MATE1 is highly expressed on the apical membrane of the hepatocytes and renal proximal tubule cells, and mediates the transport of a broad array of drugs [4
]. Functional changes in the activity or expression level of MATE1 caused by genetic variants in the coding or non-coding region of this transporter could result in changes in the levels of drugs substrates for the transporter. For example, reduction in the expression level of Mate1
in rats has been associated with reduced tubular secretion and high blood levels of the anti-ulcer drug, cimetidine [18
This study was conducted to identify and functionally characterize genetic variants in the proximal promoter region of MATE1
. By screening a large number of samples, we identified five variants that are polymorphic in four major ethnic groups. Recently Kajiwara M, et al
] described a variant, g.−32G>A, in the MATE1
promoter region that showed markedly decreased promoter activity. Their study indicated that the Sp1 transcription factor has a critical role in the basal promoter activity of MATE1
, and that the variant, g.−32G>A reduced the binding affinity of Sp1. Though the allelic frequency of g.−32G>A in the Japanese sample was 3.7%, it was not observed in our study, which included DNA samples from 68 individuals with ancestries in China. Chinese and Japanese share most alleles, but also have alleles that are population specific. Our results suggest that g.−32G>A may be a variant not shared between these two closely related populations.
The most common variant in this study, g.−66T>C showed a significant decrease in promoter activity in reporter assays in four cell lines () and in mouse liver (). Transcription factor binding site (TFBS) analyses suggested that two transcription factors could bind to the region encompassing g.−66T>C and that the binding affinity of these transcription factors was variant-dependent. In particular, AP-1 would have a higher binding affinity for the g.−66T allele whereas AP-2rep would have a higher binding affinity for the g.−66C allele.
AP-1 is a strong oncogene that mediates tumor cell invasion. The expression of AP-1 is highly elevated in many kinds of tumors such as squamous cell carcinoma, colon carcinoma, breast carcinoma and fibrosarcoma, and it has a critical role in tumor invasion [15
]. Previous studies reported that AP-1 induces the transcription of transporter genes including the human glucose transporter 1, the sodium dependent glutamate/aspartate transporter, and the sodium dependent bile acid transporter [19
]. In some cases, AP-1 may also act as a repressor; it enhances tumor cell invasion by the repression of genes that function as invasion suppressors. AP-1 is thought to bind to the DNA sequence, TGAg/cTCA [15
]. We observed that MATE1 contains the sequence, GTACT
CA, which is similar to the consensus sequence recognized by AP-1. The variant g.−66T>C results in the sequence, GTACC
CA, which is not as good of a match as the reference. Therefore we predicted that AP-1 would have binding preference to the reference DNA sequence as compared to the variant sequence. The results of EMSAs supported our hypothesis. The intensity of the DNA-AP-1 complex was decreased in the presence of g.−66T>C (). From the competition assay, we observed that g.−66T was a stronger competitor than g.−66C (), suggesting that g.−66T has a higher affinity for AP-1 than g.−66C. We also demonstrated that AP-1 regulates MATE1
transcription (), establishing that this transcription factor may be important in regulating the expression of this transporter.
In contrast to AP-1, there are few reports about AP-2rep. AP-2rep belongs to the KLFs family, which are characterized by three highly conserved classical Cys2
zinc fingers at the C-terminus. These motifs enable KLFs to bind to related GC- and CACCC-boxes of DNA. The N-termini of KLFs contain activation or repression domains. To date, a total of 17 KLFs have been identified. AP-2rep, also known as KLF 12, has a repressor domain (PVDLS motif) at its N-terminus [22
]. Schuierer M, et al
] reported that the interaction of the repressor domain of AP-2rep with co-repressor, C-terminal binding protein 1 (CtBP1), could result in the repression of the AP-2α gene that is involved in embryonic development, and malignant transformation. AP-2rep is thought to bind to the DNA sequence, CAGTGGG [25
]. The MATE1
promoter contains the sequence, CT
CACTG, and the complementary sequence, CAGTGA
G, is very similar to the consensus sequence of AP-2rep. The variant, g.−66T>C results in CAGTGG
G, an exact match to the consensus sequence of AP-2rep. We also found that over-expression of AP-2rep inhibited AP-1 binding to DNA. In particular, AP-2rep had a stronger inhibitory effect on the binding of AP-1 to the g.−66C allele than on the binding of AP-1 to the g.−66T allele (). We performed competition assays using unlabelled AP-2rep oligonucleotides to confirm that AP-1 and AP-2rep both bind competitively within the g.−66T>C region (). We also observed that AP-2rep could act as a repressor of MATE1
transcription (). In the presence of AP-2rep, the luciferase activity was reduced dramatically in the presence of the reference g.−66T (). However, in the presence of the variant g.−66C, the luciferase activity was reduced significantly only by large amounts of AP-2rep (at 50 ng) (). This may suggest that AP-2rep binds more avidly to the variant g.−66C.
Finally we examined the effect of g.−66T>C on MATE1
expression in human kidney. MATE1
expression levels in T/C or C/C genotype groups were significantly lower than in the T/T group, which is consistent with our findings in the luciferase in vitro
and in vivo
assays. T hese data suggest that g.−66T>C can affect the pharmacokinetics or pharmacodynamics of drugs that are transported by MATE1 in the body. However, the g.−66T>C was not associated with lower expression levels in liver samples (data not shown). Although AP-1 is expressed in both kidney and liver, the g.−66T>C associates with lower MATE1
expression in the kidney only [26
]. In contrast, no significant association between the g.−66T>C and MATE1
expression levels in the liver was observed. This may be explained by differences in samples and sample collection between liver and kidney. In particular, the liver samples, unlike the kidney samples, consisted of normal, post-mortem tissue. Variation in sample collection times, in particular time from death to sample collection, could have confounded the observed MATE1
expression levels in the liver. Kidney samples, on the other hand, were from surgical resection and collection times and conditions were more standardized. It is also possible that additional hepatic transcription factors (not present in HepG2 cells or in mouse liver under the experimental conditions), e.g., inducible nuclear receptors such as Pregnane X receptor (PXR), which bind to response elements elsewhere in the MATE1
gene may play a role in regulating the transcription rate of MATE1
in the liver, but not in the kidney.
In conclusion, we identified novel polymorphisms in the promoter region of MATE1. One of the polymorphisms, g.−66T>C, present at a very high allele frequency in all major ethnic groups, is associated with a significant reduction in the promoter activity of MATE1 and with lower expression levels of the transporter in the kidney. The mechanism appeared to be related to reduced binding of the transcription factor, AP-1, which acts as an activator of transcription, and to an increased binding of the repressor, AP-2rep, to the region containing the variant g.−66T>C (). Our data suggest that both transcription factors, AP-1 and AP-2rep, are involved in the transcriptional regulation of MATE1. Clinical studies examining the effect of the promoter variant, g.−66T>C, on the pharmacokinetics and pharmacodynamics of MATE1 substrates are ongoing.
Figure 7 Schematic of the interaction of the MATE1 promoter with two transcription factors that are proposed to be involved in the regulation of MATE1. The upper drawing shows the binding of the transcription factors to the reference allele and the lower drawing (more ...)