Abstract
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.
Highlights
The constitutive active/androstane receptor (CAR) is a member of the xenobiotic-sensing nuclear receptor, functioning as a ligandactivated transcription factor capable of regulating the expression of genes involved in the metabolism of both xenobiotics and endogenous chemicals produced in the organism [1,2]
It had been shown previously that cytosoplasmic CAR retention protein (CCRP) overexpression stabilizes CAR in the cytosol of HepG2 cells, and that TCPOBOP treatment is less efficacious to cause nuclear translocation of CAR [24]. These findings were based on assessment of mouse CAR (mCAR) protein levels; CCRP protein levels upon TCPOBOP treatment were not ascertained we proceeded to determine the effect of TCPOBOP on both CCRP and CAR
To test whether the ubiquitin-proteasomal pathway regulates CCRP levels, HepG2 cells were co-transfected with V5-tagged CCRP and FLAG-tagged CAR [28], which in reporter experiments responds to TCPOBOP to induce transcriptional activation like the V5-tagged CAR expression construct
Summary
The constitutive active/androstane receptor (CAR) is a member of the xenobiotic-sensing nuclear receptor, functioning as a ligandactivated transcription factor capable of regulating the expression of genes involved in the metabolism of both xenobiotics and endogenous chemicals produced in the organism [1,2]. It was identified in the late 1990s as the principal mediator of the induction by barbiturates such as phenobarbital (PB) of the human cytochrome P450 2B6 (CYP2B6), the rat CYP2B1, and the mouse Cyp2b10 genes [3,4,5,6,7]. The biological significance of these identified pathway interactions is only beginning to be understood, opening new insights into CAR signaling within the cell
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