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INTRODUCTION |
Nuclear receptors comprise a large family of ligand-regulated
transcription factors that mediate various physiological responses (1).
Nuclear receptors are composed of highly conserved structural domains,
including a DNA-binding domain
(DBD)1 and a ligand-binding
domain (LBD). In response to ligand binding, the LBDs of nuclear
receptors undergo a structural rearrangement, resulting in the
recruitment of transcriptional coactivators, such as CBP, cAMP-response
element-binding protein-binding protein (CBP)/p300, p300/CBP-associated
factor (P/CAF), steroid receptor coactivator 1, peroxisome
proliferator-activated receptor gamma (PPAR
) coactivator-1
(PGC-1
), and the thyroid hormone receptor-associated proteins/vitamin D receptor-interacting proteins/activator-recruited cofactor (TRAP/DRIP/ARC) complex, which lead to either histone acetylation or recruitment of the RNA polymerase II complex (2).
The constitutive androstane receptor (CAR, also named NR1I3 or NR1I4)
(3) and the pregnane X receptor (PXR, also named NR1I2) (3), which are
both expressed abundantly in the liver and intestine, play critical
roles in the xenobiotic response, the metabolic responses of the body
against toxic molecules and drugs (4). The activation of these nuclear
receptors induces the expression of several target genes, including
cytochromes P450. Recently, CAR gene knock-out mice exposed to
acetoaminophen were spared from the toxic effects of the drug (5).
However, natural ligands to these receptors are unknown, and thus they are included in the orphan nuclear receptors. Unlike classical nuclear
receptors, such as steroid hormone receptors, CAR shows constitutive
transcriptional activity in the absence of ligand when expressed in
cell lines and in yeast (6, 7). The constitutive activity of mouse CAR
is inhibited by sex steroids, 5
-androstan-3
-ol (androstanol) and
5
-androst-16-en-3
-ol (androstenol) (7). Yet, it is still unclear
whether or not these steroids are true natural ligands for CAR, because
a superphysiological concentration was required to inhibit the CAR
activity. Thus, the elucidation of the mechanism of CAR activation is a
current issue.
During a search for a coactivator coupled with CAR, we found that the
transcriptional coactivator PGC-1
mediated the ligand-independent activation of CAR. PGC-1
was originally identified as a
PPAR
-binding protein in a cell line derived from brown adipose
tissue (8) and subsequently was shown to couple with several nuclear
receptors (9-15). Unlike other transcriptional coactivators, PGC-1
has the distinctive structural features of a serine/arginine-rich domain (RS domain) and an RNA recognition motif (RRM) at the C-terminal end, which are both observed in many splicing factors (16). We
demonstrate here that the RS domain of PGC-1
plays an important role
for CAR activation by interacting with CAR and targeting CAR to nuclear
speckles. The significance of the interaction of CAR with PGC-1
,
which is considered to be a key modulator in glucose homeostasis, is
also discussed.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The plasmids pCMX-FLAG-mPGC1
,
pCMX-FLAG-mPGC1
, pCMX-GAL4-hERR3 (amino acids 173-436),
pCMX-GAL4-hPPAR
2 (amino acids 204-506), pCMX-GAL4-hPXR (amino acids
110-434), pCMX-GAL4-hRXR
(amino acids 222-426), pCMX-GAL4-hER
(amino acids 251-595), pCMX-GAL4 DBD, and UASx4-tk-Luc were kindly
provided by Dr. A. Kakizuka (Kyoto University). The plasmids pOZ-Tip60
and pOZ-P/CAF were obtained from Dr. T. Ikura (Hiroshima University).
The cDNA for human CAR LBD (amino acids 77-348) was obtained by
PCR from a human liver cDNA library (Origene Technologies,
Inc.) using the primers CARLs (5'-TAGATGCTGAATTC AGG AAA
GAC ATG ATA C; underlining indicates the introduced EcoRI
site) and CARLa (5'-GCGGATCC TCA GCT GCA GAT CTC CTG GAG C;
underlining indicates the introduced BamHI site). The PCR
fragment was cloned into pBluescript KS(
), and the sequence was
confirmed. The EcoRI-BamHI fragment was excised and ligated to EcoRI-BamHI-digested pCMX-GAL4
DBD.
The pCGN-TFIIB plasmid was constructed by inserting an
NheI-BamHI fragment of TFIIB, excised from phIIB
(obtained from Dr. D. Reinberg, Harvard University), into
XbaI-BamHI-digested pCGN (obtained from Dr. K. Simon). The HindIII-KpnI fragment containing a
FLAG peptide sequence was excised from pCMX-FLAG-mPGC1
and inserted
into HindIII-KpnI-digested pCMX-GAL4-RXR
,
giving rise to pCMX-FLAG-RXR
.
The pCMX-FLAG-mPGC1
plasmid was digested with NheI and
XbaI, and the large fragment was recircularized by ligation,
giving rise to the PGC-1-Nhe deletion mutant. The large fragment was blunted by Klenow DNA polymerase and was circularized by self-ligation, giving rise to the PGC-1-Nhe/Xba mutant. The pCMX-FLAG-mPGC1
plasmid
was digested with XbaI, blunted by Klenow DNA polymerase, and circularized by self-ligation, giving rise to the PGC-1-Xba mutant.
The pCMX-FLAG-mPGC1
plasmid was digested with EcoRI, and
the large fragment was circularized by self-ligation, giving rise to
the PGC-1-Eco mutant. The EcoRI-MfeI fragment
containing the RS domain of PGC-1
was inserted into the
EcoRI-digested PGC-1-Eco mutant, giving rise to the
PGC-1-Mfe mutant.
The EcoRI-NotI fragment of hCAR LBD was cloned
in-frame with the glutathione S-transferase
(GST)-encoding sequence of pGEX6P-1 (Amersham Biosciences).
Deletion mutants of GST-fused PGC-1
were constructed in a similar
manner as described above. For the S tag fusion protein, PGC-1
was
digested with the indicated restriction enzymes and cloned into pET30 (Novagen).
For the enhanced green fluorescent protein (EGFP) fusion protein, the
EcoRI-BamHI fragment of hCAR LBD was first cloned
into pBluescript KS(
), and then the SalI-BamHI
fragment was excised and cloned into pEGFP-C1 (Clontech).
Cell Culture and Transient Transfections--
COS-7 cells were
maintained in Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 10% fetal calf serum (Hyclone Laboratories). One day
before transfection, the cells were plated in 24-well plates at a
density of 6 × 104/well. The cells were transiently
transfected with 1 µg of the DNA mixture by LipofectAMINE2000
(Invitrogen), according to the manufacturer's recommendations.
Typically, we used 200 ng of nuclear receptor plasmid, 200 ng of
coactivator plasmid, 400 ng of reporter construct, 200 ng of pEYFP-C1
as an internal control, and pcDNA3 as a normalization plasmid.
After 6 h of transfection, the medium was changed to phenol
red-free Dulbecco's modified Eagle's medium (Invitrogen) containing
10% charcoal/dextran-treated fetal calf serum (Sigma), and the
ligands were added for 24 h when necessary.
Luciferase Assay--
One day after transfection, the enhanced
yellow fluorescent protein (EYFP) fluorescence was measured with a
microplate fluorescence reader, model FL500 (Bio-Tek Instruments) to
examine the transfection efficiency. Then the cells were washed once
with PBS and lysed with 150 µl of PicaGene lysis buffer (Toyo Ink
Co., Ltd.). After the cell debris was removed by centrifugation, the
luciferase activity was measured with PicaGene, using a luminometer,
model TD-20/20 (Promega). Luciferase activities were normalized to the EYFP fluorescence value and are referred to as relative light units.
All of the experiments were repeated at least three times and yielded
similar results.
Pull-down Assay--
GST fusion proteins were expressed in
DH5
cells and were purified using glutathione-Sepharose 4B (Amersham
Biosciences). If necessary, the GST tag was removed by PreScission
protease (Amersham Biosciences). His-tagged proteins were expressed in the BL21(DE3) strain (Novagen) and were purified using a
Ni2+-bound metal chelating column (Amersham Biosciences).
Ten µg of GST-fused and S-tagged PGC-1
proteins were each bound to
glutathione-Sepharose 4B and S protein-agarose (Novagen), respectively.
After the unbound proteins were removed by washing, 20 µg of GST-free
CAR proteins were added to the beads, which were incubated at 4 °C
for 2 h. The beads were extensively washed with 20 mM
Tris-Cl, pH 7.4, containing 150 mM NaCl, 0.1 mM
EDTA, 0.1% Nonidet P-40, and 10% glycerol. The bound CAR proteins
were eluted with SDS-PAGE sample buffer and were detected by Western
blotting using an anti-CAR polyclonal antibody (Santa Cruz) with the
ECL Western blotting detection reagent (Amersham Biosciences).
Purification of Recombinant CAR-PGC-1
Complex Coexpressed in
Bacteria--
The His-tagged NcoI-MfeI fragment
of PGC-1
and the GST-fused CAR LBD were coexpressed in BL21 (DE3)
cells under ampicillin and kanamycin selection. The cells were grown in
LB medium at 37 °C until an A600 of
0.8 and were induced for 18 h with 0.05 mM
isopropyl-1-thio-
-D-galactopyranoside at 18 °C. The
cells were harvested by centrifugation and frozen until use.
The cell pellets (1 liter of culture) were resuspended in PBS and
sonicated on ice. After the debris was removed by centrifugation (9000 × g at 4 °C for 20 min), the soluble extract
was loaded onto glutathione-Sepharose 4B. The column was washed with
PBS, and the bound proteins were eluted with 10 mM reduced
glutathione in 50 mM Tris-Cl, pH 8.0. The eluate was
dialyzed against loading buffer (20 mM Hepes, pH 7.4, 250 mM NaCl, and 25 mM imidazole) and was loaded
onto a Ni2+-bound metal chelating column. The column was
washed with the loading buffer and a wash buffer (20 mM
Hepes, pH 7.4, containing 150 mM NaCl). The proteins were
eluted by the wash buffer containing 50 mM EDTA, pH 7.4, and were directly transferred to a GSTrap column (Amersham
Biosciences). The column was further washed with 20 mM
Tris-Cl, pH 7.4, containing 150 mM NaCl and was
equilibrated with PreScission cleavage buffer (50 mM
Tris-Cl, pH 7.4, containing 150 mM NaCl, 10 mM
EDTA, and 1 mM dithiothreitol). Ten units of PreScission
protease (Amersham Biosciences) were then injected into the column by a
syringe. The cleavage was carried out for 2 days at 4 °C, and the
purified recombinant CAR-PGC1 complex was eluted with 20 mM
Tris-Cl, pH 7.4, containing 150 mM NaCl.
Histochemistry--
COS-7 cells were seeded onto 35-mm dishes
and were transfected with vectors expressing EGFP-fused CAR,
FLAG-tagged RXR
, or FLAG-tagged PGC-1
. After 24 h, the cells
were fixed with 3% formaldehyde for 15 min at room temperature. The
cells were washed twice with PBS, permeabilized with 0.2% Nonidet
P-40, and then incubated for 1 h in 3% bovine serum albumin in
PBS to block nonspecific binding. FLAG-tagged proteins were visualized
with anti-FLAG M2 antibody (Cosmo Bio Co. Ltd.) and
rhodamine-conjugated anti-mouse IgG (Cosmo Bio Co. Ltd.). EGFP and
rhodamine fluorescence were observed with a laser scanning microscope,
model LSM510 (Zeiss).
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RESULTS |
Functional Coupling of PGC-1
with CAR--
To find the mediator
of CAR activation, we examined the coupling of various nuclear receptor
coactivators with CAR (Fig.
1A). The CAR LBD was fused
with the GAL4 DBD and was recruited to the promoter region with the
GAL4 binding element, to drive the expression of the luciferase
reporter gene. After transfection of the respective plasmids into COS-7
cells, transcriptional activation was measured by the luciferase
activity. Without any exogenous coactivator, CAR showed the
constitutive activity (white bar). Cotransfection of various
coactivators with CAR revealed that PGC-1
strongly enhanced the CAR
activity. PGC-1
also stimulated CAR activity to a lower extent than
PGC-1
. The other coactivators, such as Tip60, TFIIB, and P/CAF, did
not stimulate the CAR activity. Next, we compared the magnitude of CAR
activation by PGC-1
with that of other nuclear receptors (Fig.
1B). PGC-1
also enhanced the ERR3 and PPAR
activities
in the absence of their ligands, as well as the CAR activity. A similar
level of transcriptional enhancement by PGC-1
was observed with
agonist-stimulated ER
. On the other hand, PGC-1
showed weak
activation of PXR, although PXR has a sequence similar to that of CAR,
and both receptors function in xenobiotic responses (4, 17). PGC-1
evoked weak activation of the retinoid X receptor
(RXR
) in the
absence of its ligand, 9-cis-retinoic acid.

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Fig. 1.
Coactivation of CAR by
PGC-1 . A, selectivity of
various coactivators in CAR activation. COS-7 cells were transiently
transfected with vectors expressing the CAR LBD fused to the GAL4
DNA-binding domain (GAL4DBD) and the indicated coactivators
together with a reporter construct. Transcriptional activation was
analyzed by the luciferase activity, normalized to EYFP fluorescence as
an internal control. The GAL4 DBD-fused CAR showed constitutive
activity as compared with the GAL4 DBD alone. Cotransfection of
PGC-1 enhanced the CAR activity, although PGC-1 alone did not
activate transcription. PGC-1 also showed slight enhancement of CAR
activity. B, coactivation of nuclear receptors by PGC-1 .
The cells were transiently transfected with vectors expressing the LBDs
of the indicated nuclear receptors fused to the GAL4 DBD, with or
without PGC-1 . In the absence of ligand, coexpression of PGC-1
greatly enhanced the CAR, ERR3, and PPAR activities. PGC-1
slightly increased the basal activities of PXR and RXR . Activation
of ER by PGC-1 was enhanced by the addition of the ER agonist
diethylstilbestrol (DES, 10 µM).
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Effects of Putative Ligands on PGC-1
-mediated CAR
Activation--
The sex steroids androstanol and androstenol
reportedly inhibit CAR activity (7, 17). In fact, we observed that
androstenol (10 µM) partially inhibited the basal
activity of CAR (Fig. 2A). In
contrast, androstenol (10 µM) weakly activated PXR, which
is consistent with the previous report (17). Next, we examined the
effect of this putative ligand on the PGC-1
-mediated CAR activation
and observed that androstenol moderately inhibited but did not abolish
the CAR activation by the coexpression of PGC-1
(Fig.
2B). We also investigated the effect of a synthetic ligand,
the antimycotic clotrimazole and observed that clotrimazole slightly
inhibited but did not abolish the PGC-1
-mediated activation of CAR
as well (data not shown).

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Fig. 2.
Effects of putative CAR ligands on the
activity. A, effects of putative ligands on CAR or PXR
activity. COS-7 cells were transiently transfected with the expression
plasmids for the GAL4 DBD, the GAL4 DBD-fused CAR, or the GAL4
DBD-fused PXR and the reporter construct. The cells were treated with
10 µM of androstenol for 24 h. Androstenol slightly
inhibited the basal CAR activity. In contrast, androstenol slightly
stimulated PXR activity. B, effects of androstenol on the
PGC-1 -dependent CAR activity. The cells were transfected
with the GAL4 DBD-fused CAR with (closed circles) or without
PGC-1 (open circles). The cells were treated with
androstenol at the indicated concentrations for 24 h. The
vertical axis indicates the activity relative to that in the
absence of androstenol (100%) in each case.
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Effects of Heterodimerization with RXR
on PGC-1
-mediated CAR
Activation--
RXR
is known to heterodimerize with CAR to enhance
the constitutive activity of CAR (7, 18) and its androstanol
sensitivity (7, 19). We therefore investigated the effect of
heterodimerization with RXR
on the PGC-1
-mediated activation of
CAR (Fig. 3). Because RXR
itself was
slightly activated by PGC-1
, as shown in Fig. 1, we used the
construct of RXR
LBD that was not fused with GAL4 DBD, to avoid the
direct recruitment of PGC-1
by RXR
to the promoter. In fact,
PGC-1
did not couple with the RXR
LBD without the GAL4 DBD
(lane 2). Coexpression of the RXR
LBD had a weak effect
on the basal activity of CAR but enhanced the PGC-1
-mediated CAR
activation (lanes 3 and 4). The RXR
LBD had no
effect on the PXR basal activity and a very weak effect on the PXR
activity when coexpressed with PGC-1
(lanes 5 and
6). Although RXR
enhanced the PGC-1
-mediated
activation of CAR, the coexpression of the RXR
LBD did not alter the
required domains of PGC-1
for CAR activation (Fig.
4 and data not shown). Then we further
characterized the PGC-1
-mediated CAR activation without RXR
.

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Fig. 3.
Enhancement of
PGC-1 -stimulated CAR activity by
heterodimerization with RXR . Although
RXR fused to the GAL4 DBD was slightly activated by PGC-1 (Fig.
1B), the RXR LBD in the absence of the GAL4 DBD did not
couple with PGC-1 (lanes 1 and 2). When the
GAL4 DBD-fused CAR was expressed with or without the RXR LBD in the
absence of PGC-1 , a slight enhancement of the basal activity of CAR
was observed (lanes 3 and 4, white
bars). In the presence of PGC-1 , the RXR LBD enhanced the
CAR activity (lanes 3 and 4, shaded
bars). The RXR LBD had no effect on the basal activity of PXR
(lanes 5 and 6, white bars) and weakly
activated the PGC-1 -mediated activation of PXR (lanes 5 and 6, shaded bars).
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Fig. 4.
PGC-1 domains
required for CAR activation. A, schematic
representation of PGC-1 domain structures and deletion mutants used
for cotransfection experiments. The putative nuclear receptor
interaction domain containing the LXXLL motif is indicated
by a gray box, the RS domain is a striped box,
and the RRM is a hatched box. The numbers at the ends of the
constructs show the amino acid positions where the stop codon was
introduced. B, PGC-1 deletion mutants were cotransfected
with the GAL4 DBD-fused CAR (panel a) and ERR3 (panel
b). The PGC-1-Nhe/Xba mutant lacking the LXXLL motif
activated CAR but not ERR3. The PGC-1-Mfe mutant lacking RRM activated
both CAR and ERR3. The CAR activation was dramatically reduced in the
PGC-1-Xba and PGC-1-Eco mutants lacking the RS domain and RRM, although
these mutants activated ERR3 to the same extent as the full length. The
PGC-1-Nhe mutant activated neither CAR nor ERR3.
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Functional Domains within PGC-1
Required for CAR
Activation--
PGC-1
has a putative nuclear receptor interaction
region containing the LXXLL motif, which is conserved in
several nuclear receptor coactivators, the RS domain, and the RRM, the
latter two of which are observed in many non-snRNP splicing factors
(16). To define the functional domains of PGC-1
required for CAR
activation, we made a series of deletion mutants of PGC-1
(Fig.
4A) and investigated their ability to activate CAR using the
luciferase assay (Fig. 4B, panel a). Removal of
the LXXLL motif (PGC-1-Nhe/Xba) decreased but did
not abolish the ability of PGC-1
to activate CAR. The deletion of
RRM (PGC-1-Mfe) did not affect the CAR activation, whereas
the deletion of both the RRM and RS domains (PGC-1-Xba and
PGC-1-Eco) further reduced the ability. A deletion mutant lacking all three regions (PGC-1-Nhe) completely lost the
ability to activate CAR. These results indicated that both the
LXXLL motif and the RS domain in PGC-1
are required for
the full activation of CAR.
Next we investigated whether the requirement of these domains for the
enhancement was specific to CAR. We took advantage of ERR3, which was
activated by cotransfection of PGC-1
(Fig. 1B) and
investigated the required domains of PGC-1
for the activation of
ERR3 (Fig. 4B, panel b). Removal of the
LXXLL motif (PGC-1-Nhe/Xba) completely abolished
the ability of PGC-1 to activate ERR3, but the deletion of the RRM and
RS domains (PGC-1-Xba and PGC-1-Eco) did not
alter the ERR3 activation. A deletion mutant lacking all three regions
(PGC-1-Nhe) completely lost the ability to activate ERR3.
These results indicated that the LXXLL motif, but not the RS
domain, is sufficient for coupling with ERR3. The difference in the
regions of PGC-1
necessary for CAR and ERR3 activation suggested
that the requirement of the RS domain for the activation is dependent
on the nuclear receptor with which it is coupled.
The Regions within PGC-1
That Interact with CAR LBD--
We
asked whether PGC-1
would physically interact with the CAR LBD and
which domains were required for the interaction. Deletion mutants of
GST-fused PGC-1
were expressed and purified from Escherichia coli (Fig. 5A,
panels a and b). The CAR LBD was purified as a GST-fused protein, and then the GST portion was removed by PreScission protease digestion. The CAR LBD was mixed with GST-PGC-1
or its mutant proteins immobilized onto glutathione-Sepharose beads, and the
bound CAR LBD was dissolved in sample buffer and visualized by Western
blotting using an anti-CAR antibody (Fig. 5A, panel c). The CAR LBD was able to bind to PGC-1-Eco (lane 5),
PGC-1-Xba (lane 6), and PGC-1-full (lane 7) but
not to PGC-1-Nhe (lane 4). The difference in the CAR binding
ability between PGC-1-Eco and PGC-1-Nhe indicated that the region
containing the LXXLL motif was required for the association
with the CAR LBD. A tighter interaction was observed in PGC-1-Xba
(lane 6) and PGC-1-full (lane 7) than in
PGC-1-Eco (lane 5). Although PGC-1-Xba and PGC-1-full were contaminated with many lower bands (Fig. 5A, panel
b), the CAR interaction was not correlated with those lower bands.
These results suggest that PGC-1
interacts with the CAR LBD through
the C-terminal region in addition to the LXXLL motif.

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Fig. 5.
Multiple PGC-1
domains interact with the CAR LBD. A, Pull-down
experiments using a series of PGC-1 mutants with C-terminal
deletions. Panel a, the GST-fused PGC-1 deletion mutants
are schematically shown. The restriction enzyme shown at the
right indicates the position where the stop codon was
introduced. The domains and the motifs indicated below the full-length
PGC-1 were described in the legend to Fig. 4A.
Panel b, PGC-1 deletion mutants, immobilized on
glutathione-Sepharose beads, were resolved by SDS-PAGE and stained by
CBB. Panel c, the CAR LBD was incubated with GST or
GST-fused PGC-1 deletion mutants. After the beads were washed, the
bound proteins were separated by SDS-PAGE. The CAR LBD interaction with
PGC-1 deletion mutants was detected by Western blotting using an
anti-CAR antibody. B, pull-down experiments using PGC-1
fragments lacking the LXXLL motif. Panel a,
S-tagged PGC-1 fragments are shown. Panel b, proteins
immobilized on S-protein beads were separated and stained by CBB.
Panel c, the CAR LBD interaction with the PGC-1 fragments
was detected by Western blotting. C, schematic
representation of the procedure to obtain the CAR-PGC-1 , complex by
serial affinity purification. The His-tagged
NcoI-MfeI fragment of PGC-1 containing the RS
domain was coexpressed and copurified with the GST-fused CAR LBD.
D, recombinant CAR LBD-PGC-1 RS domain complex. The
number above each lane indicates the fraction
eluted by PreScission cleavage. Panel a, the complex was
separated by SDS-PAGE and was visualized by silver staining.
Panel b, the PGC-1 fragment was detected by Western
blotting using an anti-His antibody.
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We produced additional deletion mutants lacking the LXXLL
motif, as His- and S-tagged (His-Stag) proteins, to locate other regions of PGC-1
interacting with the CAR LBD, besides the
LXXLL motif region (Fig. 5B, panels a
and b). The CAR LBD was mixed with the Stag-PGC-1
proteins immobilized onto S-protein beads, and the bound CAR LBD was
pulled down and visualized by Western blotting using an anti-CAR
antibody (Fig. 5B, panel c). The CAR LBD was able
to bind to these His-Stag PGC-1
proteins (Fig. 5B, panel c), indicating that multiple regions of PGC-1
directly interact with CAR. It was notable that the
NcoI-MfeI fragment, containing the RS domain,
directly bound to CAR (lane 5), which possibly reflects the
fact that the RS domain had a crucial effect on CAR activation (Fig.
4B, panel a).
Purification of the Recombinant CAR LBD-PGC-1
RS Domain
Complex--
To prove that the CAR LBD directly interacts with the RS
domain of PGC-1
, we coexpressed the GST-fused CAR LBD and the
His-tagged RS domain in E. coli and purified the complex of
these two proteins. Serial affinity purification allowed us to isolate
the CAR LBD-PGC-1
RS domain complex (Fig. 5C). The
complex was eluted from glutathione-Sepharose beads by cleaving off the
GST tag by PreScission protease, which ensured the direct binding of
PGC-1
to the CAR LBD rather than nonspecific binding to the column
(Fig. 5C). Aliquots of the eluate were subjected to SDS-PAGE
and were analyzed by silver staining (Fig. 5D, panel
a). The protein appeared as a 29-kDa band, corresponding to the
CAR LBD. The coeluted 37-kDa protein was identified as a PGC-1
fragment by Western blotting using an anti-His antibody (Fig.
5D, panel b).
PGC-1
-dependent Arrangement of CAR Subnuclear
Localization--
In the course of the experiment investigating the
colocalization of CAR with PGC-1
in cells, we found that CAR moved
to nuclear speckles when PGC-1
was coexpressed. We used the
EGFP-fused CAR LBD to determine the localization of CAR (Fig.
6A, center panel), which showed a distribution similar to that of the GAL4 DBD-fused CAR,
as determined by immunofluorescence (Fig. 6A, left
panel). Coexpression of RXR
did not affect the distribution of
CAR (Fig. 6B). When PGC-1
was coexpressed, CAR
colocalized at nuclear speckles with PGC-1
(Fig. 6C,
panel a). In all of the 50 total cells counted, colocalization in nuclear speckles was observed (100%). A significant amount of CAR was still present in the cytoplasm even when PGC-1
was
coexpressed, suggesting that the subnuclear distribution, but not the
cytoplasm-nuclear shuttling of CAR, is important for the activity.
PGC-1
expressed without CAR localized at nuclear speckles; however,
a diffusely distributed signal was observed in the entire nucleus (Fig.
6C, panel a, PGC-1 alone).
Coexpression of CAR with PGC-1
seemed to weaken the diffuse
distribution of PGC-1
in the nucleus and to intensify the signal in
nuclear speckles, suggesting that the binding of CAR and PGC-1
might
cause a mutual effect on each other.

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Fig. 6.
PGC-1 domains required for subnuclear
targeting of CAR. A, subcellular localization of CAR,
detected by either immunofluorescence or EGFP fusion. COS-7 cells were
transfected with the GAL4 DBD-fused CAR LBD or the EGFP-fused CAR LBD
and were fixed after 24 h. The GAL4 DBD-fused CAR was detected by
indirect immunofluorescence using an anti-CAR antibody and an
fluorescein isothiocyanate-conjugated secondary antibody
(GAL4DBD-CAR LBD). CAR was distributed throughout the cells
with a slight accumulation in nucleus. The EGFP-fused CAR
(EGFP-CAR) showed a pattern of distribution similar to that
detected by immunofluorescence. EGFP alone did not show any nuclear
accumulation but was distributed diffusely (EGFP).
B, the effect of coexpression of the RXR LBD on the
subcellular localization of CAR. RXR was detected by indirect
immunofluorescence, using an anti-FLAG antibody and a
rhodamine-conjugated secondary antibody (FLAG-RXR). The
pattern of CAR localization was not changed by RXR
(EGFP-CAR). C, the effect of coexpression of
PGC-1 on the subcellular localization of CAR. CAR was visualized
with an EGFP fusion (EGFP-CAR). PGC-1 was visualized with
indirect immunofluorescence using an anti-FLAG antibody and a
rhodamine-conjugated secondary antibody (FLAG-PGC-1). The
colocalization of CAR and PGC-1 was investigated by confocal
microscopy (Merge). The localization of PGC-1 without CAR
is shown on the left (PGC-1 alone). Panel
a, nuclear CAR moved to the nuclear speckles when PGC-1 was
coexpressed. PGC-1 by itself was located at the nuclear speckles.
Panel b, the PGC-1-Nhe/Xba mutant lacking the
LXXLL motif was located at the nuclear speckle and shifted
the location of CAR to the nuclear speckles when coexpressed.
Panel c, the PGC-1-Mfe mutant lacking RRM did not reside at
the nuclear speckle but was distributed throughout the nucleus.
However, the coexpression of CAR with the PGC-1-Mfe mutant induced the
colocalization of CAR and the PGC-1 mutant at the nuclear speckles.
Panel d, the PGC-1-Xba mutant lacking the RS domain and RRM
resides at the nucleus without any nuclear speckles, and coexpression
of CAR and the PGC-1-Xba mutant did not change their respective
locations. Panel e, the PGC-1-Eco mutant was distributed
throughout the cells, and the coexpression of CAR and the PGC-1-Eco
mutant did not change their respective locations. Scale bar,
20 µm. D, schematic representation of the functional
PGC-1 domains. CAR interacts with PGC-1 through multiple domains,
including the LXXLL motif in the N terminus and the RS
domain in the C terminus. PGC-1 by itself localizes at the nucleus
by its nuclear localization signal (NLS) and at the nuclear
speckles by RRM in the C terminus. The interaction with CAR induces the
CAR-dependent targeting of PGC-1 to the nuclear speckles
with CAR.
|
|
We used the deletion mutants of PGC-1
to investigate which domain of
PGC-1
was responsible for the subnuclear targeting of CAR. The
PGC-1-Nhe/Xba mutant lacking the LXXLL motif was able to
shift the localization of CAR to the nuclear speckles (Fig. 6C, panel b). Of 44 total cells counted, 30 cells
showed colocalization of CAR and PGC-1
in nuclear speckles (68%).
The PGC-1-Mfe mutant lacking RRM was not able to localize in nuclear
speckles by itself (Fig. 6C, panel c, PGC-1
alone). This observation is consistent with the previous report
(20). Interestingly, the localization of PGC-1
in nuclear speckles
was observed even in the absence of RRM but only when CAR was
coexpressed (Fig. 6C, panel c). Colocalizaton of
CAR and PGC-1
was observed in 32 cells of the 38 total cells counted
(84%). Using the PGC-1-Xba mutant lacking both the RS domain and RRM,
neither CAR nor PGC-1 localized in the nuclear speckles (Fig.
6C, panel d). The PGC-1-Eco mutant no longer
showed exclusive nuclear localization, because of the absence of a
nuclear localization signal (Fig. 6, C, panel e,
and D). A comparison between the activity and the
localization suggested that the colocalization in nuclear speckles may
be a limited process for PGC-1
-mediated CAR activation.
 |
DISCUSSION |
In this study, we report that the transcriptional coactivator
PGC-1
mediates the ligand-independent activation of the nuclear receptor CAR. Our results showed that the N-terminal region containing the LXXLL motif and the C-terminal region containing the RS
domain participate in the interaction and the activation with CAR. Both regions were necessary for full activation. In addition, the RS domain
was required for colocalization of PGC-1
in nuclear speckles with
CAR. These data indicated that the ligand-independent activation of CAR
by PGC-1
is achieved by subnuclear targeting through the RS domain
of PGC-1
.
CAR shows constitutive activity in the absence of exogenous ligand when
transfected into cell lines, such as HepG2 and CV-1 cells; however, it
is reportedly inactive in primary cultured hepatocytes because of its
cytoplasmic localization (21). This study gave us the idea that an
additional factor may be required for the constitutive activity of CAR.
In the course of searching for a coactivator coupling with CAR, we
found that PGC-1
greatly enhanced the CAR activity in the absence of
ligand. This activation was specific to PGC-1s, because several other
coactivators we examined did not show the enhancement. This link of CAR
with PGC-1
surprised us, because the liver is not the major organ of
PGC-1
expression (8). However, important experimental data have
recently described how PGC-1
expression in the liver is dynamically
regulated by the nutritional state of the body; PGC-1
is induced by
starvation or fasting and plays a critical role in gluconeogenesis (12, 22).
Recently, the crystal structures of several orphan nuclear receptors,
including ultraspiracle, hepatocyte nuclear factors (HNF4
and
HNF4
), retinoic acid-related orphan receptor
, ERR3, and PXR,
have been solved (23-28). Notably, the crystals of the ultraspiracle,
HNF4
, HNF4
, and retinoic acid-related orphan receptor
LBDs
prepared from E. coli contained lipid ligands, presumably
derived from cell lipid constituents, in the hydrophobic pocket.
According to these studies, one possible difference between the cell
lines and the primary hepatocytes was that CAR might be activated by an
unknown endogenous ligand produced by the cell lines. Alternatively, a
natural ligand, which exists in the primary hepatocytes but not in the
established cell lines, may repress the CAR activity, and its
repression might be modulated by the interaction with PGC-1
. The CAR
reverse agonist, androstenol, did not completely inhibit the
PGC-1
-mediated CAR activation, although the human CAR, which we
used, is less sensitive to androstanol than the mouse CAR (17). In
addition, the effect of a synthetic antagonist of ERR3,
diethylstilbestrol, on PGC-1
-mediated ERR3 activation was small, and
furthermore, the recombinant ERR3-PGC-1
complex was so stable that
it could not be dissociated by diethylstilbestrol stimulation (data not
shown). Thus, the binding of CAR to PGC-1
is rather stable, and the
complex might also be resistant to androstenol. The answer to this
issue awaits the development of strong and specific agonists and
antagonists, which are expected for both the clinical and experimental fields.
Because CAR forms a heterodimer with RXR
(7, 18, 19), we assessed
the effect of the heterodimerization between CAR and RXR
on the
PGC-1
coactivation. RXR
itself slightly coupled with PGC-1
in
the absence of ligand, but the enhancement of PGC-1
-mediated CAR
activation by the RXR
LBD was not additive but synergistic, suggesting that the CAR-RXR
heterodimer may stabilize PGC-1
binding to CAR rather than CAR and RXR
binding to PGC-1
independently. Using deletion mutants of PGC-1
, we observed that the
coexpression of the RXR
LBD did not change the domains of PGC-1
required for CAR activation (data not shown), supporting the idea that RXR
stabilizes the binding. These results suggest that the
heterodimerization with RXR
does not explain the constitutive
activity of CAR. This idea does not contradict the in vivo
observation that RXR
remains at the nucleus, even when CAR is
inactive in the cytosol in hepatocytes (21).
PGC-1
reportedly couples with several other nuclear receptors in
ligand-dependent and ligand-independent manners (8-15). PGC-1
interacts with these nuclear receptors through the
LXXLL motif in its N-terminal region. In this study, we
observed that CAR also directly interacted with the region containing
the LXXLL motif. In addition, we found that another region
containing RS domain in PGC-1
also interacted with CAR by pull-down
experiments and probed the complex formation using purified proteins.
The C-terminal fragment containing the RS domain and RRM reportedly interacts with an ER
hinge region in a ligand-independent manner (10). Taken together, the RS domain in PGC-1
may provide a novel
interface to nuclear receptors. The interaction between PGC-1
and
nuclear receptors through multiple domains may confer the specificity
of the coactivation.
The RS domain as well as RRM in PGC-1
is shared with SR proteins,
which are essential splicing factors that participate in mRNA
processing through their RS domains and RRMs (20, 29). PGC-1
diffusely localized at the nucleus and concentrated in the nuclear
speckles, where splicing factors are colocalized (20). Nuclear speckles
of splicing factors are considered to be a site for storage and
assembly of splicing factors (30). Nuclear speckles are highly dynamic
structures. Upon gene activation, the splicing factors, visualized by
GFP fusions, are released from the nuclear speckles to
transcriptionally active sites (31). In the absence of RRM, PGC-1
did not reside in the nuclear speckles by itself; however, when CAR was
coexpressed, the PGC-1
moved to nuclear speckles together with CAR,
suggesting that the coexpression of CAR and PGC-1
has a mutual
effect on their localization (Fig. 6D). This targeting
activity was totally dependent on the RS domain and was observed even
in the absence of the LXXLL motif in PGC-1
. The RS
domains in the splicing factors are reportedly required for both
targeting to and dissociation from nuclear speckles (32-34). These
results strongly suggest that CAR binds to PGC-1
through the RS
domain and rearranges the domain to acquire the ability to move to the
nuclear speckles for transcriptional activation or for altering
mRNA processing. It is interesting that the CAR-mediated transcriptional activation of cytochrome P450 by xenobiotics in hepatocytes is abolished by the serine/threonine-specific phosphatase inhibitor, okadaic acid (21), which is also known to prevent the
movement of splicing factors into nuclear speckles (35). The
involvement of the serine/threonine-specific phosphatases in regulating
PGC-1
-mediated CAR activation will be investigated in the future.
Although the physiological relevance of PGC-1
-mediated CAR
activation will be needed for further experiments, our data strongly suggest that PGC-1
regulates the activity of CAR in hepatocytes. Microarray data have suggested that CAR regulates several enzymes catalyzing fatty acid or carbohydrate metabolism (36). Thus, it would
be intriguing to investigate the CAR activity in the liver under
different nutritional conditions and the PGC-1
levels after exposure
to xenobiotics, which would provide clues to the relationships between
these important pathways, drug catalysis and metabolic conditions.