabeled E217 G, LTC4, cGMP, cAMP, DHEA, dehydroepiandrosterone 3-glucuronide (DHEAG), DHEAS, E1, E13S, GC, TC, chenodeoxycholate, folic acid, PGE1, PGE2, estrone 3-( -D-glucuronide) E13 G, E13S 17 -estradiol (E2); 17 -estradiol 3-( -D-glucuronide) (E23 G), 17 -estradiol 3-( -D-glucuronide) 17sulfate (E23 G17S), 17 -estradiol 3-sulfate (E23S), 17 -estradiol 3-sulfate 17-( -D-glucuronide) (E23S17 G), estriol (E3), estriol 3-( -D-glucuronide) (E33 G), estriol 3-sulfate (E33S), and estriol 546 Chen et al. at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from 16-( -D-glucuronide) (E316 G) were purchased from SigmaAldrich (St. Louis, MO). Unlabeled MTX and MTX polyglutamates were purchased from Schircks Laboratories (Jona, Switzerland). DNP-SG and [H]DNP-SG were synthesized from 1-chloro-2,4dinitrobenzene and unlabeled or labeled [H]glycine-2-glutathione (44.8 Ci/mmol; PerkinElmer Life and Analytical Sciences), as described previously (Awasthi et al., 1981). LLC-PK1-MRP8-3 and control LLC-PK1-pcDNA cells were described previously (Guo et al., 2003). HEK293 cells transduced with MRP4 and control HEK293 cells were kindly provided by Piet Borst (Netherlands Cancer Institute, Amsterdam, The Netherlands). LLC-PK1 and HEK293 cells, respectively, were grown in M-199 medium or Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine. Preparation of Membrane Vesicles. Membrane vesicles were prepared by the nitrogen cavitation method as described previously (Cornwell et al., 1986). Cells were washed with PBS and then scraped into PBS containing 1% aprotinin. Cells were then washed at 4°C in PBS, collected by centrifugation (4000g for 10 min), suspended in buffer A (10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM p-amindinophenylmethanesulfonylfluoride, and 0.2 mM CaCl2) and equilibrated at 4°C for 15 min under a nitrogen pressure of 500 psi. EDTA was added to the suspension of lysed cells to a final concentration of 1 mM, and the suspension was then diluted 1:4 with buffer B (10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, and 1 mM p-amindinophenylmethanesulfonylfluoride) and centrifuged at 4000g for 10 min at 4°C to remove nuclei and unlysed cells. The supernatant was layered onto a sucrose cushion (35% sucrose, 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA) and centrifuged for 30 min at 16,000g at 4°C. The interface was collected and centrifuged for 45 min at 100,000g for 4°C. The pellet was resuspended in buffer B by repeated passage through a 25-gauge needle. Protein concentration was determined by the method of Bradford (Bradford and Ward, 1976). Vesicles were