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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS
Virginia Commonwealth University, Medical College of Virginia Campus, Department of Pharmaceutics, Richmond, Virginia (P.M.G.); and University of Kentucky, Graduate Center for Toxicology, Lexington, Kentucky (P.M.G., W.L., V.M., M.V.)
Received August 5, 2006; accepted November 14, 2006.
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Abstract |
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-estradiol 17-(-D-glucuronide) (E217G) and -estradiol 3-(-D-glucuronide) (E23G) as substrates. MRP2 transported E23G via classic Michaelis-Menten kinetics (Km = 122 µM; Vmax = 3.0 nmol/mg/min), whereas E217G transport showed positive cooperativity (Hill slope, 2.15; Km = 75 µM; Vmax = 3.8 nmol/mg/min). UDC, tauroursodeoxycholate, and glycoursodeoxycholate (80–100 µM) maximally stimulated E23G transport 9-, 7.9-, and 3.6-fold, respectively, whereas higher concentrations (1–2 mM) inhibited transport. At low (0.3 µM) concentrations, tauroursodeoxycholate was transported only in the presence of E217G or E23G, but not other MRP2 substrates such as methotrexate, leukotriene C4, or S-methylglutathione. Kinetic analysis of higher concentrations of tauroursodeoxycholate transport by MRP2 showed positive cooperativity (Hill slope, 1.84; Km = 127 µM; Vmax = 779 pmol/mg/min). Taurocholate (2–100 µM) was not detectably transported by MRP2 either alone or in the presence of E217G but was transported in the presence of E23G. Thus, UDC, tauroursodeoxycholate, and glycoursodeoxycholate activated MRP2 transport. Tauroursodeoxycholate was transported by MRP2 and demonstrated positive cooperativity, identifying it as the second MRP2 substrate able to stimulate its own transport. The data suggest MRP2 binding sites that can require specific complementarities between substrates and modulators of MRP2-mediated transport.
; Leslie et al., 2001; Maki et al., 2003; Zelcer et al., 2003). Transport activation has been suggested to occur through allosteric activation, in which the activating agent is not transported, or by a cotransport mechanism. For example, ethinyl-estradiol-3-sulfate allosterically activates MRP2 and MRP3 transport activity but is not transported by MRP2 or MRP3 (Chu et al., 2004). In contrast, sulfinpyrazone stimulates MRP2-mediated 2,4-dinitrophenyl-S-glutathione transport, can be co-transported with glutathione by MRP2, or can be transported by MRP2 in the absence of reduced glutathione at high concentrations (Evers et al., 1998, 2000). Glycocholate and certain bile salts also activate MRP2-mediated transport of -estradiol 17-(-D-glucuronide) (E217G) (Bodo et al., 2003). Recently, Borst et al. (2006) have suggested several models that explain transport activation, favoring a model in which a broad-based binding site binds multiple ligands, thus enabling cooperative and/or competitive ligand interactions.
We recently found that ursodeoxycholate (ursodiol, UDC) can directly interact with MRP2 in vitro to activate its transport activity (Gerk et al., 2003). This finding holds special interest because of the therapeutic utility of UDC in treating cholestatic liver disease, including primary biliary cirrhosis and primary sclerosing cholangitis. UDC is also used off-label to treat other cholestatic liver diseases, including intrahepatic cholestasis of pregnancy (Paumgartner and Beuers, 2002). UDC is conjugated with glycine or taurine in the hepatocyte to glycoursodeoxycholate (GUDC) and tauroursodeoxycholate (TUDC), respectively, and these hydrophilic bile salts become a major portion of the bile salt pool upon chronic dosing with UDC. UDC protects cholangiocytes against the detergent and toxic actions of hydrophobic bile salts and protects against apoptosis induced by bile salts; it also functions to stimulate impaired hepatobiliary secretion. This latter mechanism occurs through several modes, including transcriptional up-regulation of expression of the hepatic efflux transporters bile salt export pump (Bsep) and Mrp2 and stimulation of their insertion into the canalicular membrane (particularly by TUDC) that appears dependent on Ca2+- and protein kinase C (PKC)
-dependent mechanisms (Paumgartner and Beuers, 2002).
E217G is a naturally occurring estrogen metabolite that is used widely as a model organic anion substrate, particularly for MRP2. E217G is also cholestatic, decreasing bile flow acutely and reversibly in rats (Vore, 1987); Mrp2-mediated transport of E217G is essential for its induction of cholestasis (Huang et al., 2000). E217G cholestasis is due to the retrieval of the canalicular transporters Mrp2 and Bsep into subapical vesicles (Mottino et al., 2002; Crocenzi et al., 2003) that may be triggered through its trans-inhibition of Bsep (Stieger et al., 2000). Mrp2 also transports glutathione into bile, and much of the bile salt-independent component of bile flow is attributable to the osmotic action of glutathione (Ballatori and Rebbeor, 1998). Thus, modulation of MRP2 expression and activity has important potential in treating cholestatic diseases, particularly in pregnancy, where accumulation of estrogen glucuronides is thought to contribute to decreased bile secretory function. TUDC is effective in overcoming E217G-induced cholestasis without having the adverse effects of the more hydrophobic taurocholate (Utili et al., 1990).
The purpose of the present studies was to investigate the functional nature of the activation mechanism of bile salts on MRP2 transport activity. To address this issue, we overexpressed MRP2 in Sf9 insect cells and examined MRP2 transport activity in plasma membrane vesicles, in which the inside-out membrane vesicles may access ATP. As substrates, we used E217G and its regioisomer, -estradiol 3-(-D-glucuronide) (E23G), which we recently showed to be an excellent substrate for rat Mrp2 (Gerk and Vore, 2004). The results show that a broad range of bile salts can activate MRP2 and, importantly, that TUDC is transported by MRP2 and stimulates its own transport, as evidenced by positive cooperativity. Furthermore, the presence of estrogen glucuronides also stimulates MRP2-mediated transport of low concentrations of TUDC. These data imply that certain pairs of compounds, including bile salts, can be bound and transported in tandem by MRP2.
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Materials and Methods |
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97%. Unlabeled estrogen conjugates and bile salts and all other reagents were obtained from Sigma (St. Louis, MO) or Calbiochem (La Jolla, CA); purity was verified by highperformance liquid chromatography (Roda et al., 1992). All restriction enzymes were obtained from Invitrogen (Carlsbad, CA) except BssHII (Fisher, Pittsburgh, PA) and AvrII (New England Biolabs, Beverly, MA). The plasmid (pEF6/V5-His-TOPO; Invitrogen) containing the reference MRP2 sequence (NM_000392
[GenBank]
) was a generous gift from Dr. Richard Kim (Vanderbilt University, Nashville, TN). The plasmid was sequentially digested with SalI, NotI, and AvrII to obtain a 4.3-kb fragment (5'-AvrII-NotI-3'), lacking the 5' fragment (320 bases), a region of the MRP2 coding sequence including the start codon and the AvrII site. The 5' fragment was amplified by polymerase chain reaction using the forward and reverse primers 5'-ACCGCGCGCGAATCATGCTGGAGAAGTTCTGC-3' and 5'-GATCAGCAAAACCAGGAGC-3', respectively. The 365-bp product and the pFastBac1 vector (Invitrogen) were sequentially digested with BssHII/AvrII and BssHII/NotI, respectively, isolated, and recovered. The vector and the two MRP2 fragments were ligated using T4 ligase (Promega, Madison, WI), transformed into XL-10 Gold cells, and colonies were selected by digesting miniprep DNA (Qiagen, Valencia, CA) with BamHI to find a colony yielding 0.4-, 2.5-, and 7-kb fragments. DNA from the chosen colonies was partially sequenced to verify the correct amplification and insertion of the 5' MRP2 fragment and transformed into DH10Bac Escherichia coli cells (Invitrogen) to recombine MRP2 into the baculovirus genome according to the manufacturer's instructions. The recombinant bacmids were amplified, sequenced (Elim Biopharmaceuticals, Hayward, CA) to confirm the presence (or absence) of MRP2, and transfected using Cell-Fectin (Invitrogen) into Sf9 insect cells. The supernatant containing the recombinant baculovirus was harvested, amplified, and titered by a viral plaque assay. Infection conditions in Sf9 cells were optimized with respect to MRP2 protein expression and transport activity. Sf9 cells (5 x 108 cells) in suspension culture were infected in the presence of 5% fetal bovine serum using a multiplicity of infection of six, and 48 h later, plasma membrane vesicles were isolated and stored as described previously (Ito et al., 2001b; Gerk et al., 2004). Expression of MRP2 was determined by Western blotting using 0.5 µg of membrane protein as described for rat Mrp2 (Gerk et al., 2004).
Membrane vesicles from Sf9 cells are a mixture of right side out and inside-out (ABC domain facing the extravesicular buffer) orientations; approximately 65% are inside out, permitting transport into the vesicles to occur in the presence of ATP (van Aubel et al., 1998). Transport experiments were performed in a Tris-sucrose buffer (Ito et al., 2001b), containing 5 mM ATP or AMP, 10 mM MgCl2, 10 mM phosphocreatine, 100 µg/ml creatine phosphokinase, and unlabeled estrogen conjugates in dimethylsulfoxide (0.5%) as a vehicle. ATP-dependent transport of [3H]E217G, [3H]E23G, [3H]TUDC, or [3H]TC into membrane vesicles (10 µg/20 µl) was measured in incubations at 37°C for 2 to 5 min, transport stopped with 3.5 ml of ice-cold stop buffer (Ito et al., 2001b), and the mixture was quickly filtered onto Durapore 0.4-µm filters (Millipore, Bedford, MA). The filters were selected due to their minimal binding of E217G at low (90 nM) or high (100 µM) concentrations and relatively low binding of bile salts. The filters were processed, and 3H was detected by liquid scintillation counting, as described previously (Gerk et al., 2004).
Transport corrected for that in the presence of AMP was termed ATP-dependent transport, whereas that corrected for background (EV) transport was termed MRP2-mediated transport (Gerk et al., 2004). Nonlinear regression was performed on saturation data by fitting the unweighted data to the Hill equation. Other curves were unweighted for linear or nonlinear regression using Prism version 4 computer software (GraphPad, San Diego, CA) for fitting as indicated in figure legends. Data for Fig. 4, A and B, were fitted to eq. 1 below:
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= 0.05) followed by Dunnett's multiple comparison test.
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), transport of E23G by human MRP2 was linear with respect to incubation time (Fig. 2A); furthermore, linearity with respect to membrane protein (5–20 µg) and osmotic sensitivity were established (data not shown). MRP2 transported the novel substrate E23G via classic Michaelis-Menten kinetics (Km = 122 ± 23 µM; Vmax = 2.99 ± 0.19 nmol/mg/min; Hill slope = 1), as shown in Fig. 2B.
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) (data not shown).
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MRP2-mediated transport of E217G has been shown to stimulate its own transport (homotropic activation). Kinetic analysis of E217G transport over a broad concentration range (1–250 µM) was saturable, with evidence of positive cooperativity (Hill slope = 2.1 ± 0.3; Km = 75 ± 7 µM; Vmax = 3.8 ± 0.2 nmol/mg/min), consistent with multiple interacting E217G binding sites (Fig. 5). To characterize the influence of UDC on the positive cooperativity of E217G transport, we used a low concentration (30 µM) of UDC that activated E23G transport and a high concentration (1000 µM) that inhibited E23G transport. UDC (30 µM) activated MRP2 but increased the apparent Km and Vmax estimates to 360 ± 120 µM and 13 ± 2 nmol/mg/min, respectively; positive cooperativity was abolished as the Hill slope was decreased to 1.0 ± 0.1. In contrast, 1000 µM UDC competitively inhibited E217G transport (Km = 140 ± 14 µM; Vmax = 3.5 ± 0.2 nmol/mg/min), with a decrease in the Hill slope (1.2 ± 0.1) (Fig. 5).
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Nonsulfated and nonglucuronidated bile salts, as monoanionic bile salts, have been shown not to be transported by MRP2 (Stieger et al., 2000). However, their ability to stimulate MRP2 transport could be explained by an apparent cotransport model, as proposed for sulfinpyrazone and glutathione (Evers et al., 2000) and for glycocholate and E217G (Bodo et al., 2003). We therefore tested the ability of MRP2 to mediate transport of taurocholate and TUDC. Data are reported as the clearance, or flux divided by concentration, which permits comparison of initial MRP2 transport activities for substrates of differing specific activities. [3H]taurocholate (2–100 µM) was not transported alone or in the presence of E217G (20 µM); surprisingly, [3H]taurocholate (2 µM) was transported to a small extent in the presence of E23G (50 µM) (10.4 ± 1.9 µl/mg/min, mean ± S.D.). MRP2 did not significantly transport [3H]TUDC (0.3 µM) alone or in the presence of S-methylglutathione (3 mM, a nonreducing glutathione analog; Rius et al., 2003) or the MRP2 transport substrates leukotriene C4 (1 µM), taurolithocholate-3-sulfate (10 µM), and methotrexate (100 µM) (Table 1). However, in the presence of E217G (20 µM), E23G (50 µM), or at a higher concentration of TUDC (100 µM), significant transport of [3H]TUDC was observed (Table 1). The clearance of [3H]TUDC in the presence of E23G (14.4 ± 1.9 µl/mg/min) thus approached the MRP2-mediated clearance of E23G (20–25 µl/mg/min).
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To verify MRP2-mediated transport of TUDC, we demonstrated that [3H]TUDC transport was linear with time (Fig. 6A) and occurred into an osmotically sensitive space (Fig. 6B), consistent with transport rather than binding. [3H]TUDC transport was saturable and best fit to the Hill equation (Fig. 6C), yielding Vmax of 779 ± 106 pmol/mg/min and Km of 127 ± 26 µM, and showed clear evidence of positive cooperativity (Hill slope = 1.84 ± 0.36). These data indicate that TUDC is a substrate of MRP2 and is also able to stimulate its own transport.
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Discussion |
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). The effects of UDC, TUDC, and GUDC on transport were biphasic. At low concentrations (
100 µM), these bile salts strongly increased MRP2-mediated transport activity; however, at higher concentrations, these bile salts decreased transport activity, even below baseline values, as seen with GUDC. This latter effect is consistent with competitive inhibition because UDC increased the Km for E217G transport, without decreasing Vmax (Fig. 2D). These data indicate that UDC and its amidates are both able to stimulate transport and to compete with transport substrates at one (or more) site(s) on MRP2. The effects of TUDC, GUDC, and UDC on MRP2-mediated transport of E23G were qualitatively similar, with TUDC having the greatest maximal activation. These effects are unlikely to reflect nonspecific perturbations on membranes since TUDC does not directly stabilize membranes (Heuman et al., 1996), and neither 100 µM nor 2 mM TUDC significantly altered sucrose permeability under the present experimental conditions (P. M. Gerk, unpublished data). The ability to competitively inhibit MRP2-mediated transport suggested that these monoanionic bile salts might therefore also be MRP2 substrates. Consistent with these findings, we showed directly that TUDC is an MRP2 substrate.
These data are consistent with the current model of multiple overlapping binding sites on MRP2 (Ito et al., 2001a; Bodo et al., 2003; Zelcer et al., 2003), where transport activity can be markedly stimulated by numerous endogenous and exogenous compounds. Thus, taurocholate, glycocholate, glycochenodeoxycholate, taurochenodeoxycholate, and taurodeoxycholate can activate transport of E217G by MRP2 expressed in Sf9 cell membrane vesicles; glycocholate may also be transported in the presence of E217G (Bodo et al., 2003; Zelcer et al., 2003). Here, we examined a range of concentrations of UDC, TUDC, and GUDC to provide an in-depth mathematical assessment of the nature of the binding and to obtain good fits to a pharmacologically meaningful model. We also corrected for endogenous transport (i.e., transport in EV membranes) to obtain more accurate kinetic parameters of the modulation of MRP2-mediated transport. The present data confirm the previous studies (Bodo et al., 2003; Zelcer et al., 2003) and provide additional insight regarding the mechanism of activation of MRP2 by the bile salts.
The literature is replete with examples of compounds that stimulate MRP2 transport, whereas the number of substrates whose transport can be stimulated is more restricted. To date, E217G is the only substrate also able to stimulate its own transport; the present data clearly identify TUDC as a second such substrate. Borst et al. (2006) have proposed a model whereby compounds can be transported and/or stimulate MRP2-mediated transport. In this model, MRP2 contains a substrate binding site (S) to which a substrate binds and is transported. An additional site can accommodate compounds (M) that modulate/stimulate transport at the S site, thus leading to heterotropic activation. If the substrate can also bind the modulatory site, it can stimulate its own transport, termed homotropic activation, which is detected as positive cooperativity, as seen here for TUDC. The present data require a more complex model in that some MRP2 substrates (e.g., methotrexate, leukotriene C4) must bind to a distinct site(s) that does not participate in the interaction between these bile salts and the estrogen glucuronides. The present data also indicate that there are specific complementarities between modulators and substrates such that a modulator can stimulate transport of some substrates, but not others. Thus, E23G does not stimulate its own transport but does stimulate that of taurocholate and TUDC. Although E217G can stimulate its own transport and that of TUDC, it does not stimulate transport of taurocholate.
The physical nature of the modulatory and substrate binding sites is not known, and it is difficult to predict whether modulators and substrates bind to sites spatially relatively far apart or very close, such as in different areas of a single large site. Three ligand molecules bind simultaneously in a single large binding site of the E. coli multidrug efflux pump AcrB due to their aromatic stacking (Yu et al., 2003). In a potentially analogous manner, our data show that estrogen glucuronides and amidated bile salts can bind simultaneously to MRP2 site(s), resulting in a fully occupied transporter that then operates more efficiently.
Although MRP2 has classically been considered unable to transport bile salts containing a single anionic charge, e.g., taurocholate (Stieger et al., 2000), other members of the Mrp family of transporters, i.e., Mrp3 and MRP4, transport taurocholate (Ito et al., 2001b; Rius et al., 2003). Site-directed mutagenesis studies of Mrp2 demonstrated that cationic Arg residues in transmembrane domains 11 (Arg586) and 14 (Arg1096) prevent Mrp2-mediated transport of TC; mutation of these Arg to a neutral amino acid such as Leu led to acquisition of TC transport activity (Ito et al., 2001b). Conversely, substitution of Leu at the comparable 1084 of rat Mrp3 with Lys resulted in the loss of TC transport activity. MRP4 also transports TC, but only in the presence of glutathione or its analogs (Rius et al., 2003). These authors further demonstrated that TC stimulated transport of [3H]glutathione. UDC competitively inhibited MRP4-mediated TC transport in the presence of S-methyl-glutathione, whereas cGMP and cAMP, MRP4 substrates, had no effect, suggesting that bile salts and the cyclic nucleotides are transported from distinct transport sites. Taken together, these data indicate that subtle but important differences in the substrate binding sites for MRP2, Mrp3, and MRP4 confer the ability to transport bile salts.
UDC stimulates hepatobiliary secretion in several model systems (Paumgartner and Beuers, 2002). Treatment of mice or rats with UDC increases expression of Bsep and Mrp2 mRNA; however, other bile salts have similar effects (Fickert et al., 2001). In addition, TUDC stimulates the exocytic insertion of carrier proteins Bsep and Mrp2 into the apical membrane of the hepatocyte, thus stimulating biliary secretion. Roles for cytosolic free intracellular calcium and translocation of the Ca2+-sensitive -isoform of PKC to hepatocyte membranes and activation of membrane-bound PKC have been demonstrated in TUDC-induced stimulation of Mrp2 transport in the cholestatic liver (Beuers et al., 2001). Our data are the first to suggest that UDC, TUDC, and GUDC increase hepatobiliary secretion by directly enhancing the transport activity of MRP2.
The present results have important implications for the understanding and treatment of cholestatic liver disease. UDC is effective in the treatment of a number of cholestatic liver diseases, such as primary biliary cirrhosis (Paumgartner and Beuers, 2002), and is effective in resolving or improving the liver function and clinical status of children with progressive familial intrahepatic cholestasis (Jacquemin et al., 1997). Early studies noted decreased clearance of bile acids following administration of UDC to subjects with Dubin Johnson Syndrome, an inherited autosomal recessive disorder characterized by the absence of functional MRP2 protein (Kawasaki et al., 1981). In a recent case study, Corpechot et al. (2006) noted a sharp increase in serum bile acid levels and no improvement in bilirubin concentration following UDC treatment of a patient with confirmed Dubin Johnson Syndrome (Corpechot et al., 2006). These data support the hypothesis of an important role for MRP2 in the biliary excretion of bile salts.
UDC is also administered to patients with intrahepatic cholestasis of pregnancy and has been shown to improve maternal symptoms, without adverse fetal effects (Mullally and Hansen, 2002). Intracellular accumulation of cholestatic estrogen glucuronide conjugates, such as E217G and estriol-16-glucuronide, has been postulated to contribute to intrahepatic cholestasis of pregnancy (Vore, 1987). E217G cholestasis is due to the retrieval of the canalicular transporters Mrp2 and Bsep (Mottino et al., 2002; Crocenzi et al., 2003) and may be facilitated by E217G trans-inhibition of Bsep (Stieger et al., 2000). UDC and its amidates could activate MRP2-mediated efflux of E217G present in the hepatocyte into bile, while simultaneously increasing bile flow, thus decreasing intracellular concentrations and preventing retrieval of MRP2 and BSEP (Mottino et al., 2002; Crocenzi et al., 2003), as well as diluting biliary concentrations of E217G so as to overcome any trans-inhibition of BSEP (Stieger et al., 2000). Although the free concentration of bile acids in the hepatocyte under physiological conditions or following UDC therapy is not known, they are thought to be extremely low (Bachrach and Hofmann, 1982), and certainly well below the maximal activation concentration of 100 µM.
In summary, the present studies demonstrate that UDC, TUDC, and GUDC stimulate MRP2-mediated transport of E23G and E217G. Importantly, MRP2 transports TUDC, and this transport demonstrates homotropic activation, as shown by a Hill coefficient significantly greater than 1. Further mechanistic studies are required to understand the structural features of MRP2 that lead to its activation, as well as clinical studies that exploit this property to develop additional therapeutic approaches for cholestatic liver disease.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: MRP, multidrug resistance protein; ABC, ATP-binding cassette; E217G, -estradiol 17-(-D-glucuronide); UDC, ursodiol (ursodeoxycholic acid); GUDC, glycoursodeoxycholate; TUDC, tauroursodeoxycholate; BSEP, bile salt export pump; PKC, protein kinase C; E23G, -estradiol 3-(-D-glucuronide); TC, taurocholate; EV, empty vector.
Address correspondence to: Dr. Mary Vore, University of Kentucky, Graduate Center for Toxicology, Room 306 HSRB, Lexington, KY 40536-0305. E-mail: maryv{at}uky.edu
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