Hepatocyte nuclear factor-4 regulates the human organic anion transporter 1 gene in the kidney

【摘要】  Human organic anion transporter 1 (OAT1, SLC22A6), which is localized to the basolateral membranes of renal tubular epithelial cells, plays a critical role in the excretion of anionic compounds. OAT1 is regulated by various pathophysiological conditions, but little is known about the molecular mechanisms regulating the expression of OAT1. In the present study, we investigated the transcriptional regulation of OAT1 and found that hepatocyte nuclear factor (HNF)-4 markedly transactivated the OAT1 promoter. A deletion analysis of the OAT1 promoter suggested that the regions spanning -1191 to -700 base pairs (bp) and -140 to -79 bp were essential for the transactivation by HNF-4. These regions contained a direct repeat separated by two nucleotides (DR-2), which is one of the consensus sequences binding to HNF-4, and an inverted repeat separated by eight nucleotides (IR-8), which was recently identified as a novel element for HNF-4, respectively. An electrophoretic mobility shift assay showed that HNF-4 bound to DR-2 and IR-8 under the conditions of HNF-4 overexpression. Furthermore, under normal conditions, HNF-4 bound to IR-8, and a mutation in IR-8 markedly reduced the OAT1 promoter activity, indicating that HNF-4 regulates the basal transcription of OAT1 via IR-8. This paper reports the first characterization of the human OAT1 promoter and the first gene in the kidney whose promoter activity is regulated by HNF-4.

【关键词】  SLCA proximal tubule promoter inverted repeat

THE ORGANIC ANION TRANSPORTER (OAT) family plays an important role in the renal excretion of endogenous and exogenous organic anions, including drugs, toxins, and hormones ( 8, 18, 33, 41 ). Among the OAT family, OAT1 (SLC22A6) and OAT3 (SLC22A8) are localized to the basolateral membranes of the renal proximal tubular epithelial cells ( 26 ), and mRNA levels of both transporters were higher than those of other members of organic ion transporter (SLC22A) family in the human kidney cortex ( 42 ). So far, the molecular natures of OAT1 and OAT3 have been well characterized with regard to transport characteristics, structure-function relationships, and regulation ( 1, 8, 42, 47 ). As for regulation, various pathophysiological conditions, including hyperuricemia ( 14 ), renal failure ( 25, 36 ), bilateral ureteral obstruction ( 43 ), and acute biliary obstruction ( 6 ), affected the expression of OAT1 and/or OAT3. Although the elucidation of their regulatory mechanisms is quite important, studies that address the point are limited.

Recently, Kikuchi et al. ( 23 ) and we ( 28 ) reported the transcriptional regulation of the OAT3 gene. In contrast, as for the OAT1 promoter, a computational analysis but not a functional analysis has been conducted ( 2 ). In the present study, therefore, we attempted to characterize the transcriptional regulation of OAT1 by focusing on two kinds of transcription factors: hepatocyte nuclear factor (HNF)-1 / and HNF-4. The former is reported to stimulate the promoter activity of OAT3 ( 23 ), while the latter transactivates the OAT2 and OCT1 genes, which are other members of the SLC22A family mainly expressed in the liver ( 31, 34 ).

HNF-1 / are homeodomain-containing transcription factors that are expressed in the liver, kidney, intestine, stomach, and pancreas ( 3, 10 ). In the kidney, HNF-1 is reported to control the expression of transporters such as sodium/glucose cotransporter 2 and sodium/phosphate cotransporter 1 besides OAT3 ( 9, 30 ). Furthermore, Oat1 had markedly lower renal mRNA levels in Hnf-1 -null mice than in wild-type mice ( 24 ). On the other hand, HNF-4 is an orphan member of the nuclear receptor superfamily that is expressed in the liver, kidney, intestine, stomach, and pancreas ( 27, 40 ). Although few reports are available on the physiological role of HNF-4 in the kidney, HNF-4 protein has been found only in proximal tubular epithelial cells in the kidney ( 22 ), where OAT1 protein is located ( 26 ). Accordingly, it is possible that the OAT1 gene is regulated by HNF-1 / or HNF-4. In the present study, we cloned the human OAT1 promoter region and examined whether the OAT1 gene is regulated by these transcription factors.

MATERIALS AND METHODS

Materials. [ - 32 P]ATP was obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Restriction enzymes were from New England Biolabs (Beverly, MA). An antibody against HNF-4 (H-171) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The human HNF-1 and HNF-1 expression vectors were kindly supplied by Dr. Marco Pontoglio (Institute Pasteur, Paris, France). The human HNF-4 (transcript variant 2) expression vector was from OriGene Technologies (Rockville, MD).

5'-Rapid amplification of cDNA ends. To identify the transcription start site of human OAT1, 5'-rapid amplification of cDNA ends (5'-RACE) was carried out using Human Kidney Marathon-Ready cDNA (Clontech, Mountain View, CA) according to the manufacturer's instructions. The primers for 5'-RACE were as follows: a gene-specific primer for OAT1 (accession number NM_004790 ), 5'-GGTCCCACTCAGTCACGATGGTAGATGG-3' (683 to 656); and a nested gene-specific primer for OAT1, 5'-CGACACCCCCCACCTGCTGCAGGAGG-3' (347 to 322). The PCR products were subcloned into the pGEM-T Easy Vector (Promega, Madison, WI) and sequenced using a multicapillary DNA sequencer RISA384 system (Shimadzu, Kyoto, Japan).

Cloning of the 5'-regulatory region of the OAT1 gene. Based on the human genomic sequence (accession number NT_033903 ), the 2,747-base pair (bp) flanking region upstream of the transcription start site was cloned by PCR using the primers listed in Table 1 and human genomic DNA (Promega). The PCR product was isolated by electrophoresis and subcloned into the firefly luciferase reporter vector pGL3-Basic (Promega) at the Nhe I and Xho I sites. This full-length reporter plasmid is hereafter referred to as -2,747/+88.

Table 1. Oligonucleotide sequence of primers

Preparation of deletion reporter constructs. The 5'-deleted constructs (-1851/+88, -1191/+88) were generated by digestion of the -2747/+88 construct with Mlu I and either Nde I or Pst I. The ends were blunted with T4 DNA polymerase (Takara Bio, Otsu, Japan) and then self-ligated. The other 5'-deleted constructs were generated by PCR with primers containing an Nhe I site and Xho I site ( Table 1 ). The site-directed mutation in direct repeat (DR)-2 was introduced into the -2747/+88 construct and the mutation in inverted repeat (IR)-8 was introduced into the -2747/+88 construct and -140/+88 construct with a QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) using the primers listed in Table 1. The nucleotide sequences of these deleted or mutated constructs were verified.

Cell culture, transfection, and luciferase assay. Opossum kidney (OK) cells were cultured in medium 199 (Invitrogen, Carlsbad, CA) containing 10% FBS (Invitrogen) without antibiotics, in an atmosphere of 5% CO 2 -95% air at 37°C, and subcultured every 7 days using 0.02% EDTA and 0.05% trypsin. OK cells were plated into 24-well plates (4 x 10 6 cells/well) and transfected the following day with the reporter constructs and expression vector, and 25 ng of the Renilla reniformis vector pRL-TK (Promega), using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendation. The firefly and Renilla activities were determined 48 h after the transfection using a dual luciferase assay kit (Promega) and a LB940 luminometer (Berthold, Bad Wildbad, Germany). The firefly activity was normalized to Renilla activity.

EMSA. Nuclear extract was prepared from OK cells transiently transfected with HNF-4 or not, according to the method of Shimakura et al. ( 37 ). The double-stranded oligonucleotides used in the EMSA are listed in Table 1. The OAT1 probes (-882/-853) and (-127/-100) were end-labeled with [ - 32 P]ATP using T4 polynucleotide kinase (Takara Bio) and purified through a Sephadex G-25 column (GE Healthcare). EMSA was performed according to Prieur et al. ( 32 ) but with some modifications. The OK nuclear extract (10 µg) was incubated in binding buffer [40 mM KCl, 10 mM Tris·HCl (pH 8.0), 1 mM DTT, 6% glycerol, and 0.05% Nonidet P-40] for 10 min at 4°C. Thereafter, one of the labeled probes was added and the mixture was incubated for a further 10 min at 4°C. For competition experiments and supershift assays, excess (50-fold) unlabeled oligonucleotides and antibody (2 µg) were added 10 min and 1 h before the addition of the labeled probe, respectively. The volume of the binding mixture was 20 µl throughout the experiment. The DNA-protein complex was then separated on a 4% polyacrylamide gel for 1.5 h at 200 V and room temperature in 0.5 x Tris borate-EDTA buffer. Gels were dried and exposed to X-ray film for autoradiography.

Data analysis. The results were expressed relative to pGL3-Basic and represent the mean ± SD of three replicates. Two or three experiments were conducted, and representative results are shown.

RESULTS

Determination of the transcription start site of OAT1. To identify the transcription start site of human OAT1, 5'-RACE was performed. Sequencing of the longest RACE product showed that the terminal position of OAT1 cDNA was located 265 nucleotides above the start codon, which was 43 bp downstream of the 5'-end of OAT1 cDNA registered in the National Center for Biotechnology Information (NCBI) database (accession number NM_004790 ). No unreported intron was identified in the 5'-flanking region of OAT1, so the 5'-end of OAT1 cDNA registered in the NCBI database was numbered with +1 as the transcription start site in this study.

OAT1 promoter is transactivated by HNF-4. To perform the functional analysis of the human OAT1 promoter, the 2,747-bp flanking region upstream of the transcription start site of OAT1 was subcloned into the pGL3-Basic vector. At first, the OAT1 promoter construct -2747/+88 was transiently transfected into a renal epithelial cell line derived from OK, a cell line derived from human embryonic kidney (HEK293), and a human intestinal cell line (Caco-2), and luciferase activity was measured. As shown in Fig. 1, the -2747/+88 construct showed a significant increase in luciferase activity compared with pGL3-Basic in OK cells, which have many characteristics of renal proximal tubule cells, including an organic anion transport system ( 17 ). However, this construct had little promoter activity in HEK293 and Caco-2 cells, which lack an organic anion transport system. Therefore, OK cells were used in the subsequent experiments.

Fig. 1. Functional analysis of the human organic anion transporter 1 (OAT1) promoter in kidney-derived cell lines [opposum kidney (OK) and HEK293] and a human intestinal cell line (Caco-2). The longest OAT1 construct -2747/+88 (500 ng) was transfected into these cells for luciferase assays. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are reported as the relative fold-increase compared with pGL3-Basic and represent the means ± SD of 3 replicates.

Next, to investigate whether the OAT1 promoter is regulated by HNF-1 / or HNF-4, the -2747/+88 construct was transiently transfected into OK cells simultaneously with the HNF-1, HNF-1, or HNF-4 expression plasmid. Only HNF-4 could transactivate the OAT1 promoter activity markedly; HNF-1 and HNF-1 could not ( Fig. 2 ). These results suggested that the OAT1 promoter is regulated by HNF-4.

Fig. 2. Effects of hepatocyte nuclear factor (HNF)-1, HNF-1, and HNF-4 overexpression on human OAT1 promoter activity. OK cells were transiently transfected with 500 ng of a -2747/+88 construct and 500 ng of the expression vector for HNF-1, HNF-1, HNF-4, or empty vector. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are reported as the relative fold-increase compared with pGL3-Basic and represent the means ± SD of 3 replicates.

Identification of HNF-4 response elements. To determine the elements required for the transactivation of OAT1 promoter activity by HNF-4, a series of deletion constructs of OAT1 were transfected into OK cells simultaneously with HNF-4, and luciferase activity was measured ( Fig. 3 ). With the -2747/+88 to -1191/+88 constructs, the promoter activity in the presence of HNF-4 increased seven- to ninefold compared with that in the absence of HNF-4. Deletion of the fragment spanning -1191 to -700 bp reduced the activation of the OAT1 promoter by HNF-4, and the -536/+88 to -140/+88 constructs showed the same level of activation by HNF-4 as the -700/+88 construct. Further deletion of the fragment spanning -140 to -79 bp almost abolished the responsiveness. These results suggested that the regions spanning -1191 to -700 and -140 to -79 bp contain the elements important for the transactivation of OAT1 promoter activity by HNF-4.

Fig. 3. Identification of the HNF-4 -responsive region in the human OAT1 promoter. A series of deleted promoter constructs [equimolar amounts of the -2747/+88 construct (500 ng)] and 500 ng of the HNF-4 expression vector or empty vector were transiently transfected into OK cells for luciferase assays. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are reported as the relative fold-increase compared with pGL3-Basic ( A ) or as the ratio of HNF-4 expression vector to empty vector ( B ) and represent the means ± SD of 3 replicates.

HNF-4 binds as a homodimer to a DNA sequence consisting of a direct repeat of AGGTCA-like hexamers separated by one or two nucleotides, which were designated as DR-1 and DR-2, respectively ( 12, 21 ). We investigated whether the regions mentioned above had DR-1 and DR-2 sites using NUBIScan, an in silico tool to identify nuclear receptor response elements ( 29 ). As a result, a DR-2-like element was identified between -875 and -862 bp in the -1191/-700 fragment, while neither a DR-1 nor DR-2 site was found in the -140/-79 fragment. Between -123 and -104, however, the -140/-79 fragment contained an inverted repeat of hexamers separated by eight nucleotides (IR-8), which was recently identified as a HNF-4 response element in the human apolipoprotein AV promoter ( 32 ).

HNF-4 binds to DR-2 and IR-8 elements. To confirm that HNF-4 was able to bind to the DR-2 and IR-8 elements within the OAT1 promoter, EMSA was performed using an OAT1 probe (-882/-853) containing the DR-2 element or OAT1 probe (-127/-100) containing the IR-8 element and nuclear extract from OK cells transiently transfected with HNF-4 (OK-HNF-4 NE). Both OAT1 probes formed a DNA-protein complex ( Fig. 4 B, lanes 2 and 9 ), and the formation of the complex was completely impaired by the addition of an excess amount of the unlabeled wild-type oligonucleotides ( Fig. 4 B, lanes 3 and 10 ). Mutations in DR-2 ( Fig. 4 A, mutDR-2-B, mutDR-2-AB) and in IR-8 ( Fig. 4 A, mutIR-8-A, mutIR-8-B, mutIR-8-AB) abolished the ability to compete with the formation of the complex, while mutDR-2-A weakly competed with the formation of the complex ( Fig. 4 B, lanes 4-6, 11-13 ). Furthermore, the DNA-protein complex was supershifted on the addition of the HNF-4 antibody ( Fig. 4 B, lanes 7 and 14 ). These results indicate that HNF-4 binds to DR-2 and IR-8 elements within the OAT1 promoter.

Fig. 4. EMSA using nuclear extract from OK cells transiently transfected with HNF-4 (OK-HNF-4 NE) and two human OAT1 probes, (-882/-853) and (-127/-100). A : sequence of the oligonucleotides used in EMSA. Mutations introduced into the oligonucleotides are shown in bold. B : OK-HNF-4 NE was incubated with the 32 P-labeled OAT1 oligonucleotide probe alone ( lanes 2 and 9 ), or in the presence of excess unlabeled wild-type oligonucleotides ( lanes 3 and 10 ), excess mutated oligonucleotides ( lanes 4-6 and 11-13 ), or antibody against HNF-4 ( lanes 7 and 14 ). In lanes 1 and 8, nuclear extract was not added.

Mutagenesis of DR-2 and IR-8 elements. To examine the contribution of the DR-2 and IR-8 elements to the responsiveness to HNF-4, mutations at these sites were introduced into the -2747/+88 construct, and OK cells were transfected. Mutations in DR-2-B and IR-8-B reduced the transactivation of OAT1 promoter activity by HNF-4 more than mutations in DR-2-A and IR-8-A, respectively (data not shown); hereafter, mutations in DR-2-B and IR-8-B are regarded as mutations in DR-2 and IR-8, respectively. As shown in Fig. 5, mutations in DR-2 and IR-8 reduced the responsiveness to HNF-4 to two-thirds and one-half of the wild-type level, respectively. Furthermore, mutations in both DR-2 and IR-8 reduced the responsiveness to one-third that of the wild-type. These results suggest that the DR-2 and IR-8 elements in the OAT1 promoter play an important role in the responsiveness to HNF-4 and that IR-8 contributed to the activation by HNF-4 more than did DR-2.

Fig. 5. Effects of mutation in the direct repeat (DR)-2 and inverted repeat (IR)-8 elements on the responsiveness to HNF-4. The mutated -2747/+88 construct (500 ng) and 500 ng of the HNF-4 expression vector or empty vector were transiently transfected into OK cells for luciferase assays. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are reported as the relative fold-increase compared with pGL3-Basic or as the ratio of HNF-4 expression vector to empty vector and represent the means ± SD of 3 replicates. X, Presence of site-directed mutations.

HNF-4 is responsible for basal OAT1 promoter activity. Focusing on the promoter activity in the absence of HNF-4 ( Fig. 5 ), we found that mutations in IR-8 also reduced OAT1 promoter activity, suggesting that IR-8 is essential for basal promoter activity of OAT1. To investigate the influence of IR-8 on basal activity, we carried out a progressive deletion analysis and a mutational analysis. Transfection of the -2747/+88 construct resulted in a fivefold increase in luciferase activity compared with that of pGL3-Basic, and serial 5'-deletions of the construct from -2747 to -1851, -1191, -700, -536, -270, and -140 gradually increased luciferase activity ( Fig. 6 A ). However, deletion of the fragment spanning -140 to -79 bp significantly reduced the luciferase activity ( Fig. 6 A ). These results suggested that the -140/-79 region containing IR-8 is essential for basal transcriptional activity.

Fig. 6. Identification of the elements important for basal promoter activity of human OAT1. A : deletion analysis of the human OAT1 promoter in OK cells. A series of deleted promoter constructs [equimolar amounts of the -2747/+88 construct (500 ng)] were transfected into OK cells for luciferase assays. B : mutational analysis of the IR-8 in the human OAT1 promoter. The deleted or mutated constructs [equimolar amounts of the -140/+88 construct (330 ng)] were transfected into OK cells for luciferase assays. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are reported as the relative fold-increase compared with pGL3-Basic and represent the means ± SD of 3 replicates. X, Presence of site-directed mutations.

Next, a mutation in IR-8 was introduced into the -140/+88 construct, and luciferase activity was measured. The mutation reduced luciferase activity to one-third of the wild-type level, which was the same level of activity as the -79/+88 construct ( Fig. 6 B ), suggesting that IR-8 is responsible for basal promoter activity of OAT1.

To examine whether endogenous HNF-4 binds to IR-8 without the overexpression of HNF-4, EMSA was performed using an OAT1 probe (-127/-100) and nuclear extract from intact OK cells (OK-intact NE). Like OK-HNF-4 NE, OK-intact NE also formed a DNA-protein complex which was supershifted on the addition of the HNF-4 antibody ( Fig. 7, lanes 2 and 7 ), suggesting that endogenous HNF-4 binds to IR-8 without the overexpression of HNF-4. In competition experiments, mutIR-8-B and mutIR-8-AB did not impair the formation of the DNA-protein complex, while mutIR-8-A weakly competed with the formation of the complex ( Fig. 7, lanes 4-6 ).

Fig. 7. EMSA using nuclear extract from intact OK cells (OK-intact NE) and a human OAT1 probe (-127/-100). OK-intact NE was incubated with the 32 P-labeled OAT1 oligonucleotide probe (-127/-100) alone ( lane 2 ), or in the presence of excess unlabeled oligonucleotide (-127/-100; lane 3 ), excess mutated oligonucleotides ( lanes 4-6 ), or antibody against HNF-4 ( lane 7 ). In lane 1, nuclear extract was not added.

DISCUSSION

HNF-4 transactivates human OAT1 promoter activity, and this effect occurs mainly via DR-2 and IR-8 elements, as demonstrated by the deletion analysis, mutational analysis, and EMSA. Furthermore, it was found that HNF-4 is also responsible for basal promoter activity of OAT1 via IR-8 ( Fig. 8 ). Prieur et al. ( 32 ) proposed that IR-8 is a novel HNF-4 response element, and our results support this. These findings may be useful for further understanding gene regulation by HNF-4.

Fig. 8. Schematic model of transcriptional regulation of the human OAT1 gene. In the abundance of HNF-4, HNF-4 transactivates human OAT1 promoter activity via DR-2 and IR-8 elements. IR-8 contributes to the activation by HNF-4 more than DR-2 (HNF-4 overexpression). Under normal conditions, IR-8 is responsible for the basal promoter activity of OAT1, and HNF-4 binds to IR-8 (constitutive expression).

HNF-4 is an orphan member of the nuclear receptor superfamily that is expressed in the liver, kidney, intestine, stomach, and pancreas ( 27, 40 ). Liver-specific disruption of the HNF-4 gene indicated that HNF-4 is central to the maintenance of hepatocyte differentiation and lipid homeostasis ( 16 ). On the other hand, mutations in the gene encoding HNF-4 cause maturity-onset diabetes of the young (MODY), which is a genetically heterogeneous monogenic form of type 2 diabetes, associated with pancreatic -cell dysfunction ( 46 ). In contrast to the roles of HNF-4 in the liver and pancreas, little has been reported on the physiological implications of HNF-4 in the kidney. In the present study, we clearly indicated that the renal OAT1 gene is transcriptionally regulated by HNF-4. Because HNF-4 and OAT1 protein showed the restricted intrarenal localization, that is, in the renal proximal tubular epithelial cells ( 22, 26 ), it is strongly suggested that HNF-4 is a key factor regulating the intrarenal expression of OAT1 protein.

In the EMSA using OK-HNF-4 NE, the mutDR-2-A oligonucleotide containing an intact DR-2-B competed more with the formation of the DNA-protein complex than did the mutDR-2-B oligonucleotide containing an intact DR-2-A ( Fig. 4 B, lanes 4 and 5 ). Similarly, in the EMSA using OK-intact NE, HNF-4 bound to the mutIR-8-A oligonucleotide containing an intact IR-8-B with higher affinity than the mutIR-8-B oligonucleotide containing an intact IR-8-A ( Fig. 7, lanes 4 and 5 ). These results indicate that DR-2-B and IR-8-B had greater activity to bind HNF-4 than did DR-2-A and IR-8-A, respectively, which are consistent with the finding that mutations in DR-2-B and IR-8-B reduced the responsiveness to HNF-4 more than mutations in DR-2-A and IR-8-A, respectively (data not shown). These findings suggest that DR-2-B and IR-8-B are important to the transactivation of the OAT1 promoter via HNF-4 compared with DR-2-A and IR-8-A. In the EMSA using OK-HNF-4 NE, both oligonucleotides mutIR-8-A and mutIR-8-B failed to impair the formation of the DNA-protein complex ( Fig. 4 B, lanes 11 and 12 ). It is possible that the abundance of HNF-4 caused the mutIR-8-A oligonucleotide to fail to completely compete with the formation of the complex.

It has been demonstrated that individual variation in the expression levels of drug transporters is responsible for the individual variation in pharmacokinetics by experiments using human tissue ( 15, 35, 36 ) and laboratory animals ( 11, 14, 20 ). Single nucleotide polymorphisms in the coding region [coding SNPs (cSNPs)] of drug transporter genes are also thought to be responsible for the variation in drug responses among individuals ( 19 ). Although there have been few reports on cSNPs of the human OAT1 gene, allele frequencies of these cSNPs were very low ( 5, 13, 45 ). In addition, Fujita et al. ( 13 ) reported that there was no significant decrease in the renal secretory clearance of adefovir in subjects with R454Q, which resulted in a complete loss of OAT1 function in vitro. These reports suggest that cSNPs of human OAT1 are unlikely to influence the interindividual variation in the drug responses and that OAT1 expression is an alternative candidate for the cause of the variation in pharmacokinetics among individuals. Recent studies suggest that SNPs in the promoter region [regulatory SNPs (rSNPs)] can alter the transcription of genes ( 7 ). In the OAT1 gene, Bhatnagar et al. ( 4 ) found one rSNP 3 kb upstream of the transcription start site, but it is unclear whether this rSNP affects the mRNA level of OAT1. We previously reported that the OAT1 mRNA level varies and is significantly lower in the kidney of patients with renal diseases than in the normal kidney cortex ( 36 ). Accordingly, it is important to clarify the cause of the interindividual differences in OAT1 mRNA expression and the reduction in renal diseases. The present study suggests that HNF-4 regulates OAT1 expression at the mRNA level. Regulatory SNPs of OAT1, the HNF-4 expression level, or cSNPs in the HNF-4 gene that led to a loss of function may be the cause of the interindividual variation in OAT1 mRNA levels.

Tissue-specific gene expression is generally regulated by transcription factors ( 48 ). For example, we previously demonstrated that the intestine-specific transcription factor Cdx2 is responsible for the intestine-specific expression of H + /peptide cotransporter 1 ( 38 ). OAT1 mRNA is mainly expressed in the kidney ( 1 ), and we found that HNF-4 is responsible for the transcriptional regulation of the OAT1 gene. Interestingly, the major expression site of HNF-4 is the liver, but OAT1 mRNA is not expressed in the liver. Furthermore, the human OCT1 gene is also under the control of HNF- 4 ( 34 ), but OCT1 mRNA is expressed in the liver but not in the kidney. Thus it is difficult to explain the renal-specific expression of OAT1 by HNF-4 itself. Other transcription factors that suppress the hepatic expression of OAT1 (or renal expression of OCT1) may contribute to tissue-specific expression. Alternatively, microRNA and DNA methylation of the promoter may be involved in the tissue-specific expression of genes ( 39, 44 ). Further studies are needed to elucidate the mechanism of tissue-specific expression of the SLC22A family.

In conclusion, the present study indicates that the human OAT1 gene is regulated by HNF-4. This is the first report on the transcriptional regulation of OAT1. In addition, OAT1 is the first gene in the kidney whose promoter activity has been demonstrated to be regulated by HNF-4.

GRANTS

This work was supported in part by the 21st Century COE program "Knowledge Information Infrastructure for Genome Science," a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and a Grant-in-Aid for Research on Advanced Medical Technology from the Ministry of Health, Labor and Welfare of Japan. J. Asaka is supported as a research assistant by the 21st Century COE program "Knowledge Information Infrastructure for Genome Science."

ACKNOWLEDGMENTS

We thank Dr. Marco Pontoglio (Institute Pasteur, Paris, France) for kindly providing the human HNF-1 and HNF-1 expression vectors.

【参考文献】
  Anzai N, Kanai Y, Endou H. Organic anion transporter family: current knowledge. J Pharmacol Sci 100: 411-426, 2006.

Bahn A, Prawitt D, Buttler D, Reid G, Enklaar T, Wolff NA, Ebbinghaus C, Hillemann A, Schulten HJ, Gunawan B, Fuzesi L, Zabel B, Burckhardt G. Genomic structure and in vivo expression of the human organic anion transporter 1 (hOAT1) gene. Biochem Biophys Res Commun 275: 623-630, 2000.

Baumhueter S, Mendel DB, Conley PB, Kuo CJ, Turk C, Graves MK, Edwards CA, Courtois G, Crabtree GR. HNF-1 shares three sequence motifs with the POU domain proteins and is identical to LF-B1 and APF. Genes Dev 4: 372-379, 1990.

Bhatnagar V, Xu G, Hamilton BA, Truong DM, Eraly SA, Wu W, Nigam SK. Analyses of 5' regulatory region polymorphisms in human SLC22A6 (OAT1) and SLC22A8 (OAT3). J Hum Genet 51: 575-580, 2006.

Bleasby K, Hall LA, Perry JL, Mohrenweiser HW, Pritchard JB. Functional consequences of single nucleotide polymorphisms in the human organic anion transporter hOAT1 (SLC22A6). J Pharmacol Exp Ther 314: 923-931, 2005.

Brandoni A, Villar SR, Picena JC, Anzai N, Endou H, Torres AM. Expression of rat renal cortical OAT1 and OAT3 in response to acute biliary obstruction. Hepatology 43: 1092-1100, 2006.

Buckland PR. The importance and identification of regulatory polymorphisms and their mechanisms of action. Biochim Biophys Acta 1762: 17-28, 2006.

Burckhardt BC, Burckhardt G. Transport of organic anions across the basolateral membrane of proximal tubule cells. Rev Physiol Biochem Pharmacol 146: 95-158, 2003.

Cheret C, Doyen A, Yaniv M, Pontoglio M. Hepatocyte nuclear factor 1 controls renal expression of the Npt1-Npt4 anionic transporter locus. J Mol Biol 322: 929-941, 2002.

De Simone V, De Magistris L, Lazzaro D, Gerstner J, Monaci P, Nicosia A, Cortese R. LFB3, a heterodimer-forming homeoprotein of the LFB1 family, is expressed in specialized epithelia. EMBO J 10: 1435-1443, 1991.

Deguchi T, Takemoto M, Uehara N, Lindup WE, Suenaga A, Otagiri M. Renal clearance of endogenous hippurate correlates with expression levels of renal organic anion transporters in uremic rats. J Pharmacol Exp Ther 314: 932-938, 2005.

Fraser JD, Martinez V, Straney R, Briggs MR. DNA binding and transcription activation specificity of hepatocyte nuclear factor 4. Nucleic Acids Res 26: 2702-2707, 1998.

Fujita T, Brown C, Carlson EJ, Taylor T, de la Cruz M, Johns SJ, Stryke D, Kawamoto M, Fujita K, Castro R, Chen CW, Lin ET, Brett CM, Burchard EG, Ferrin TE, Huang CC, Leabman MK, Giacomini KM. Functional analysis of polymorphisms in the organic anion transporter, SLC22A6 (OAT1). Pharmacogenet Genomics 15: 201-209, 2005.

Habu Y, Yano I, Okuda M, Fukatsu A, Inui K. Restored expression and activity of organic ion transporters rOAT1, rOAT3 and rOCT2 after hyperuricemia in the rat kidney. Biochem Pharmacol 69: 993-999, 2005.

Hashida T, Masuda S, Uemoto S, Saito H, Tanaka K, Inui K. Pharmacokinetic and prognostic significance of intestinal MDR1 expression in recipients of living-donor liver transplantation. Clin Pharmacol Ther 69: 308-316, 2001.

Hayhurst GP, Lee YH, Lambert G, Ward JM, Gonzalez FJ. Hepatocyte nuclear factor 4 (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis. Mol Cell Biol 21: 1393-1403, 2001.

Hori R, Okamura M, Takayama A, Hirozane K, Takano M. Transport of organic anion in the OK kidney epithelial cell line. Am J Physiol Renal Fluid Electrolyte Physiol 264: F975-F980, 1993.

Inui K, Masuda S, Saito H. Cellular and molecular aspects of drug transport in the kidney. Kidney Int 58: 944-958, 2000.

Ishikawa T, Tsuji A, Inui K, Sai Y, Anzai N, Wada M, Endou H, Sumino Y. The genetic polymorphism of drug transporters: functional analysis approaches. Pharmacogenomics 5: 67-99, 2004.

Ji L, Masuda S, Saito H, Inui K. Down-regulation of rat organic cation transporter rOCT2 by 5/6 nephrectomy. Kidney Int 62: 514-524, 2002.

Jiang G, Nepomuceno L, Hopkins K, Sladek FM. Exclusive homodimerization of the orphan receptor hepatocyte nuclear factor 4 defines a new subclass of nuclear receptors. Mol Cell Biol 15: 5131-5143, 1995.

Jiang S, Tanaka T, Iwanari H, Hotta H, Yamashita H, Kumakura J, Watanabe Y, Uchiyama Y, Aburatani H, Hamakubo T, Kodama T, Naito M. Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4 (HNF4 ) isoforms in human and rats. Nucl Recept 1: 5, 2003.

Kikuchi R, Kusuhara H, Hattori N, Shiota K, Kim I, Gonzalez FJ, Sugiyama Y. Regulation of the expression of human organic anion transporter 3 by hepatocyte nuclear factor 1 / and DNA methylation. Mol Pharmacol 70: 887-896, 2006.

Maher JM, Slitt AL, Callaghan TN, Cheng X, Cheung C, Gonzalez FJ, Klaassen CD. Alterations in transporter expression in liver, kidney, and duodenum after targeted disruption of the transcription factor HNF1. Biochem Pharmacol 72: 512-522, 2006.

Monica Torres A, Mac Laughlin M, Muller A, Brandoni A, Anzai N, Endou H. Altered renal elimination of organic anions in rats with chronic renal failure. Biochim Biophys Acta 1740: 29-37, 2005.

Motohashi H, Sakurai Y, Saito H, Masuda S, Urakami Y, Goto M, Fukatsu A, Ogawa O, Inui K. Gene expression levels and immunolocalization of organic ion transporters in the human kidney. J Am Soc Nephrol 13: 866-874, 2002.

Nakhei H, Lingott A, Lemm I, Ryffel GU. An alternative splice variant of the tissue specific transcription factor HNF4 predominates in undifferentiated murine cell types. Nucleic Acids Res 26: 497-504, 1998.

Ogasawara K, Terada T, Asaka J, Katsura T, Inui K. Human organic anion transporter 3 gene is regulated constitutively and inducibly via a cAMP-response element. J Pharmacol Exp Ther 319: 317-322, 2006.

Podvinec M, Kaufmann MR, Handschin C, Meyer UA. NUBIScan, an in silico approach for prediction of nuclear receptor response elements. Mol Endocrinology 16: 1269-1279, 2002.

Pontoglio M, Prie D, Cheret C, Doyen A, Leroy C, Froguel P, Velho G, Yaniv M, Friedlander G. HNF1 controls renal glucose reabsorption in mouse and man. EMBO Rep 1: 359-365, 2000.

Popowski K, Eloranta JJ, Saborowski M, Fried M, Meier PJ, Kullak-Ublick GA. The human organic anion transporter 2 gene is transactivated by hepatocyte nuclear factor-4 and suppressed by bile acids. Mol Pharmacol 67: 1629-1638, 2005.

Prieur X, Schaap FG, Coste H, Rodriguez JC. Hepatocyte nuclear factor-4 regulates the human apolipoprotein AV gene: identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor- coactivator-1, AMP-activated protein kinase, and mitogen-activated protein kinase pathway. Mol Endocrinology 19: 3107-3125, 2005.

Russel FG, Masereeuw R, van Aubel RA. Molecular aspects of renal anionic drug transport. Annu Rev Physiol 64: 563-594, 2002.

Saborowski M, Kullak-Ublick GA, Eloranta JJ. The human organic cation transporter-1 gene is transactivated by hepatocyte nuclear factor-4. J Pharmacol Exp Ther 317: 778-785, 2006.

Sakurai Y, Motohashi H, Ogasawara K, Terada T, Masuda S, Katsura T, Mori N, Matsuura M, Doi T, Fukatsu A, Inui K. Pharmacokinetic significance of renal OAT3 (SLC22A8) for anionic drug elimination in patients with mesangial proliferative glomerulonephritis. Pharm Res 22: 2016-2022, 2005.

Sakurai Y, Motohashi H, Ueo H, Masuda S, Saito H, Okuda M, Mori N, Matsuura M, Doi T, Fukatsu A, Ogawa O, Inui K. Expression levels of renal organic anion transporters (OATs) and their correlation with anionic drug excretion in patients with renal diseases. Pharm Res 21: 61-67, 2004.

Shimakura J, Terada T, Katsura T, Inui K. Characterization of the human peptide transporter PEPT1 promoter: Sp1 functions as a basal transcriptional regulator of human PEPT1. Am J Physiol Gastrointest Liver Physiol 289: G471-G477, 2005.

Shimakura J, Terada T, Shimada Y, Katsura T, Inui K. The transcription factor Cdx2 regulates the intestine-specific expression of human peptide transporter 1 through functional interaction with Sp1. Biochem Pharmacol 71: 1581-1588, 2006.

Shiota K. DNA methylation profiles of CpG islands for cellular differentiation and development in mammals. Cytogenet Genome Res 105: 325-334, 2004.

Sladek FM, Zhong WM, Lai E, Darnell JE Jr. Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev 4: 2353-2365, 1990.

Sweet DH. Organic anion transporter (Slc22a) family members as mediators of toxicity. Toxicol Appl Pharmacol 204: 198-215, 2005.

Terada T, Inui K. Gene expression and regulation of drug transporters in the intestine and kidney. Biochem Pharmacol 73: 440-449, 2007.

Villar SR, Brandoni A, Anzai N, Endou H, Torres AM. Altered expression of rat renal cortical OAT1 and OAT3 in response to bilateral ureteral obstruction. Kidney Int 68: 2704-2713, 2005.

Wienholds E, Plasterk RH. MicroRNA function in animal development. FEBS Lett 579: 5911-5922, 2005.

Xu G, Bhatnagar V, Wen G, Hamilton BA, Eraly SA, Nigam SK. Analyses of coding region polymorphisms in apical and basolateral human organic anion transporter (OAT) genes [OAT1 (NKT), OAT2, OAT3, OAT4, URAT (RST)]. Kidney Int 68: 1491-1499, 2005.

Yamagata K, Furuta H, Oda N, Kaisaki PJ, Menzel S, Cox NJ, Fajans SS, Signorini S, Stoffel M, Bell GI. Mutations in the hepatocyte nuclear factor-4 gene in maturity-onset diabetes of the young (MODY1). Nature 384: 458-460, 1996.

You G. Towards an understanding of organic anion transporters: structure-function relationships. Med Res Rev 24: 762-774, 2004.

Yu X, Lin J, Zack DJ, Qian J. Computational analysis of tissue-specific combinatorial gene regulation: predicting interaction between transcription factors in human tissues. Nucleic Acids Res 34: 4925-4936, 2006.

作者单位：Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto, Japan