Human organic cation transporter 1 (OCT1/SLC22A1) plays important roles in the hepatic uptake of cationic drugs. The functional characteristics of this transporter have been well evaluated, but molecular information regarding transcriptional regulation is limited. In the present study, therefore, we examined the gene regulation of OCT1 gene focusing on basal core expression. An ∼2.5-kb fragment of the OCT1 promoter region was isolated, and promoter activity was measured by luciferase assay in the human liver cell lines Huh7 and HepG2. Deletion analysis suggested that the region spanning −141/−69 was essential for the basal core transcriptional activity and that this region contained the sequence of a cognate E-box (CACGTG). The E-box is known to be bound by the basal transcription factors, upstream stimulating factors (USFs), and the functional involvements of USF1 and USF2 were confirmed by a transactivation effect, a mutational analysis of the E-box, and an electrophoretic mobility shift assay. The transactivation effect of USFs on the OCT1 promoter was further stimulated by hepatocyte nuclear factor 4α, a liver-enriched transcription factor. There were no polymorphisms in the proximal promoter region (about 400 bp) of OCT1 gene (n = 109). These findings indicated that both USF1 and USF2 bind to an E-box sequence located in the OCT1 core promoter region and are required for the basal gene expression of this transporter.
organic cation transporters (OCTs) play important roles in the disposition and elimination of numerous cationic compounds, including clinically used drugs. Organic cation transporters are functionally classified into two types: membrane potential-dependent OCTs and H+/organic cation antiporters (5, 10, 18). There are three isoforms of membrane potential-dependent OCTs (OCT1-3/SLC22A1-3). Human OCT1 and OCT2 are expressed predominantly in the hepatic and renal basolateral membranes, respectively, whereas OCT3 is expressed in multiple tissues and organs such as skeletal muscle, the liver, the placenta, and the brain (11, 13). In vivo studies using Oct1−/− mice and clinical pharmacokinetic/pharmacogenetic analyses have demonstrated that hepatic OCT1 is responsible for the therapeutic efficacy and toxicity of a biguanide agent, metformin (21, 22, 25). For H+/organic cation antiporters, there are two isoforms, multidrug and toxin extrusion 1 (MATE1)/SLC47A1 and MATE2-K/SLC47A2, and their tissue expression and functional characteristics have been demonstrated (24). MATE1 is expressed in the kidney and liver, whereas MATE2-K is exclusively expressed in the kidney, and both transporters mediate the transport of cationic drugs such as metformin and cimetidine (H2-blocker).
In addition to the information described above, the transcriptional mechanisms of these transporters have been recently characterized. For example, basal transcription of the OCT2 and MATE1 genes was stimulated by upstream stimulating factor (USF) 1 and Sp1, respectively (3, 12). Although hepatic expression of OCT1 was shown to be mediated by hepatocyte nuclear factor-4α (HNF-4α) via two direct repeat (DR)-2 sites (19), the core promoter region and basal transcriptional mechanisms of OCT1 gene remain unclear. The core promoter and/or proximal promoter regions contain elements that control the initiation of transcription, and, therefore, essential regions that harbor functionally relevant polymorphisms may have significant effects on gene expression (4). We have recently identified a single nucleotide polymorphism (SNP) in the regulatory region: regulatory SNP (rSNP) of the MATE1 gene (−32G>A), which belongs to a Sp1 binding site (12). This rSNP caused a decrease in Sp1 binding and promoter activity of about 50%.
In the present study, we characterized the promoter activity of OCT1 to identify the core promoter region and performed rSNP analyses for OCT1 gene in the core promoter region.
MATERIALS AND METHODS
[γ-32P]ATP was obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Restriction enzymes were from New England BioLabs (Beverly, MA). Antibodies against USF1 (sc-8983X) and USF2 (sc-862X) used for Western blot analyses and supershift assays were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals used were of the highest purity available.
Determination of the putative transcription start site.
To identify the transcription start site of OCT1, 5′-rapid amplification of cDNA ends (5′-RACE) was carried out using human liver 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 OCT1 (GenBank accession number NM_003057), 5′-CCAGGAGTCAGCACACACCAGGTTGAAC-3′ (543/516), and a nested gene-specific primer for OCT1, 5′-CTGCTCCAGAATGTCATCCACGGTGGG-3′ (135/109). The PCR products were subcloned into the pGEM-T Easy Vector (Promega, Madison, WI). All PCR products were sequenced using a multicapillary DNA sequencer RISA384 system (Shimadzu, Kyoto, Japan).
Reporter constructs for the OCT1 promoter.
The OCT1 promoter was isolated from the human genomic DNA (Promega) by a PCR-based method using primers designed on the basis of the human genomic DNA (Table 1). The PCR product was isolated by electrophoresis and subcloned into the firefly luciferase reporter vector, pGL3-Basic (Promega), at NheI and XhoI sites. This full-length reporter plasmid is hereafter referred to as −2,516/+42. The 5′-deleted constructs (−1,686/+42, −886/+42, −653/+42, and −229/+42) were generated by digestion with appropriate restriction enzymes. 5′-Deleted constructs (−141/+42, −69/+42, and −36/+42) were prepared by PCR with the primers listed in Table 1. The site-directed mutations in the putative E-box were introduced into the OCT1 (−141/+42) construct with a QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the primers listed in Table 1. All PCR products and deletion constructs for reporter assays were sequenced using a multicapillary DNA sequencer RISA384 system (Shimadzu).
The preparation of cDNAs for USF1 and HNF-4α was described previously (3, 16). cDNA for USF2 (GenBank accession number: NM_003367) was isolated by PCR using primers listed in Table 1. The PCR product was subcloned into the expression vector pcDNA 3.1 (+) (Invitrogen, Carlsbad, CA), and its sequence was verified.
The human hepatoma cell lines Huh7 and HepG2 were obtained from the Health Science Research Resources Bank (Osaka, Japan) and American Type Culture Collection (Rockville, MD), respectively. Huh7 cells were maintained in DMEM (Sigma, St. Louis, MO) with 10% fetal calf serum (Invitrogen). HepG2 cells were grown in DMEM supplemented with 10% fetal calf serum, 1% nonessential amino acids (Invitrogen), and 1% pyruvic acid (Invitrogen). Both cells were plated into 24-well plates (1 × 105 cells/well) and transfected the following day for luciferase assays. Transfection and luciferase assays were carried out as described previously (2, 12, 16).
Western blot analysis.
The preparation of nuclear extracts from cultured cells was described previously (20). With the use of the nuclear extracts of Huh7 (60 μg), Western blot analyses were carried out as described previously (12) with some modifications. Namely, blots were blocked with 5% nonfat dry milk and 2% bovine serum albumin (Serological Proteins, Kankakee, IL) in Tris-buffered saline with 0.3% Tween 20 (TBS-T) for 1 h at room temperature. Blots were washed in 0.5% TBS-T and followed by overnight incubation at 4°C with the anti-USF1 or anti-USF2 polyclonal antibody (1 μg/ml in the same buffer with blocking).
A nuclear extract prepared from Huh7 cells was used for the EMSA. The probes listed in Fig. 2B were prepared by annealing complementary sense and antisense oligonucleotides, followed by end-labeling with [γ-32P]ATP using T4 polynucleotide kinase (Takara Bio, Otsu, Japan) and purification through a Sephadex G-25 column (GE Healthcare). The binding mixture consisted of 10 μg of Huh7 nuclear extract and unlabeled competitor probes in a buffer solution as reported by Dimova et al. (7). After preincubation at 4°C for 30 min, the labeled probe was added and the binding mixture was incubated for a further 30 min. For supershift assays, 2 μg of USF1 antibody (sc-8983X) or USF2 antibody (sc-862X) was added 30 min before the addition of the labeled probe. The volume of the binding mixture was 20 μl throughout the experiment. The DNA-protein complex was then separated on a 4% polyacrylamide gel at room temperature in 0.5× Tris-borate-EDTA buffer. The gels were dried and exposed to X-ray film for autoradiography.
Genomic DNA was extracted from noncancerous parts of the liver from 109 patients with hepatectomy using a Wizard Genomic DNA Purification Kit (Promega). The promoter region of the OCT1 gene was amplified by PCR using as a forward primer (GenBank accession number: NT_007422), 5′-ACCTTGTTGTGCTTGTATTCCATTGTTCA-3′ (−1,247/−1,219), and as a reverse primer, 5′-TAAGATGAGGAAGGCTTGCTTCTG-3′ (180/157). PCR conditions were as follows: denaturation at 95°C for 3 min, annealing and synthesis at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, 40 cycles, followed by a single additional 10-min extension at 72°C. All PCR products were sequenced using a multicapillary DNA sequencer RISA384 system (Shimadzu) by the following primer: 5′-ATCTCACAAATCACCACTAAAGA-3′ (−440/−418).
This study was conducted in accordance with the Declaration of Helsinki and its amendments and was approved by the Kyoto University Graduate School and Faculty of Medicine, Ethics Committee. All patients gave their written informed consent.
The results were relative to pGL3-Basic and represent the means ± SD for three replicates. Two or three experiments were conducted, and representative results are shown. Data were analyzed statistically with the one-way ANOVA, followed by Dunnett's test.
Determination of the transcription start site of OCT1.
Sequencing of the longest RACE product showed that the terminal position of OCT1 cDNA was located 105 nucleotides upstream of the start codon, which is the same as the 5′-end of OCT1 cDNA in the database (GenBank accession number NM_003057). Therefore, the 5′-end of OCT1 cDNA was numbered +1 as the transcription start site in this study.
Determination of the minimal OCT1 promoter.
To determine the minimal region required for basal activity of the core promoter, a series of OCT1 deletion constructs were transfected into the human hepatoma cell lines Huh7 and HepG2, and luciferase activity was measured. The luciferase activity decreased in the −886/+42 construct (Fig. 1). This may be due to the deletion of DR-2 sites, which are binding sites for HNF-4α. Further deletion from −653 to −141 resulted in an increase in promoter activity, and the luciferase activity of the OCT1 reporter construct in Huh7 cells was abolished with −69/+42. In HepG2 cells, luciferase activity was also reduced with −69/+42, suggesting that the region between −141 and −69 is important for the core promoter activity of the OCT1 gene. We then performed a computational sequence analysis of this region using TRANSFAC 6.0 software (http://www.gene-regulation.com) and found a completely matched sequence of an E-box (5′-CACGTG-3′) (Fig. 2A). The E-box is known to be stimulated by USF1 and USF2. These transcription factors are members of the eukaryotic evolutionarily conserved basic-helix-loop-helix-leucine zipper transcription factor family and are involved in the basal promoter activity of various genes (6). The protein expression of USF1 and USF2 in the nuclear extract of Huh7 cells was confirmed by Western blot analyses (Fig. 2C).
Functional involvement of USF1 and USF2 in OCT1 promoter activity.
We then evaluated the functional involvement of USFs via E-box in the OCT1 promoter activity. As shown in Fig. 3, mutations introduced into the E-box of the −141/+42 construct remarkably reduced promoter activity in both Huh7 and HepG2 cells. We next investigated the effect of the overexpression of USF1 or USF2 on the promoter activity of OCT1. The −141/+42 construct was cotransfected into Huh7 cells with the USF1 or USF2 expression vector. As shown in Fig. 4, the promoter activity of OCT1 (−141/+42) was increased by the coexpression of USF1 or USF2 in a dose-dependent manner, providing direct evidence that USF1 and USF2 enhanced promoter activity.
To determine whether USF1 and USF2 directly bind to the E-box, we performed an EMSA using the OCT1 probe (−107/−78) containing the E-box (Fig. 2B). The OCT1 probe formed a DNA-protein complex (Fig. 5, lane 2), and the formation of the complex was completely impaired by the addition of an excess amount of the unlabeled probe with a wild-type sequence but not by a mutated probe (Fig. 5, lanes 3 and 4). Furthermore, the DNA-protein complex was supershifted on addition of the USF1 or USF2 antibody (Fig. 5, lanes 5 and 6). These results indicate that USF1 and USF2 bind to the E-box sequence of the OCT1 promoter.
Transactivation of the promoter activity by USF1, USF2, and HNF-4α.
Saborowski et al. (19) reported that OCT1 promoter activity was stimulated by a liver-enriched transcription factor, HNF-4α, via DR-2 sites located at around −1.5 kb. To examine the synergetic effects of HNF-4α and USF1 or USF2 on OCT1 promoter activity, the OCT1 construct (−1,686/+42) was coexpressed with either USF1, USF2, HNF-4α, or a combination. As shown in Fig. 6, coexpression of USF1 or USF2 and HNF-4α showed additive effects to increase the promoter activity of OCT1, suggesting that these transcription factors coordinately function for OCT1 promoter activity.
rSNP analyses of OCT1 gene.
Finally, we sequenced the proximal promoter region (about 400 bp) of the OCT1 gene in 109 patients with hepatectomy and found no polymorphisms.
In the present study, we performed a functional promoter assay of the OCT1 gene, and clearly demonstrated that USF1 and USF2 play significant roles as core transcription factors through an E-box to the OCT1 promoter. This conclusion is supported by results of experiments involving the overexpression of USF1 and USF2, mutagenesis of the E-box, and EMSA. USF1 and USF2 are members of the eucaryotic evolutionarily conserved basic helix-loop-helix-leucine zipper transcription factor family (6). They interact with cognate E-box regulatory elements (CANNTG), which occur across the entire genome in eukaryotes, with the two central nucleotides (NN) in most cases either GC or CG (6). Because the E-box of the OCT1 promoter is CACGTG, a perfectly conserved consensus sequence, it is reasonable that both transcription factors play pivotal roles to the basal promoter activity of the OCT1 gene.
Previously, we reported that OCT2 promoter activity is also regulated by USF1 via the cognate E-box regulatory element (3), suggesting that OCT1 and OCT2 genes share the same molecules for basal expression. Although USF1 and USF2 are ubiquitously expressed (23), the mRNA expression profiles of OCT1 and OCT2 are completely different; that is, OCT1 mRNA is mainly expressed in the liver, whereas OCT2 mRNA is predominantly expressed in the kidney (9, 15). HNF-4α plays a crucial role in hepatocyte differentiation and maintenance of the hepatic gene expression profile and is involved in the transcriptional regulation of OCT1 gene (19). Although precise mechanisms remain to be clarified, the liver-enriched transcription factor HNF-4α may be mainly responsible for the liver-specific expression of OCT1 mRNA. Actually, we demonstrated that USF1 or USF2 coordinately regulates OCT1 promoter activity with HNF-4α (Fig. 6).
It has been demonstrated that methylation at the CpG site located centrally within the E-box motif (CACpGTG) strongly inhibits the formation of a transcription factor complex and negatively regulates gene expression (17, 26). Very recently, we have demonstrated that the renal-specific expression of OCT2 gene is also regulated by methylation of E-box (1). As an example of the liver-specific expression, Fujii et al. (8) reported that the E-box of the promoter of the hibernation-specific protein HP-27 is hypomethylated in the liver but highly methylated in the kidney and heart and is therefore involved in the liver-specific expression. However, we found that methylation status of E-box in the OCT1 promoter region is not different between the liver and kidney (1). These findings suggested that different epigenetic regulation of OCT1 and OCT2 genes may be involved in the incomprehensible gene regulation of both transporters via E-box.
It has been demonstrated that SNPs within the E-box core motif can modulate gene expression. For example, a single G to C base transition within the E-box of the thymidylate synthase gene prevents the USF complex from binding to its cognate sequence (14). Because a high frequency of G to C polymorphisms in all major racial and ethnic groups has been observed, a knowledge of this SNP would be useful to tailor individual chemotherapy with 5-fluorouracil, an inhibitor of thymidylate synthase. To find such SNPs in the promoter region of OCT1, we sequenced the proximal promoter region (about 400 bp) of the OCT1 gene in 109 individuals. However, we could not find any polymorphisms in the promoter region, including the E-box of the OCT1 gene. Recently, it has been demonstrated that several SNPs in the coding region (cSNPs) of the OCT1 gene reduce transport activity and that these cSNPs had a significant effect on the pharmacokinetics and pharmacodynamics of metformin, an anti-diabetic agent (21, 22). Although ethnic differences should be considered, rSNPs for the OCT1 gene may not be involved in the interindividual variation in the pharmacokinetics and pharmacodynamics of metformin.
In conclusion, the present study clearly indicated that USF1 and USF2 function as core transcriptional regulators of the OCT1 gene through an E-box. These findings should serve as a basis for future investigations into the molecular regulation of the transport of organic cations and some pharmaceuticals in the human liver.
This work was supported in part by 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.
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