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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS
Departments of Biopharmaceutical Sciences (N.A., T.N., L.W.C., L.L, K.M.G., D.L.K) and Pharmaceutical Chemistry (C.C.H, S.J.J., M.K., D.S., T.R.T., T.E.F.), University of California, San Francisco, California
Received January 14, 2008; accepted March 24, 2008.
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; Adachi et al., 2002; Anderson et al., 2006; Imaoka et al., 2007), anticancer drugs [methotrexate, 6-mercaptopurine, 6-thioguanine, camptothecins (Lee et al., 2000; Chen et al., 2002; Wielinga et al., 2002; Leggas et al., 2004; Tian et al., 2005)], as well as endogenous molecules, such as prostaglandins, steroids, bile acids, cyclic nucleotides, and folate (Chen et al., 2002; van Aubel et al., 2002; Reid et al., 2003; Zelcer et al., 2003; Denk et al., 2004).
MRP4 is ubiquitously expressed, with a high expression in the prostate and in hematopoietic stem cells and blood cells (Su et al., 2004) (http://symatlas.gnf.org/SymAtlas/). It is also present in the kidney proximal tubules (van Aubel et al., 2002), in the liver (Rius et al., 2003), and in the brain (Leggas et al., 2004). It is interesting that its localization in most tissues is apical, but basolateral localization has been demonstrated in brain choroid plexus epithelial cells, in prostate, and in hepatocytes (Lee et al., 2000; van Aubel et al., 2002; Rius et al., 2003; Leggas et al., 2004). Similar to most efflux transporters, no disease has been directly linked to altered MRP4 activity. Recently, a study with Abcc4-/- mice showed that the absence of this transporter did not induce obvious abnormalities. However, it resulted in acute PMEA toxicity, suggesting a protective role of MRP4 in the bone marrow, spleen, thymus, and gastrointestinal tract. Moreover, these data suggested that MRP4 may reduce the passage of PMEA and probably other nucleotide analogs into the brain (Belinsky et al., 2007). This is in agreement with a previous report stating that Abcc4-/- mice accumulated more topotecan in the brain and cerebrospinal fluid than wild-type mice (Leggas et al., 2004). Therefore, its physiological role could include detoxification of drugs, as well as that of endogenous molecules. With respect to endogenous substrates, up-regulation of MRP4 in the liver of rats and humans with obstructive cholestasis provides a mechanism to eliminate excess bile salts (Denk et al., 2004; Gradhand et al., 2008).
Although ABCC4 is a highly polymorphic gene (Saito et al., 2002), few data are available concerning the function of its variants. Recent studies have investigated the functional effects of several ABCC4 single-nucleotide polymorphisms (SNPs) on drug disposition. Anderson et al. (2006) showed a 20% increase in lamivudine-triphosphate intracellular concentrations in patients carrying the 4131T>G variant, whereas the 3724G>A variant was associated with a trend for elevated AZT-triphosphate, suggesting a reduced MRP4 efflux function (Anderson et al., 2006). It is interesting that the 4131T>G variant is in the 3'-untranslated region (UTR) of the gene, whereas the 3724G>A variant is synonymous and there is no clear mechanism explaining these effects. In another study, no association was observed between two nonsynonymous and seven synonymous ABCC4 variants and tenofovir disoproxil fumarate-induced renal proximal tubulopathy (Izzedine et al., 2006). Most recently, 74 genetic variants in ABCC4 were shown to have no effect on MRP4 mRNA and protein expression in Caucasian cholestatic and noncholestatic patients (Gradhand et al., 2008).
The aims of this study were to identify genetic variants of ABCC4 in a cohort of healthy individuals of different ethnic groups and to functionally characterize selected nonsynonymous variants in vitro. AZT and PMEA, the first nucleoside/nucleotide analogs reported to be substrates of MRP4 (Schuetz et al., 1999), have been selected for this study. AZT and PMEA are used in the treatment of human immunodeficiency virus and hepatitis B infections, respectively, and altered function of MRP4 might contribute to interindividual variability in the response to these antivirals.
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(the proportion of nucleotide sites that are expected to be polymorphic in a sample of sequences drawn at random from a population), nucleotide diversity 
(average proportion of nucleotide differences between all possible pairs of sequences in a sample), and Tajima's D statistic (used to detect departures from the standard neutral model) were calculated in each ethnic group for synonymous, nonsynonymous, and noncoding sites according to Tajima (Tajima, 1989, 1993).
Haplotype Analysis. Haplotypes were statistically inferred in each ethnic group using the PHASE method (Stephens et al., 2001) for variants with a minor allele frequency >5%. The analysis was run 10 times, and haplotypes estimated in at least seven runs were reported. Analyses were carried out using all variable sites of ABCC4 above the mean allele frequency cut-off. A separate analysis excluded the intronic sites.
Construction of ABCC4 Variants and Nonfunctional Mutant. The ABCC4 cDNA was cloned from human kidney using the following primers: forward 5'-AGCATCCCTGCTTGAGGTCCA-3' and reverse 5'-ACGGACTTGACATTTTGGTTGG-3'. The cDNA was originally inserted into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA), and subsequently into the pcDNA5/FRT vector (Invitrogen). This plasmid contained two synonymous ABCC4 SNPs (rs1678339 and rs1189466), which were reversed by site-directed mutagenesis to obtain the reference ABCC4. The previously described reference sequence (GenBank accession number NM_005845 [GenBank] ) contains two SNPs (rs11568681/Leu18Ile and rs1557070) with low frequencies (<10%) that are not found in our reference sequence. This reference plasmid was used as a template for constructing the 10 nonsynonymous variants selected for this study, as well as a nonfunctional mutant. Variant constructs were obtained by site-directed mutagenesis, using the Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) according to the manufacturer's protocol and the primers reported in Table 1. The mutations were confirmed by sequencing, and complete sequencing of each plasmid was also performed to check that no new mutations were introduced during the mutagenesis procedure.
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Functional Assays of MRP4 Variants. Human embryonic kidney epithelial cells transformed with SV40 T antigen (HEK 293T) were obtained from the Gladstone Institute of Virology and Immunology (San Francisco, CA). They were maintained in minimal essential Eagle's medium with Earle's balanced salt solution, supplemented with sodium pyruvate, penicillin, streptomycin, and nonessential amino acids (Cell Culture Facility, University of California, San Francisco, CA), as well as fetal bovine serum (Invitrogen). HEK 293T cells were seeded onto poly-D-lysine-coated 24-well plates (BD Biosciences Discovery Labware, Bedford, MA). The next day, the cells were transfected with 0.8 µg of DNA (ABCC4 reference, variant or nonfunctional mutant) and 1 µl of Lipofectamine 2000 (Invitrogen) in each well, according to the manufacturer's protocol. Accumulation studies were performed 24 h after transfection. Cells were washed with phosphate-buffered saline and incubated with 100 nM [3H]bis(pivaloyloxymethyl) [9-(2-phosphonylmethoxyethyl)-adenine]/[2-(6-aminopurin-9-yl)ethoxymethyl-(2,2-dimethylpropanoyloxymethoxy)phosphoryl]oxymethyl 2,2-dimethylpropoanoate (bis-POM-PMEA) or [3H]AZT (Moravek Biochemicals, Inc., Brea, CA) at 37°C. The effect of endogenous MRP4 was minimized by running the assay for 30 min for AZT and 60 min for PMEA. After 30 min of accumulation, the ratio of accumulation of AZT between transfected and untransfected cells reaches its peak, whereas the assay with PMEA needs to be longer to allow the intracellular release of PMEA from bis-POM-PMEA by esterases. For studies of accumulation as a function of concentration, higher concentrations of substrates were obtained by adding radiolabeled substrates to different concentrations of unlabeled AZT (Toronto Research Chemicals Inc., North York, ON, Canada) and bis-POM-PMEA (Moravek Biochemicals, Inc.). Accumulation was stopped by removing the substrate and washing the cells three times with ice-cold phosphate-buffered saline. The cells were lysed with 800 µl/well of an aqueous solution containing 0.1 N NaOH and 0.1% sodium dodecyl sulfate. An aliquot of the lysate (775 µl) was added to 3.25 ml of Ecolite scintillation fluid (MP Biomedicals, Irvine, CA), and intracellular amounts of the radiolabeled substrates were determined by scintillation counting. These data were then normalized to protein concentration in each well, measured using a BCA protein assay kit (Pierce Biotechnology Inc., Rockford, IL).
RNA and Protein Expression. Cells were seeded onto 12-well plates and transfected. Twenty-four hours after transfection, RNA or proteins were extracted from the cells. For RNA extraction, TRIzol reagent was added to the wells (Invitrogen), and RNA was extracted according to the manufacturer's protocol. ABCC4 mRNA was quantified in each sample by real-time reverse transcription-polymerase chain reaction (TaqMan) and compared to its basal expression in cells transfected with the empty vector (endogenous ABCC4). For protein extraction, the cells were lysed, and proteins were extracted using centrifugation. Protein concentrations were quantified by the BCA assay, and these samples were used for Western blot analysis. A monoclonal antibody to MRP4 (M4-I10) and a polyclonal antibody to rat IgG (Axxora, LLC, San Diego, CA) were used to visualize MRP4 using the Immobilon Western detection reagent (Millipore Corporation, Billerica, CA). Lysates from three separate transfections were analyzed by Western blotting, and the density of the MRP4 signal was quantified using image analysis and expressed relative to that of GAPDH.
Subcellular Localization (Immunocytochemistry). Cells were grown on poly-D-lysine-coated coverslips (BD Biosciences Discovery Labware) in a 24-well plate. They were transfected the next day and stained 24 h later. The same antibody to MRP4 that was used for Western blotting was used for immunocytochemistry. A fluorescent goat anti-rat secondary antibody (Invitrogen) was used for the detection of MRP4. Staining was visualized under a Zeiss Axioskop epifluorescence microscope.
Design of a Nonfunctional Mutant. In addition to the naturally occurring variants studied, a nonfunctional mutant was designed as a functional negative control. This mutant, G538D, has an aspartate instead of a glycine at the fourth position in the ABC signature of the first nucleotide binding domain (NBD; Fig. 1). A corresponding mutation was previously reported to lead to nonfunctional, although stable, P-glycoprotein (Bakos et al., 1997) and MRP1 (Ren et al., 2004).
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). Nonsynonymous SNPs are well distributed throughout the protein, with the majority of them located in the intracellular regions (17 of 22) (Fig. 1). Most high-frequency SNPs (>5% in at least one population) are either synonymous (9 SNPs) or noncoding (25 SNPs). Only three nonsynonymous variants (G187W, K304N, and M744V) have frequencies higher than 5% in a given ethnic group. A relatively high number of singletons was found among the nonsynonymous variants (14 of 22) compared to synonymous variants (6 of 21).
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Values for and were calculated for coding and noncoding sites in the different populations studied (Fig. 2). The same pattern of genetic variation is observed with each ethnic group, with slightly higher values for African Americans. Specifically, synonymous sites are highly variable, whereas nonsynonymous sites have very low and values. On average, the values are slightly higher for noncoding compared with coding regions. The values for the entire sequenced region in each population are in the same range as the previously reported average value of 10.4 x 104 for 24 human membrane transporters (not including ABCC4), whereas values are higher than the reported value of 5.09 x 104 (Leabman et al., 2003). The ratio between nonsynonymous and synonymous values (NS/S = 0.07) is lower than the average value for membrane transporters (0.23), indicating a high degree of negative selection (Leabman et al., 2003). The Tajima's D values associated with these parameters are negative with the exception of noncoding and synonymous sites in Asian Americans and synonymous sites in African Americans (data not shown).
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Variant Selection for Functional Studies. Ten nonsynonymous variants were chosen for this study based on a frequency of 
5% in the populations studied (G187W, K304N, and M744V), high evolutionary conservation (all variants with the exception of M744V), or a high Grantham value (G187W > C956S > P403L = G487E > K304N). Evolutionary conservation was determined by alignment of orthologous amino acid sequences from seven mammalian species. Variants referred to as "evolutionarily conserved" show a complete conservation across the seven species, with the exception of an arginine to lysine change for variant K304N in two species. The majority of these variants are localized in the intracellular region of the protein (Fig. 1).
Transport of AZT and PMEA by MRP4. Accumulation data confirmed previous reports indicating that AZT and PMEA are substrates of MRP4 (Schuetz et al., 1999; Imaoka et al., 2007). Similar to other nucleoside/nucleotide analogs, these molecules require phosphorylation for antiviral activity. AZT undergoes three sequential phosphorylations inside the cell, and its monophosphate form is effluxed by MRP4. PMEA is a monophosphate, and only two phosphorylation steps are required for its activity. PMEA is effluxed before being phosphorylated (Schuetz et al., 1999). The intracellular accumulation of both drugs was decreased when the cells were transfected with the reference ABCC4 cDNA compared to the cells transfected with the empty pcDNA5/FRT vector (
55 and 84% decrease for AZT and PMEA, respectively; Fig. 4A) due to MRP4-mediated efflux. As expected, a mutation in a highly conserved glycine of NBD1 leads to a nonfunctional MRP4. This is illustrated by the fact that the accumulation of both substrates in the cells transfected with the G538D construct is not statistically different from the vector-transfected cells (Fig. 4A). Therefore, this mutant can be used as a negative control to validate the data obtained in the functional studies and confirms that MRP4 is responsible for the decrease observed in intracellular concentrations of AZT and PMEA.
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To characterize the functional difference observed with some of the variants, we measured the intracellular accumulation of AZT and PMEA as a function of the extracellular concentration for the reference and the variants that showed a reduced function with one or both substrates (P78A, G187W, and G487E), as well as the C956S variant, which was more functional with respect to PMEA transport (Fig. 5). For each MRP4 variant or the reference protein, the efflux transport velocity was estimated at each concentration by subtracting the concentration measured in the transfected cells from the intracellular accumulation in empty vector-transfected cells. The transport velocity curves (Fig. 5) suggest that the differences reported with some of the variants (G187W and G487E) are real and concentration-independent, and this effect seems more pronounced with AZT.
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100-fold. Negligible differences were observed between the RNA levels of the ABCC4 variants and reference.
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MRP4 protein was also highly expressed in the cells, and variability in expression was observed among the variants and reference protein. The MRP4 and GAPDH signals were quantified using image analysis, and the G187W variant was expressed at a significantly lower level than that of the reference MRP4 (data not shown). None of the other differences was significant when lysates from three separate transfections were blotted and quantified. It should be noted that the MRP4 protein was detected in the empty vector control cells, with a longer exposure of the blot.
To check that the functional differences were not due to differences in localization of the variants at the cell membrane, immunocytochemistry studies were carried out with the less functional variants (G187W, G487E, and P78A), the nonfunctional mutant (G538D) and the more functional variant (C956S). The variant MRP4 proteins are all expressed at the membrane, and no significant difference in cell membrane localization can be seen between MRP4 reference and these variants (Fig. 7). A negative control with empty vector shows almost no signal with the same exposure time.
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Genetic variation at synonymous sites is high, although it is relatively low and similar to other ABC transporters at nonsynonymous sites. The low NS/S ratio suggests that this gene has a low tolerance for nonsynonymous compared to synonymous variations. This could explain why most nonsynonymous variants are rare. However, one may infer from this low ratio that a mutation affecting the protein structure would be deleterious, but this has not been confirmed clinically. Similar to most ABC efflux transporters, no disease has been associated with a nonfunctional MRP4.
This study confirms the role of MRP4 in transporting AZT and PMEA by a direct measurement of transport in cells transfected with a known ABCC4 sequence. The lipophilic ester prodrug of PMEA, bis-POM-PMEA, has been used to increase passive intracellular uptake (Srinivas et al., 1993; Hatse et al., 1998). PMEA is then rapidly released intracellularly from the prodrug by esterases (Srinivas et al., 1993) and is effluxed unchanged by MRP4 (Schuetz et al., 1999). Endogenous MRP4 protein expressed in HEK 293T could contribute to the cellular efflux. This contribution seems greater in the case of AZT, which shows a less dramatic difference between empty vector and MRP4-transfected cells than PMEA. Other transporters are probably involved in AZT and PMEA uptake and efflux [OAT1 and MRP5 for PMEA (Cihlar et al., 1999; Wijnholds et al., 2000); OAT1–4 and MXR for AZT (Takeda et al., 2002; Wang et al., 2004)], and passive diffusion of these antivirals will be determined by their lipophilicity. Differences in these alternate pathways for AZT and PMEA transport may explain the differences observed between these two substrates. MRP4 expression was also high in other cell lines, including MDCKII (Madin-Darby canine kidney II), Chinese hamster ovary-K1, HepG2, and CV-1 cells. Moreover, because MRP4 is highly expressed in the kidney (van Aubel et al., 2002), HEK 293T cells provide a realistic model for studying the function of this transporter.
MRP4-mediated transport of AZT and PMEA was observed with concentrations as low as 20 nM. Reported Cmax values are 3.7 µM following a 200-mg dose of AZT (Singlas et al., 1989) and 67 nM for a 10-mg dose of PMEA (http://www.hepsera.com). Therefore, an intermediate donor concentration of 100 nM was used in the first screen of the variants. All 10 nonsynonymous variants of MRP4 that were tested are functional. Four variants have significantly lower function, with the G187W variant displaying the greatest reduction in function. In contrast, the C956S variant had a significantly higher transport of PMEA. In all cases, a similar trend was observed with both substrates; this strongly suggests that the functional differences observed are real and substrate-independent within this same chemical group. The velocity curves can in theory be used to estimate and compare kinetic parameters (Vmax and Km) related to the transport of AZT and PMEA by MRP4 variant and reference proteins. However, the assay described in this study is an indirect measurement of the efflux and is based on a difference in accumulation between transfected and untransfected cells. The sensitivity of the analytical method did not allow direct measurement of the amount of substrate being effluxed following accumulation. Therefore, whereas the Vmax values could in theory reflect efflux by MRP4 (because the transport velocities related to MRP4 variants and reference have been normalized to empty vector transfected cells), the Km values would only be apparent and correspond to the extracellular concentration to which the cells are exposed (instead of the intracellular concentration to which the transporter is really exposed).
A mutant designed to be nonfunctional by replacing a highly conserved glycine in NBD1 with an aspartate showed no difference in intracellular accumulation compared to the empty vector-transfected cells, confirming that the reduction of substrate accumulation seen with the reference and the variants is due to efflux by MRP4. This validates the MRP4 efflux data for AZT and PMEA and is consistent with earlier results with P-glycoprotein and MRP1, where a mutation in a specific conserved amino acid in one of the two NBDs is sufficient to lead to a total loss of function (Bakos et al., 1997; Ren et al., 2004). The glycine at the fourth position of the ABC signature is almost fully conserved in the ABC transporter family (it is present in 99% of 1000 transporters analyzed in different species).
The functional differences observed with most of the variants cannot be attributed to differences in expression of the proteins at the membrane or total MRP4 expression levels. One exception to this is the G187W variant, which had lower levels of antiretroviral transport and showed deceased expression by Western blotting. A similar observation has been made for several MRP2, OATP1B1, and OATP1A2 variants (Tirona et al., 2001; Hirouchi et al., 2004; Lee et al., 2005). It is noteworthy that the nonfunctional mutant is also correctly expressed at the membrane, showing that its loss of function is not due to a decreased stability or impaired trafficking to the membrane. What are other possible mechanisms to explain the functional differences? One hypothesis implicates the modifications in the chemical structure of the protein when replacing one amino acid with another. The Grantham value for G187W is the greatest among the nonsynonymous variants of MRP4 (D = 184), indicating that this variant has the greatest structural change, with respect to composition, polarity, and molecular volume (Grantham, 1974). This could contribute to a certain extent to the reduction in function. It is supported by the fact that the G487E and C956S variants also have high Grantham values (D = 98 and 102, respectively) and altered functions. However, this estimator probably can not account completely for these data, because another variant with a high Grantham value (K304N) does not show any functional difference.
The 50% reduction in function observed with the G187W variant could be clinically relevant, whereas the small differences observed with the other variants (G487E, P78A, P403L, and C956S) are less likely to be significant. The G187W variant is present at a frequency of 2.5 to 13% in various ethnic groups. A decrease in MRP4 transport of antivirals could be beneficial as a result of higher target cell concentrations but could also result in a higher incidence of toxicity related to increased systemic and/or tissue levels of MRP4 substrates. The G187W MRP4 variant could also play a role at the organism level, because MRP4 is expressed in the kidney and the liver where it may be involved in antiviral elimination (Zamek-Gliszczynski et al., 2006; Imaoka et al., 2007). The expression of MRP4 variants in the liver has been evaluated in a recent clinical study, which showed no association between MRP4 polymorphism and RNA or protein expression in human liver (Gradhand et al., 2008). It is interesting that there was a trend toward decreased protein levels of 187W MRP4 in normal livers, consistent with our Western blot data. AZT is primarily eliminated by hepatic metabolism before being excreted in the urine, whereas PMEA is mainly renally excreted (http://us.gsk.com/products/assets/us_retrovir.pdf; http://www.hepsera.com). Therefore, the consequences of altered MRP4 transport on AZT and PMEA pharmacokinetics should be investigated.
Previous reports have shown a certain degree of substrate specificity associated with variants in membrane transporters (Erdman et al., 2006; Jeong et al., 2007), including a different specificity between endogenous substrates and drugs (Urban et al., 2006). This specificity should be further investigated for MRP4, which has both physiological and xenobiotic substrates. Inhibitory interactions of xenobiotics with MRP4 nonsynonymous variants should also be considered. This is particularly relevant for nucleoside/nucleotide analogs used for treating human immunodeficiency virus infection that are always administered as combination therapy.
In summary, ABCC4 is a highly polymorphic gene, as illustrated by the large number of variants and haplotypes identified in this study. Convincing evidence of AZT and PMEA transport by MRP4 is provided in this first functional characterization of MRP4 nonsynonymous variants. None of the 10 nonsynonymous variants of MRP4, which were studied, is completely deleterious. The G187W variant shows the greatest decrease in function and lower expression in this in vitro system. The clinical consequences of this altered function (e.g., increased response or higher incidence of side effects) require further investigation.
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Acknowledgements |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: MRP4, multidrug resistance protein 4; ABC, ATP-binding cassette; AZT, azidothymidine, 1-[4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione; HEK 293T, human embryonic kidney epithelial cells transformed with SV40 T antigen; NBD, nucleotide binding domain; PMEA, adefovir, 9-(2-phosphonylmethoxyethyl)adenine; bis-POM-PMEA, bis(pivaloyloxymethyl)PMEA; SNP, single nucleotide polymorphism; UTR, untranslated region; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FRT, Flp recombinase target.
Address correspondence to: Professor Deanna L. Kroetz, Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, 1550 4th Street, Box 2911, San Francisco, California. E-mail: deanna.kroetz{at}ucsf.edu
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