| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 29, 26356-26363, July 19, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
§,, andFrom the Department of Molecular & Integrative Physiology, College of Medicine at Urbana-Champaign, University of Illinois, Urbana, Illinois 61801
Received for publication, January 3, 2002, and in revised form, April 24, 2002
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Phenobarbital (PB) induction of
CYP2B genes is mediated by translocation of the
constitutively active androstane receptor (CAR) to the nucleus.
Interaction of CAR with p160 coactivators and enhancement of CAR
transactivation by the coactivators have been shown in cultured cells.
In the present studies, the interaction of CAR with the p160
coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) was
examined in vitro and in vivo. Binding of GRIP1
to CAR was shown by glutathione S-transferase (GST)
pull-down and affinity DNA binding. N- or C-terminal fragments of GRIP1 that contained the central receptor-interacting domain bound to GST-CAR, but the presence of ligand increased the binding to GST-CAR of
only the fragments containing the C-terminal region. In gel shift
analysis, binding to CAR was observed only with GRIP1 fragments containing the C-terminal region, and the binding was increased by a
CAR agonist and decreased by a CAR antagonist. Expression of GRIP1
enhanced CAR-mediated transactivation in cultured hepatic-derived cells
2-3-fold. In hepatocytes transfected in vivo, expression of exogenous GRIP1 alone induced transactivation of the
CYP2B1 PB-dependent enhancer 15-fold, whereas
CAR expression alone resulted in only a 3-fold enhancement in untreated
mice. Remarkably, CAR and GRIP1 together synergistically transactivated
the enhancer about 150-fold, which is approximately equal to activation
by PB treatment. In PB-treated mice, expression of exogenous CAR alone
had little effect, expression of GRIP1 increased transactivation about
2-fold, and with CAR and GRIP, a 4-fold activation was observed. In
untreated mice, expression of GRIP resulted in nuclear translocation of
green fluorescent protein-CAR. These results strongly suggest that a
p160 coactivator functions in CAR-mediated transactivation in
vivo in response to PB treatment and that the synergistic
activation of CAR by GRIP in untreated animals results from both
nuclear translocation and activation of CAR.
In response to treatment with drugs or other xenobiotics,
metabolism of the administered drug or other drugs is often increased (1). Underlying the increase in most cases is an induction of the
expression of cytochrome P450 genes. Different subsets of
cytochrome P450 genes are induced by different chemicals. Recently, members of the nuclear receptor family that form heterodimers with RXR,
including peroxisomal proliferator activating receptor CAR has been identified as the mediator of induction of
CYP2B genes by the classical inducer of drug metabolism, PB.
CAR was implicated in PB induction of CYP2B genes by the
observation that CAR was selectively present in nuclear extracts from
PB-treated animals and could bind to site with direct repeats separated
by 4 base pairs (NR-1 and NR-2) in the CYP2B PB-responsive
enhancer, termed PBRU or PB-responsive module (3). In cultured cells, expression of CAR by transient or stable transfection could
transactivate the PBRU or induce the expression of the endogenous
CYP2B6 gene in HepG2 cells (4-7). The loss of PB induction
of Cyp2b genes in transgenic mice with a disrupted
CAR gene provided conclusive evidence of an essential role for CAR in
PB induction (8).
CAR is unusual among the nuclear receptors in that it has relatively
high constitutive activity (9). The initial ligands identified for CAR,
androstanes, inhibited rather than activated CAR so that CAR was
considered constitutively active (10). The concentrations of
androstanes required for inhibition were higher than physiological
concentrations, so it was initially unclear how PB induction could be
mediated by such a constitutive nuclear receptor. This was clarified by
the observation that CAR in untreated animals or primary cultures of
hepatocytes is predominantly located in the cytoplasm of hepatocytes in
contrast to continuously cultured cells where it is located in the
nucleus (11). Treatment with PB resulted in translocation into the
nucleus, which should be sufficient for transactivation of the PBRU
because of its constitutive activity. Binding of PB to CAR was not
detected, but binding of other PB-like ligands, such as TCPOBOP, was
detected by several techniques, and activation of CAR by these ligands
was implied by an increase in interaction with the coactivator SRC-1 in
the presence of TCPOBOP (12, 13). This led to a two-stage model for CAR
activation in which (i) translocation into the nucleus was induced by
PB-like inducers and (ii) CAR was directly activated by some of the
inducers. The translocation was inhibited by okadaic acid, suggesting
that phosphatase activity is required for the translocation (11), and
activation in the nucleus could be blocked by a
Ca2+/CaM-dependent protein kinase inhibitor,
suggesting a role for phosphorylation in the activation (14).
The mechanism by which CAR transactivates the PBRU is not clear. In
transient transfections, in addition to the NR-1 and NR-2 CAR-binding
sites, a nuclear factor 1 site between and sequences flanking these NR
sites are required for maximal PB induction (4, 15-17). Like other
nuclear receptors, CAR transactivation probably involves coactivator
proteins. The p160 coactivator SRC-1 has been shown to bind to CAR both
biochemically and in two-hybrid studies, and the binding was decreased
by antagonists, androstanes, and increased by the agonist, TCPOBOP (10,
12). In primary cultures of hepatocytes, SRC-1 expression alone
increased transactivation of the PBRU but not a synthetic enhancer with
two of the CAR NR-1 sites about 3-fold in untreated cells and similarly
increased transcription 2-3-fold in cells expressing exogenous CAR
(7). These results suggest that p160 coactivators interact with and enhance transactivation by CAR, but the relatively small increases in
transactivation mediated by overexpressed SRC-1 and the assay of
activity in cultured hepatocytes in which the relative concentrations of regulatory factors may differ from hepatocytes in vivo
fall short of establishing a role for these coactivators in PB induction.
SRC-1 is a member of a family of related p160 coactivators that
includes SRC-1, TIF2/GRIP1, and RAC3/ACTR/pCIP/AIB-1 (18). GRIP1
has been shown to coactivate hepatic nuclear receptors, for example
hepatic nuclear factor 4 (19, 20), and thus is a potential coactivator
for CAR in the liver. We now show that GRIP1 interacts with CAR and
with DNA-bound CAR·RXR and that the binding is modulated by
ligands. GRIP1 modestly enhances CAR-mediated activation in
continuously cultured cells. Remarkably, in untreated mice, exogenous
expression of GRIP1 in hepatocytes transfected in vivo
increases transactivation of the PBRU more than expression of CAR does,
and coexpression with CAR results in a dramatic synergistic activation
equal to that resulting from PB treatment. In PB-treated animals,
exogenous expression of GRIP1 also results in a 2-fold increase in
transactivation, whereas CAR has little effect and a 4-fold increase is
observed if both exogenous CAR and GRIP are expressed.
Plasmids--
Vectors for expression of CAR in mammalian cells
(pcDNA3-CAR) and bacteria (pETCAR) have been described (6).
For expression of GST-CAR, a
BamH1/EcoR1 fragment containing the CAR cDNA
was inserted into pGEX2TK digested with the same enzymes. The
expression vector, pSG5.HA-GRIP1, encoding full-length GRIP1 and the
bacterial expression vectors pGEX.GRIP5-766,
pGEX.GRIP530-1121, and pGEX.GRIP730-1121 were
obtained from M. R. Stallcup. The vector pCMX-RXR was obtained
from R. Evans. The expression vector for GFP-CAR, pEGFPCARC1, was
constructed by inserting a BamHI/EcoR1 fragment
from pcDNA3-CAR containing the CAR cDNA into the
BglII/EcoR1 site of pEGFP-C1 (BD Biosciences CLONTECH).
Expression and Purification of CAR, RXR, and
GRIP1--
His-tagged CAR and FLAG-tagged RXR were expressed in
bacteria and isolated as described (6).
GST Pull-downs--
GST fusion proteins were expressed in
Escherichia coli BL21(DE3 (pLys)) and purified by binding to
glutathione-Sepharose (Pharmacia Corp.) according to the
manufacturer's protocols. 35S-Labeled proteins were
synthesized by transcription of the mRNA and translation in
reticulocyte lysates (Promega Corp.) according to the manufacturer's
instructions, and the lysate was precleared by incubation with GST
bound to glutathione-Sepharose for 30 min at 4 °C. One µg of GST
or GST fusion proteins bound to glutathione-Sepharose were incubated at
4 °C for 2 h with 4 µl of precleared reticulocyte lysate
containing the 35S-labeled proteins in 200 µl of binding
buffer (25 mM KOH-HEPES, pH 7.6, 100 mM
NaCl, 10% glycerol, 0.1 mM EDTA, 1 mM
dithiothreitol, 0.5 mg/ml BSA, and 0.05% Nonidet P-40). In some cases,
5 µM TCPOBOP or 50 µM androstenol dissolved
in Me2SO or Me2SO alone (0.25% volume)
were added to the reactions. After incubation, the samples were washed
by centrifugation and resuspension in binding buffer without BSA
five times. The proteins were eluted from the Sepharose by incubation
with 20 mM reduced glutathione in the same buffer, the
eluted proteins were separated by SDS-PAGE, and the radioactive proteins were detected by autoradiography.
Affinity DNA Binding--
A biotinylated DNA fragment containing
four copies of the CYP2B1 NR1 site was synthesized by PCR
using biotinylated oligonucleotide primers. CAR and RXR were purified
from bacteria, and 35S-labeled coregulator proteins were
synthesized by transcription and translation in reticulocyte lysates.
The lysates were precleared by incubation at room temperature for
1 h with streptavidin-agarose in DNA binding buffer (15 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM MgCl2, 1 mM EDTA) that contained
10% glycerol, 1 mM dithiothreitol, and protease
inhibitors. To reduce nonspecific interactions, streptavidin-agarose was coated with BSA by incubation with 0.5 mg/ml BSA at room
temperature for 30 min. About 250 ng of the biotinylated DNA fragment
was incubated with 100-250 ng of purified CAR and/or RXR in DNA
binding buffer at room temperature for 25 min. BSA-coated
streptavidin-agarose was added to the binding reactions, and the
incubation was continued for 30 min at room temperature. This agarose
complex was collected by centrifugation and washed two times by
resuspension in 500 µl of DNA binding buffer and centrifugation. The
streptavidin-agarose DNA-protein complex was resuspended in 100 µl of
DNA binding buffer and incubated for 10 min at room temperature with 5 µM TCPOBOP, 50 µM androstenol, or
Me2SO. Then 4 µl of precleared reticulocyte lysate with
the 35S-labeled coregulator proteins were added, and the
sample was incubated for 30 min at room temperature. The agarose
complex was washed five times by centrifugation and resuspension in 500 µl of DNA binding buffer that contained 250 mM KCl and
0.5% Nonidet P-40. The proteins were eluted from the agarose by the
addition of 0.2% sarkosyl in the same buffer, and the eluted proteins
were analyzed by SDS-PAGE and autoradiography.
Gel Mobility Shift Analysis--
Gel mobility shift analysis was
carried out as described (6). The DNA probe was a 32P
end-labeled oligonucleotide containing the CYP2B1 NR-1 site, and 5,000-10,000 cpm were added to each reaction. 5 µM
TCPOBOP, 50 µM androstenol, or 1 µM
9-cis-retinoic acid was added to the binding reactions as
indicated under "Results."
Cell Culture and Transfection--
Mouse Hepa1c1c7 and human
HepG2 cells were cultured and transfected using LipofectAMINE as
described (6). For transfections, the cells were seeded in 24-well
plates. 1 µg/well of reporter plasmid containing either the
CYP2B1 PBRU or four copies of the NR-1 site from the PBRU
fused to the minimal CYP2C1 promoter and firefly luciferase
reporter gene, 10 ng/well of pRL-SV40 plasmid DNA, containing the SV40
promoter and Renilla luciferase gene, and varying amounts as
indicated of expression vectors for CAR and GRIP1 were added to each
well. In experiments in which androstenol or TCPOBOP were added, the
cells were incubated for 24 h after transfection, fresh medium
containing the ligands was added, and the cells were incubated for an
additional 24 h. The luciferase activities were determined by the
dual luciferase reporter assay system (Promega Biotech), and the
firefly luciferase values were normalized to the Renilla
values for each sample.
Tail Vein Injection--
Plasmid DNAs used for injection into
mouse tail veins were purified by two rounds of CsCl density gradient
centrifugation. 6-8-week-old (20-25 g) BALB/c male mice (Harlan Labs)
were injected via the tail vein using the TransIT In
Vivo Gene Delivery System as described by the manufacturer
(Panvera Corp., Madison, WI). DNA was mixed with Mirus polymer solution
at a concentration of 1 µg/µl, diluted to 200 µl with water, and
incubated for 5 min at room temperature. Just prior to injection, the
DNA-polymer mixture was diluted in Mirex delivery solution to a volume
equal to Expression of GFP-CAR--
5 µg of an expression vector for
CAR-GFP, pEGFPCARC1, with or without 25 µg of an expression vector
for GRIP1, pSG5.HA-GRIP1, was injected into mouse tail veins as
described above. Two animals were used for each experimental group.
Either isotonic saline or 100 mg/kg body weight of PB was injected
intraperitoneally 2 h after injection of the DNA, and the animals
were sacrificed 4 h later. The livers were cut into small pieces,
placed in Tissue-Tek O.C.T. compound (Miles, Inc.), and frozen in
liquid N2. Frozen sections of 10 µm were prepared with a
cyrostatic microtome and fixed with 2% paraformaldehyde. The cells
were permeabilized by incubation for 5 min in 0.1% Triton X-100 in
phosphate-buffered saline, and DNA was stained by incubation with 0.5 µg/ml propidium iodide in phosphate-buffered saline. Fluorescence was
detected with a Zeiss LSM confocal microscope. Images of 100-nm optical sections through the center of the nucleus were used for analysis. To
determine the relative intensities of green fluorescence from the GFP
in the nucleus and cytoplasm, a line was drawn across the center of
cell, and the sum of the fluorescence intensities for the line over the
nucleus or cytoplasm was divided by the length of the line to derive
IN or IC, respectively.
The data were expressed as
IN/(IN + IC) so that a value of 1 is obtained when the
fluorescence is 100% nuclear and a value of 0 is obtained when the
fluorescence is 100% cytoplasmic.
GRIP1 Interacts with CAR--
To determine whether GRIP1 interacts
with CAR in vitro, the binding of 35S-labeled
GRIP1 to GST-CAR was analyzed by GST pull-downs. Full-length GRIP1 was
bound to GST-CAR, whereas little or no binding of GRIP1 to GST was
observed (Fig. 1B). The CAR
antagonist androstenol (10) had little effect on the binding, whereas
the agonist TCPOBOP (12) increased the binding. In the converse
experiment with 35S-labeled CAR and GST-GRIP1, three
fragments of GRIP1, GRIP1-1 (5-766), GRIP1-2 (530-1121), and
GRIP1-3 (730-1121) fused to GST were examined (Fig. 1A).
GRIP1 contains three LXXLL sequences within a central
nuclear receptor-interacting domain, which extends from amino acids 641 to 749 (21). GRIP1-1 and GRIP1-2 contain all three LXXLL
motifs in this domain, whereas GRIP1-3 contains only the third motif.
Strong binding of 35S-CAR was observed to GRIP1-1 (Fig.
1C, lane 2), and weaker binding was observed for
the other two fragments (Fig. 1C, lanes 5 and 8), whereas no binding to GST was observed (Fig.
1C, lane 11). Interestingly, the CAR ligands had
little effect on binding to GRIP1-1, but androstenol modestly
decreased, and TCPOBOP increased the binding of CAR to GRIP1-2 and
GRIP1-3. These results are consistent with binding of GRIP1 to CAR
through the central nuclear receptor-interacting domain and suggest
that changes in interaction of GRIP1 with CAR by ligand binding are
mediated by the C-terminal region.
GRIP1 Interacts with DNA-bound CAR·RXR--
Although the
previous results showed that GRIP1 could interact with CAR, to mediate
transactivation of CAR, GRIP1 must bind to CAR when it is part of a
heterodimer with RXR and is bound to DNA. Binding of CAR to RXR and to
DNA could either mask binding sites for GRIP1 that are observed when
binding to CAR alone is studied or alter the conformation of CAR so
that binding of GRIP1 is altered. Further, GRIP1 has been shown to
interact with RXR (22), so that GRIP1 potentially could interact with
both CAR and RXR in an additive or synergistic manner. To determine
whether GRIP1 could bind to a CAR·RXR·DNA complex, CAR·RXR, bound
to biotinylated DNA containing four copies of the CYP2B1
NR1, was incubated with 35S-labeled GRIP1, and the complex
was isolated by binding to streptavidin-agarose. Some nonspecific
binding of 35S-GRIP1 to the streptavidin-agarose was
observed if RXR was omitted from the reaction (Fig.
2, lane 2). However, specific
binding of 35S-GRIP1 was observed as an increase in binding
when both CAR and RXR were present (Fig. 2, compare lanes 2 and 4). Binding was further increased if the agonist TCPOBOP
was added to the reaction (Fig. 2, lane 5), and the
antagonist androstenol modestly reduced the binding (Fig. 2, lane
6), results that are consistent with the GST pull-down studies
above. Approximately the same amount of CAR·RXR was bound to the
biotinylated DNA in each reaction as shown by the Western blot of RXR
bound to the DNA (Fig. 2, bottom panel).
The interaction of GRIP1 with CAR·RXR bound to DNA was also examined
by gel mobility shift analysis. As shown previously (6, 11), CAR·RXR
binds to the CYP2B1 NR-1 (Fig.
3, lane 1). The addition of
GST or GST-GRIP1-1 to reactions did not result in a supershift of the
CAR·RXR complex (Fig. 3, lanes 2 and 3). In contrast, some radioactivity migrated more slowly than the CAR·RXR complex for GRIP1-2 and GRIP1-3, although not as discrete bands, suggesting that these fragments of GRIP1 interact with the CAR·RXR complex but that the binding is not stable under the conditions for gel
electrophoresis. The addition of ligands had little effect on the
binding of GST or GST-GRIP1-1 (Fig. 3, lanes 7,
8, 12, 13, 17, and
18). In contrast, TCPOBOP strongly increased the
supershifted complex when GRIP1-2 and GRIP1-3 were added to the
reactions (Fig. 3, lanes 9 and 10), whereas in
the presence of androstenol, no supershift of the CAR·RXR complex was
observed (Fig. 3, lanes 14 and 15). The reason
for the difference in the mobility of the supershifted complexes for
GRIP1-2 and GRIP1-3 is not known. These results are consistent with the
GST pull-down and DNA affinity studies above, indicating that TCPOBOP
increases and androstenol decreases the binding of GRIP1 to CAR and
that the dependence of the binding on ligands requires the C-terminal
sequence. Even though the strongest binding to CAR alone in the GST
pull-down experiments was observed with the GRIP-1 fragment, little or
no binding with GRIP-1 was detected in these gel shift studies. This suggests that the C-terminal region is required for stable binding of
GRIP1 to CAR·RXR·DNA complexes but not for stable binding to CAR
alone. Further, the influence of the C-terminal region of GRIP1 on
binding is modulated by ligand binding.
The modulation of the interaction of GRIP1 with CAR·RXR by ligands
raised the question of whether the RXR ligand 9-cis-retinoic acid would have any effect on the interaction. Interestingly, addition
of cis-retinoic acid to the binding reaction resulted in
increases in GRIP1 binding to CAR·RXR analogous to that observed with
TCPOBOP (Fig. 3, lanes 17-20). Similar results have been observed with thyroid hormone receptor-RXR heterodimers (22). These
results suggest that conformational changes induced in one nuclear
receptor subunit of the CAR·RXR heterodimer can alter the
conformation of the partner nuclear receptor and affect its interaction
with a coactivator.
GRIP1 Enhances CAR-mediated Transactivation in Cultured
Cells--
To examine the functional consequences of the CAR and GRIP1
interactions, the effects of cotransfection of GRIP1 with CAR on
transactivation of an enhancer with four copies of the
CYP2B1 NR1 site fused to the CYP2C1 proximal
promoter and a luciferase reporter were examined. In two hepatic cell
lines, CAR transactivation was enhanced by expression of GRIP1.
Transfection of 1 ng of CAR expression vector alone resulted in about a
2-fold increase in luciferase activity in HepG2 cells, and coexpression
of GRIP1 resulted in an additional 2-fold increase (not shown). In
Hepa1c1c7 cells, 1 ng of CAR increased luciferase activity about
3-fold, and a dose-dependent enhancement of the CAR
activation by GRIP-1 was observed (Fig.
4). Androstenol nearly abolished the
effect of exogenous CAR expression in the absence of exogenous GRIP1, but this inhibition was partially reversed by expression of GRIP, suggesting that the androstenol decreased the interaction between CAR
and GRIP (Fig. 4). TCPOBOP increased CAR-mediated transactivation by an
additional 2-fold, and GRIP1 increased the transactivation further, but
the fold-induction was less than in the untreated cultures (Fig. 4).
This result is consistent with an increased affinity of GRIP1 for CAR
as a result of TCPOBOP binding so that increased activation is observed
with the lower concentrations of endogenous GRIP. These ligand effects
on transcription are thus consistent with the modulation of in
vitro binding of CAR and GRIP by ligands.
Exogenous Expression of CAR in Hepatocytes in Situ Has Little or No
Effect on Transactivation of the CYP2B1 PBRU--
The previous
experiments examined CAR-mediated transactivation and the influence of
GRIP1 in cultured cells. In vitro studies and disruption of
the CAR gene in transgenic mice have demonstrated that CAR is critical
for PB induction of Cyp2b genes in mice (8). Although
CAR transactivation in continuously cultured cells was used above as a
model for the induction of CYP2B genes by PB, the PB
response is not seen in these cells, and therefore the observed
transactivation by CAR may not accurately reflect the induction of the
genes in vivo. To examine the effects of expression of
exogenous CAR and GRIP1 in vivo, transactivation of the PBRU was studied by transfection of mouse hepatocytes in vivo by
injection of DNA into the tail vein. Among organs taking up the DNA,
highest expression is observed in liver, and between 5 and 25% of
hepatocytes are transfected (23). PB treatment has been shown to result in a >100-fold increase in expression of luciferase from a vector in
which the PBRU of CYP2B1 and a minimal CYP2C1
promoter is fused to the luciferase gene using this
method.2
In untreated animals, the expression of exogenous CAR in hepatocytes
increased transactivation of the PBRU by about 3-fold (Fig.
5, inset). Because CAR is
predominantly a cytoplasmic protein in untreated animals (11), the
small increase indicates that overexpression of CAR in the hepatocytes
results in some leakage into the nucleus. In PB-treated animals, the
expression of luciferase is increased about 100-fold. If CAR is
expressed exogenously in these PB-treated animals, there is essentially
no additional increase. The lack of effect suggests that a component of
the transactivation mechanism other than CAR is limiting in PB-treated
animals.
Expression of GRIP1 in Vivo Synergistically Activates Transcription
with CAR and Enhances the PB Response--
To examine the effect of
GRIP1 on PBRU transactivation, liver cells were transfected in
situ by the tail vein injection procedure with expression vectors
for GRIP1 and/or CAR. In the untreated animal, CAR is primarily
cytoplasmic so the overexpression of GRIP1 would be expected to have
little effect on transactivation of the PBRU. Surprisingly, expression
of GRIP1 in hepatocytes in vivo increased luciferase
expression about 15-fold in untreated animals, much more than
expression of CAR alone did (Fig. 5, inset). Even more
remarkably, coexpression of CAR with GRIP1 resulted in a synergistic
increase of 150-fold in transactivation of the PBRU, which is similar
to the induction observed with PB treatment. Although expression of
exogenous CAR had little effect in PB-treated mice, expression of GRIP1
resulted in a 2-fold increase. CAR and GRIP1 together were modestly
synergistic in PB-treated animals compared with untreated animals and
resulted in a 4-fold increase (nearly 500-fold compared with untreated
animals). The 2-fold increase observed with expression of CAR in the
presence of exogenous GRIP1 suggests that GRIP1 is limiting relative to
CAR in the PB-treated animal. This synergistic increase in
transactivation of the PBRU in hepatocytes in vivo provides
strong evidence that CAR-mediated transactivation of the PBRU involves
a p160 coactivator like GRIP1.
Exogenous Expression of GRIP1 Results in Nuclear Translocation of
CAR in Untreated Animals--
One possible explanation for the
synergistic effect of GRIP1 and CAR in the untreated animals is that
exogenous expression of GRIP1 both directly activates nuclear CAR and
causes nuclear translocation of CAR. In this case, in cultured cells
and in PB-treated animals, CAR is already nuclear so only a direct
activation of CAR by GRIP1 of several-fold is observed. To examine the
effect of expression of GRIP1 on localization of CAR, hepatocytes were transfected in vivo with an expression vector for GFP-CAR.
The cells were examined 6 h after transfection and 4 h after
PB treatment. Nuclear localization was examined at earlier times after
transfection than the transcriptional luciferase assays because nuclear
translocation is an early step in the regulation of the gene and the
synthesis of the luciferase protein product is the end result. The
localization of fluorescence in representative cells is shown in Fig.
6. In all groups, the distribution of
fluorescence between the nucleus and cytoplasm was heterogenous, but in
the untreated mice (Fig. 6, CONTROL) without GRIP1,
expressed GFP-CAR was absent from the nucleus in nearly all cells with
occasional cells containing some nuclear green fluorescence (Fig. 6,
bottom row, GFP-CAR). In the other three
groups, all cells had detectable nuclear green fluorescence, but the
relative nuclear localization was greater in the PB-treated groups than
in the untreated (CONTROL) group in which exogenous GRIP1
was expressed (Fig. 6). To provide a semiquantitative estimate of
nuclear localization, the relative intensity of fluorescence in the
nucleus was quantified, and a histogram of the nuclear intensity in 20 cells in each group was plotted (Fig. 7).
A value of 0 or 1 indicates 100% cytoplasmic or 100% nuclear
localization, respectively. In untreated animals, 75% of cells had
less than 0.25 relative nuclear fluorescence (Fig. 7). In contrast, in
PB-treated animals, 75% of cells had greater than 0.75 relative
nuclear fluorescence, and all cells had greater than 0.25 nuclear
fluorescence, which is consistent with earlier studies showing nuclear
localization after PB treatment (11, 24). The expression of GRIP1 in
untreated animals resulted in nuclear fluorescence intermediate between these two groups, but all cells had relative nuclear intensities greater than 0.25, and 40% of the cells had relative intensities greater than 0.75 (Fig. 7). GRIP1 expression did not detectably change
localization of CAR in the PB-treated mice. These results show that
exogenous expression of GRIP1 causes nuclear translocation of CAR in
hepatocytes in vivo in the absence of ligand.
Previous studies have implicated p160 coactivators in the
transactivation mediated by CAR, but these studies rested on showing interactions of SRC-1 with CAR alone (10, 12) and modest increases in
CAR transactivation resulting from overexpression of SRC-1 in cultured
cells (7, 24). The present studies confirm the interaction of CAR with
a p160 coactivator, GRIP1, and further show that GRIP1 interacts with
CAR·RXR heterodimers bound to DNA, which is a more functional form of
these nuclear receptors. CAR-mediated transactivation of either the
PBRU (data not shown) or four copies of the CYP2B1 NR-1 was
enhanced 2-3-fold by expression of GRIP1 in cultured cells. These
results are similar to those obtained for the PBRU or The binding of GRIP1 to GST-CAR in vitro was increased by
the agonist TCPOBOP and decreased by the antagonist androstenol, which
is consistent with earlier studies in which ligands modulated binding
of SRC-1 to CAR (10, 12). Interesting differences were observed in the
binding between CAR and different fragments of GRIP1 containing the
central receptor interacting sites and either the N-terminal portion or
the C-terminal portion of molecule. Both types of fragments bound to
CAR alone, but binding was modulated by ligand only for the C-terminal
fragment. In gel shift assays, little binding of GRIP1 to
CAR·RXR·DNA complexes was observed with the N-terminal fragment,
but binding was observed with the C-terminal fragment even if only one
of the three LXXLL motifs in the nuclear interaction domain
was present. This binding was increased by the CAR agonist TCPOBOP and
decreased by the antagonist androstenol. The role of LXXLL
in binding of GRIP1 to CAR has not been established, but there appears
to be little specificity for individual LXXLL motifs because mutation
individually of each of the three LXXLL motifs in the
nuclear receptor interaction domain did not alter the interaction of
CAR and the related p160 coactivator from Xenopus, xSRC-3
(29). These results suggest that effects on the binding of GRIP1 to CAR
by ligands is mediated through the C-terminal portion of the molecule,
analogous to ligand-dependent nuclear receptors, such as
the steroid hormone receptors, and that the C-terminal portion is
required for stable binding of GRIP1 to CAR under the conditions of the
gel shift assay.
It has been reported that exogenous expression of CAR inhibited PB
induction of CYP2B1 in primary cultures of hepatocytes and
that changing the PBRU NR-1 site to a The most surprising result in this study was the dramatic synergistic
effects of exogenous CAR and GRIP1 expression in untreated animals
resulting in a 150-fold increase in transactivation. The basis for the
synergistic effect is most likely due to two effects of the expression
of exogenous GRIP1, translocation of CAR to the nucleus, and direct
activation of CAR by GRIP1. Exogenous expression of GRIP1 resulted in
nuclear localization of GFP-CAR in essentially all of the transfected
hepatocytes, although the extent of translocation was heterogenous and
less complete than that observed in PB-treated cells. Direct activation
of CAR by GRIP1 is about 2-fold based on the effects of GRIP1 in
cultured cells and in PB-treated mice, so that most of the synergistic 150-fold effect in untreated animals is the result of translocation of
CAR to the nucleus. Because in cultured cells, CAR is always present in
the nucleus, the difference in localization of CAR explains most of the
dramatic difference in magnitude of the GRIP1 effect in cultured cells
and in vivo. In addition, the PBRU is a complex enhancer
with DNA-binding proteins other than CAR contributing to the PB
response, (4, 15-17) and coactivators or cosuppressors other than the
p160 coactivators may be also recruited to the PBRU. The differences in
concentration of these factors in cultured cells and in vivo
may contribute to the differences observed for the effects of GRIP1
expression in these two systems.
These studies raise the possibility that PB activation of GRIP1 might
contribute to the translocation of CAR to the nucleus after PB
treatment. Although the action of PB is poorly understood, the
phosphatase inhibitor, okadaic acid, inhibits CAR nuclear translocation
(11). The target of the phosphatase, presumed to be CAR, has not been
directly identified. Precedents exist for modulation of GRIP1 activity
by phosphorylation (33) and for translocation of regulatory proteins by
coregulators, for example translocation of histone deacetylase-4 by
SMRT (silencing mediator for
retinoic acid receptor and thyroid hormone
receptor) (34), so that it is possible that dephosphorylation of a p160 coactivator could contribute to the PB response. An activated GRIP1
could mediate translocation of CAR either by interacting with
cytoplasmic CAR and inducing nuclear translocation or by interacting
with nuclear CAR and enhancing of nuclear retention of CAR. The latter
mechanism would be possible if CAR is continuously shuttling between
the nucleus and cytoplasm even in untreated animals as has been
proposed for nuclear receptors (35). Any GRIP effect on CAR nuclear
translocation would have to be independent of its effect on activation
of CAR because CAR with the C-terminal transactivation domain
inactivated by deletion is still translocated to the nucleus (24).
Further studies will be required to establish the mechanism by which
p160 coactivators induce CAR nuclear translocation and the role of this
effect in PB induction of CYP genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
pregnane X receptor/steroid X receptor, and
CAR,1 have been identified as
mediators of the cellular response to xenobiotics (reviewed in Ref. 2).
These nuclear receptors have relatively low specificity and affinity
for their ligands so that they can be activated by a wide range of
structurally diverse chemicals and thus comprise a broad response
mechanism to xenobiotics.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (42K):
[in a new window]
Fig. 1.
Interaction of GRIP1 with CAR and effects of
CAR ligands assessed by GST pull-down assays. GST-CAR and GST-GRIP
fragments were expressed in bacteria and purified, 35S-GRIP
and 35S-CAR were synthesized by transcription/translation
in reticulocyte lysates, and binding of the radioactive proteins to GST
fusion proteins was determined as described under "Materials and
Methods." Radioactive proteins were eluted from the
glutathione-Sepharose complexes with glutathione, and the eluted
proteins were analyzed by SDS-PAGE and autoradiography. The
lanes marked Input contain 20% of the
radioactive proteins present in each binding reaction. In B,
binding of full-length 35S-GRIP1 (5-1121) with GST or
GST-CAR is shown, and in C binding of 35S-CAR to
GST or the indicated GST-GRIP1 fragments is shown. GRIP1-1, GRIP1-2,
and GRIP1-3, which are fusions to GST of fragments of GRIP1, 5-766,
530-1121, and 730-1121, respectively, are shown schematically in
A. 50 µM androstenol (A) or 5 µM TCPOBOP (T) was added to the reactions as
indicated. The expected positions of GRIP1 and CAR are indicated by
arrows. These results were consistently observed in five
independent experiments.
View larger version (36K):
[in a new window]
Fig. 2.
Binding of 35S-GRIP1 to
CAR·RXR·DNA complexes. CAR alone or RXR and CAR were incubated
with a biotinylated DNA fragment containing four copies of the
CYP2B1 NR-1-binding site for CAR·RXR. The complex of
proteins with the DNA was purified by binding to streptavidin-agarose
and was incubated with full-length 35S-GRIP1 (5-1121)
synthesized by transcription/translation in the presence or absence of
TCPOBOP (T) or androstenol (A) as indicated. The
radioactive proteins bound to the streptavidin-agarose complex were
eluted with 0.2% sarkosyl and analyzed by SDS-PAGE and
autoradiography. The expected position for GRIP1 is indicated. At the
bottom, a Western blot of the proteins in the
streptavidin-agarose complex detected with antiserum to RXR
demonstrated that similar amounts of CAR·RXR were binding to the DNA
for each sample. These results were consistently observed in three
independent experiments.
View larger version (62K):
[in a new window]
Fig. 3.
Interaction of GRIP with CAR·RXR detected
by gel mobility supershifts. A 32P-labeled
oligonucleotide containing a CYP2B1 NR-1 site was incubated
with CAR·RXR and either GST or fragments of GRIP1 fused to GST. An
agonist, TCPOBOP, or an antagonist, androstenol (ANDRO.),
for CAR or an agonist for RXR, 9-cis-retinoic acid
(9-cis-RA), was added as indicated. The samples were
separated by nondenaturing gel electrophoresis, and radioactivity was
detected by autoradiography. The position of CAR·RXR complexed to DNA
is indicated, as is the region of supershifts containing GRIP1
fragments bound to the CAR·RXR complexes. These results were
reproducibly observed in three independent experiments.
View larger version (19K):
[in a new window]
Fig. 4.
Enhancement of CAR-mediated transactivation
by GRIP1 in Hepa1c1c7 cells and effect of ligands. Hepa1c1c7 cells
were transfected with a firefly luciferase reporter, which contained
four copies of the CYP2B1 NR-1 site fused to the
CYP2C1 proximal promoter, an SV40
promoter/Renilla luciferase reporter as an internal
standard, and with expression vectors for CAR and GRIP1, as indicated.
The values for firefly luciferase were normalized by dividing by the
Renilla luciferase values. Me2SO ( 
), 50 µM androstenol (ANDRO.), or 5 µM
TCPOBOP were added to the cultures as indicated. The standard errors of
the mean are indicated for six transfected wells from two independent
experiments.
View larger version (18K):
[in a new window]
Fig. 5.
Transactivation by CAR and GRIP1 in murine
hepatocytes transfected in situ. DNA of
expression vectors for CAR and/or GRIP1, a firefly luciferase reporter
with the CYP2B1 PBRU fused to the CYP2C1
promoter, and a Renilla luciferase reporter containing the
SV40 enhancer/promoter were injected into mouse tail veins as described
under "Materials and Methods." 6 h after the DNA injections
the mice were injected intraperitoneally with either saline or 100 mg/kg body weight PB. 24 h after injection, the mice were
sacrificed, and the luciferase activities in liver extracts were
determined. The firefly luciferase activities were normalized by
dividing by the Renilla luciferase activities. The
inset shows the first three bars with the scale
on the abscissa expanded 10×. The standard errors of the
mean are indicated for four to eight independent determinations.
View larger version (49K):
[in a new window]
Fig. 6.
Effect of exogenous expression of GRIP1 on
the cellular location of GFP-CAR in hepatocytes in
situ. Expression vectors for GFP-CAR (5 µg) and GRIP1
(25 µg) as indicated were injected into tail veins of mice.
2 h after injection, the mice were treated intraperitoneally with
saline (CONTROL) or 100 mg/kg body weight PB and were
sacrificed 4 h later. Frozen sections were prepared, and DNA was
stained with propidium iodide as described under "Materials and
Methods." Fluorescence was detected with a laser scanning confocal
microscope. Four representative cells are shown for each group with
both red fluorescence from propidium iodide and
green fluorescence from GFP shown in the left
panel, and only green fluorescence shown in the
right panel for each cell.
View larger version (16K):
[in a new window]
Fig. 7.
Distribution of relative nuclear localization
of GFP-CAR. Expression of GFP-CAR and GRIP, treatment with PB, and
imaging with a confocal microscope were as described in the legend to
Fig. 6. Relative nuclear green fluorescence was estimated by drawing a
line across the center of cell and determining the sum of the green
fluorescence intensities for the line over the nucleus or cytoplasm
divided by the length of the line to derive IN
or IC, respectively. Relative nuclear
fluorescence was calculated as
IN/(IN + IC), so that a value of 1 is obtained when green
fluorescence is 100% nuclear and a value of 0 is obtained when green
fluorescence is 100% cytoplasmic. The numbers of cells in four groups
with relative nuclear green fluorescence of >0.25, 0.25-0.50,
0.50-0.75, or >0.75 were plotted.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RARE sites
when SRC-1 was coexpressed with CAR in primary cultures of hepatocytes
or CV-1 cells, respectively (7, 10), although transactivation of 2 NR-1
sites was not increased by SRC-1 in primary hepatocytes in contrast to
the present results with four copies of the NR-1. Although CAR
transactivation in cultured cells was only modestly enhanced by GRIP1,
exogenous expression of GRIP1 in vivo in untreated mice
increased transactivation by 15-fold and increased transactivation by
50-fold in cells exogenously expressing CAR. In PB-treated animals,
exogenous expression of GRIP1 enhanced CAR transactivation about
2-fold. The dramatic 50-fold increase in CAR transactivation mediated
by GRIP1 expression in vivo in the untreated animals,
compared with 2-3-fold increases in cell culture, provides strong
additional evidence for the role of p160 coactivators in PB induction
of CYP2B genes. GRIP1 has been shown to activate hepatic
nuclear receptors, and either the protein or mRNA was reported to
be present in human or mouse liver (25-27), but it was recently
reported that GRIP1 was not detectable in hepatic parenchymal cells
using immunocytochemical techniques (28). Thus, either SRC-1 or
AIB1/p/CIP/SRC-3, the latter of which is predominantly expressed in
Xenopus liver (29), may function as the p160 form
interacting with CAR in the liver.
RARE site, which still binds
CAR, eliminated PB induction (30). These results led to the proposal
that CAR was not involved in PB induction of CYP2B genes. An
alternate explanation of these results is that CAR is saturating in the
nucleus after PB treatment, so that expression of additional CAR has
little effect and may be inhibitory. The present result that exogenous
expression of CAR in PB-treated animals does not increase
transactivation unless GRIP1 is expressed exogenously as well provides
support for this alternate explanation and suggests that the p160
coactivator is limiting relative to CAR in the PB-treated hepatocyte.
The loss of PB induction in transgenic mice with disrupted CAR genes
(8) and other studies showing that exogenous CAR expression increased
transactivation of the PBRU in PB-treated primary cultures of
hepatocytes or hepatocytes transfected in situ by bolistic
particles (7) also support a role for CAR in PB induction. Possible
explanations for the loss of PB induction observed with the conversion
the PBRU NR-1 site to a RARE site (30) are that the conformation of
CAR·RXR may be different when bound to the RARE site (31) or the
alignment of CAR·RXR with other proteins binding to the PBRU may be
changed (32), resulting in a loss of transactivation.
| |
ACKNOWLEDGEMENTS |
|---|
We thank M. R. Stallcup and R. Evans for supplying plasmids and Jun Xia for assistance in collecting and analyzing images of GFP expressing cells.
| |
FOOTNOTES |
|---|
* This work was supported by Grant GM39360 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ Present address: Chilam-Dong 150, Department of Microbiological Engineering, Jinju National University, Jinju-City 660-758, Gyeongsangnam-Do, Korea.
¶ To whom correspondence should be addressed: Dept. of Molecular & Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-333-1146; Fax: 217-333-1133; E-mail: byronkem@life.uiuc.edu.
Published, JBC Papers in Press, May 8, 2002, DOI 10.1074/jbc.M200051200
2 Rivera-Rivera, I., Kim, J., and Kemper, B. (2002) Biochim. Biophys. Acta, in press.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
CAR, constitutive
androstane receptor;
RXR, retinoid X receptor;
PB, phenobarbital;
PBRU, PB-responsive unit;
SRC-1, steroid hormone receptor coactivator-1;
TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene;
GRIP, glucocorticoid receptor interacting protein;
GST, glutathione
S-transferase;
BSA, bovine serum albumin;
NR, nuclear
receptor;
RARE, -retinoic acid-responsive element.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Gonzalez, F. J. (1990) Pharmacol. Ther. 45, 1-38[CrossRef][Medline] [Order article via Infotrieve] |
| 2. |
Xie, W.,
and Evans, R. M.
(2001)
J. Biol. Chem.
276,
37739-37742 |
| 3. |
Honkakoski, P.,
Zelko, I.,
Sueyoshi, T.,
and Negishi, M.
(1998)
Mol. Cell. Biol.
18,
5652-5658 |
| 4. |
Honkakoski, P.,
Moore, R.,
Washburn, K. A.,
and Negishi, M.
(1998)
Mol. Pharmacol.
53,
597-601 |
| 5. |
Sueyoshi, T.,
Kawamoto, T.,
Zelko, I.,
Honkakoski, P.,
and Negishi, M.
(1999)
J. Biol. Chem.
274,
6043-6046 |
| 6. |
Kim, J.,
Min, G.,
and Kemper, B.
(2001)
J. Biol. Chem.
276,
7559-7567 |
| 7. | Muangmoonchai, R., Smirlis, D., Wong, S. C., Edwards, M., Phillips, I. R., and Shephard, E. A. (2001) Biochem. J. 355, 71-78[CrossRef][Medline] [Order article via Infotrieve] |
| 8. | Wei, P., Zhang, J., Egan-Hafley, M., Liang, S., and Moore, D. D. (2000) Nature 407, 920-923[CrossRef][Medline] [Order article via Infotrieve] |
| 9. |
Baes, M.,
Gulick, T.,
Choi, H.-S.,
Grazia, M.,
Martinoli, G.,
Simha, D.,
and Moore, D. D.
(1994)
Mol. Cell. Biol.
14,
1544-1552 |
| 10. | Forman, B. M., Tzameli, I., Choi, H.-S., Chen, J., Simha, D., Seol, W., Evans, R. M., and Moore, D. D. (1998) Nature 395, 612-615[CrossRef][Medline] [Order article via Infotrieve] |
| 11. |
Kawamoto, T.,
Sueyoshi, T.,
Zelko, I.,
Moore, R.,
Washburn, K.,
and Negishi, M.
(1999)
Mol. Cell. Biol.
19,
6318-6322 |
| 12. |
Tzameli, I.,
Pissios, P.,
Schuetz, E. G.,
and Moore, D. D.
(2000)
Mol. Cell. Biol.
20,
2951-2958 |
| 13. |
Moore, L. B.,
Parks, D. J.,
Jones, S. A.,
Bledsoe, R. K.,
Consler, T. G.,
Stimmel, J. B.,
Goodwin, B.,
Liddle, C.,
Blanchard, S. G.,
Willson, T. M.,
Collins, J. L.,
and Kliewer, S. A.
(2000)
J. Biol. Chem.
275,
15122-15127 |
| 14. | Zelko, I., and Negishi, M. (2000) Biochem. Biophys. Res. Commun. 277, 1-6[CrossRef][Medline] [Order article via Infotrieve] |
| 15. |
Honkakoski, P.,
and Negishi, M.
(1997)
J. Biol. Chem.
272,
14943-14949 |
| 16. |
Stoltz, C.,
Vachon, M.-H.,
Trottier, E.,
Dubois, S.,
Paquet, Y.,
and Anderson, A.
(1998)
J. Biol. Chem.
273,
8528-8536 |
| 17. | Liu, S., Park, Y., Rivera-Rivera, I., Li, H., and Kemper, B. (1998) DNA Cell Biol. 17, 461-470[Medline] [Order article via Infotrieve] |
| 18. | Leo, C., and Chen, J. D. (2000) Gene (Amst.) 245, 1-11[CrossRef][Medline] [Order article via Infotrieve] |
| 19. |
Dong, Y.,
McGurie, J.,
Okret, S.,
Poellinger, L.,
Makino, I.,
and Gustafsson, J.
(1993)
J. Biol. Chem.
268,
1854-1859 |
| 20. | Estabrook, R. W., Hilderbrandt, A. G., Baron, J., Netter, K. J., and Leibman, K. (1971) Biochem. Biophys. Res. Commun. 42, 132-139[CrossRef][Medline] [Order article via Infotrieve] |
| 21. | Heery, D., Kalkhoven, E., Hoare, S., and Parker, M. (1997) Nature 387, 733-737[CrossRef][Medline] [Order article via Infotrieve] |
| 22. |
Leers, J.,
Treuter, E.,
and Gustafsson, J. A.
(1998)
Mol. Cell. Biol.
18,
6001-6013 |
| 23. | Hagstrom, J. E., Machnik, K. J., Noble, M. A., Loomis, A. G., and Rozema, D. B. (1999) Panvera Postings 5, 2-3 |
| 24. |
Zelko, I.,
Sueyoshi, T.,
Kawamoto, T.,
Moore, R.,
and Negishi, M.
(2001)
Mol. Cell. Biol.
21,
2838-2846 |
| 25. |
Gervois, P., Vu-,
Dac, N.,
Kleemann, R.,
Kockx, M.,
Dubois, G.,
Laine, B.,
Kosykh, V.,
Fruchart, J. C.,
Kooistra, T.,
and Staels, B.
(2001)
J. Biol. Chem.
276,
33471-33477 |
| 26. | Hong, H., Kohli, K., Garbedian, M. J., and Stallcup, M. R. (1997) Mol. Cell. Biol. 17, 2735-2744[Abstract] |
| 27. | Voegel, J. J., Heine, M. J., Zechel, C., Chambon, P., and Gronemeyer, H. (1996) EMBO J. 15, 3667-3675[Medline] [Order article via Infotrieve] |
| 28. | Puustinen, R., Sarvilinna, N., Manninen, T., Tuohimaa, P., and Ylikomi, T. (2001) Eur. J. Endocrinol. 145, 323-333[Abstract] |
| 29. |
Kim, H.-J.,
Lee, S.-K., Na, S.-Y.,
Choi, H.-S.,
and Lee, J. W.
(1998)
Mol. Endocrinol.
12,
1038-1047 |
| 30. |
Paquet, Y.,
Trottier, E.,
Beaudet, M.-J.,
and Anderson, A.
(2000)
J. Biol. Chem.
275,
38427-38436 |
| 31. |
Wood, J. R.,
Likhite, V. S.,
Loven, M. A.,
and Nardulli, A. M.
(2001)
Mol. Endocrinol.
15,
1114-1126 |
| 32. | Kim, T. K., and Maniatis, T. (1997) Mol. Cell. 1, 119-1129[CrossRef][Medline] [Order article via Infotrieve] |
| 33. |
Lopez, G. N.,
Turck, C. W.,
Schaufele, F.,
Stallcup, M. R.,
and Kushner, P. J.
(2001)
J. Biol. Chem.
276,
22177-22182 |
| 34. |
Wu, X., Li, H.,
Park, E. J.,
and Chen, J. D.
(2001)
J. Biol. Chem.
276,
24177-24185 |
| 35. | Freeman, B. C., and Yamamoto, K. R. (2001) Trends Biochem. Sci. 26, 285-290[CrossRef][Medline] [Order article via Infotrieve] |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Yamamoto and M. Negishi The Antiapoptotic Factor Growth Arrest and DNA-Damage-Inducible 45 {beta} Regulates the Nuclear Receptor Constitutive Active/Androstane Receptor-Mediated Transcription Drug Metab. Dispos., July 1, 2008; 36(7): 1189 - 1193. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Pascual, M. J. Gomez-Lechon, J. V. Castell, and R. Jover ATF5 Is a Highly Abundant Liver-Enriched Transcription Factor that Cooperates with Constitutive Androstane Receptor in the Transactivation of CYP2B6: Implications in Hepatic Stress Responses Drug Metab. Dispos., June 1, 2008; 36(6): 1063 - 1072. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Guo, J. Sarkar, K. Suino-Powell, Y. Xu, K. Matsumoto, Y. Jia, S. Yu, S. Khare, K. Haldar, M. S. Rao, et al. Induction of Nuclear Translocation of Constitutive Androstane Receptor by Peroxisome Proliferator-activated Receptor {alpha} Synthetic Ligands in Mouse Liver J. Biol. Chem., December 14, 2007; 282(50): 36766 - 36776. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Ponugoti, S. Fang, and J. K. Kemper Functional Interaction of Hepatic Nuclear Factor-4 and Peroxisome Proliferator-Activated Receptor-{gamma} Coactivator 1{alpha} in CYP7A1 Regulation Is Inhibited by a Key Lipogenic Activator, Sterol Regulatory Element-Binding Protein-1c Mol. Endocrinol., November 1, 2007; 21(11): 2698 - 2712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xia and B. Kemper Subcellular Trafficking Signals of Constitutive Androstane Receptor: Evidence for a Nuclear Export Signal in the DNA-Binding Domain Drug Metab. Dispos., September 1, 2007; 35(9): 1489 - 1494. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Stoner, S. S. Auerbach, S. M. Zamule, S. C. Strom, and C. J. Omiecinski Transactivation of a DR-1 PPRE by a human constitutive androstane receptor variant expressed from internal protein translation start sites Nucleic Acids Res., April 1, 2007; 35(7): 2177 - 2190. [Abstract] [Full Text] [PDF] |
