From the Program in Molecular and Cellular Biology
and § Laboratory of Molecular Pharmacology, College of
Pharmacy, Oregon State University, Corvallis, Oregon 97331
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ABSTRACT |
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Nuclear receptor corepressor (NCoR) was
demonstrated to interact strongly with peroxisome
proliferator-activated receptor Members of the steroid/thyroid hormone receptor superfamily
function by binding to specific DNA response elements within the regulatory regions of target genes and modulating expression of these
genes at the transcriptional level (1-3). Regulation of target gene
expression mediated by nuclear receptors may occur in response to
activation of the receptors by ligands (4) or by phosphorylation (5) or
by a combination of both events (5).
The mammalian PPAR1 family is
composed of at least three genetically and pharmacologically distinct
subtypes, PPAR Retinoid X receptors (RXRs) serve as obligate heterodimeric partners
for all PPAR subtypes, and the PPAR-RXR heterodimeric complex binds
most efficiently to degenerate direct repeats of the hexameric
nucleotide sequence, AGGTCA, separated by 1 base pair (DR1; Refs.
14-17). PPAR/RXR heterodimeric binding sites, known as peroxisome
proliferator response elements (PPREs), have been identified within
the regulatory regions of several genes that encode proteins
implicated in lipid metabolism (8, 9, 14, 16, 18) and adipocyte
function (19, 20).
PPAR In addition to activating expression of target genes upon binding
cognate ligands, receptors for all-trans retinoic acid (RAR) and thyroid hormone (TR) actively repress genes in the absence of
ligand (26, 27). The related corepressor proteins, NCoR and silencing
mediator of retinoid and thyroid hormone receptors (SMRT), have been
demonstrated to interact with and mediate the repression functions of
unliganded RAR and TR (28, 29). NCoR and SMRT are associated with a
multiprotein corepressor complex that minimally contains Sin3a and the
histone deacetylase, HDAC1/Rpd3 (30, 31). Ligand binding by RAR and TR
promotes dissociation of the receptor-corepressor complex (28, 29) and
subsequent interaction of the receptor with one or more coactivator
proteins that possess intrinsic histone acetyltransferase activity
(32-35). Several nuclear receptor-associated coactivator proteins that exhibit histone acetyltransferase activity have been identified including p300/CREB binding protein (CBP; Refs. 35 and 36), steroid
receptor coactivator-1 (SRC-1; Ref. 37), and p300/CBP-interacting protein (p/CIP; Ref. 34). These findings, when considered together with
previous observations that hyperacetylated histones are associated with
actively transcribed chromatin (reviewed in Ref. 38), offer a molecular
explanation for ligand-dependent transcriptional modulation by nuclear receptors. Thus, ligand binding may serve as a molecular switch between transcriptional repression and activation by promoting exchange of a receptor-associated deacetylase-containing corepressor complex with that of a histone acetyltransferase-containing coactivator complex.
PPAR-dependent transcriptional activation of many genes is
well documented, and direct, ligand-enhanced interactions between PPARs
and the coactivators, p300/CBP (39), SRC-1 (39-41), PPAR-binding protein (PBP; Ref. 42), and PGC-1 (43) are thought to play a role in
such activation. In contrast, PPAR-mediated transcriptional repression
of target genes, as observed for RAR and TR (see above), is relatively
unexplored or at best controversial. PPAR Plasmids and Receptor Constructs--
Plasmids encoding the
receptors described below were used either directly or as templates for
polymerase chain reaction to assemble all constructs described herein
using standard techniques. All plasmids were kind gifts from the
following individuals: mouse PPAR
The parental bait vector for the yeast two-hybrid screen (pBTM16) and
the yeast reporter strain L40 (Refs. 49 and 50, respectively) were kind
gifts from Drs. R. Losson and P. Chambon (Institute de
Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France). The yeast expression vectors encoding p300 amino
acids 39-221 and the following amino acids of the indicated receptors
have been described previously (39): PPAR
GST/p300 has been described previously as GST/p300 (39-221; Ref. 39).
GST-NCoR (
A (PPRE)3-tk-CAT reporter construct was prepared by
insertion of a multimerized acyl-CoA oxidase PPRE (8) into the
XbaI site of pBL2CAT2 (52). Additional information
concerning any of the above constructs described herein can be obtained
upon request.
Yeast Two-hybrid Screening--
The yeast two-hybrid screen was
conducted as described (39) except that a mouse brain cDNA library
inserted into the GAL4 activation domain-encoded yeast expression
vector, pACT2 (CLONTECH), was used in place of the
mouse embryo library and approximately 2 × 106 yeast
transformants were screened. Plasmid DNA from positive clones was then
isolated, and the resulting cDNAs were re-tested for interaction
with PPAR baits, bait DBDs alone, and non-PPAR-related baits. cDNA
clones, which exhibited a specific interaction with the PPAR baits,
were then sequenced using the standard dideoxynucleotide chain
termination method.
Protein-Protein Interaction Assay--
GST pulldown experiments
were conducted as described previously (39), and
ligand-dependent assays were carried out by inclusion of 1 µM 9-cis-RA, 1 mM clofibrate, 100 µM troglitazone, 100 µM LY-171883, 100 µM ETYA, 100 µM WY-14,643 or vehicle in
binding buffers except where otherwise noted. GST and GST fusion
proteins were produced as described previously (39).
Mammalian Cell Transfection Experiments--
Human embryonic
kidney 293 (HEK293) cells were maintained and transiently transfected
as described previously (53). Dose-response curves were fit using
GraphPad Prism software as described above.
Chemicals and Reagents--
WY-14,643 and clofibrate were
purchased from Chemsyn Science Laboratories (Lenexa, KS) and Sigma,
respectively. LY-171883 and ETYA were obtained from Biomol (Plymouth
Meeting, PA). Troglitazone was kindly supplied by Dr. S. Kliewer
(Glaxo Wellcome). All radioisotopes were purchased from NEN Life
Science Products.
A previously described yeast two-hybrid system (49) was used to
isolate a carboxyl-terminal NCoR (29) fragment as a PPAR (PPAR
), and PPAR
ligands
suppressed this interaction. In contrast to the interaction of PPAR
with the coactivator protein, p300, association of the receptor with
NCoR did not require any part of the PPAR
ligand binding domain.
NCoR was found to suppress PPAR
-dependent
transcriptional activation in the context of a PPAR
·retinoid X
receptor
(RXR
) heterodimeric complex bound to a peroxisome
proliferator-responsive element in human embryonic kidney 293 cells.
This repression was reversed agonists of either receptor
demonstrating a functional interaction between NCoR and PPAR
·RXR
heterodimeric complexes in mammalian cells. NCoR
appears to influence PPAR
signaling pathways and, therefore, may
modulate tissue responsiveness to peroxisome proliferators.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, -
, and -
/
(also referred to as NUCI;
reviewed in Refs. 6 and 7). The primary physiological roles for the
and
subtypes of PPAR appear to be regulation of lipid metabolism
and adipogenesis, respectively, and both subtypes have been implicated
in modulating inflammatory responses (8-12). A physiological role for
PPAR
/
has not been elucidated but this receptor subtype is
expressed ubiquitously in the mouse, possibly suggesting a more general function (13).
was initially identified in a search for novel superfamily
members and was shown to be activated by a group of compounds known to
elicit proliferation of hepatic peroxisomes in rodents (21).
Structurally diverse peroxisome proliferators include phthalate ester
plasticizers (di(-2-ethylhexyl)-pthalate), herbicides (2,4,5-trichlorophenoxyacetic acid), and several fibric acid
anti-hyperlipidemic agents (WY-14, 643, clofibric acid, gemfibrozil;
Ref. 22). Additional compounds have since been shown to activate
PPAR
, including the fatty acid, arachidonic acid (23), and the
corresponding synthetic analog 5,8,11,14-eicosotetraynoic acid (ETYA;
Ref. 24), the leukotriene D4 antagonist, LY-171883 (25), and the
arachidonic acid derivative, leukotriene B4, which has been
proposed to be an endogenous ligand for PPAR
(12).
has been shown to interact
in solution with the corepressors, NCoR and SMRT, but weakly if at all
when bound to DNA, possibly suggesting that neither of these
corepressors mediate putative PPAR-dependent gene
repression (44). However, Lavinsky and co-workers (45) demonstrated
SMRT-dependent gene repression mediated by a phosphorylated form of PPAR
. Such findings illustrate the need for a more complete mechanistic understanding of potential PPAR-dependent
repression of gene expression. We report here the isolation of NCoR
from a yeast two-hybrid screen using PPAR
as a bait. We describe
results from studies in yeast, in mammalian cells, and in
vitro that were conducted to characterize PPAR
-NCoR
interactions and to examine the influence of PPAR ligands upon such interactions.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(21) from Drs. S. Green and
J. D. Tugwood (Zeneca, Macclesfield, United Kingdom); mouse RXR
(RXR
; Ref. 46) and human RAR
(RAR
; Ref. 47) from Drs. P. Kastner, A. Krust and P. Chambon (Institute de Génétique et
de Biologie Moléculaire et Cellulaire, Illkirch, France); human
p300 (48) from Dr. D. Livingston (Dana Farber Cancer Institute, Boston,
MA), NCoR (29) from Dr. T. Heinzel (German Cancer Research Center,
Heidelberg, Germany), SMRT (28) from Dr. R. Evans (Salk Institute, La
Jolla, CA). The integrity of all constructs was verified by restriction
digest and/or sequence analysis.
(91-468 of mPPAR
),
PPAR
D/E (166-468 of mPPAR
), RXR
(132-467 of mRXR
), and
RAR
(90-454 of hRAR
). PPAR
448 (91-447 of mPPAR
),
PPAR
425 (amino acids 91-424 of mPPAR
), PPAR
E (282-468 of
mPPAR
), and identical fragments of all receptors and mutants thereof
listed above were subcloned in pTL1 (46) and have been described
previously (39, 51). PPAR
288 (amino acids 91-287 of mPPAR
),
PPAR
227 (amino acids 91-226 of mPPAR
), PPAR
202
(amino acids 91-201 of mPPAR
), and PPAR
180 (amino acids
91-179 of mPPAR
) were constructed by polymerase chain reaction
amplification of the indicated receptor regions with primers containing
appropriate restriction sites for insertion into pTL1. Receptor
proteins and derivatives thereof were expressed by in vitro
transcription/translation for use in GST pulldown experiments as
described previously (39, 51). Note that all PPAR
constructs encode
receptors truncated in the amino-terminal (
AB).
ID II) and GST-NCoR, encoding GST fusions with NCoR amino
acids 2218-2381 and 2110-2453, respectively, were prepared by
polymerase chain reaction amplification with primers containing
BamHI and EcoRI sites using the original
pACT2/NCoR plasmid isolated in the yeast two-hybrid screen as a
template. The resulting fragments were digested and subcloned into
BamHI/EcoRI-digested pGEX2T (Amersham Pharmacia Biotech).
-Galactosidase Assays and Data
Analysis--
-Galactosidase assays were conducted as described
previously (39). All titration data were analyzed using an iterative, curve-fitting routine (GraphPad Prism) and the four-parameter logistic
equation. Yeast
-galactosidase results were analyzed using a
two-tailed Student's t test.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-interacting protein from an oligo(dT)-primed, mouse brain cDNA library (NCoR amino acids 2110-2453, Fig. 1). Previous
studies have identified two domains, ID I and ID II, that mediate
interactions between NCoR and nuclear receptors (29, 54). The NCoR
fragment isolated in this screen encompasses the last 33 amino acids of
ID II and the entirety of ID I (Fig. 1).
View larger version (55K):
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Fig. 1.
Isolation of NCoR as a
PPAR -interacting protein.
Carboxyl-terminal NCoR amino acid sequence. The encoded fragment
isolated in the screen is indicated by bold,
underlined text (amino acids
Lys2110-Asp2453 of NCoR; Ref. 29). Previously
identified nuclear receptor interaction domains, ID I
(Asp2239-Met2300) and ID II
(Arg2062-Ser2142), are delineated by
boxed amino acids (see Ref. 54). Note that amino acids
Lys2110-Asp2453 of NCoR are identical to
Lys319-Asp662 of the originally reported
RXR-interacting protein (RIP) 13 isolate (55). For clarity we have
referred to our clone throughout the text as NCoR amino acids
2110-2453.
PPAR and interaction domains of the coactivators, p300 and SRC-1,
exhibit strong ligand-independent association when examined in a yeast
two-hybrid system (39). When examined in vitro, however, these interactions are strictly ligand-dependent,
suggesting that yeast may contain endogenous PPAR
activating ligands
(39). The isolation of NCoR in a yeast two-hybrid screen was therefore unexpected as we (39) had hypothesized that PPAR
existed in a
liganded state in yeast that may not facilitate receptor interactions with potential corepressor proteins as has been observed for other liganded nuclear receptors (28, 29). Toward the goal of understanding the influence of NCoR on PPAR
signaling mechanisms, we chose to
conduct a more thorough analysis of the interaction between these two proteins.
WY-14,643 Inhibits PPAR-NCoR Interaction in a
Dose-dependent Manner--
The yeast two-hybrid system was
used to compare NCoR interactions with PPAR
, RAR
, and RXR
baits. PPAR
interacted specifically and robustly with amino acids
2110-2453 of NCoR, and the PPAR
ligand, WY-14,643, potently
suppressed this interaction, while the retinoid receptor ligand,
9-cis-RA, had no influence (Fig. 2A). RAR
also interacted
with NCoR and 9-cis-RA abolished this interaction (Fig.
2A). In contrast, a relatively weak interaction was observed
between RXR
and NCoR, which was enhanced by 9-cis-RA (Fig. 2A, see below).
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To confirm the observed interactions in yeast, in vitro
protein-protein interaction assays were conducted. A GST/NCoR fusion protein containing amino acids 2110-2453 of NCoR was examined for the
ability to interact with radioactively labeled PPAR, RAR
, and
RXR
prepared by in vitro translation. PPAR
interacted strongly with GST/NCoR in vitro, but not GST alone, in the
absence of ligand (Fig. 2B, lanes 2 and
10), and the presence of WY-14,643 did not significantly
affect this interaction (Fig. 2B, lane 3). Similarly, RAR
interacted with GST/NCoR in vitro, and
this interaction was not significantly affected by the presence of
9-cis-RA (Fig. 2B, lanes 5 and
6). As observed in yeast, RXR
interacted weakly with
GST/NCoR and 9-cis-RA appeared to stimulate this interaction modestly (3.4-fold; Fig. 2B, lanes 8 and
9). A weak, ligand-enhanced interaction between RXR and the
related corepressor, SMRT, has also been observed by Chen and
co-workers (28). In addition, Seol and collaborators (55) observed an
approximately 3-fold increased interaction between NCoR/RIP13 (see Fig.
1 legend) and RXR in the presence of 9-cis-RA.
To probe the ligand dependence of PPAR-NCoR interactions further,
several additional PPAR ligands were examined using the yeast
two-hybrid system as described above. Clofibrate, LY-171883, and ETYA,
as well as the PPAR
-specific ligand, troglitazone, did not
significantly affect PPAR
-NCoR interactions in yeast (Fig.
3A). The only ligand examined
in yeast that efficiently promoted dissociation of PPAR
and NCoR was
WY-14,643, and this effect was dose-dependent with an
apparent IC50 of 134 nM (Fig. 3B).
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NCoR and p300 Require Distinct PPAR Regions for
Interaction--
To determine which regions of PPAR
are required
for association with NCoR, we examined GST/NCoR interactions with
several carboxyl- and amino-terminal PPAR
truncation mutants
in vitro using standard GST pulldown methodology (Fig.
4A). The results of these
in vitro studies are depicted schematically in Fig.
4A. As shown above, PPAR
interacted strongly with both
GST/NCoR and GST/p300 in vitro (Fig. 4B,
lanes 4-7). The former interaction was only modestly
inhibited by ligand, while the latter interaction was strictly
ligand-dependent. In contrast, PPAR
448, which lacks 21 carboxyl-terminal amino acids, efficiently interacted with GST/NCoR
but ligand-dependent interaction between this truncation mutant and GST/p300 was abolished (Fig. 4B, lanes
11-14). The hinge/LBD region of PPAR
, PPAR
D/E, interacted
in vitro with GST/NCoR and GST/p300 in a manner similar to
that of PPAR
(Fig. 4B, lanes
18-21), although the WY-14,643-induced dissociation of
receptor·GST/NCoR complexes was more apparent (lanes
18-19). In contrast, PPAR
E, which lacks residues contained
within the hinge region of PPAR
(amino acids 166-281), did not
interact with either GST/NCoR or GST/p300 (Fig. 4B,
lanes 25-28). These results demonstrate that both NCoR and
p300 require the hinge region of the receptor for efficient
interaction, while only the latter requires an intact PPAR
LBD.
Therefore, the regions of PPAR
required for interaction with NCoR
and p300 are partially overlapping but largely distinct.
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The carboxyl-terminal PPAR truncation mutants, PPAR
425,
PPAR
288, PPAR
227, and PPAR
202, all interacted
efficiently with GST/NCoR (Fig. 4C, lanes 3,
6, 9, and 12). PPAR
180, which lacks the entire LBD, also interacted with GST/NCoR (Fig.
4C, lane 15). PPAR
amino acids 166-179 within
the hinge region are common to all receptor proteins observed to
interact with GST/NCoR, suggesting that these residues may play a role
in PPAR
interactions with NCoR. However, this isolated region of the
receptor did not interact with NCoR either in yeast or in
vitro under a variety of conditions (data not shown), suggesting
that this region of PPAR
is necessary but not sufficient to mediate
interaction with NCoR.
NCoR ID II Is Not Necessary for PPAR-NCoR
Interactions--
Protein-protein interaction studies carried out with
a GST/NCoR fusion protein lacking the entire ID II, GST/NCoR (
ID
II), demonstrated that the ID II region of NCoR was not required for interaction with PPAR
(Fig. 5). As
observed for interactions between PPAR
and GST/NCoR (amino acids
2110-2453, see Fig. 2B, lanes 2 and
3), WY-14,643 did not significantly affect receptor-NCoR interactions in vitro (Fig. 5, lanes 4 and
5).
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PPAR Ligands Promote Both PPAR
-NCoR Dissociation and
PPAR
-p300 Association--
The yeast two-hybrid system was used to
compare the influence of several PPAR ligands on receptor interactions
with the corepressor, NCoR (amino acids 2110-2453), and the
coactivator, p300 (amino acids 39-221). PPAR
D/E was used as the
receptor component in this series of experiments because we previously
observed a readily detectable WY-14,643-enhanced interaction in yeast
between the D/E region of PPAR
and p300 amino acids 39-221 (39). As
observed for yeast two-hybrid analyses using PPAR
(see Fig. 3),
WY-14,643, but not troglitazone, clofibrate, LY-171883, or ETYA,
significantly repressed the interaction between PPAR
D/E and NCoR
(Fig. 6A). Similarly,
troglitazone, clofibrate and LY-171883 had no significant influence on
the strong ligand-independent PPAR
D/E interaction with p300, while
both ETYA and WY-14,643 modestly, but significantly, enhanced this
interaction (Fig. 6A). Thus, while both ETYA and WY-14,643
promoted p300-PPAR
interactions in yeast and in vitro, only WY-14,643 was observed to induce dissociation of NCoR-PPAR
complexes, and this was only significantly apparent in yeast.
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To confirm the observed interactions in yeast, in vitro
protein-protein interaction assays were conducted as described above. GST/NCoR (amino acids 2110-2453) and GST/p300 (amino acids 39-221) were examined for interaction with both PPAR D/E and PPAR
in the
absence and presence of several, above-mentioned PPAR ligands. None of
the ligands tested significantly affected the in vitro interaction of NCoR with either PPAR
D/E (Fig. 6B) or
PPAR
(Fig. 6C). In contrast, in vitro association between
GST/p300 and both PPAR
D/E (Fig. 6B) and PPAR
(Fig.
6C) was observed only in the presence of ETYA or WY-14,643
(lanes 18 and 19, respectively).
NCoR Represses PPAR/RXR
-mediated Transcriptional Activation
from a PPRE in HEK293 Cells--
Cotransfection experiments in HEK293
cells were conducted to determine the physiological significance of the
strong interaction between PPAR
and NCoR observed in yeast and
in vitro. The reporter construct used for these studies,
(PPRE)3-tk-CAT, exhibited low basal or ligand-stimulated
activity in the absence of cotransfected receptor (Fig.
7, lanes 1-6). However,
cotransfection of PPAR
and RXR
resulted in a strong, constitutive
activity of this reporter that was only minimally stimulated by PPAR
(WY-14,643 and ETYA) or RXR
(9-cRA) agonists or both types of
ligands together (Fig. 7, lanes 7-12). Cotransfection of
full-length NCoR (2 µg) dramatically reduced constitutive,
PPAR
·RXR
-dependent transcriptional activation (compare lanes 7 and 13 of Fig. 7),
and this repression was reversed by treating the cells with either
PPAR
agonists alone or in combination with 9-cis-RA
(lanes 13-18). Cotransfection of a SMRT expression vector
(28) similarly repressed PPAR
·RXR
-dependent
transcriptional activation (data not shown). NCoR-dependent
repression of transcriptional activation mediated by PPAR
·RXR
was also reversed by 9-cis-RA alone (Fig. 7, lane
15), suggesting that RXR
may be competent to bind ligand
and/or activate transcription in the context of a PPAR
·RXR
heterodimeric complex bound to the acyl-CoA oxidase PPRE (8). The
potencies with which WY-14,643, 9-cis-RA, and ETYA activated
PPAR
·RXR
in the presence of cotransfected NCoR were determined
in a series of concentration-response experiments using HEK293 cells.
WY-14,643 (EC50 = 288 ± 81 nM;
n = 5, Fig. 8A) and ETYA (EC50 = 189 ± 108 nM, n = 5; Fig.
8C) were roughly equipotent, while 9-cis-RA
activated this reporter construct with an EC50 = 40 ± 8 nM (n = 3, Fig. 8B). These
EC50 values are in general agreement with previous
estimates of the affinity of PPAR
for WY-14,643 (21, 51) as well as
that of RXR
for 9-cis-RA (56). These findings demonstrate
that NCoR interacts with and represses transcriptional activation
mediated by PPAR
·RXR
heterodimeric complexes bound to a PPRE in
mammalian cells and this repression is reversed by agonists of either
receptor. From these results, we conclude that the cellular interaction
between NCoR and PPAR
and/or RXR
is likely to be of physiological
relevance and may influence tissue responsiveness to both peroxisome
proliferators and retinoic acids.
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DISCUSSION |
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We have demonstrated that PPAR interacts strongly with NCoR,
and the PPAR
ligand, WY-14,643, inhibits this interaction in yeast
and in mammalian cells. The PPAR
ligand, ETYA, also inhibits PPAR
-NCoR interaction in mammalian cells but fails to influence this
interaction in yeast. Neither ligand significantly affected NCoR-PPAR
interactions in vitro under the conditions
examined in these studies. ETYA does, however, promote interaction of
the receptor with the coactivator, p300, in yeast, suggesting that the
strain of yeast used for these studies is permeable to ETYA. Thus, the
simplest explanation for these results is that ETYA may be metabolized
in yeast to compounds which, while capable of promoting p300·PPAR
complex formation, are incapable of inducing dissociation of
NCoR·PPAR
complexes. It is also possible that ETYA may elicit
production of an endogenous yeast compound that promotes PPAR
-p300
interaction but fails to influence PPAR
-NCoR complexes. Furthermore,
we cannot rule out the possibility that a combination of these two
possibilities may be responsible for our observations in yeast using
ETYA. Given that treatment of transfected HEK293 cells with ETYA
activates a PPRE-containing reporter construct, the anomalous results
that we obtained in yeast with this compound do not appear to have
direct applicability to function of PPAR
in mammalian cells.
We previously hypothesized the existence of putative, endogenous
agonists in yeast that may disfavor PPAR-corepressor interactions (39). However, if putative, endogenous ligands contribute to the
constitutive PPAR
-coactivator interactions observed in yeast (39),
the presence of these agonists clearly does not preclude PPAR
interaction with NCoR. In this case, endogenous yeast agonists may
function in a manner similar to that of ETYA or metabolic derivatives
thereof which promote PPAR
-p300 association but do not inhibit
PPAR
-NCoR interaction. These hypotheses may provide an explanation
for how NCoR was unexpectedely isolated as a PPAR
-interacting protein in our yeast two-hybrid screen.
Previously we demonstrated that the coactivators, p300 and SRC-1,
require 21 carboxyl-terminal residues of the PPAR LBD for interaction with PPAR
(39). By analogy with other nuclear receptors, this region of PPAR
is predicted to contain the putative core of the
ligand dependent-transcriptional activation function (57). We
demonstrate herein that the PPAR
truncation mutant, PPAR
448, which lacks the 21 carboxyl-terminal amino acids encompassing the
putative AF-2 core, interacts with NCoR but not p300. Therefore, the
corepressor, NCoR, and the coactivators, p300 and SRC-1, appear to
interact with the receptor in mechanistically distinct manners that
utilize different regions of PPAR
as protein-protein interaction surfaces. However, simultaneous interaction between the receptor and
both NCoR and p300/SRC-1 are unlikely, because both types of
interaction require common amino acid residues within the hinge region
of the receptor.
Deletion of the hinge region of PPAR (amino acids 166-281)
abolished NCoR-PPAR
interaction, and amino acids 166-179 within the
amino-terminal portion of the PPAR
hinge region were common to all
receptor fragments that exhibited interaction with NCoR. The PPAR
mutant, PPAR
180 (amino acids 91-179), which interacted efficiently with NCoR, lacks the entirety of both the putative CoR box
(29) and the ligand binding domain. These results suggest that PPAR
amino acids 166-179 may mediate interactions with NCoR. However,
extensive analyses in yeast and in vitro have failed to
demonstrate that PPAR
166-179 are sufficient to mediate interaction with NCoR (data not shown), possibly indicating that additional contacts are required for efficient interaction. Nonetheless, our
results suggest that PPAR
likely contains a NCoR interaction surface
that is clearly not contained within the LBD of the receptor and, thus,
may be distinct from that present in either RAR or TR (28, 29).
Zamir and collaborators (44) have shown that PPAR can interact with
the corepressors, SMRT and NCoR, in solution, but weakly if at all when
bound to DNA. Similarly, we were unable to demonstrate the formation of
a DNA bound PPAR
·RXR
·NCoR complex in vitro (data
not shown). However, in contrast to the findings of Zamir and
colleagues (44) who observed no corepressor-dependent
PPAR
-mediated repression, our transient transfection studies clearly
demonstrate a functional interaction between a PPRE-bound,
PPAR
·RXR
complex and NCoR. Such discrepant results could simply
be a result of either differing PPAR subtypes (
versus
) or cell lines (HEK 293 versus 293T) or a combination of
these two possibilities. The inability to observe a DNA-bound
PPAR
·RXR
·NCoR complex in vitro may be due to an
inherent instability of such complexes, and indeed, other groups have
reported that cross-linking reagents are required to stabilize similar
complexes in vitro (58). NCoR clearly associates with and
represses the transcriptional activity of PPRE-bound, PPAR
·RXR
heterodimeric complexes in HEK293 (Fig. 7). However, we cannot
exclude the possibility that additional cellular factor(s) present in
HEK293 cells, but lacking in vitro, are required for the
assembly of a DNA-bound, PPAR
·RXR
·NCoR complex.
Finally, it is conceivable that interaction of NCoR or SMRT with either
PPAR or PPAR
·RXR
complexes may influence other signaling
pathways by titration of limiting amounts of these corepressors. This
form of receptor cross-talk may serve to relieve transcriptional repression mediated by other nuclear receptors, such as RAR, TR, or
RevErb, that utilize common corepressors. Results presented herein
raise the possibility that PPAR interactions with corepressors in
solution or on DNA may play a prominent role in regulating PPAR-dependent transcriptional regulation of target genes.
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ACKNOWLEDGEMENTS |
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We thank P. Chambon, R. Losson, P. Kastner, T. Lufkin, J. D. Tugwood, S. Green, and D. Livingston for plasmid constructs and reagents; S. Kliewer and Glaxo Wellcome for generously providing troglitazone; Drs. W. Wahli and B. Desvergne for useful discussion and suggestions; and J. Webster for expert technical assistance.
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FOOTNOTES |
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* This work was supported by American Heart Association Grant 9640219N and National Institute of Environmental Health Science Grant ES00040.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.
¶ Supported by a predoctoral fellowship from the American Foundation for Pharmaceutical Education. Current address: The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Laboratory of Molecular Pharmacology, College of Pharmacy, Oregon State University, Corvallis, OR 97331. Tel.: 541-737-5809; Fax: 541-737-3999; E-mail:
mark.leid{at}orst.edu.
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ABBREVIATIONS |
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The abbreviations used are:
PPAR, peroxisome
proliferator-activated receptor;
RXR, retinoid X receptor;
DR1, direct
repeat separated by one nucleotide;
ETYA, 5,8,11,14-eicosatetraynoic
acid;
RAR, retinoic acid receptor;
TR, thyroid hormone receptor;
NCoR, nuclear receptor corepressor;
SMRT, silencing mediator of retinoid and
thyroid hormone receptors;
CBP, CREB-binding protein;
SRC-1, steroid
receptor coactivator-1;
p/CIP, p300/CBP-interacting protein;
mPPAR, mouse PPAR
;
ID, interaction domain;
9-cis-RA, 9-cis-retinoic acid;
RIP13, RXR-interacting protein 13;
GAD, GAL4 activation domain;
PPRE, peroxisome proliferator response element;
GST, glutathione S-transferase;
CAT, chloramphenicol
acetyltransferase;
LBD, ligand binding domain.
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REFERENCES |
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