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.

Figure 1. Gene targeting and conditional deletion of exon 2 to 4 of the mouse Ccrp gene.

(A) Restriction maps of the wild type allele and knockout construct. After Cre recombination, Ccrp gene exons 2–4 deleted allele (ΔEx) was generated as described in material and methods. (B) Determination and confirmation of genotype for CCRP KO mice by Southern blot analyses. Expected size of digested fragments was shown. (left) KpnI digested, and (right) BglI digested genomic DNA derived from CCRP KO mice were detected by indicated probes. (C) CCRP protein expression in the cytoplasm and the nuclei. The cytoplasm and nuclear proteins were extracted from the livers of WT and KO males and 10 µg proteins were subjected to western blotting as described in materials and method section. HSP90 and TBP were used as loading controls of the cytoplasm and the nuclei, respectively. (D) Genotyping result of DKO mice. Genomic DNA was extracted from ear tissue. CAR primer set generated 266 bp and 466 bp PCR products for WT and KO, respectively. CCRP primer set generated 150 bp and 450 bp products for WT and KO, respectively.

Figure 2. CAR protein levels after PB treatment.

Fifteen microgram aliquots of liver nuclear extract or whole lysate were used for western blot analysis as described in materials and method section. Nuclear accumulation of CAR after 6 h PB treatment in WT and KO males on the left. On the right, total CAR protein levels in liver whole lysate prepared from PB-treated WT and KO males. We used TBP and β-Actin as loading control of nuclear extract and whole lysate, respectively. Columns denote mean ± SE determined in at 3 individual animals. Opened and closed columns represent control and PB-treated animals, respectively. Unpaired t-test was used to compare the relative CAR levels between genotypes (*WT vs KO, p<0.05). This experiment was repeated twice with different sample set prepared from different animals.

Figure 3. The mRNA expressions of CAR target genes in the livers after 6 h PB treatment.

(A) CYP2B10 and CYP2C55 mRNA levels relative to GAPDH mRNA were measured by qRT-PCR. The relative levels were expressed as a ratio to that of PBS-treated WT mice. Columns denote mean ± SE determined in at 6-8 individual animals. Opened and closed columns represent control and PB-treated animals, respectively. One way-ANOVA was used to compare delta Ct values among groups (*control vs PB-treated, p<0.05; #WT vs KO, p<0.05). (B) Delta Ct values of CYP2B10 mRNA levels measured by qRT-PCR. The value was calculated by subtracting GAPDH Ct value from CYP2B10 Ct value in each animal.

Figure 4. The recruitment of transcription factors and histone de-methylation after PB treatment.

(A) The mRNA expressions of CYP2B10 in the livers of WT, KO and DKO mice after 3 h PB treatment. Columns denote mean ± SD determined in triplicates. Opened and closed columns represent control and PB-treated animals, respectively. (B) PB-induced recruitment of CAR/RXRα and RNA polymerase II in the Cyp2b10 promoter in the livers of WT and KO; WT and DKO mouse liver. Precipitation of DNA fragments by anti-RXRα and anti-RNA polymerase II antibodies at PBREM and TATA box-containing region, respectively, was determined. (C) Histone de-methylation after PB treatment in the Cyp2b10 promoter of WT and KO; WT and DKO mouse liver. Precipitation of DNA fragments by antibodies against the repressive mark H3K27me3. For both, precipitation of DNA fragments by normal IgG was used as negative control and DNA fragments without immunoprecipitation (Input) were used as positive control. Data shown are representative of results from two individual experiments.

Figure 5. Development of steatotic liver in non-treated KO males.

(A) HE or Oil-red-O staining of livers from WT and KO males fasted for 24 h. C: central vein; P: portal vein. Magnification, 200x. (B) Serum levels of total cholesterol, HDL and LDL in males. Columns denote mean ± SE determined in 6 individual animals. Opened and closed columns represent WT and KO males, respectively. Unpaired t-test was used to compare the levels between genotypes (*WT vs KO, p<0.05).