(Received for publication, November 4, 1996, and in revised form, December 12, 1996)
From the Department of Medicine, University of
Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom
and the ¶ Division of Endocrinology, Diabetes and Metabolism,
Departments of Medicine, Genetics, and Pharmacology, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
The nuclear receptor peroxisome
proliferator-activated receptor (PPAR
) regulates transcription
in response to prostanoid and thiazolidinedione ligands and promotes
adipocyte differentiation. The amino-terminal A/B domain of this
receptor contains a consensus mitogen-activated protein kinase site in
a region common to PPAR
1 and -
2 isoforms. The A/B domain of human
PPAR
1 was phosphorylated in vivo, and this was abolished
either by mutation of serine 84 to alanine (S84A) or coexpression of a
phosphoprotein phosphatase. In vitro, this domain was
phosphorylated by ERK2 and JNK, and this was markedly reduced in the
S84A mutant. A wild type Gal4-PPAR
(A/B) chimera exhibited weak
constitutive transcriptional activity. Remarkably, this was
significantly enhanced in the S84A mutant fusion.
Ligand-dependent activation by full-length mouse PPAR
2 was also augmented by mutation of the homologous serine in the A/B
domain to alanine. The nonphosphorylatable form of PPAR
was also
more adipogenic. Thus, phosphorylation of a mitogen-activated protein
kinase site in the A/B region of PPAR
inhibits both
ligand-independent and ligand-dependent transactivation
functions. This observation provides a potential mechanism whereby
transcriptional activation by PPAR
may be modulated by growth factor
or cytokine-stimulated signal transduction pathways involved in
adipogenesis.
Adipocyte differentiation is a complex process regulated by
extracellular hormone and cytokine stimulation (1). Cultured preadipocyte cell lines differentiate into lipid-laden adipocytes following exposure to insulin, glucocorticoid, and inducers of intracellular cyclic AMP (2). Conversely, epidermal growth factor
(EGF)1 and transforming growth factor act via the EGF receptor to inhibit both primary and preadipocyte cell
line differentiation (3, 4). Tumor necrosis factor
(TNF
) is also
a potent inhibitor of differentiation. In addition, this cytokine
promotes lipolysis and down-regulates adipocyte-specific gene
expression in mature adipocytes (5, 6).
Adipocyte differentiation is driven by the coordinate expression of a
range of transcription factors, including C/EBP, -
, and -
(7)
and ADD1 (8), which lead to the expression of adipocyte-specific genes.
In addition, the peroxisome proliferator-activated receptor
(PPAR
) has been shown to be selectively expressed in adipocytes (9,
10) and to modulate adipocyte-specific gene expression (10).
Alternative splicing generates two receptor isoforms such that mouse
PPAR
2 has a 28-residue extension at its amino terminus compared with
human PPAR
1. PPAR
2 mRNA is highly expressed in murine
adipocyte cell lines (10), whereas both receptor isoforms are abundant
in freshly isolated mouse adipocytes (11). The early induction of
PPAR
mRNA expression during adipogenesis, combined with the
ability of retrovirally overexpressed PPAR
to induce lipid
accumulation and the expression of adipocyte-specific genes (12),
suggests that this receptor plays an important role in preadipocyte
differentiation. This notion is strengthened by the observation that
specific high affinity ligands for PPAR
including thiazolidinediones
(which act as insulin sensitizers in vivo), as well as
eicosanoids, promote the differentiation of murine preadipocyte cell
lines (13-15).
PPAR is an orphan member of the nuclear receptor family. These
receptors share a conserved domain structure and modulate gene
transcription through two transcription activation mechanisms: a
hormone-dependent transcription activation function (AF-2)
is located in the carboxyl-terminal hormone-binding domain, whereas the
amino-terminal A/B domain contains a ligand-independent activation function (AF-1). Recently, the AF-1 activity of the estrogen receptor (ER), another member of this family, has been shown to be modulated following phosphorylation by mitogen-activated protein (MAP) kinase (16, 17). Three MAP kinase pathways have been identified in mammalian
cells. Members of the extracellular signal-regulated kinases, ERK1 and
ERK2, are activated predominantly by growth factor stimulation via a
Ras-dependent signal transduction cascade (18). In
contrast, activity of Jun NH2-terminal kinase (JNK, also
known as SAPK) and p38 kinase is increased by exposure of cells to
environmental stress or to cytokines including TNF
(19, 20). In
turn, activated MAP kinases have been shown to regulate the activity of
specific transcription factors including Elk-1, ATF-2, and c-Jun by
phosphorylation of serine or threonine residues in the appropriate
context (21).
Interestingly, it has been reported that PPAR1 and PPAR
2 are
similarly adipogenic but that a truncated receptor in which the
amino-terminal domain of PPAR
2 is deleted is a more potent inducer
of adipocyte differentiation (12). We noted that
NH2-terminal domain of PPAR
contains a consensus MAP
kinase site in a region conserved between PPAR
1 and PPAR
2
isoforms. Furthermore, we and others (11, 22) have observed that
PPAR
proteins migrate on immunoblots as closely spaced doublets, a
pattern suggestive of phosphorylation. In this report we show that this
putative MAP kinase site is phosphorylated in vivo and also
in vitro by ERK2 and JNK. Furthermore, we demonstrate that
phosphorylation significantly inhibits both ligand-independent and
ligand-dependent transcriptional activation by PPAR
.
Finally, we show that mutation of this MAP kinase site in PPAR
increases its adipogenic activity. These findings provide a potential
pathway by which extracellular hormones and cytokines might regulate
adipocyte differentiation by phosphorylation-dependent
modulation of PPAR
activity.
The GAL4 UAS-TKLUC luciferase reporter
contains two copies of the GAL4 17-mer binding site (23) and the
BOS-gal and CMV
-gal reference plasmids have been described
previously (23, 24). The GAL4-hPPAR
(A/B) wild type and S84A mutant
expression vectors contain residues 1-108 of human PPAR
1 cloned
into the EcoRI site of pSG424 (25). These domains were also
cloned into pGEX4T for expression of glutathione
S-transferase (GST) fusion proteins. pCMV-flag-p38, which
contains the Flag epitope between codons 1 and 2 of p38, was obtained
from J. Han (Dept. of Immunology, Scripps Research Institute, San
Diego, CA). RSV-PP1 is an expression vector encoding protein
phosphatase 1
(26). pSG-CL100 is an expression vector encoding a
dual specificity MAP kinase phosphatase (27). pSPORT-mPPAR
2 encodes
full-length mouse PPAR
2 (10), and the S112A point mutation was
introduced into it by polymerase chain reaction-directed mutagenesis.
Both PPAR
2 and PPAR
2-SA cDNAs were then subcloned into the
SalI site of pCMX. All mutations and ligation junctions were
confirmed by sequencing. The acyl-CoA × 3-TK-LUC construct
contains three copies of the acyl-CoA oxidase PPAR response element
(5
-GATCTGGACCAGGACAAAGGTCACGTTCA) in pTK-luciferase.
For functional
studies, JEG-3 cells were cultured in DMEM containing 10% fetal bovine
serum and transferred to DMEM plus charcoal-stripped fetal calf serum
immediately prior to transfection. Cells were transfected with
luciferase reporter, receptor expression vector, -gal expression
vector, and phosphatase expression vector where indicated using the
calcium phosphate precipitation method. 5 µM BRL49653 (in
Me2SO) or vehicle control was added 16 h after transfection. Cells were lysed 24 h later, and luciferase and
-gal was measured as described (23, 28). Luciferase values were
normalized to
-gal activity and fold activation was calculated.
JEG-3 cells, plated on 10-cm plates, were transfected
with 30 µg of GAL4-hPPAR plus 10 µg of PP1 expression vector as
indicated. 40 h later cells were incubated in phosphate-free DMEM
for 30 min, followed by a 4-h incubation with 1 mCi/ml
[32P]orthophosphate (DuPont NEN). Cells were lysed by a
30-min incubation at 4 °C in RIPA buffer (1% Nonidet P-40, 1%
deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M
NaPO4, pH 7.2, 2 mM EDTA, 50 mM
NaF, 0.2 mM sodium vanadate, 1 µg/ml leupeptin, and 1 µg/ml aprotinin). Extracts were cleared by centrifuging at
26,000 × g for 30 min at 4 °C.Samples were
precleared with 10 µl of whole rabbit serum (Cappel) in a 50% slurry
of protein A-agarose (Life Technologies, Inc.). The resulting
supernatant was incubated with 10 µl of GAL4 DBD rabbit polyclonal
antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at
4 °C. Immune complexes were then precipitated with a 50% slurry of
protein A-agarose and washed five times with RIPA buffer. The
immunoprecipitate was analyzed by 12% SDS-PAGE and
autoradiography.
For immunoprecipitation of p38 kinase, JEG-3 cells were maintained on
10-cm plates and transfected with pCMV-flag-p38 by a 4-h exposure to
calcium phosphate. After 48 h cells were UV irradiated (40 J/m2 for 30 s) and lysed by a 5-min incubation at
4 °C in buffer containing 20 mM Hepes, 2 mM
EGTA, 50 mM -glycerophosphate, pH 7.4, 1% Triton X-100,
10% glycerol, 1 mM dithiothreitol, 1 mM
orthovanadate, 2 µM leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 0.1 µg/ml okadaic
acid. Lysates were cleared by centrifugation at 10000 rpm for 10 min at
4 °C. Flag-p38 was immunoprecipitated by a 1-h incubation of cleared
cell lysates with anti-FLAG M2 monoclonal antibody (Scientific Imaging
Systems, New Haven, CT) followed by a 1-h incubation with protein
G-Sepharose. Precipitates were washed three times with lysis buffer
followed by three washes in lysis buffer containing 150 mM
NaCl and a further three washes in kinase buffer (20 mM
MOPS, pH 7.0, 1 mM EDTA, 5% glycerol, 0.1%
-mercaptoethanol, and 0.1 µg/ml okadaic acid).
Whole cell extracts were prepared from JEG-3 cells transfected with
wild type or mutant mPPAR2 as described above and analyzed by
SDS-PAGE and Western blotting followed by chemiluminescent detection
(Amersham Corp.) with anti-PPAR
IgG at a dilution of 1:1000.
GST fusion proteins were
expressed in Escherichia coli, induced with 1 mM
isopropylthio--D-galactosidase, and purified using glutathione-Sepharose 4B affinity resin (Pharmacia Biotech Inc.) but
not removed from the matrix. Following purification, proteins were
resuspended in NETN buffer (20 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, 0.5% Nonidet P-40, pH 8.0),
and their concentrations were determined.
Phosphorylation of 2 µg of GST
fusion proteins by ERK2 (New England Biolabs, Bishop's Stortford, UK)
or JNK (Calbiochem-Novabiochem, Nottingham, UK) were carried out as
recommended by the manufacturers. p38 kinase assays were performed with
2 µg of substrate at 30 °C for 30 min in kinase buffer containing
18 mM magnesium acetate, 90 µM ATP, and 2 µCi of [-32P]ATP. Myelin basic protein (Life
Technologies, Inc.) was used as a control substrate for ERK2, and a GST
fusion protein containing a truncated form of activating transcription
factor-2 (GST-ATF2, residues 1-96, Santa Cruz Biotechnology, Wembley,
UK) provided positive controls for JNK and p38. 32P
incorporation was determined following SDS-PAGE fractionation. Gels
were stained with Coomassie Blue to check equal loading of GST fusion
proteins and autoradiographed.
3T3-L1 cells were made to ectopically express PPAR2 or
PPAR
2-S112A using retroviral gene transduction as described
previously (22). Infected 3T3-L1 cells were selected in G418 and grown to confluence in DMEM containing 10% iron-enriched fetal bovine serum.
2 days after reaching confluency, cells were treated with BRL49653
dissolved in Me2SO or Me2SO alone. After 7 days, cells were washed three times with phosphate-buffered saline,
fixed by 10% formalin in phosphate buffer for 1 h at room
temperature, washed once again with phosphate-buffered saline, then
stained with 60% filtered Oil Red O stock solution (0.5 g of Oil Red O (Sigma) in 100 of ml isopropanol) for 15 min, washed
four times with water, and photographed.
Sequence alignment of the amino-terminal A/B region that is common
to PPAR1 and PPAR
2 isoforms indicated that a consensus MAP kinase
site is conserved between species (Fig. 1A).
To determine whether the NH2-terminal region of PPAR
is
phosphorylated in vivo, an expression vector encoding the
the A/B domain of human PPAR
1 (residues 1-108) fused to the
heterologous DBD of GAL4 (residues 1-147) was transfected into JEG-3
cells. Orthophosphate labeling followed by immunoprecipitation with an
antibody directed against GAL4 revealed that the GAL4 PPAR
(A/B)
fusion protein was highly phosphorylated, whereas Gal4 DBD alone was
not (Fig. 1B, lanes 2 and 3). In
comparison, phosphorylation of transfected GAL4 PPAR
(A/B S84A), in
which a putative phosphoserine at position 84 has been mutated to
alanine (S84A) was markedly reduced (Fig. 1B, lane
4), although this fusion is expressed and even more
transcriptionally active than wild type Gal4-PPAR
(A/B) (see below).
Furthermore, cotransfection of PP1, an activated form of a serine
phosphatase, led to near complete loss of phosphorylation of the wild
type PPAR
construct (Fig. 1B, compare lanes 3 and 6).
Several MAP kinase cascades have been defined, each with a slightly
different substrate specificity and activation pathway (21). In view of
this, we tested the ability of three kinases, ERK2, JNK, and p38, to
phosphorylate the isolated A/B domains of wild type and mutant
hPPAR1 expressed as GST fusion proteins in E. coli.
Purified recombinant ERK2 was able to phosphorylate the wild type
GST-PPAR
1 fusion protein (Fig. 2A). This
phosphorylation was abolished by mutation of the serine at position 84 in hPPAR
1, which corresponds to the MAP kinase site to alanine
(S84A) under conditions in which myelin basic protein was a good
substrate for this kinase. Similarly, purified recombinant JNK was also able to phosphorylate wild type GST-PPAR
(A/B) in addition to a
control GST-ATF2 fusion protein (Fig. 2B). Mutation of
serine 84 to alanine markedly reduced but did not abolish
phosphorylation by JNK, suggesting weak phosphorylation of a second
kinase site in vitro, which is not phosphorylated in
vivo (Fig. 1B). Similar experiments have been performed
with p38 kinase and phosphorylation of wild type or mutant GST PPAR
was not detected under conditions in which GST-ATF2 was a good
substrate for this enzyme (data not shown).
These results suggested that phosphorylation of a MAP kinase site by
either ERK2 or JNK might regulate the ligand-independent transcriptional activity (AF1) of hPPAR. Transient transfection assays in JEG-3 cells showed that the A/B domain of hPPAR
acts as a
weak, ligand-independent transcriptional activator when fused to the
DBD of GAL4 (Fig. 3). Mutation of serine 84 to alanine, which abolished phosphorylation of the GAL4-PPAR
fusion protein (see
above and Fig. 1B), markedly enhanced the AF1
transactivation function of the PPAR
A/B domain. Similar results
were obtained following transfection of wild type and mutant
GAL4-PPAR
fusions in COS-7 cells (data not shown). Furthermore,
coexpression of CL100, the human homologue of murine MAP kinase
phosphatase (MKP-1), also augmented the transcriptional activity of
wild type Gal4-PPAR
A/B (Fig. 3) but had no effect on the mutant
GAL4-PPAR A/B S84A fusion. Thus, two independent paradigms that either
prevent (S84A mutation) or reduce (CL-100 coexpression) phosphorylation
of the PPAR
A/B domain markedly enhance AF1 activity.
In addition to the constitutive AF1 function in the
amino-terminal domain, PPAR also contains a
ligand-dependent transcription activation function (AF2).
We therefore investigated whether phosphorylation of the MAP kinase
site in the A/B domain could influence the ligand-dependent AF2 activity of full-length PPAR
. Because PPAR
is conserved between species and the residues encompassing the amino-terminal MAP
kinase site are identical in PPAR
1 and PPAR
2 isoforms (Fig. 1A), murine PPAR
2, which is induced specifically during
adipocyte differentiation, was used in these studies. Transfection of
wild type mPPAR
2 expression vector together with a reporter
construct containing three copies of the PPAR-response element from the acyl-CoA oxidase gene and the thiazolidenedione ligand BRL49653 was
associated with significant transcriptional activation (Fig. 4). Mutation of the homologous serine within the
putative MAP kinase site in mPPAR
2 to alanine (S112A) significantly
increased ligand-dependent transactivation to approximately
double that of the wild type receptor. These observations indicate that
in addition to inhibiting the inherent AF1 activity of PPAR
,
phosphorylation of the MAP kinase site within the A/B domain also
attenuates ligand-induced transcription activation by this
receptor.
We next tested whether the enhanced transactivation by PPAR2-S112A
resulted in increased adipogenic activity. Wild type PPAR
2 or
PPAR
2-S112A were ectopically expressed in 3T3-L1 preadipocytes using
a retroviral expression strategy that we have previously described
(22). In the absence of adipogenic stimulation, confluent preadipocytes
differentiate into adipocytes at a very low frequency (<1%). Ectopic
expression of PPAR
2 resulted in ~10% adipose conversion, as shown
by Oil Red O staining of accumulated intracellular lipid (Fig.
5). This effect is presumably mediated by low levels of an endogenous PPAR
2 ligand, although the possibility that this was a
ligand-independent effect of PPAR
overexpression cannot be
discounted. Remarkably, ectopic expression of PPAR
2-S112A at levels
similar to those of the ectopically expressed wild type PPAR
2 (data
not shown) induced adipocyte differentiation much more dramatically. In
fact, Fig. 5 shows that the degree of adipocyte differentiation due to
ectopic PPAR
2-S112A expression was comparable with that achieved by
cells expressing wild type PPAR
and treated with the ligand BRL49653
(50 nM). At this concentration BRL49653 alone is a
relatively weak adipogenic stimulus for control cells (13, 15, 30),
resulting in ~10% adipose conversion. These results show clearly
that mutation of the MAP kinase consensus site produces a more
adipogenic form of PPAR
2, consistent with the increased
transcriptional activity of this mutant receptor.
Our studies indicate that a consensus MAP kinase site located
within the conserved amino-terminal A/B domains of PPAR1 and PPAR
2 regulates the transcriptional activity of these adipogenic nuclear receptor isoforms. Phosphorylation of this site reduces the
inherent transcriptional activity of the AF1 transactivation domain. In
addition, ligand-dependent transactivation by the
full-length receptor is also inhibited. This observation contrasts with
previous studies of the ER showing that MAP kinase-mediated
phosphorylation of the amino-terminal A/B domain enhanced the
transcriptional activity of this receptor (16, 17) and indicates that
constitutive activity of the NH2-terminal domains of
nuclear receptors can be modulated in response to other signal
transduction pathways. Furthermore, the divergent transcriptional
effects of phosphorylation in PPAR
versus ER suggest that
the different sequences around the consensus MAP kinase site in each
A/B domain may also influence the way AF1 activity is modulated.
Although the A/B domain sequence motif that we have identified
represents a consensus MAP kinase site, we have demonstrated that this
residue is amenable to phosphorylation by both ERK2 and JNK.
Interestingly, both TNF and EGF, which are potent inhibitors of
adipocyte differentiation (3, 4, 5, 6) are known to activate these
pathways. In addition to enhancing JNK activity, TNF
stimulation has
also been reported to activate MAP kinase (31, 32), and EGF stimulation
enhances MAP kinase activity in a variety of cell types (18). The
present work suggests that activation of either pathway could
phosphorylate and hence inhibit PPAR
activity, thus contributing to
the anti-adipogenic effects of these agents.
It is also possible that other signal transduction pathways may be
involved. For example, it is interesting to note that adipocyte differentiation is promoted by culture in conditions that raise intracellular cAMP (33, 34), and elevated cAMP levels have been
reported to inhibit ERK activity in rat adipocytes (35, 36). In some
cell types, cAMP-dependent stimulation of protein kinase A
leads to direct inhibition of Raf-1, which in turn inhibits EGF-stimulated MAP kinase activity (37). Given the convergence of these
pathways, it is tempting to speculate that altered PPAR phosphorylation might contribute to the abnormal adipogenesis seen in
mice harboring a mutant protein kinase A regulatory subunit (38).
Phosphorylation has been shown to regulate the activity of
transcription factors by a number of different mechanisms (21). Because
equal levels of mutant and wild type PPAR2 were detected in
transfected cells (Fig. 4), phosphorylation does not appear to alter
PPAR
protein stability, as has been shown for the transcription factor Fos (39). Furthermore, because the inhibitory effect of
NH2-terminal PPAR
phosphorylation is transferable to the
heterologous DNA binding domain of GAL4, we also consider it unlikely
that phosphorylation alters the ability of PPAR
to bind to DNA. A third possibility is that phosphorylation inhibits the activity of
PPAR
by altering receptor interaction with other transcription intermediary proteins. For example, an interaction between the A/B
domain of thyroid hormone receptors TR
2 and TR
1 and TFIIB, which
might influence transcription by altering preinitiation complex
formation or stability, has recently been described (40, 41). This
raises the possibility of analogous interactions between basal
transcription factors and the A/B region of PPAR
that are phosphorylation-sensitive. Alternatively, phosphorylation at this site
might influence PPAR
interaction with specific coactivator or
corepressor proteins, in the same way that phosphorylation of c-Jun
enhances recruitment of the CREB-binding coactivator protein (42).
Our finding of increased adipogenicity of a PPAR2 point mutant that
is not phosphorylated by ERK and related MAP kinases in its A/B domain
strongly suggests that NH2-terminal phosphorylation inhibits the adipogenic activity of the wild type receptor. This also
provides a molecular basis for an earlier observation that retroviral
expression of NH2-terminally truncated PPAR
2 induces murine preadipocyte differentiation more strongly than its wild type
counterpart (12). The precise role of PPAR
phosphorylation in
regulation of adipocyte differentiation remains to be elucidated. However, the ability to modulate the activity of this adipogenic transcription factor by altering its phosphorylation state might provide a novel strategy for development of anti-adipogenic therapeutic agents to treat obesity.
We thank Sam Krakow for technical assistance.