Fibrates Increase Human REV-ERB
Expression in Liver via a Novel Peroxisome Proliferator-Activated Receptor Response Element
Philippe Gervois1,
Sandrine Chopin-Delannoy1,
Abdessamad Fadel,
Guillaume Dubois,
Vladimir Kosykh,
Jean-Charles Fruchart,
Jamila Najïb,
Vincent Laudet and
Bart Staels
U.325 INSERM Département dAthérosclérose
(P.G., A.F., G.D., J.-C.F., J.N., B.S.) Institut Pasteur de Lille
and The Faculté de Pharmacie Université de Lille
II 59019 Lille, France
Endocrinos Group (S.C.-D.)
CNRS UMR 319 Institut de Biologie de Lille 59019 Lille,
France
Cardiology Research Complex (V.K.) 721552 Moscow,
Russia
E.N.S. (V.L.) 69364 Lyon cedex 07,
France
 |
ABSTRACT
|
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Fibrates are widely used hypolipidemic drugs that
act by modulating the expression of genes involved in lipid and
lipoprotein metabolism. Whereas the activation of gene transcription by
fibrates occurs via the nuclear receptor peroxisome
proliferator-activated receptor-
(PPAR
) interacting with response
elements consisting of a direct repeat of the AGGTCA motif spaced by
one nucleotide (DR1), the mechanisms of negative gene regulation by
fibrates and PPAR
are largely unknown. In the present study, we
demonstrate that fibrates induce the expression of the nuclear receptor
Rev-erb
, a negative regulator of gene transcription. Fibrates
increase Rev-erb
mRNA levels both in primary human hepatocytes and
in HepG2 hepatoblastoma cells. In HepG2 cells, fibrates furthermore
induce Rev-erb
protein synthesis rates. Transfection studies with
reporter constructs driven by the human Rev-erb
promoter revealed
that fibrates induce Rev-erb
expression at the transcriptional level
via PPAR
. Site-directed mutagenesis experiments identified a PPAR
response element that coincides with the previously identified
Rev-erb
negative autoregulatory Rev-DR2 element. Electromobility
shift assay experiments indicated that PPAR
binds as
heterodimer with 9-cis-retinoic acid receptor to a subset
of DR2 elements 5' flanked by an A/T-rich sequence such as in the
Rev-DR2. PPAR
and Rev-erb
bind with similar affinities to the
Rev-DR2 site. In conclusion, these data demonstrate human Rev-erb
as
a PPAR
target gene and identify a subset of DR2 sites as novel
PPAR
response elements. Finally, the PPAR
and Rev-erb
signaling pathways cross-talk through competition for binding to those
response elements.
 |
INTRODUCTION
|
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Fibrates are hypolipidemic drugs that lower plasma cholesterol and
triglycerides (1). Fibrates exert their effects primarily via the liver
by regulating the expression of several genes implicated in lipid
metabolism. On the one hand, fibrates stimulate the expression of the
human apo A-I (2), rat lipoprotein lipase (3), rat acyl-CoA synthetase
(4), rat acyl-CoA oxidase (5), rat multifunctional enzyme (6), and
human muscle-type carnitine palmitoyltransferase I (7) genes in the
liver. On the other hand, fibrates repress the expression of the rat
apo A-I (8), rat apo A-IV (9), human, rat, and mouse apo C-III
(10, 11, 12, 13), rat hepatic lipase (14), and rat lecithin-cholesterol acyl
transferase (15) genes in the liver. Fibrates have been shown to
activate specific receptors, termed peroxisome proliferator-activated
receptors (PPARs), belonging to the nuclear receptor gene superfamily
(16, 17, 18). So far, three different PPAR forms,
, ß(
), and
,
have been identified, of which the PPAR
form mediates the effects of
fibrates on liver gene expression (13, 19). After activation, PPARs
heterodimerize with the 9-cis-retinoic acid receptor (RXR)
and subsequently bind to DNA on specific response elements termed
peroxisome proliferator response elements (PPRE), located in regulatory
regions of target genes, thereby modulating their transcriptional
activity. All PPREs identified so far consist of the juxtaposition of
two derivatives of the canonical hexamer sequence PuGGTCA spaced by one
nucleotide and commonly called direct repeat 1 (DR1).
Whereas PPAR
mediates fibrate action on lipoprotein metabolism
through PPREs identified in the regulatory sequences of positively
regulated genes, the mechanisms of negative gene regulation by fibrates
are unclear. Studies using PPAR
knockout mice demonstrated that
PPAR
is a mediator of the negative regulation by fibrates, at least
with respect to the mouse apo A-I and apo C-III genes (13). Fibrates
may repress transcription by interfering negatively with the expression
and activity of positive transcription factors, such as hepatocyte
nuclear factor-4 (HNF-4) (11, 20). However, not all
fibrate-regulated genes are under transcriptional control by HNF-4. For
instance, although fibrates repress rat apo A-I gene transcription,
HNF-4 is not considered to be a major regulator of apo A-I gene
transcription (21, 22). Alternatively, fibrates may actively repress
transcription by activating a negative transcription factor.
Interestingly, we recently identified in the rat apo A-I gene promoter
a response element for the nuclear receptor Rev-erb
, an orphan
receptor of the nuclear receptor family that acts as a negative
transcription factor (23). Furthermore, we have shown that Rev-erb
gene expression is induced by fibrates in rat liver, indicating that
Rev-erb
may be a mediator of negative gene transcription by
fibrates.
The goal of the present study was to determine whether fibrates also
regulate human Rev-erb
expression and to investigate the molecular
mechanisms involved. Our results demonstrate that fibrates increase
Rev-erb
expression in human hepatocytes and in HepG2 cells.
Furthermore, we show that the induction of Rev-erb
gene expression
occurs at the transcriptional level in hepatocytes and is mediated by
PPAR
. Finally, we demonstrate that PPAR
binds to a DR2 site
coinciding with the Rev-DR2 site in the human Rev-erb
promoter (24),
which constitutes a novel PPAR
response element mediating a
cross-talk between the PPAR
and Rev-erb
pathways.
 |
RESULTS
|
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Fibrates Increase Rev-erb
mRNA Expression and Protein Synthesis
in Human Liver Cells
The regulation of Rev-erb
by fibrates was analyzed in
human primary hepatocytes. Treatment of cells with fenofibric acid or
Wy 14,643 induced a pronounced increase of Rev-erb
mRNA levels,
whereas control 36B4 mRNA levels did not change (Fig. 1A
). In addition, in HepG2 cells
treatment with Wy 14,643 increased Rev-erb
mRNA levels in a
dose-dependent fashion (Fig. 1B
). To analyze whether the induction of
Rev-erb
mRNA by fibrates is associated with increased synthesis of
Rev-erb
protein, HepG2 cells were cultured for 24 h in the
presence of Wy 14,643 or vehicle, labeled with
35S-methionine, and Rev-erb
was subsequently
immunoprecipitated. Compared with control, treatment with Wy 14,643
resulted in a significant increase in Rev-erb
protein synthesis
(Fig. 1C
). By contrast, as a control, apolipoprotein E secretion
was not influenced by fibrate treatment (data not shown). These
experiments demonstrate that fibrates increase Rev-erb
mRNA levels
as well as protein synthesis in human hepatocytes.

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Figure 1. Fibrates Increase Rev-erb mRNA Expression and
Protein Synthesis in Primary Human Hepatocytes and HepG2 Cells
Total RNA (10 µg) was subjected to Northern blot analysis using
hRev-erb (top panel) or 36B4 (bottom
panel) cDNA probes as described in Materials and
Methods. A, Human hepatocytes were isolated and treated for
24 h with 100 µM fenofibric acid, 50
µM Wy 14,643, or vehicle (DMSO). B, HepG2 cells were
treated for 24 h with increasing concentrations of Wy 14,643 (0,
10, 50, 100, 150, 200, 250, 500 µM). C, HepG2 cells were
treated for 24 h with 500 µM Wy 14,643 or DMSO.
Left, RNA analysis from the same plates used for
immunoprecipitation experiments. Right, Cell lysates
were subjected to immunoprecipitation using serum depleted of
anti-Rev-erb antibody (A83 ads) or polyclonal anti-Rev-erb
antibody (A83 spe) as described in Materials and
Methods.
|
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Fibrate Induction of Rev-erb
Gene Expression Occurs at the
Transcriptional Level via PPAR
Interacting with the Rev-DR2 Site of
the Human Rev-erb
Promoter
To investigate whether the effect of fibrates on Rev-erb
expression occurred at the transcriptional level, the 1.7 kb containing
Rev-erb
promoter was transiently transfected in HepG2 cells in the
presence of a human PPAR
expression vector (pSG5hPPAR
) or empty
vector (pSG5) (Fig. 2A
). Rev-erb
promoter-driven luciferase activity increased significantly after
cotransfection with PPAR
, an effect that was increased in the
presence of fenofibric acid (Fig. 2A
), indicating that Rev-erb
gene
transcription is increased by PPAR
. Two putative nuclear
receptor-binding sites containing AGGTCA-like motifs were
previously identified in the human Rev-erb
promoter and called
distal (Rd) and proximal sites (Rp) (24). To delineate whether one of
these putative binding sites mediated PPAR
transactivation,
unilateral deletion and site- directed mutagenesis experiments were
performed. Hence, a 0.7-kb 5'-deletion of the Rev-erb
promoter
containing only the Rp site was transfected in the presence or absence
of human PPAR
(Fig. 2A
). This deleted Rev-erb
promoter construct
was induced by PPAR
. Since both the 1.7-kb and 0.7-kb Rev-erb
promoter constructs responded to the same extent to PPAR
, we
hypothesized the existence of a PPRE located near the Rp site of the
human Rev-erb
promoter (24). Thus, to determine the role of this
site in the transcriptional regulation of Rev-erb
by PPAR
, we
explored the influence of PPAR
on various mutations around this
region (Fig. 2A
). Mutations affecting either the 5'-AGGTCA motif
(pGL2hRev-erb
) or the A/T-rich region (pGL2hRev-erb
CCC) of
the Rp site resulted not only in a loss of Rev-erb
promoter
inductibility by PPAR
, but also in an increase in baseline reporter
activity (Fig. 2A
). These results indicate that the PPAR
response
element colocalizes with the proximal Rev-erb
binding site, referred
to as Rev-DR2 (24).
To ascertain that the Rev-DR2 site could function as a
PPAR-responsive element, we performed transient transfection
experiments using wild-type and mutated versions of the Rev-DR2 site
cloned in front of the heterologous SV40 promoter (Rev-DR2 SV40,
M5'Rev-DR2 SV40, and M3'Rev-DR2 SV40) (Fig. 2B
). Upon cotransfection
with pSG5hPPAR
in HepG2 cells, it was evident that the Rev-DR2 could
transmit PPAR
responsiveness to the heterologous SV40 promoter, an
effect that was enhanced in the presence of fenofibric acid. By
contrast, PPAR
did not activate the SV40 promoter. Furthermore,
PPAR
did not induce the activity of the SV40 promoter driven by the
Rev-DR2 site mutated in its 5'-half-site (Fig. 2B
), confirming the
importance of this motif in the structure of the PPAR
-responsive
element. Interestingly, mutation of the 3'-half-site of the DR2 also
abolished transactivation by PPAR
(Fig. 2B
), indicating that the
3'-AGGTCA half-site is also implicated in mediating induction of human
Rev-erb
gene transcription by PPAR
. Taken together, these data
strongly argue that the human Rev-erb
promoter contains a bona fide
PPAR-responsive element that coincides with the Rev-DR2 site, which is
constituted of two AGGTCA motifs separated by two nucleotides (DR2) and
5'-flanked by an A/T-rich region (Rev-DR2).
PPAR Binds as a Heterodimer with RXR to a DR2 Site Containing an
A/T-Rich 5'-Flanking Region, but Not to a Standard DR2 Site
To investigate direct interaction of PPAR
with the
Rev-DR2 site, we performed electromobility shift assays (EMSAs) using
in vitro synthesized PPAR
and RXR
protein. RXR
or
PPAR
alone did not bind to the Rev-DR2 site oligonucleotides (Fig. 3B
, lanes 1012 and 14 and lanes 1517
and 19). Furthermore, PPAR
did not bind to an oligonucleotide
containing a monomer binding site for Rev-erb
(G8A) (Fig. 3B
, lane
18) (24), indicating that PPAR
cannot bind as a monomer. By
contrast, binding was observed when PPAR
was incubated in the
presence of RXR with the Rev-erb
promoter Rev-DR2 as well as the
consensus Rev-DR2 sites (direct repetition of the AGGTCA motif
separated by two nucleotides), which both contain an A/T-rich region at
their 5'-extremities (Fig. 3B
, lanes 5 and 9). This binding was
specific since it was competed out by excess of unlabeled
oligonucleotide (Fig. 3
, panel C, lanes 48, and panel D, lanes
48).
Next, we characterized the structural requirements for PPAR/RXR binding
by performing competition EMSAs on the promoter (Fig. 3C
) as well as on
the consensus (Fig. 3D
) oligonucleotides. PPAR/RXR binding to wild-type
promoter Rev-DR2 (Fig. 3C
, lane 4) or to consensus Rev-DR2 (Fig. 3D
, lane 4) oligonucleotides could not be competed either by the promoter
Rev-DR2 site carrying a mutation in the 5'-half-site (M5') (24) or in
the 3'-half-site (M3') (Fig. 3
, C and D, lanes 9, 11, and 12).
Interestingly, at high excess (100-fold) of competitor, the
3'-half-site-mutated oligonucleotide started competing for PPAR/RXR
binding, indicating that the 3'-part of the Rev-DR2 site is of lesser
importance (Fig. 3
, C and D, lane 10). Similar results were obtained
with an oligonucleotide containing the promoter Rev-DR2 site completely
lacking the second half-site (1/2A) (Fig. 3
, C and D, lanes 13 and 14),
whereas an oligonucleotide lacking the promoter Rev-DR2 A/T-rich
sequence and 5'-half-site (1/2B) did not compete at all (Fig. 3
, C and
D, lanes 15 and 16). These results indicate that 5'- and 3'-half-sites
are both implicated in PPAR/RXR binding, with the 5'-half-site, to
which PPAR
presumably binds (25), being most important.
To investigate the role of the 5'-A/T-rich flanking sequence in
PPAR/RXR binding to a DR2 site, competition experiments were performed
with oligonucleotides in which the 5'-flanking sequence was substituted
by C nucleotides (911C and 79C; Fig. 3A
). Neither 911C nor 79C
oligonucleotides competed for PPAR/RXR binding to the consensus Rev-DR2
sequence (Fig. 3E
), indicating absolute requirement of the 5'-A/T-rich
flanking sequence for PPAR/RXR binding to a DR2 site.
To ensure that Rev-DR2 sites are high-affinity response elements for
PPAR
, we compared the relative affinities of PPAR/RXR binding to
either DR1 or Rev-DR2 sites by competition EMSA (Fig. 4
). Using oligonucleotides labeled to
similar specific activities and under identical experimental
conditions, a higher intensity shift with PPAR/RXR was obtained on the
Rev-DR2 oligonucleotide compared with the naturally occurring DR1 PPRE
site of the human apo A-II promoter, which has been shown to drive its
regulation by fibrates (26) (Fig. 4
, lanes 1 and 10). When increasing
amounts of unlabeled oligonucleotide were added, cold Rev-DR2
oligonucleotide competed more efficiently than cold DR1 oligonucleotide
for binding of PPAR/RXR to the Rev-DR2 site (Fig. 4
, lanes 25 and
69). Reciprocally, binding of PPAR/RXR to labeled DR1 oligonucleotide
was more rapidly competed by cold Rev-DR2 than by cold DR1
oligonucleotide (Fig. 4
, lanes 1114 and 1518).
These binding experiments demonstrate that PPAR
binds as a
heterodimer with RXR, but not as monomer, to DR2 sites containing a
Rev-erb
-type 5'-flanking region and that Rev-DR2 constitutes a novel
PPAR
-binding site of higher affinity than the natural apo A-II DR1
PPRE site.
Rev-DR2 Mediates a Cross-Talk between PPAR
and Rev-erb
To test directly whether PPAR
and Rev-erb
could functionally
compete on a Rev-DR2 element, transient cotransfection experiments were
performed. As expected, Rev-erb
was able to repress Rev-DR2-driven
SV40 promoter activity (24) (Fig. 5A
).
Cotransfection of PPAR
in increasing proportions against a constant
amount of Rev-erb
led to a progressive abolishment of
Rev-erb
-mediated repression resulting in a transcriptional
activation of the reporter gene at a 3:2 ratio of PPAR
to
Rev-erb
, respectively, as evidenced by Western blot analysis of
transfected cell extracts (Fig. 5A
and inset). Furthermore,
in the absence of cotranfected Rev-erb
, reporter
transcription activity was even further enhanced by PPAR
(Fig. 5A
).
Thus, PPAR
and Rev-erb
are able to functionally cross-compete for
the same Rev-DR2 element.
Finally, to estimate the relative affinities of PPAR/RXR and Rev-erb
binding to a Rev-DR2 site, EMSAs were performed using Rev-DR2 as probe
(Fig. 5B
). As expected, PPAR/RXR formed a heterodimeric complex whereas
Rev-erb
bound both as monomer and as heterodimer (Fig. 5B
, lanes 1
and 6). When competition was performed using cold Rev-DR2
oligonucleotide, PPAR/RXR binding decreased in a manner similar to
Rev-erb
monomer (Fig. 5B
, compare lanes 25 and 710). However,
Rev-erb
homodimer binding appeared slightly more sensitive to
competition with unlabeled Rev-DR2 (Fig. 5B
, lanes 710). Altogether,
these results indicate that PPAR/RXR binds to Rev-DR2 sites with
similar affinity as Rev-erb
monomer, whereas Rev-erb
homodimers appear to bind with higher affinity.
 |
DISCUSSION
|
---|
In the present report we studied the regulation of Rev-erb
by
fibrates in human liver cells and the molecular mechanisms involved.
Our results on human primary hepatocytes and HepG2 cells demonstrate
that fibrates induce Rev-erb
mRNA expression, an effect that is
associated with induction of Rev-erb
protein synthesis in HepG2
cells. In addition, transfection studies revealed that the regulation
of Rev-erb
expression by fibrates occurs at the transcriptional
level via PPAR
. Using deleted and mutated Rev-erb
promoter
constructs, we localized the fibrate-responsive region in the human
Reverb
promoter to the previously identified negative Rev-erb
autoregulation site. Moreover, mutations in the Rev-DR2 site of the
Rev-erb
promoter abolished basal Rev-erb
-mediated repression as
well as PPAR
-mediated activation. EMSA experiments proved that
fibrate signaling occurs through direct interaction of PPAR/RXR
heterodimers to the Rev-DR2 site of the human Rev-erb
promoter.
Since all PPREs described so far consist of the juxtaposition
of the degenerated hexamer AGGTCA sequence separated by one
nucleotide (DR1) (4, 6, 11, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38), these results represent the first
demonstration of a DR2 site as a PPAR-responsive element.
Interestingly, a specific structure of the DR2 is required for
high-affinity PPAR/RXR binding. In addition to the 5'- and
3'-AGGTCA half-sites, the 5'-flanking region is required for binding of
PPAR/RXR to a DR2 site. Thus, several fundamental characteristics of
protein-DNA interaction, such as the contact of the receptor with the
5'-A/T-rich flanking sequences of the response element, are conserved
among a number of the superfamily members. Taken together, our data
suggest that nuclear receptors are more flexible for recognition of
responsive elements than previously anticipated.
Rev-erb
belongs to a subfamily of orphan receptors that are
repressors of target gene transcription (for review see Ref. 39).
Rev-erb
appears to be ubiquitously expressed (40, 41), but its
functions are ill defined. Several observations suggest a role for
Rev-erb
in metabolic control and energy homeostasis. First,
Rev-erb
mRNA levels increase during differentiation of preadipocytes
into adipocytes (42). Second, Rev-erb
has been suggested to act as a
modulator of thyroid hormone signaling (40, 41, 43, 44). Indeed,
Rev-erb
has been shown to bind a subset of thyroid hormone-response
elements (45). Interestingly, a significant level of cross-talk exists
also between peroxisome proliferator and thyroid hormone-signaling
pathways (46, 47, 48, 49, 50, 51, 52). Our present data identify Rev-erb
as a fibrate
target gene and reveal the existence of cross-talk between the PPAR
and Rev-erb
-signaling pathways. This cross-talk is governed via two
mechanisms (see Fig. 6
for
overview). First, PPAR
induces Rev-erb
expression by interfering
with the negative autoregulatory loop of Rev-erb
expression via the
Rev-DR2 site. Therefore, genes regulated by Rev-erb
, such as
N-myc (53) and rat apo A-I (23), will be negatively
regulated by PPAR
via an indirect mechanism. Second, PPAR
and
Rev-erb
may compete for binding to similar DR2 sites. Rev-erb
itself is an example of a gene containing a response element recognized
by both PPAR
and Rev-erb
. Hence, target genes containing Rev-DR2
sequences to which PPAR
, as heterodimer with RXR, and Rev-erb
compete for binding will be derepressed by fibrates. By contrast, genes
carrying monomeric Rev-RE, to which Rev-erb
binds exclusively as
monomer, will be further repressed after fibrate treatment. Therefore,
whether a gene will be predominantly regulated by PPAR
or Rev-erb
will depend on the relative levels of ligands for each receptor, the
relative concentrations of each receptor, and on the structure of the
target gene DR2 sequence that determines the relative binding
affinities of PPAR
and Rev-erb
. The fact that PPAR
and
Rev-erb
bind to similar DR2 subset sites is most likely due to the
similarity of their T/A boxes (25), which are involved in the
recognition of the 5'-half-site extension (24, 45, 54, 55, 56). Therefore,
PPAR
could repress genes by inducing Rev-erb
while simultaneously
activating its own target genes via either DR1 or DR2 sites.
In a previous study, we demonstrated that differences between human and
rat apo A-I gene regulation in response to fibrates are due to a
combination of two distinct mechanisms implicating the nuclear
receptors PPAR
and Rev-erb
(23). Our data indicated that the
species-distinct regulation of apo A-I gene expression by fibrates is
due to sequence differences in cis-acting elements. In man,
apo A-I transcription is induced via PPAR
binding to a positive PPRE
located in the A site footprint (2). This site is not conserved in
rats, resulting in a lack of binding of PPAR to the rat apo A-I A
promoter site. By contrast, rat apo A-I gene transcription is repressed
by Rev-erb
, the expression of which is induced by fibrates and which
binds to a Rev-RE site adjacent to the TATA-box in the rat, but not in
the human apo A-I gene promoter. The identification of rat apo A-I as a
target gene for Rev-erb
suggests an implication of Rev-erb
in
lipoprotein metabolism (23). Although the rat apo A-I Rev-RE is not
conserved in man, Rev-erb
expression is controlled by fibrates both
in rats and in man, which may point to a role for this nuclear receptor
as a modulator in lipid and lipoprotein metabolism and possibly in
atherosclerosis susceptibility in both species. It will be of interest,
therefore, to identify target genes involved in lipid metabolism that
are also under control of Rev-erb
in man.
In conclusion, our data indicate that the human Rev-erb
gene is
regulated at the transcriptional level by fibrates in liver.
Furthermore, this regulation is mediated by PPAR
, which binds to a
novel response element consisting of a 5'-A/T-rich preceded DR2
sequence. Finally, we provide evidence that PPAR
and Rev-erb
bind
to the same regulatory site, indicating the existence of a cross-talk
between PPAR
and Rev-erb
-signaling pathways.
 |
MATERIALS AND METHODS
|
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Cell Culture
Human hepatocytes, isolated by collagenase perfusion, and HepG2
cells were cultured exactly as described previously (12).
RNA Analysis
RNA extraction and Northern blot analysis were performed as
described (3) using human Rev-erb
(43) and human acidic ribosomal
phosphoprotein 36B4 (57) cDNA probes.
Construction of Recombinant Plasmids and Transfection
Cloning of the human Rev-erb
promoter fragments into pGL2
promoterless or SV40pGL2 reporter vectors (Promega, Madison, WI) and
site-directed mutagenesis of Rev-erb
response elements were as
described (24). Human hepatoma HepG2 cells were obtained from European
Collection of Animal Cell Culture (Porton Down, Salisbury, UK). Cells
were grown in DMEM, supplemented with 2 mM glutamine and
10% (vol/vol) FCS, in a 5% CO2 humidified atmosphere at
37 C. Stimuli were dissolved in dimethylsulfoxide (DMSO). Control cells
received vehicle only. All transfections were performed with a mixture
of plasmids containing reporter (2 µg) and expression vectors (0.3 to
1 µg). The luciferase activity in cell extracts was determined using
a luciferase assay system (Promega) following the suppliers
instruction. Transfection experiments were performed in
triplicate and repeated at least three times.
In Vitro Translation and EMSAs
pSG5hPPAR
, pSG5mRXR
, and pSG5hRev-erb
were in
vitro transcribed with T7 polymerase and translated using the
rabbit reticulocyte lysate sytem (Promega). EMSAs with Rev-erb
,
PPAR
, and/or RXR
were performed exactly as described previously
(2, 58). For competition experiments, increasing amounts of indicated
cold probe were added just before the labeled oligonucleotide. The
complexes were resolved on 5% polyacrylamide gels in 0.25x TBE buffer
(90 mM Tris-borate, 2.5 mM EDTA, pH 8.3) at 4
C. Gels were dried and exposed overnight at -70 C to x-ray film
(XOMAT-AR, Eastman Kodak, Rochester, NY).
Coimmunoprecipitation from Cell Extracts
HepG2 cells incubated in DMEM + 0.2% BSA were treated with
fenofibric acid (0.5 mM) or vehicle (DMSO) for 24 h.
Cells were subsequently washed in PBS and incubated in methionine-free
DMEM supplemented with 35S-labeled methionine (0.1 mCi/ml
medium) for 5 h. Cells were lysed in 1 ml RIPA buffer [20
mMTris, pH 7.5, 150 mM sodium chloride, 2
mM EDTA, 1% (wt/vol) sodium deoxycholate, 1% (vol/vol)
Triton X-100, 0.25% (wt/vol) SDS]. Lysates were centrifuged at
100,000 x g for 30 min, and the supernatant was
subsequently incubated with polyclonal anti-Rev-erb
antibody (S.
Chopin-Delannoy and V. Laudet, manuscript in preparation)
overnight at 4 C in RIPA buffer. Immune complexes were collected using
protein A-Sepharose (Pharmacia, Piscataway, NJ) and washed six times in
RIPA buffer. Protein complexes were separated on 10%
SDS-polyacrylamide gels under reducing conditions. Gels were dried and
exposed at -70 C to BIOMAX-MS film (Kodak).
 |
ACKNOWLEDGMENTS
|
---|
We thank Olivier Chassande, Jean-Marc Vanacker, and Franck
Delaunay for critical reading.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Bart Staels, U.325 INSERM, Département dAthérosclérose, Institut Pasteur, 1 Rue Calmette, 59019 Lille, France. E-mail
Bart.Staels{at}pasteur-lille.fr
This research was sponsored by grants from INSERM, Fondation pour la
Recherche Médicale, and the Région Nord-Pas de Calais.
1 Both authors have equally contributed to this work. 
Received for publication July 13, 1998.
Revision received November 5, 1998.
Accepted for publication November 23, 1998.
 |
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