Nuclear Receptor CAR Suppresses GADD45B-P38 MAPK Signaling to Promote

Author Manuscript Published OnlineFirst on May 1, 2018; DOI: 10.1158/1541-7786.MCR-18-0118 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Nuclear Receptor CAR Suppresses GADD45B-p38 MAPK Signaling to Promote

Phenobarbital-induced Proliferation in Mouse Liver

Takeshi Hori1, Kosuke Saito1, Rick Moore1, Gordon P. Flake2, and Masahiko Negishi1*

1 Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National

Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle

Park, North Carolina, 27709, USA

2 Cellular and Molecular Pathology Branch, Division of the National Toxicology Program,

National Institute of Environmental Health Sciences, National Institutes of Health, Research

Triangle Park, North Carolina, 27709, USA

* To whom correspondence should be addressed. Tel: +1 919 541 2404; Fax: +1 919 541

0696; Email: [email protected]

Running title: CAR Suppresses Hepatic p38 MAPK via GADD45B

Keywords: Phenobarbital, Chemical carcinogenesis, Constitutive active/androstane

receptor

Funding: This work was supported by the Intramural Research Program of the National

Institutes of Health and National Institute of Environmental Health Sciences [Grant numbers

Z01ES71005-01].

Conflicts of interest: The authors declare no potential conflicts of interest.

Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 1, 2018; DOI: 10.1158/1541-7786.MCR-18-0118 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Phenobarbital (PB), a non-genotoxic hepatocarcinogen, induces hepatic proliferation and

promotes development of hepatocellular carcinoma (HCC) in rodents. Nuclear receptor

constitutive active/androstane receptor (NR1I3/CAR) regulates the induction and promotion

activities of PB. Here, it is demonstrated that PB treatment results in dephosphorylation of a

tumor suppressor p38 mitogen-activated protein kinase (MAPK) in the liver of C57BL/6 and

C3H/HeNCrlBR mice. The molecular mechanism entails CAR binding and inhibition of the

growth arrest and DNA-damage-inducible 45 beta (GADD45B)-MAPK kinase 6 (MKK6)

scaffold to repress phosphorylation of p38 MAPK. PB-induced hepatocyte proliferation, as

determined by BrdU incorporation, was significantly reduced in both male and female livers

of GADD45B knockout (KO) mice compared with the wild-type mice. The PB-induced

proliferation continued until 48 h after PB injection in only the C57BL/6 males, but not in

males of GADD45B KO mice nor females of C57BL/6 and GADD45B KO mice. Thus, these

data reveal nuclear receptor CAR interacts with GADD45B to repress p38 MAPK signaling

and elicit hepatocyte proliferation in male mice.

Implications:

This GADD45B-regulated male-predominant proliferation can be expanded as a

phenobarbital promotion signal of HCC development in future studies.

Introduction

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death in the

world (1). PB promotes HCC development in rodents. A two-stage mouse model - initiation

by a genotoxic carcinogen diethylnitrosamine (DEN) and subsequent promotion by chronic

treatment with phenobarbital (PB) - has long been utilized for mechanistic studies of drug-

induced development of HCC (2). Following the discovery that nuclear receptor constitutive

active/androstane receptor (CAR; NR1I3) is essential for PB-promoted HCC (3), the

molecular mechanism of the HCC promotion has been intensively investigated. Our previous

studies demonstrated that activated CAR represses phosphorylation of c-Jun amino-terminal

kinase 1 (JNK1) in mouse primary hepatocytes (4). CAR directly bound to growth arrest and

DNA-damage-inducible 45 beta (GADD45B) and accelerated the ability of GADD45B to

inhibit MAPK kinase 7 (MKK7) that phosphorylates JNK1. Moreover, tumor necrosis factor α

(TNFα)/actinomycin D (ActD)-induced cell death was attenuated by a CAR activator, and the

attenuation was not observed in GADD45B knock-out (KO) mice as well as CAR KO mice.

Thus, GADD45B appears to be a crucial factor that regulates CAR-mediated repression of

JNK1 signaling and of cell growth in mouse primary hepatocytes. However, the function of

GADD45B and its molecular mechanisms have not been fully elucidated.

GADD45B is a member of the GADD45 family with the other members GADD45A

and GADD45G. GADD45 proteins are stress inducible signal scaffolds that interact with

various protein kinases and phosphatases to regulate diverse cellular functions (5). In

addition to JNK1, GADD45B is also known to regulate p38 mitogen-activated protein kinase

(MAPK). GADD45B indirectly activates p38 MAPK through a GADD45B-MTK1-MKK6-p38

MAPK pathway (6), while it directly represses JNK1 activation. Upon activation, p38 MAPK

activates a tumor suppressor p53 and cell cycle checkpoint-related proteins such as MAPK-

activated protein kinase 2 (MAPKAPK2) and induces apoptosis and cell cycle arrest (7-9).

Moreover, p38 MAPK prevents accumulation of reactive oxygen species (ROS) (10,11).

Through these regulations, p38 MAPK functions as a tumor suppressor (12,13). In fact,

hepatocyte-specific knock-out of p38 MAPK increased susceptibility to the genotoxin DEN,

predisposing mice to HCC (10,14). Furthermore, restoring p38 MAPK activity by ablation of

the BH3-Only protein (BID) significantly delayed tumor development in mice (15). Therefore,

these findings prompted us to investigate whether PB and CAR regulate p38 MAPK activity

and, if they do, its molecular mechanism should help us to understand the PB-

promoted/CAR-regulated HCC development in mouse livers.

In the present study, we first utilized C57BL/6, C3H/HeNCrlBR, CAR KO, and

GADD45B KO mice to demonstrate that PB induces dephosphorylation of p38 MAPK in

mouse livers and that this dephosphorylation requires both CAR and GADD45B.

Subsequently, the molecular mechanism of p38 MAPK dephosphorylation was examined in

cell-based assays. GADD45B, acting as a scaffold protein, interacted with p38 MAPK and

potentiated phosphorylation of p38 MAPK by upstream MAPK kinase 6 (MKK6). CAR was

shown to interact with GADD45B, preventing GADD45B to form a complex with p38 MAPK

and MKK6. Finally, GADD45B KO mice were treated with PB, and BrdU-positive

hepatocytes were counted in the liver of these mice. Here, with these experimental

observations, we will discuss the hypothesis that GADD45B is a cornerstone that enables

CAR to repress p38 MAPK signaling and to promote hepatocyte proliferation, implicating PB-

induced promotion of HCC development.

Materials and methods

Materials

Phenobarbital sodium salt, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP),

anisomycin, diethylnitrosamine (DEN), anti-FLAG M2 affinity gel, an anti-FLAG M2-

horseradish peroxidase (HRP) antibody, 3x FLAG peptides, and 5-Bromo-2'-deoxyuridine

(BrdU) were purchased from Sigma-Aldrich (St. Louis, MO, USA); an anti-GFP (HRP)

antibody from Abcam (Cambridge, MA, USA); antibodies against phospho-p38 MAPK (p-

p38) (Thr180/Tyr182; #4511), p38 MAPK (#8690), phospho-MAPKAPK2 (#3007S), and

MAPKAPK2 (#3042) from Cell Signaling Technology (Danvers, MA, USA); an anti-V5

antibody from Invitrogen (Carlsbad, CA, USA); Lipofectamine 2000 from Life Technologies

(Grand Island, NY, USA); TaqMan Gene Expression Assays (probe and primer sets) for

CYP2B10 (AssayID: Mm00456591_m1), GADD45A (Mm00432802_m1), GADD45B

(Mm00435123_m1), GADD45G (Mm00442225_m1), and mouse glyceraldehyde-3-

phosphate dehydrogenase (GAPDH) (FAM) from Applied Biosystems (Foster, CA, USA);

cOmplete mini protease inhibitor cocktail tablets from Roche Diagnostics Corp. (Indianapolis,

IN, USA); a Vectastain Elite ABC kit from Vector Laboratories (Burlingame, CA, USA).

Plasmids

CAR cDNA (GenBank accession no. NM_009803.5) was previously cloned into pCR3 vector

(Invitrogen) or pcDNA V5-His vector (Invitrogen). The FLAG tag was inserted within the 5’-

flanking region of CAR/pCR3 (16). p38α cDNA (NM_011951.3) was cloned into pcDNA3.1

vector. GADD45B cDNA (NM_008655.1) was cloned into pcDNA3.1 vector harboring a

3xFLAG tag or pEYFP-c1 vector. The p38α cDNA or GADD45B cDNA was cloned into

pGEX-4T-3 vector (GE Healthcare, Piscataway, NJ, USA) for glutathione S-transferase

(GST)-tagged proteins. FLAG-MKK6 active mutant (Ser207Glu and Thr211Glu)/pcDNA

vector (Addgene ID: 13518) was purchased from Addgene (Cambridge, MA, USA). A

GADD45B 1-92 mutant/pEYFP and a GADD45B 93-160/pEYFP were constructed by a

deletion method.

Animals and drug treatments

C3H/HeNCrlBR and C57BL/6 mice were obtained from Charles River Laboratories

(Wilmington, MA, USA) and Jackson Laboratories (Bar Harbor, ME, USA), respectively. CAR

knock-out (KO) and CAR wild-type (WT) in the C3H/HeNCrlBR background (3) and in the

C57BL/6 background and also GADD45B KO and GADD45B WT in the C57BL/6

background (4,17) were maintained at the National Institute of Environmental Health

Sciences (NIEHS, USA). Phenobarbital (PB) in PBS (100 mg/kg body weight), TCPOBOP in

dimethyl sulfoxide (DMSO)/corn oil (3 mg/kg body weight), or a control solution was

intraperitoneally injected. For a two-stage model of HCC development, five-week-old

C57BL/6 male mice were intraperitoneally injected with DEN (90 mg/kg body weight) and

housed for two weeks, followed by drinking water containing PB (500 ppm) for additional two

weeks. Animal experiments were conducted according to protocols approved by the Animal

ethics committee at NIEHS/National Institutes of Health.

BrdU immunohistochemistry

Starting 48 h before PB treatment, mice received BrdU (0.5 mg/ml) in their drinking water to

analyze hepatocyte proliferation. PB in PBS (100 mg/kg body weight) was intraperitoneally

injected 48 h later, and four mice from each of the four groups (C57BL/6 male and female

and GADD45B KO male and female; around 9 weeks of age) were then sacrificed at 0, 24,

36, and 48 h. A section of liver was taken at necropsy from each animal, fixed in 10%

neutral buffered formalin, processed, sectioned at 5 μm, and stained with hematoxylin and

eosin. Sections were also stained by an anti-BrdU antibody (1:500) to assess cell

proliferation. Counting of hepatocytes stained for BrdU was performed with an Olympus

BX51 light microscope, using a 40x objective and an ocular grid, according to the method of

Ton et al. (18). BrdU-stained hepatocytes were identified as cells located within the

hepatocyte plates and having rounded nuclei with abundant cytoplasm. Binucleate

hepatocytes were often seen and were counted singly. The labeling index for each liver was

calculated by dividing the number of BrdU-stained hepatocytes in 20 fields by the total

number of BrdU-positive and negative hepatocytes in the same 20 fields, expressed as a

percentage.

Cell culture and transient transfection

Human hepatoma cell line Huh-7 cells were obtained from Japan Collection of Research

Bioresources (JCRB) Cell Bank (Osaka, Japan). Huh-7 cells were authenticated by short

tandem repeat (STR) analysis at ATCC (American Type Culture Collection) in January 2018.

Cells were cultured in minimum essential media (Invitrogen) supplemented with 10% FBS

and penicillin/streptomycin at 37ºC with 5% CO2. Plasmids were transfected into Huh-7 cells

with a Lipofectamine 2000 reagent according to the manufacturer’s instructions.

Immunoprecipitation assays

Huh-7 cells were lysed in a lysis buffer [20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 100 mM

NaCl, 1% Triton X-100, 10% glycerol, a protease inhibitor cocktail, and phosphatase inhibitor

cocktails 2 and 3], sonicated, and centrifuged. Obtained supernatants were used as extracts

for immunoprecipitation assays. Protein extracts were incubated with anti-FLAG M2 affinity

gels or anti-GFP antibody-conjugated agarose beads. These gels or beads were washed

three times with Tris-buffered saline (TBS) [25 mM Tris-HCl (pH 7.4), 140 mM NaCl, and 2.7

mM KCl]. Washed gels or beads were heat-treated in an SDS sample buffer (2x) [157 mM

Tris-HCl (pH 6.8), 4% SDS, 25% glycerol, and 0.01% bromophenol blue] and subjected to

Western blot analyses.

For double immunoprecipitation assays, immunoprecipitation was sequentially

repeated with anti-FLAG M2 affinity gels at the 1st step and anti-GFP antibody-conjugated

agarose beads at the 2nd step; each step of the immunoprecipitation was performed as

described above. Proteins bound at the 1st step were eluted by incubation with FLAG

peptides (100 µg/mL) in TBS for 2-4 h at 4ºC and subjected to the 2nd step of

immunoprecipitation with anti-GFP antibodies and to subsequent Western blot analyses.

In vitro kinase assays

FLAG-MKK6 was expressed in Huh-7 cells, followed by treatment with 200 nM anisomycin

for 15-30 min to activate MKK6. Cells were lysed in the above-mentioned lysis buffer

containing 2.5 mM Na4P2O7 and 1 mM Na3VO4. FALG-MKK6 was bound to anti-FLAG M2

affinity gels for 3 h at 4°C, washed by TBS and utilized as an active MKK6 enzyme. GST-

tagged p38 MAPK was expressed in and purified from E. coli BL21 (DE3) (Agilent

Technologies, Palo Alto, CA, USA) using GSH Sepharose 4B (GE Healthcare, Marlborough,

MA, USA). After the GST tag was removed by thrombin digestion, p38 MAPK was used as a

substrate. Recombinant GADD45B was obtained by the same way that p38 MAPK was

purified.

In in vitro phosphorylation assays, recombinant p38 MAPK was phosphorylated by

active MKK6 with or without the presence of recombinant GADD45B in a kinase buffer [25

mM Tris-HCl (pH 7.5), 2.5 mM Na4P2O7, 1 mM Na3VO4, 10 mM MgCl2, and 1 mM

dithiothreitol] with ATP (130-200 µM). Recombinant GST protein was used as a control for

GADD45B. Kinase reaction was continued for 20 min at 30ºC. The reaction was stopped by

adding 1x SDS sample buffer. Phosphorylation levels of p38 MAPK were determined by

Western blot analysis.

Western blot and immunohistochemistry

Western blot analysis was performed as described previously (16) and

immunohistochemistry was performed as described previously (19).

Modeling of CAR-GADD45G complexes

The CAR ligand-binding domain (LBD) and GADD45G were docked using ZDOCK (3.0.2)

(20). Since the crystal structure of GADD45B is unavailable, GADD45G (PDB ID: 3CG6)

was used to dock with CAR LBD (PDB ID: 1XNX). Stability was calculated using a protein

interfaces, surfaces and assemblies' service PISA at the European Bioinformatics Institute

(21).

Statistical Analysis

mRNA expression levels were analyzed by Student’s t-test. A value of P < 0.05 was

considered statistically significant.

Results

CAR regulates PB-induced p38 MAPK dephosphorylation in mouse livers

Liver extracts were prepared from CAR WT and CAR KO mice in the C3H/NeCrIBR

background after PB treatment and subjected to Western blot analysis to examine

phosphorylation levels of p38 MAPK. Hepatic p38 MAPK was phosphorylated and remained

phosphorylated 12 h after PB treatment (Fig.1A). However, phosphorylation levels of p38

MAPK was greatly decreased in CAR WT but not CAR KO mice 24 + 4 h after PB treatment

(Fig. 1B). This PB-induced dephosphorylation was equally observed in both male and female

mice (Fig. 1C). Although C3H/NeCrIBR mice are susceptible in developing HCC and

C57BL/6 mice are resistant, p38 MAPK was equally phosphorylated in livers of both strains

(Fig. 1D). PB-induced dephosphorylation was also observed in C57BL/6 mice and was

dependent on CAR (Fig. 1E). This CAR-mediated dephosphorylation was not specific to PB

and also observed after treatment with TCPOBOP, a mouse CAR ligand (Fig. 1F). These

results indicate that PB-induced dephosphorylation of p38 MAPK is regulated by CAR in

strain- and sex-independent manners. Immunohistochemistry provided further evidence that

phosphorylated p38 MAPK are present in the nuclei of hepatocytes and decreased after PB

treatment in a CAR-dependent manner (Fig. 1G).

GADD45B promotes p38 MAPK phosphorylation by MKK6

To understand how PB induces dephosphorylation, p38 MAPK phosphorylation by MKK6

was first investigated. p38 MAPK, which was barely phosphorylated in Huh-7 cells, became

more phosphorylated as GADD45B was increased in the cells (Fig. 2A). A purified active

MKK6 phosphorylated p38 MAPK in in vitro kinase assays (Supplemental Fig. 1A), and

recombinant GADD45B protein greatly increased this phosphorylation (Fig. 2B). Thus,

GADD45B was capable of directly enhancing MKK6 to phosphorylate p38 MAPK. A FLAG-

tagged active MKK6 mutant and an untagged p38 MAPK were expressed with or without

EYFP-tagged GADD45B in Huh-7 cells. An anti-FLAG antibody co-precipitated p38 MAPK

with MKK6, and this co-immunoprecipitation was dramatically increased in the presence of

GADD45B (lane 4 in Fig. 2C). These three proteins were confirmed to interact with each

other (Supplemental Fig. 1B-D). Subsequently, double co-immunoprecipitation assays were

employed to examine a complex consisting of GADD45B, p38 MAPK, and MKK6. To this

end, EYFP-GADD45B, the FALG-MKK6 mutant, and untagged p38 MAPK were co-

expressed in Huh-7 cells from which extracts were prepared for sequential

immunoprecipitation with an anti-FLAG antibody and then an anti-GFP antibody. p38 MAPK

was co-precipitated with MKK6 and GADD45B (Fig. 2D). These results indicated that

GADD45B forms a complex with p38 MAPK and MKK6 to stimulate phosphorylation of p38

MAPK.

CAR interferes with GADD45B regulating MKK6

Co-immunoprecipitation assays were performed to examine whether CAR alters interactions

between p38 MAPK and MKK6. As demonstrated in Fig. 2C (lane 4), co-expressed

GADD45B increased co-precipitation of p38 MAPK with MKK6 (Fig. 3A, lane 6), and

ectopically expressed CAR nearly abolished this co-immunoprecipitation (Fig. 3A, lane 8).

Ectopic expression of CAR is known to enable the cells to contain active CAR proteins

(22,23). Ectopically expressed CAR was confirmed to bind to GADD45B (Supplemental Fig.

2A). Subsequent double co-immunoprecipitation assays provided evidence that a complex

formed by GADD45B, MKK6, and p38 MAPK was dissociating in the presence of co-

expressed CAR (Fig. 3B). Under these conditions in which CAR dissociated the complex,

CAR interacted with GADD45B and p38 MAPK, forming another complex consisting of

GADD45B, p38 MAPK, and CAR (Fig. 3C). In addition, GADD45B can also form a complex

with CAR and MKK6 (Supplemental Fig. 2B). These results indicated that CAR binds to

GADD45B and prevents interactions between p38 MAPK and MKK6 by forming a complex

with p38 MAPK or MKK6.

Structural basis for interactions between CAR and GADD45B

Given the finding that CAR disrupted a GADD45B-schaffoled p38 MAPK-MKK6 interaction,

the molecular basis of this CAR-GADD45B interaction was examined by co-expressed CAR

and a GADD45B fragment in Huh-7 cells, from which the N-terminal half (1/92) of GADD45B,

but not the C-terminal half (93/160), was co-precipitated with CAR (Fig. 4A). An internal

deletion mutant GADD45B deletion (Δ41/92) was not coprecipitated with CAR (Fig. 4B). The

2nd and 3rd helices constitute this N-terminal region and a CAR-binding site. Similar co-

immunoprecipitation assays revealed that p38 MAPK or MKK6 bound the same region of

GADD45B (Supplemental Figs. 2C and D). Numerous deletion mutants were constructed

with CAR (Supplemental Fig. 3A). Co- immunoprecipitation assays showed a preferential

binding of GADD45B to CAR LBD over the CAR DBD (Supplemental Fig. 3B). Furthermore,

GADD45B was co-precipitated with CAR when its mutants included helix 3, helix 7, or both

(Fig. 4D). Having these potential binding regions in CAR and GADD45B, ZDOCK docking

program and PISA program were utilized to visualize a heterodimer between CAR (PDB ID:

1XNX) and GADD45G (PDB ID: 3CG6). Since a GADD45B structure has not been

determined, the GADD45G was used instead. The most stable heterodimer interface was

formed between the helix 7 of CAR and helices 2 to 3 of GADD45G (Supplemental Fig. 4).

PB-induced p38 MAPK dephosphorylation depends on GADD45B

GADD45B WT and GADD45B KO male mice were treated with PB as CAR WT and CAR

KO males were treated in experiments shown in Fig. 1B. Liver extracts from these

GADD45B mice were subjected to Western blot analyses. PB-induced dephosphorylation of

p38 MAPK was significantly attenuated in GADD45B KO mice over that in GADD45B WT

mice (Fig. 5A), which was reminiscent of CAR-dependent dephosphorylation of p38 MAPK in

experiments with CAR KO mice (Fig. 1B).

p38 MAPK dephosphorylation in a two-stage model of HCC development

It was decided to explore whether PB induces p38 MAPK dephosphorylation in livers after

HCC initiation. C57BL/6 males were pre-treated with a single injection of DEN for two weeks,

prior to drinking PB for additional two weeks. Western blot analysis of liver extracts from

these mice revealed that PB was capable of inducing dephosphorylation in the conditions of

two-stage model of HCC development (Fig. 5B). In addition to p38 MAPK, its downstream

kinase MAPKAPK2 (MK2) was also dephosphorylated.

BrdU labeling index

Baseline BrdU hepatocyte labeling indices (LI), prior to PB treatment (0 h), were 0.43% and

0.39% in C57BL/6 males and females, respectively, and 1.41% and 1.44% in GADD45B KO

males and females, respectively (Table. 1). These findings indicate a 3 times higher resting

proliferation rate in the GADD45B KO mice, probably reflecting the function of GADD45B as

a tumor suppressor. Following PB treatment for 24 h LIs of both strains and both sexes

increased. The C57BL/6 males and females increased the LI values a 1.53- and 9.72-fold

over their values at 0 hr, respectively. These increases were a 1.72- and 6.03-folds in the

GADD45B KO mice. By 36 h following PB injection, the LI of both the males and females of

each strain continued to rise. At this point, the increase was particularly notable in the

C57BL/6 males with a 9.35-fold over the 24 h values, while this increase was only 3.23-fold

in the GADD45B KO males. On the other hand, the females declined these folds to 2.05 and

1.28 in the C57BL/6 and GADD45B KO mice, respectively, indicating that the females

peaked the increase of LI values by this time. Thus, proliferation had increased most

significantly in the C57BL/6 males during this period of PB treatment. However, the LIs of the

GADD45B KO males and females exceeded the LI of their C57BL/6 counterparts, but only

slightly. By 48 h the average LI of the C57BL/6 males continued to rise by 1.22-fold from

6.17% at 36 h to a 7.54%. Conversely, the LI of the GADD45B KO males fell 2.13-fold from

7.82% at 36 h to 3.01% at 48 h. As a result, the LI values of C57BL/6 males exceeded that

of GADD45B KO males. These observations suggested that PB-induced hepatocyte

proliferation depends on GADD45B and that these GADD45B-dependent increases continue

in males but not in females.

Sequence of CAR regulation

Dephosphorylation of p38 MAPK was observed from 24 h after PB treatment (Fig. 1A and

Supplemental Figs. 6A and 6B). This p38 MAPK dephosphorylation at late stages should

negatively affect PB-activated transcription of the Cyp2b10 gene, a representative target

gene of CAR, since PB/CAR requires phosphorylated-p38 MAPK for transcription of the

Cyp2b10 gene (16). In fact, expression levels of CYP2B10 mRNA peak 12-18 h after the 1st

injection of PB and then begin to decline. The 2nd PB injection at 24 h after the 1st injection

was unable to restore the induction, even though nuclear levels of CAR increased after this

2nd injection (Supplemental Fig. 6C), probably because p38 MAPK activity became low

levels. During the time when CAR/GADD45B induces p38 MAPK dephosphorylation,

CAR/GADD45B-dependent hepatocyte proliferation continued to rise in male mice (Table. 1).

The molecular mechanism that regulates these sequential processes is an important subject

in future research.

Discussion

Phosphorylated p38 MAPK can be a tumor suppressor that represses hepatic proliferation.

PB, a non-genotoxic carcinogen, repressed p38 MAPK by inducing dephosphorylation in

mouse livers. CAR regulated this dephosphorylation by binding to GADD45B that scaffolds

p38 MAPK and MKK6 (Fig. 6). While PB induced hepatocyte proliferation through both

GADD45B-depedent and -independent mechanisms, GADD45B-dependent proliferation

continued in only male mice. PB may utilize this GADD45B-mediated p38 MAPK pathway as

an essential cell signal to promote HCC development.

Livers to develop HCC require chronic PB exposures with genotoxic mutations of β-

catenin gene (24,25). In a two-stage HCC model, DEN mutates the Ctnnb1 gene, which

sustains the activity of β-catenin. Consequently, PB selectively proliferates hepatocytes that

bear these mutations of the Ctnnb1 gene (26,27). Notably, β-catenin seemed to manifest

hepatocyte proliferation in a sex-dependent manner, as indicated by the fact that hepatic β-

catenin KO mice attenuated proliferation only in males (24). p38 MAPK signaling can

suppress downstream factors of β-catenin proliferation signaling. Thus, PB treatment may

attenuate the p38 MAPK-mediated suppression of those factors.

Since hepatocyte-specific ablation of either p38 MAPK or JNK1 signaling promotes

DEN-initiated HCC (10,28), CAR/GADD45B-mediated ablation of these signaling can be a

causing factor of PB-induced HCC promotion, although with the caveat that interactions of

CAR-GADD45B-p38 MAPK or JNK1 remain to be demonstrated with endogenous proteins

in livers in future investigations. PB is known to promote HCC development in both mouse

strain- and sex-dependent manners in C3H over C57BL/6 mice and males over females.

However, the GADD45B-mediated repression of p38 MAPK signaling was equally observed

in C3H and C57BL/6 and their males and females. On the other hand, PB-induced and

GADD45B-dependnet BrdU-positive hepatocytes continued to rise in only male mice.

Therefore, sex-related factors appear to render the GADD45B-dependent proliferation male-

predominant. If the GADD45B-dependent proliferation is sustained, it may become a

dominant proliferation signal, since GADD45B-independent proliferation was transient in

male mice. GADD45B-mediated regulation of p38 MAPK and JNK1 signaling should be

expanded to be an essential determinant that promotes PB-induced HCC development in

future investigations. PB appears to prototypically alter MAPK signaling (e.g. ERK1/2, JNK1,

and p38 MAPK signaling). To regulate ERK1/2, PB binds and represses the epidermal

growth factor (EGF) receptor and/or the insulin receptor (29,30). GADD45B is positioned as

a key factor to regulate JNK1 and p38 MAPK signaling through its interaction with CAR in

response to phenobarbital. In addition to the stress-activated protein kinases, it is possible

that other MAPKs also can be regulated by CAR/GADD45B. Furthermore, GADD45B is

involved in regulation of numerous biological processes including DNA methylation and cell

migration. PB may also affect GADD45B on these additional regulations that become critical

factors in HCC development.

In conclusion, GADD45B is characterized as a target of CAR to repress p38 MAPK

activity and to promote cell proliferation in mouse livers, providing us with an experimental

basis for understanding the molecular mechanism of PB-promoted HCC development.

Acknowledgements

We thank the DNA sequencing and histology core facilities (NIEHS/NIH). We thank the

protein expression core facility (NIEHS/NIH) for anti-GFP antibody agarose beads.

Abbreviations

BrdU, 5-Bromo-2'-deoxyuridine; CAR, constitutive active/androstane receptor; CYP2B,

cytochrome P450 2B; DBD, DNA-binding domain; DEN, diethylnitrosamine; GADD45B,

growth arrest and DNA-damage-inducible 45 beta; HCC, hepatocellular carcinoma; JNK1, c-

Jun amino-terminal kinase 1; KO, knock-out; LBD, ligand-binding domain; MAPK, mitogen-

activated protein kinase; MAPKAPK2 or MK2, MAPK-activated protein kinase 2; MKK6,

MAPK kinase 6; PB, phenobarbital; PCR, polymerase chain reaction; TCPOBOP, 1,4-bis[2-

(3,5-dichloropyridyloxy)]benzene; WT, wild-type.

References

1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55(2):74-108. 2. Heindryckx F, Colle I, Van Vlierberghe H. Experimental mouse models for hepatocellular carcinoma research. Int J Exp Pathol 2009;90(4):367-86 doi 10.1111/j.1365-2613.2009.00656.x. 3. Yamamoto Y, Moore R, Goldsworthy TL, Negishi M, Maronpot RR. The orphan nuclear receptor constitutive active/androstane receptor is essential for liver tumor promotion by phenobarbital in mice. Cancer research 2004;64(20):7197-200 doi 10.1158/0008-5472.can-04-1459. 4. Yamamoto Y, Moore R, Flavell RA, Lu B, Negishi M. Nuclear receptor CAR represses TNFalpha-induced cell death by interacting with the anti-apoptotic GADD45B. PloS one 2010;5(4):e10121 doi 10.1371/journal.pone.0010121. 5. Tamura RE, de Vasconcellos JF, Sarkar D, Libermann TA, Fisher PB, Zerbini LF. GADD45 proteins: central players in tumorigenesis. Current molecular medicine 2012;12(5):634-51. 6. Miyake Z, Takekawa M, Ge Q, Saito H. Activation of MTK1/MEKK4 by GADD45 through induced N-C dissociation and dimerization-mediated trans autophosphorylation of the MTK1 kinase domain. Mol Cell Biol 2007;27(7):2765-76 doi 10.1128/MCB.01435-06. 7. She QB, Chen N, Dong Z. ERKs and p38 kinase phosphorylate p53 protein at serine 15 in response to UV radiation. The Journal of biological chemistry 2000;275(27):20444-9 doi 10.1074/jbc.M001020200. 8. Manke IA, Nguyen A, Lim D, Stewart MQ, Elia AE, Yaffe MB. MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation. Mol Cell 2005;17(1):37-48 doi 10.1016/j.molcel.2004.11.021. 9. Olson JM, Hallahan AR. p38 MAP kinase: a convergence point in cancer therapy. Trends Mol Med 2004;10(3):125-9 doi 10.1016/j.molmed.2004.01.007. 10. Sakurai T, He G, Matsuzawa A, Yu GY, Maeda S, Hardiman G, et al. Hepatocyte necrosis induced by oxidative stress and IL-1 alpha release mediate carcinogen- induced compensatory proliferation and liver tumorigenesis. Cancer cell 2008;14(2):156-65 doi 10.1016/j.ccr.2008.06.016. 11. Sakurai T, Kudo M, Umemura A, He G, Elsharkawy AM, Seki E, et al. p38alpha inhibits liver fibrogenesis and consequent hepatocarcinogenesis by curtailing accumulation of reactive oxygen species. Cancer research 2013;73(1):215-24 doi 10.1158/0008-5472.CAN-12-1602. 12. Kennedy NJ, Cellurale C, Davis RJ. A radical role for p38 MAPK in tumor initiation. Cancer cell 2007;11(2):101-3 doi 10.1016/j.ccr.2007.01.009. 13. Wagner EF, Nebreda AR. Signal integration by JNK and p38 MAPK pathways in cancer development. Nature reviews Cancer 2009;9(8):537-49 doi 10.1038/nrc2694. 14. Hui L, Bakiri L, Mairhorfer A, Schweifer N, Haslinger C, Kenner L, et al. p38alpha suppresses normal and cancer cell proliferation by antagonizing the JNK-c-Jun pathway. Nature genetics 2007;39(6):741-9 doi 10.1038/ng2033. 15. Orlik J, Schungel S, Buitrago-Molina LE, Marhenke S, Geffers R, Endig J, et al. The BH3-only protein BID impairs the p38-mediated stress response and promotes hepatocarcinogenesis during chronic liver injury in mice. Hepatology 2015;62(3):816- 28 doi 10.1002/hep.27888. 16. Hori T, Moore R, Negishi M. p38 MAP Kinase Links CAR Activation and Inactivation in the Nucleus via Phosphorylation at Threonine 38. Drug Metab Dispos 2016;44(6):871-6 doi 10.1124/dmd.116.070235.

17. Lu B, Ferrandino AF, Flavell RA. Gadd45beta is important for perpetuating cognate and inflammatory signals in T cells. Nat Immunol 2004;5(1):38-44 doi 10.1038/ni1020. 18. Ton T, Foley J, Flagler N. Feasibility of administering 5′Bromo-2-Deoxyuridine (BrdU) in drinking water for labeling S-phase hepatocytes in mice and rats. Toxicology Methods 1997;7:123-36. 19. Saito K, Moore R, Negishi M. Nuclear receptor CAR specifically activates the two- pore K+ channel Kcnk1 gene in male mouse livers, which attenuates phenobarbital- induced hepatic hyperplasia. Toxicological sciences : an official journal of the Society of Toxicology 2013;132(1):151-61 doi 10.1093/toxsci/kfs338. 20. Pierce BG, Wiehe K, Hwang H, Kim BH, Vreven T, Weng Z. ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics 2014;30(12):1771-3 doi 10.1093/bioinformatics/btu097. 21. Krissinel E, Henrick K. Inference of macromolecular assemblies from crystalline state. J Mol Biol 2007;372(3):774-97 doi 10.1016/j.jmb.2007.05.022. 22. Mutoh S, Osabe M, Inoue K, Moore R, Pedersen L, Perera L, et al. Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3). The Journal of biological chemistry 2009;284(50):34785-92 doi 10.1074/jbc.M109.048108. 23. Timsit YE, Negishi M. Coordinated regulation of nuclear receptor CAR by CCRP/DNAJC7, HSP70 and the ubiquitin-proteasome system. PloS one 2014;9(5):e96092 doi 10.1371/journal.pone.0096092. 24. Braeuning A, Heubach Y, Knorpp T, Kowalik MA, Templin M, Columbano A, et al. Gender-specific interplay of signaling through beta-catenin and CAR in the regulation of xenobiotic-induced hepatocyte proliferation. Toxicological sciences : an official journal of the Society of Toxicology 2011;123(1):113-22 doi 10.1093/toxsci/kfr166. 25. Rignall B, Braeuning A, Buchmann A, Schwarz M. Tumor formation in liver of conditional beta-catenin-deficient mice exposed to a diethylnitrosamine/phenobarbital tumor promotion regimen. Carcinogenesis 2011;32(1):52-7 doi 10.1093/carcin/bgq226. 26. Aydinlik H, Nguyen TD, Moennikes O, Buchmann A, Schwarz M. Selective pressure during tumor promotion by phenobarbital leads to clonal outgrowth of beta-catenin- mutated mouse liver tumors. Oncogene 2001;20(53):7812-6 doi 10.1038/sj.onc.1204982. 27. Dong B, Lee JS, Park YY, Yang F, Xu G, Huang W, et al. Activating CAR and beta- catenin induces uncontrolled liver growth and tumorigenesis. Nature communications 2015;6:5944 doi 10.1038/ncomms6944. 28. Das M, Garlick DS, Greiner DL, Davis RJ. The role of JNK in the development of hepatocellular carcinoma. Genes & development 2011;25(6):634-45 doi 10.1101/gad.1989311. 29. Mutoh S, Sobhany M, Moore R, Perera L, Pedersen L, Sueyoshi T, et al. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling. Science signaling 2013;6(274) doi 10.1126/scisignal.2003705. 30. Yasujima T, Saito K, Moore R, Negishi M. Phenobarbital and insulin reciprocate the nuclear receptor CAR activation through insulin receptor. J Pharmacol Exp Ther 2016 doi 10.1124/jpet.116.232140. 31. Tornatore L, Marasco D, Dathan N, Vitale RM, Benedetti E, Papa S, et al. Gadd45 beta forms a homodimeric complex that binds tightly to MKK7. J Mol Biol 2008;378(1):97-111 doi 10.1016/j.jmb.2008.01.074.

Figure Legends

Fig. 1. Dephosphorylation of p38 MAPK by phenobarbital-activated CAR. (A-G) p38

phosphorylation levels in the mouse liver. (A) C3H/HeNCrlBR (C3H) males were

intraperitoneally injected with phenobarbital (PB) or control phosphate-buffered saline (PBS).

After 12 h of injection, livers were collected and subjected to Western blot analysis with

phospho- or total-p38 MAPK antibodies. (B) CAR wild-type (WT) or knock-out (KO) males in

the C3H background were treated with PB or PBS for 24 h (the 1st injection) and additional 4

h (the 2nd injection) (24 + 4 h), and livers were used for Western blot analysis. (C) C3H

males or females were treated with PB or PBS for 24 + 4 h to examine sex difference. (D)

Basal phosphorylation levels of p38 MAPK were compared in C3H and C57BL/6 mice. (E)

CAR WT or KO males in the C57BL/6 background were treated with PB or PBS at the same

condition of Fig. 1B. (F) A CAR ligand TCPOBOP or a control solution was injected into C3H

males (24 + 6 h). (G) Immunohistochemistry was performed in C3H CAR WT or CAR KO

males treated with PB or PBS (24 + 4 h).

Fig. 2. GADD45B enhances MKK6-catalized p38 MAPK phosphorylation. (A) EYFP-tagged

GADD45B was expressed in human hepatoma Huh-7. Cell lysates were used to determine

p38 MAPK phosphorylation levels. (B) Recombinant p38 MAPK was phosphorylated by

active MKK6 in the presence or absence of recombinant GADD45B in vitro. After kinase

reaction, reaction mixtures were subjected to Western blot analysis. (C) EYFP-GADD45B,

FLAG-MKK6 Glu, and p38 MAPK were expressed in Huh-7 cells. Cells were lysed and the

lysates were subjected to co-immunoprecipitation assays with anti-FLAG antibodies.

Immunoprecipitated proteins were used for Western blot analysis. MKK6 Glu indicates an

active MKK6 mutant, a phosphomimetic mutant (Ser207Glu and Thr211Glu). (D) EYFP-

GADD45B, FLAG-MKK6 Glu, and p38 MAPK were expressed in Huh-7 cells. Cells were

lysed and the lysates were subjected to double co-immunoprecipitation assays as described

in Materials and methods.

Fig. 3. CAR inhibits association of p38 MAPK with MKK6. (A) CAR V5-His, EYFP-GADD45B,

FLAG-MKK6 Glu, and p38 MAPK were expressed in Huh-7 cells. Cells were lysed and the

lysates were subjected to co-immunoprecipitation assays with anti-FLAG antibodies.

Immunoprecipitated proteins were subjected to Western blot analysis. MKK6 Glu indicates

an active MKK6 mutant (Ser207Glu and Thr211Glu). (B) In the same condition as Fig. A,

cellular lysates were subjected to double co-immunoprecipitation assays as described in

Materials and methods. (C) EYFP-GADD45B, FLAG-CAR, and p38 MAPK were expressed

in Huh-7 cells. Cellular lysates were subjected to double co-immunoprecipitation assays.

Fig. 4. Interaction regions between CAR and GADD45B. (A) An EYFP-GADD45B 1-92

mutant or the 93-160 mutant was expressed along with FLAG-CAR in Huh-7 cells. Cells

were lysed and the lysates were subjected to co-immunoprecipitation assays with anti-GFP

antibodies. Immunoprecipitated proteins were used for Western blot analysis. (B) EYFP-

GADD45B wild-type (WT), an EYFP-GADD45B Δ41/92 mutant, or control EYFP was

expressed with FLAG-CAR in Huh-7 cells. Co-immunoprecipitation was performed in the

same conditions as Fig. A. (C) The structure of mouse GADD45B with five α-helices. (D)

Overlapping-fragments of hCAR were expressed along with FLAG-GADD45B in Huh-7 cells.

Co-immunoprecipitation was performed using anti-GFP antibodies. Structure-based amino

acid sequence information of these fragments is presented in Supplemental Fig. 3A.

Fig. 5. GADD45B-dependent p38 MAPK dephosphorylation and its emergence in livers after

tumor initiation. (A) GADD45B dependency in phenobarbital-mediated p38 MAPK

dephosphorylation. GADD45B wild-type (WT) and knock-out (KO) males in the C57BL/6

background were intraperitoneally injected with phenobarbital (PB) or control phosphate-

buffered saline (PBS). After 24 h, mice were again injected with the same solution for

additional 6 h. Livers were collected and subjected to Western blot analysis. Band intensities

(p-p38/p38) were analyzed with ImageJ 1.51s (NIH, USA). (B) PB-mediated p38 MAPK

dephosphorylation in the mouse liver with tumor initiation. Diethylnitrosamine (DEN) (90

mg/kg) was injected into C57BL/6J males of 5 weeks of age. After 2 weeks, PB (500 ppm) or

control water was given to mice for 2 weeks. Phosphorylation levels of p38 MAPK or

MAPKAPK2 (MK2) were determined by Western blot analysis. Band intensities for p-

p38/p38 or p-MK2/MK2 were analyzed.

Fig. 6. Cross-talk between CAR and p38 MAPK signaling on GADD45B scaffolds. Stresses

such as DNA damage induce GADD45B in the liver. GADD45B forms a homodimer (31) and

enhances MKK6-catalized phosphorylation of p38 MAPK by scaffolding them. The activated

p38 MAPK stimulates downstream molecules to induce cell-cycle arrest and apoptosis.

Phenobarbital also induces GADD45B through activating CAR (Supplemental Fig. 5) (4),

and CAR inhibits formation of the p38 MAPK-MKK6-GADD45B complex by interacting with

GADD45B, resulting in repression of p38 MAPK activity.

Table 1. BrdU-positive hepatocytes following PB injection.

C57BL/6 GADD45B KO

Male Female Male Female

% Fold % Fold % Fold % Fold change change change change 0 hr 0.43 ± - 0.39 ± - 1.41 ± - 1.44 ± - 0.07 0.06 0.20 0.58 24 0.66 ± 1.53 3.79 ± 9.72 2.43 ± 1.72 8.69 ± 6.03 hr 0.25 1.39 0.24* 3.20 36 6.17 ± 14.3 7.75 ± 19.9 7.82 ± 5.55 11.1 ± 7.71 hr 1.09* (9.35a) 2.40 (2.05a) 3.24 (3.23a) 3.08 (1.28a) 48 7.54 ± 17.5 5.63 ± 14.4 3.01 ± 2.13 5.23 ± 3.63 hr 0.70** (1.22b) 1.66 (0.72b) 0.97 (0.38b) 2.01 (0.47b) Each value is shown as the mean ± SE (n=4). **P < 0.01, *P < 0.05, PB vs. control (0 h), by

Student’s t-test. a fold over values at 24 h. b fold over values at 36 h.

Nuclear Receptor CAR Suppresses GADD45B-p38 MAPK Signaling to Promote Phenobarbital-induced Proliferation in Mouse Liver

Takeshi Hori, Kosuke Saito, Rick Moore, et al.

Mol Cancer Res Published OnlineFirst May 1, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-18-0118

Supplementary Access the most recent supplemental material at: Material http://mcr.aacrjournals.org/content/suppl/2018/05/04/1541-7786.MCR-18-0118.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mcr.aacrjournals.org/content/early/2018/05/01/1541-7786.MCR-18-0118. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.