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.

使用大鼠肾脏cDNA文库，通过5'-Race方法扩增RoCT2基因的5'Temnial区域。我们发现ROCT2的转录启动位点位于翻译起始位点上方的306个基部。接下来，我们尝试使用对应于翻译起始位点上游区域的3.0 Kb上游区域的cDNA探针筛选大鼠基因组培养基来分离ROCT2启动子区域。分离出约3 kb的cDNA并将其置入PGL3载体中，然后转染到LLC-PK_1细胞中。启动子活性的测量表明，在存在睾丸激素的情况下，转录得到了增强。此外，观察到启动子活性的饱和度具有10 nm或更高浓度的睾丸激素，这等同于雄性大鼠中睾丸激素的生理浓度。在ROCT2的启动子区域中，碱基序列的启动子区域被发现与Androgen受体反应元素相似，与ROCT2转录的相关作用相关。因此，我们生成了含有ROCT2启动子截短产物的contracts，然后与大鼠雄激素受体一起引入LLC-PK_1细胞中。启动子测定的结果表明，位置-3,036和-819之间的核酸序列参与了ROCT2转录的调节。此外，将突变引入五个ARE，然后进行启动子测定。由于结果，提议-3,000和-1,200周围的两个ARE参与ROCT2转录的调节。这是第一个证据表明，有机阳离子转运蛋白2在转录水平下受到调节，并被认为有助于理解阳离子药物的肾脏消除活性中的性别差异。