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. 2010 Apr 12;5(4):e10121.
doi: 10.1371/journal.pone.0010121.

Nuclear receptor CAR represses TNFalpha-induced cell death by interacting with the anti-apoptotic GADD45B

Yukio Yamamoto  1 , Rick Moore, Richard A Flavell, Binfeng Lu, Masahiko Negishi
Affiliations

Nuclear receptor CAR represses TNFalpha-induced cell death by interacting with the anti-apoptotic GADD45B

Yukio Yamamoto et al. PLoS One. .

Abstract

Background: Phenobarbital (PB) is the most well-known among numerous non-genotoxic carcinogens that cause the development of hepatocellular carcinoma (HCC). PB activates nuclear xenobiotic receptor Constitutive Active/Androstane Receptor (CAR; NR1I3) and this activation is shown to determine PB promotion of HCC in mice. The molecular mechanism of CAR-mediated tumor promotion, however, remains elusive at the present time. Here we have identified Growth Arrest and DNA Damage-inducible 45beta (GADD45B) as a novel CAR target, through which CAR represses cell death.

Methodology/principal findings: PB activation of nuclear xenobiotic receptor CAR is found to induce the Gadd45b gene in mouse liver throughout the development of HCC as well as in liver tumors. Given the known function of GADD45B as a factor that represses Mitogen-activated protein Kinase Kinase 7 - c-Jun N-terminal Kinase (MKK7-JNK) pathway-mediated apoptosis, we have now demonstrated that CAR interacts with GADD45B to repress Tumor Necrosis Factor alpha ( TNFalpha)-induced JNK1 phosphorylation as well as cell death. Primary hepatocytes, prepared from Car(+/+), Car(-/-), Gadd45b(+/+) and Gadd45b(-/-) mice, were treated with TNFalpha and Actinomycin D to induce phosphorylation of JNK1 and cell death. Co-treatment with the CAR activating ligand TCPOBOP (1,4 bis[2-(3,5-dichloropyridyloxy)]benzene) has resulted in repression of both phosphorylation and cell death in the primary hepatocytes from Car(+/+) but not Car(-/-) mice. Repression by TCPOBOP was not observed in those prepared from Gadd45b(-/-) mice. In vitro protein-protein interaction and phosphorylation assays have revealed that CAR interacts with MKK7 and represses the MKK7-mediated phosphorylation of JNK1.

Conclusions/significance: CAR can form a protein complex with GADD45B, through which CAR represses MKK7-mediated phosphorylation of JNK1. In addition to activating the Gadd45b gene, CAR may repress death of mouse primary hepatocytes by forming a GADD45B complex and repressing MKK7-mediated phosphorylation of JNK1. The present finding that CAR can repress cell death via its interaction with GADD45B provides an insight for further investigations into the CAR-regulated molecular mechanism by which PB promotes development of HCC.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CAR-mediated repression of cell death.
Approximately the same number (1×105) of primary hepatocytes prepared from the livers of Car+/+ and Car−/− mice were plated on plastic dishes (24 wells). Cells were pretreated with DMSO or 250 nM TCPOBOP for 24 hours and were subsequently treated with or without TNFα (20 ng/ml) plus ActD (0.2 µg/ml) for 16 hours. Release of lactate hydrogenase (LDH) in culture media was determined as described in the Materials and Methods. Each value represents the mean +/− S.D as fold changes relative to DMSO without TNFα and ActD, which was independently reproduced 3 times. Symbol # indicates statistical significance between DMSO-pretreated cells and TCPOBOP-pretreated cells (p<0.05).
Figure 2
Figure 2. Screening of Gadd45b for a CAR-regulated gene in chronic PB-treated tissues.
RNAs were prepared from non-tumor tissues of the PB-treated livers for 23- and 32- weeks and from tumor tissues of the PB-treated livers for 32 weeks. At least, six animals were used for each group. Each value represents the mean +/− S.D as fold changes relative to the RNAs from the non-tumor liver tissues of wild type mice treated with diethylnitrosamine (DEN) for 23 weeks.
Figure 3
Figure 3. CAR-mediated repression of JNK phosphorylation.
Primary hepatocytes (1×105 cells/well) were prepared from Car+/+ and Car−/− mice, were pretreated with either DMSO (DM) or TCPOBOP (TC; 250 nM) and were co-treated with TNFα and ActD to induce cell death. Subsequently cell extracts were prepared for Western blot analysis for phosphorylated JNK (P-JNK) and total JNK.
Figure 4
Figure 4. GADD45B facilitating the ability of CAR to form a complex with MKK7.
A. GST pull down assays showing direct binding of GADD45B to CAR and MKK7. GST-GADD45B was incubated with the in vitro translated MKK7 and/or CAR, which was subjected for SDS polyacrylamide gel analysis as described in the Materials and Methods. B. Co-immunoprecipitation (Co-IP) assays to show formation of a CAR complex with MKK7. CAR-V5 was co-expressed with Flag-MMK7 in the presence and absence of GADD45B-V5 in HEK293T cells, co-precipitated by anti-Flag antibody and was analyzed by Western blot using anti-V5 and anti-Flag antibodies.
Figure 5
Figure 5. CAR potentiating the ability of GADD45B to inhibit MKK7 activity.
A. GST-CAR was pre-incubated with recombinant GADD45B and the active kinase MKK7 or MKK4 for 20 min at room temperature. Kinase reactions were initiated by adding ATP and purified substrate JNK1. Following incubation for 30 min at room temperature, incubation mixtures were applied on a SDS gel and were subjected to Western blotting analysis using anti-phospho-JNK antibody. Arabic numbers indicate the µg amounts of recombinant proteins in pre-reaction mixtures: 1 means approximately 1 µg of protein. B. CAR required its AF2 domain for the inhibitory activity. GST-pull down assays shows AF2 domain of CAR was required to bind to GADD45B. Purified GST-CAR mutants were incubated with purified GADD45B protein. Western blotting analysis was performed using anti-GADD45B antibody to detect binding. C. MKK7 kinase assays showing requirement of the AF2 domain for inhibition. The GST-CARΔAF2 mutant was used to measure its inhibitory activity of MKK7 kinase, which was analyzed Western blotting using anti-Phospho-JNK antibody as described in Experimental Procedures.
Figure 6
Figure 6. No CAR-dependent repression of cell death in the absence of GADD45B.
Primary hepatocytes were prepared from Gadd45b+/+ and Gadd45b−/− mice and were subjected to cell death assays as described in Materials and Methods and in the legend of Figure 1. Each value represents the mean +/− S.D as fold changes relative to DMSO without TNFα and ActD. The data shown reproduced in 3 independent experiments. Symbol # indicates statistical significance between DMSO-pretreated cells and TCPOBOP-pretreated cells (p<0.05).
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