Kawasaki Yuki, Nemoto Nobuo, Sakuma Tsutomu
Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan; Department of Cancer Cell Biology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan; Laboratory of Public Health, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki, Gunma, 370-0033, Japan.
Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Drug Metab Pharmacokinet. 2023 Feb;48:100485. doi: 10.1016/j.dmpk.2022.100485. Epub 2022 Dec 5.
The mouse cytochrome P450 1A2 (Cyp1a2) gene is one of the constitutive androstane receptor (CAR, NR1I3) activator-inducible genes, and the regions involved in induction were examined herein. A reporter gene assay indicated the involvement of the -0.2-kb region in the induction of transcriptional activation by the mouse CAR agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). Some putative nuclear receptor-binding elements were identified in this region, and mutations in the elements located at -160/-155 or -153/-148 abolished this induction. An electrophoretic mobility shift assay demonstrated that a fragment comprised of three elements was capable of binding to the CAR/retinoid X receptor alpha (RXRα) heterodimer. The three elements comprise the two elements indicated above and one located at -146/-141. A chromatin immunoprecipitation assay confirmed CAR binding to the region including these elements in chromatin after treatment with TCPOBOP. These results indicate that mouse Cyp1a2 is the direct target of CAR, and binding of the CAR/RXRα heterodimer to the newly identified region in the promoter may be involved in transcriptional activation. Binding motifs were estimated as ER1 (everted repeat with a spacing of 1 nucleotide, -160/-155 and -153/-148) and ER8 (everted repeat with a spacing of 8 nucleotides, formed with -160/-155 and -146/-141).
小鼠细胞色素P450 1A2（Cyp1a2）基因是组成型雄甾烷受体（CAR，NR1I3）激活剂诱导型基因之一，本文研究了参与诱导的区域。报告基因分析表明，-0.2 kb区域参与了小鼠CAR激动剂配体1,4-双-[2-(3,5-二氯吡啶氧基)]苯（TCPOBOP）对转录激活的诱导作用。在该区域鉴定出一些假定的核受体结合元件，位于-160/-155或-153/-148的元件发生突变会消除这种诱导作用。电泳迁移率变动分析表明，由三个元件组成的片段能够与CAR/视黄酸X受体α（RXRα）异二聚体结合。这三个元件包括上述两个元件和一个位于-146/-141的元件。染色质免疫沉淀分析证实，用TCPOBOP处理后，CAR与染色质中包括这些元件的区域结合。这些结果表明，小鼠Cyp1a2是CAR的直接靶标，CAR/RXRα异二聚体与启动子中新鉴定区域的结合可能参与转录激活。结合基序估计为ER1（间隔1个核苷酸的反向重复序列，-160/-155和-153/-148）和ER8（间隔8个核苷酸的反向重复序列，由-160/-155和-146/-141形成）。
