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Regulation of the Expression of Human Organic Anion Transporter 3 by Hepatocyte Nuclear Factor 1/ and DNA Methylation

Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences (R.K., H.K., Y.S.), and Laboratory of Cellular Biochemistry, Department of Animal Resource Sciences/Veterinary Medical Sciences (N.H., K.S.), University of Tokyo, Tokyo, Japan; and Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (I.K., F.J.G.)

Human organic anion transporter 3 (hOAT3/SLC22A8) is predominantly expressed in the proximal tubules of the kidney and plays a major role in the urinary excretion of a variety of organic anions. The promoter region of hOAT3 was characterized to elucidate the mechanism underlying the tissue-specific expression of hOAT3. The minimal promoter of hOAT3 was identified to be located approximately 300 base pairs upstream of the transcriptional start site, where there are canonical TATA and hepatocyte nuclear factor (HNF1) binding motifs, which are conserved in the rodent Oat3 genes. Transactivation assays revealed that HNF1 and HNF1 markedly increased hOAT3 promoter activity, where the transactivation potency of HNF1 was lower than that of HNF1. Mutations in the HNF1 binding motif prevented the transactivation. Electrophoretic mobility shift assays demonstrated binding of the HNF1/HNF1 homodimer or HNF1/HNF1 heterodimer to the hOAT3 promoter. It was also demonstrated that the promoter activity of hOAT3 is repressed by DNA methylation. Moreover, the expression of hOAT3 was activated de novo by forced expression of HNF1 alone or both HNF1 and HNF1 together with the concomitant DNA demethylation in human embryonic kidney 293 cells that lack expression of endogenous HNF1 and HNF1, whereas forced expression of HNF1 alone could not activate the expression of hOAT3. This suggests a synergistic action of the HNF1/HNF1 homodimer or HNF1/HNF1 heterodimer and DNA demethylation for the constitutive expression of hOAT3. These results indicate that the tissue-specific expression of hOAT3 might be regulated by the concerted effect of genetic (HNF1 and HNF1) and epigenetic (DNA methylation) factors.

Address correspondence to: Dr. Yuichi Sugiyama, Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: sugiyama{at}mol.f.u-tokyo.ac.jp

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