A Truncated Human Peroxisome Proliferator-Activated Receptor
Splice Variant with Dominant Negative Activity
Philippe Gervois,
Inés Pineda Torra,
Giulia Chinetti,
Thilo Grötzinger,
Guillaume Dubois,
Jean-Charles Fruchart,
Jamila Fruchart-Najib,
Eran Leitersdorf1 and
Bart Staels
U.325 INSERM Département
dAthérosclérose Institut Pasteur de Lille 59019
Lille, France
Faculté de Pharmacie Université
de Lille II 59006 Lille, France
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ABSTRACT
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The peroxisome proliferator-activated receptor
(PPAR
) plays a key role in lipid and lipoprotein metabolism.
However, important inter- and intraspecies differences exist in the
response to PPAR
activators. This incited us to screen for PPAR
variants with different signaling functions. In the present study,
using a RT-PCR approach a variant human PPAR
mRNA species was
identified, which lacks the entire exon 6 due to alternative splicing.
This deletion leads to the introduction of a premature stop codon,
resulting in the formation of a truncated PPAR
protein
(PPAR
tr) lacking part of the hinge region
and the entire ligand-binding domain. RNase protection analysis
demonstrated that PPAR
tr mRNA is expressed
in several human tissues and cells, representing between 2050% of
total PPAR
mRNA. By contrast, PPAR
tr mRNA
could not be detected in rodent tissues. Western blot analysis using
PPAR
-specific antibodies demonstrated the presence of an
immunoreactive protein migrating at the size of in vitro
produced PPAR
tr protein both in human
hepatoma HepG2 cells and in human hepatocytes. Both in the presence or
absence of 9-cis-retinoic acid receptor,
PPAR
tr did not bind to DNA in gel shift
assays. Immunocytochemical analysis of transfected CV-1 cells indicated
that, whereas transfected PPAR
wt was mainly
nuclear localized, the majority of PPAR
tr
resided in the cytoplasm, with presence in the nucleus depending on
cell culture conditions. Whereas a chimeric
PPAR
tr protein containing a nuclear
localization signal cloned at its N-terminal localized into the nucleus
and exhibited strong negative activity on
PPAR
wt transactivation function,
PPAR
tr interfered with
PPAR
wt transactivation function only under
culture conditions inducing its nuclear localization. Cotransfection of
the coactivator CREB-binding protein relieved the
transcriptional repression of PPAR
wt by
PPAR
tr, suggesting that the dominant
negative effect of PPAR
tr might occur
through competition for essential coactivators. In addition,
PPAR
tr interfered with transcriptional
activity of other nuclear receptors such as PPAR
, hepatic nuclear
factor-4, and glucocorticoid receptor-
, which share
CREB-binding protein/p300 as a coactivator. Thus, we have
identified a human PPAR
splice variant that may negatively interfere
with PPAR
wt function. Factors regulating
either the ratio of PPAR
wt vs.
PPAR
tr mRNA or the nuclear entry of
PPAR
tr protein should therefore lead to
altered signaling via the PPAR
and, possibly also, other nuclear
receptor pathways.
 |
INTRODUCTION
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Peroxisome proliferator (PP)-activated receptors (PPARs) are
ligand-activated transcription factors belonging to the superfamily of
nuclear receptors (1, 2). After activation, PPARs heterodimerize with
the 9-cis-retinoic acid receptor (RXR) as a preferential
partner. The PPAR/RXR complex subsequently binds to DNA on specific
response elements termed peroxisome proliferator response elements
(PPREs), located in regulatory regions of target genes, thereby
modulating their transcriptional activity. PPREs consist of the
juxtaposition of two derivatives of a canonical hexamer sequence
PuGGTCA spaced by one or two nucleotides and commonly called direct
repeat (DR) 1 or 2. Three distinct PPARs,
, ß, and
, have been
described in several species such as Xenopus (3), rat (4),
mouse (5, 6, 7, 8, 9, 10), hamster (11), and human (12, 13, 14, 15, 16, 17), each encoded by
different genes and exhibiting distinct tissue distribution
patterns.
Mouse PPAR
was the first identified PPAR family member, and
its expression is principally detected in tissues exhibiting high rates
of ß-oxidation, i.e. in liver, kidney, heart, and muscle
(5). In rodents, PPAR
mediates a pleiotropic response to a wide
variety of compounds, termed peroxisome proliferators (PPs), leading to
peroxisome proliferation, hepatomegaly, and possibly
hepatocarcinogenesis (see Ref. 18 for review). PPs include various
xenobiotics such as plasticizers, herbicides, some dietary compounds
(fatty acids), pharmacological drugs (e.g. fibrates), and
eicosanoids, some of which have been shown to be PPAR
ligands
(19, 20, 21, 22, 23, 24). The implication of PPAR
in peroxisome proliferation in
mice has been unequivocally demonstrated in PPAR
knockout mice that
become resistant to PPs (25). Interestingly, PPAR
also mediates the
hypolipidemic effect of PPs by regulating the transcription of key
genes in lipoprotein metabolism. For instance, the hypolipidemic
fibrates act by repressing the transcription of the apo A-I and apo
A-II (26), apo A-IV (27), apo C-III (28, 29), hepatic lipase (30), and
LCAT (lecithin-cholesterol acyltransferase) (31) genes in rodents, an
effect that is mediated by PPAR
(32). Moreover, fibrates induce the
transcription of the lipoprotein lipase gene in rat liver (33). PPAR
also plays an important role in intracellular lipid metabolism by
regulating gene expression of peroxisomal ß-oxidation enzymes such as
acyl-coenzyme A (CoA) oxidase (34, 35), multifunctional enzyme (36),
and 3-ketoacyl-CoA thiolase (37) in rodents. Moreover, PPREs have been
identified in the 5'-upstream region of several extraperoxisomal genes
such as microsomal CYP4A6 (38) and 3-hydroxy-3-methylglutaryl-CoA
synthase, a key mitochondrial enzyme in ketogenesis (39). In man also,
PPAR
mediates fibrate action on lipoprotein metabolism since
functional PPREs have been identified in the regulatory sequences of
genes implicated in lipid transport such as apo A-I (40), apo A-II
(41), apo C-III (28), and lipoprotein lipase (42). Thus, PPAR
can be
considered as a major regulator of intra- and extracellular lipid
metabolism.
Although most of the PPAR
functions seem to be conserved,
some important differences in response exist. Although treatment with
fibrates as well as with other PPs leads in rodents to a marked
peroxisome proliferation and hepatomegaly (43, 44), such a response has
never been shown to occur in man (45, 46, 47). Whereas in the majority of
fibrate-treated patients plasma high-density lipoprotein-cholesterol
concentrations increase, a significant fraction of patients respond to
fibrates with no change or even with a decrease in their plasma
high-density lipoprotein-cholesterol levels (48, 49). Such different
responses to fibrates among species and within a species among
individuals may be linked to variations in the PPAR
signaling
pathways, possibly due to the presence of different PPAR
variants.
In this report, we therefore searched for the existence of
different PPAR
variants in human tissues. Using a RT-PCR approach,
we identified, in addition to the full-length transcript, a transcript
lacking exon 6 due to alternative splicing. This splicing leads to the
generation of a premature stop codon, giving rise to a C-terminal
truncated protein (hPPAR
tr), which lacks part of the
hinge region and the entire ligand-binding domain (LBD). We furthermore
demonstrate that the variant transcript is expressed in various cell
lines and tissues of human origin but not in rodents. Furthermore,
results from Western blot experiments indicate both in vitro
and in vivo expression of hPPAR
tr protein.
Moreover, we show that hPPAR
tr has a repressive activity
on the hPPAR
wt transactivation function, only when it
translocates to nucleus. Together, these data indicate that
hPPAR
tr, if produced in sufficient amounts in
vivo, may play a regulatory role on hPPAR
wt
signaling function.
 |
RESULTS
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The hPPAR
Gene Gives Rise to Two Distinct Transcripts
To determine the existence of different hPPAR
variants, RT-PCR experiments were performed using a set of specific
primers covering the entire hPPAR
-coding region (Fig. 1A
). These primers were designed to
hybridize to the extremities of putative exons in the hPPAR
gene deduced from sequence comparison of the mouse, rat, and human
cDNAs and the mouse gene of PPAR
(5, 4, 13, 14, 50). RT-PCR analysis
was first performed on RNA extracted from HepG2 cells. For each primer
pair tested, a product of the expected size was detected (Fig. 1B
).
Surprisingly, using primer pairs 35/83, 45/83, and 15/73, a second,
approximately 200-bp shorter product was obtained in the same PCR
reaction (Fig. 1B
). However, whenever primer combinations including a
putative exon 6 primer were used, only one fragment of a size
corresponding to the hPPAR
wt transcript was obtained.
These observations suggest the existence of two transcripts for
hPPAR
, the shorter one lacking at least one portion of approximately
200 bp around the putative exon 6 region. To determine the identity of
the shorter transcript, the shorter PCR fragment was isolated, cloned,
and sequenced (Fig. 2A
). Sequence
analysis revealed that the hPPAR
variant lacked 203 bp between bp
632834 (numbering based on the nucleotide sequence described in Ref.
14), resulting in a frame shift introducing a premature stop codon 3'
of the deletion (Fig. 2A
). The shorter transcript should result in the
production of a truncated form of hPPAR
containing the first 170
N-terminal amino acids of the hPPAR
wt protein and 4
different C-terminal amino acids (Fig. 2B
), including the
ligand-independent transactivation and DNA-binding domains and part of
the hinge region. To determine whether this deleted transcription
variant was generated by alternative splicing of exon 6, the
intron-exon boundaries of the hPPAR
gene were determined by direct
sequencing of a hPPAR
gene containing BAC clone. Sequence comparison
showed that the deleted fragment localized exactly at the boundaries of
exon 6, indicating that it is generated by an alternative splicing
event skipping exon 6 (Fig. 2C
). Therefore, the hPPAR
gene gives
rise in HepG2 cells to two transcripts of distinct size, which are
generated by alternative splicing.

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Figure 1. The hPPAR Gene Gives Rise to Two Distinct
Transcripts
A, Schematic representation of the hPPAR coding sequence and
position of the primers (arrows) used for RT-PCR
analysis. Numbers below arrows indicate the first
nucleotide of the 5'-termini of primers. Numbers above the
schematic coding sequence represent the number of the first
5'-nucleotide of corresponding primers. B, RT-PCR analysis on RNA
isolated from HepG2 cells. Total RNA was extracted, reverse
transcribed, and amplified using the indicated pairs of primers as
described in Materials and Methods. Control corresponds
to PCR reaction without RT product.
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The Variant hPPAR
Transcript Is Widely Expressed in Human Cell
Lines and Tissues, but Not in Rodents
RT-PCR analysis was performed next to test whether the
alternative splicing underlying formation of the hPPAR
variant
transcript occurs in different human cell lines and tissues. RT-PCR
analysis was therefore performed using the 35 sense primer and
either the 73 or 83 antisense primers that cover the alternatively
spliced region. Two PCR products of the expected size for wt and
variant transcripts were detected in all cells analyzed, indicating a
wide distribution of both messages in different human transformed cell
lines (Fig. 3A
). Furthermore, both wt and
variant transcripts were detected in human tissues such as liver and
adipose tissue (Fig. 3B
). Therefore, both hPPAR
transcripts appear
to be widely distributed in different human tissues and cells.
We next tested whether the variant transcript is also
expressed in liver and in adipose tissue of two rodent species, namely
rats and mice. RT-PCR was carried out on RNA isolated from these
tissues using the conserved primer pair 35/73 (5, 4, 13, 14) and
resulted in detection of only one fragment of predicted size of 922 bp
for the PPAR
wt transcript (Fig. 3C
). Thus, the PPAR
variant transcript appears to be specifically expressed in man but not
in rodents.
To quantify the relative level of variant and wt hPPAR
mRNA, a RNase protection assay was developed that allows simultaneous
quantification of both mRNA species. Using a riboprobe that includes
exon 6 and part of exon 7 of hPPAR
, protected fragments
corresponding to wt and variant hPPAR
mRNA were detected in human
liver and adipose tissue (Fig. 4
). When
liver samples from different subjects were compared, absolute mRNA
levels of hPPAR
wt and hPPAR
tr varied from
2.42 to 6.97 relative absorbance units (R.A.U.) and from 1.57 to 2.67
R.A.U., respectively. In adipose tissue, the hPPAR
wt
transcript level ranged from 1.78 to 2.00 R.A.U. whereas the
hPPAR
tr varied from 1.18 to 1.60 R.A.U. Moreover, in
liver the wt-variant mRNA ratio also varied among individuals from 1:1
to 4:1, whereas it was almost constant and near 1:1 in each adipose
tissue sample analyzed. Interestingly, whereas hPPAR
wt
levels in liver varied substantially between individuals,
hPPAR
tr levels appeared to be more constant and similar
in liver and in adipose tissue. These results indicate that both
transcripts are widely expressed in human cell lines and tissues and
that the wt-variant PPAR
mRNA ratio seems to vary substantially
among individuals in human liver. On the contrary, expression level of
each PPAR
transcript appears fairly constant among individuals in
adipose tissue.
hPPAR
tr Protein Can Be Produced in
Vitro and in Vivo
To determine whether the alternatively spliced hPPAR
mRNA
could give rise to the production of a protein, in vitro and
in vivo translation experiments were performed next. To
detect effective synthesis of both hPPAR
wt and
hPPAR
tr protein, we performed Western blot analysis. An
anti-hPPAR
antibody was generated using as antigen a peptide that
covers a part of the A/B domain with 95% identity between mouse and
human PPAR
. This polyclonal anti-PPAR
antibody recognizes
specifically the PPAR
subtype (51). A specific signal was observed
for both PPAR
forms from in vitro translated
plasmids (Fig. 5A
). Cos-1 cells
transfected with either pSG5hPPAR
wt or
pSG5hPPAR
tr produced proteins of the expected size,
respectively (Fig. 5A
). Although the size of the detected signal in
SDS-PAGE was larger than theoretically calculated, the PPARs
synthesized either with the in vitro translation rabbit
reticulocyte system or in vivo in Cos-1 cells were of
similar size. Furthermore, when whole-cell protein extracts from human
hepatoma HepG2 cells and from human primary hepatocytes were analyzed
for PPAR
protein expression, specific bands were detected, the size
of which was comparable to those using in vitro
translated hPPAR
wt and hPPAR
tr protein
(Fig. 5
, B and D). To ensure that the shorter protein detected in HepG2
cells and in human primary hepatocytes corresponds to
hPPAR
tr, the membranes were reprobed using an antibody
raised against the C-terminal extremity of hPPAR
, which is lacking
in hPPAR
tr. As expected, a specific band was observed
for hPPAR
wt, whereas no signal was detected for
hPPAR
tr (Fig. 5
, C and E), strongly suggesting that
hPPAR
tr protein may be produced in both HepG2 cells and
human primary hepatocytes. These results indicate that the
hPPAR
tr transcript may lead to the in vivo
expression of a corresponding protein containing the ligand-independent
transactivation and the DNA-binding domains, but lacking the
ligand-dependent transactivation domain.
The hPPAR
tr Isoform Does Not Bind to a
PPRE
Since hPPAR
tr still contains the DNA-binding
domain, we next sought to determine whether it could bind a
PPRE-containing DNA fragment. Therefore, gel retardation assays were
performed using in vitro translated hPPAR
wt
and hPPAR
tr and the apo A-II PPRE-containing
oligonucleotide as probe (41). mRXR
, hPPAR
wt, and
hPPAR
tr alone did not bind to the labeled apoA-II PPRE
site (Fig. 6
). However, in the presence
of mRXR
, hPPAR
wt formed a DNA-protein complex. This
binding was specific since excess amounts of cold probe could compete
for PPAR/RXR binding. By contrast, even in the presence of mRXR
,
hPPAR
tr did not bind to DNA (Fig. 6
). The same results
were obtained using different PPREs of the ACO or the apo A-I genes
(data not shown). These results demonstrate that hPPAR
tr
protein is not able to interact with DNA either as dimer with RXR or
even as monomer. However, it cannot be excluded that
hPPAR
tr heterodimerizes with mRXR
in solution,
leading to the formation of an inactive protein complex. To test this
mechanism, hPPAR
wt was incubated with increasing amounts
of hPPAR
tr in the presence of mRXR
. The
hPPAR
wt/mRXR
complex could be detected, but signal
intensity did not vary for a constant amount of hPPAR
wt
even when large amounts of hPPAR
tr were added (data not
shown). These results indicate that the hPPAR
tr form
does not bind to DNA and is unable to heterodimerize efficiently with
mRXR
in vitro.
hPPAR
tr Affects Transcriptional Activity
of hPPAR
wt
Given the structure of the hPPAR
tr protein
(without transactivation domain), it is tempting to speculate that this
isoform may interfere with wild-type function in a dominant negative
manner. To test the transactivation activity of both
hPPAR
tr and hPPAR
wt and possible mutual
interference, transient cotransfection experiments were performed in
HepG2 cells using a apoA-II PPRE containing luciferase vector as a
reporter (J3-TK-luc). Basal level of luciferase activity was unchanged
by cotransfection with hPPAR
tr whereas
hPPAR
wt activated transcription of the J3-TK-luc (Fig. 7
). When increasing amounts of the
hPPAR
tr were cotransfected with constant amounts of
hPPAR
wt, a slight decrease of J3-TK promoter activation
by hPPAR
wt was observed in the presence of high amounts
of hPPAR
tr (Fig. 7
). Surprisingly, the repressive
activity of hPPAR
tr on hPPAR
wt
transactivation function was influenced by cell culture conditions
using two different batches of FCS (see FCS1 and FCS2 in Fig. 7
). Indeed, whereas neither batch of FCS tested influenced basal
or hPPAR
wt-stimulated reporter activity,
hPPAR
tr-repressive activity on hPPAR
wt
was more pronounced depending on the FCS tested (Fig. 7
). These results
indicate that the hPPAR
tr isoform is unable to
transactivate a luciferase reporter construct driven by the
PPRE-containing thymidine kinase (TK) promoter and that it can
efficiently repress hPPAR
wt activity depending on the
presence of certain serum factors.
Nuclear Localized hPPAR
tr Is a Potent
Repressor of Nuclear Receptor Activity
To determine whether the repressive activity of
hPPAR
tr on hPPAR
wt was linked to its
subcellular localization, immunocytochemistry studies were performed
next. When CV-1 cells were transfected with the
pSG5hPPAR
wt vector, hPPAR
wt was
predominantly detected in the nucleus with only minor staining in the
cytoplasm (Fig. 8B
). Signals were
specific since pSG5-transfected cells did not give a significant
fluorescence signal (Fig. 8A
). In
pSG5hPPAR
tr-transfected cells, the fluorescence signal
was predominantly detected in the cytoplasm when cells were cultured in
FCS1 (Fig. 8C
), which yielded low repressive activity. By contrast, a
stronger signal was observed in the nucleus when cells were
cultured in FCS2 (Fig. 8D
) in which hPPAR
tr behaved as a
strong repressor. These differences in subcellular localization were
confirmed by subcellular fractionation followed by immunoblotting
analysis (data not shown). These results indicate that
hPPAR
wt is predominantly localized in the nucleus and
that the extent of the repressive effect of hPPAR
tr is
correlated with nuclear localization.
To demonstrate unequivocally that the subcellular localization
influenced hPPAR
tr-repressive activity, the SV40 large T
antigen nuclear localization signal (NLS) (52) was inserted 5' of the
hPPAR
tr cDNA to produce a chimeric
NLShPPAR
tr protein that should be able to translocate
into the nucleus. To determine the subcellular localization of this
protein, CV-1 cells were transfected with
pSG5-NLShPPAR
tr and subjected to immunocytochemistry
analysis. A strong signal was detected inside the nucleus, indicating a
massive translocation of NLShPPAR
tr into the nucleus
(Fig. 8F
). The signal was specific since transfection with pSG5-NLS
resulted in undetectable fluorescence intensity (Fig. 8E
). Thus, the
NLS system seems to be able to efficiently direct
hPPAR
tr into the nucleus. Next, we analyzed the
transcriptional activity of this NLS-hPPAR
tr by
transient cotransfection assays in HepG2 cells (cultured in
FCS1-containing medium) using a Luc reporter construct that contains
the apo A-II PPRE (J site) in three copies in front of the heterologous
TK promoter (J3-TK-Luc). The J3-TK-Luc vector was not
activated by NLS-hPPAR
tr either in the presence or in
the absence of Wy 14,643 (Fig. 9A
).
Cotransfection of hPPAR
wt resulted in a stronger
induction of Luc activity, which was repressed when
NLS-hPPAR
tr was cotransfected at a 1:1 ratio. Western
blot analysis cell extracts of pSG5hPPAR
wt- and
pSG5hPPAR
tr-cotransfected cells at a 1:1 ratio
demonstrated that both proteins were at approximately equimolar levels
(Fig. 9A
, inset). Interestingly, this transcriptional
activation was repressed by increasing amounts of
NLS-hPPAR
tr (Fig. 9A
). The repressive effect of
NLS-hPPAR
tr was similar both in the absence and in the
presence of Wy 14,643. To ensure that the repressive effect of
NLS-hPPAR
tr was not due to a saturation effect of the
protein import pathway caused by high levels of NLS peptide, the
pSG5-NLS expression vector was cotransfected in increasing amounts
against a constant amount of the hPPAR
wt. As shown in
the right part of Fig. 9A
, the induction of Luc activity by
hPPAR
wt was unaffected, indicating that there was no
effect of the NLS peptide itself on hPPAR
wt-induced Luc
activity. Consequently, these experiments confirm that nuclear
localized hPPAR
tr has a repressive activity on
hPPAR
wt transactivation function, independent of the
presence of the NLS peptide. Given the fact that the nuclear
translocation of hPPAR
tr has to be induced, we can
consider that it acts as a negative modulator on hPPAR
wt
function. Finally, to define the specificity of the negative effect of
hPPAR
tr, we assessed its ability to alter the
transactivation of other nuclear receptor family members, namely
PPAR
, hepatic nuclear factor-4 (HNF-4), related orphan
receptor-
(ROR
), and glucocorticoid receptor-
(GR
).
HepG2 cells were transfected with the appropriate response
element-driven reporter plasmids in the presence of expression vectors
expressing either PPAR
, HNF-4, ROR
, and GR
in the presence of
hPPAR
tr (Fig. 9B
). Cotransfection of
hPPAR
tr almost completely repressed
hPPAR
wt and HNF-4, whereas PPAR
and GR
activity
was less affected and ROR
activity was only marginally repressed.
These experiments indicate that hPPAR
tr, if synthetized
in significant amounts in vivo, interferes with several
nuclear receptor pathways, albeit to a different extent, and suggest
that one mechanism through which hPPAR
tr exerts its
inhibitory effect may be by sequestration of a common coactivator
necessary for nuclear receptor transcriptional activity.

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Figure 9. Nuclear Localized hPPAR tr Is a
Potent Repressor of Nuclear Receptor Activity
A, HepG2 cells, cultured in FCS1-containing medium, were cotransfected
with 1 µg of Luc reporter construct driven by the heterologous TK
promoter containing three copies of the apo A-II PPRE (J3). Activity of
the reporter plasmid J3-TK-Luc was set at 1. Increasing amounts (1x,
5x, 10x) of pSG5-NLShPPAR tr were transfected in the
presence or in the absence of pSG5hPPAR wt (200 ng).
Proteins extracted from HepG2 cells transfected at a 1:1 ratio of
pSG5hPPAR wt:pSG5-NLShPPAR tr were used for
immunoblot analysis (inset). Increasing amounts of
pSG5-NLS (1x, 5x, 10x) were added to constant amounts of
pSG5hPPAR wt (right of the figure). Cells
were treated with either Wy 14,643 (1 µM) or vehicle
(dimethylsulfoxide), and luciferase activity was measured as described
in Materials and Methods. B, HepG2 cells, cultured in
FCS2-containing medium, were cotransfected with 1 µg of J3-TK-Luc,
and 200 ng of pSG5, pSG5hPPAR , pSG5hPPAR , or pSG5HNF-4.
pSG5ROR or GR (200 ng) was transfected in the presence of 1 µg
of RORE-TK-Luc and MMTV-TK-Luc, respectively. Increasing amounts (1x,
5x) of pSG5hPPAR tr were cotransfected as indicated.
hPPAR , PPAR , and GR were activated using Wy 14,643 (1
µM), BRL 49653 (0.1 µM) and
dexamethasone (1 µM), respectively. Empty vector plasmid
was added to obtain equal amounts of DNA per well. Activity of the
reporter plasmid J3-TK-Luc, RORE-TK-Luc, and MMTV-TK-Luc was set at 1.
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Dominant Negative Effect of hPPAR
wt May
Occur through Titration of the Coactivator CREB-Binding Protein
(CBP)
Since it was reported that CBP or p300 is a coactivator
of PPAR
, PPAR
, HNF-4, and GR
, as well as ROR
(53, 54, 55, 56, 57), we
hypothesized that titration of CBP could constitute a mechanism by
which hPPAR
tr might exert its inhibitory effect. To test
this possibility, HepG2 cells, cultured in FCS2-containing medium, were
transfected with increasing amounts of CBP expression vector in the
presence of hPPAR
wt alone or both hPPAR
wt
and hPPAR
tr (Fig. 10
).
As expected, transcriptional activity of hPPAR
wt was
markedly enhanced by increasing amounts of CBP. Interestingly,
transcriptional repression of hPPAR
wt activity by
hPPAR
tr was restored by CBP cotransfection to levels
attained by hPPAR
wt in the absence of
hPPAR
tr. These data indicate that hPPAR
tr
may exert its dominant negative activity through a mechanism involving
the titration of common coactivator, such as CBP.
 |
DISCUSSION
|
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PPAR
plays an important role in lipid and lipoprotein
metabolism by regulating a number of genes implicated in ß-oxidation
and plasma lipid transport (18). It has been well documented that
treatment with hypolipidemic drugs results in distinct effects
depending on species (humans vs. rodents) (18). Furthermore,
among patients, differential responses to these drugs have been
reported as well (48, 49). In an attempt to determine whether these
phenomena could be linked to differences in expression level or to the
existence of variant forms of hPPAR
, we performed expression
analysis of PPAR
in humans. In this report we identified a
PPAR
-truncated protein (hPPAR
tr) encoded by a mRNA
derived from an alternative splicing event, results that extend
observations published while this work was in progress (58). We
furthermore show that the variant transcript appears to be present in
all human cells and tissues expressing hPPAR
wt, but not
in rodent liver. The hPPAR
tr transcripts might result in
both in vitro and in vivo production of a protein
as shown by immunoblot analysis of extracts from transfected cells or
from HepG2 cells and human primary hepatocytes. Moreover,
immunocytochemistry studies of transfected cells revealed that whereas
transfected hPPAR
wt is constitutively imported into the
nucleus, hPPAR
tr cellular localization is influenced by
cell culture conditions. Furthermore, transient transfection studies
with an NLS-containing hPPAR
tr expression vector
showed that this variant can alter hPPAR
wt
transactivation capacities once translocated in the nucleus. Finally,
cotransfection experiments with CBP suggested cofactor competition as a
potential mechanism of hPPAR
tr dominant negative
activity.
PPAR
function may be regulated at the transcriptional, the
posttranscriptional, and at the protein level. Transcriptional
regulation of PPAR
expression has been shown to occur in rodents. In
rat, PPAR
expression is regulated by hormones, such as
glucocorticoids and insulin, or by physiological stimuli such as stress
(59, 60, 61). However, very little is known about the regulation of PPAR
gene expression in man. In the present study, we analyzed the variation
of hPPAR
wt expression in human liver, the principal site
of PPAR
expression, among several individuals. Our results indicate
that hPPAR
wt mRNA levels in the liver vary substantially
(from 1 to 3) among individuals. These data suggest that PPAR
is strongly regulated at the gene level in human liver by genetic
and/or environmental factors. By contrast, in adipose tissue, the level
of expression of hPPAR
mRNA appears to be lower and more constant
among the individuals analyzed. It will be interesting to determine
whether transcriptional regulation of PPAR
also occurs through the
use of alternative promoters, as has been described for another member
of the PPAR family, PPAR
, which results both in mouse and man (9, 62, 63) in the production of two distinct proteins, PPAR
1 and
PPAR
2, with distinct activation capacities due to differences only
in their N-terminal amino acid sequence (64).
Our results furthermore demonstrate that the hPPAR
variant, identified in this and a previous study (58), is generated by
a posttranscriptional mechanism involving alternative splicing
resulting in the skipping of exon 6. Generation of isoforms by
alternative splicing has already been reported for other nuclear
receptors such as the estrogen receptor (65), the glucocorticoid
receptor (GR) (66), the thyroid hormone receptor (67), or the vitamin D
receptor (68). Alternative splicing of these receptors results in the
production of isoforms with repressive activity on the wild-type (wt)
receptor. Therefore, the use of alternative splicing may constitute a
general mechanism to diversify and to adapt receptor action upon
physiological changes.
Alternative splicing could regulate wt receptor activity both
at the gene expression level and at the protein level. Our results
obtained from the quantitative RNase protection assay demonstrate that
the ratio of wt to variant PPAR
mRNA varies substantially in liver
among individuals. The variation in this ratio appears, however, to be
mainly due to variation in hPPAR
wt mRNA levels.
Nevertheless, factors that influence this splicing event may interfere
with the PPAR
-signaling pathway by posttranscriptional regulation of
the RNA level of hPPAR
wt. By contrast, in adipose
tissue, wt-variant mRNA levels appear to be fairly constant and close
to 1:1. Therefore, high levels of hPPAR
tr mRNA may
result in lesser activity of PPAR
and its ligands, such as fibrates,
in this tissue.
In addition, if present in significant amounts in
vivo, hPPAR
tr may interfere with PPAR
-signaling
pathways at the protein level. Immunoblot experiments revealed that
both wt and truncated PPAR
proteins can be produced in
vivo by HepG2 cells and by human primary hepatocytes. Moreover,
transient expression assays suggest that hPPAR
tr may
display a repressive effect on hPPAR
wt transactivation
function. As shown by immunocytochemistry experiments, modulation of
this effect might be explained by the amount of hPPAR
tr
that translocates into the nucleus. Furthermore, when
hPPAR
tr protein is fused to a NLS peptide, nuclear
translocation of hPPAR
tr is induced, and its repressive
effect on hPPAR
wt transactivation function is strongly
enhanced. This repressive activity of nuclear hPPAR
tr
could be due to several mechanisms, such as competition for DNA
binding, sequestering of RXR by the formation of inactive heterodimers,
or titration of nuclear factors necessary for transcription activation
(69). The first two mechanisms can be excluded since, as shown by gel
retardation assays, hPPAR
tr does not bind to a PPRE
sequence nor does it inhibit DNA binding of the
PPAR
wt/RXR
complex even when added at a 4-fold
excess. These data are in line with recently published findings that
demonstrate that the C-terminal part of PPAR
is required for
heterodimerization and DNA binding (70). The third mechanism of
inhibitory effect on hPPAR
wt transactivation might be
due to the titration of a cofactor binding to the N-terminal half of
the protein, thereby limiting the amounts of common coactivators
available for transactivation by hPPAR
wt and other
nuclear receptors. Such a mechanism is probable since cotransfection of
CBP could completely revert the inhibition of hPPAR
wt
transcriptional activity by hPPAR
tr. Furthermore,
hPPAR
tr was also found to exert transcriptional
repressive activity on nuclear receptors such as PPAR
, HNF-4, GR
,
and ROR
, which all share CBP/p300 as a common coactivator (53, 54, 55, 56, 57).
Interestingly, hPPAR
tr interfered only marginally with
ROR
transcription activity, suggesting that a selectivity of
hPPAR
tr toward certain nuclear receptors exists. The
hPPAR
tr protein structure consists of the A/B and C
domains. Similarly, an estrogen receptor isoform with identical
structure has been previously described also being derived from
alternative splicing and interfering with wt receptor function (71).
Although most cofactors thus far identified interact with the LBD, the
existence of cofactors binding to the A/B domain has been also
demonstrated, such as for thyroid hormone receptor (72). Thus, the
expression level of hPPAR
tr, as well as the selectivity
of interaction with cofactors such as CBP, will determine the
specificity of hPPAR
tr action on transcription signaling
by hPPAR
wt and other transcription factors. However,
hPPAR
tr dominant negative action was more effective on
hPPAR
wt, suggesting that hPPAR
tr may also
compete specifically for cofactors other than CBP alone binding the N
terminus of hPPAR
.
To exert its repressive activity, hPPAR
tr needs
to be transported into the nucleus. Transport of transcription factors
and nuclear receptors into the nucleus is an essential step in target
gene transcription regulation. In eukaryotes, the nuclear membrane
constitutes a barrier and thereby offers a way to regulate gene
expression. Transport of nuclear proteins across the nuclear pore
complex (73) is a regulated process, but the mechanisms by which
proteins enter the nucleus are not yet fully understood. To date, the
best studied mechanism is the NLS-mediated system. Many nuclear
proteins carry NLS. Given the fact that hPPAR
tr was
observed both in the cytoplasm and in the nucleus, with higher
intensity of hPPAR
wt in the nucleus, the constitutive
NLS of hPPAR
wt is probably comprised in the E/F domain.
However, immunocytochemistry experiments, showing that the cellular
distribution of hPPAR
tr is influenced by cell culture
conditions, suggest the existence of an inducible NLS in
hPPAR
tr that could influence receptor trafficking. Such
a regulated nuclear entry has been demonstrated for a number of
proteins, including GR (74), for which two NLS appear to mediate
regulated nuclear import. Certain proteins, such as mitogen-activated
protein kinase kinase, are able to translocate rapidly into the nucleus
upon mitogenic stimulation and thus to regulate gene expression through
phosphorylation of transcription factors (75, 76). It is therefore
possible that the nuclear translocation of hPPAR
tr may
constitute a specific and controlled mechanism that modulates its
activity. Several mechanisms of induced nuclear translocation may be
suggested. First, protein phosphorylation constitutes a mechanism by
which a cryptic NLS may be unmasked. Such mechanism has been shown to
occur for the Xenopus nuclear factor Xnf7 (77) implicated in
gene regulation during development. Second, PPAR
might show high
affinity for a cytoplasmic retention protein. Such cytoplasmic
retention occurs for NF-KB, which is sequestered in the cytoplasm by
its inhibitor IK-B. Under activation by a variety of cytokines and
mitogenic factors (e.g. interleukin-1, tumor necrosis
factor-
), the NFK-B/IK-B complex dissociates and NFK-B is imported
into the nucleus (78). Finally, hPPAR
tr might be
translocated to the nucleus as a part of a cell cycle-dependent process
as shown for viral Jun (79). In this respect, it is interesting to note
that, upon transfection with hPPAR
tr, a small number of
cells consistently stained more pronouncedly in the nucleus. It will
therefore be of interest to determine whether nuclear import of
hPPAR
tr is subject to regulation, for instance during
different stages of the cell cycle.
In conclusion, we have described a PPAR
variant transcript
that is expressed in human cell lines and tissues, but not in rodents.
This transcript may give rise to the production of a truncated receptor
form of hPPAR
in human liver cells. In addition, upon nuclear
translocation, hPPAR
tr becomes a potent inhibitor of
hPPAR
wt as well as other transcription factors via a
mechanism implicating CBP sequestration. The generation of PPAR
variant transcript by alternative splicing may regulate PPAR
signaling both at the mRNA and at the protein level. Regulation of
variant transcript generation, together with its powerful inducible
repressive effect on hPPAR
wt function upon induction of
nuclear translocation, might represent a new approach for modulation of
nuclear receptor function. It will be of interest to determine whether
hPPAR
tr also contributes to the species-specific
differential response to PPAR
activators. Moreover, differences in
the level of expression of hPPAR
tr might be implicated
in the heterogeneity in response to fibrates among different patients.
Further studies are warranted to determine the physiological role of
hPPAR
tr protein in humans.
 |
MATERIALS AND METHODS
|
---|
Tissue and Cell RNA Extraction and RT-PCR Analysis
Total cellular RNA from tissues and cells was prepared by the
guanidinium thiocyanate/phenol-chloroform method (80). For analysis of
hPPAR
wt expression by RT-PCR, total RNA was reverse
transcribed using random hexamer primers (Amersham Pharmacia Biotech, Buckinghamshire, UK) and Superscript reverse
transcriptase (Life Technologies, Inc., Paisley, UK), and
the resulting cDNA product was subsequently PCR amplified (35 cycles of
1 min at 94 C, 1 min at 55 C, 1 min at 72 C) using the following
primers: oligo 15, 5'-GAA GTT CAA GAT CAA AGT GCC AGC-3'; oligo 35,
5'-TCT GAA GAG TTC CTG CAA GAA ATG G-3'; oligo 45, 5'-ACA CGC TTT CAC
CAG CTT CGA CCC-3'; oligo 65, 5'-GAC GAA TGC CAA GAT CTG AGA AAG C-3';
oligo 33, 5'-GTG ATG ACC GAG CCA TCT GAG C-3'; oligo 63, 5'-TGT ATT GTT
ACT GGC CTT TCC TGA GAG G-3'; oligo 73, 5'-AGC ATC CCG TCT TTG TTC
ATC-3'; oligo 83, 5'-CGT CTC CTT TGT AGT GCT GTC AGC-3'. Each primer
corresponds to a region well conserved between the mouse and human
PPAR
-coding sequence (5, 13, 14). The resulting products were
separated alongside suitable molecular markers on a 1.5% agarose gel
and visualized by ethidium bromide staining.
Cloning and Construction of Recombinant Plasmids
The hPPAR
wt cDNA containing the entire open
reading frame of wt hPPAR
was excised from the pCMX vector (14) by
NotI digestion and blunt ending followed by NruI
digestion, and was subsequently cloned into the blunt-ended
BamHI site of the pSG5 expression vector
(Stratagene, La Jolla, CA), giving
pSG5hPPAR
wt. The hPPAR
tr-containing pSG5
expression vector was constructed by insertion of the shorter fragment,
lacking exon 6, produced by RT-PCR amplification using the 35/73 pair
of primers (Fig. 1
). The PCR fragment was digested with MluI
and SphI and inserted into the MluI and
SphI sites of the pSG5hPPAR
wt vector
resulting in the pSG5hPPAR
tr construct. To increase
translation efficiency of both hPPAR
wt and
hPPAR
tr, a Kozak consensus sequence (81) was inserted at
the 5'-ATG of both pSG5hPPAR
wt and
pSG5hPPAR
tr constructs using a PCR method. Briefly, the
sense primer 5'-C CAT GGA TCC ACC ATG GTG GAC ACG GAA AGC-3', which
includes a BamHI site upstream from the Kozak consensus
sequence, and the antisense primer 73 were used to amplify a fragment
of 1026 bp, which was subsequently digested with BamHI and
MluI. After agarose gel purification, this fragment was
inserted into the BamHI and MluI sites of the
pSG5hPPAR
wt or pSG5hPPAR
tr plasmids. To
introduce a nuclear localization signal (NLS) into
hPPAR
tr, a cDNA encoding the SV40 large T antigen NLS
(52) was inserted in frame 5' of the hPPAR
tr cDNA. The
Kozak consensus containing pSG5hPPAR
tr was digested with
NcoI, blunt ended, and then BglII digested. The
resulting fragment was inserted into pSG5-NLS linearized by
BamHI digestion followed by blunt ending and
BglII digestion yielding pSG5-NLShPPAR
tr.
Determination of Intron-Exon Boundaries and DNA
Sequencing
The BAC clone containing the hPPAR
gene was a kind gift from
B. Wilkison (Glaxo Wellcome Inc., Research Triangle
Park, NC). Sequencing reactions were performed using the Dye
Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Corp., Foster City, CA) and an automatic ABI prism 377 sequencer
(Perkin Elmer Corp.). 5'- And 3'-intron-exon
boundaries of exon 6 were sequenced using the oligonucleotides 5'-GAC
CCG GGC TTT GAC CTT GTT G-3' and 5'-GAC GAA TGC CAA GAT CTG AGA AAG
C-3', respectively, as primers.
RNase Protection Assay
The hPPAR
antisense probe used was designed to distinguish
between the wt and the variant transcripts. A pBSSKhPPAR
clone
containing the full-length cDNA sequence was SmaI and
SacI digested, and the resulting fragment (which spans a
region located between bp 800983 according to nucleotide numbering
used in Ref. 14 was isolated and subcloned into a pBSKS vector
(Stratagene). The resulting hPPAR
riboprobe covers
54 bp of vector sequence plus 152 bp that are common to both
hPPAR
wt and hPPAR
tr and 36 bp of exon 6
that are only present in the wt transcript. A 36B4 riboprobe was used
as an internal control (82). Therefore, the 36B4 cDNA was
PstI/Sau3AI digested and subcloned in the
PstI/BamHI sites of the pBSKS vector. The
construct was further linearized with XhoI. This control
probe contains 140 bp of vector sequence and 170 bp of 36B4 sequence.
Identity and orientation of both clones were confirmed by sequencing
analysis. Both PPAR
and 36B4 riboprobes were subsequently produced
by in vitro transcription of the respective
XhoI-linearized pBSKS plasmid in the presence of
[32P]-CTP (800 Ci/mmol) using T3 RNA
Polymerase and the RNA transcription Kit (Stratagene).
Different molar ratios of [32P]CTP to cold CTP were used
(1:166 for 36B4 and 1:0 for hPPAR
) to synthesize each riboprobe,
which allowed signal comparison of the corresponding mRNA on the same
gel. The RNase protection assay was carried out using the HybSpeed
RNase protection Kit (Ambion, Inc. Austin, TX). Twenty
micrograms of total RNA were hybridized simultaneously to hPPAR
(6 x 104 cpm) and 36B4 (103 cpm)
antisense probes. RNA samples were electrophoresed on a 5% denaturing
polyacrylamide gel and visualized by autoradiography. Protected
fragments representing hPPAR
wt, hPPAR
tr,
and 36B4 mRNA were quantified on a GS525 Phosphorimager (Bio-Rad Laboratories, Inc. Hercules, CA). The radioactivity was
corrected for differences in the radiolabeled nucleotide content
between hPPAR
wt and hPPAR
tr and
subsequently normalized to the internal 36B4 control.
Cell Culture and Transfections
Human hepatoma Hep G2 and Cos-1 cells were obtained from the
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. Medium was changed every other day. Stimuli were
dissolved in dimethylsulfoxide. Control cells received vehicle only.
All transfections were performed with a mixture of plasmids, which
contained in addition to the reporter (1 µg) and expression vector
(0.22 µg), 1 µg of Cytomegalovirus-driven ß-galactosidase
expression vector as control for transfection efficiency. All samples
were complemented to an equal amount of plasmid DNA using empty pSG5
vector. Cells were transfected at 5060% confluence in 60-mm dishes
by the calcium phosphate coprecipitation procedure. After a 4-h
incubation period, cells were washed with PBS and then refed with fresh
medium and treated with Wy 14,643 (Chemsyn, Lenexa, KS), vehicle, or
dexamethasone (Sigma, St. Louis, MO) as indicated. Cells
were harvested after 24 h incubation. The luciferase activity in
cell extracts was determined using a luciferase assay system
(Promega Corp., Madison, WI) following the suppliers
instruction. Transfection experiments were performed in triplicate and
repeated at least three times.
Protein Extract Preparation
Total cellular extracts were made from 510 x
106 cells. Cells were washed twice with ice-cold PBS,
scraped off in 5 ml ice-cold PBS, and collected by centrifugation for 5
min at 800 rpm at 4 C. The pellet was resuspended in 100 µl ice-cold
lysis buffer (1% Nonidet P-40, 0.5% sodium desoxycholate, 0.1% SDS
in PBS), and protease inhibitors were freshly added (5 µg/ml
leupeptin, 5 µg/ml pepstatin, 5 mg/ml EDTA-Na2, 1
mM benzamidine, 5 µg/ml aprotinin, and 0.5 mM
phenylmethylsulfonyl fluoride). Cells were immediately transferred into
1.5-ml tubes, vigorously but quickly (10 sec) vortexed, and allowed to
swell on ice for 15 min. The cell extract was then obtained by
centrifugation (5 min at 13000 rpm at 4 C), and the supernatant was
transferred into new tubes, aliquoted, and stored at -80 C. The
samples were continuously kept at 4 C during the entire extraction
procedure.
SDS-PAGE and Western Blotting
Electrophoresis of samples (100 µg of total protein) and
prestained mol wt markers was performed on 10% SDS-polyacrylamide gels
(Minigel system, Bio-Rad Laboratories, Inc.) under
reducing conditions (sample buffer containing 10 mM
dithiothreitol). Proteins were electrophoretically blotted onto a
nitrocellulose membrane. Nonspecific binding sites were blocked with
10% skim milk powder in TNT buffer (20 mM Tris, 55
mM NaCl, 0.1% Tween 20), overnight at 4 C. The membrane
was probed with a PPAR
rabbit polyclonal antibody developed against
either a N-terminal PPAR
peptide (amino acids 1056) or a
C-terminal PPAR
peptide and diluted in 5% skim milk-TNT, for 4
h at room temperature. After washes, membranes were incubated with
peroxidase-conjugated antirabbit antibody (Diagnostics
Pasteur, Marnes- La-Coquette, France) diluted 1:4000 and
visualized using a chemiluminescent system (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK).
Immunocytochemistry
Cells were cultured on microscopy coverslips of 14 mm
diameter (sterilized with ethanol) under standard conditions. Cells
were washed with a solution of 0.01 M Tris, 0.5
M NaCl (pH 7.4) and then fixed with 3% paraformaldehyde,
2% sucrose in PBS, pH 7.5, for 15 min at room temperature. After three
washes in Tris-NaCl, cells were incubated with 0.1 M lysine
for 4 h at room temperature to avoid nonspecific fluorescent
signals. Cells were permeabilized using methanol-acetone (1:1 vol/vol)
for 5 min at room temperature. The nonspecific staining was blocked by
incubation in Tris-NaCl, 0.5% ovalbumin (TNO), and 1% of normal goat
serum for 30 min at room temperature. The preparations were incubated
overnight at 4 C with primary antibody (N-terminal PPAR
antibody)
diluted in TNO and followed by three washes with TNO (3 x 30 min)
and by incubation with secondary fluorescein
isothiocyanate-conjugated antirabbit IgG antibody (dil.1:100 in
TNO) for 2 h at room temperature. After successive washes in TNO,
Tris-NaCl, and distilled water, the preparations were assembled on
microscope slides for analysis with a Leitz DMR
fluorescence microscope (Leica Corp., Nussloch,
Germany).
In Vitro Translation and Gel Retardation Assays
pSG5hPPAR
wt, pSG5hPPAR
tr
and pSG5mRXR
were in vitro transcribed with T7 polymerase
and translated using the rabbit reticulocyte lysate sytem
(Promega Corp.). For gel retardation assay, a synthetic
double stranded oligonucleotide, which spans nucleotides -737 to -715
of the human apo A-II gene upstream regulatory sequences and contains a
PPRE, was end labeled and used as probe (41). The PPAR and RXR proteins
were incubated in a total volume of 20 µl for 15 min on ice with 2.5
µg of poly-dI-dC and 1 µg of herring sperm DNA in TM buffer (10
mM Tris-HCl, pH 7.9, 40 mM KCl, 10% glycerol,
0.05% Nonidet P-40, and 1 mM dithiothreitol). The
32P-radiolabeled oligonucleotide was then added, and the
mixture was incubated 20 min on ice. For competition experiments,
100-fold molar excess of cold probe was 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).
 |
ACKNOWLEDGMENTS
|
---|
Helpful discussions with Gérard Torpier, Ngoc
Vu-Dac, and Piet De Vos are gratefully acknowledged. We thank Odile
Vidal, Lluis Fajas, and Isabelle Saves for expert technical assistance.
The BAC clone, hepatoma cell line RNA, and human liver samples were
kind gifts from B. Wilkison, H. Will/S.F. Chang, and U. Beisiegel/V.
Kosykh, respectively. We thank R. Mukherjee for providing a hPPAR
cDNA clone. The CBP clone is the kind gift of D. Hum. M. Dauça is
acknowledged for providing anti-PPAR
antibody raised against the
C-terminal part of PPAR
. We are grateful to J. J. Berthelon for
providing BRL 49653.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Bart Staels, U.325 INSERM, Département dAthérosclérose, Institut Pasteur, 1 Rue Calmette, 59019 Lille, France.
This research was sponsored by grants from INSERM, ARCOL (Comité
Français de Coordination des Recherches sur
lAthérosclérose et le Cholestérol), Biomed 2
Concerted action (Grant PL963324), Fondation pour la Recherche
Médicale, the Région Nord-Pas de Calais, and the European
Community (Grant ERBFMBICT983214).
1 On sabbatical leave from the Center for Research, Prevention and
Treatment of Atherosclerosis, Department of Medicine, Hadassah
University Hospital, Jerusalem, Israel. 
Received for publication July 13, 1998.
Revision received April 22, 1999.
Accepted for publication May 26, 1999.
 |
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