Elucidation of gene expression mechanisms of kidney-specific organic cation transporter OCT2 by promoter analyses.

利用大鼠肾脏cDNA文库，采用5′-RACE法扩增rOCT2基因的5′-末端区。我们发现rOCT2的转录起始位点位于翻译起始位点上方306个碱基处。接下来，我们尝试利用翻译起始位点上游3.0 kb区域对应的cDNA探针筛选大鼠基因组文库，分离rOCT2启动子区域。分离出长约3kb的cDNA，将其转入pGL3载体，转染到lc - pk_1细胞中。启动子活性的测量显示，在睾酮存在下，转录增强。此外，在10 nM或更高浓度的睾酮（相当于雄性大鼠生理睾酮浓度）下观察到启动子活性饱和。在rOCT2的启动子区域，发现有5个碱基序列与雄激素受体反应元件ARE相似，可能在调控rOCT2转录中发挥相关作用。因此，我们制作了含有rOCT2启动子截断产物的构建物，然后与大鼠雄激素受体一起导入LLC-PK_1细胞。启动子分析结果显示，- 3036和-819位点之间的核酸序列参与了rOCT2转录的调控。此外，将突变引入5个AREs，然后进行启动子试验。结果表明，- 3000和- 1200位点附近的两个AREs参与了rOCT2转录的调控。这是第一个证明有机阳离子转运体2在转录水平上受到调控的证据，并被认为有助于理解阳离子药物肾消除活性的性别差异。

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.