Characterization of the basal promoter element of human organic cation transporter 2 gene
Characterization of the basal promoter element of human organic cation transporter 2 gene
复制标题
DOI：
10.1124/jpet.106.118695
复制
发表时间：
2007-05-01
影响因子：
3.5
通讯作者：
Inui, Ken-ichi
中科院分区：
文献类型：
--
作者：
Asaka, Jun-ichi;Terada, Tomohiro;Inui, Ken-ichi
人有机阳离子转运蛋白2 （hOCT2; SLC22A2）在肾脏中大量表达，在肾小管分泌阳离子药物中起重要作用。虽然hOCT2的转运特性已经被广泛研究，但没有关于hOCT2转录调控的信息。本研究旨在确定hOCT2基础表达的顺式元件和反式因子。利用cDNA末端的5′-快速扩增，转录起始位点位于翻译起始位点上方385个核苷酸。分离出一个大约4千碱基的hOCT2启动子区域片段，并在肾上皮细胞系LLC-PK1中测量了启动子活性。缺失分析表明，跨越-91至-58碱基对的区域对基础转录活性至关重要。该区域缺少一个tata盒，但有一个CCAAT盒和一个E-box。电泳迁移量转移实验表明，E-box中存在特异性的DNA/蛋白复合物，而CCAAT中不存在；超转移实验显示，上游刺激因子1 （USF-1）与E-box结合，该因子属于基本的螺旋-环-螺旋-亮氨酸拉链转录因子家族。E-box突变导致hOCT2启动子活性降低，USF-1过表达以剂量依赖性方式增强hOCT2启动子活性。本文首次报道了hOCT2启动子的特征，并表明USF-1通过E-box作为hOCT2基因的基础转录调节因子发挥作用。
Human organic cation transporter 2 (hOCT2; SLC22A2) is abundantly expressed in the kidney, and it plays important roles in the renal tubular secretion of cationic drugs. Although the transport characteristics of hOCT2 have been studied extensively, there is no information available for the transcriptional regulation of hOCT2. The present study was undertaken to identify the cis-element and trans-factor for basal expression of hOCT2. The transcription start site was located 385 nucleotides above the translation start site by using 5'-rapid amplification of cDNA ends. An approximately 4-kilobase fragment of the hOCT2 promoter region was isolated and the promoter activities were measured in the renal epithelial cell line LLC-PK1. A deletion analysis suggested that the region spanning -91 to -58 base pairs was essential for basal transcriptional activity. This region lacked a TATA-box but contained a CCAAT box and an E-box. Electrophoretic mobility shift assays showed that specific DNA/protein complexes were present in the E-box but not in the CCAAT box, and supershift assays revealed that upstream stimulatory factor 1 ( USF-1), which belongs to the basic helix-loop-helix-leucine zipper family of transcription factors, bound to the E-box. Mutation of the E-box resulted in a decrease in hOCT2 promoter activity, and overexpression of USF-1 enhanced the hOCT2 promoter activity in a dose-dependent manner. This article reports the first characterization of the hOCT2 promoter and shows that USF-1 functions as a basal transcriptional regulator of the hOCT2 gene via the E-box.