From the § Center for Molecular Toxicology and
Carcinogenesis and the Department of Veterinary Science, and the
Graduate Program in Molecular Toxicology, Pennsylvania
State University, University Park, Pennsylvania 16802
Received for publication, November 4, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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The peroxisome proliferator-activated
receptor The ability of peroxisome proliferators to activate a receptor in
the steroid receptor superfamily was first discovered in 1990, and the
cognate protein was designated as
PPAR1 (1). The PPARs are
soluble transcription factors that are activated by a diverse class of
lipophilic compounds (2). With the activation of PPAR, a concomitant
induction of a number of genes that code for peroxisomal fatty acid
metabolizing enzymes was observed in mouse liver. Among these, the
peroxisomal enzyme AOx is the most broadly used indicator of peroxisome
proliferator action. Transcription of the AOx gene is increased by
exposure to the hypolipidemic peroxisome proliferator WY-14,643, and
this effect is mediated by a PPRE located 570 base pairs upstream of the transcriptional start site (3). This PPRE contains a direct repeat
of the sequence motifs TGACCT and TGTCCT, which is separated by a
single nucleotide. A heterodimer of PPAR and RXR binds to PPREs located
in upstream regulatory regions of various target genes and the RXR
ligand, 9-cis-retinoic acid, increases PPAR/RXR transcriptional activity (4).
There are three PPAR subtypes, designated as PPAR Many steroid hormone receptors exist in a complex with the chaperone
hsp90 and certain co-chaperones, such as immunophilins or
immunophilin-like proteins. The highly abundant chaperone hsp90 aids in
protein folding and modulates the function of a wide range of proteins.
In particular, hsp90 stabilizes certain steroid hormone receptors by
aiding the folding of the ligand binding domain of the receptor to a
high affinity ligand binding conformation. There are two classes of
immunophilins: one group designated as FKBPs, which bind drugs like
FK506 and rapamycin; and the other, referred to as cyclophilins, which
are known to bind cyclosporin A. The larger immunophilins, FKBP51,
FKBP52, and CyP40, are associated with steroid hormone receptors (11).
Mature untransformed glucocorticoid receptors and progesterone
receptors are in a heterocomplex with hsp90, CyP40 or FKBP52, and p23
(12). The precise role of FKBP52 or CyP40 in mature steroid receptor
complexes has been difficult to determine.
Besides steroid hormone receptors, many other proteins are in a complex
with hsp90 including serine/threonine kinases such as Raf, tyrosine
kinases such as v-Src, and tumor suppressor p53 (13-17). The AhR, a
basic helix loop helix/Per-Arnt-Sim transcription factor, is also known
to be associated with hsp90, and hsp90 appears to be required for the
AhR to maintain a proper conformation to bind ligand (18). Mapping of
the hsp90 binding site on the AhR reveals that there are two separate
domains complexed with hsp90, corresponding to amino acids 1-166 and
289-347 of AhR, the latter sequence contains part of the ligand
binding domain (19). The ability of hsp90 to associate with different
classes of receptors suggests the possibility that hsp90 could
associate with PPAR Type II steroid hormone receptors (e.g. PPAR, RXR, pregnane
X receptor, etc.) are presumed to be localized to the nucleus, and the
inactive receptors have not been demonstrated to associate with the
hsp90 co-chaperone complexes (20). We hypothesized that PPAR Antibody Production--
The rat PPAR Plasmid Production--
The rPPAR
PPAR Isolation of Tissue Cytosolic Extracts--
Each tissue was
quickly removed and minced in the presence of MENGM + protease
inhibitors (Sigma) at a 1/10 ratio (w/v) and homogenized with a Dounce
homogenizer. The homogenate was centrifuged at 10,000 × g for 20 min; the resulting supernatant was centrifuged at
100,000 × g for 60 min. Cytosol was removed, carefully
avoiding the upper lipid layer. Protein concentration of the cytosol
was measured using the bicinchoninic acid (BCA) assay (Pierce).
In Vitro Translation--
In vitro translations of
PPAR subtypes and XAP2 were performed using a TNT rabbit
reticulocyte lysate coupled transcription/translation kit, as described
by the manufacturer (Promega Biotech).
Immunoprecipitations--
In vitro translated samples
were immunoprecipitated with the mAb
3B6/PPAR2 pre-bound to
Protein G-Sepharose. The hsp90 was immunoprecipitated from 300 µg of
C57BL/6N mouse liver cytosol using the mAb 3G3p90 pre-bound to 100 µl
of Protein L-Sepharose (Pierce). PPAR Analysis of PPAR In Vitro Translation Binding Assay--
The constructs pCI/XAP2
and pBK/CMV/PPAR Cell Culture--
COS-1 cells were maintained in Mammalian Two-hybrid Assay--
Mammalian two-hybrid studies
were performed using the Matchmaker Mammalian-2-Hybrid system
(Clontech). COS-1 cells were plated in 24-well
plates and allowed to recover overnight. Transfection was carried out
with LipofectAMINE reagent (Invitrogen) according to the
protocol of the manufacturer. Each well was transfected with 200 ng of
pM/PPAR Transient Transfections and Luciferase Reporter
Assays--
COS-1 cells were transfected at 80% confluence in
six-well tissue culture plates. The LipofectAMINE transient
transfection procedure was employed according to the instructions from
the manufacturer (Invitrogen). Each transfection included 500 ng of receptor (or pCI), 500 ng of PPRE-driven reporter construct
pAOx(x2)-luciferase, 200 ng of the internal transfection control vector
pDJM/ The mAb 3B6/PPAR Is Capable of Immunoprecipitating and Visualizing
PPAR PPAR PPAR hsp90 Interacts Predominantly with the E/F Domain of
PPAR XAP2 Represses Reporter Gene Activity Mediated by PPAR To visualize possible co-immunoprecipitated proteins complexed
with PPAR PPARs belong to the type II steroid receptor family (20), which also
includes members such as RXR and thyroid hormone receptor. These
receptors are generally considered to be localized to the nucleus and
appear not to be bound to other proteins in inactive complexes, as has
been extensively described for type I receptors (e.g.
glucocorticoid receptor, progesterone receptor, androgen receptor).
Although oncogenic v-Erb A (NR1A1), a derivative of the thyroid hormone
receptor, stably interacts with hsp90 in the cytoplasm (29), endogenous
type II receptors are not associated with hsp90. For example, neither
RXR nor thyroid hormone receptor is associated with hsp90 (30, 31).
Although PPAR The molecular chaperone hsp90 appears to aid in folding and maintenance
of the appropriate conformation of the ligand binding domain of certain
soluble receptors. In addition, hsp90 prevents protein aggregation as
well as aids in the stability and function of a wide variety of client
proteins (11). During heat stress hsp90 exhibits both elevated levels
and a heat stress protective function (34). hsp90 is composed of an
N-terminal dimerization domain and a charged domain, which binds
calmodulin and functions in intramolecular interactions, as well as a
C-terminal dimerization and TPR-binding domain (11). Both the
N-terminal and C-terminal domains of hsp90 can associate with
co-chaperones, whereas the middle domain binds the client proteins such
as steroid hormone receptors (35, 36). Receptors bound to hsp90 are
considered to be inactive, unable to either dimerize or bind DNA. Upon
ligand binding, hsp90 dissociates from the complex leading to receptor transformation to the DNA binding state in the nucleus. In addition, hsp90 appears to play a major role in receptor trafficking between cytoplasm and nucleus. Untransformed progesterone and glucocorticoid receptors exist in heterotetrameric 8-9 S complexes containing one
subunit of the steroid binding receptor and two subunits of the hsp90
with a hsp90 co-chaperone protein (e.g. FKBP52) (37). It is
logical to hypothesize that hsp90 serves similar functions bound to
PPAR The hsp90 co-chaperone proteins, which are found in a mature
hsp90-receptor complex, include p23 and immunophilins Cyp40 and FKBP52.
For example, CyP40 was associated with estrogen receptor (39),
progesterone receptor (40), and the glucocorticoid receptor (41)
complexes, whereas FKBP52 has been identified as a component of the
estrogen receptor (39), progesterone receptor (42), and glucocorticoid
receptor (43) oligomeric complexes. Both FKBP52 and CyP40 contain
C-terminal TPR motifs, which are necessary for their interactions with
the hsp90-receptor complex. FKBP52 is predominantly localized in the
nucleus and may function by targeting receptors to this compartment,
whereas CyP40 localizes in the nucleoli and may also function in
protein trafficking. However, the exact role of immunophilins in
receptor complexes is currently not clear. The hsp90 co-chaperone p23
was first discovered as a protein that complexes with purified
receptors and appears to stabilize receptor-hsp90 association. Whether
p23 also interacts with PPAR In addition to hsp90, the immunophilin-like protein XAP2
co-immunoprecipitated with PPAR (PPAR
) is a ligand-inducible transcription
factor, which belongs to the nuclear receptor superfamily. PPAR
mediates the carcinogenic effects of peroxisome proliferators in
rodents. In humans, PPAR
plays a fundamental role in regulating
energy homeostasis via control of lipid metabolism. To study the
possible role of chaperone proteins in the regulation of PPAR
activity, a monoclonal antibody (mAb) was made against PPAR
and
designated as 3B6/PPAR. The specificity of mAb 3B6/PPAR in recognizing
PPAR
was tested in immunoprecipitations using in vitro
translated PPAR subtypes. The mAb 3B6/PPAR recognized PPAR
, failed
to bind to PPAR
or PPAR
, and is efficient in both immunoprecipitating and visualizing the receptor on protein blots. The
immunoprecipitation of PPAR
in mouse liver cytosol using mAb
3B6/PPAR has resulted in the detection of two co-immunoprecipitated proteins, which are heat shock protein 90 (hsp90) and the hepatitis B
virus X-associated protein 2 (XAP2). The concomitant depletion of
PPAR
in hsp90-depleted mouse liver cytosol was also detected. Complex formation between XAP2 and PPAR
/FLAG was also demonstrated in an in vitro translation binding assay. hsp90 interacts
with PPAR
in a mammalian two-hybrid assay and binds to the E/F
domain. Transient expression of XAP2 co-expressed with PPAR
resulted in down-regulation of a peroxisome proliferator response element-driven reporter gene activity. Taken together, these results indicate that
PPAR
is in a complex with hsp90 and XAP2, and XAP2 appears to
function as a repressor. This is the first demonstration that PPAR
is stably associated with other proteins in tissue extracts and the
first nuclear receptor shown to functionally interact with
XAP2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(NR1C1), -
(NR1C2), and -
(NR1C3); and each subtype is capable of binding to
DNA after heterodimerizing with RXR (NR2B1) (5). Many of the genes
regulated by PPAR
are involved in fatty acid metabolism. PPAR
isoforms are activated by both exogenous and endogenous chemicals, including phthalates used as plasticizers, fibrate-type hypolipidemic drugs, and naturally occurring polyunsaturated fatty acids. PPAR
is
highly expressed in the liver, kidney, and cardiac smooth muscle. However, most studies examining PPAR
function have been performed in
the liver, where PPAR
is responsible for the tumor promotional properties of peroxisome proliferators such as DEHP. Following exposure
to DEHP and other peroxisome proliferators, rodents demonstrate biological and biochemical responses such as peroxisome proliferation, increased microsomal fatty acid oxidation, increased hepatic hydrogen peroxide formation, hepatomegaly, hyperplasia, and subsequent neoplasia
(6). The involvement of PPAR
in cell proliferation and tumor
promotion has been examined using PPAR
knockout mice (7, 8). In
contrast to wild type mice, following exposure to chemicals such as
clofibrate, DEHP, and WY-14,643, PPAR
knockout mice were unable to
demonstrate the characteristic effects of peroxisome proliferators,
which are hepatomegaly, peroxisome proliferation, and transcriptional
activation of various target genes (7). PPAR
null mice were also
refractory to the WY-14,643-induced replicative DNA synthesis (8).
These results show that PPAR
is the major isoform required for
mediating the responses resulting from the actions of peroxisome
proliferators in liver. PPAR
is expressed in human liver at low
levels, and humans appear to be insensitive to the carcinogenic
response of peroxisome proliferators (6, 9, 10), although controversy
exists and further studies are needed.
.
could
complex with hsp90 or other chaperones. One protein that has been found
to associate with PPAR
is heat shock protein 72; however, the
functional significance of this association has not been determined yet
(21). The only other proteins reported to associate with PPAR
are
co-activators such as PPAR
-binding protein (designated PBP) (22),
the steroid receptor co-activator 1 (23), p300 (24), peroxisome
proliferator- activated receptor interacting protein (designated PRIP)
(25), and nuclear receptor co-repressor (26). Thus, little is known
about the regulation of PPAR
through protein-protein interactions.
In this report PPAR
was found to associate with hsp90 and the
immunophilin-like protein XAP2. In addition, evidence is provided that
suggests XAP2 represses PPAR
-mediated transcriptional activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cDNA (obtained from
Frank Gonzalez, National Cancer Institute, National Institutes of
Health, Bethesda, MD) was subcloned into the pMALc2 plasmid by PCR, and
the fusion protein PPAR/maltose binding protein was expressed in
the bacterial strain BL21. The fusion protein was purified on amylose
resin and the purified protein eluted with maltose. The fusion protein
was cleaved with Xa protease, after cleavage PPAR
precipitated out
of solution and was collected by ultracentrifugation. The precipitated
protein was dissolved in performic acid as previously described,
lyophilized, dissolved in 0.1 N NaOH, and neutralized with
0.1 N HCl (15). This protein was used as an antigen, and
BALB/c mice were immunized as described (27). An ELISA was developed
using the PPAR
/maltose binding protein. Sera from mice was tested by
ELISA, and positive mice were used in hybridoma production essentially
as previously described (27). The wells that were positive in the ELISA
assay were then screened on a Western blot of PPAR
mounted in a
Miniblotter45 (Immunetics, Cambridge, MA). The hybridomas that were
positive in this assay were cloned by limiting dilution and further characterized.
cDNA was subcloned into
mammalian expression vector pCI (Promega Biotech, Madison, WI). A PCR
primer was designed to amplify the 3' half of PPAR and a primer
containing the 3' end of the cDNA plus a nucleotide sequence
corresponding to the FLAG sequence, along with a stop codon, and a
KpnI site. This PCR product was digested with
BstEII and KpnI and subcloned into the
pCI/rPPAR
vector digested with the same restriction enzymes. The
SP163 enhancer sequence was amplified by PCR using primers containing
BamHI sites with pcDNA4/HisMax C vector as a template, and was inserted into pBK/CMV digested with BamHI. The
rPPAR
/FLAG cDNA was subcloned from pCI/rPPAR
/FLAG using
XhoI and KpnI restriction sites into
pBK/CMV/SP163. The vector pSG5/mPPAR
was obtained from Jonathan
Tugwood (Astra Zeneca), whereas pSG5/mPPAR
and pSG5/mPPAR
vectors
were obtained from Paul Grimaldi (INSERM). The SP163 enhancer sequence
was amplified by PCR using primers containing SacI and
SpeI sites, with pcDNA4/HisMax C vector as a template,
and was inserted into pBK/CMV digested with SacI and SpeI. PCR primers were designed to amplify full-length
mPPAR
, -
, and -
and to add a FLAG sequence to the 5' end of
the cDNA. The forward and reverse primers contained SpeI
and ClaI sites, respectively. The PCR products were digested
with SpeI and ClaI and subcloned into the
corresponding sites in pBK/CMV/SP163. PPRE-driven reporter
pAOx(x2)-luciferase, which is under the control of rat AOx promoter,
was a gift from Dr. David Waxman (Boston University, Boston, MA). The
vector pDJM/
gal, which consists of
-galactosidase reporter gene
under the control of the murine phosphoglycerate kinase 1 promoter, was
kindly provided by M. W. McBurney (University of Ottawa, Ottawa,
Canada). The construction of pCI/XAP2 was previously described
(28).
was fused to the GAL4 DNA binding domain cDNA in the vector
pM (Clontech), and human hsp90 was fused to the
VP16 activation domain cDNA in the vector pVP16. Domains of PPAR
were cloned into the pM vector as well as all the combination of
domains using standard techniques. A GAL4-responsive reporter vector,
pFR-luciferase (Stratagene), was used to assess transcriptional
activity mediated by pM and pVP16 after transfection into cells.
Transfection efficiency was assessed by co-transfection of the control
Renilla vector, pRLTK (Promega).
was immunoprecipitated from
300 µg of liver cytosol using mAb 3B6/PPAR pre-bound to goat
anti-mouse IgG-agarose. Mouse IgM (Rockland, Gilbertsville, PA) and
mouse IgG (Jackson Immunoresearch, West Grove, PA) bound resins were
used in the hsp90 and PPAR control IP, respectively. In all IP,
antibody binding to resin was carried out in PBS at 4 °C with
mixing. After 1 h, the PBS was removed and the resin was washed
twice in PBS and then twice in MENGM buffer. In both hsp90 and PPAR IP,
liver cytosol was immunoprecipitated in the presence of IP buffer,
which consists of MENGM supplemented with 100 mM NaCl, 10 mg/ml bovine serum albumin (Fisher Scientific, Springfield, NJ), and 5 mg/ml ovalbumin (Sigma), for 1 1/2 h at 4 °C with mixing. In hsp90
IP, the immune-depleted cytosol was isolated after centrifugation of
the resin for 1 min at 113 × g. Before SDS-PAGE
analysis, all IP were washed five times with MENGM supplemented with
100 mM NaCl. Samples (either pellet or immune depleted
cytosol) were then mixed with 2× Tricine sample buffer, heated at
95 °C for 5 min, and resolved by SDS-PAGE. After SDS-PAGE, proteins
were transferred from the gel to a PVDF membrane (Millipore, Bedford,
MA) at 16 V for 3 h at 4 °C in a Genie blotter (Idea Scientific
Co., Minneapolis, MN).
Interaction with hsp90 and
XAP2--
Co-immunoprecipitation of XAP2 and hsp90 with PPAR
was
detected on membranes using anti-ARA9 (Novus Biological, Littleton, CO)
and anti-hsp84/hsp86 (Affinity Bioreagents), respectively. Peroxidase-conjugated donkey anti-rabbit and goat anti-mouse antibodies were used as the secondary antibodies (Jackson Immunoresearch). Depletion of PPAR
in hsp90-depleted cytosol was visualized using mAb
3B6/PPAR and peroxidase-conjugated Protein L. As a loading control, the
membrane was probed for the 35-kDa protein, lactase dehydrogenase. To
detect each protein, SuperSignal West Pico chemiluminescent substrate
(Pierce) was used. To quantify the amount of proteins, antibodies were
stripped from the blot using 0.1 M glycine, pH 2.5, and
reprobed with primary antibody followed by incubation with
125I-labeled secondary antibodies (Amersham
Biosciences). The amount of PPAR
in hsp90-depleted cytosol
relative to control was evaluated using a Cyclone phosphorimager
(Packard, Meriden, CT).
/FLAG were in vitro translated in the
presence of [35S]methionine using the TNT
rabbit reticulocyte lysate system according to the instructions from
the manufacturer. In vitro translated pCI/XAP2 and
pBK/CMV/PPAR
/FLAG constructs were mixed and incubated for 1 h
at 4 °C. For the control, an in vitro translation, which is carried out without the receptor construct, was mixed with in
vitro translated pCI/XAP2. After incubation, PPAR
/FLAG was immunoprecipitated using anti-FLAG M2-agarose (Sigma). After a 1-h
incubation with mixing, the IP were washed five times using MENGM + 100 mM NaCl, samples were subjected to SDS-PAGE, and proteins were transferred from the gel to a PVDF membrane. Co-immunoprecipitated XAP2 was detected by autoradiography.
-minimal
essential medium (Sigma) supplemented with 8% fetal bovine serum
(HyClone Laboratories, Logan, UT), 100 IU/ml penicillin, and 0.1 mg/ml
streptomycin (Sigma), and grown at 37 °C in 5% CO2.
or pM containing various domains of PPAR
, 200 ng of pVP16
or pVP16/hsp90, 100 ng each of pFR-luciferase and pRL-TK. Transfected
cells were treated with 50 µM WY-14,643 or
Me2SO for 6 h and assayed for luciferase activity.
Relative luciferase activity was corrected using the internal
transfection control (pRL-TK) and the Dual Luciferase Kit (Promega).
The corrected luciferase values were also corrected for changes in
protein levels.
gal, and 0-1000 ng of pCI/XAP2. The vector pCI was used to
maintain the total amount of plasmid at 2 µg of DNA/well. Transfected
cells were harvested 24 h after the start of the transfection, and
PPRE-driven luciferase reporter activity was assayed with a luciferase
assay system (Promega Biotech) using a Turner TD-20e luminometer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
To meaningfully study the ability of PPAR
to associate
with other proteins, a highly specific antibody that was efficient at
immunoprecipitating native PPAR
needed to be produced. To achieve
this goal, we chose to produce monoclonal antibodies to bacterially
expressed PPAR
. Three different clones that were positive in the
initial screen using an ELISA procedure were tested for the ability to
recognize PPAR
. Among the three monoclonal antibodies examined, only
3B6/PPAR specifically recognized PPAR
(Fig.
1A). The specificity of mAb
3B6/PPAR in both immunoprecipitating and visualizing mouse PPAR
was
tested using in vitro translated mouse PPAR constructs.
Among the three isoforms of PPAR, mAb 3B6/PPAR only immunoprecipitated
PPAR
. Rat PPAR
can also be recognized by mAb 3B6/PPAR (Fig.
1B). The mAb 3B6/PPAR was also the only antibody capable of
recognizing PPAR
on membranes (Fig. 1C). The
ability of mAb 3B6/PPAR to visualize PPAR
in mouse tissue extracts
was tested using the cytosolic extracts from various C57BL/6N mouse
tissues. The mAb 3B6/PPAR recognized PPAR
in liver, kidney, heart,
and lung mouse cytosolic extracts (Fig. 1D). These data
demonstrate that a monoclonal antibody has been made that is not only
highly specific for PPAR
but also works well in
immunoprecipitations and visualization of PPAR
on
membranes.
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Fig. 1.
The mAb 3B6/PPAR is able to both
immunoprecipitate and visualize PPAR on
protein membranes. A, the ability of mAb 3B6/PPAR to
immunoprecipitate PPAR
was tested using in vitro
translation lysate containing [35S]methionine and
mPPAR
, mPPAR
, or mPPAR
. Three different clones that were
positive in the initial screen using an ELISA procedure, and protein
blots of PPAR
fusion protein were tested. B, the ability
of mAb 3B6/PPAR to immunoprecipitate rat PPAR
was tested using
in vitro translation lysate containing
[35S]methionine and rat PPAR construct. C,
mouse PPAR
, mPPAR
, and mPPAR
expression constructs were
in vitro translated in the presence of
[35S]methionine, subjected to SDS-PAGE, transferred to a
PVDF membrane, and probed with mAb 3B6/PPAR and a secondary antibody
peroxidase-conjugated Protein L. The presence of radioactivity was
visualized by autoradiography (lower panel). The
protein blot probed with mAb 3B6/PPAR was visualized using a secondary
antibody peroxidase-conjugated Protein L (upper
panel). D, the ability of mAb 3B6/PPAR to
visualize PPAR
in cytosolic extracts of various mouse tissues.
Approximately 150 µg of protein from mouse liver, kidney, heart, and
lung cytosol was subjected to SDS-PAGE followed by immunoblotting.
PPAR
was detected using mAb 3B6/PPAR and peroxidase-conjugated
Protein L.
Is in a Complex with hsp90 and XAP2--
The chaperone
protein hsp90 was immunoprecipitated from C57BL/6N mouse liver cytosol
using the anti-hsp90 mAb 3G3p90, which has previously been shown to be
efficient at immunoprecipitating hsp90 complexes (26). Approximately
50% depletion of PPAR
and ~40% depletion of hsp90 was obtained,
compared with the control IP (Fig.
2A). This result suggests that
a significant proportion of PPAR
is complexed with hsp90. A
recurring theme for many proteins that complex with hsp90 is the
presence of hsp90 co-chaperone proteins, such as FKBP52, CyP40, XAP2,
and p50cdc37. Thus, it was logical to screen for
the presence of these previously characterized hsp90 co-chaperones on
protein blots. Immunoprecipitation of PPAR
with the mAb 3B6/PPAR has
yielded two co-immunoprecipitated proteins, identified as hsp90 and
XAP2 (Fig. 2B). FKBP52 and CyP40 were not detected in a
complex with PPAR
(data not shown). In the rabbit reticulocyte
lysate system, XAP2 was in vitro translated in the presence
of [35S]methionine and was able to bind to in
vitro translated PPAR
/FLAG (Fig.
3). These data demonstrate that PPAR
from C57BL/6N mouse liver exists in a complex with hsp90 and XAP2.
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Fig. 2.
PPAR is in a complex
with hsp90 and XAP2. A, depletion of PPAR
in
hsp90-depleted cytosol. The hsp90 from mouse liver cytosol was
immunoprecipitated using mAb 3G3p90 pre-bound to Protein L-agarose.
After incubating 300 µg of mouse liver cytosol with mAb 3G3p90
pre-bound to Protein L-agarose, the cytosol was isolated and subjected
to SDS-PAGE followed by immunoblotting. Mouse IgM bound to Protein
L-agarose was used in the control experiment. The 35-kDa protein
lactase dehydrogenase was used as a loading control. B,
PPAR
from mouse liver cytosol was immunoprecipitated using mAb
3B6/PPAR pre-bound to goat anti-mouse IgG. Mouse IgG pre-bound to goat
anti-mouse IgG-agarose was used in the control experiment. PPAR
was
immunoprecipitated, subjected to SDS-PAGE, transferred to a PVDF
membrane, and probed using mAb 3B6/PPAR and peroxidase-conjugated
Protein L. The presence of hsp90 and XAP2 was detected using standard
techniques.
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Fig. 3.
Binding of in vitro
translated XAP2 to PPAR . Both XAP2 and PPAR
/FLAG were
in vitro translated and mixed, and PPAR
was
immunoprecipitated using anti-FLAG M2-agarose, subjected to SDS-PAGE,
and transferred to a PVDF membrane. PPAR
and co-immunoprecipitated
XAP2 were detected by autoradiography. Lane 1, control
in vitro translation; lane 2, in vitro
translated XAP2; lane 3, in vitro translated
PPAR
; lane 4, FLAG IP of control in vitro
translation mixed with in vitro translated XAP2; lane
5, FLAG IP of in vitro translated PPAR
mixed with
in vitro translated XAP2.
Interacts with hsp90 in a Mammalian Two-hybrid
Assay--
hsp90 can interact with PPAR
in cells as determined
using a mammalian two-hybrid assay in COS-1 cells (Fig.
4). A reduced level of activity was
observed with VP16/hsp90 expression in the presence of a WY-14,643 when
compared with the level of activity in the presence of VP16 expression
(control). This result suggests that hsp90/VP16 binds to the liganded
GAL4-PPAR
and partially represses transcriptional activity (Fig. 4).
Thus, ligand binding alone appears not to result in hsp90 dissociation
from PPAR
.
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Fig. 4.
Influence of ligand on binding of hsp90 to
PPAR in mammalian two-hybrid assay. COS-1
cells were transfected with pM/GAL4/PPAR
and pVP16/hsp90. Cells were
treated with 50 µM WY-14,643 or carrier solvent for
6 h and assayed for luciferase activity. Activity was corrected
for transfection efficiency as described under "Experimental
Procedures." All bars were standardized to the VP16
carrier solvent bar (n = 3). This
graph is representative of two independent experiments.
DMSO, Me2SO.
--
The ability of a given domain of PPAR
to interact with
hsp90 was tested using a mammalian two-hybrid assay in COS-1 cells. The
D and E/F domains, when expressed in the absence of other domains of
PPAR
, are capable of interacting with hsp90 (Fig. 5). However, the D domain in combination
with other domains appears not to significantly interact with hsp90,
suggesting that binding to the D domain in the context of the
full-length PPAR
may not be significant. Thus, it appears that the
E/F domain is the primary domain of PPAR
mediating hsp90
interaction.
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Fig. 5.
Determination of the PPAR
domain that interacts with hsp90. COS-1 cells were
transfected with the appropriate plasmids to express each PPAR
domain or combination of domains fused to the GAL4 DNA binding domain.
Cells were lysed and assayed for luciferase activity. Activity was
corrected for transfection efficiency and expressed as -fold increase
in activity relative to values obtained with expression of VP16.
Induction ratio is expressed as the ratio of induction between pVP16
and pVP16-hsp90.
--
In
a COS-1 cell-based reporter assay, transient expression of XAP2 with
PPAR
resulted in a decrease in PPRE-driven luciferase reporter
activity (Fig. 6A). PPAR
activity was induced by WY-14,643 and was significantly suppressed by
expression of XAP2 in a dose-dependent manner. The
constitutive activity of PPAR
was also moderately repressed by XAP2.
In contrast, the transient expression of FKBP52 did not affect either
the ligand-inducible activity or the constitutive activity of PPAR
(Fig. 6B). This result suggests that XAP2 represses PPAR
transcriptional activity and this effect is unique to XAP2.
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Fig. 6.
XAP2 suppresses PPAR
transcriptional activity. Upper panel, COS-1 cells
were co-transfected with pBK/CMV/PPAR
/FLAG and increasing
concentrations of pCI/XAP2. A subset of cells were treated with 50 µM WY-14,643 for 8 h. Twenty-four h after the start
of transfection, cells were lysed and PPRE-driven reporter activity was
measured. Lower panel, COS-1 cells were co-transfected with
PPAR
/FLAG and increasing concentrations of FKBP52. Twenty-four h
after the start of transfection, cells were lysed and PPRE-driven
reporter activity was measured. Relative luciferase activity was
corrected relative to protein levels.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, an antibody that is highly specific to PPAR
needed to
be produced. Because of the high level of sequence similarity between
the PPAR subtypes, most antibodies have been produced as peptide
antibodies to unique amino acid sequences. We instead took the approach
of making monoclonal antibodies to bacterially expressed full-length
PPAR
and screening potential clones for their specific recognition
of PPAR
relative to PPAR
and PPAR
. This approach has led to
the isolation of one highly specific PPAR
mAb. The results in Fig. 1
indicate that mAb 3B6/PPAR only recognizes PPAR
and is efficient at
both immunoprecipitating and visualization of PPAR
on membranes. The
mAb 3B6/PPAR is also able to efficiently immunoprecipitate PPAR
from
mouse liver cytosolic extracts (Fig. 2B) and thus should be
useful to test for the presence of PPAR
-associated proteins.
interacts with hsp72 in vitro (21), the
association of a hsp90 molecular chaperone with PPAR
has not been
previously established. Co-immunoprecipitation with the hsp90 mAb
3G3p90 has demonstrated that hsp70, p60, FKBP52, p50cdc37 (32), as well as AhR (19) and the
glucocorticoid receptor (33), all associated with this molecular
chaperone. We utilized this approach, and our data suggest that a
strong association exists between hsp90 and PPAR
in mouse liver
cytosol (Fig. 2). Thus, the results in this report are the first
evidence that a type II receptor is bound to a hsp90/co-chaperone complex.
, as has been observed with other steroid receptors. hsp90 binds
near the ligand binding domain of the glucocorticoid receptor (11), and
evidence in this report would indicate that hsp90 is also complexed
near the ligand binding domain of PPAR
. These data suggest that
hsp90 may influence the ligand binding pocket of PPAR
.
Interestingly, because most of the hsp90 resides in the cytoplasm, its
association with a nuclear receptor found predominantly in the nucleus
would appear to require that a significant amount of hsp90 is present
in the nucleus, which has previously been observed (38). Although the
subcellular localization of PPAR
-bound hsp90 is currently not clear,
it is reasonable to hypothesize that the PPAR
-hsp90 complex may also
exist in the nucleus.
remains to be tested. Interestingly,
the molecular chaperone p50cdc37 bound to hsp90
is involved in the folding of a number of protein kinases (13-15) and
has recently been identified in a complex with the androgen receptor
(44). This demonstrates the ability of hsp90 co-chaperones to interact
with different classes of hsp90 client proteins, as has been observed
here with XAP2 and PPAR
.
(Fig. 2B). The ability of
XAP2 to associate with PPAR
was also established in the reticulocyte lysate system (Fig. 3). XAP2 was originally discovered as a protein associated with the X protein of the hepatitis B virus (45). It
contains three TPR motifs and regions of homology to immunophilins FKBP12 and FKBP52. However, unlike FKBP52, XAP2 is unable to bind to
FK506 (46). XAP2 associates with the hepatitis B-virus protein X, the
AhR (28, 47), and the Epstein-Barr virus nuclear protein (EBNA-3) (48),
which is a nuclear antigen that is necessary for B-cell transformation.
Interestingly, XAP2 enhances AhR levels (35, 46) and plays a role in
the cytoplasmic localization of the dioxin receptor (49). XAP2, also
known as ARA-9 and AIP, is highly expressed in spleen and thymus and
minimally expressed in liver, kidney, and lung (28, 50). There are low
levels of XAP2 in the liver, in contrast to the high levels of PPAR
in this organ, which is logical from a biological standpoint when one
considers that the liver is a target tissue for PPAR
activity. Several groups have reported XAP2 as a transcriptional activator for
the AhR (28, 47, 51). In contrast, data presented here clearly suggest
that XAP2 is a potent repressor of PPAR
activity. However, this
result is consistent with the inhibition of hepatitis B virus X protein
transcriptional activity by XAP2 (45). Interestingly, this effect has
not been observed for FKBP52 in glucocorticoid receptor or
progesterone receptor complexes (11). Future studies will examine
whether or not XAP2 and/or hsp90 bind to other members of the PPAR
family of receptors, as well as whether XAP2 plays a role in modulating
tissue-specific activity of PPAR
.
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ACKNOWLEDGEMENT |
---|
We thank Marcia H. Perdew for critically reviewing this article.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant ES07799 from the NIEHS, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom all correspondence should be addressed: Center for Molecular Toxicology and Carcinogenesis, 226 Fenske Laboratory, Pennsylvania State University, University Park, PA 16802. Tel.: 814-865-0400; Fax: 814-863-1696; E-mail: ghp2@psu.edu.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M211261200
2 mAb 3B6/PPAR is available from Affinity Bioreagents (Golden, CO).
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ABBREVIATIONS |
---|
The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; AOx, fatty acyl-CoA oxidase; PPRE, peroxisome proliferator response element; RXR, retinoid X receptor; DEHP, diethylhexylphthalate; AhR, aryl hydrocarbon receptor; FKBP, FK506-binding protein; hsp90, heat shock protein 90; CyP40, cyclophilin 40; XAP2, hepatitis B virus X-associated protein 2; MENGM, MENG buffer + 20 mM sodium molybdate; MENG buffer, 20 mM MOPS, 2 mM EDTA, 0.02% NaN3, 10% glycerol, pH 7.4; MOPS, 4-morpholinepropanesulfonic acid; mAb, monoclonal antibody; IP, immunoprecipitation; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TPR, tetraricopeptide repeat.
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