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Am J Physiol Renal Physiol 295: F165-F170, 2008. First published May 28, 2008; doi:10.1152/ajprenal.90257.2008
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Kidney-specific expression of human organic cation transporter 2 (OCT2/SLC22A2) is regulated by DNA methylation

Masayo Aoki,1 Tomohiro Terada,1 Moto Kajiwara,1 Ken Ogasawara,1 Iwao Ikai,2 Osamu Ogawa,3 Toshiya Katsura,1 and Ken-ichi Inui1

1Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, 2Department of Surgery, Graduate School of Medicine, and 3Department of Urology, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto, Japan

Submitted 18 April 2008 ; accepted in final form 23 May 2008


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Human organic cation transporter 2 (OCT2/SLC22A2), which is specifically expressed in the kidney, plays critical roles in the renal secretion of cationic compounds. Tissue expression and membrane localization of OCT2 are closely related to the tissue distribution, pharmacological effects, and/or adverse effects of its substrate drugs. However, the molecular mechanisms underlying the kidney-specific expression of OCT2 have not been elucidated. In the present study, therefore, we examined the contribution of DNA methylation of the promoter region for the OCT2 gene to its tissue-specific expression using human tissue samples. In vivo methylation status of the proximal promoter region of OCT2 and that of OCT1, a liver-specific organic cation transporter, were investigated by bisulfite sequencing using human genomic DNA extracted from the kidney and liver. All CpG sites in the OCT2 proximal promoter were hypermethylated in the liver, while hypomethylated in the kidney. On the other hand, the promoter region of OCT1 was hypermethylated in both the kidney and liver. The level of methylation of the OCT2 promoter was especially low at the CpG site in the E-box, the binding site of the basal transcription factor upstream stimulating factor (USF) 1. In vitro methylation of the OCT2 proximal promoter dramatically reduced the transcriptional activity, and an electrophoretic mobility shift assay showed that methylation at the E-box inhibited the binding of USF1. These results indicate that kidney-specific expression of human OCT2 is regulated by methylation of the proximal promoter region, interfering with the transactivation by USF1.

drug transporter; renal secretion; tissue-specific gene expression; epigenetics

THE ORGANIC CATION TRANSPORTER (OCT) family plays critical roles in the renal secretion and hepatic uptake of endogenous and exogenous organic cations. Among the OCT family, the tissue distribution of each isoform is quite different. Human (h) OCT1 is primarily expressed at the basolateral membrane of hepatocytes (12, 14). On the other hand, OCT2 is mainly expressed at the basolateral membrane of renal tubular cells as a major transporter involved in the secretion of organic cations in the kidney (12, 18). In the kidney, cationic drugs such as cimetidine, metformin, and procainamide, and endogenous organic cations such as N1-methylnicotineamide are recognized by OCT2 and are excreted into the urine (11, 13). Thus tissue expression and membrane localization of OCT2 are closely related to the tissue distribution, pharmacological effects, and/or adverse effects of its substrates. For example, cisplatin, a platinum-based anticancer drug widely used in chemotherapies of various solid tumors, is also a substrate of OCT2 (7, 30). Despite the effectiveness of cisplatin, severe nephrotoxicity limits its clinical application, although other platinum agents show little nephrotoxicity. We reported that the kidney-specific expression of OCT2 is involved in the renal distribution and accumulation of cisplatin, leading to the nephrotoxicity (29–31). However, the mechanism underlying the kidney-specific expression of OCT2 has not been elucidated.

In a previous study, we demonstrated that upstream stimulating factor (USF) 1 functions as a basal transcriptional regulator of the OCT2 gene via the E-box-regulatory element (CACGTG) (2). As USF1 is ubiquitously expressed (26), this transcription factor may not be responsible for the tissue-specific expression of OCT2. Alternatively, we were interested in DNA methylation, a common epigenetic process of gene regulation, for the kidney-specific expression of OCT2. Methylation of the cytosine residue in a CpG dinucleotide is involved in imprinting, X inactivation, and oncogenesis (5, 9, 23). It has also been reported that DNA methylation at the promoter region plays important roles in the regulation of tissue-specific expression of various genes. For example, the tissue-specific expression of the mouse Abcc6 gene is regulated by DNA methylation; high and moderate levels of methylation correlate with low levels of Abcc6 expression in the tail extremity, skin, and kidney, while a low level of methylation correlates with a high level of Abcc6 expression in the liver (10). Like this, there have been several reports of culture cell lines and tissue samples from a rat or mouse showing such differences in the methylation status among tissues. However, there have been few reports of epigenetic gene regulation of drug transporters using human tissue samples.

In the present study, we focused on DNA methylation of the OCT2 proximal promoter and investigated the tissue-specific methylation status and effects on the basal transcriptional regulation via USF1.


   MATERIALS AND METHODS
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Materials. Restriction enzymes were obtained from New England Biolabs (Beverly, MA). Oligonucleotides were purchased from Sigma-Aldrich Japan (Tokyo, Japan). T4 polynucleotide kinase was purchased from Takara Bio (Otsu, Japan). {gamma}-[32P]ATP was obtained from GE Healthcare Life Sciences (Buckinghamshire, UK). Anti-USF1 antibody (SC-8983X) used for a supershift assay was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals used were of the highest purity available.

Tissue samples. Noncancerous parts of renal and hepatic tissues were obtained respectively from 12 patients with renal cell carcinoma (11 men and 1 woman) and 11 patients with hepatocellular carcinoma (5 men and 6 women) at Kyoto University Hospital. 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.

Quantification of OCT1, OCT2, and USF1 mRNA expression. Total RNA from various human tissues was purchased from BioChain (Hayward, CA). Reverse transcription of the total RNA (500 ng/20 µl reaction) and real-time PCR were carried out as described previously (18). The primer-probe sets used for OCT1 and OCT2 were designed previously (18). The primer-probe sets used for USF1 were predeveloped TaqMan Assay Reagents (Applied Biosystems, Foster City, CA). GAPDH mRNA was also measured as an internal control with GAPDH Control Reagent (Applied Biosystems).

Bisulfite sequencing. Genomic DNA was extracted from the kidney and liver using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Sodium bisulfite treatment using the CpGenome DNA Modification Kit (Chemicon International) was performed according to the manufacturer's instructions. Converted DNA was amplified by PCR using primers listed in Table 1. The PCR conditions were as follows: denaturation at 94°C for 3 min, annealing and synthesis at 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, 40 cycles, followed by a single additional 10-min extension at 72°C. PCR products were purified with the Wizard DNA clean-up System (Promega) and subcloned into pCR4-TOPO (Invitrogen, Carlsbad, CA), and 10 clones for each tissue were sequenced with M13 forward and reverse primers using the multicapillary DNA sequencer RISA384 system (Shimadzu, Kyoto, Japan).


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  		Table 1. Sequences of primers

 
In vitro methylation of OCT2 promoter. The plasmid carrying the OCT2 promoter (–91/+23) and firefly luciferase reporter was constructed previously (2). The transcription start site is numbered as +1, which is located at the –377 nucleotide above the translation start site. The nucleotide number was incorrectly counted as –385 in our previous report (2). The construct was methylated by SssI methylase (New England Biolabs) according to the procedure recommended by the manufacturer. Complete methylation was confirmed by resistance to HpaII digestion (New England Biolabs). For the unmethylated control (mock), pGL3-Basic (Promega) and the –91/+23 construct were treated with all components required for in vitro methylation except S-adenosylmethionine.

Cell culture and luciferase assay. The porcine kidney epithelial cell line LLC-PK1 was obtained from American Type Culture Collection (ATCC CRL-1392, Rockville, MD). Cell culture, transfection, and the luciferase assay were performed as described previously (3).

EMSA. Nuclear extract (NE) was prepared from LLC-PK1 cells according to the method of Shimakura et al. (24). The oligonucleotides listed in Table 1 were used as probes. EMSAs and supershift assays were carried out as described previously (2).

Data analysis. For the luciferase assay, the results were expressed relative to the mock-methylated pGL3-Basic and represent means ± SD of three replicates. Three experiments were conducted, and representative results are shown. Data were analyzed statistically with one-way ANOVA followed by Dunnett's test.


   RESULTS
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Quantification of OCT1, OCT2, and USF1 mRNA expression. To confirm the tissue-specific expression of OCT1 and OCT2, we first determined the mRNA expression levels of OCT1, OCT2, and USF1 in various human tissues. USF1 has been demonstrated to be the basal transcription factor for the OCT2 gene (2). As reported, OCT1 and OCT2 were predominantly expressed in the liver and kidney, respectively, while USF1 was expressed at similar levels in all tissues examined (Fig. 1).


Figure 1
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  		Fig. 1. Tissue distribution of organic cation transporter (OCT) 1, OCT2, and upstream stimulating factor (USF) 1 mRNA. RNA from various tissues was reverse transcribed, and OCT1, OCT2, USF1, and GAPDH mRNA levels were determined by real-time PCR using an ABI PRISM 7700 sequence detector. The mRNA expression levels of OCT1, OCT2, and USF1 are represented as a ratio to that of GAPDH in each tissue.

 
Bisulfite sequencing. To investigate the level of in vivo methylation of the OCT2 proximal promoter, we performed bisulfite sequencing using genomic DNA from two groups of human tissues, kidney and liver. There were nine CpG sites in the 5'-flanking sequence up to –250. As shown in Figs. 2 and 3, these CpG sites were hypermethylated in the liver, while in the kidney the methylation levels were relatively low. Notably, the CpG site within the USF1 binding site (E-box) at –85 was hypomethylated in the kidney. These results suggested that the methylation of the OCT2 promoter is involved in the kidney-specific expression of OCT2. We also analyzed the methylation status of the OCT1 proximal promoter (the 5'-flanking sequence up to –300, containing 7 CpG sites). OCT1 is expressed specifically in the liver, and our recent study suggests that the basal transcription of OCT1 is also regulated by USF1 via the E-box spanning –95 to –90. Interestingly, the methylation patterns of all CpG sites in the OCT1 promoter analyzed were similar between the two tissues (Figs. 4 and 5), suggesting that the methylation of the proximal promoter region of OCT1 is not involved in the tissue-specific expression.


Figure 2
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  		Fig. 2. Methylation status of nine CpG sites in the OCT2 promoter. Genomic DNA extracted from human kidney (n = 10) and liver (n = 10) was bisulfite-modified, amplified by PCR, and then subcloned into pCR4-TOPO, and 10 clones from each individual were sequenced. Each block of circles represents the methylation status of 9 CpG sites from individual clones. Open circles and closed circles represent unmethylated and methylated CpG sites, respectively. Numbers at the left indicate the CpG sites and are relative to the OCT2 transcription start site determined by our previous study (2).

 

Figure 3
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  		Fig. 3. Frequency of methylation of individual CpG sites in the OCT2 promoter. Frequency of methylation was calculated by dividing the total number of methylated clones by the total number of clones analyzed in bisulfite sequencing (100 clones each for the kidney and liver from Fig. 2). The CpG site at –85 is located in the E-box.

 

Figure 4
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  		Fig. 4. Methylation status of 7 CpG sites in the OCT1 promoter. Each block of circles represents the methylation status of 7 CpG sites from individual clones (kidney, n = 6; liver, n = 6). Open circles and closed circles represent unmethylated and methylated CpG sites, respectively. Numbers at the left indicate the CpG site and are relative to the OCT1 transcription start site reported previously (GenBank Accession No. NM_003057).

 

Figure 5
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  		Fig. 5. Frequency of methylation of individual CpG sites in the OCT1 promoter. Frequency of methylation was calculated by dividing the total number of methylated clones by the total number of clones analyzed in bisulfite sequencing (60 clones each for the kidney and liver from Fig. 4). The CpG site at –93 is located in the E-box.

 
In vitro methylation and luciferase assay. To examine the effect of CpG methylation on the transcriptional activity of OCT2 in vitro, the OCT2 reporter construct (–91/+23) containing the E-box was methylated in vitro, and its promoter activity was assessed in LLC-PK1 cells. We compared the relative luciferase activity of the methylated and mock-methylated constructs with a mock-methylated pGL3-Basic vector. The luciferase activity of the mock-methylated reporter construct (–91/+23) was as strong as that of the construct (–91/+23) without any treatment used in our previous study (2). On the other hand, the methylated construct (–91/+23) exhibited little luciferase activity (Fig. 6). These results suggest that CpG methylation of the OCT2 proximal promoter reduces the transcriptional activity.


Figure 6
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  		Fig. 6. Effect of methylation on OCT2 transcriptional activity. The luciferase constructs [pGL3-Basic and OCT2 (–91/+23)] were in vitro methylated (Met) or mock-methylated (mock) with SssI methylase and transfected into LLC-PK1 cells for luciferase assays. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are shown as the relative fold-increase compared with mock-methylated pGL3-Basic and are means ± SD of 3 replicates. **P < 0.01, significantly different from the value of mock-methylated OCT2 (–91/+23).

 
EMSA. To confirm whether CpG methylation at the E-box affected the binding of USF1, EMSA was carried out using nuclear extract from LLC-PK1 cells and the methylated and unmethylated OCT2 probes (–101/–74), which contain the E-box (Fig. 7). As we reported previously (2), the OCT2 (–101/–74) probe formed a DNA-protein complex (Fig. 7, lane 3) and this complex was supershifted by the addition of the anti-USF1 antibody (Fig. 7, lane 5). On the other hand, when the cytosine in the E-box was selectively methylated, the formation of a DNA-protein complex was prevented (Fig. 7, lane 4) and the supershifted band was not observed (Fig. 7, lane 6). These results indicate that CpG methylation at the E-box impedes the binding of USF1.


Figure 7
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  		Fig. 7. Effect of methylation at the E-box on USF1 binding. Nuclear extract from LLC-PK1 cells was incubated with the 32P-labeled oligonucleotide probes (OCT2 –101/–74) alone (lanes 3 and 4) or in the presence of anti-USF1 antibody (lanes 5 and 6). In lanes 1 and 2, nuclear extract was not added. In lanes 2, 4, and 6, the oligonucleotide probe (OCT2 –101/–74) was in vitro methylated specifically at cytosine in the E-box.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
OCT2 is specifically expressed in the kidney and plays important roles in the renal secretion of various cationic compounds (13). In a previous study, we demonstrated that USF1 functions as a basal transcriptional regulator of the OCT2 gene via the E-box (2). As USF1 is ubiquitously expressed, it is not likely to explain the kidney-specific expression of OCT2 by USF1. On the other hand, we previously reported that H+/peptide cotransporter 1 is mainly expressed in the small intestine and that its limited expression in the small intestine is determined by the intestinal-specific transcription factor Cdx2 (25). So far, kidney-specific transcription factors have not been identified. Therefore, in the case of OCT2, other mechanisms rather than the regulation by tissue-specific transcription factors may contribute to the kidney-specific expression.

In the present study, we investigated whether DNA methylation contributed to the kidney-specific expression of OCT2 and demonstrated that 1) the methylation status of CpG sites in the OCT2 proximal promoter, especially the CpG site in the E-box, varies between the kidney and liver; 2) in vitro methylation of the OCT2 proximal promoter dramatically reduces the transcriptional activity; and 3) site-specific methylation at the E-box inhibits the binding of USF1. These results clearly indicate that CpG methylation of the OCT2 proximal promoter plays important roles in the kidney-specific expression. Namely, in the kidney, USF1 binds to the E-box at the hypomethylated OCT2 promoter and enhances the level of basal transcription, and in other tissues, such as the liver, CpG methylation prevents the binding of USF1, leading to the repression of OCT2 gene expression. In rat or mouse genes such as the rat N-cadherin (1), mouse urate transporter 1 (16), and mouse Abcc6 (10) genes, it has been reported that the methylation status of the promoter region differs between the kidney and liver. However, to our knowledge, there have been no reports of such differences in vivo in human drug transporter genes. Thus the present study is the first to demonstrate that kidney-specific human OCT2 gene expression is caused by the tissue-specific methylation status of the promoter region in vivo and in vitro.

It is generally thought that DNA methylation affects gene expression through two mechanisms. First, methylation of cytosine residues directly interrupts the recognition and binding of transcription factors and inhibits transcriptional activation. Second, methyl-CpG-binding proteins recognize methylated DNA and recruit corepressor molecules to induce chromatin structure condensation and gene-silencing (17, 23). In cases of some transcription factors like AP-2 (8), c-Myc (21), CTCF (4), and CREB (28), the first mechanism has been shown to be involved in the methylation-related transcriptional repression. In the present study, we demonstrated that the methylation status of individual CpG sites in the OCT2 proximal promoter, not only in the E-box, varied between in the kidney and in the liver. In the case of regulation of the kidney-specific expression of OCT2 by methylation, both mechanisms may be involved. Although the degree of CpG methylation of the OCT2 promoter in the kidney is relatively low, there were individual differences. Whether the individual differences in the degree of CpG methylation affect the expression level of OCT2 remains to be elucidated. Recently, we identified one deletion polymorphism in the promoter region, which reduces OCT2 promoter activity (19). The individual differences in the expression levels of OCT2 may be caused by multiple factors affecting coordinately.

OCT1, another member of the OCT family, is specifically expressed in the liver (12, 14). OCT1 is transactivated by hepatocyte nuclear factor (HNF)-4{alpha} via two direct repeat-2 elements around –1500 (22). In the proximal promoter of OCT1, the E-box is located in the region from –95 to –90, and we recently found that USF1 and USF2 bind to the E-box as basal transcription factors. In this study, we demonstrated that the methylation profile of the proximal promoter of OCT1 was quite similar in the kidney and liver. Thus the liver-specific expression of OCT1 may not be regulated by methylation. HNF-4{alpha} has been demonstrated to be involved in the regulation of hepatic expression of various genes such as apolipoproteins, albumin (6), cytochrome P-450 (CYP) 3A4 and CYP3A5 (15), and organic anion transporter 2 (20). In the case of OCT1, HNF-4{alpha} may play a role in liver-specific expression, but because the tissue distribution of HNF-4{alpha} is not restricted to the liver (27), other factors might also be involved in OCT1 gene expression in the liver.

It is of interest why the methylation status is similar for OCT1 between the liver and kidney, but different for OCT2. The mechanisms of tissue-specific and sequence-specific DNA methylation by DNA methyltransferases are still unknown. The activities and recognition of the target sequences of DNA methyltransferases may be regulated by interaction with various factors. Therefore, the tissue distribution of these factors can affect the tissue specificity of DNA methylation.

In conclusion, the present study clearly indicates that kidney-specific expression of human OCT2 is regulated by methylation of the proximal promoter region, leading to inhibition of transactivation by USF1.


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This work was supported 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 form the Ministry of Health, Labor, and Welfare of Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Inui, Dept. of Pharmacy, Kyoto Univ. Hospital, Sakyo-ku, Kyoto 606-8507, Japan (e-mail: inui{at}kuhp.kyoto-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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