Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences and WPI Nano Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences and WPI Nano Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa, Japan
J Pharmacol Exp Ther. 2019 Sep;370(3):408-415. doi: 10.1124/jpet.119.260109. Epub 2019 Jul 3.
Adenosine deaminases acting on RNA (ADARs) enzymes-catalyzing adenosine-to-inosine RNA editing possibly modulates gene expression and function. In this study, we investigated whether ADARs regulate the expression of human constitutive androstane receptor (CAR), which controls the expression of various drug-metabolizing enzymes. CAR mRNA and protein levels in human hepatocellular carcinoma-derived HepG2 cells were increased by knockdown of ADAR1 and slightly increased by ADAR2, indicating that ADARs negatively regulate CAR expression. Increased luciferase activity of a reporter plasmid containing the CYP3A4 promoter region by phenobarbital was augmented by transfection of siRNA for ADAR1 (siADAR1) but not by siADAR2. In addition, the knockdown of ADAR1 resulted in the enhanced induction of CYP2B6 and CYP3A4 mRNA by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde -(3,4-dichlorobenzyl)oxime and phenobarbital, respectively. These results suggest that ADAR1-mediated downregulation of CAR affects its downstream cytochrome P450 expression. When the transcription was inhibited by -amanitin, the degradation of CAR mRNA was attenuated by knockdown of ADAR1, suggesting that the increase in CAR mRNA level by ADAR1 knockdown is a post-transcriptional event. Finally, we found that ADAR1 knockdown promotes the splicing of CAR as a mechanism of the increased expression of CAR by ADAR1 knockdown. In conclusion, this study revealed that ADAR1 plays a role in modulating xenobiotic metabolism potency via regulation of CAR. SIGNIFICANCE STATEMENT: This study revealed that adenosine deaminase acting on RNA 1 (ADAR1) and ADAR2, which catalyze adenosine-to-inosine RNA editing, downregulate the expression of constitutive androstane receptor (CAR) in human liver-derived cells by attenuating splicing. The downregulation of CAR by ADARs affected its downstream cytochrome P450 expression. ADARs would play a role in modulating xenobiotic metabolism potency via regulation of CAR.
腺苷脱氨酶作用于 RNA（ADAR）酶催化的腺苷到肌苷 RNA 编辑可能调节基因表达和功能。在这项研究中，我们研究了 ADAR 是否调节人组成型雄烷受体 (CAR) 的表达，CAR 控制着各种药物代谢酶的表达。人肝癌衍生的 HepG2 细胞中 CAR mRNA 和蛋白水平通过 ADAR1 的敲低而增加，ADAR2 的敲低略有增加，表明 ADAR 负调节 CAR 表达。含有 CYP3A4 启动子区域的报告质粒的荧光素酶活性通过 ADAR1 的 siRNA（siADAR1）转染而增强，但 ADAR2 的 siRNA 则不然。此外，ADAR1 的敲低导致 CYP2B6 和 CYP3A4 mRNA 分别由 6-(4-氯苯基)咪唑并[2,1-b][1,3]噻唑-5-甲酰基-(3,4-二氯苄基)肟和苯巴比妥诱导增强。这些结果表明，ADAR1 介导的 CAR 下调影响其下游细胞色素 P450 的表达。当转录被鹅膏蕈碱抑制时，CAR mRNA 的降解被 ADAR1 的敲低减弱，这表明 ADAR1 敲低导致 CAR mRNA 水平增加是一种转录后事件。最后，我们发现 ADAR1 的敲低促进 CAR 的剪接，这是 ADAR1 敲低导致 CAR 表达增加的机制。总之，这项研究表明，ADAR1 通过调节 CAR 在调节异生物代谢效力方面发挥作用。意义声明：这项研究表明，RNA 上的腺苷脱氨酶 1（ADAR1）和 ADAR2 通过减弱剪接，下调人肝源细胞中组成型雄烷受体（CAR）的表达，它们催化腺苷到肌苷 RNA 编辑。ADARs 下调 CAR 影响其下游细胞色素 P450 的表达。ADARs 通过调节 CAR 在调节异生物代谢效力方面发挥作用。
