(Received for publication, April 8, 1997, and in revised form, May 19, 1997)
From the Department of Medicine, Division of Endocrinology, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215
Peroxisome proliferator-activated receptor
(PPAR
) is a member of the nuclear hormone receptor superfamily,
and is an important regulator of adipogenesis and adipocyte gene
expression. PPAR
exists as two isoforms, PPAR
1 and PPAR
2, that
differ only in their N termini. Both isoforms are activated by ligands
that include the antidiabetic thiazoladinedione drugs and
15-deoxy-
12, 14-prostaglandin J2, and potential
differences in their function have yet to be described. We report that,
in addition to a ligand-activated transcriptional activity, when
studied under conditions of ligand depletion, intact PPAR
has a
ligand-independent activation domain. To identify the basis for this
ligand-independent activation, we used GAL4-PPAR
chimeric expression
constructs and UAS-TK-LUC in CV1 cells and isolated rat adipocytes. In
both cell systems, isolated PPAR
1 and PPAR
2 N termini have
activation domains, and the activation function of PPAR
2 is
5-6-fold greater than that of PPAR
1. Insulin enhances the
transcriptional effect mediated by both PPAR
1 and PPAR
2
N-terminal domains. These data demonstrate that 1) PPAR
has an
N-terminal (ligand-independent) activation domain; 2) PPAR
1 and
PPAR
2 N termini have distinct activation capacities; and 3) insulin
can potentiate the activity of the N-terminal domain of PPAR
.
The peroxisome proliferator-activated receptor (PPAR
)1 is a member of the
nuclear receptor superfamily that plays a pivotal role in the molecular
determination of adipogenesis and the regulation of adipocyte gene
expression (1-5). Under appropriate conditions, expression of PPAR
through retroviral infection of fibroblastic cell lines is sufficient
to cause differentiation along an adipocyte lineage, as assessed by
expression of adipocyte-specific genes, accumulation of lipid, and
acquisition of adipocyte morphology (6). Recently, it has been shown
that 15-deoxy-
12, 14-prostaglandin J2 (PG J2) is a high
affinity ligand for PPAR
(7, 8) and that PPAR
is also the
receptor for the thiazoladinedione class of insulin-sensitizing drugs
(7, 8). PPAR
resembles other members of the nuclear receptor
superfamily in that ligand-dependent receptor activation
alters the rates of transcription of genes, specifically those that
have peroxisome proliferator response elements (PPREs) within their
promoters (e.g. aP2, phosphoenolpyruvate carboxykinase, and
uncoupling protein) (9-11).
PPAR exists as two isoforms, PPAR
1 and PPAR
2, that differ only
in their N termini, with PPAR
2 having an additional 30 amino acids
that are encoded by a single exon (9, 12). Expression of mRNA
encoding the two isoforms is driven by alternative promoters within a
single PPAR
gene (12), and their expression is differentially regulated in a tissue-specific manner. PPAR
2 is most abundantly expressed in adipocytes and is relatively specific for this tissue (9,
13). In contrast, while PPAR
1 is also expressed at a high level in
adipocytes, it is also found at significant but lower levels in a
number of other tissues, including muscle (13-15). Considering the
relative abundance of PPAR
1 in many nonadipose tissues, it is likely
that this isoform is capable of subserving roles apart from regulation
of adipogenesis. In addition, although no functional differences
between the
1 and
2 isoforms have been described to date, it is
possible that these isoforms subserve different functions under some
conditions. However, deletion of the N-terminal 129 amino acids of
PPAR
did not diminish the adipogenic potency of PPAR
that was
introduced into 3T3 fibroblasts by retroviral infection (6), and it has
therefore been viewed as unlikely that the N terminus of PPAR
subserves a functionally important role.
Here, we provide evidence that PPAR, in addition to being activated
in a ligand-dependent manner, can also be activated in a
ligand-independent manner, and we define a ligand-independent activation domain within the N terminus of PPAR
. We also demonstrate the first potential functional difference between the two PPAR
isoforms, wherein the N terminus of PPAR
2 more potently activates a
heterologous promoter than does PPAR
1. We have mapped the overall activation domain to a region common to the two isoforms and
demonstrate that the 30 amino acids unique to PPAR
2 can activate a
heterologous promoter only in concert with the main N-terminal
activation domain. Finally, we provide evidence that the
ligand-independent activation function of the N terminus of PPAR
is
augmented when cells are treated with insulin. We propose a model for
the activation of PPAR
and discuss its possible implications.
The PPRE reporter construct consisted
of two copies of the DR1 element upstream of the TK109 promoter in the
vector pA3Luc (16). All GAL4 constructs were constructed by
inserting a PCR-generated fragment into the GAL4 vector (17). The 1
N terminus,
2 N terminus, each of the GAL4 constructs made to map
the activation domain, and the PPAR
N terminus were constructed in a
similar manner. 5
primers were designed to contain an EcoRI
(GAATTC) site after a random pentamer (CGCGG) to ameliorate restriction digestion. 3
primers were designed to contain a stop codon before a
PstI (CTATAG) site and the same pentamer. The
C-terminal-GAL4 construct was designed in a similar manner except both
primers contained a BamHI (GGATCC) site instead of
EcoRI and PstI. All restriction sites were in
frame with the template, which kept an open reading frame from GAL4
through the entire PCR fragment. PCR was performed under the following
conditions: after heating to 94 °C, buffer and enzyme (Takara) were
added, and 30 PCR cycles were performed, each as follows: 94 °C for
30 s, 54 °C for 1 min, and 72 °C for 1.5 min. Each PCR was
purified from the buffer and enzyme using Qiaquick (Qiagen) and
digested with appropriate restriction enzymes, run on an agarose gel,
purified, and ligated into the GAL4 vector, which has been previously
linearized using the same enzymes, thus keeping the GAL4 DNA binding
domain (DBD) in frame with the appropriate PCR fragment. UAS-TK-Luc
contains five copies of the 17-base pair upstream activating sequence
upstream of TK luciferase (14). The integrity of each construct was
confirmed by restriction endonuclease digestion and dideoxy sequencing. All plasmids for transfection were prepared using column (Qiagen) purification. The murine PPAR
2 plasmid and the murine PPAR
plasmid, which were used as templates for PCR reactions, were a gift
from B. M. Spiegelman.
Adipocytes were isolated from male Sprague-Dawley rats
(8-10 weeks) using a procedure previously described with modifications (18). Briefly, epididymal adipose tissue was minced and digested in
Krebs Ringer buffer containing 1.5 mg/ml collagenase (Worthington) and
1% bovine serum albumin (Intergen) at 37 °C for 30 min. The adipocytes were strained through nylon mesh and washed three times with
Krebs Ringer buffer containing 1% bovine serum albumin and three more
times with Dulbecco's modified Eagle's medium containing 2 mM glutamine, 400 mM
N6-[R-()-1-methyl-2-phenyl]adenosine,
25 mM HEPES, 100 units/ml penicillin, and 100 µg/ml
streptomycin (Life Technologies, Inc.). After the final wash, cells
were resuspended as a 50% solution in Dulbecco's modified Eagle's
medium. Adipocytes were transfected by electroporation as described
previously (19), with some modifications (electroporation at 400 V, 500 microfarads). We used 6 µg of UAS-TK-Luc and 1.5 µg of empty GAL4,
PPAR
1 N terminus GAL4, or
2 N terminus GAL4. The incubation was
carried out at 37 °C in 5% CO2. 2 h after electroporation, Dulbecco's modified Eagle's medium with 3.5% bovine
serum albumin (final concentration) was added, and the indicated cells
were treated with 10 nM insulin. The cells were harvested
for luciferase activity 20 h after transfection.
CV-1 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
L-glutamine, 10% fetal calf serum, 100 µg/ml penicillin,
and 0.25 µg/ml streptomycin. Transient transfections were performed
using the calcium phosphate technique in six-well plates, with each
well receiving 1.60 µg of reporter and 80 ng of the 1 N terminus,
the
2 N terminus, each of the indicated constructs containing a part
of the N terminus, or empty GAL4 vector. 20 h after transfection,
the cells were washed with phosphate-buffered saline and refed with
Dulbecco's modified Eagle's medium with 10% fatty acid/growth
factor-depleted fetal bovine serum and the indicated concentration of
BRL49653. To deplete PPAR ligands and serum growth factors, fetal
bovine serum was treated for 24 h at 4 °C with 5 mg/ml of
activated charcoal (Sigma) and 30 mg/ml of anion exchange resin (type
AGX-8, analytical grade, Bio-Rad). After centrifugation, anion exchange
resin was added again for an additional 5 h. The resulting fetal
bovine serum was centrifuged again and filtered before use. 44-48 h
after transfection, the cells were harvested in extraction buffer and
assayed for luciferase activity (20). All experiments were performed in
triplicate and repeated between 2-6 times. The results shown are the
mean ± the S.E.
CV-1 cells were passed to six-well dishes the day before transfection and transfected in duplicates as described above. Cells were harvested 72 h post-transfection by aspirating the medium, rinsing in ice-cold phosphate-buffered saline, and scraping into 250 µl of ice-cold lysis buffer (1% Nonidet P-40, 0.5% Triton X-100, 10% glycerol, 150 mM NaCl, 2 mM Na3VO4, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 50 mM Tris-HCl, pH 7.4). The lysate was clarified by centrifugation at 23,000 × g for 15 min, and finally 125 µl of 3 × Laemmli buffer (21) was added to the supernatant.
Proteins were boiled for 5 min and subjected to SDS-polyacrylamide gel electrophoresis (21), followed by transfer of the resolved polypeptides to nitrocellulose membranes using the system of Towbin et al. (22). The membranes were blocked with 10% nonfat dried milk in Towbin buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) for 2 h at room temperature and then incubated with a polyclonal anti-yeast GAL4 DNA binding domain antibody (Upstate Biotechnology, Inc., Lake Placid, NY) (1:1000) in 5% milk overnight at 4 °C. After removal of unbound antibodies by three washes of 20 min in Towbin buffer at room temperature, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (1:1000) in 2.5% milk for 1.5 h at room temperature and washed five times in Towbin buffer. The targeted proteins were detected using enhanced chemiluminescence (ECL) as described by the manufacturer (Amersham International, Buckinghamshire, UK).
We first assessed the capacity of PPAR to mediate
transcriptional activation under conditions of varying availability of endogenous ligand. To do this, we transfected CV-1 cells with an
expression plasmid for PPAR
2 and assayed its ability to activate a
reporter plasmid consisting of a PPRE upstream of a thymidine kinase
(TK) promoter and the luciferase reporter gene. In an attempt to limit
the availability of PPAR
ligand, we charcoal-stripped the serum
twice before use (Fig. 1A).
Despite this treatment of the serum, PPAR
transactivated the
reporter plasmid in the absence of added ligand. Activity was further
enhanced by the addition of the thiazoladinedione BRL49653, which is
known to bind to and activate PPAR
(7, 8). Since this basal
activation could have been due to residual ligand in the serum or to
ligand produced by the cells during the course of the experiment, we
constructed a plasmid containing the yeast GAL4 DBD upstream of the
isolated PPAR
ligand binding domain (amino acids 193-505). This
construct was co-transfected with a reporter gene containing the yeast
GAL4 response element upstream of the TK promoter and luciferase as a
reporter (UAS-TK-LUC), under serum and cell conditions identical to
those of the previous experiment (Fig. 1B). Although BRL was able to activate this construct very efficiently, indicating the intactness of the ligand binding domain, there was no activation in the
absence of BRL, suggesting that the cellular level of endogenous ligand
was extremely minimal under these conditions. The results additionally
suggest that the high level of reporter activation mediated by the
full-length PPAR
in the presence of the same stripped serum and
without any added ligand was indeed ligand-independent and did not map
to the ligand binding domain. To further explore the possible existence
of a ligand-independent activation domain in PPAR
, we constructed
two separate plasmids containing sequence encoding the N termini of
PPAR
1 or -2 (amino acids 1-98 and amino acids 1-128, respectively)
downstream of GAL4's DNA binding domain. Upon cotransfecting each of
these constructs with UAS-TK-LUC, reporter activation was seen with
both N termini, with the activation by the PPAR
2 N terminus being
about 5-fold greater than that of PPAR
1 (Fig.
2). In Western blotting experiments,
using antibodies against the GAL4 DBD of the proteins, the expression
levels of the two fusion proteins were the same (Fig.
3). PPAR
1 DBD and PPAR
2 DBD
migrated with molecular masses of ~32 and ~36 kDa, respectively.
It is possible that the N-terminal 30 amino acids that are unique to
PPAR2 define a self-contained transactivation domain that might
fully account for the difference between the activation potency of the
two isoforms. A construct containing amino acids 1-30 of PPAR
2
downstream of the GAL4 DBD was therefore tested by co-transfection with
UAS-TK-LUC. This construct failed to show any transactivation ability
on its own (Fig. 4). We therefore further
dissected the N terminus of PPAR
into several constructs, each
containing a unique segment of the PPAR
N terminus in the same GAL4
fusion construct (Fig. 4). The activation domain maps to the region
common to the two isoforms. Although the 30 N-terminal amino acids had
no activation ability on their own, constructs lacking those 30 amino
acids were consistently less active, suggesting a role for this region
in the overall active conformation of the domain. Interestingly, amino
acids 99-129 seemed to repress the ability of these constructs to
activate transcription. Together, these results suggest a complex basis
for the structural determinants of the PPAR
ligand-independent
activation domain.
To test whether N-terminal activation ability was present in a
structurally homologous molecule, we constructed a plasmid containing
the A/B domain of PPAR (amino acids 1-72) downstream of GAL4's
DBD, and this construct was unable to activate transcription of the
reporter gene (Fig. 2B). Since the extent of transcriptional activation mediated by the PPAR
N termini might be dependent on the
presence of tissue-specific co-activators, we transfected the PPAR
2
N-terminal constructs together with UAS-TK-LUC into freshly isolated
rat adipocytes (Fig. 5A). The
PPAR
2 N terminus showed a greater fold activation in adipocytes, the
cell most relevant for PPAR
2 function, than in CV-1 cells. To
determine whether the ligand-independent transcriptional activation
mediated by PPAR
2 is regulated by insulin, we added 10 nM insulin to the transfected cells (Fig. 5B).
Insulin enhanced the ligand-independent activation mediated by both
PPAR
1 and -2 constructs, while having little or no effect on the
vector containing the GAL4 DBD alone.
In this report we show that PPAR resembles other members of the
nuclear receptor superfamily in having the capacity to activate transcription in a ligand-independent manner. We have shown this in a
number of ways. First, by co-transfecting intact PPAR
with a
reporter plasmid under conditions designed to limit the availability of
endogenous PPAR
ligand, we found that PPAR
activated
transcription constitutively. To determine whether this activation was
caused by residual endogenous ligand in the cells or media, and to
identify a possible activation domain in the N terminus, we utilized
the sensitive GAL4/UAS system. First, we created a construct that included PPAR
's C terminus with both the LBD and its activation domain, but without the DNA binding and N-terminal domains. By cotransfecting this plasmid with the reporter UAS-TK-LUC, we created a
sensitive assay for the presence of PPAR
ligand. Using this paradigm, we showed that, under identical cell and serum conditions to
those of the experiment with intact PPAR
, this construct showed no
transactivation activity. However, the addition to these cell transfectants of the PPAR
ligand BRL49653 produced more than 50-fold
activation. Taken together, these results suggest that the activation
seen with intact PPAR
in the presence of charcoal-stripped serum and
without added ligand is due neither to residual ligand nor to a
constitutively active ligand binding domain.
To pursue this possibility further, we created chimeras containing the
GAL4 DBD with PPAR1 and -2 A/B domains, in the absence of their DNA
binding, ligand-binding, and C-terminal activation domains. Using these
constructs, it is apparent that the N termini of both PPAR
1 and -2 do indeed contain activation domains, as assessed by the ability of
these constructs to activate transcription of a GAL4-responsive
reporter gene. Thus, we have shown that PPAR
can enhance
transcription in both a ligand-dependent and
ligand-independent manner.
The potential importance of this ligand-independent activation is worthy of discussion. Many members of the nuclear receptor superfamily, including receptors for estrogen, progesterone, thyroid hormone, glucocorticoids, vitamin D3, and retinoic acid contain two distinct domains responsible for activating transcription of specific genes (23). A critical activation domain is present in the receptor C terminus (the E domain), and this is activated upon binding of the cognate hormone/ligand to the adjacent ligand binding domain (reviewed in Ref. 24). In addition to this ligand-activated function, these receptors can also activate transcription independent of ligand binding, via an activation domain that resides within their N termini (the A/B domain). This domain can be constitutively active and/or be regulated independently of ligand via phosphorylation (25, 26). Ligand-independent activation (AF-1) domains have been shown to have several attributes. In some cases they act synergistically with the ligand-dependent activation domain, as with the human estrogen receptor in HeLa cells (27), where neither activation domain in isolation was capable of activating transcription significantly but both domains together produced marked activation. The ligand-independent activation domain may also be necessary for the regulation of complex cellular events, as in the case of glucocorticoid-induced apoptosis in lymphocytes (28). Transfection of glucocorticoid receptors in which the N terminus was deleted ablated the apoptosis caused by the intact receptor, while the same receptor remained responsive to a number of other dexamethasone effects to induce gene expression, suggesting that regulation of different genes may be mediated by distinct activation domains of the same receptor (29). In the case of the vitamin D3 receptor, it has been shown that mutating serine 51 and thus preventing phosphorylation reduces activity (30), suggesting that phosphorylation/dephosphorylation may be involved in regulation of this activation domain.
These data also provide evidence that the two PPAR isoforms may have
differential abilities to activate target genes. To date, there have
been no reports of functional differences between the PPAR
1 and 2 isoforms, which have been described as having very similar capacities
to be activated by known activators in co-transfection assays (14) and
to be fully capable of bringing about the adipogenic program of
differentiation in 3T3 cells under appropriate conditions. Here, we
show that the N terminus of PPAR
2 is much more potent at activating
a reporter independent of ligand than is the N terminus of PPAR
1.
Interestingly, this difference between the two PPAR
isoforms is even
more pronounced after transient transfection into isolated rat
adipocytes, cells that normally express markedly higher levels of
PPAR
2 than other tissues (1, 13). Whether this observation is due to
higher expression in adipocytes of relevant but currently unknown
co-activators is not known at this time. It is interesting to note that
in adipocytes from both rodents (13) and humans (15), PPAR
2 is the
isoform whose expression is more influenced by obesity and nutritional perturbations, while the expression of PPAR
1 is relatively stable under these conditions in vivo. The preferential regulation
of PPAR
2 expression in physiologic states such as starvation is likely to serve a physiologic purpose that would not be evident if the
two isoforms had identical profiles of biologic activity.
Tontonoz et al. (6) have shown that deletion of amino acids
1-127 in the N terminus of PPAR2 does not reduce, but actually increases, the capacity of retrovirally expressed PPAR
2 to induce adipocyte differentiation of NIH-3T3 cells, as assessed by lipid accumulation and expression of adipose-specific genes. This could be
viewed as conflicting with our results by suggesting that the N
terminus does not play a physiological role in vivo. This
apparent conflict can be resolved in several ways, however. First,
Tontonoz et al. (6) performed their studies under conditions
of excess ligand, including ETYA, which was at the time the best
PPAR
activator, and they used nonstripped serum, another potential
source of PPAR
ligand. Thus, while their results demonstrate that
under conditions of abundant ligand, the N terminus of PPAR
is not
necessary for fat cell differentiation, they do not conflict with the
possibility that the N-terminal activation domain of PPAR
plays an
important role under distinctly different conditions, i.e.
when ligand is not abundant. Studies of differentiation induced by
isolated PPAR
N termini using the retroviral approach will be
necessary to further resolve the ability of the PPAR
N terminus to
bring about the full program of adipogenesis or perhaps reveal an
adipogenesis-unrelated role.
Finally, we observed in both CV-1 cells and isolated rat adipocytes
that insulin is capable of regulating the ligand-independent transcriptional activity of PPAR through its N-terminal activation domain. To understand the possible mechanism for this finding, it is
necessary to review recent observations on the regulation of PPAR
activity by covalent modification. Shalev et al. (31) demonstrated that PPAR
is a phosphoprotein and that phosphorylation is capable of enhancing its transcriptional activation potency, although mapping of the responsible sites was not carried out. Zhang
et al. (32) showed that insulin treatment of cells enhanced the ability of full-length PPAR
2 to stimulate aP2 gene expression and speculated that this might be mediated by the mitogen-activated protein kinase-dependent phosphorylation of PPAR
that
they also demonstrated. In contrast, Hu et al. (33) clearly
demonstrated that mitogen-activated protein
kinase-dependent phosphorylation of PPAR
2 took place on
serine 112 in the N-terminal activation domain in response to mitogens,
but this phosphorylation inhibited, rather than stimulated, the ability
of full-length PPAR
2 to promote specific gene expression and the
process of adipogenesis. Very recently, Adams et al. (34)
reported that mutation of the consensus mitogen-activated protein
kinase site (serine 82) of PPAR
1 to alanine resulted in increased
ligand-dependent transcriptional activity. In addition,
these authors also described a weak constitutive transcriptional
activity of the isolated PPAR
1 N terminus, and this was also
increased when serine 82 was mutated. Whether or not the activity of
insulin to enhance transcription by intact PPAR
2 (32) or PPAR
2 N
terminus as reported here is mediated by phosphorylation of PPAR
2,
it is clear that signaling by PPAR
and insulin produce a number of
common effects, including the ability to promote adipocyte
differentiation (35, 36). Another indication of convergent pathways for
these molecules is the fact that the insulin-sensitizing
thiazoladinediones are now known to act by binding to and activating
PPAR
(7, 8). The data presented here suggest a novel molecular basis
for such a link, i.e. through convergence of their signaling
pathways by an ability of insulin to enhance the function of the AF-1
activation domain of PPAR
, whether by direct phosphorylation or more
likely through some other mechanism, such as an ability of insulin to
modify a co-activator protein.
Interestingly, our mapping experiments of the N-terminal activation
domain provide evidence for a complex mechanism by which PPAR's N
terminus can influence transactivation. Thus, the PPAR
2 GAL4-(1-128) construct was less active than the PPAR
2
GAL4-(1-99), and the PPAR
2 GAL4-(1-66) construct was less active
than the PPAR
2 GAL4-(31-99). Although such results could be the
consequence of a number of factors, they could be consistent with the
existence of an N-terminal repression moiety under some conditions.
Prior to these studies and the report of Adams et al. (34),
a ligand-independent N-terminal activation domain had not been described in the PPAR gene family. Our results raise many questions about the functional roles of the ligand-independent and
-dependent PPAR activation domains under varying
metabolic conditions that are likely to confront adipocytes and other
cells that express these receptors. For example, it is possible that,
under conditions of abundant PPAR
ligand, PPAR
would favor
differentiation by causing growth arrest and transcription of adipocyte
genes via the ligand-dependent AF-2 domain, whereas when
ligand is limiting, as might be predicted to occur during starvation,
PPAR
would act on promoters of genes needed for basal adipocyte
homeostasis via the ligand-independent AF-1 domain. Such speculations
on the possible functional domains of PPAR
will now need to be
tested experimentally.
In summary, we have demonstrated that PPAR, like many other members
of the steroid receptor superfamily, contains a ligand-independent activation domain in its N terminus. We have further defined a functional difference between the two PPAR
isoforms; PPAR
2, which
is expressed mainly in adipose tissue, activates ~5-10-fold more
potently via its AF-1 activation domain than does PPAR
1. Lastly, we
have shown that the N-terminal ligand-independent activation domain can
be regulated by insulin, providing a new basis for the convergent
signaling pathways of these molecules. Together, these results suggest
a more complex regulation of PPAR
action than previously known and
raise the possibility that differential regulation of PPAR
isoforms
in vivo might account for different molecular activities and
phenotypes.