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

通过启动子分析阐明肾脏特异性有机阳离子转运蛋白 OCT2 的基因表达机制。

基本信息

批准号：
15590128
负责人：
OKUDA Masahiro
金额：
$ 2.3万
依托单位：
Mie University (2004)Kyoto University (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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.

利用大鼠肾脏cDNA文库，采用5'-RACE方法扩增rOCT 2基因的5'-末端区域。我们发现rOCT 2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，我们尝试通过使用对应于翻译起始位点的3.0kb上游区域的cDNA探针筛选大鼠基因组文库来分离rOCT 2启动子区域。将约3kb的cDNA克隆到pGL 3载体中，转染LLC-PK_1细胞。启动子活性的测量显示，在睾酮的存在下，转录增强。此外，当睾酮浓度为10 nM或更高时，观察到启动子活性饱和，相当于雄性大鼠睾酮的生理浓度。在rOCT 2的启动子区域中，发现有五部分碱基序列与雄激素受体反应元件ARE相似，可能在rOCT 2转录的调节中发挥相关作用。因此，我们制备了含有rOCT 2启动子截短产物的质粒，然后将其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和-819位之间的核酸序列参与了rOCT 2转录的调控。此外，将突变引入五个ARE中，然后进行启动子测定。结果表明，位于-3，000和-1，200位的两个ARE参与了rOCT 2转录的调控，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的证据，并被认为有助于理解阳离子药物肾清除活性的性别差异。