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

以大鼠肾脏cDNA文库为模板，采用5‘-RACE方法扩增出rOCT2基因的5’端序列。我们发现rOCT2的转录起始点位于翻译起始点上方的306个碱基上。接下来，我们试图通过筛选大鼠基因组文库来分离rOCT2启动子区域，方法是使用对应于翻译起始点3.0kb上游区域的cDNA探针。扩增出约3kb的c DNA片段，将其克隆入pGL3载体，再转入LLC-PK_1细胞。启动子活性的测定表明，在睾酮存在的情况下，转录被增强。此外，在10 nM或更高浓度的睾酮作用下，启动子活性达到饱和，相当于雄性大鼠生理浓度的睾酮。在rOCT2的启动子区域，发现5个碱基序列与雄激素受体反应元件ARE相似，可能在rOCT2转录调控中发挥相关作用。因此，我们构建了含有rOCT2启动子截短产物的真核表达载体，并与大鼠雄激素受体一起导入LLC-PK_1细胞。启动子分析结果表明，-3,036-819位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ARE，然后进行启动子分析。结果表明，-3,000位和-1,200位附近的两个ARE参与了rOCT2的转录调控，这是首次证明有机阳离子转运蛋白2在转录水平上受到调控，有助于理解阳离子药物肾清除活性的性别差异。

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.