Coordinated regulation of nuclear receptor CAR by CCRP/DNAJC7, HSP70 and the ubiquitin-proteasome system

• Published in: PloS one Vol. 9; no. 5; p. e96092
• Main Authors: Timsit, Yoav E, Negishi, Masahiko
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 02.05.2014; Public Library of Science (PLoS)
• Subjects: Animals; Benzene; Bile acids; Bilirubin; Biology and Life Sciences; CAR protein; CYP2B6 gene; Cytochrome; Cytoplasm; Cytoplasm - drug effects; Cytoplasm - metabolism; Deoxyribonucleic acid; DNA; Dose-Response Relationship, Drug; Drugs; Environmental health; Fatty acids; Gene regulation; Genes; Health sciences; Heat shock; Heat shock proteins; Heat-Shock Response - drug effects; Hep G2 Cells; Hsp60 protein; HSP70 Heat-Shock Proteins - metabolism; Hsp70 protein; Hsp90 protein; Humans; Immunoprecipitation; Laboratories; Leupeptins - pharmacology; Ligands; Liver cancer; Medicine and Health Sciences; Metabolic pathways; Mice; Nuclei; Penicillin; Phenobarbital; Phenobarbital - pharmacology; Phosphatase; Physiology; Plasmids; Proteasome Endopeptidase Complex - metabolism; Proteasome Inhibitors - pharmacology; Proteasomes; Protein interaction; Protein-protein interactions; Pyridines - pharmacology; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Cytoplasmic and Nuclear - metabolism; Regulation; Toxicology; Transcription activation; Transcriptional Activation - drug effects; Ubiquitin; Ubiquitin - metabolism; Ubiquitination; Ubiquitination - drug effects; North Carolina; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0096092

The constitutive active/androstane receptor (CAR) plays an important role as a coordinate transcription factor in the regulation of various hepatic metabolic pathways for chemicals such as drugs, glucose, fatty acids, bilirubin, and bile acids. Currently, it is known that in its inactive state, CAR is retained in the cytoplasm in a protein complex with HSP90 and the tetratricopeptide repeat protein cytosoplasmic CAR retention protein (CCRP). Upon activation by phenobarbital (PB) or the PB-like inducer 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP), CAR translocates into the nucleus. We have identified two new components to the cytoplasmic regulation of CAR: ubiquitin-dependent degradation of CCRP and protein-protein interaction with HSP70. Treatment with the proteasome inhibitor MG132 (5 µM) causes CAR to accumulate in the cytoplasm of transfected HepG2 cells. In the presence of MG132, TCPOBOP increases CCRP ubiquitination in HepG2 cells co-expressing CAR, while CAR ubiquitination was not detected. MG132 treatment of HepG2 also attenuated of TCPOBOP-induced CAR transcriptional activation on reporter constructs which contain CAR-binding DNA elements derived from the human CYP2B6 gene. The elevation of cytoplasmic CAR protein with MG132 correlated with an increase of HSP70, and to a lesser extent HSP60. Both CCRP and CAR were found to interact with endogenous HSP70 in HepG2 cells by immunoprecipitation analysis. Induction of HSP70 levels by heat shock also increased cytoplasmic CAR levels, similar to the effect of MG132. Lastly, heat shock attenuated TCPOBOP-induced CAR transcriptional activation, also similar to the effect of MG132. Collectively, these data suggest that ubiquitin-proteasomal regulation of CCRP and HSP70 are important contributors to the regulation of cytoplasmic CAR levels, and hence the ability of CAR to respond to PB or PB-like inducers.