The Roles of Co-Chaperone CCRP/DNAJC7 in Cyp2b10 Gene Activation and Steatosis Development in Mouse Livers

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.

Phosphorylated Nuclear Receptor CAR Forms a Homodimer To Repress Its Constitutive Activity for Ligand Activation

“…Although HSP90 appeared to be able to interact with a surface that forms a dimer interface, HSP90 was not critically involved in CAR homodimerization (our unpublished observation). Studies with CCRP KO mice found that while CCRP regulates PB-induced nuclear accumulation of CAR, CCRP also regulates CAR-mediated transcription in the nucleus (11). Active ERK1/2 binds to the XRS peptide near the C terminus of the CAR LBD to keep CAR phosphorylated (7).…”

Nuclear receptors and nonalcoholic fatty liver disease

“…Phenobarbital, is a classically described indirect CAR activator, and it works by modulating the threonine-38 phosphorylation site downstream of its direct interaction with the epidermal growth factor receptor (EGFR) [125]. Cytoplasmic CAR retention protein (CCRP) is speculated to have an alternate role as a co-activator of CAR in the nucleus, and CCRP −/− mice develop steatosis which implies that CAR activity may prevent steatosis [126]. …”

Phenobarbital Meets Phosphorylation of Nuclear Receptors

“…4). Apparently, nuclear translocation is essential but not sufficient for CAR to activate transcription and CAR activation requires distinct processes in the nucleus (Ohno et al, 2014). Recent studies with cytoplasmic CAR retention protein (CCRP) knockout (KO) mice showed that CCRP does not regulate basal intracellular localization of CAR (Ohno et al, 2014).…”

“…Although CCRP (the cochaperone DNAJC7) was first characterized for accumulating ectopic CAR in the cytoplasm of HepG2 cells , CCRP is now known to regulate CAR in the nucleus (Ohno et al, 2014). CAR-mediated transcription is diverse but tightly regulated in the nucleus.…”