MOLECULAR AND CELLULAR BIOLOGY, Sept. 2004, p. 7931–7940
0270-7306/04/$08.00?0 DOI: 10.1128/MCB.24.18.7931–7940.2004
Vol. 24, No. 18
Nuclear Receptors CAR and PXR Cross Talk with FOXO1 To
Regulate Genes That Encode Drug-Metabolizing
and Gluconeogenic Enzymes
Susumu Kodama, Chika Koike, Masahiko Negishi,* and Yukio Yamamoto
Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of
Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
Received 23 February 2004/Returned for modification 18 March 2004/Accepted 16 June 2004
The nuclear receptors CAR and PXR activate hepatic genes in response to therapeutic drugs and xenobi-
otics, leading to the induction of drug-metabolizing enzymes, such as cytochrome P450. Insulin inhibits the
ability of FOXO1 to express genes encoding gluconeogenic enzymes. Induction by drugs is known to be
decreased by insulin, whereas gluconeogenic activity is often repressed by treatment with certain drugs, such
as phenobarbital (PB). Performing cell-based transfection assays with drug-responsive and insulin-responsive
enhancers, glutathione S-transferase pull down, RNA interference (RNAi), and mouse primary hepatocytes, we
examined the molecular mechanism by which nuclear receptors and FOXO1 could coordinately regulate both
enzyme pathways. FOXO1 was found to be a coactivator to CAR- and PXR-mediated transcription. In contrast,
CAR and PXR, acting as corepressors, downregulated FOXO1-mediated transcription in the presence of their
activators, such as 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and pregnenolone 16?-carboni-
trile, respectively. A constitutively active mutant of the insulin-responsive protein kinase Akt, but not the
kinase-negative mutant, effectively blocked FOXO1 activity in cell-based assays. Thus, insulin could repress the
receptors by activating the Akt-FOXO1 signal, whereas drugs could interfere with FOXO1-mediated tran-
scription by activating CAR and/or PXR. Treatment with TCPOBOP or PB decreased the levels of phos-
phoenolpyruvate carboxykinase 1 mRNA in mice but not in Car?/?mice. We conclude that FOXO1 and the
nuclear receptors reciprocally coregulate their target genes, modulating both drug metabolism and
Liver plays a major role in the metabolism of therapeutic
drugs and environmental contaminants. It is endowed with a
mechanism to induce hepatic enzymes, leading to increased
detoxification and elimination of those xenobiotics. Drug in-
duction is generally regulated by transcriptional activation of
hepatic genes encoding drug-metabolizing enzymes, such as
cytochrome P450s (CYPs) and specific transferases. Acting as
the principal transcription factors which also form complexes
with RXR, the nuclear receptors CAR and PXR play a central
role in induction by binding to the phenobarbital (PB)- and
xenobiotic-responsive enhancer modules PBREM and XREM
(5, 6, 11, 12, 18, 46, 53), respectively, and activating transcrip-
tion of their target genes, such as CYP genes, in response to a
distinct but overlapping group of xenobiotics (23, 36, 50). How-
ever, induction is heavily influenced by endocrine conditions;
glucocorticoid hormone, for example, augments CYP induc-
tion by PB (31). In contrast, insulin is known to repress the
induction of drug-metabolizing activity by certain drugs and in
diabetic livers (44, 49, 56). Insulin treatment either eliminated
or significantly reduced PB induction of CYP2B in rat primary
hepatocytes (17, 42, 58). Hepatic CYP2B, CYP3A, and
CYP4A were increased in experimentally generated diabetic
rats and mice and were reduced to normal levels by insulin
treatment (37, 56). While insulin is involved in regulating drug
metabolism, drug treatment influences insulin-responsive glu-
cose metabolism. For instance, both PB and 1,4-bis[2-(3,5-
dichloropyridyloxy)]benzene (TCPOBOP) decreased hepatic
enzymes, such as phosphoenolpyruvate carboxykinase 1
(PEPCK1) and glucose-6-phosphatase (G6P) (1, 24, 50).
Chronic PB treatment decreased plasma glucose and improved
insulin sensitivity in diabetic patients (20). Here we investi-
gated the molecular mechanism of insulin repression by focus-
ing on whether CAR and/or PXR could be coregulated by
insulin response transcription factor FOXO1 and vice versa.
A forkhead transcription factor, FOXO1, is an activator of
gluconeogenic genes, such as PEPCK1, G6P, and insulin-like
growth factor-binding protein 1 (29, 32, 38, 57). Insulin inhibits
FOXO1 activity, leading to repression of these genes. In fact,
the insulin receptor knockout mouse partially restored serum
glucose to normal levels when it was crossed with a Foxo1?/?
mouse (28). These gluconeogenic genes contain an insulin
response sequence (IRS) to which FOXO1 can bind directly
and activate in the absence of insulin (7, 32, 38, 51, 57). Insulin
treatment triggers the phosphorylation of FOXO1 via a phos-
phatidylinositol 3-kinase (PI3K)-Akt pathway (7, 30, 35). Phos-
phorylation inactivates FOXO1 by decreasing the binding af-
finity of FOXO1 to IRS and/or exporting FOXO1 from the
nucleus (2, 25, 34, 59). FOXO1 has been implicated as a
coregulator for various nuclear receptors, coactivating or core-
pressing the estrogen, thyroid, and retinoid A receptors (39,
60), whereas glucocorticoid and progesterone receptors (60),
* Corresponding author. Mailing address: Pharmacogenetics Sec-
tion, Laboratory of Reproductive and Developmental Toxicology, Na-
tional Institute of Environmental Health Sciences, National Institutes
of Health, Research Triangle Park, NC 27709. Phone: (919) 541-2404.
Fax: (919) 541-0696. E-mail: firstname.lastname@example.org.
hepatic nuclear factor 4 (HNF4) (9), and peroxisome prolif-
erator-activated receptor ? (PPAR?) (4) are corepressed. In
addition, the androgen receptor prevents the binding of
FOXO1 to IRS, leading to a repression of FOXO1-IRS activity
(21). Our yeast two-hybrid screening identified FOXO1 as a
CAR-binding protein. These observations prompted us to in-
vestigate whether FOXO1 mediates the insulin-dependent re-
pression of CAR and/or PXR-mediated trans-activation of
drug-induced genes, such as CYP genes. Moreover, we also
investigated whether CAR and/or PXR receptors reciprocally
coregulate FOXO1-IRS activity.
By employing glutathione S-transferase (GST) pull-down,
mammalian two-hybrid, and transient luciferase reporter assays
and in vivo animal experiments, we demonstrate here that the
nuclear receptors and FOXO1 cross-talk to coregulate their re-
sponse elements. Drug and glucose metabolisms, which are two
major liver functions that can be regulated independently, ap-
peared to be coordinately regulated by this cross-talk.
MATERIALS AND METHODS
Materials. 5?-Androst-16-en-3?-ol (androstenol) was purchased from Ster-
aloids, Inc. Insulin, insulin-like growth factor 1 (IGF1), TCPOBOP, and preg-
nenolone 16?-carbonitrile (PCN) were purchased from Sigma-Aldrich. Restric-
tion endonucleases and DNA-modifying enzymes were purchased from New
England Biolabs, Inc. [35S]methionine was purchased from Amersham.
LY294002 was purchased from Calbiochem.
Plasmid constructions. The following plasmids were kindly provided: XREM-
3A4-Luc plasmid by Brian Goodwin at GlaxoSmithKline (5); rPKB?/pECE and
rPKB?K179 M/pECE by Ushio Kikkawa at Kobe University; myr-rPKB?/pcDL-
SRM by Kiyoshi Hidaka at the National Institute of Environmental Health
Sciences. In all plasmids, m, r, and h in front of the insert denote mouse, rat, and
human, respectively. The pGL3/1.8 kbp-2B6-Luc, pGL3/1.6 kbp-2B6-Luc (52),
pGEX/mCAR, pCMX/hRXR (11), pCR3/mCAR (47), and pBIND/mCAR (55)
plasmids were described previously. The CAR mutant T176V in pCR3 plasmid
and more details of its characterization will be published elsewhere (A. Ueda, K.
Matsui, Y. Yamamoto, L. Pedersen, T. Sueyoshi, and M. Negishi, unpublished
data). Full-length PXR (18) was amplified from a mouse liver cDNA library and
was cloned into pcDNA3-V5-His (Invitrogen) and pGEX-4T-1 (Stratagene)
vectors designated pcDNA3/mPXR and pGEX/mPXR, respectively. cDNA en-
coding residues 425 to 652 was amplified from a FOXO1 clone selected from the
yeast two-hybrid screening and was inserted into pACT (Promega) to generate
pACT/mFOXO1Ct. Using proper sets of primers, the first and second exons were
amplified from mouse genomic DNA using LA taq DNA polymerase with GC
buffer (TaKaRa Shuzo, Otsu, Japan) and were cloned into pCR2.1-TOPO (In-
vitrogen). Inserts were cut out by double digestion with BamHI and EcoRI from
the TOPO plasmids, ligated, and recloned into pcDNA3-V5-His to obtain the
full-length cDNA of FOXO1, designated pcDNA3/mFOXO1. Using pcDNA3/
mFOXO1 as a template, proper mutated primers, and the QuikChange mu-
tagenesis kit (Stratagene), Thr24, Ser253, and Ser316 were mutated to alanine to
generate pcDNA3/mFOXO13A. To clone the full-length FOXO1 cDNA into
pGEX-4T-1, the cDNA was amplified to include an additional XhoI site at the
5? end, digested with XhoI, and inserted at the SalI site of pGEX-4T-1 to create
pGEX/mFOXO1. For the various Akt expression vectors, the full-length cDNAs
of wild-type Akt (AktWt), kinase-negative Akt (AktKN), and constitutively acti-
vated Akt (AktCA) were amplified from rPKB?/pECE, rPKB?K179 M/pECE,
and myr-rPKB?/pcDL-SRM, respectively. These cDNAs were cloned into
pcDNA3-V5-His plasmids designated pcDNA3/AktWt, pcDNA3/AktKN, and
pcDNA3/AktCA, respectively. To construct the pGL3/IRS-tk-Luc plasmid, a
complementary pair of oligonucleotides containing three tandem copies of IRS
GTA-3? from the human IGFBP1 gene) (48) was synthesized. The underlined
sequence indicates an additional overhanging XhoI restriction site. Oligonucle-
otides were annealed and cloned into the XhoI-digested site of the pGL3-Basic
vector containing a 160-bp thymidine kinase (tk) promoter (47), and a plasmid
was selected containing six repeats of IRS. For amplification of FOXO1 from its
genomic clone, Pfu Turbo DNA polymerase (Stratagene) was used. Insert se-
quences of newly constructed vectors were verified by DNA sequencing.
Yeast two-hybrid screen. The ligand-binding domain (residues 87 to 358) of
the mouse CAR T176V mutant was cloned into the pGBKT7 plasmid (Clon-
tech). This plasmid was cotransfected with the mouse liver MATCHMAKER
cDNA library (Clontech) into the Y190 yeast strain. Positive yeast colonies were
selected in the presence of TCPOBOP (1 ?M), and the binding activity was
measured by ?-galactosidase assay according to the manufacturer’s instructions.
One-hundred five yeast colonies were isolated from 2.2 ? 106primary transfor-
mants. The plasmids encoding the activation domain fusion proteins were re-
covered from these yeast transformants, and the inserts were sequenced. Three
of these inserts were found to encode FOXO1 and were cloned as a fusion
protein in frame with the GAL4 activation domain.
GST pull-down assay. The GST fusion proteins GST-mCAR, GST-mPXR,
and GST-mFOXO1 were expressed in Escherichia coli strain BL21 cells and were
purified with glutathione-Sepharose. The cDNAs of FOXO1, CAR, and PXR in
pcDNA3-V5-His were in vitro translated in the presence of [35S]methionine
using the TNT-coupled reticulocyte lysate system (Promega) according to the
manufacturer’s instructions. GST pull-down assays were carried out as described
Cell culture and transfection. HepG2 cells were cultured in minimum essential
medium (MEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine,
100 U of penicillin per ml, and 100 ?g of streptomycin per ml in an atmosphere
of 5% CO2at 37°C. Primary hepatocytes were prepared from male Cr1:CD-
1(ICR)BR mice (Charles River Laboratories) as described previously (13).
HepG2 cells plated in 24 wells at 50 to 60% confluence were transfected with
lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.
pRL-CMV (10 ng) (Promega) was used as an internal control for all transfection
assays. Mouse primary hepatocytes were cotransfected with pGL3/1.8 kbp-2B6-
Luc (10 ?g) and pRL-CMV (5 ?g) by the electroporation method (16). For
experiments with RNAi, SMARTpool (M-003006-01-05; Dharmacon) for human
FOXO1 or an unrelated RNA (5?-NNUGGUAGGCAGGAUGAGUAC-3?) as
nonspecific control was transfected into HepG2 cells using lipofectamine 2000.
After 24 h of transfection the cells were treated with drug for an additional 48 h
Animal treatment. Cr1:CD-1(ICR)BR male mice (7 to 8 weeks old) main-
tained on a 12-h light and 12-h dark cycle were randomly divided into two groups
that were intraperitoneally injected with TCPOBOP (0.3 mg/kg of body weight)
or dimethyl sulfoxide (DMSO). Twenty-four hours after injection, mice were fed
until sacrificed to prepare liver RNAs. The original background strain of Car?/?
mice used in a previous work (50) was changed to C3H/HeNCrlBR mice
(Charles River) by repeated backcrossing until microsatellite analysis showed
that mice contained 98% of the C3H markers. Car?/?and Car?/?male mice
were treated with a single injection of 90 mg of diethylnitrosamine/kg, followed
by chronic treatment with PB in drinking water (500 ppm) for 32 weeks. These
treatments were originally for investigating liver tumor promotion. Livers and
blood samples were collected and used for analyses.
Real-time PCR. TRIZOL reagent (Invitrogen) was used to extract total RNAs
from HepG2 cells, mouse livers, and primary mouse hepatocytes, and cDNAs
were prepared from total RNAs using Super-Script II reverse transcriptase
(Invitrogen). Real-time PCR was performed with an ABI prism 7700 sequence
detection system (Applied Biosystems) with the following primers and probes:
Hs00231106_m1 (Applied Biosystems) for the human FOXO1 gene; 5?-6FAM-
CTCTTCCA-3?, and 5?-CAGCAGGCGCAAGAACTGA-3? for CYP2B10 (14);
CATCCGCAAGCTGAAG-3?, and 5?-TTCGATCCTGGCCACATCTC-3? for
the mouse Pepck1 gene. The TaqMAN rodent GAPDH control reagent (Applied
Biosystems) was used as internal control.
Gel shift assay. CAR, RXR, PXR, and FOXO1 proteins were produced by in
vitro translation as described above. For probes, three sets of double-stranded
oligonucleotides containing NR1 sequence (47), wild-type IRS (5?-GATCGCT
AGATGCAAAACAACTTGTGACGATC-3?), and mutant IRS (5?-GATCGC
TAGATGCAACAGAACTTGTGACGATC-3?) were synthesized and end-la-
beled by using [32P]dATP and DNA polymerase Klenow fragment (New England
Biolab). Gel shift assays were performed as described previously (11). For com-
petition or antibody supershift assays, unlabeled oligonucleotide, rabbit immu-
noglobulin G (Santa Cruz), or FOXO1 antibody (Santa Cruz) was added 15 min
before adding radioactive oligonucleotide to start the binding reaction.
FOXO1 as a CAR-binding protein. Yeast two-hybrid screen-
ing of a mouse liver cDNA library was performed using the
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