Abstract
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.
Highlights
Constitutive active receptor (CAR) was originally characterized as a drugactivated nuclear receptor that induces hepatic drug metabolism and secretion by activating genes that encode enzymes such as cytochrome P450s (CYP), sulfotransferases and UDP-glucuronosyltransferases as well as drug transporter genes [1,2,3,4,5,6,7]
Southern hybridization analysis confirmed the deletion of the Ccrp gene in the mouse genome (Fig. 1-B), whereas Western blot analysis using anti-mouse Cytoplasmic CAR Retention Protein (CCRP) polyclonal antibody demonstrated the presence of CCRP at protein levels within both the cytoplasm and nucleus of wild type (WT) but not in those of KO males (Fig. 1-C)
Our present work with livers of CCRP KO mice has confirmed that CCRP regulates CAR-mediated activation of the Cyp2b10 gene as well as the intracellular localization of CAR
Summary
Constitutive active receptor (CAR) was originally characterized as a drugactivated nuclear receptor that induces hepatic drug metabolism and secretion by activating genes that encode enzymes such as cytochrome P450s (CYP), sulfotransferases and UDP-glucuronosyltransferases as well as drug transporter genes [1,2,3,4,5,6,7]. Regulation by CAR has been extended far beyond drug metabolism to hepatic energy metabolism and cell growth and death, thereby becoming a critical factor in the development of diseases including diabetes and hepatocellular carcinoma [8, 9]. Threonine 38 is phosphorylated when epidermal growth factor receptor (EGFR) signaling is stimulated, while repression of this signaling results in dephosphorylation that activates CAR [12, 13]. CAR is, in principle, a cell signal-regulated nuclear receptor and this signal-mediated mechanism is demonstrated in both mouse and human liver cells [13, 14]. Phosphorylation of threonine 38 is an essential factor that regulates CAR activation and nuclear translocation
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