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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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Thermal stress attenuates TCPOBOP-induced CAR transcriptional activation in HepG2 cells, similar to the effect of proteasomal inhibition.Luciferase activity of each sample was normalized to Renilla activity, and expressed as means ± SD of triplicate determinations. Shown is representative of three independent experiments. (A and B) Experiments were performed as described in Fig. 3, however empty vector or pcDNA3.1/V5-His-mCCRP was cotransfected with pcDNA3.1/V5-His-mCAR, -1.8-kb-luc, and phRL-tk (as normalization control). After transfection, cells were treated for 24 hr with DMSO (0.1% v/v), TCPOBOP (250 nM dissolved in 0.1% DMSO, final concentrations), MG132 (5 µM in 0.1% DMSO, final concentrations), or MG132 plus TCPOBOP, after which cells were harvested and luciferase activity measured. For fold-change determinations (B), each group was normalized to its corresponding DMSO-treated control (set as 1). Statistics are based on one-way ANOVA with post-hoc Tukey multiple comparisons test. *Significantly different (p<0.0001) compared to DMSO; and #significantly different (p<0.002) compared to TCPOPOP without CCRP overexpression. (C and D) Experiments and data analysis were performed as in A and B, with an incubation step at 42°C for 1 hr for heat-shock-designated cells performed prior to 24 hr treatment with DMSO (0.1% v/v) or TCPOBOP (250 nM in 0.1% DMSO, final concentrations). For fold-change determinations (D), each group was normalized to its corresponding DMSO-treated control (set as 1). Statistics are based on one-way ANOVA with post-hoc Tukey multiple comparisons test. *Significantly different (p<0.0001) compared to corresponding DMSO control treatment; #significantly different (p<0.001) to TCPOPOP alone (without both CCRP overexpression and heat shock).
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pone-0096092-g006: Thermal stress attenuates TCPOBOP-induced CAR transcriptional activation in HepG2 cells, similar to the effect of proteasomal inhibition.Luciferase activity of each sample was normalized to Renilla activity, and expressed as means ± SD of triplicate determinations. Shown is representative of three independent experiments. (A and B) Experiments were performed as described in Fig. 3, however empty vector or pcDNA3.1/V5-His-mCCRP was cotransfected with pcDNA3.1/V5-His-mCAR, -1.8-kb-luc, and phRL-tk (as normalization control). After transfection, cells were treated for 24 hr with DMSO (0.1% v/v), TCPOBOP (250 nM dissolved in 0.1% DMSO, final concentrations), MG132 (5 µM in 0.1% DMSO, final concentrations), or MG132 plus TCPOBOP, after which cells were harvested and luciferase activity measured. For fold-change determinations (B), each group was normalized to its corresponding DMSO-treated control (set as 1). Statistics are based on one-way ANOVA with post-hoc Tukey multiple comparisons test. *Significantly different (p<0.0001) compared to DMSO; and #significantly different (p<0.002) compared to TCPOPOP without CCRP overexpression. (C and D) Experiments and data analysis were performed as in A and B, with an incubation step at 42°C for 1 hr for heat-shock-designated cells performed prior to 24 hr treatment with DMSO (0.1% v/v) or TCPOBOP (250 nM in 0.1% DMSO, final concentrations). For fold-change determinations (D), each group was normalized to its corresponding DMSO-treated control (set as 1). Statistics are based on one-way ANOVA with post-hoc Tukey multiple comparisons test. *Significantly different (p<0.0001) compared to corresponding DMSO control treatment; #significantly different (p<0.001) to TCPOPOP alone (without both CCRP overexpression and heat shock).

Mentions: As heat stress elevates cytosolic HSP70 levels, with concomitant elevation of CAR and to a smaller extent CCRP, we sought to determine whether ligand-activated CAR transcriptional activation is altered by heat stress. Firstly, it was important to establish the effect of increased cytosolic retention of CAR by means other than proteasomal inhibition. With the observations described earlier that CCRP overexpression reduces the ability of TCPOBOP to translocate accumulated CAR to the nucleus, TCPOBOP-induced CAR transcriptional activation was assessed by overexpressing mCCRP with mCAR in HepG2 cells and using the PBREM-containing -1.8-kb-luc reporter. As a control, experiments were performed using a -1.6-kb-luc reporter lacking the distal CYP2B6 phenobarbital responsive enhancer module (PBREM), and TCPOBOP-induced reporter activity was absent (data not shown). Normalized luciferase (Fig. 6A) and fold-change (Fig. 6B) activities are shown under the various conditions. TCPOBOP induced CAR transcriptional activity both in the absence and presence of overexpressed CCRP (Figs. 6A and 6B); CCRP overexpression attenuated slightly the induction by TCPOBOP. Treatment with MG132 robustly attenuated TCPOBOP-induced reporter activity (absolute and fold-change) irrespective of CCRP overexpression (Figs 6A and 6B), demonstrating again the potent effect of proteasome inhibition on CAR-mediated transcriptional activation.


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

Timsit YE, Negishi M - PLoS ONE (2014)

Thermal stress attenuates TCPOBOP-induced CAR transcriptional activation in HepG2 cells, similar to the effect of proteasomal inhibition.Luciferase activity of each sample was normalized to Renilla activity, and expressed as means ± SD of triplicate determinations. Shown is representative of three independent experiments. (A and B) Experiments were performed as described in Fig. 3, however empty vector or pcDNA3.1/V5-His-mCCRP was cotransfected with pcDNA3.1/V5-His-mCAR, -1.8-kb-luc, and phRL-tk (as normalization control). After transfection, cells were treated for 24 hr with DMSO (0.1% v/v), TCPOBOP (250 nM dissolved in 0.1% DMSO, final concentrations), MG132 (5 µM in 0.1% DMSO, final concentrations), or MG132 plus TCPOBOP, after which cells were harvested and luciferase activity measured. For fold-change determinations (B), each group was normalized to its corresponding DMSO-treated control (set as 1). Statistics are based on one-way ANOVA with post-hoc Tukey multiple comparisons test. *Significantly different (p<0.0001) compared to DMSO; and #significantly different (p<0.002) compared to TCPOPOP without CCRP overexpression. (C and D) Experiments and data analysis were performed as in A and B, with an incubation step at 42°C for 1 hr for heat-shock-designated cells performed prior to 24 hr treatment with DMSO (0.1% v/v) or TCPOBOP (250 nM in 0.1% DMSO, final concentrations). For fold-change determinations (D), each group was normalized to its corresponding DMSO-treated control (set as 1). Statistics are based on one-way ANOVA with post-hoc Tukey multiple comparisons test. *Significantly different (p<0.0001) compared to corresponding DMSO control treatment; #significantly different (p<0.001) to TCPOPOP alone (without both CCRP overexpression and heat shock).
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getmorefigures.php?uid=PMC4008524&req=5

pone-0096092-g006: Thermal stress attenuates TCPOBOP-induced CAR transcriptional activation in HepG2 cells, similar to the effect of proteasomal inhibition.Luciferase activity of each sample was normalized to Renilla activity, and expressed as means ± SD of triplicate determinations. Shown is representative of three independent experiments. (A and B) Experiments were performed as described in Fig. 3, however empty vector or pcDNA3.1/V5-His-mCCRP was cotransfected with pcDNA3.1/V5-His-mCAR, -1.8-kb-luc, and phRL-tk (as normalization control). After transfection, cells were treated for 24 hr with DMSO (0.1% v/v), TCPOBOP (250 nM dissolved in 0.1% DMSO, final concentrations), MG132 (5 µM in 0.1% DMSO, final concentrations), or MG132 plus TCPOBOP, after which cells were harvested and luciferase activity measured. For fold-change determinations (B), each group was normalized to its corresponding DMSO-treated control (set as 1). Statistics are based on one-way ANOVA with post-hoc Tukey multiple comparisons test. *Significantly different (p<0.0001) compared to DMSO; and #significantly different (p<0.002) compared to TCPOPOP without CCRP overexpression. (C and D) Experiments and data analysis were performed as in A and B, with an incubation step at 42°C for 1 hr for heat-shock-designated cells performed prior to 24 hr treatment with DMSO (0.1% v/v) or TCPOBOP (250 nM in 0.1% DMSO, final concentrations). For fold-change determinations (D), each group was normalized to its corresponding DMSO-treated control (set as 1). Statistics are based on one-way ANOVA with post-hoc Tukey multiple comparisons test. *Significantly different (p<0.0001) compared to corresponding DMSO control treatment; #significantly different (p<0.001) to TCPOPOP alone (without both CCRP overexpression and heat shock).
Mentions: As heat stress elevates cytosolic HSP70 levels, with concomitant elevation of CAR and to a smaller extent CCRP, we sought to determine whether ligand-activated CAR transcriptional activation is altered by heat stress. Firstly, it was important to establish the effect of increased cytosolic retention of CAR by means other than proteasomal inhibition. With the observations described earlier that CCRP overexpression reduces the ability of TCPOBOP to translocate accumulated CAR to the nucleus, TCPOBOP-induced CAR transcriptional activation was assessed by overexpressing mCCRP with mCAR in HepG2 cells and using the PBREM-containing -1.8-kb-luc reporter. As a control, experiments were performed using a -1.6-kb-luc reporter lacking the distal CYP2B6 phenobarbital responsive enhancer module (PBREM), and TCPOBOP-induced reporter activity was absent (data not shown). Normalized luciferase (Fig. 6A) and fold-change (Fig. 6B) activities are shown under the various conditions. TCPOBOP induced CAR transcriptional activity both in the absence and presence of overexpressed CCRP (Figs. 6A and 6B); CCRP overexpression attenuated slightly the induction by TCPOBOP. Treatment with MG132 robustly attenuated TCPOBOP-induced reporter activity (absolute and fold-change) irrespective of CCRP overexpression (Figs 6A and 6B), demonstrating again the potent effect of proteasome inhibition on CAR-mediated transcriptional activation.

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