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

Timsit YE, Negishi M - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.

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.

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Proposed model for the regulation of CAR by the ubiquitin-proteasome system.An equilibrium between a stable CAR cytoplasmic complex (at top left) and a weaker associated complex exists (at top right), and HSP70 functions to favor the stable cytoplasmic complex. Activator treatment signals for ubiquitination of CCRP, resulting in its degradation by the proteasome. This results in the release of CAR from the cytosolic complex, followed by translocation into the nucleus.
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pone-0096092-g007: Proposed model for the regulation of CAR by the ubiquitin-proteasome system.An equilibrium between a stable CAR cytoplasmic complex (at top left) and a weaker associated complex exists (at top right), and HSP70 functions to favor the stable cytoplasmic complex. Activator treatment signals for ubiquitination of CCRP, resulting in its degradation by the proteasome. This results in the release of CAR from the cytosolic complex, followed by translocation into the nucleus.

Mentions: These findings suggest a simple model for CAR retention and regulation by the ubiquitin-proteasomal system (Fig. 7). In our model, we propose that CAR exists in equilibrium between a complexed and dissociated state with its cytoplasmic partners. With increased CCRP expression and in concert with HSP70, a heteromeric protein complex that includes CAR, CCRP, and HSP70 is favored resulting in cytosolic stabilization of CAR. CCRP ubiquitination would therefore regulate the amount of stabilized CAR in the cytosol. Upon treatment with a CAR activator, CCRP ubiquitination and degradation would increase (over basal levels) resulting in the release of CAR and its subsequent translocation to the nucleus. Consistent with this model, increased cytoplasmic retention of CAR by proteasomal inhibition would reduce transcriptional activity, and this was indeed our observation and consistent with that observed for human CAR [44].


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

Timsit YE, Negishi M - PLoS ONE (2014)

Proposed model for the regulation of CAR by the ubiquitin-proteasome system.An equilibrium between a stable CAR cytoplasmic complex (at top left) and a weaker associated complex exists (at top right), and HSP70 functions to favor the stable cytoplasmic complex. Activator treatment signals for ubiquitination of CCRP, resulting in its degradation by the proteasome. This results in the release of CAR from the cytosolic complex, followed by translocation into the nucleus.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4008524&req=5

pone-0096092-g007: Proposed model for the regulation of CAR by the ubiquitin-proteasome system.An equilibrium between a stable CAR cytoplasmic complex (at top left) and a weaker associated complex exists (at top right), and HSP70 functions to favor the stable cytoplasmic complex. Activator treatment signals for ubiquitination of CCRP, resulting in its degradation by the proteasome. This results in the release of CAR from the cytosolic complex, followed by translocation into the nucleus.
Mentions: These findings suggest a simple model for CAR retention and regulation by the ubiquitin-proteasomal system (Fig. 7). In our model, we propose that CAR exists in equilibrium between a complexed and dissociated state with its cytoplasmic partners. With increased CCRP expression and in concert with HSP70, a heteromeric protein complex that includes CAR, CCRP, and HSP70 is favored resulting in cytosolic stabilization of CAR. CCRP ubiquitination would therefore regulate the amount of stabilized CAR in the cytosol. Upon treatment with a CAR activator, CCRP ubiquitination and degradation would increase (over basal levels) resulting in the release of CAR and its subsequent translocation to the nucleus. Consistent with this model, increased cytoplasmic retention of CAR by proteasomal inhibition would reduce transcriptional activity, and this was indeed our observation and consistent with that observed for human CAR [44].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.

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.

Show MeSH
Related in: MedlinePlus