From the George Whipple Laboratory for Cancer Research, Departments of Pathology, Urology, and Radiation Oncology, University of Rochester Medical Center, Rochester, New York, 14642
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ABSTRACT |
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In an effort to understand transcriptional
regulation by the peroxisome proliferator-activated receptor The peroxisome proliferator-activated receptors
(PPARs)1 are structurally
similar members of the nuclear hormone receptor superfamily that become
transcriptionally active in response to a diverse group of chemical
compounds, including fibrate hyperlipidemic drugs, thiazolidinedione
anti-diabetic drugs, arachidonic acid derivatives, fatty acids, and
peroxisome-proliferating chemicals (reviewed in Ref. 2). These
receptors are considered to play key roles in lipid metabolism and
storage (3). The three mammalian PPAR members ( The PPAR isoforms regulate target gene transcription through binding to
PPAR response elements (PPREs), a DR1-type hormone response element.
This binding is enhanced by heterodimerization to the retinoid X
receptor (RXR) (16, 17). The PPAR-RXR heterodimer is responsive to both
PPAR ligands and the RXR ligand 9-cis-retinoic acid (17,
18). Functional PPREs have been identified in the promoters of a number
of genes including acyl-CoA oxidase (6), phosphoenolpyruvate
carboxykinase (19), CYP4A6 (5), lipoprotein lipase (20), and ap2
(8).
Transcriptional activation or repression by nuclear hormone receptors
can be augmented by transcriptional coactivators and corepressors,
which can serve as a bridge between the nuclear receptor and the basal
transcriptional machinery. A number of coactivators, including ARA70
(1), RIP140 (21), CBP/p300 (22, 23), TIF2 (24), SRC-1 (25), and ARA54
(26) have recently been identified as interacting with one or more
nuclear hormone receptors. These interactions are typically dependent on the presence of ligand. Recently a number of cofactors for the PPAR
isoforms have been identified. SRC-1, PBP, CBP, and PGC-1 (27-30) have
been shown to interact with PPAR Plasmids--
The plasmid expressing the GAL4-DNA binding domain
(DBD) and the mPPAR Coimmunoprecipitation--
Recombinant mPPAR Cell Culture and Transfection--
Human prostate DU145 cells
were grown in Dulbecco's minimal essential medium containing 5% fetal
calf serum at 37 °C. The cells were transfected by modified
BES-calcium phosphate procedure (25). Cells were plated 4 × 105/60-mm Petri dish 1 day before transfection.
Transfection medium contained a constant amount of reporter plasmid and
indicated amounts of pSG5-mPPAR Interaction of ARA70 with PPAR
Interaction between PPAR Site-directed Mutation of ARA70 Attenuates Its Interaction with
PPAR ARA70 Enhances the Transcriptional Activity of PPAR AR Can Squelch ARA70 from PPAR Previously, we have demonstrated that ARA70 is a relatively
specific transcriptional coactivator of the androgen receptor that
shows only a very marginal induction of transcriptional activity by the
estrogen, glucocorticoid, and progesterone receptors in DU145 cells
(1). Here we show that ARA70 is also a coactivator of PPAR Ligand-independent activation by PPAR ARA70 contains a single LXXLL motif at amino acids 92-96.
This motif has been identified in several nuclear receptor coactivators as being involved in the interaction with the receptor AF2 domain. For
example, mutation of the four LXXLL boxes of SRC-1 abolished its ability to interact with the estrogen receptor (35), and the
crystal structure of PPAR The heterodimeric partner of PPAR The relative specificity of ARA70 could allow cross-talk between
PPAR (PPAR
), we have investigated its potential interaction with
coregulators and have identified ARA70 as a ligand-enhanced
coactivator. ARA70 was initially described as a coactivator for the
androgen receptor (AR) and is expressed in a range of tissues including
adipose tissue (Yeh, S., and Chang, C. (1996) Proc. Natl. Acad.
Sci. U. S. A. 93, 5517-5521). Here we show that ARA70 and
PPAR
specifically interact by coimmunoprecipitation and in a
mammalian two-hybrid assay. PPAR
and ARA70 interact in the absence
of the PPAR
ligand 15-deoxy-
12,14-prostaglandin J2, although the
addition of exogenous ligand enhances this interaction. Similarly, in
transient transfection of DU145 cells, cotransfection of PPAR
and
ARA70 induces transcription from reporter constructs driven by either
three copies of an isolated PPAR response element or the natural
promoter of the adipocyte fatty acid-binding protein 2 in the absence
of exogenous 15-deoxy-
12,14-prostaglandin J2. However, this
PPAR
-ARA70 transactivation is enhanced by the addition of ligand.
Thus, ARA70 can function as a ligand-enhanced coactivator of PPAR
.
Finally, we show that AR can squelch PPAR
-ARA70 transactivation, which suggests that cross-talk may occur between PPAR
- and
AR-mediated responses in adipocytes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
) are
each encoded by a separate gene and show a distinct but overlapping
tissue distribution (4). PPAR
is highly expressed in the liver,
kidney, and adrenal gland and has been shown to regulate expression of
genes involved in hepatic lipid metabolism, including the P450 fatty
acid
-hydroxylase gene and genes involved in the peroxisomal
-oxidation pathway (5, 6). PPAR
(also referred to as NUC1 or
FAAR) is expressed ubiquitously and has been reported to repress
transcription by PPAR
and the thyroid hormone receptor (7). PPAR
exists as two isoforms encoded by a single gene, PPAR
1 and PPAR
2
(8). PPAR
2 is transcribed from an alternative promoter and is 30 amino acids longer than PPAR
1. Although PPAR
2 is the predominant
isoform in adipocytes (8), PPAR
1 is also expressed in a number of other tissues including the adrenal gland and spleen (4). Consistent with its adipocyte localization, PPAR
is involved in the regulation of adipocyte-specific genes, including the adipocyte fatty acid binding
protein 2 (ap2) (8). PPAR
is also implicated in adipogenesis (9).
Stable transfection of PPAR
1 or PPAR
2 into fibroblasts induces
them to differentiate into adipocytes (3, 8). Ligands identified for
PPAR
include the insulin-sensitizing thiazolidinedione antidiabetic
drugs and the naturally occurring prostaglandin derivative, 15-deoxy-
12,14-prostaglandin J2 (15dJ2), each of which can promote adipocyte differentiation (10-12). In addition to its role in
adipocyte differentiation and function, PPAR
has recently been
implicated in a number of pathological conditions including
atherosclerosis and colorectal cancer (13-15).
, and p300 has been shown to be a
coactivator for PPAR
(31). The rat dUTPase is a potential
corepressor for all the PPAR isoforms (32). To further understand
transcriptional regulation by PPAR
, we have investigated the
potential interaction between PPAR
and ARA70. ARA70 was originally described as a coactivator of the androgen receptor and shows a broad
tissue distribution of expression, including adipose tissue (1). Here
we present evidence to demonstrate that ARA70 can induce PPAR
target
genes through a PPAR
-ARA70 complex. We also show that PPAR
- and
AR-mediated pathways may be linked through their common use of ARA70.
Unlike previously described receptor-coactivator interactions, ARA70
can confer transcriptional activity to PPAR
in the absence of
ligand, although the presence of ligand enhances PPAR
-ARA70 transactivation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ligand binding domain (pGAL4DBD-mPPAR
) was
constructed by inserting the mouse PPAR
1 ligand binding domain (from
amino acids 162-475), isolated as a ScaI/BamHI
fragment from pGBTmPPAR
1 in-frame into pCMXGal4 DBD. The plasmid
expressing VP16-ARA70 was constructed by inserting a fragment of ARA70
cDNA encoding amino acids 1-401 into the VP16 activation domain
plasmid pCMX-VP16. The site-directed mutagenesis was generated
by using the following four primers: 5'-CCGGAATTCTCAGTCCACCCAAGGTCT-3',
5'-GCTCTACTCGGCAGCGGGCCAGTTCAATTG-3', 5'-GAACTGGCCCGCTGCCGAGTAGAGCGCTG-3', 5'-CGCGGATCCCTCTACCTTACATGGGTC-3'. Mutagenesis was carried out on the ARA70 cDNA fragment
encoding amino acids 1-401 by polymerase chain reaction (33). The
mutated fragment was then reinserted in-frame into pCMX-VP16. The
construction of pSG5-PPAR was done by inserting the
Asp718-SalI fragment from pGBTmPPAR
1 into pSG5.
1, RXR
, AR,
and ARA70 were expressed by the TNT Coupled Reticulocyte Lysate System
(Promega) incorporating [35S]methionine according to the
manufacturer's instructions. Fifteen µl of labeled receptors were
mixed with 50 µl of ARA70 and incubated with 15-20 µl of antibody
to PPAR
(Santa Cruz), RXR
(Santa Cruz), or with the AR antibody
NH27 (34). Proteins and antibody were incubated in 70 µl of HC400 (20 mM Hepes-KOH, pH 7.9, 400 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.1 mg/ml bovine serum albumin) for 1 h at 4 °C before adding 10 µl of protein A-Sepharose beads
(Amersham Pharmacia Biotech). DHT was added to the AR + ARA70 reaction
at a final concentration of 100 nM. The reaction was then
incubated while rocking overnight at 4 °C. Immunoprecipitated
complexes were collected by centrifugation at 2000 rpm at 4 °C for 1 min. The pelleted beads were washed three times with HC400, four times with 10 mM K3PO4, pH 8.0, 0.1 M KCl, and then mixed with SDS sample buffer, boiled, and
separated by 8% SDS-polyacrylamide gel electrophoresis.
, pSG5-hRXR
, andpSG5-hARA70 using
pSG5 as a carrier to provide equal amounts of transfected DNA. One h
before transfection, the medium was changed to Dulbecco's minimal essential medium with 5% charcoal-stripped fetal calf serum, and the
medium was changed again 20 h post-transfection and treated with
steroid hormone or 15dJ2 for another 14-16 h. Cell extracts were
prepared and assayed for CAT or luciferase activity (Promega) and
normalized against
-galactosidase or Renilla luciferase
activity as indicated. All data were the average results from three to six independent experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
To investigate the
potential interaction between ARA70 and PPAR
, we first analyzed
their interaction in a mammalian two-hybrid assay in DU145 cells. DU145
cells were used because they do not express AR, and the subline in our
hands shows a low level of endogenous ARA70 activity to coactivate
AR-mediated transcription. The ligand binding domain of AR, PPAR
,
and RXR were fused to the GAL4 DNA binding domain (GAL4DBD). These were
cotransfected with a vector expressing the VP16 activation domain alone
or linked with ARA70. In agreement with our previous results (1),
coexpression of GAL4DBD-AR with VP16-ARA70 induced CAT activity by
11-fold in the presence of DHT (Fig.
1A, lanes 7 and
8). ARA70 is also able to interact with RXR in the presence
of its ligand, 9-cis-retinoic acid. In contrast,
GAL4DBD-PPAR
cotransfected with VP16-ARA70, did not require the
presence of ligand to induce CAT activity. In this case, CAT activity
was induced 36-fold in the absence of the PPAR
ligand 15dJ2 (10, 11)
and 52-fold in the presence of exogenous ligand (Fig. 1A,
lanes 4 and 10). It has previously been reported
that SRC-1 is a ligand-dependent cofactor of PPAR
(28).
Here we also find that VP16-SRC1 interacts with GAL4DBD-PPAR
to
induce luciferase activity. A slight interaction between SRC-1 and
PPAR
is observed in the absence of ligand, but this is significantly weaker than the ligand-independent activation seen with ARA70 (Fig.
1B, lanes 2 and 3). In the presence of
15dJ2, the interaction between PPAR
and ARA70 is more than twice
that of PPAR
and SRC-1 (Fig. 1B, lanes 5 and
6).
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Fig. 1.
Interaction of ARA70 with hAR,
mPPAR 1, and hRXR
in
ligand-dependent or -independent manner. A,
DU145 cells were transfected with an expression vector expressing a
chimeric protein consisting of the GAL4 DBD linked the hAR, mPPAR
1
or hRXR
nuclear receptor ligand binding domain (2 µg). These cells
were cotransfected with a vector expressing VP16 activation domain
alone or linked to the N-terminal 401 amino acids of ARA70 (4 µg).
Plates were also transfected with the pG5-CAT reporter (3.5 µg) and
pCMV-
-galactosidase as the internal control. After 24 h of
transfection, cells were treated with ethanol or ligand for AR,
PPAR
1, or RXR, that is, 1 nM 5
-DHT, 1 µM 15dJ2, and 1 µM
9-cis-retinoic acid, respectively. Cells were harvested
24 h after treatment of ligands for determination of
-galactosidase and CAT activity. The values are the means ±S.D. of
three experiments. B, DU145 cells were cotransfected with
pGAL4 DBD-mPPAR
(2 µg), the pG5-tk-luciferase reporter plasmid
(2.5 µg), and 4 µg of the VP16 activation domain alone or linked to
either the N terminus of ARA70 (70) or the C terminus of SRC-1 (SRC).
Transfected cells were either mock-treated with ethanol or with 1 µM 15dJ2 as indicated. Renilla luciferase
activity from 2 µg of transfected pRL-tk-luciferase was used as an
internal control. The activity of pG5-tk-luciferase when cotransfected
with GVAL4DBD-mPPAR
,and the VP16 activation domain was set as one.
The results are the mean ±S.D. of three experiments. C,
TNT-translated (Promega) [35S]methionine-labeled
ARA70 full-length protein, hAR, mPPAR
1, and hRXR
were
incubated with the appropriate receptor antibody and protein
A-Sepharose beads as described under "Experimental Procedures."
Input receptor-loaded represents 1/3 of that used in the
immunoprecipitation reaction, and input ARA70-loaded represents 1/10 of
the final used.
and RXR with ARA70 was further confirmed by
coimmunoprecipitation. The receptors and ARA70 were expressed in
vitro using rabbit reticulocyte lysate and coimmunoprecipitated using antibodies specific for AR, RXR, or PPAR
as indicated (Fig. 1C). Although AR coimmunoprecipitated ARA70 only in the
presence of DHT, PPAR
and RXR were able to bind ARA70 even in the
absence of their respective ligands.
and RXR--
A number of nuclear receptor coactivators contain
a LXXLL motif (or NR box) that is considered to play an
important role in nuclear receptor-coactivator interaction (35, 36).
ARA70 contains one NR box (LYSLL) at amino acids 92-96 (Fig.
2A). To investigate whether
the ARA70 NR box influences the PPAR
-ARA70 and RXR-ARA70 interactions, we mutated this region. Mutation of the leucine doublet
to alanines (LYSLL
LYSAA) of ARA70 (mtARA70) was tested in a
mammalian two-hybrid assay in DU145 cells. The VP16-mtARA70 showed
reduced transactivation of both GAL4DBD-RXR and GAL4DBD-PPAR
(Fig.
2B). In the presence of the appropriate ligand, the
VP16-mtARA70 significantly reduced the transactivation of both
receptors compared with the wild type ARA70. In the case of
GAL4DBD-PPAR
, the mutant ARA70 also reduced transactivation in the
absence of ligand (Fig. 2B, lanes 3 and
6). This demonstrates the importance of the ARA70 NR box in
the interaction of ARA70 with both PPAR
and RXR.
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Fig. 2.
Site-direct mutation of ARA70 attenuate the
interaction with nuclear receptors. A, site-directed
mutation of ARA70 was generated by polymerase chain reaction
("Experimental Procedures") to mutate LYSLL, the single
LXXLL motif of ARA70 located at 92-96 amino acids, to
LYSAA. This fragment, containing 401 amino acids of N-terminal ARA70,
was then fused to VP16 for a mammalian two-hybrid assay. B,
DU145 cells were cotransfected with pGAL4DBD-hAR, -mPPAR 1, or -hRXR
as described in Fig. 1A. Cells were cotransfected with
VP16-ARA70 (wt ARA70), the mutated ARA70 construct (mt
ARA70), or the VP activation domain alone. After 24 h, cells
were treated with ethanol or ligand for AR, PPAR
1, or RXR, that is,
1 nM 5
-DHT, 1 µM 15dJ2, and 1 µM 9-cis-retinoic acid, respectively. Cells
were harvested after 16 h of treatment with ligand and assayed for
-galactosidase activity and CAT activity.
--
To
investigate the functional relevance of the interaction between PPAR
and ARA70, we coexpressed PPAR
and ARA70 in DU145 cells. We examined
PPAR
-mediated transcriptional activity on two luciferase reporter
genes, one driven by three copies of a PPRE linked to a TK promoter
(Fig. 3) and another containing 5.4 kilobases of the ap2 gene promoter (Fig.
4). The addition of ARA70 increases the
PPAR
-mediated transcription of both reporter constructs. The
transfection of ARA70 with PPAR
alone or with PPAR
and RXR did
not show significant differences, probably because of abundant endogenous RXR expression in DU145 cells. RXR, like PPAR, binds to a
DR-1-type response element and therefore in theory could activate our
reporter constructs. Transfection of RXR with ARA70 or ARA70 alone did
not result in reporter gene expression, showing that both reporter
genes are activated through PPAR
. In agreement with previous reports
(17, 37), we show that transcriptional activation occurs in response to
either 15dJ2 or 9-cis-retinoic acid and that maximal
transcription by PPAR
·RXR occurs in the presence of both ligands
(Fig. 3). In the presence of both ligands, PPAR
·RXR-ARA70 elicits
a stronger transcriptional response than PPAR
·RXR-SRC-1 (Fig. 4).
Consistent with the mammalian two-hybrid assay and the
coimmunoprecipitation data, we observe that ARA70 can enhance
PPAR
-mediated expression in the absence of exogenous ligand,
although this expression is significantly less than that seen when
ligand is added (Figs. 3A and 4A). This is
potentially because of the presence of endogenous ligand, as has been
suggested for PPAR
transactivation by SRC-1 observed in the absence
of added ligand (28). However, transfection of PPAR
with SRC-1 in
the absence of ligand in our hands shows only background
transcriptional activity (Fig. 4, lane 3), whereas
cotransfection of PPAR
·RXR with SRC-1 shows ligand-independent
activation comparable with that observed for ARA70 (Fig. 4, lane
6). It is unclear whether this indicates an absence of endogenous
ligand in our system or an unanticipated regulatory affect of
overexpressed RXR with SRC-1 on the ap2 promoter. It has recently been
suggested that PPAR
1 and PPAR
2 may have different activation
capabilities, which is determined by their different N termini (38). We
find that ARA70 shows similar increases in transcription with both
PPAR
isoforms (data not shown).
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Fig. 3.
ARA70 increases
PPAR -mediated transactivation of the
(PPRE)3-tk-luciferase reporter construct in
DU145 cells. A, DU145 cells were cotransfected with 3 µg (PPRE)3-tk-luciferase, 1 µg of pSG5-mPPAR
(PPAR), 0.5 µg pSG5-mPPAR
and 0.5 µg pSG5-hRXR
(PPAR:RXR), or 1 µg of pSG5-hRXR
(RXR).
PSG5-hARA70 was transfected at 5 µg, and pSG5 was used to provide
equal amounts of transfected DNA. Cells were mock-treated with ethanol.
B, DU145 cells were transfected as in A but were
treated with 3 µM 15dJ2 (P), 1 µM 9-cis-retinoic acid (R) or both
(P+R). All ligands were dissolved in ethanol. Transfection
efficiency was normalized against the internal control of
Renilla luciferase activity. The activity of
(PPRE)3-tk-luciferase transfected with pSG5-mPPAR
and
mock-treated with ethanol was taken as one. Results are the mean ±S.D.
of three to six independent experiments.
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Fig. 4.
ARA70 and SRC-1 increase the
PPAR -mediated transcription of the
ap2(-5.4)-luciferase reporter construct. A, DU145 cells
were cotransfected as in Fig. 3A using 3 µg of the
ap2(
5.4)-luciferase reporter construct and 5 µg of pSG5-hARA70 or
pSG5SRC-1 as indicated. Cells were mock-treated with ethanol.
B, DU145 cells were transfected as in Fig. 3B
using 5 µg of pSG5-hARA70 or pSG5SRC-1 as indicated with the
ap2(
5.4)-luciferase reporter gene. Results are the mean ±S.D. of
three to six independent experiments.
--
In a previous report (1),
we demonstrated that ARA70 and AR physically interact and that ARA70
can function as an androgen-dependent coactivator for AR.
We were interested to know if AR and PPAR
could compete for ARA70.
Cotransfection of PPAR
, RXR, and AR does not significantly influence
PPAR
·RXR transcription (Fig. 5).
However, AR is able to significantly reduce PPAR
·RXR-ARA70 transcriptional activity even in the presence of 15dJ2 and
9-cis-retinoic acid (Fig. 5, lanes 15 and
16). Potentially, cross-talk between AR and PPAR
could
occur through their functional association with ARA70.
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Fig. 5.
Effect of AR on the
PPAR ·(PPRE)x3-tk-luciferase reporter in the
presence or absence of androgen. DU145 cells were cotransfected
with 3 µg of (PPRE)x3-tk-luciferase reporter, 0.5 µg each of
pSG5-mPPAR
and pSG5-hRXR
, 5 µg of pSG5-hARA70, and 1 µg
pSG5-AR as indicated. Cells were either mock-treated with ethanol, with
3 µM 15dJ2 and 1 µM
9-cis-retinoic acid (P+R), or with 10 nM DHT as
indicated. The data are the mean ±S.D. of three independent
experiments. P+R, 9-cis-retinoic acid and
15dJ2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Unlike
the AR-ARA70 interaction, which requires the presence of androgen, the
PPAR
-RXR-ARA70 interaction occurs in the absence of exogenous ligand
as determined by a mammalian two-hybrid assay and by
coimmunoprecipitation. Cotransfection of PPAR
and ARA70 also results
in low level constitutive expression from an ap2 promoter and an
isolated PPRE-driven construct. However, in both the mammalian
two-hybrid system and the transfection studies, the transcriptional
activation of the reporter genes could be further enhanced by the
addition of the appropriate ligands, demonstrating that the binding of
ligand is still necessary for maximal transcriptional response.
Interestingly, in the presence of ligand, ARA70 mediates a greater
increase in transcriptional activity than SRC-1, suggesting that in
adipose tissue it may be the more important coactivator of PPAR
.
Recently, CBP and PBP have been identified as cofactors of PPAR
(27,
30). CBP and PBP demonstrate ligand-independent physical interaction
with PPAR
but require ligand to show a transcriptional effect. This
suggests that although unliganded PPAR
may be permissive to some
cofactor interactions, the nature of its interaction with ARA70 is
different in that it allows constitutive transcription.
in the presence of a cofactor
has also been observed for SRC-1 (28). It is possible that these
effects are due to the presence of an endogenous ligand. However,
transfection of PPAR
and SRC-1 in our system does not lead to
transcription above background, suggesting that ARA70 alone induces a
low level of PPAR
-mediated transcription that is further enhanced in
the presence of ligand. Ligand-independent transcription is not seen
between AR and ARA70 (1), suggesting that the manner in which ARA70
interacts with PPAR
and AR may be different. ARA70 may have a
limited ability to stabilize the AF-2 helix of PPAR
in an
"active" conformation (similar to a liganded receptor) to allow
transcription to occur but may be unable to do this with AR.
Ligand-independent activation has been shown for several nonmutated
steroid receptors. Both the estrogen and progesterone receptors can
induce transcription in the absence of ligand when phosphorylated (39,
40). Alternatively, the unliganded receptor may bind to a
transcriptional repressor and allow constitutive expression, as is the
case when the thyroid hormone receptor binds p53 to reverse repression
in some cell types (41). The role of phosphorylation of PPAR
via the
mitogen-activated protein kinase pathway is unclear, with some authors
showing phospho-PPAR
having a negative transcriptional effect (42)
and others showing phospho-PPAR
can activate transcription in the
absence of endogenous ligand (43). However, in DU145 cells,
transfection of PPAR
carrying a mutated mitogen-activated protein
kinase target site or incubation with inhibitors of the
mitogen-activated protein kinase pathway does not alter the
ligand-independent activation of PPAR
in the presence of ARA70, but
it does decrease the ligand enhanced
effect.2 The possibility that
ARA70 itself may possess kinase activity is currently being
investigated. It is possible that the PPAR
·ARA70 complex may bind
to a repressor and thus allow constitutive expression of our reporter
constructs. COUP-TF has been identified as a potential repressor of
some PPAR
target genes and could possibly be subject to this
interaction (2, 44).
with SRC-1 has been determined (45). The
importance of this motif in PPAR
-ARA70 and RXR-ARA70 interactions
was confirmed by mutating the conserved leucine doublet to alanines
(LYSLL
LYSAA). The mutant ARA70 showed a decreased interaction with
both PPAR and RXR in a mammalian two-hybrid assay. In the case of
PPAR
, the mutant ARA70 showed a decreased interaction in the
presence and absence of 15dJ2. This suggests that
ligand-dependent and -independent interactions between
PPAR
and ARA70 both occur through the same ARA70 domain. However,
more detailed interaction data between PPAR
and ARA70 is needed to
confirm this possibility.
, RXR, also interacts with ARA70
but in a ligand-dependent manner. PPAR
-RXR-mediated
transcription can be activated by the addition of RXR ligands (Fig. 3
and Ref. 28). It is therefore possible that in vivo PPAR
and RXR may independently recruit ARA70 to contribute to
transactivation by the heterodimer, as has been reported for SRC-1
(28). Whether ARA70 can enhance transactivation by RXR-RAR heterodimers
is currently under investigation.
and AR in tissues where they are coexpressed with ARA70, as is
the case in adipocytes. We have shown that when PPAR and AR are
cotransfected with ARA70, DHT treatment reduces the expression of a
PPAR
reporter gene. This may indicate that liganded AR is able to
compete with PPAR
for ARA70 to differentially regulate their
respective target genes. PPAR
and AR are both known to mediate
adipose tissue metabolism and have both been implicated in relation to
insulin sensitivity. It has been demonstrated that activators of PPARs,
and particularly of PPAR
, promote adipocyte differentiation in
preadipocytes (46) and in the multipotential cell line C3H10T1/2 (10).
Stable transfection of PPAR
can induce differentiation of
fibroblastic NIH 3T3 cells to adipocytes in the presence of PPAR
ligands (9, 11). PPAR
is also known to transcriptionally activate a
number of adipocyte-expressed genes including lipoprotein lipase (20)
and ap2 (8). Testosterone affects the regulation of lipolysis by
increasing
-adrenoreceptors and regulating adenylate cyclase
activity (47). Testosterone in some studies downregulates lipoprotein
lipase (48-50). The differential expression of lipoprotein lipase may
contribute to gender-specific regional fat distribution (47, 48). The
thiazolidinedione class of drugs increase insulin sensitivity in
patients with noninsulin-dependent diabetes and are known
to be PPAR
ligands (11, 12), although the mechanism of this affect
is unknown. Aberrant levels of testosterone have been associated with
insulin resistance. Female rats treated with testosterone or human
females with polycystic ovary syndrome (which results in higher levels
of circulating androgens) show an increased incidence of insulin
resistance (51, 52). In males, low levels of androgens have been
correlated with insulin resistance. Castrated male rats or hypogonadal
men have increased insulin resistance, which can be improved by low
level testosterone treatment (49, 50). That PPAR
and AR share a
coactivator suggests that cross-talk between the two receptor-mediated
pathways may occur. However, further investigation is necessary to
establish the degree of this possible interaction in
vivo.
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ACKNOWLEDGEMENTS |
---|
SRC-1 was a gift from Dr. B. W. O'Malley (Baylor College of Medicine, TX) and the ap2(-5.4)-luciferase reporter plasmid was a gift from Dr. B. Zhang (Merck, NJ). We also thank Karen Wolf for the critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA55638, CA68568, and CA77532.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: George Whipple Laboratory for Cancer Research, Dept. of Pathology, Urology, and Biochemistry, University of Rochester Medical Center, Box 626, 601 Elmwood Ave., Rochester, NY, 14642. Tel.: 716-273-4500; Fax: 716-756-4133; E-mail: chang{at}pathology.rochester.edu.
2 C. A. Heinlein and C. Chang, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
PPAR, peroxisome
proliferator-activated receptor;
PPRE, peroxisome proliferator response
element;
mPPAR, mouse PPAR;
DBD, DNA binding domain;
15dJ2, 15-deoxy-12,14 prostaglandin J2;
AR, androgen receptor;
hAR, human
AR;
RXR, retinoid X receptor;
SRC-1, steroid receptor coactivator 1;
ap2, adipocyte fatty acid-binding protein 2;
DHT, dihydroxytestosterone;
CAT, chloramphenicol acetyltransferase;
BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid;
TK, thymidine
kinase;
CBP, cAMP response element-binding protein-binding protein;
PBP, PPAR
-binding protein.
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REFERENCES |
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