| Research Abstract |
Using rat kidney cDNA library, 5'-temnial region of rOCT2 gene was amplified by 5'-RACE method. We found that the transcription initiation site of rOCT2 was located at 306 bases above translation initiation site. Next, we tried to isolate rOCT2 promoter region by screening rat genomic library using cDNA probes corresponding to 3.0 kb upstream region of the translation initiation site. A cDNA about 3 kb long was isolated and harbored into pGL3 vector, and then transfected into LLC-PK_1 cells. Measurement of promoter activity revealed that the transcription was enhanced in the presence of testosterone. Furthermore, saturation of promoter activity was observed with 10 nM or higher concentrations of testosterone, which was equivalent to the physiological concentration of testosterone in male rat.
In the promoter region of rOCT2, five portions of base sequences were found to be similar to androgen receptor response elements, ARE, which could play a relevant role in the regulation of rOCT2 transcription. Therefore, we generated constracts containing truncated products of rOCT2 promoter, and then introduced into LLC-PK_1 cells together with rat androgen receptor. The results of promoter assay revealed that nucleic acid sequences between positions -3,036 and -819 were involved in the regulation of rOCT2 transcription. Furthermore, mutations were introduced into five AREs, and then subjected to promoter assay. As the results, two AREs around positions -3,000 and -1,200 were suggested to be involved in the regulation of rOCT2 transcription.
This is the first evidence demonstrating that the organic cation transporter 2 are regulated at the level of transcription, and considered to be useful for understanding gender differences in the renal elimination activity of cationic drugs.
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