Multidrug Resistance Associated Protein Family Mrp Abcc

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The human MRP gene encodes an MRP polypeptide with an apparent mass of 170 kDa, which is posttranslationally converted to a 190 kDa form by the addition of N-linked complex oligosaccharides (Almquist et al., 1995). To date, nine members within the MRP family have been identified, delineated as MRP1 to MRP6 (ABCC1 to ABCC6) and MRP7 to MRP9 (ABCC10 to ABCC12) (Belinsky et al., 1998; Bera et al., 2001, 2002; Kubota et al., 2001; Kruh and Belinsky, 2003). Membrane topology of MRP1 identified three transmembrane spanning domains (two are of 6 and the third is of 5 helices) (Fig. 7.1) (Bakos et al., 1996; Hipfner et al., 1997). Additionally, MRP1 has two NBDs and two intracellular loops with the first linker segment located between two transmembrane domains (TMD) whereas the second is between the transmembrane and a nucleotide binding domains. MRP4 (ABCC4), MRP5 (ABCC5), MRP8 (ABCC11), and MRP9 (ABCC12) lack the five helices of the third membrane spanning domain, but possess intracellular loops whereas MRP2 (ABCC2), MRP3 (ABCC3), MRP6 (ABCC6), and MRP7 (ABCC10) resemble MRP1 (Bera et al., 2001, 2002; Hopper et al., 2001; Yabuuchi et al., 2001; Kruh and Belinsky, 2003). The human genome nomenclature details and chromosomal localization of important intestinal MRP transporters are listed in Table 7.1.

7.2.2.1 The Expression of MRPs

MRP1 was first cloned by Cole et al. (1992) from a multidrug-resistant human lung cancer cell line. MRP1 is highly expressed in the intestine, where it is localized in the basolateral membrane of intestinal epithelial cells and is involved in the absorptive efflux of its substrates into blood (Peng et al., 1999). MRP1 expression has also been demonstrated in brain, liver, lung, kidney, and testis (Flens et al., 1996; Zhang et al., 2000; Cherrington et al., 2002).

Comparatively, MRP2 was first cloned from human liver as a canalicular multi-specific organic anion transporter (cMOAT) (Paulusma et al., 1996) and was found to be expressed on the apical membrane of the intestine, liver, and kidney tubules (Schaub et al., 1997; Fromm et al., 2000; Scheffer et al., 2002). Furthermore, MRP2 expression was localized in the brush-border membrane of villi in rabbit small intestine with expression decreasing from the villous tip to the crypt (Van Aubel et al., 2000). Moreover, MRP2 expression was shown to vary substantially along the human GI tract, with higher expression levels found in small intestine and minimal expression observed in colonic segments (Zimmermann et al., 2005).

MRP3 expression is also evident in the basolateral membrane of the small intestine, as well as the liver, colon, lung, spleen, and kidney (Kool et al., 1997;

Scheffer et al., 2002). Rost et al. (2002) identified MRP3 mRNA expression throughout the rat intestine by RT-PCR, with lower expression observed in the duodenum and jejunum and markedly increased expression observed in the ileum and colon. MRP4 is expressed in several tissues including jejunum, kidney, brain, lung, and gall bladder (Kool et al., 1997; Zhang et al., 2000; Taipalensuu et al., 2001; Van Aubel et al., 2002). MRP5 expression is found to be in colon, liver, kidney, skeletal muscle, and brain (Kool et al., 1997; McAleer et al., 1999; Zhang et al., 2000), while MRP6 is highly expressed in the kidney and liver, with low expression in several other tissues, including duodenum, colon, brain, and salivary gland (Zhang et al., 2000). The functional activity and expression of MRP7, MRP8, and MRP9 have not been as well characterized, although they are gaining increasing attention due to their involvement in conveying multidrug resistance (Bera et al. 2002; Kruh and Belinsky, 2003; Chen et al., 2003, 2005; Hopper-Borge et al., 2004).

The comparative mRNA expression of MRPs 1-5 and P-gp studied in ten healthy human intestinal tract biopsy samples (Zimmermann et al., 2005) demonstrated a rank order expression of MRP3 » MDR1 > MRP2 > MRP5 > MRP4 > MRP1 and MDR1 > MRP3 » MRP1 in the duodenum and the terminal ileum, respectively. The comparative rank order of mRNA expression remains consistent throughout the colon (ascending, transverse, descending, and sigmoid colon) with MRP3 » MDR1 > MRP4 = MRP5 > MRP1 » MRP2. Total RNA analysis by Taipalensuu et al. (2001) showed a ranking of BCRP = MRP2 > MDR1 = MRP3 = MRP6 = MRP5 = MRP1 > MRP4 > MDR3, as determined by quantitative polymerase chain reaction in jejunal biopsies from 13 healthy human subjects. Direct comparison between the expression of BCRP, MDR1, MDR3, and MRP1-6 found that BCRP exhibited a 100-fold lower transcript level in Caco-2 cells compared with jejunum (Taipalensuu et al., 2001). Furthermore, based upon the ratio of MRP:18S rRNA expression, the rank order for MRP expression in Caco-2 cells was MRP2 > MRP6 > MRP4 > MRP3 > MRP1 = MRP5 (Prime-Chapman etal., 2004).

Our laboratory also observed a variable intestinal expression of P-gp, MDR3, S-P-gp, LRP, MRP1-4 in tissue slides and intestinal protein lysates obtained from normal adult small and large intestinal epithelium of Chinese and Caucasian donors (Wang et al., 2004). Furthermore, the expressions of P-gp, MDR3, LRP, MRP1, and MRP2 were found to be higher in the small intestine of Chinese when compared to Caucasian samples, suggesting distinct expression profiles of these transporters (Wang et al., 2004). Interestingly, there has been concern over the variable expression of these isoforms in traditional cell screening models (e.g., Caco-2 cells), as compared with normal physiological expression observed in patients. Given the widespread use of these screening models, these differences in transporter expression may not properly translate to the actual physiological condition resulting in selection of lead candidates based on erroneous data. Finally, immunofluorescent staining of Caco-2 cells revealed the apical and basolateral localizations of MRP2 and MRP3, respectively, whereas MRP1 was not observed on either membrane (Prime-Chapman et al., 2004).

7.2.2.2 The Regulation of MRP Isoform Expression

Nuclear receptors that regulate transcription of MRP2 proteins include pregnane X receptor (PXR, NR1I2), farnesoid X receptor (FXR, NR1H4), and the constitutive androstane receptor (CAR, NR1I3) (Tanaka et al., 1999; Kast et al., 2002). Data indicate that steroid-based compounds, such as bile acids, cholesterol, hormones, as well as other drugs, including rifampin, dexamethasone, and phenobarbital, all affect the above nuclear receptors. For example, the mRNA expression of MRP2 was up regulated in the presence of rifampin in the rhesus monkey liver, human small intestine, and primary cultures of human hepatocytes (Kauffmann et al., 1998; Fromm et al., 2000; Dussault et al., 2001). Moreover, rats treated with known PXR ligands, such as the antiglucocorticoid/antiprogestin RU486 and the antifungal clotrimazole, showed upregulation of MRP2 gene expression, suggesting a role of PXR ligands in regulation of MRP2 expression (Courtois et al., 1999). Naturally occurring chenodeoxycholic acid, synthetic GW4064, and known FXR ligands have induced MRP2 mRNA expression in both human and rat hepatocytes. Finally, the CAR agonist, phenobarbital also induced MRP2 mRNA expression (Kast etal., 2002).

Intestinal MRP3 mRNA has been shown to increase in a dose- and time-dependent manner in Caco-2 cells after treatment with a series of bile salts, chenodeoxycholic acid, taurochenodeoxycholic acid, taurocholic acid, and taurolithocholic acid (Inokuchi et al., 2001). Additionally, the glucocorticoids, dexamethasone, and hydrocortisone have also been shown to transcriptionally upregulate human MRP3 mRNA in a non-small-cell lung cancer cell line, which was correlated to an increase in MRP3 protein (Pulaski et al., 2005).

Recently, the involvement of various transcriptionally mediated pathways aryl hydrocarbon receptor (AhR), PXR, CAR, peroxisome proliferator-activated receptor a (PPARa), and nuclear factor-E2-related factor 2 (Nrf2) were studied on the induction ofMRPs (Maher etal., 2005). The mRNA expression ofMRP2,3,5, and 6 were shown to be induced using AhR ligands [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinatedbiphenyl 126 (PCB126), and p -naphthoflavone], while the CAR activator 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) induced MRP2, 3, 4, 6, and 7 mRNA expression. In addition, Nrf2 activators (butylated hydroxyanisole, oltipraz, and ethoxyquin) induced MRP2-6, further supporting the relevance of transcription factors with respect to MRP regulation (Maher et al., 2005).

7.2.2.3 The Substrate Specificity of MRP's

The substrate specificity and function of MRP family members have been extensively reviewed by Kruh and Belinsky (2003), and will only be briefly discussed here. MRP1 is involved in the transport of anionic drugs (e.g., methotrexate), drug or metabolite conjugates (glutathione, glucuronate, or sulfate), leukotriene C4 (LTC4), 2,4-dinitrophenyl-5-glutathione(DNP-SG), bilirubin glucuronides, estradiol-17-glucuronide, and dianionic bile salts (Leier et al., 1994; Jedlitschky

Table 7.3. Partial list of substrates that have shown to interact with Multidrug Resistance-Associated protein

Transporter

Drugs/xenobiotics

Physiological substrates

MRP1

Vinca alkaloids, epipodophyllotoxins,

LTC4, estrogen glucuronide, leukotriene

anthracyclines, camptothecin, MTX

E4, S-Glutathionyl prostaglandin A2,

bilirubin (monoglucuronosyl,

bisglucuronosyl)

MRP2

Vinca alkaloids, anthracylines,

Bilirubin glucuronide DNP-SG,

vincristine, etoposide camptothecin,

S-Glutathionyl ethacrynic acid,

MTX, ochratoxin A,

17ß-glucuronosyl estradiol

p-Aminohippurate

MRP3

Etoposide, MTX

Glycocholic acid

MRP4

6-MP, MTX, PMEA

Cyclic nucleotides, DHEAS

MRP5

6-MP, PMEA

Cyclic nucleotides

MRP6

Anthracycline, etoposide

?

MRP7

?

17ß-estradiol 17-(ß-D-glucuronide)?

MRP8

5-FU, ddC, PMEA

Cyclic nucleotides

MRP9

?

?

LTC4, cysteinyl leukotriene; MTX, methotrexate; 6-MP, 6-mercaptopurine; PMEA, 9-(2-phosphonylmethoxyethyl)adenine; 5-FU, 50-fluorouracil; ddC, zalcitabine; DHEAS, dehy-droepiandrosterone sulfate; DNP-SG, S-glutathionyl 2,4-dinitrobenzene. Table modified from the information reported by Konig et al. (1999) and Kruh and Belinsky (2003)

LTC4, cysteinyl leukotriene; MTX, methotrexate; 6-MP, 6-mercaptopurine; PMEA, 9-(2-phosphonylmethoxyethyl)adenine; 5-FU, 50-fluorouracil; ddC, zalcitabine; DHEAS, dehy-droepiandrosterone sulfate; DNP-SG, S-glutathionyl 2,4-dinitrobenzene. Table modified from the information reported by Konig et al. (1999) and Kruh and Belinsky (2003)

et al., 1996; Nies et al., 1998; Hooijberg et al., 1999). Representative substrates of MRP1 are listed in Table 7.3 and include anthracyclines, vinca alkaloids, pipodophyllotoxins, and camptothecins (Cole et al., 1994; Konig et al., 1999; Kruh and Belinsky, 2003). Several compounds are also known to be either direct or indirect inhibitors of MRP1. Verapamil and its analogs, in the presence of reduced glutathione (GSH) or its nonreducing S-methyl derivative, inhibited LTC4 transport in membrane vesicles prepared from MRP1-transfected cells. Verapamil itself was not transported by MRP1 in either intact cells or membrane vesicles, suggesting that verapamil modulates MRP1 activity through enhancing MRP1 mediated GSH transport (Loe et al., 2000a,b). Leslie et al. (2003) demonstrated a similar effect on MRP1 mediated GSH transport in the presence of bioflavonoids, possibly through an interaction(s) with one of the several multiple flavonoid binding sites (Trompier et al., 2003).

Similarly, MRP2 transports leukotrienes C4, D4, and E4 and various glutathione conjugates, including oxidized glutathione, 2,4-dinitrophenyl-S-glutathione, bro-mosulfophthalein glutathione, as well as those conjugates of heavy metals including arsenic and cadmium (Jedlitschky et al., 1997; Madon et al., 1997; Suzuki and Sugiyama, 1998, 1999). The glucuronide conjugates of bilirubin, estradiol, acetaminophen, grepafloxacin, triiodo-L-thyronine, and SN-38 have also been demonstrated to be MRP2 substrates (Suzuki and Sugiyama, 1998, 1999). Additionally, MRP2 also transports glucuronide and sulfate conjugates of several bile salts, a range of unconjugated organic anions such as methotrexate, reduced folates, bromosulfophthalein, irinotecan and its metabolite SN-38, pravastatin, ceftriaxone, temocaprilat, ampicillin as well as Fluo-3 and p-aminohippurate (Konig et al., 1999; Suzuki and Sugiyama, 1998, 1999; Kusuhara and Sugiyama, 2002). Keppler et al. (1997) have suggested the transport efficiency (Vmax/^m) of MRP2 substrates in the rank order of leukotriene C4 > leukotrieneD4 > S-(2,4-dinitrophenyl)-glutathione > monoglucuronosyl bilirubin > estradiol-17|3-D-glucuronide > taurolithocholate sulfate > oxidized glutathione.

The overlapping substrate specificity of MRP2 with P-gp, coupled with their intestinal and cellular colocalization to the apical membrane, suggests a concerted function between these two transporters that would comprise a significant barrier to the intestinal absorption of many xenobiotics. interestingly, grepafloxacin uptake was observed to be directly influenced by the combined effect of P-gp and MRP2. However, the secretory efflux of grepafloxacin was shown to be predominantly a function of MRP2 activity as opposed to P-gp, both in vitro and in vivo, suggesting that in spite of some overlapping substrate specificity, MRP members may also act independently as a functional barrier to bioavailability (Naruhashi etal., 2002).

Recently, MRP2 has also been shown to be involved in the efflux of a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and the food carcinogen PhIP, reducing their oral bioavailabilities, thus protecting against food-derived carcinogenesis (Dietrich et al., 2001; Leslie et al., 2001). In addition, bioavailability of fungal toxin ochratoxin A and the tea flavonoid epicatechin were inhibited by MK571, an antagonist of MRP2, suggesting these as MRP2 substrates (Leier et al., 2000; Vaidyanathan and Walle, 2001).

Similar to both MRP1 and MRP2, MRP3 also has the capacity to transport organic anionic drugs and glucuronate-conjugated drugs; however, MRP3 exhibits a reduced capacity for GSH conjugates (Hirohashi et al., 1999), as well as a wide range of bile salts such as glycocholate, taurolithocholate-3-sulfate, and taurochenodeoxycholate-3-sulfate (Hirohashi et al., 2000; Zeng et al., 2000; Zelcer et al., 2001). In addition MRP3, in contrast to MRP1 and MRP2, does not require glutathione for mediating the transport of natural products (Zelcer et al., 2001).

Substrates for MRP4 include folic acid, folinic acid (leucovorin), and methotrexate (Chen et al., 2002), as well as cAMP, cGMP, estradiol-17|3-D-glucuronide, bile acids (Chen et al., 2001; Lai and Tan, 2002; Van Aubel et al., 2002; Zelcer et al., 2003), and thiopurines (Wielinga et al. 2002). Recent literature also indicates that MRP4 is involved in the efflux of camptothecins, as shown in human MRP4 stably transfected HepG2 cells (Tian et al., 2005). MRP5 has an affinity for nucleotide-based substrates including anticancer thiopurine and thiogua-nine drugs, as well as the anti-HIV drug 9-(2-phosphonylmethoxyethyl)adenine (Wijnholds et al., 2000; Wielinga et al. 2002; Reid et al., 2003). Interestingly, organic anion such as benzbromarone and sulfinpyrazone inhibit MRP5, suggesting an affinity for anionic phosphate/phosphonate moieties (Wijnholds et al., 2000). With respect to the remaining MRP family members, MRP6 was shown to be involved in the transport of different natural cytotoxic agents such as etoposide, doxorubicin, and cisplatin in MRP6-transfected Chinese hamster ovary (CHO) cells (Belinsky et al. 2002), while MRP7 is also a lipophilic anion efflux transporter with an affinity for docotaxel and 17ß-estradiol-(17ß-D-glucuronide) (Chen et al., 2003; Hopper-Borge et al., 2004). MRP8 has been demonstrated to mediate the efflux of cyclic nucleotides (Guo et al., 2003).

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