AJP - Renal Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 293: F1564-F1570, 2007. First published September 12, 2007; doi:10.1152/ajprenal.00322.2007
0363-6127/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/F1564    most recent
00322.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kajiwara, M.
Right arrow Articles by Inui, K.-i.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kajiwara, M.
Right arrow Articles by Inui, K.-i.

Critical roles of Sp1 in gene expression of human and rat H+/organic cation antiporter MATE1

Moto Kajiwara,1 Tomohiro Terada,1 Jun-ichi Asaka,1 Ken Ogasawara,1 Toshiya Katsura,1 Osamu Ogawa,2 Atsushi Fukatsu,3 Toshio Doi,4 and Ken-ichi Inui1

1Department of Pharmacy, 2Department of Urology, and 3Division of Artificial Kidneys, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto; and 4Department of Clinical Biology and Medicine, University of Tokushima, Tokushima, Japan

Submitted 13 July 2007 ; accepted in final form 10 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A H+/organic cation antiporter (multidrug and toxin extrusion 1: MATE1/SLC47A1) plays important roles in the tubular secretion of various clinically important cationic drugs such as cimetidine. We have recently found that the regulation of this transporter greatly affects the pharmacokinetic properties of cationic drugs in vivo. No information is available about the regulatory mechanisms for the MATE1 gene. In the present study, therefore, we examined the gene regulation of human (h) and rat (r) MATE1, focusing on basal expression. A deletion analysis suggested that the regions spanning –65/–25 and –146/–38 were essential for the basal transcriptional activity of the hMATE1 and rMATE1 promoter, respectively, and that both regions contained putative Sp1-binding sites. Functional involvement of Sp1 was confirmed by Sp1 overexpression, a mutational analysis of Sp1-binding sites, mithramycin A treatment, and an electrophoretic mobility shift assay. Furthermore, we found a single nucleotide polymorphism (SNP) in the promoter region of hMATE1 (G–32A), which belongs to a Sp1-binding site. The allelic frequency of this rSNP was 3.7%, and Sp1-binding and promoter activity were significantly decreased. This is the first study to clarify the transcriptional mechanisms of the MATE1 gene and to identify a SNP affecting the promoter activity of hMATE1.

tubular secretion; basal transcriptional activity; single nucleotide polymorphism; multidrug and toxin extrusion 1

RENAL ELIMINATION, including glomerular filtration and tubular secretion, is a major clearance mechanism for many drugs. The renal tubular secretion of drugs takes place primarily in the proximal tubules as an interplay of drug transporters located in the basolateral and brush-border membranes, respectively. For example, organic cations are taken up by membrane potential-dependent organic cation transporters and then extruded into the lumen by H+/organic cation antiporters (14). Molecular cloning of membrane potential-dependent organic cation transporter 1 (OCT1) was reported in 1994 (12), and at present, three kinds of organic cation transporters (OCT1–3) from various species have been identified and characterized (7, 14, 36). In contrast to OCTs, attempts at the molecular identification of apical H+/organic cation antiporters have failed for more than a decade.

Recently, mammalian orthologs of the multidrug and toxin extrusion (MATE) family, which confers multidrug resistance on bacteria, have been identified in humans (19, 25), mice (25), rats (23, 34), and rabbits (37). Two functional isoforms, MATE1/SLC47A1 (human, mouse, rat, and rabbit) and MATE2-K/SLC47A2 (human and rabbit), have been characterized in terms of tissue distribution, membrane localization, and transport characteristic and have been demonstrated to work as H+/organic cation antiporters (2, 19, 20, 23, 25, 33–35, 37). MATE1 is able to transport various organic cations such as tetraethylammonium, cimetidine, and metformin and the zwitterion cephalexin using an oppositely directed H+ gradient (33–35). Human (h) MATE1 is primarily expressed in the kidney and liver and is located at the membranes of the luminal side (19, 25). Rat (r) MATE1 is also present in the brush-border membranes of renal proximal tubules (20), and the intrarenal expression of rMATE1 is limited to the proximal tubules (34). Our group (20) also demonstrated that the expression level of rMATE1 was remarkably reduced in 5/6 nephrectomized rats and that the level of rMATE1, but not of rOCT2, correlated well with the tubular secretion of cimetidine in female rats (r = 0.74). Furthermore, very recently, our group identified cysteine and histidine residues essential to the function of rat and human MATE1 (2). Hence, functional and expressional aspects of MATE1 have been clarified in much detail, but there has been no information about the transcriptional regulation including basal promoter activity of this transporter.

The core promoter and 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 (6). When such polymorphisms occur in the promoter region of pharmacokinetic-related genes, drug disposition, efficacy, and toxicity can be influenced. For example, a genetic polymorphism of the uridine diphosphate-glucuronosyltransferase 1A1 gene, UGT1A1*28, a variant sequence in the promoter region, contributed to individual variation for adverse events in patients administered the anti-cancer agent irinotecan (13). Thus a search for single nucleotide polymorphisms in the promoter region (rSNP) of drug transporters may provide useful information on interindividual differences in drug disposition.

In the present study, therefore, we characterized the promoter activity of hMATE1 and rMATE1 to identify the minimal region and cis-regulatory elements required for the basal expression of MATE1. Furthermore, we performed rSNP analyses for hMATE1 and found that SNP at G–32A, which belongs to a Sp1-binding site, affected the promoter activity of the gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. [{gamma}-32P]ATP was obtained from GE Healthcare (Little Chalfont, UK). Restriction enzymes were obtained from New England BioLabs (Beverly, MA). Antibody for Sp1 (H-225) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mithramycin A was purchased from Sigma-Aldrich (St. Louis, MO). The plasmid CMV-Sp1 was kindly provided by Dr. Robert Tjian (University of California, Berkeley, CA). All other chemicals used were of the highest purity available.

Determination of putative transcriptional start site. To identify the transcription start site of hMATE1 and rMATE1, we carried out 5'-rapid amplification of cDNA ends (5'-RACE) using human and rat kidney Marathon-Ready cDNA (Clontech, Mountain View, CA), respectively, according to the manufacturer's instructions. The primers for 5'-RACE were as follows: a gene-specific primer for hMATE1 (GenBank accession no. FLJ10847), 5'-CAGGACCAAGAGCGCCCGCAGCTCTTCT-3' (209/182); a nested gene-specific primer for hMATE1, 5'-CTGGCGCGGGCTCCTCAGGAGCTTCCAT-3' (114/87); a gene-specific primer for rMATE1 (GenBank accession no. NM_001014118), 5'-GAGCTGGGCCAAGAACGCAGGACCC-3' (170/146); and a nested gene-specific primer for rMATE1, 5'-CTGGTCCCGGCGCAGGCTCCTCCAAGA-3' (57/31). The polymerase chain reaction (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).

Construction of reporter plasmids for hMATE1 and rMATE1 promoters. The human or rat promoter region upstream of the transcription start site was amplified by PCR using human genomic DNA (Promega) or rat genomic DNA (Clontech) with the primers listed in Table 1. The PCR products were subcloned into the firefly luciferase reporter vector pGL3-Basic (Promega). The full-length reporter plasmids are hereafter referred to as hMATE1 (–2408/+13) and rMATE1 (–2422/+24). The 5'-deleted constructs were generated by digestion with appropriate restriction enzymes. The hMATE1 (–65/+13) and rMATE1 (–38/+24) constructs were generated by PCR with the primers listed in Table 1. The site-directed mutations in putative Sp1-binding sites were introduced into the hMATE1 (–65/+13) construct with a QuickChange 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 or rSNP analyses were sequenced using a multicapillary DNA sequencer RISA384 system (Shimadzu).


View this table:
[in this window]
[in a new window]

  		Table 1. Primer sequences

 
Cell culture, transfection, and reporter gene assays. LLC-PK1 and HEK293 cells were obtained from the American Type Culture Collection (ATCC CRL-1392 and -1573). Cell culture, transfection, and reporter gene assays were carried out as described previously (1, 22, 29).

Western blot analysis. Preparation of nuclear extracts from LLC-PK1 cells was described previously (29). The nuclear extracts of LLC-PK1 cells (125 ng) were solubilized in SDS sample buffer, separated on a 10% polyacrylamide gel, and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA) by semidry electroblotting. Blots were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS; 20 mM Tris, 137 mM NaCl, pH 7.5) with 0.1% Tween 20 (TBS-T) for 1 h at room temperature. Blots were washed in TBS-T and then incubated with the anti-Sp1 polyclonal antibody (1 µg/ml, overnight at 4°C). The bound antibody was detected on X-ray film by enhanced chemiluminescence with a horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare).

EMSA. Recombinant human Sp1 (rhSp1) and recombinant human Sp3 (rhSp3) were obtained from Promega and Alexis (Lausen, Switzerland), respectively. The probes shown in Table 1 were prepared by annealing complementary sense and antisense oligonucleotides, followed by end labeling with [{gamma}-32P]ATP using T4 polynucleotide kinase (Takara Bio, Otsu, Japan) and purification through a Sephadex G-25 column (GE Healthcare). The composition of the binding mixture containing rhSp1 was based on Martín et al. (17), and compositions of rhSp3 binding buffer were as follows: 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris·HCl (pH 7.5), and 4% glycerol. Labeled probes were added to the binding mixture and incubated for 0.5–1 h at room temperature. The DNA-protein complexes were then separated on a 4% polyacrylamide gel at room temperature in 0.5x Tris-borate-EDTA buffer. The gel was dried and exposed to X-ray film for autoradiography.

rSNP analyses. Genomic DNA was extracted from peripheral blood from 88 patients with renal diseases (47 men and 41 women; range 14–78 yr) and normal parts of the kidney from 21 renal cell carcinoma patients (21 men; range 39–77 yr) using a Wizard Genomic DNA purification kit (Promega). About 120 bp of the promoter region of the hMATE1 gene were amplified by PCR using the forward primer 5'-CGCAGTGGTGCAGAGAGAGGTGCAA-3' (–154/–130) and the reverse primer 5'-AGTCACCCGCGGAGGCAGAAATCAC-3' (297/273). PCR conditions were as follows: denaturation at 94°C for 30 s, annealing and synthesis at 68°C for 3 min, for 35 cycles. All PCR products were sequenced using a multicapillary DNA sequencer RISA384 system (Shimadzu).

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.

Data analysis. The results are mainly expressed relative to pGL3-Basic and represent means ± SE of three replicates. Two or three experiments were conducted, and representative results are shown. Data were analyzed statistically with one-way ANOVA followed by Dunnett's test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Determination of the transcription start site of hMATE1 and rMATE1. Sequencing of the longest RACE product showed that the terminal position of hMATE1 and rMATE1 cDNA was located 22 and 20 nucleotides upstream of the start codon, which was 64 and 3 bp downstream of the 5'-end of hMATE1 and rMATE1 cDNA, respectively, registered in the National Center for Biotechnology Information database [GenBank accession no. FLJ10847 (hMATE1) and NM_001014118 (rMATE1)]. Therefore, the 5'-end of hMATE1 and rMATE1 cDNA in the database was numbered with +1 as the transcription start site in this study.

Determination of the minimal hMATE1 and rMATE1 promoter. To determine the minimal region required for basal activity of the promoter, a series of hMATE1 (Fig. 1A) or rMATE1 (Fig. 1B) deletion constructs were transfected into LLC-PK1 cells, and luciferase activity was measured. In LLC-PK1 cells, the H+/organic cation antiport system is expressed in the apical membrane (9, 15, 28), and we hypothesized that transcription factors and/or cofactors required for the expression of MATE1 exist intrinsically in these cells. The longest reporter constructs of hMATE1 and rMATE1 showed an approximately three- to fivefold increase in luciferase activity compared with pGL3-Basic. The luciferase activity of both constructs was completely abolished with the –25/+13 (hMATE1) and –38/+24 (rMATE1) deleted promoter constructs, respectively, suggesting that the region between –65 and –25 for hMATE1 or between –146 and –38 for rMATE1 was important for basal promoter activity. We then performed a computational sequence analysis of these regions using TFSEARCH (www.cbrc.jp/research/db/TFSEARCH.html). This analysis revealed that the region proximal to the transcription start site lacks canonical TATA or CCAAT boxes. Instead, two GC-rich sites were observed, suggesting a possible contribution of Sp1 to the basal promoter activity of MATE1 (Fig. 2, A and B).


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 1. Deletion analysis of human multidrug and toxin extrusion 1 (hMATE1) and rat (r)MATE1 promoter activity in LLC-PK1 cells. A: a series of hMATE1 deleted promoter constructs (equimolar amounts of the –2408/+13 construct; 600 ng) were transfected into LLC-PK1 cells for luciferase assays. B: a series of rMATE1 deleted promoter constructs (equimolar amounts of the –2422/+24 construct; 600 ng) were transfected into LLC-PK1 cells for luciferase assays. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are reported as the relative increase compared with pGL3-Basic and are means ± SE of 3 replicates.

 

Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 2. Nucleotides sequence of the hMATE1 (A) and rMATE1 (B) promoter. Numbering is relative to the transcription start site. The putative binding sites for Sp1 are indicated on the sequence (arrows indicate the direction). C: detection of Sp1 by Western blot analysis. Nuclear extracts from LLC-PK1 cells (125 ng) were separated on a 10% SDS-polyacrylamide gel and blotted onto a polyvinylidene difluoride membrane. Sp1 antibody (1 µg/ml) was used as a primary antibody. Horseradish peroxidase-conjugated anti-rabbit IgG antibody was used for detection of bound antibody. The arrowhead indicates the position of Sp1.

 
Western blot analysis. To confirm the expression of Sp1 in LLC-PK1 cells, we carried out Western blot analysis for Sp1. As shown in Fig. 2C, an immunoreactive protein with a molecular mass of 106 kDa corresponding to Sp1 was detected in LLC-PK1 cells.

Functional involvement of Sp1 in MATE1 promoter activity. To evaluate the functional involvement of Sp1 in MATE1 promoter activity, we performed experiments for the inhibition and transactivation of Sp1. Mithramycin A is known to bind to the GC box and inhibit Sp1 binding (4, 27). As shown in Fig. 3, treatment with mithramycin A led to a significant decrease in the promoter activity in a dose-dependent manner for both reporter constructs. Next, we investigated the effect of Sp1 overexpression on the MATE1 promoter activity in HEK293 cells (Fig. 4). Transfection of the CMV-Sp1 expression vector in LLC-PK1 cells remarkably reduced the activity of the pGL3-Basic (control) via unknown mechanisms, and therefore, HEK293 cells were used for Sp1 overexpression experiments. The promoter activity of hMATE1 (–65/+13) or rMATE1 (–146/+24) showed a dose-dependent increase in the cotransfection of CMV-Sp1, providing direct evidence that Sp1 enhanced the activity.


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 3. Inhibition of hMATE1 (A) and rMATE1 (B) transcriptional activity by mithramycin A. LLC-PK1 cells were transiently transfected with the hMATE1 (–65/+13) or rMATE1 (–146/+24) construct (same amount as in the deletion analysis). Mithramycin A was added to the cells 5 h after the transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are reported as the relative increase compared with pGL3-Basic and are means ± SE of 3 replicates. **P < 0.01, significantly different from the values without mithramycin A.

 

Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 4. Effect of Sp1 overexpression on hMATE1 (A) and rMATE1 (B) transcriptional activity. HEK293 cells were transiently transfected with 250 ng of hMATE1 (–65/+13) or rMATE1 (–146/+24) construct with the CMV-Sp1 expression vector (500, 750, and 1,000 ng). The total amount of transfected DNA was kept constant by adding empty vector. Data are reported as the relative increase compared with that without CMV-Sp1 and are means ± SE of 3 replicates. *P < 0.05; **P < 0.01, significantly different from the values without CMV-Sp1.

 
Identification of Sp1-binding site of hMATE1 promoter. In the proximal hMATE1 promoter region, there are two putative Sp1-binding sites located between –65 and –25, and these sites were designated as Sp-A (–64/–55) and Sp-B (–40/–31), respectively. To determine whether these sites were important for hMATE1 promoter activity, we introduced a mutation at each site of the hMATE1 (–65/+13) construct (mutation A and mutation B; Fig. 5A). As shown in Fig. 5B, both mutants remarkably reduced the luciferase activity compared with the wild type. These results suggest that both Sp-A and Sp-B are important for regulating the hMATE1 promoter activity. Finally, to confirm that Sp1 binds to Sp-A and Sp-B sites, we performed EMSAs with rhSp1 and wild-type or mutational probes of Sp-A and Sp-B (Fig. 5A). Both wild probes A and B formed DNA-protein complexes (Fig. 6A, lanes 3 and 7), whereas no complex was formed in the absence of rhSp1 or mutational probes A and B (Fig. 6A, lanes 1, 2, 4–6, and 8). These results suggest that Sp1 stimulates the basal promoter activity of hMATE1 via Sp-A and Sp-B regions. It was reported that Sp3 as well as Sp1 also recognizes classic Sp1-binding sites (5). We then further examined the interaction of Sp-A and Sp-B with rhSp3. As observed in rhSp1, specific DNA-protein complexes in both wild probes A and B were detected (Fig. 6B).


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 5. Mutational analysis of the putative Sp1-binding sites of the hMATE1 promoter. A: the nucleotide sequence of the promoter region from –77 to –18 is shown with the putative Sp1-binding elements (Sp-A and Sp-B underlined). Site-directed mutations that destroy Sp1-binding elements were introduced individually and designated mutation A and mutation B. The nucleotides altered for mutational analysis are shown italicized above the wild-type sequence. The regions used for oligonucleotide probes for EMSA are also indicated. B: the mutated –65/+13 constructs (same amount as in the deletion analysis) were transiently expressed in LLC-PK1 cells for luciferase assays. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are reported as the relative increase compared with pGL3-Basic and are means ± SE of 3 replicates. **P < 0.01, significantly different from the wild type.

 

Figure 6
View larger version (54K):
[in this window]
[in a new window]

  		Fig. 6. EMSA of recombinant human (rh)Sp1 (A) or rhSp3 (B) proteins binding to the hMATE1 probes containing putative Sp1-binding elements. rhSp1 (30 ng) or rhSp3 (10 ng) was incubated with the wild or mutated [{gamma}-32P]ATP-labeled oligonucleotide probes [probe A (–77/–48) and probe B (–48/–18)] in lanes 3, 4, 7, and 8. In lanes 1, 2, 5, and 6, rhSp1 (A) or rhSp3 (B) was not added.

 
rSNP Analyses of hMATE1. We then sequenced the promoter region (~120 bp) of the MATE1 gene in 88 patients with renal diseases and 21 patients with renal cell carcinoma and found the G–32A SNP at the Sp-B site. The frequency of this rSNP is summarized in Table 2. To evaluate the effect of the G–32A SNP on the promoter activity of hMATE1, we carried out an EMSA and a reporter assay. Both wild and SNP probes formed DNA-protein complexes, although less complex was formed with the SNP probe (–32A) (Fig. 7, lanes 4 and 8) than with the wild probe (–32G) (Fig. 7, lanes 3 and 7). No complex was formed in the absence of rhSp1 (Fig. 7, lanes 1, 2, 5, and 6). Furthermore, as shown in Fig. 8, the SNP construct (–32A) remarkably reduced the luciferase activity compared with the wild type (–32G). These results indicate that the G–32A substitution downregulates the basal promoter activity of hMATE1 by weakening the binding of Sp1.


View this table:
[in this window]
[in a new window]

  		Table 2. Allelic frequency of G–32A of hMATE1 for 109 patients with renal diseases and renal cell carcinoma

 

Figure 7
View larger version (69K):
[in this window]
[in a new window]

  		Fig. 7. Effect of single nucleotide polymorphism (SNP; G–32A) on EMSA for rhSp1. rhSp1 (90 or 120 ng) was incubated with the wild probe (–32G) in lanes 3 and 4) or the SNP probe (–32A) in lanes 7 and 8. In lanes 1, 2, 5, and 6, rhSp1 was not added.

 

Figure 8
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 8. Effect of rSNP (G–32A) of the putative Sp1-binding sites on the hMATE1 promoter activity. A: the nucleotide sequence of the promoter region from –48 to –18 is shown. B: the SNP –65/+13 constructs were transiently expressed in LLC-PK1 cells for luciferase assays. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are reported as the relative increase compared with pGL3-Basic and are means ± SE of 3 replicates. **P < 0.01, significantly different from the wild type.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we performed a functional promoter assay of human and rat MATE1 genes and obtained convincing evidence of the involvement of Sp1 in the regulation of the basal expression of these transporters. This conclusion is supported by results of experiments involving the inhibition of mithramycin A, the overexpression of Sp1, mutagenesis of the GC-rich region, and EMSAs using rhSp1. In the proximal promoter region of the human and rat MATE1 genes, two GC-rich sites exist instead of a TATA box, and these features are conserved among species (Fig. 9). In a TATA-less promoter, Sp1 binds to the GC-rich region to recruit TATA-binding protein (26) and to fix the transcription start site (3). Furthermore, it was reported that the promoter activity is enhanced if multiple Sp1-binding sites exist (18). These findings suggested that Sp1 plays a significant role as a basal transcription factor through these GC-rich sites in the MATE1 promoter.


Figure 9
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 9. Nucleotide sequences of the human, rat, and mouse MATE1 promoter. The nucleotide sequences of the promoter region of the human (–69/–23), rat (–86/–39), and mouse (–91/–44) MATE1 are shown with the putative Sp1-binding elements. All of the promoter sequences have the Sp1 consensus sequence (GGGCGG). The putative Sp1-binding sites are shown in bold.

 
Furthermore, we found a SNP at a Sp1-binding site (G–32A, belonging to the Sp-B site) of the hMATE1 promoter for the first time and demonstrated that this substitution affects hMATE1 promoter activity by disrupting the binding of Sp1. It is strongly suggested that this rSNP influences the mRNA level of MATE1.

To date, many large-scale screenings of SNPs of drug transporters (mainly focusing on SNPs in the coding region; cSNPs) have been carried out to identify genetic factors involved in the interindividual differences of pharmacokinetics. So far, clinical implications for cSNPs in the genes for organic anion transporting polypeptides (16) and OCT1 (31, 32) have been demonstrated, but significant results have not been obtained for other SLC-type drug transporters. In the present study, we found an rSNP of Sp1-binding sites affecting the MATE1 gene. This type of SNP may be involved in the interindividual differences of pharmacokinetics. There are reports that rSNPs of Sp1-binding sites of the resistin gene contribute to type 2 diabetes mellitus susceptibility (24) and that those of the collagen type I {alpha}1 gene contribute to low bone mass and vertebral fracture (11). Further studies of the relationship between gene polymorphisms of MATE1 and the pharmacokinetic properties of MATE1 substrate drugs may clarify the clinical implications of this SNP.

Human and rat MATE1 mRNA is abundantly expressed in the kidney (19, 34), whereas Sp1 is expressed ubiquitously. The present study revealed the contribution of Sp1 to the transcriptional regulation of MATE1, but the mechanism of this tissue-specific expression has not been clarified. In the case of intestinal H+-coupled peptide transporter 1, the basal promoter activity was also determined by Sp1, but its tissue specific expression was regulated by the intestinal specific transcription factor Cdx2 (29, 30). Currently, there is little information about the renal specific transcription factor. It has been reported that Sp3 as well as Sp1 also recognizes classic Sp1-binding sites, and there are a number of reports that the relative abundance of Sp1 and Sp3 should allow regulation of gene activities (5). We confirmed by EMSA that a recombinant Sp3 protein bound to the Sp-A and Sp-B sites of the hMATE1 promoter (Fig. 6B). Another possible mechanism is the methylation of the GC box in the promoter region. Methylation of the cytosine residue in the sequence of 5'-CpG-3' is an epigenetic modification, and recent reports have revealed that this epigenetic modification contributes to the tissue-specific expression (8, 10, 21). Further studies are needed to clarify the tissue-specific expression of MATE1.

In conclusion, the present study clearly indicates that Sp1 functions as a basal transcriptional regulator of the human and rat MATE1 gene through the two GC boxes, and this system may be conserved among species. Furthermore, we have identified an rSNP of the hMATE1 gene (G–32A) (belonging to a Sp1-binding site) that affects the promoter activity.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the 21st Century Center of Excellence 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.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Robert Tjian (University of California, Berkeley) for the Sp1 expression vector.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Prof. 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Asaka J, Terada T, Okuda M, Katsura T, Inui K. Androgen receptor is responsible for rat organic cation transporter 2 gene regulation but not for rOCT1 and rOCT3. Pharm Res 23: 697–704, 2006.[CrossRef][Web of Science][Medline]
  2. Asaka J, Terada T, Tsuda M, Katsura T, Inui K. Identification of essential histidine and cysteine residues of H+/organic cation antiporter, multidrug and toxin extrusion (MATE). Mol Pharmacol 46: 1487–1493, 2007.
  3. Blake MC, Jambou RC, Swick AG, Kahn JW, Azizkhan JC. Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter. Mol Cell Biol 10: 6632–6641, 1990.[Abstract/Free Full Text]
  4. Blume SW, Snyder RC, Ray R, Thomas S, Koller CA, Miller DM. Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo. J Clin Invest 88: 1613–1621, 1991.[Web of Science][Medline]
  5. Bouwman P, Philipsen S. Regulation of the activity of Sp1-related transcription factors. Mol Cell Endocrinol 195: 27–38, 2002.[CrossRef][Web of Science][Medline]
  6. Buckland PR. The importance and identification of regulatory polymorphisms and their mechanisms of action. Biochim Biophys Acta 1762: 17–28, 2006.[Medline]
  7. Burckhardt G, Wolff NA. Structure of renal organic anion and cation transporters. Am J Physiol Renal Physiol 278: F853–F866, 2000.[Abstract/Free Full Text]
  8. Douet V, Heller MB, Le Saux O. DNA methylation and Sp1 binding determine the tissue-specific transcriptional activity of the mouse Abcc6 promoter. Biochem Biophys Res Commun 354: 66–71, 2007.[CrossRef][Web of Science][Medline]
  9. Fouda AK, Fauth C, Roch-Ramel F. Transport of organic cations by kidney epithelial cell line LLC-PK1. J Pharmacol Exp Ther 252: 286–292, 1990.[Abstract/Free Full Text]
  10. Fujii G, Nakamura Y, Tsukamoto D, Ito M, Shiba T, Takamatsu N. CpG methylation at the USF-binding site is important for the liver-specific transcription of the chipmunk HP-27 gene. Biochem J 395: 203–209, 2006.[CrossRef][Web of Science][Medline]
  11. Grant SFA, Reid DM, Blake G, Herd R, Fogelman I, Ralston SH. Reduced bone density and osteoporosis associated with a polymorphic Sp1 binding site in the collagen type I {alpha}1 gene. Nat Genet 14: 203–205, 1996.[CrossRef][Web of Science][Medline]
  12. Gründemann D, Gorboulev V, Gambaryan S, Veyhl M, Koepsell H. Drug excretion mediated by a new prototype of polyspecific transporter. Nature 372: 549–552, 1994.[CrossRef][Medline]
  13. Hasegawa Y, Ando Y, Ando M, Hashimoto N, Imaizumi K, Shimokata K. Pharmacogenetic approach for cancer treatment-tailored medicine in practice. Ann NY Acad Sci 1086: 223–232, 2006.[CrossRef][Web of Science][Medline]
  14. Inui K, Masuda S, Saito H. Cellular and molecular aspects of drug transport in the kidney. Kidney Int 58: 944–958, 2000.[CrossRef][Web of Science][Medline]
  15. Inui K, Saito H, Hori R. H+-gradient-dependent active transporter of tetraethylammonium cation in apical-membrane vesicles isolated from kidney epithelial cell line LLC-PK1. Biochem J 227: 199–203, 1985.[Web of Science][Medline]
  16. Maeda K, Ieiri I, Yasuda K, Fujino A, Fujiwara H, Otsubo K, Hirano M, Watanabe T, Kitamura Y, Kusuhara H, Sugiyama Y. Effects of organic anion transporting polypeptide 1B1 haplotype on pharmacokinetics of pravastatin, valsartan, and temocapril. Clin Pharmacol Ther 79: 427–439, 2006.[CrossRef][Web of Science][Medline]
  17. Martín MG, Wang J, Solorzano-Vargas RS, Lam JT, Turk E, Wright EM. Regulation of the human Na+-glucose cotransporter gene, SGLT1, by HNF-1 and Sp1. Am J Physiol Gastrointest Liver Physiol 278: G591–G603, 2000.[Abstract/Free Full Text]
  18. Mastrangelo IA, Courey AJ, Wall JS, Jackson SP, Hough PVC. DNA looping and Sp1 multimer links: a mechanism for transcriptional synergism and enhancement. Proc Natl Acad Sci USA 88: 5670–5674, 1991.[Abstract/Free Full Text]
  19. Masuda S, Terada T, Yonezawa A, Tanihara Y, Kishimoto K, Katsura T, Ogawa O, Inui K. Identification and functional characterization of a new human kidney-specific H+/organic cation antiporter, kidney-specific multidrug and toxin extrusion 2. J Am Soc Nephrol 17: 2127–2135, 2006.[Abstract/Free Full Text]
  20. Nishihara K, Masuda S, Ji L, Katsura T, Inui K. Pharmacokinetic significance of luminal multidrug and toxin extrusion 1 in chronic renal failure rats. Biochem Pharmacol 73: 1482–1490, 2007.[CrossRef][Web of Science][Medline]
  21. Nomura J, Hisatsune A, Miyata T, Isohoma Y. The role of CpG methylation in cell type-specific expression of the aquaporin-5 gene. Biochem Biophys Res Commun 353: 1017–1022, 2007.[CrossRef][Web of Science][Medline]
  22. Ogasawara K, Terada T, Asaka J, Katsura T, Inui K. Hepatocyte nuclear factor-4{alpha} regulates the human organic anion transporter 1 gene in the kidney. Am J Physiol Renal Physiol 292: F1819–F1826, 2007.[Abstract/Free Full Text]
  23. Ohta K, Inoue K, Hayashi Y, Yuasa H. Molecular identification and functional characterization of rat multidrug and toxin extrusion type transporter 1 as an organic cation/H+ antiporter in the kidney. Drug Metab Dispos 34: 1868–1874, 2006.[Abstract/Free Full Text]
  24. Osawa H, Yamada K, Onuma H, Murakami A, Ochi M, Kawata H, Nishimiya T, Niiya T, Shimizu I, Nishida W, Hashiramoto M, Kanatsuka A, Fujii Y, Ohashi J, Makino H. The G/G genotype of a resistin single-nucleotide polymorphism at –420 increases type 2 diabetes mellitus susceptibility by inducing promoter activity through specific binding of Sp1/3. Am J Hum Genet 75: 678–686, 2004.[CrossRef][Web of Science][Medline]
  25. Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H, Moriyama Y. A human transporter protein that mediates the final excretion step for toxic organic cations. Proc Natl Acad Sci USA 102: 17923–17928, 2005.[Abstract/Free Full Text]
  26. Pugh BF, Tjian R. Transcription from a TATA-less promoter requires a multisubunit TFIID complex. Genes Dev 5: 1935–1945, 1991.[Abstract/Free Full Text]
  27. Ray R, Snyder RC, Thomas S, Koller CA, Miller DM. Mithramycin blocks protein binding and function of the SV40 early promoter. J Clin Invest 83: 2003–2007, 1989.[Web of Science][Medline]
  28. Saito H, Yamamoto M, Inui K, Hori R. Transcellular transport of organic cation across monolayers of kidney epithelial cell line LLC-PK1. Am J Physiol Cell Physiol 262: C59–C66, 1992.[Abstract/Free Full Text]
  29. 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.[Abstract/Free Full Text]
  30. 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.[CrossRef][Web of Science][Medline]
  31. Shu Y, Brown C, Castro RA, Shi RJ, Lin ET, Owen RP, Sheardown SA, Yue L, Burchard EG, Brett CM, Giacomini KM. Effect of genetic variation in the organic cation transporter 1, OCT1, on metformin pharmacokinetics. Clin Pharmacol Ther. In press.
  32. Shu Y, Sheardown SA, Brown C, Owen RP, Zhang S, Castro RA, Ianculescu AG, Yue L, Lo JC, Burchard EG, Brett CM, Giacomini KM. Effect of genetic variation in the organic cation transporter 1 (OCT1) on metformin action. J Clin Invest 117: 1422–1431, 2007.[CrossRef][Web of Science][Medline]
  33. Tanihara Y, Masuda S, Sato T, Katsura T, Ogawa O, Inui K. Substrate specificity of MATE1 and MATE2-K, human multidrug and toxin extrusions/H+-organic cation antiporters. Biochem Pharmacol 74: 359–371, 2007.[CrossRef][Web of Science][Medline]
  34. Terada T, Masuda S, Asaka J, Tsuda M, Katsura T, Inui K. Molecular cloning, functional characterization and tissue distribution of rat H+/organic cation antiporter MATE1. Pharm Res 23: 1696–1701, 2006.[CrossRef][Web of Science][Medline]
  35. Tsuda M, Terada T, Asaka J, Ueba M, Katsura T, Inui K. Oppositely directed H+ gradient functions as a driving force of rat H+/organic cation antiporter MATE1. Am J Physiol Renal Physiol 292: F593–F598, 2007.[Abstract/Free Full Text]
  36. Wright SH. Role of organic cation transporters in the renal handling of therapeutic agents and xenobiotics. Toxicol Appl Pharmacol 204: 309–319, 2005.[CrossRef][Web of Science][Medline]
  37. Zhang X, Cherrington NJ, Wright SH. Molecular identification and functional characterization of rabbit MATE1 and MATE2-K. Am J Physiol Renal Physiol 293: F360–F370, 2007.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
M. Tsuda, T. Terada, T. Mizuno, T. Katsura, J. Shimakura, and K.-i. Inui
Targeted Disruption of the Multidrug and Toxin Extrusion 1 (Mate1) Gene in Mice Reduces Renal Secretion of Metformin
Mol. Pharmacol., June 1, 2009; 75(6): 1280 - 1286.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
M. L. Becker, L. E. Visser, R. H.N. van Schaik, A. Hofman, A. G. Uitterlinden, and B. H.Ch. Stricker
Genetic Variation in the Multidrug and Toxin Extrusion 1 Transporter Protein Influences the Glucose-Lowering Effect of Metformin in Patients With Diabetes: A Preliminary Study
Diabetes, March 1, 2009; 58(3): 745 - 749.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M. Kajiwara, T. Terada, J.-i. Asaka, M. Aoki, T. Katsura, I. Ikai, and K.-i. Inui
Regulation of basal core promoter activity of human organic cation transporter 1 (OCT1/SLC22A1)
Am J Physiol Gastrointest Liver Physiol, December 1, 2008; 295(6): G1211 - G1216.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/F1564    most recent
00322.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kajiwara, M.
Right arrow Articles by Inui, K.-i.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kajiwara, M.
Right arrow Articles by Inui, K.-i.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.