Ohno Marumi, Kanayama Tomohiko, Moore Rick, Ray Manas, Negishi Masahiko
Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.
Knockout Core, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.
PLoS One. 2014 Dec 26;9(12):e115663. doi: 10.1371/journal.pone.0115663. eCollection 2014.
Cytoplasmic constitutive active/androstane receptor (CAR) retention protein (CCRP and also known as DNAJC7) is a co-chaperone previously characterized to retain nuclear receptor CAR in the cytoplasm of HepG2 cells. Here we have produced CCRP knockout (KO) mice and demonstrated that CCRP regulates CAR at multiple steps in activation of the cytochrome (Cyp) 2b10 gene in liver: nuclear accumulation, RNA polymerase II recruitment and epigenetic modifications. Phenobarbital treatment greatly increased nuclear CAR accumulation in the livers of KO males as compared to those of wild type (WT) males. Despite this accumulation, phenobarbital-induced activation of the Cyp2b10 gene was significantly attenuated. In ChIP assays, a CAR/retinoid X receptor-α (RXRα) heterodimer binding to the Cyp2b10 promoter was already increased before phenobarbital treatment and further pronounced after treatment. However, RNA polymerase II was barely recruited to the promoter even after phenobarbital treatment. Histone H3K27 on the Cyp2b10 promoter was de-methylated only after phenobarbital treatment in WT but was fully de-methylated before treatment in KO males. Thus, CCRP confers phenobarbital-induced de-methylation capability to the promoter as well as the phenobarbital responsiveness of recruiting RNA polymerase II, but is not responsible for the binding between CAR and its cognate sequence, phenobarbital responsive element module. In addition, KO males developed steatotic livers and increased serum levels of total cholesterol and high density lipoprotein in response to fasting. CCRP appears to be involved in various hepatic regulations far beyond CAR-mediated drug metabolism.
细胞质组成型活性/雄烷受体（CAR）滞留蛋白（CCRP，也称为DNAJC7）是一种辅助伴侣蛋白，先前的研究表明它能将核受体CAR滞留在HepG2细胞的细胞质中。在此，我们制备了CCRP基因敲除（KO）小鼠，并证明CCRP在肝脏中细胞色素（Cyp）2b10基因激活的多个步骤中对CAR进行调控：核积累、RNA聚合酶II募集和表观遗传修饰。与野生型（WT）雄性小鼠相比，苯巴比妥处理显著增加了KO雄性小鼠肝脏中CAR的核积累。尽管有这种积累，但苯巴比妥诱导的Cyp2b10基因激活明显减弱。在染色质免疫沉淀（ChIP）分析中，CAR/视黄酸X受体-α（RXRα）异二聚体与Cyp2b10启动子的结合在苯巴比妥处理前就已增加，处理后进一步增强。然而，即使在苯巴比妥处理后，RNA聚合酶II也几乎未被募集到启动子上。WT小鼠中，Cyp2b10启动子上的组蛋白H3K27仅在苯巴比妥处理后去甲基化，而在KO雄性小鼠中，处理前就已完全去甲基化。因此，CCRP赋予启动子苯巴比妥诱导的去甲基化能力以及募集RNA聚合酶II的苯巴比妥反应性，但不负责CAR与其同源序列苯巴比妥反应元件模块之间的结合。此外，KO雄性小鼠在禁食后出现脂肪变性肝脏，血清总胆固醇和高密度脂蛋白水平升高。CCRP似乎参与了远远超出CAR介导的药物代谢的各种肝脏调节。
