A Novel Human Hepatic Organic Anion Transporting Polypeptide (OATP2)

A novel human organic transporter, OATP2, has been identified that transports taurocholic acid, the adrenal androgen dehydroepiandrosterone sulfate, and thyroid hormone, as well as the hydroxymethylglutaryl-CoA reductase inhibitor, pravastatin. OATP2 is expressed exclusively in liver in contrast to all other known transporter subtypes that are found in both hepatic and nonhepatic tissues. OATP2 is considerably diverged from other family members, sharing only 42% sequence identity with the four other subtypes. Furthermore, unlike other subtypes, OATP2 did not transport digoxin or aldosterone. The rat isoform oatp1 was also shown to transport pravastatin, whereas other members of the OATP family, i.e.rat oatp2, human OATP, and the prostaglandin transporter, did not. Cis-inhibition studies indicate that both OATP2 and roatp1 also transport other statins including lovastatin, simvastatin, and atorvastatin. In summary, OATP2 is a novel organic anion transport protein that has overlapping but not identical substrate specificities with each of the other subtypes and, with its liver-specific expression, represents a functionally distinct OATP isoform. Furthermore, the identification of oatp1 and OATP2 as pravastatin transporters suggests that they are responsible for the hepatic uptake of this liver-specific hydroxymethylglutaryl-CoA reductase inhibitor in rat and man.

The liver functions in the clearance of a large variety of metabolic products, drugs, and other xenobiotics by transporting them across the sinusoidal membrane into the hepatocyte. Several classes of transport systems have been described that mediate these processes including the Na ϩ /taurocholate cotransporter polypeptide, in rat and human liver (1,2), and a family of organic anion transporting polypeptides (OATPs) 1 that are principally expressed in liver, kidney, and brain. These transport a broad spectrum of substrates in a sodium-independent manner (3,4). The distribution of this latter family of transporters in liver, kidney, and choroid plexus in the brain is thought to reflect common physiological requirements of these organs for the clearance of a multitude of organic anions. There are three OATP isoforms identified to date in the rat: roatp1 (5), roatp2 (6), and roatp3 (7). Rat oatp1 and oatp2 are abundantly expressed in liver and kidney and much less in brain, whereas oatp3 is absent in liver but highly expressed in kidney. In addition to bile acids, OATPs are known to transport a variety of other compounds. These include, depending on the transporter, unconjugated and conjugated steroids, such as estrone sulfate, estradiol-17B-glucuronide, and aldosterone, and cardiac glycosides (6, 8 -11). Bromosulfophthalien (5), mycotoxin (12), leukotriene C 4 (13), and thyroid hormone (7) are additional substrates.
In contrast to the rat, only one transporter of this family, OATP, has been identified in humans (14). It is expressed most abundantly throughout the brain and in much lower amounts in liver, kidney, and lung. In addition to bile acids and the above steroids, human OATP has recently been shown to transport the adrenal androgen dehydroepiandrosterone sulfate (DHEAS) (15).
HMG-CoA reductase inhibitors, or statins, are members of an important class of lipid lowering drugs that have been demonstrated to decrease risk for myocardial infarction (16 -18) and stroke (19,20). This is due, at least in part, to their ability to lower low density lipoprotein cholesterol through the inhibition of cholesterol synthesis in the liver. This results in an induction of hepatic low density lipoprotein receptors, thereby increasing the catabolism of circulating low density lipoprotein. The majority of statins, including lovastatin, simvastatin, and atorvastatin, are lipophilic molecules with high octanol:water partition coefficients, and, as such, they freely diffuse across membranes (21). Consequently, they are potent inhibitors of HMG-CoA reductase in a broad spectrum of tissues. In contrast, the reductase inhibitor pravastatin is hydrophilic in nature, is relatively membrane impermeable, and exhibits much greater tissue selectivity than the other statins. It is taken up, primarily in the liver, by a transport mechanism that is sodium-independent and is competitively inhibited by bile acids and dibromosulfophthaein in isolated rat hepatocytes (22,23). It is the presence or absence of this transporter that is likely the basis for the tissue selectivity of pravastatin. However, the protein that is responsible for this pravastatin transport activity has not been identified. Because of their substrate specificities and tissue localization, members of the OATP fam-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF205071.
¶ To whom all correspondence should be addressed: ily are good pravastatin transporter candidates.
Clearly the OATP transporter class plays a critical role in hepatic organic anion uptake mechanisms. However, only a single human isoform has been identified, and it has very low hepatic expression. Consequently, we sought to identify additional human OATPs that are found in liver. The novel transporter, OATP2, was cloned, and its substrate specificity was examined in expression studies. It transports taurocholate, DHEAS, and pravastatin and is the only known OATP whose expression is confined to liver. Furthermore, we have identified oatp1 as a pravastatin transporter in the rat.
The novel oatp family member, human OATP2, was identified by searching the public EST data bases for sequences homologous to human OATP. One EST sequence, GenBank TM accession number T73863, shared 41% sequence identity with OATP, and a clone containing fulllength coding sequence was obtained from a human liver library using the Gene Trapper cDNA Positive Selection System (LifeTechnologies, Inc.). Briefly, oligonucleotide PY79 (5Ј-ACCCTGTCTAGCAGGTTGCA-3Ј), complementary to the EST sequence, was biotinylated at the 3Ј-end and used to hybridize to a single-stranded human liver cDNA library constructed in pCMVSport2 (LifeTechnologies, Inc.). Hybrids between the biotinylated oligonucleotides and single-stranded DNA were captured on streptavidin-coated paramagnetic beads. After washing, the captured single-stranded DNA target was released from the biotinylated oligonucleotides and converted to dsDNA by DNA polymerase using unbiotinylated PY79. Following transformation and plating, several positive clones were identified by PCR analysis. Full-length cDNA clones were identified by sequencing.
For expression studies, all cDNAs were cloned into the expression vector pCEP4␤R, a modified form of pCEP4 (Invitrogen, Inc.) in which the cytomegalovirus promoter-driven expression cassette has been inverted and used in transient transfections. Rat oatp1 and oatp2 PCRamplified fragments were prepared for cloning by digestion with KpnI and NotI. Human OATP2 cDNA in pCMVSport2, corresponding to nucleotides 59 -2361 in Fig. 3, was excised by digestion with KpnI and NotI. All of the above cDNAs were cloned into the KpnI and NotI sites of pCEP4␤R. Human OATP amplified cDNA was digested with BglII and KpnI and cloned into the BglII and KpnI sites of pCEP4␤R.
Transport Assays-For transient transfections, 293c18 cells (43) were plated in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum and penicillin-streptomycin in poly-D-lysine coated dishes and co-transfected with transporter expression plasmids using LipofectAMINE Plus (Life Technologies, Inc.) according to the manufacturer. The cells and media were assayed for substrate transport 24 h later. To measure transport, medium was removed, and monolayers were assayed in triplicate by washing once in serum-free DMEM and adding the same medium containing 3 H-labeled substrate alone or in the presence of various concentrations of unlabeled compounds. Monolayers were incubated at room temperature for 5-10 min depending on the transporter. Then the cells were rapidly washed once with ice-cold DMEM containing 5% bovine serum albumin and three times with ice-cold DMEM. Cells were lysed in 0.1 N NaOH, a fraction of the lysate was used to determine radiolabel incorporation by liquid scintillation counting, and another fraction was used to determine protein concen-tration in the lysate using the Bradford assay with bovine serum albumin as a standard.
Northern Blotting-A Northern blot containing 2 g each of human poly(A) ϩ RNA (CLONTECH) was prehybridized in Church buffer (0.5 M sodium phosphate, pH 7.0, 7% SDS, 1% bovine serum albumin, 2 mM EDTA) and hybridized overnight at 65°C in the same buffer with a 32 P-labeled cDNA containing the full-length coding sequence of OATP2. The blot was washed to a final stringency of 0.1ϫ SSC, 0.1% SDS at 65°C and exposed to film at Ϫ80°C with an intensifying screen.
Sequence Analysis-Data base searches for OATP family members were performed using the BLAST and tBLAST algorithms (24). Protein transmembrane domains were predicted using the TMPred algorithm. Multiple sequence alignments were conducted using the CLUSTALW (25)

RESULTS
roatp1 Transports Pravastatin-293c18 cells transfected with pCEP4 expression plasmids containing roatp1, roatp2, and hOATP cDNAs were assayed for the uptake of pravastatin. Specific transport of [ 3 H]pravastatin was observed in roatp1 transfected cells (Fig. 1A). A time course experiment showed pravastatin transport to be linear for approximately 6 min with uptake reaching equilibrium after 10 min (Fig. 1B), whereas uptake was saturable with an apparent K m of 30 M (Fig. 1C). In contrast, no pravastatin transport was observed in 293c18 cells transfected with roatp2, hOATP, or the empty vector, pCEP4 (Fig. 1A). In control experiments all three transporters transported taurocholate (data not shown), in agreement with earlier studies demonstrating uptake of taurocholate by oatp1 expressing Xenopus laevis oocytes and HeLa cells (5,10); this verifies that all three transporters are active in this system.
Pravastatin transport in isolated rat hepatocytes is competitively inhibited by taurocholate and the statins lovastatin and simvastatin (22). To further characterize statin interactions with roatp1, the cis-inhibition of pravastatin and taurocholate uptake by statins and other compounds was examined. Both [ 3 H]pravastatin and [ 3 H]taurocholate transport was inhibited by 50 M (100-fold molar excess over tracer) of pravastatin, taurocholate, and several other HMG-CoA reductase inhibitors including simvastatin, lovastatin, atorvastatin, BMS241423, and BMS243887 (Fig. 2). The latter are two hydroxylated atorvastatin metabolites that are found in plasma from treated subjects. The rank order of statin potency for inhibition of pravastatin uptake was BMS243887 Ͼ BMS241423 ϭ lovastatin ϭ simvastatin Ͼ atorvastatin Ͼ pravastatin. A similar pattern was observed for statin inhibition of taurocholate transport. Taurocholate also blocked pravastatin uptake, consistent with it being a substrate of roatp1 ( Fig. 2 and Ref. 5) and its inhibition of pravastatin transport in hepatocytes. DHEAS, a substrate for human OATP, was also an inhibitor of pravastatin and taurocholate transport by roatp1. In contrast, digoxin and prostaglandin E 2 , substrates for the OATP family members roatp2 and PGT, respectively, are not roatp1 substrates, and these molecules did not inhibit pravastatin transport. 2 Identification of a Novel Human Organic Anion Transporter-The lack of pravastatin transport activity by hOATP plus the existence of multiple OATP isoforms in the rat suggested that an additional OATP exists that could account for the hepatic uptake of 2 B. Hsiang and T. Kirchgessner, unpublished work. pravastatin in humans. A number of PCR-based approaches utilizing degenerate PCR primers based on conserved sequences within the OATP family failed to identify any novel sequences from hu-man adult and fetal liver or kidney cDNA or from human genomic DNA. However, a search of the GenEMBL EST data base revealed a sequence (GenBank TM accession number T73863) obtained from a human liver library with significant homology to hOATP. The insert of this clone did not contain full-length coding sequence. Thus, an oligonucleotide based on EST T73863 was used to screen for full-length clones using the Gene Trap method. A 2.8-kilobase cDNA was identified containing 134, 2076, and 620 nucleotides of 5Ј-untranslated, coding, and 3Ј-untranslated sequences, respectively (Fig. 3). Conceptual translation of the cDNA sequence predicts a 691-amino acid protein with the following putative structural features: (i) 12 transmembrane domains (TMDs) ranging from 18 to 28 amino acids each (Fig. 4) and (ii) 11 potential Nglycosylation sites. Only two of the latter (Asn 134 and Asn 617 ) are predicted to be in extracellular loops. No sequence prior to the first TMD that strongly conforms to a signal peptide consensus predicted by the SPScan algorithm (27) could be found. This new transporter will be referred to as OATP2 (organic anion transporting polypeptide 2. Four OATPs expressed in liver have been previously reported; three isoforms have been identified in the rat (oatp1, 2, and 3) and one in human liver (OATP) (5)(6)(7)14). The alignment of OATP2 with these other transporters is shown in Fig. 4. 32% of the amino acids are conserved, and twelve TMDs are found in all five proteins predicted by the algorithm TMPred. The sequence identities of all pairwise comparisons are shown in Table I. There is high homology among human OATP and the three rat oatp isoforms, ranging from 67 to 82% identical residues. However, OATP2 is substantially diverged in sequence; its sequence is only 42% identical to each of the four other proteins. Furthermore, the lengths of all of the inter-TMD loops are identical among the transporters shown in Fig. 4 with the exception of OATP2. Loops between TMDs 3 and 4, TMDs 6 and 7, and TMDs 9 and 10 are all four to six residues longer in OATP2 compared with the others. In addition, OATP2 is considerably more basic than the other proteins, in particular hOATP; the predicted pI of OATP2 is 8.51 compared with 7.75, 7.74, 7.70, and 5.80 for roatp1, roatp2, roatp3, and hOATP, respectively. OATP2 has overlapping but not identical sub- strate specificities with roatp1, roatp2, and hOATP (see below), and these substrate preferences are presumably reflected in this sequence divergence.
Tissue and Cellular Distribution of OATP2-The tissue distribution of OATP2 expression was determined by Northern blotting of poly(A) ϩ RNA from a variety of human tissues. OATP, roatp1, roatp2, and roatp3 are all expressed in liver, kidney, and brain. In contrast, the expression OATP2 is very hepato-specific; a major 3.2-kilobase band and several minor hybridizing bands were observed only in RNA from liver and no other tissue (Fig. 5). A potential alternative polyadenylation signal was likely utilized in one of the OATP2 cDNA clones that was isolated (Fig. 3), which could account for the smaller 2.4kilobase mRNA species seen in the Northern blot. In situ hybridization of OATP2 probe to human liver showed signal in hepatocytes throughout the liver lobule, whereas no signal was observed in bile ducts, Kupffer cells, or vessels. 3 OATP2 Is a Human Pravastatin Transporter-The transport of pravastatin by OATP2 was investigated in transfected 293c18 cells. Specific uptake of [ 3 H]pravastatin was observed in cells transfected with OATP2 but not with empty vector (pCEP4) (Fig. 6A). Transport was linear for approximately 5 min (Fig. 6B) and was saturable with an apparent K m of 35 M (Fig. 6C), similar to that of roatp1 for pravastatin. Thus, OATP2 is a liver-specific human pravastatin transporter.
Other Substrates of OATP2-The ability of OATP2 to transport other molecules was examined using additional radiolabeled compounds in addition to assaying the cis-inhibition of [ 3 H]pravastatin uptake by unlabeled substrates. Known substrates of other transporters in this family include taurocholate (all OATPs), DHEAS (human OATP), and thyroid hormone (T3/T4) (roatp2 and roatp3). Of these, taurocholate, DHEAS, and T4, were shown to be transported by OATP2 (Fig. 7A). Uptake of T4 was only modestly higher in OATP2 compared with mock transfected cells (2.38 Ϯ 0.58 and 1.3 Ϯ 0.18 pmol/ min/mg, respectively; p Ͻ 0.05) because of a high endogenous uptake by 293 cells. However, this uptake was repeatable and statistically significant. The apparent K m of OATP2 for taurocholate uptake was 33.8 M (Fig. 7B), compared with 60, 50, 34, and 18 M for uptake by human OATP (14), roatp1 (28), roatp2 (6), and roatp3 (7), respectively. Additional substrates tested but not transported include aldosterone and digoxin.
The ability of unlabeled compounds to inhibit OATP2 mediated [ 3 H]pravastatin and [ 3 H]taurocholate uptake was also determined. Specific uptake was inhibited 36 and 89% by 50 and 500 M unlabeled pravastatin, respectively (Fig. 8). All other statins tested were much more potent than pravastatin in this regard: 50 M each of simvastatin, atorvastatin, the two hydroxylated atorvastatin metabolites BMS241423 and BMS243887, and lovastatin inhibited [ 3 H]pravastatin transport by greater than 95%. Taurocholate and DHEAS also inhibited pravastatin transport, consistent with these two compounds being substrates of OATP2 (Fig. 8A). The cis-inhibition of [ 3 H]taurocholate uptake by the same compounds showed a similar pattern with simvastatin, atorvastatin, and its metabolites, with lovastatin being the most potent inhibitors, whereas taurocholate and DHEAS showed more modest effects. DISCUSSION The present work describes a new member of the organic anion transporter polypeptide family, OATP2. It was obtained during efforts to uncover additional hepatic transporters and to define the molecular mechanism that mediates the transport of HMG-CoA reductase inhibitors. In addition to the novel human OATP2, oatp1 was also shown to transport pravastatin in the rat.
OATP2 is unique among OATPs in several ways. It is substantially diverged in sequence from the other family members; it is only 42% identical to each of the other OATPs, compared with 67-82% identity seen among the other transporters. Furthermore, although all of the loops between TMDs are strictly conserved among the other transporters, OATP2 has additional amino acids in three of eleven loops. Our inability to clone the human hepatic pravastatin transporter by PCR using degenerate oligonucleotides that were conserved among the known OATPs is likely due to this divergence.
Another distinguishing feature of OATP2 is its restricted pattern of expression. Out of 16 tissues examined, only liver was found to express OATP2 mRNA. This is in marked contrast to the other family members, all of which are found in multiple 3 V. Sasseville and T. Kirchgessner, unpublished work.

FIG. 3. Nucleotide and amino acid sequence of human OATP2.
The nucleotide sequence of the longest OATP2 cDNA clone is shown with the predicted amino acid sequence underneath. Nucleotide numbering and amino acid numbering are indicated on the left and right sides, respectively. Potential N-linked glycosylation sites are indicated by an asterisk below the residue. Underlined letters indicate a putative alternative polyadenylation signal utilized in a shorter cDNA that was also cloned from the same library. tissues in addition to liver. This includes kidney, skeletal muscle and colon for roatp1 (5), and kidney brain and retina for roatp2 and roatp3 (6,7). Human OATP mRNA, although found in liver, kidney, and lung, is expressed much more in brain than in these other tissues (14).
Although there is evidence for a single multivalent hepatocellular uptake site for amphipathic compounds (29,30), the discovery of a second OATP isoform in human suggests that, as in the rat, such uptake is in fact mediated by more than one carrier with partially overlapping substrate specificities. A comparison of OATP2 with human OATP reveals that the two proteins are functionally distinct with respect to the types of substrates transported by each. Both OATP2 (Fig. 8) and OATP (14,15) transport taurocholate and DHEAS. However, only OATP2 transports HMG-CoA reductase inhibitors. Furthermore, the two appear to differ in their transport of steroids; OATP mediates the uptake of conjugates such as estradiol 17␤-D-glucuronide (3) and estrone-3-sulfate (9), whereas OATP2 transports neither the former nor the neutral steroid, aldosterone. OATP has been demonstrated to transport bile acid. However it does so with a 60-fold lower transport rate in Xenopus oocytes compared with roatp1. This suggests that OATP may not mediate the majority of hepatic bile acid uptake in humans. Indeed, the much higher level of expression in brain compared with other tissues indicates that OATP may be more important in CNS transport processes than in liver uptake mechanisms (14). Thus, OATP2 may be the predominant sodium-independent transporter of bile acids in human liver, whereas OATP might be a relatively minor hepatic carrier.
DHEAS along with the nonsulfated DHEA are the major  circulating adrenal steroids, serving as precursors for endogenous sex steroid synthesis (31). Furthermore, they have been suggested to have positive neuropsychiatric, immune, and metabolic effects (32). OATP2 is the second DHEAS transporter described to date and the only hepato-specific carrier. The majority of DHEAS is formed in the liver from the sulfation of DHEA, after which it is effluxed back into the circulation across the sinusoidal surface of the hepatocyte (33). Furthermore, taurocholate inhibitable DHEAS uptake into isolated rat hepatocytes is also known to occur (34). Because members of this family are bi-directional transporters, OATP2 along with OATP are candidates for either of these transport processes in humans.
A search for pravastatin transport activity in the rat revealed that rat oatp1 but not oatp2 mediates pravastatin uptake in vitro. In addition to this transport activity in transfected cells, several other lines of evidence suggest that this is the protein responsible for pravastatin uptake observed in rat hepatocytes. Yamazaki et al. (22) demonstrated that uptake in liver cells was sodium-independent, appeared to be a single component, and was competitively inhibited by taurocholate and other cholephils as well as the lipophilic statins, lovastatin, and simvastatin. The order of potency for inhibition of radiolabeled pravastatin transport into rat hepatocytes was lovastatin ϭ simvastatin Ͼ Ͼ taurocholate Ͼ pravastatin. The above characteristics of hepatic pravastatin uptake mechanisms are consistent with the known properties of roatp1 and its behavior in mediating pravastatin transport in this study: (i) roatp1 expression is relatively restricted, being expressed in relatively few organs, including liver, consistent with the relative hepatoselectivity of pravastatin; (ii) roatp1 is a sodium-independent transporter of bile acid; and (iii) in the present studies, [ 3 H]pravastatin transport was inhibited by lovastatin, simvastatin, taurocholate, and pravastatin in the same rank order as in isolated hepatocytes. Furthermore, pravastatin inhibits the sodium-independent uptake of taurocholate in rat hepatocytes (22,23) and in roatp1-transfected 293c18 cells.
Liver and kidney are the two principle organs of distribution when pravastatin is administered intravenously to rats. This clearance occurs by uptake on the sinusoidal or basolateral side of the liver cell followed by biliary excretion across the canilicular membrane. Rat oatp1 has been localized to the basolateral surface of the hepatocyte (34), consistent with a role in hepatic pravastatin clearance (35), whereas the unrelated multi-drug resistance protein, cMOAT (canilicular multi-specific organic anion transporter), is thought to be the canilicular transporter (36). With regard to renal elimination of this drug, there is uptake from the basolateral side of the renal tubular epithelial cell in addition to glomerular filtration (37). However, in the kidney, in contrast to the situation in liver, roatp1 resides on the apical surface of the epithelial cell, and thus, it cannot be responsible for the pravastatin uptake that occurs on the basolateral side. This implies that there is yet another pravastatin transporter in rat kidney that has this activity. It could be the recently reported roatp3, the unrelated rat OAT1 (38), or cMOAT, all of which are expressed in kidney. Because OATP transporters are bi-directional in nature (39), roatp1 may transport pravastatin and other statins from the inside of the epithelial cell through the apical membrane to the lumen of the renal tubule in rat kidney. Alternatively it could be an as yet undescribed transporter.
Likewise, because significant elimination of pravastatin also occurs via tubular epithelial cell transport in human kidneys (40), an additional pravastatin transporter must exist in humans other than the liver-specific OATP2. Because the uptake of pravastatin into tissues is absolutely dependent on carrier mediated transport, the difference in the tissues expressing human and rat pravastatin transporters (oatp1 in rat liver, kidney, brain, lung, and skeletal muscle, and OATP only in human liver) could result in pharmacodynamic differences in the distribution of this drug between the two species.
The importance of heptocellular uptake in the elimination of any drug is largely determined by its physiochemical properties. Polar organic hydrophilic compounds require specific carriers for uptake into hepatocytes, whereas lipophilic compounds are freely permeable to the basolateral hepatocyte membrane and exhibit flow-dependent distribution in the liver. Even though such drugs do not require specific uptake mechanisms, they can act as substrates for transporters and can potentially alter the hepatic handling of other drugs or endogenous compounds (41). There are examples of both kinds of drugs among HMG-CoA reductase inhibitors. Pravastatin is an example of the former class, in which oatp1 and OATP2 are likely required for rat and human hepatic uptake, respectively. The lipophilic statins simvastatin, lovastatin, and atorvastatin are thought to be in the second class; they do not require carriers for extraction by the liver. However, the data presented here suggest that they are nevertheless very potent inhibitors of radiolabeled pravastatin and taurocholate transport by oatp1 and OATP2, showing much greater inhibition than the unlabeled substrates themselves. Although the mode of this inhibition will require more detailed mechanistic studies, it suggests that the lipophilic statins may utilize these carriers as well.
In summary, we have identified a liver-specific member of the organic anion transport protein family in humans that mediates the transport of bile acid, the adrenal androgen DHEAS, and HMG-CoA reductase inhibitors, a clinically important class of hypolipidemic agents. The divergent sequence of OATP2, its substrate specificity, and its unique tissue distribution indicate that it is not the human orthologue of any previously described OATP, and thus, it represents a new functional entity. This should further facilitate the understanding of hepatic transport and clearance mechanisms for the molecules described as well as additional substrates.