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。

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590128/
• 关键词: Organic Cation Transporter; Renal Tubules; OCT2; Promoter; Sex-related differences; Testosterone; Transcriptional regulation; Basic drugs

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，通过5'-RACE方法扩增rOCT2基因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，我们尝试通过使用对应于翻译起始位点3.0 kb上游区域的cDNA探针筛选大鼠基因组文库来分离rOCT2启动子区域。分离出约3kb长的cDNA并将其携带至pGL3载体中，然后转染至LLC-PK_1细胞中。启动子活性的测量表明，在睾酮存在的情况下转录得到增强。此外，在10nM或更高浓度的睾酮下观察到启动子活性饱和，该浓度相当于雄性大鼠睾酮的生理浓度。在rOCT2的启动子区域中，发现5个部分的碱基序列与雄激素受体相似。反应元件 ARE，可能在 rOCT2 转录调控中发挥相关作用。因此，我们生成了含有rOCT2启动子截短产物的合同，然后与大鼠雄激素受体一起引入LLC-PK_1细胞中。启动子检测结果显示-3,036和-819位点之间的核酸序列参与rOCT2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，-3,000和-1,200位点附近的两个ARE被认为参与了rOCT2转录的调节。这是证明有机阳离子转运蛋白2在转录水平上受到调节的第一个证据，并被认为是有用的用于了解阳离子药物的肾脏消除活性的性别差异。

项目成果

Decreased function of genetic variants, Pro283Leu and Arg287Gly, in human organic cation transporter hOCT1.

人类有机阳离子转运蛋白 hOCT1 中遗传变异 Pro283Leu 和 Arg287Gly 的功能降低。

• 发表时间: 2003
• 期刊: Drug Metab.Pharmacokinet. 18
• 作者: Takeuchi;A.
• 通讯作者: A.

Increased protein level of PEPT1 intestinal H+-peptide cotransporter upregulates absorption of glycylsarcoisine and ceftibuten in 5/6 nephrectomized rats.

PEPT1肠H肽协同转运蛋白的蛋白质水平增加上调5/6肾切除大鼠中甘氨酰肌氨酸和头孢布烯的吸收。

• 发表时间: 2005
• 期刊: Am.J.Physiol.Gastrointest.Liver Physiol. 288・4
• 作者: Shimizu;Y.
• 通讯作者: Y.

Takeuchi et al.: "Decreased function of genetic variants, Pro283Leu and Arg287Gly, in human organic cation transporter hOCT1"Drug Metab.Pharmacokin.. 18・6. 409-412 (2003)

Takeuchi等人：“人类有机阳离子转运蛋白hOCT1中遗传变异Pro283Leu和Arg287Gly的功能降低”Drug Metab.Pharmacokin.. 18・6 (2003)。

土井俊夫: "改訂 腎機能別薬剤使用マニュアル"じほう. 194 (2003)

Toshio Doi：“根据肾功能修订用药手册”Jiho 194（2003）。

Expression levels of renal organic anion transporters (OATs) and their correlation with anionic drug secretion in patients with renal diseases.

肾病患者肾脏有机阴离子转运蛋白（OAT）的表达水平及其与阴离子药物分泌的相关性。

• 发表时间: 2004
• 期刊: Pharm.Res.C 21
• 作者: Sakurai;Y.
• 通讯作者: Y.