Nuclear Receptor CAR Represses TNFa-Induced Cell
Death by Interacting with the Anti-Apoptotic GADD45B
Yukio Yamamoto1¤, Rick Moore1, Richard A. Flavell2, Binfeng Lu3, Masahiko Negishi1*
1Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park,
North Carolina, United States of America, 2Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, United States of America,
3Department of Immunology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, United States of America
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 45b (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 a ( TNFa)-
induced JNK1 phosphorylation as well as cell death. Primary hepatocytes, prepared from Car+/+, Car2/2, Gadd45b+/+and
Gadd45b2/2mice, were treated with TNFa 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 Car2/2mice. Repression by TCPOBOP
was not observed in those prepared from Gadd45b2/2mice. 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.
Citation: Yamamoto Y, Moore R, Flavell RA, Lu B, Negishi M (2010) Nuclear Receptor CAR Represses TNFa-Induced Cell Death by Interacting with the Anti-
Apoptotic GADD45B. PLoS ONE 5(4): e10121. doi:10.1371/journal.pone.0010121
Editor: Ulrich Zanger, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Germany
Received January 27, 2010; Accepted March 15, 2010; Published April 12, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This study was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Environmental Health:
Z01ES71005-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: Medical Top Track Program, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Nuclear receptor CAR was originally characterized as a PB-
activated transcription factor that up-regulates the cytochrome
P4502B genes [1,2]. Soon after this, CAR became established as
the nuclear receptor that regulates hepatic drug metabolism and
excretion by coordinately activating various hepatic genes that
encode cytochrome P450s, conjugation enzymes such as UDP-
glucuronosyltransferases and sulfotransferases and drug transport-
ers [3,4,5]. Recent work has extended the biological function of
CAR far beyond the regulation of drug metabolism: for example,
CAR is now implicated in the regulation of hepatic energy
metabolism by cross talking with insulin or glucagon response
transcription factors; FoxO1, FoxA2, PGC-1 and CREB [6,7,8,9].
CAR is also found to play an essential role in nongenotoxic
carcinogenesis; drug activation of CAR by PB resulted in the
promotion of HCC development in mice, while no such
promotion occurred in the absence of CAR [10,11]. Thus, CAR
has been suggested to regulate a cellular signal leading to cell
growth and death.
GADD45B is a signal molecule inducible to response through
external stimuli such as oxidative stress, inflammation and UV
irradiation [12,13,14]. GADD45B is an anti-apoptotic factor that
directly binds to MKK7 and inhibits MKK7-dependent phos-
phorylation of JNK1/2 to repress apoptosis [15,16] and,
moreover, a possible role of GADD45B in hepatocarcinogenesis
and hepatocyte proliferation has been suggested [17,18]. To
search for a CAR-regulated signal molecule that may be involved
in promotion of HCC development, we hypothesized that this
factor should exhibit CAR-dependent induction by nongenotoxic
carcinogens long before the liver develops tumors and should be
further induced in tumor tissues. c-JUN, MDM2 and FoxM1B
PLoS ONE | www.plosone.org1 April 2010 | Volume 5 | Issue 4 | e10121
were previously suggested as signal molecules critical for HCC
development [10,19,20]. While none of these genes fit with this
hypothesis, the expression pattern of the Gadd45b gene is consistent
with the hypothesis. In addition, the Gadd45b gene has been known
to be induced after short-term treatment with CAR activator
TCPOBOP in mouse livers [21,22]. Therefore, here we have
further investigated GADD45B as a candidate of CAR-regulated
For present investigations, we have used mouse primary
hepatocytes prepared from Car+/+, Car2/2, Gadd45b+/+and
Gadd45b2/2mice. Cell death was induced by treatment of
primary hepatocytes with TNFa and actinomycin D (ActD) in the
presence or absence of TCPOBOP to examine whether or not
CAR could repress cell death and as to whether GADD45B could
mediate the CAR-mediated repression of cell death. Moreover,
GST-pull down and co-immunoprecipitation assays were per-
formed to characterize the binding nature of CAR to GADD45B.
Given the finding that TCPOBOP treatment decreased the
phosphorylation of JNK in the primary hepatocytes from Car+/+
but not from Car2/2, in vitro kinase assays were employed to
demonstrate CAR potentiating GADD45B-dependent inhibition
of JNK1 phosphorylation by MKK7. Here we present the
experimental considerations consistent with the conclusion that
CAR represses the death of TNFa-induced primary hepatocytes
by increasing GADD45B activity and inhibiting the MKK7-
dependent phosphorylation of JNK.
Results and Discussion
CAR repressing cell death
To examine whether or not CAR could play a role in cell death,
mouse primary hepatocytes were treated with either TNFa or
ActD or were co- treated with both TNFa and ActD to induce cell
death. TNFa is known to induce apoptosis of hepatocytes [23,24].
In our experimental conditions, only the co-treatment of TNFa
and ActD increased the cell death of primary hepatocytes
prepared from Car+/+mice by approximately 3-fold (Figure 1).
Pre-treatment with CAR activator TCPOBOP resulted in an
approximately 55% reduction of the TNFa/ActD induced cell
death. In the primary hepatocytes prepared from Car2/2mice, the
TNFa/ActD induced cell death was already elevated approxi-
mately 1.5-fold over the corresponding cell death of Car+/+
primary hepatocytes in the absence of TCPOBOP (Figure 1).
Consistent with the lack of CAR, TCPOBPO treatment did not
affect the cell death sensitivity of Car2/2primary hepatocytes. The
results indicated that CAR repressed the cell death: TCPOBOP
activation of CAR attenuated the TNFa/ActD induced cell death
of mouse primary hepatocytes and that cell death was intrinsically
higher in Car2/2primary hepatocytes.
Gadd45b as the CAR-regulated gene
Given the fact that CAR repressed cell death of mouse
hepatocytes, we searched for a CAR-regulated gene that encodes
a signal molecule involved in cell death using the liver samples
collected during our previous studies of PB promotion of HCC
development in the Car+/+and Car2/2mice . For the search,
two criteria were placed: the CAR-regulated gene should be
induced by PB before the liver developed HCC and this gene
should be continuously induced further in the tumor tissues. The
Gadd45b gene was reported to be induced by a 24 hr treatment
with TCPOBOP in the mouse liver . The expression of the
Gadd45b gene was determined in the liver samples of the Car+/+
and Car2/2treated with PB for 23 weeks and for 32 weeks. No
tumors developed in the livers treated 23 weeks. From the livers
treated 32 weeks, tumor tissues and the non tumor tissues were
separately dissected out for analysis. Real-time PCR assays showed
that GADD45B mRNA had increased in the 23 weeks of PB
treatment in the livers of the Car+/+mice but not of Car2/2mice
(Figure 2). This mRNA was further increased in the tumor tissues.
Conversely, the other members of the GADD45 family,
GADD45A and GADD45G mRNAs were not induced by PB
The two signal molecules c-JUN and FOXM1B have been
suggested as being the factors responsible for HCC development in
PB-treated mouse liver based on the fact that the c-Jun2/2and
Foxm1b2/2mice exhibited attenuated HCC development [19,20].
In addition, MDM2 was implicated as the CAR-regulated gene
that was important for PB induced HCC development . Real
time PCR was performed to determine the expression of these
three genes. Neither c-JUN, FOXM1B nor MDM2 mRNA were
Figure 1. CAR-mediated repression of cell death. Approximately the same number (16105) of primary hepatocytes prepared from the livers of
Car+/+and Car2/2mice 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 TNFa (20 ng/ml) plus ActD (0.2 mg/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 +/2 S.D as fold changes relative to DMSO without TNFa and
ActD, which was independently reproduced 3 times. Symbol # indicates statistical significance between DMSO-pretreated cells and TCPOBOP-
pretreated cells (p,0.05).
CAR and GADD45B in Cell Death
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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 +/2 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.
CAR and GADD45B in Cell Death
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induced by PB either for 23 or 32 weeks treatments, although they
were up-regulated in the tumor tissues, c-JUN and FOXM1B in
particular (Figure 2). Thus, none of these three genes appeared to
be regulated by CAR, implying that they are not directly
responsible for CAR-mediated tumor promotion by PB, although
they are critical for maintaining tumor status. Therefore, we chose
GADD45B to further investigate its potential at being the CAR-
regulated signal molecule that may regulate the TNFa-dependent
cell death of mouse primary hepatocytes.
CAR repressing JNK phosphorylation in mouse primary
GADD45B is known to directly bind to MKK7 inhibiting its
activity to phosphorylate JNK, resulting in the repression of
apoptosis [16,25]. Based on the finding that CAR up-regulated the
Gadd45b gene, we examined whether or not the activation of CAR
could down-regulate JNK phosphorylation in mouse primary
hepatocytes (Figure 3). TCPOBOP treatment attenuated JNK
phosphorylation by TNFa/ActD in the primary hepatocytes of the
Car+/+but not Car2/2mice. The levels of JNK protein remained
unchanged by TCPOBOP treatment in all primary hepatocytes.
Although the effect was not as large as that observed with the cell
death assays (Figure 1), the JNK phosphorylation level was higher
in the DMSO-treated Car2/2hepatocytes when compared with
the corresponding Car+/+hepatocytes. Thus, CAR was clearly
capable of attenuating the phosphorylation of JNK in mouse
primary hepatocytes, tempting us to investigate the molecular
mechanism of CAR-mediated repression of JNK phosphorylation.
CAR potentiating GADD45B-mediated inhibition of MKK7
Confirming previous observations [15,16], GST-pull down
assays showed the binding of GADD45B to MKK7 (Figure 4A).
We also found that GADD45B binds directly to CAR.
Subsequently, the interactions between CAR, GADD45B and
MKK7 were examined by co-expressing combinations of
GADD45B-V5, CAR-V5 and Flag-MKK7 in HEK293T cells
and by co-immunoprecipitating the GADD45B and CAR with
anti-Flag antibody from the cell extracts. Flag antibody precipi-
tated MKK7 as shown by the Western blot with anti-Flag
antibody and co-precipitated GADD45B-V5 when it was co-
expressed with Flag-MKK7 (Figure 4B). CAR-V5 was also co-
precipitated by Flag antibody, suggesting that CAR could form a
complex with MKK7. With co-expression of all three proteins,
CAR-V5 profoundly increased its binding to Flag-MKK7 in the
presence of GADD45B-V5, whereas GADD45B binding to
MKK7 remained unchanged (Figure 4B). These results indicated
that GADD45B could increase the formation of a CAR-MKK7
complex. This complex might be a triple complex of CAR-
GADD45B-MKK7, in which the molecular ratio of three proteins
remained to be determined by future investigations.
Figure 3. CAR-mediated repression of JNK phosphorylation.
Primary hepatocytes (16105cells/well) were prepared from Car+/+and
Car2/2mice, were pretreated with either DMSO (DM) or TCPOBOP (TC;
250 nM) and were co-treated with TNFa 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. 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.
CAR and GADD45B in Cell Death
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To examine whether or not forming a complex with CAR
affects MKK7 kinase ability to phosphorylate JNK1, an in vitro
assay system of the MKK7-dependent JNK phosphorylation was
reconstituted using purified active MKK7 and substrate JNK1
(Figure 5A). As expected, the JNK phosphorylation was inhibited
in a concentration-dependent manner by addition of GADD45B
to the reaction mixture. CAR had no effect on the levels of JNK
phosphorylation at any concentrations tested. However, in the
presence of CAR, GADD45B greatly enhanced its inhibition of
JNK1 phosphorylation by MKK7 (Figure 5A). The JNK1
phosphorylation by MKK4 was not affected by GADD45B,
CAR or GADD45B plus CAR, serving as a negative control for
the specific GADD45B-MKK7 reaction. Thus, these results were
consistent with the hypothesis that CAR, by forming a complex
with GADD45B and MKK7, strongly potentiated GADD45B-
dependent inhibition of MKK7-mediated phosphorylation of
JNK1. Moreover, CAR required the AF2 domain for binding to
GADD45B in GST-pull down assays (Figure 5B) and the CAR
lacking the AF2 domain was not capable of potentiating the
GADD45B-mediated inhibition of JNK1 phosphorylation by
MKK7 (Figure 5C).
CAR not repressing cell death in the absence of
Given the fact that CAR inhibited MKK7 activity via its
interaction with GADD45B, we hypothesized that CAR may
require the presence of GADD45B to repress cell death. To test
this hypothesis, we performed cell death assays with mouse
primary hepatocytes prepared from Gadd45b+/+and Gadd45b2/2
mice. Co-treatment with TNFa and ActD induced the cell death
of the Gadd45b+/+hepatocytes approximately 2-fold (Figure 6).
This rate of induction was smaller than that observed with the
Car2/2hepatocytes (Figure 1), which appeared to be due to the
fact that these cells originate from different strains with different
sensitivities to TNFa and ActD: the genetic backgrounds of
Gadd45b2/2and Car2/2mice are C57BL/6 and C3H, respec-
tively. Similar to the cell death observed with Car+/+hepatocytes
(Figure 1), TCPOBOP activation of CAR attenuated the cell
death approximately 45%. The cell death of the Gadd45b2/2
hepatocytes was significantly elevated in the absence of TCPO-
BOP and was not attenuated by TCPOBOP treatment (Figure 6).
These cell death responses of the Gadd45b2/2hepatocytes were
reminiscent of those with the Car2/2hepatocytes. Thus, CAR
could not repress the TNFa/ActD induced cell death in the
absence of GADD45B.
Using mouse primary hepatocyetes
Gadd45b2/2mice, here we have determined the molecular
mechanism by which CAR represses TNFa-induced cell death
through its interactions with GADD45B. GADD45B is an anti-
apoptotic factor that directly interacts with MKK7 and inhibits
MKK7-dependent phosphorylation of JNK to repress apoptosis.
CAR can increase GADD45B-dependent inhibition of the MKK7
kinase activity by transcriptional up-regulation of the Gadd45b
gene as well as by directly binding to GADD45B, thereby
potentiating its inhibitory activity and repressing TNFa-induced
cell death in the Gadd45b2/2primary hepatocytes. Various types
of crosstalk between signal transduction pathway and nuclear
receptor have been reported. For example, to control various
cellular responses, nuclear receptors regulate gene transcription by
acting as cofactors (e.g. GR to AP1, CAR to Fox proteins) [7,8,26].
In addition to genomic regulation, several nuclear steroid
hormone receptors exhibit non-genomic function to regulate cell
signaling; ERa associates with the epidermal growth factor
receptor mediated-signaling to activate extracellular signal-regu-
lated kinase . Compared with these known mechanisms, the
direct interaction of CAR with GADD45B to potentiate the anti-
apoptotic activity of GADD45B is a unique mechanism.
Many CAR activators such as PB and TCPOBOP are non-
genotoxic carcinogens and CAR itself has been implicated as
being the determining factor in PB/TCPOBOP promotion of
HCC development in mice [10,11]. Tumor promotion considers
the pathological incidence in which non-genotoxic carcinogens,
such as chronic PB treatment, determine tumor development in
organs pre-initiated by genotoxic carcinogens such as diethylni-
trosamine . The pathology of tumor promotion is not well
defined and its molecular mechanism is virtually unknown at the
present time. However, PB must promote HCC development
through activation of a CAR-regulated gene since CAR is a target
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 mg amounts of recombinant proteins in pre-
reaction mixtures: 1 means approximately 1 mg 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-CARDAF2 mutant was used to
measure its inhibitory activity of MKK7 kinase, which was analyzed
Western blotting using anti-Phospho-JNK antibody as described in
CAR and GADD45B in Cell Death
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of PB action and because PB does not promote HCC development
in the absence of CAR. In this respect, previously suggested c-
JUN, FOXM1B and MDM2 do not appear to be the critical
factor responsible for PB promotion since these genes are not
regulated by CAR in our experimental system. The question is
whether or not CAR-mediated repression of apoptosis through the
GADD45B-MKK7-JNK pathway is, in fact, responsible for PB
promotion of HCC development. We determined caspase-3
positive hepatocytes of the mouse livers during PB promotion
(unpublished observation). No PB- and CAR-dependent increase
of apoptosis was detected. Thus, not only the role of the
GADD45B pathway but also that of general apoptosis in PB
tumor promotion remains elusive. Now, future tumor promotion
studies using Gadd45b2/2and Jnk2/2mice may provide us with
clues to understand the molecule mechanism of how CAR
regulates PB promotion of HCC development.
Materials and Methods
Phenobarbital, TCPOBOP, anti-Flag M2 antibody and anti-
Flag M2-agarose were purchased from Sigma-Aldrich; recombi-
nant mouse TNFa from R&D systems; actinomycin D from
Biomol Reserch Labs., Inc; William’s medium E, penicillin,
streptomycin, L-glutamate, HEPES, anti-V5 antibody, Hepato-
ZYME-SFM and Superscript First-Strand Synthesis System from
Invitrogen; fetal bovine serum from Atlanta biological; anti-
SAPK/JNK (56G8) antibody from Cell Signaling Technology;
GADD45B (N-19) antibody from Santa Cruz Biotechnology;
CytoTox 96 kit from Promega; Complete mini protease inhibitor
cocktail tablet from Roche. All other reagents were purchased
from Sigma, unless indicated otherwise.
Car2/2and Gadd45b2/2mice were previously generated and
characterized [11,29]. Car2/2mice used in these studies were
maintained in C3H genetic background. Gadd45b2/2mice used in
these studies were maintained in C57BL/6 genetic background.
All animals were housed in a temperature-controlled environment
with 12 hr light/dark cycles with access to standard chow and
water ad libitum. Age-matched groups of 8 to 12-week-old mice
were used for preparation of primary hepatocytes. All protocols
and procedures were approved by the National Institutes of
Health Animal Care and Use Committee and were in accordance
with National Institutes of Health guidelines.
Mouse primary hepatocytes
Mouse primary hepatocytes were prepared from male mice by a
two-step collagenase perfusion method and cultured as previously
described . Hepatocytes were suspended in Williams medium E
supplemented with 7% fetal bovine serum, 1 X Liquid Media
Supplement (5 mg/ml insulin, 5 mg/ml transferrin, 5 mg/ml
selenite; Sigma-Aldrich), 2 mM L-glutamine and 30 mM pyruvate
and were allowed to adhere to 24-well plates (16105cells) for
4 hours in a CO2 incubator at 37uC. These hepatocytes were
maintained in HepatoZYME-SFM for 16 h and were subsequent-
ly pretreated with DMSO or 250 nM TCPOBOP for 24 hours.
To induce cell death, hepatocytes were incubated with or without
20 ng/ml TNFa plus 200 ng/ml Actinomycin D in Hepato-
ZYME-SFM for 16 hours. Release of lactate dehydrogenase
(LDH) was determined in the supernatant of hepatocyte cultures
using the CytoTox 96 kit according to the manufacturer’s
recommendations. DMSO treated cells were used to normalize
the results. Significant differences were analyzed by student t-test.
Protein was separated by SDS-PAGE and transferred to PVDF
membrane (Immobilon P, Millipore). The membranes were
probed with specified primary antibodies; anti-phospho-SAPK/
JNK (Thr183/Thy185) antibody and anti-SAPK/JNK (56G8)
antibody (Cell Signaling Technology), anti-GADD45B (N-19)
antibody (Santa Cruz Biotechnology), anti-Flag antibody (Sigma-
Aldrich) and anti-V5 antibody (Invitrogen).
Real time PCR
All Real Time PCR data were obtained using RNA isolated
from tissues of individual animals. Total RNAs were isolated from
mouse hepatic tissues using the RNeasy (QIAGEN), from which
cDNAs were synthesized from total RNAs with the Superscript
First-Strand Synthesis System and random hexamer primers. Real
Time PCR measurements of individual cDNAs were performed
with the ABI prism 7700 sequence detection system. Gene-specific
Figure 6. No CAR-dependent repression of cell death in the absence of GADD45B. Primary hepatocytes were prepared from Gadd45b+/+
and Gadd45b2/2mice 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 +/2 S.D as fold changes relative to DMSO without TNFa 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).
CAR and GADD45B in Cell Death
PLoS ONE | www.plosone.org6 April 2010 | Volume 5 | Issue 4 | e10121
primers and probes were purchased as pre-designed TaqMan
Gene Expression Assays gene-specific probe and primers mixture
(PE Applied Biosystems). Assay ID number of pre-designed
TaqMan Gene expression Assay (gene, assay ID number) used
in this study were as follows: Gadd45b, Mm00435123_m1;
Cyp2b10, Mm00456591_m1; c-Jun, Mm00495062_s1; Mdm2,
Mm00487656_m1; Foxm1b, Mm00514924_m1. The TaqMan
rodent GAPDH control reagent (PE Applied Biosystems) was used
as an internal control.
GST pull-down assays with in vitro translated proteins or purified
proteins were performed as previously reported . Glutathione
S-transferase (GST) fusion proteins, GST-GADD45B, GST-CAR,
GST-CAR-DBD, GST-CAR-LBD, GST-CAR-LBDDAF2, GST-
CARDAF2 were expressed in Escherichia coli strain BL21 cells
and purified with glutathione-Sepharose 4B (Amersham Biosci-
ences). MKK7 and CAR were labeled with [35S] methionine
using the TNT T7 quick-coupled transcription/translation system
(Promega). GST-GADD45B protein was purified with glutathi-
one-Sepharose 4B and digested with thrombin (Sigma-Aldrich).
Bound proteins were detected by autoradiography after SDS-
PAGE separation for MKK7 and CAR. GADD45B proteins were
detected by Western blotting using GADD45B (N-19) antibody
(Santa Cruz Biotechnology). Western blots with similar results
were reproduced twice.
HEK293T cells were transfected with Flag-tagged MKK7 and
V5-tagged CAR and/or V5-tagged GADD45B for 16 hr, then
were rinsed once with Tris-buffered saline (TBS) and lysed with
ice-cold buffer (50 mM Tris-HCl, pH 7.5 containing 150 mM
NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1% Triton X-100,
0.5% NP40, and Complete mini protease inhibitor cocktail tablet).
Resulting cell lysates were sonicated and centrifuged for 20 min at
20,0006g at 4uC. The supernatants were incubated with anti-Flag
M2-agarose for 1 h at 4uC. The agarose were washed with ice-
cold buffer (50 mM Tris-HCl, pH 7.5 containing 150 mM NaCl,
1 mM EDTA, 1 mM dithiothreitol, 0.4% Triton X-100, 0.2%
NP40 and Complete mini protease inhibitor cocktail tablet), from
which bound proteins were eluted in LDS (lithium dodecyl sulfate)
sample buffer (Invitrogen) and analyzed by SDS-PAGE and
western blotting with anti-Flag or anti-V5 antibody. Western blots
with similar results were reproduced 3 times.
In vitro kinase assays
GST-JNK1, GST-GADD45B, GST-CAR, GST-CARDAF2
and GST were expressed in Escherichia coli BL21 cells and purified
using glutathione-Sepharose 4B. GST-JNK1, GST-GADD45B,
GST-CAR and GST-CARDAF2 proteins were excised by
thrombin. Flag-MKK7 and Flag-MKK4 were purified from
HEK293T cells by using anti-Flag M2-agarose. Given GST-
proteins were pre-incubated in a kinase buffer containing 25 mM
Tris-HCl, pH 7.5, 5 mM b-glycerolphosphate, 0.1 mM Na3VO4,
10 mM MgCl2, 1 mM dithiothreitol for 20 min at room
temperature. Kinase reaction was the initiated by adding GST-
JNK1 as kinase substrate and 200 mM ATP and was continued for
30 min at room temperature. The reaction mixtures were
subjected to SDS-PAGE and to Western blotting analysis using
anti-phospho-JNK antibody. Western blots with similar results
were independently reproduced 3 times.
We thank for the sequencing core in NIEHS.
Conceived and designed the experiments: YY MN. Performed the
experiments: YY RM. Analyzed the data: YY. Contributed reagents/
materials/analysis tools: RAF BL. Wrote the paper: YY MN.
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