(Received for publication, May 25, 1996, and in revised form, October 15, 1996)
From the Division of Endocrinology, Beth Israel
Hospital, Harvard Medical School, Boston, Massachusetts 02215, the
§ Department of Molecular Endocrinology, Merck Research
Laboratories, Rahway, New Jersey 07065, and the ¶ Dana-Farber
Cancer Institute and Department of Cell Biology, Harvard Medical
School, Boston, Massachusetts 02115
The ob gene product,
leptin, is a major hormonal regulator of appetite and fat cell mass.
Recent work has suggested that the antidiabetic agents, the
thiazolidinediones (TZ), which are also high affinity ligands of
peroxisome proliferator-activated receptor- (PPAR
), inhibit
leptin expression in rodents. To examine the effects of this class of
drug on the leptin gene in adipocytes we performed Northern analysis on
primary rat adipocytes cultured in the presence or absence of TZ. TZ
reduced leptin mRNA levels by 75%. To determine whether this
effect was mediated at the transcriptional level, we isolated 6510 base
pairs of 5
-flanking sequence of the leptin promoter and studied
reporter constructs in primary rat adipocytes and CV-1 cells. Sequence
analysis demonstrated the presence of a consensus direct repeat with a
1-base-pair gap site between
3951 and
3939 as well as a consensus
CCAAT/enhancer binding protein (C/EBP) site between
55 and
47. Our
functional analysis in transfected primary rat adipocytes demonstrates
that, despite the presence of a canonical direct repeat with a
1-base-pair gap site, TZ alone decreases reporter gene expression of
leptin promoter constructs ranging from
6510 to +9 to
65 to +9. In CV-1 cells, which contain endogenous PPAR
, TZ treatment alone had
little effect on these constructs. However, TZ treatment did inhibit
C/EBP
-mediated transactivation of the leptin promoter. This
down-regulation of leptin reporter constructs mapped to a
65 to +9
promoter fragment which binds C/EBP
in gel-mobility shift assays but
does not bind PPAR
2 alone or as a heterodimer with
9-cis-retinoic acid receptor. Conversely, the promoter
(
5400 to +24 base pairs) of the aP2 gene, another adipocyte-specific gene, was induced 7.3-fold by TZ. Co-transfection with C/EBP
minimally stimulated the aP2 promoter from basal levels but notably blocked activation by TZ. These data indicate that PPAR
and C/EBP
can functionally antagonize each other on at least two separate promoters and that this mechanism may explain the down-regulation of
leptin expression by thiazolidinediones.
The identification of the leptin gene product (1) as a major
hormonal regulator of appetite and fat cell mass provides a novel
paradigm to explore the regulation of a fat-specific gene product which
may be influenced by nutritional intake as well as other metabolic
mediators. Recent studies have clearly demonstrated that the leptin
gene is down-regulated by fasting (2) and up-regulated by obesity (3).
Studies on fat cell gene expression have been enhanced by the
identification of two specific classes of transcription factors: the
CCAAT/enhancer binding proteins (C/EBPs)1
(4, 5), members of the b-ZIP (basic DNA binding domain and a leucine
zipper domain required for dimerization) family and PPAR, a member
of the peroxisomal proliferator activated receptor family of nuclear
hormone receptors (6). The C/EBP isoforms are expressed at high levels
in adipocytes and are induced during adipogenesis (7). Furthermore,
C/EBP
has been demonstrated to play an important role in the
differentiation of preadipocytes to adipocytes (8-10) and can convert
fibroblasts into adipocytes (11). C/EBP
can also induce adipocyte
differentiation (12), possibly by inducing PPAR
(13), which contains
C/EBP sites in its promoter (14). PPAR
isoforms are also potent
triggers of the adipocyte differentiation cascade (15) and can
synergize with C/EBP
to promote adipocyte differentiation (15) or
the differentiation of myoblasts into adipocytes (16). In addition, C/EBP
and PPAR
can bind to the promoters and activate
adipose-specific genes such as aP2 (6, 15, 17-18) and PEPCK
(19-20).
From these previous observations logical candidate regulators of the
leptin promoter include C/EBP isoforms and PPAR. Recently, C/EBP
(21-22) has been identified as a transactivator of the leptin promoter
working through a consensus C/EBP binding site in the proximal leptin
promoter. This site mediates activation of the leptin promoter by
co-transfected C/EBP
in primary rat adipocytes (21) and 3T3-L1
preadipocytes (22). Also, it has been recently established that the
administration of the antidiabetic thiazolidinedione, which is a high
affinity ligand for the PPAR
isoforms (23), down-regulates leptin
expression in rodents (24) and in 3T3-L1 adipocytes (25). These data
suggest that unlike other adipose tissue-specific genes such as aP2 and
PEPCK, which are up-regulated by PPAR
(6, 20) in the presence of
ligand, leptin may in fact be negatively regulated by PPAR
in the
presence of its ligand.
The PPAR isoforms mediate positive effects on gene expression by
binding to the hexamer sequence in a direct repeat formation spaced by 1 nucleotide (26, 27) with the retinoid X receptor
(RXR) as a heterodimer. Both ligands for the PPARs and RXR can activate
transcription from this complex (28). Recently, a putative ligand for
the PPAR
isoform has been identified as a metabolite of prostenoid
J2 (29, 30), which can activate PPAR
in transient transfection
assays and as well induce adipocyte differentiation in fibroblasts. In
contrast to positive regulation by PPAR isoforms, little is known about
negative regulation. The PPARs can down-regulate thyroid
hormone-responsive promoters by competing for available RXR (31, 32);
furthermore, fibrates, which can activate gene expression through
PPARs, have been shown to down-regulate the human apolipoprotein A-1
promoter, but the mechanism remains unclear (33). Whether specific
negative peroxisome proliferator-activated receptor response elements
or crosscompetition between PPARs with other positively acting
transcription factors occurs is unknown.
Other members of the steroid/thyroid hormone receptor superfamily mediate negative regulation by 1) binding to specific negative response elements, as is the case for the thyroid hormone receptor on a number of specific genes (34-38); 2) interfering through protein-protein interactions or direct competition for DNA binding with positively acting transcription factors such as c-jun or C/EBP (39-42); and 3) as recently reported, by competition for limiting co-factors such as CREB-binding protein (CBP)/p300 (43).
In this paper we examine the role of PPAR in the regulation of the
leptin promoter and demonstrate that, despite the presence of a
consensus DR+1 binding site located between
3951 and
3939 of the
mouse 5
-flanking sequence, PPAR
2 mediates down-regulation of the
leptin promoter by inhibiting C/EBP
-mediated transactivation. This
mechanism may explain the down-regulation of leptin expression in
rodents by the thiazolidinediones.
Adipocytes were isolated from male Wistar rats using the procedure previously described with modifications (44). Briefly, epididymal adipose tissue was minced and digested in Dulbecco's modified Eagle's (DME)-Ham's F-12 medium (v:v, 1:1) containing 1 mg/ml collagenase (Worthington) and 1% bovine serum albumin at 37 °C for 1 h. The adipocytes were strained through nylon mesh and washed three times with the DME-Ham's F-12 medium containing 1% bovine serum albumin. Packed adipocytes (4 ml) were added to 20 ml of DME-Ham's F-12 medium containing 2% fetal bovine serum (Hyclone), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.), and the incubation was carried out at 37 °C in 5% CO2 for 16 h. Insulin, AD-5075 (5-methyl-2-phenyl-4-oxazolyl-2-hydroxyethoxy-benzyl-2,4-thiazolidinedione) were added to the culture medium, and the incubation was continued for 24 h.
Isolation of RNA and Northern Blot AnalysisTotal RNA was prepared from cells using the method of Chomczynski and Sacchi (45). RNA concentrations were quantitated by absorbance at 260 nM. For Northern analyses, 10-µg aliquots of RNA were denatured in formamide/formaldehyde and electrophoresed in formaldehyde-containing 1% agarose gels. RNA was transferred to HybondTM-N membranes (Amersham Corp.) by capillary blotting. Prehybridization and hybridization with 32P-labeled cDNA probes (for leptin or aP2) were carried out as described (24). After washing the membranes, the hybridization signals were analyzed with a PhosphorImager (Molecular Dynamics). Each time point was repeated at least two times in duplicate.
Isolation of the Leptin Promoter and Construction of Luciferase ConstructsTwo overlapping P1 clones (Genome Systems, St. Louis,
MO) representing the mouse leptin gene and 5 and 3
sequences were isolated. 6.5 kb of 5
-flanking sequence including the transcription start site and TATAA box were identified. The transcription start site
was confirmed by 5
rapid amplification of cDNA ends from adipose
tissue. A similar transcription start site has been identified by other
investigators (21, 22). The promoter region was subjected to dideoxy
sequencing between
6.51 kb and +9 bp.
Leptin promoter-luciferase constructs were created by initially
shuttling a BamHI fragment containing 765 bp of the promoter into PGEM 3Z. Subsequently 454 to +9 and
765 to +9 fragments were
introduced into the luciferase vector pA3Luc, which
contains a trimerized SV40 poly(A) termination to prevent
transcriptional readthrough (46, 47) as
KpnI-HindIII and
SmaI-HindIII fragments, respectively. The
6510
bp construct was created by excising the region from
454 to
6510 as
a KpnI fragment from a genomic subclone and introducing it
into the KpnI site of the
454 to +9 construct. The
3821
bp construct was created using an internal HindIII site and
a HindIII site in pA3Luc. The
159 to +9
construct was created by XhoI digestion of the
454
construct followed by blunt-ending using Klenow and ligating to the
SmaI site of pA3Luc. The
65 to +9 construct
was cloned using a similar strategy employing EaeI.
Site-directed mutagenesis of the C/EBP site (see below for oligonucleotide sequence) between
55 and
47 was performed in context of the
454 to +9 construct using synthetic oligonucleotides and restriction enzyme selection (48). The aP2 promoter (
5400 bp to
+24) was cloned into the SmaI site of pA3Luc
using HincII and SmaI sites. To create the
249
aP2 construct, the parental aP2 plasmid was digested using Ppu101 and
HindIII, and the insert was cloned into the SmaI
and HindIII sites of pA3Luc. The RSV 180 construct contains 180 bp of the Rous sarcoma virus long terminal repeat in pA3Luc. The sequences encompassing the leptin
DR+1 (from
3951 to
3939, see below) were inserted as a
HindIII fragment either as one or two copies upstream of 109 bp of the thymidine kinase promoter (TK109) in pA3Luc.
TK109 pA3Luc was constructed by inserting the TK promoter
from PT109 (49) into pA3Luc (a gift of T. Nagaya). All
promoter constructs were confirmed by restriction endonuclease
digestion and dideoxy sequencing. Plasmids for transfection were
prepared using column purification (Qiagen) and were generally
subjected to at least two separate plasmid preparations.
The MSV rat C/EBP
expression vector was a gift from Alan Friedman (4). An
EcoRI-BssHII fragment encompassing the complete C/EBP cDNA sequence was subsequently cloned into PGEM 3Z, pSV-SPORT (BioResearch Laboratories), and pBK-CMV (Stratagene). The mouse PPAR
2 isoform was cloned into pSV-SPORT. All expression vectors were
confirmed by restriction endonuclease digestion and dideoxy sequencing.
Plasmids for transfection were prepared using column purification
(Qiagen) and were generally subjected to at least two separate plasmid
preparations.
CV-1 and NIH-3T3
cells were maintained in Dulbecco's modified Eagle's medium
supplemented with L-glutamine, 10% fetal calf serum, 100 µg/ml penicillin, 0.25 µg/ml streptomycin, and amphotericin. Transient transfections were performed in six-well plates with each
well receiving 1.67 µg of reporter and 417 ng of expression vector
using the calcium phosphate technique. Amount of expression vector DNA
was kept constant within experiments using pKCR2 (similar SV40 early promoter to pSV-SPORT). Each well also received 0.4 µg of
a -galactosidase expression plasmid to control for transfection efficiency. 15-18 h after transfection the cells were washed with phosphate-buffered saline and refed with Dulbecco's modified Eagle's medium with 10% steroid hormone-depleted fetal bovine serum and the
indicated concentration of BRL 49653. To remove steroid and thyroid
hormones, fetal bovine serum was treated for 24 h at 4 °C with
50 mg/ml activated charcoal (Sigma) and 30 mg/ml anion
exchange resin (type AGX-8, analytical grade, Bio-Rad). After
centrifugation, anion exchange resin was added again for 5 more hours.
The resulting fetal bovine serum was spun again and filtered before
use. 40-44 h after transfection the cells were harvested in extraction
buffer (38) and assayed for both luciferase and
-galactosidase
activity (Tropix). Luciferase activity was corrected for
-galactosidase activity as indicated. All points were repeated in
triplicate, and each experiment was repeated at least twice.
Primary rat adipocytes were harvested by isolating white adipose tissue
cells from epididymal fat pads of Sprague-Dawley rats. Isolated
adipocytes were transfected by electroporation as described previously
(50) with 6 µg of either RSV 180 or leptin promoter luciferase
constructs. A -galactosidase expression vector was not introduced
into the primary rat adipocytes. Two hours after transfection
Dulbecco's modified Eagle's medium with 3.5% bovine serum albumin
(final concentration) was added, and the cells were treated with 1 µM BRL 49653 or dimethylsulfoxide carrier alone. 20 h after transfection the cells were harvested for luciferase activity.
Statistical analysis was performed using Student's t test
comparing treated groups to dimethylsulfoxide carrier alone.
Wild type and
mutant overlapping oligonucleotides encompassing the C/EBP site from
67 to
36 (wild type
5
-TGGCCGGACAG
TGGCACTGGGG-3
: mutant
5
-TGGCCGGACAG
TGGCACTGGGG-3
)
and the DR+1 site at
3951 kb (wild type
5
-ACGTAGAAGCTTGAAATG
A
GAGTCCAAGCTT-3
) were radiolabeled by filling in with Klenow and
[32P]deoxycytodine triphosphate (3000 µCi/mmol). The
65 to +9 fragment was prepared by BamHI digestion of the
pA3Luc reporter construct and was radiolabeled with Klenow.
Unincorporated 32P was removed by Sephadex G-25
chromatography, and each oligonucleotide was subsequently gel-purified.
In vitro-translated C/EBP
, PPAR
2, and RXR
were
prepared in rabbit reticulocyte lysate, and protein production was
analyzed by 35S incorporation and direct visualization on
SDS-polyacrylamide gel electrophoresis. 3-5 µl of in
vitro-translated protein were incubated in 10 µl of binding
buffer (20% glycerol, 20 mM Hepes, pH 7.6, and 50 mM KCl), 1 mM dithiothreitol, 1 µg of
poly(dI-dC), and 0.1 µg of salmon sperm DNA. Incubations were carried
out at room temperature for 25 min. Gel-mobility shifts were resolved on 5% nondenaturing polyacrylamide gels and visualized after
autoradiography.
CV-1 cells were transfected with 15 µg
of different C/EBP expression vectors as described above. Cells were
treated in triplicate with either BRL 49653 or dimethylsulfoxide
carrier for 18-24 h. Whole cell extracts were generated in 600 µl of
RIPA buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 10 µl/ml phenylmethylsulfonyl fluoride (10 mg/ml) and 10 µl/ml sodium orthovanadate (100 mM)). 40 µg of protein from each well was subjected to SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose. Primary antibody
(C/EBP rabbit polyclonal, Santa Cruz Biotechnology) probing was
performed according to the manufacturer's instructions, and the
secondary antibody specificity was elicited by Amersham ECL.
C/EBP-specific bands were quantified with a PhosphorImager. Each
experiment was performed in triplicate.
The b-ZIP
region of human C/EBP was cloned in frame with the glutathione
S-transferase moiety in PGEX-2TK (a gift of D.-E. Zhang).
The recombinant protein and GST itself were induced in Escherichia coli DH5
at 32 °C in the presence of 1 mM isopropyl-
-D-thiogalactopyranoside. The
GST proteins were purified on glutathione- agarose beads
(Sigma) as described elsewhere (51) and analyzed on
SDS-polyacrylamide gel electrophoresis. Interaction assays were
performed by incubating equal amounts of GST-C/EBP or GST protein alone
immobilized on glutathione-agarose beads with 5 µl of
35S-labeled in vitro-translated PPAR
2 or
C/EBP
for 2 h in 500 µl of interaction buffer (150 mM NaCl, 20 mM Tris (pH 7.5), 0.3% Nonidet
P-40, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride) at 4 °C with gentle
rocking. The protein-GST beads were washed four times with the same
buffer. The resulting bound proteins were analyzed by
SDS-polyacrylamide gel electrophoresis followed by autoradiography.
In previously reported work (24) it was demonstrated
that the chronic administration of a TZ down-regulates leptin mRNA in rodent models. To confirm that this effect was mediated by a direct
effect of TZ on adipocytes, we studied the regulation of leptin gene
expression using primary rat adipocytes. In the absence of added
insulin, leptin mRNA levels were low and not significantly affected
by the TZ agonist AD-5075. The effect of insulin to augment leptin
expression (Fig. 1) was consistent with data reported by
Saladin et al. (52). In the context of insulin incubation,
AD-5075 caused a 75% decrease in leptin mRNA levels (Fig. 1).
Similar data on the regulation of leptin mRNA by the thiazolidinediones were seen by Kallen and Lazar in 3T3-L1 adipocytes (25). Furthermore, these authors demonstrated that this effect was not
due to changes in mRNA stability. In contrast to the effects on
leptin, TZ stimulated aP2 expression by approximately 2-fold in the
absence of insulin and 6-fold in insulin-treated cells (Fig. 1,
panel 2). In that TZs are high affinity ligands for PPAR and that the aP2 promoter contains two positive peroxisome
proliferator-activated receptor response elements between
5.4 and
4.9 kb (6), these data indicate that liganded PPAR
has
differential effects on aP2 and leptin gene expression.
Structure of the Mouse Leptin Promoter
To facilitate our
analysis of the transcriptional regulation of leptin we isolated two
overlapping P1 clones which include the entire leptin gene as well as
5- and 3
-flanking sequences. Our analysis agrees with other reports
concerning the genomic structure and initiation of transcription (21,
22). We subsequently analyzed the 5
-flanking sequence of the leptin
gene. As outlined in Fig. 2, a consensus C/EBP site
exists between
55 and
47 and a consensus DR+1 between
3951 and
3939 bp. Other sites with partial homology to C/EBP sites were also
identified. No other exact consensus sites for members of the
steroid/thyroid receptor superfamily were found between
6510 and +9
of the leptin promoter.
The Leptin Promoter Is Down-regulated by Thiazolidinediones in Primary Rat Adipocytes
In that the TZ compounds down-regulate
leptin mRNA levels in primary rat adipocytes, we then determined
the transcriptional effects of TZ on leptin promoter-constructs
transiently transfected into primary rat adipocytes. As Fig.
3A shows, leptin promoter constructs were
expressed well above background in transfected rat adipocytes
(background, 250-300 light units). However, their expression was
markedly below the RSV control (70,508 light units). Also, another
full-length fat-specific gene promoter, aP2, was expressed at levels
slightly above the 454 to +9 leptin promoter construct. Mutagenesis
of the proximal C/EBP binding site in context of either the
454
construct (Fig. 3A) or the
6510 bp leptin construct (data
not shown) caused reporter gene activity to be dramatically reduced
indicating that this C/EBP site is essential for full promoter activity
in transiently transfected primary rat adipocytes. The addition of 1 µM BRL 49653, a potent and selective TZ and PPAR
ligand, caused significant negative regulation of all the leptin
promoter constructs (Fig. 3B). In contrast, the
249 aP2
construct was unaffected while the full-length aP2 construct which
contains the two DR+1 elements was slightly induced. These data
indicate that negative transcriptional regulation of the leptin gene by
PPAR
2 appears to be mediated by elements within the proximal leptin
promoter.
Leptin Promoter Function in Heterologous Cells
To ascertain
the effect of C/EBP and PPAR
2 on the leptin promoter, we
performed transient transfections in CV-1 cells, an African green
monkey kidney cell line used by many investigators to study steroid
hormone action. Recently, these cells were found to contain active
PPAR
(30). All leptin promoter constructs had very low basal
activity (300-1000 light units) and did not respond appreciably to the
thiazolidinedione BRL 49653 (data not shown). We hypothesized that the
leptin DR+1 site at position
3951 to
3939 may not support basal
activity in these cells and that the lack of reporter activity of
leptin promoter constructs was due to an absence of C/EBP
from this
cell line. In contrast, the full-length aP2 promoter demonstrated a
much higher basal activity (70,000-75,000 light units) and was induced
considerably in response to BRL 49653 consistent with the presence of
endogenous PPAR
and RXR within this cell line (see Fig.
6C). These data support the use of CV-1 cells as a model
system to study regulation of PPAR
-responsive genes. When C/EBP
was co-transfected into CV-1 cells, leptin promoter constructs from
6510 to +9 down to
65 to +9 were induced from 17-73-fold (Fig.
4). Absolute levels of expression of the leptin promoter
constructs were similar (10,000-25,000 light units) when induced by
C/EBP
except for the full-length construct whose induced expression
was never above 5000 light units. Mutagenesis of the proximal C/EBP
site in context of the
454 to +9 construct (
454 to +9 C/EBP MUT,
Fig. 4) caused a near complete loss of C/EBP induction. The viral
control, RSVLuc, was not up-regulated by C/EBP
. Surprisingly, the
full-length aP2 promoter was only mildly induced (1.5-fold) by C/EBP
despite the presence of a functional C/EBP site within the proximal
promoter (17, 18).
Functional antagonism between C/EBP and
PPAR
2 on the leptin and aP2 promoters. CV-1 cells were
transfected as described and treated for 16-20 h with 10 µM BRL 49653 before being assayed for luciferase and
-galactosidase activity. A, effect of BRL 49653 on C/EBP
activation of leptin promoter-luciferase constructs. The indicated
reporters (1.6 µg) were co-transfected with the pSV-C/EBP
(417 ng)
expression vector as described previously and treated with BRL 49653. The results are quantified as the percent maximal stimulation by C/EBP,
where 100% is the maximum expression of the indicated construct. The
data presented are the means ± S.E. of at least two separate
transfections performed in triplicate. Empty expression vector was
included in certain experiments with no effect on down-regulation by
BRL 46953. B, effect of BRL 49653 on C/EBP activation of
leptin promoter-luciferase constructs in the presence of additional
PPAR
2. A similar paradigm was used, except that the cells were also
co-transfected with equal amounts of a pSV-PPAR
2 expression vector.
The data are expressed as above where 100% is the maximum expression
of the indicated construct in the absence of ligand and the presence of
co-transfected C/EBP
and PPAR
2 and represents the means ± S.E. of at least two separate transfections performed in triplicate. C, effect of C/EBP or additional PPAR
2 on the
aP2-luciferase construct (
5400 to +24) in the presence or absence of
BRL 49653. The full-length aP2 reporter (
5400 to +24) was
co-transfected with blank expression vector, pSV-C/EBP
, or
pSV-PPAR
2. The data are shown as relative luciferase activity, where
basal expression in the presence of blank expression vector is shown at
a value of 1. The data are from a representative experiment performed in triplicate ± S.E. which was repeated at least twice with
similar results.
These data demonstrate that the proximal C/EBP site within the leptin promoter is critical for C/EBP-induced activation and that the far upstream DR+1 site does not appear to contribute to basal activity of the leptin promoter.
PPARGiven the consensus
nature of the leptin DR+1 site at 3951 bp, we were surprised at its
lack of ability to functionally direct transcription in response to BRL
49653 in context of the
6510 to +9 leptin construct. To assess its
ability to interact with PPAR
2, we performed gel-mobility shift
assays on the DR+1 site and surrounding sequences. Because of its
structural similarity to other DR+1s, we were not surprised to see
strong binding of PPAR
2/RXR heterodimers to this element (Fig.
5). Given this binding, we then inserted the
corresponding oligonucleotides upstream of TK109 luciferase. A single
copy of the leptin DR+1 was induced up to 3-fold by 10 µM
BRL 49653, while two copies directed up to 8-fold induction of the
reporter construct (data not shown). The TK109 promoter itself was not
induced by 10 µM BRL 49653. These data indicate that the
leptin DR+1 is able to function as a PPAR response element when
positioned close to a heterologous promoter.
Thiazolidinediones Inhibit C/EBP-Induced Activation of the Leptin Promoter
PPAR2 and C/EBP
are important determinants of fat
cell differentiation and both bind to the promoters of a number of
fat-specific genes including leptin, as our data demonstrate. Therefore
we studied the effects of C/EBP
and PPAR
2 coexpression on the
leptin promoter. When 10 µM BRL 49653 was added to CV-1
cells co-transfected with pSV-C/EBP
expression vector, leptin
promoter induction by C/EBP was blocked by 40-50% on constructs from
3821 bp to +9 down to
65 to +9 (Fig. 6A).
Similar data were obtained with BRL 49653 concentrations ranging from 1 µM to 50 µM. The full-length reporter
construct, which includes the DR+1 site, was not as greatly reduced
(its activation was attenuated by 10-15%). The
454 to +9 construct
with the mutant C/EBP site was expressed at near background levels,
making any determination of the effect of BRL 49653 difficult to
interpret. RSV 180 Luc was not appreciably affected in the presence of
co-transfected C/EBP. The addition of equal amounts of pSV-PPAR
2
increased the negative effect of BRL 49653 on all constructs (Fig.
6B). In another heterologous cell line, NIH-3T3 cells, which
have little PPAR
, an effect of TZ on both positive and negative
regulation on the aP2 and leptin promoters, respectively, could not be
seen unless PPAR
2 was co-transfected (data not shown). This implies
that the effect of TZ is mediated principally through PPAR
. Thus,
these data strongly suggest that PPAR
, in the presence of its
ligand, can inhibit C/EBP induction of reporter activity through a
direct protein-protein interaction, competition for a common co-factor,
or by binding to a non-canonical PPAR site. We also examined the effect
of 9-cis-retinoic on C/EBP activation of the
454 to +9
leptin construct in order to examine the effect of ligand-activated
endogenous RXR on the C/EBP effect. In contrast to BRL 49653, 1 µM 9-cis-retinoic acid failed to decrease leptin promoter activity (data not shown), indicating that the negative
regulation of the leptin promoter appears to be specific to endogenous
PPAR
.
Further confirmation of mutual antagonism between C/EBP and PPAR
was gained by assessing the effect of co-transfected C/EBP
on the
full-length aP2 reporter construct, in that co-transfected C/EBP should
be able to block the positive effect of endogenous PPAR
. As shown in
Fig. 6C, the aP2 promoter was induced 7.3-fold in CV-1 cells
by BRL 49653 through endogenous PPAR
receptors (top).
Co-transfection of C/EBP
(Fig. 6C, center)
reduced BRL 49653-induced activation both in terms of fold activation
(only 1.4-fold) and overall activity (by approximately 65%).
Co-transfected PPAR
2 increased both the overall response to BRL
49653 (by 2.5 times) and also substantially increased basal activity in
the absence of added ligand (Fig. 6C, bottom).
To rule out an effect of BRL 49653 on the pSV-C/EBP expression plasmid we performed Western blots on CV-1 whole cell extracts in the presence and absence of BRL 49653 and found no difference in C/EBP amount in the presence or absence of ligand (data not shown). Similar experiments performed using a MSV-C/EBP expression plasmid demonstrated a down-regulation of C/EBP expression by BRL 49653. Thus, the MSV C/EBP expression plasmid was not used.
C/EBPAs our functional data
demonstrate inhibition of C/EBP activity by PPAR2 and its ligand, we
examined the ability of PPAR
2 and C/EBP to bind to the region of the
promoter which mediates inhibition by BRL 49653. We performed
gel-mobility shift assays using either the immediate region surrounding
the C/EBP site (data not shown) or the entire region spanning
65 to
+9 as probes (Fig. 2B), which encompasses the canonical
C/EBP site (between
55 and
47). As shown in Fig. 7,
in vitro-translated C/EBP
bound this region of the
promoter and all binding was lost when the site was mutated (data not
shown). Furthermore, PPAR
2 and RXR did not bind to this region of
the promoter (Fig. 7) as a heterodimer (lanes 4 and
5) or alone (lanes 3 and 6). When
C/EBP
and PPAR
2 were added together, C/EBP
binding was
decreased (data not shown). However, this finding was most likely not
specific, as both in vitro-translated RXR and thyroid
hormone receptor caused a similar decrease in C/EBP binding.
The inability of PPAR2 to bind to the proximal leptin promoter in
the gel-mobility shift studies suggests that PPAR
2-mediated inhibition occurs through direct interactions with C/EBP or by competition for a critical co-factor. To examine further the
possibility of a direct protein-protein interaction we performed GST
pull-down experiments using the b-ZIP domain of C/EBP
fused to GST
(51). Our data demonstrate that radiolabeled PPAR
2 could not
interact with GST-C/EBP either in the presence or absence of its
ligand, while radiolabeled C/EBP could interact with GST-C/EBP,
consistent with its ability to form a homodimer (data not shown).
Further evidence of a lack of interaction between these two
transcription factors was garnered from a yeast two-hybrid system where
no interaction was seen between a PPAR
bait construct (containing
the C terminus) and the full C/EBP
protein used as the
prey.2
Leptin is an adipocyte-secreted hormone which communicates the
status of fat stores to the brain, and as such leptin is the afferent
signal for a feedback loop whose function is to regulate fat stores.
For this reason, regulation of leptin gene expression is critical in
maintaining normal body fat content. However, little is known about the
transcriptional regulation of leptin gene expression. Previous studies
have demonstrated an important role for C/EBP in regulating the
leptin promoter (21-22). The 5
-flanking sequence of leptin possesses
a C/EBP
binding site (mouse promoter,
55 to
47) and
co-transfection of C/EBP
expression plasmids transactivates leptin
promoter-reporter constructs (21-22). Mutation of the C/EBP
site
within the leptin promoter results in a loss of transactivation by
C/EBP
(Ref. 21 and this study). Finally, this C/EBP
site appears
to be critical for expression in transiently transfected primary rat
adipocytes, as promoter activity is almost completely lost when the
site is destroyed (53) (Fig. 3A). These data imply that the
C/EBP
site may be necessary for adipocyte-specific expression of the
leptin gene.
PPAR, an important cell-specific regulator of adipocyte gene
expression, is another transcription factor which could control leptin
gene expression. Of note, PPARs are activated directly and indirectly
by metabolites of fat (29, 30, 54-57). This is potentially
significant, as many studies have demonstrated that leptin mRNA
levels are proportional to the size of fat stores, suggesting that
leptin gene expression is somehow linked to adipocyte triglyceride
content (58, 59). Of interest, treatment of rodents in vivo
with a thiazolidinedione (TZ), a PPAR
agonist, reduces leptin
mRNA and serum protein levels demonstrating a role for PPAR
in
regulating leptin gene expression (24). Recent work has extended
these studies and demonstrated that leptin mRNA levels in 3T3-L1
adipocytes are down-regulated at the transcriptional level by the
thiazolidinediones (25), and 3 kb of the human leptin promoter has been
shown to be negatively regulated by BRL 49653 in primary adipocytes and
3T3-L1 preadipocytes (60). However, neither of these studies examined
the possible mechanisms by which PPAR
mediates negative regulation
of the leptin promoter.
In the present study, TZ treatment of cultured primary rat adipocytes
was found to reduce leptin mRNA levels, indicating that negative
regulation by TZ is due to a direct effect on adipocytes. Furthermore,
the reduction in leptin mRNA levels appears to result at least in
part from negative effects on gene transcription as TZ reduced activity
of leptin promoter-reporter gene constructs in primary rat adipocytes.
Of note, negative regulation by TZ mapped to the proximal leptin
promoter (65 to +9), a region which binds C/EBP
but not PPAR
and RXR, suggesting that negative regulation by PPAR
occurred
without DNA binding. Negative regulation by TZ was also observed in
CV-1 cells, a heterologous cell line which expresses endogenous PPAR
but not C/EBP
. However, this effect required co-transfection of a
C/EBP
expression plasmid suggesting that negative regulation by
liganded PPAR
might be mediated via functional antagonism of
C/EBP
. C/EBP
, as shown above, is required for leptin promoter
activity in primary rat adipocytes. In addition to the effects of
C/EBP
and PPAR
on the leptin promoter, we also found functional
antagonism between C/EBP
and liganded PPAR
on the aP2 promoter in
CV-1 cells, where co-transfection of a C/EBP
expression plasmid
markedly blunted the sizable stimulatory effect of TZ on aP2 promoter
activity.
Functional antagonism between PPAR and C/EBP
could be mediated by
a direct protein-protein interaction between these two transcription
factors or by competition for a limiting critical co-factor such as
CBP/p300 (43). As GST-fusion protein pull-down assays and yeast
two-hybrid analyses failed to detect a direct interaction between
PPAR
and C/EBP
, we favor the latter hypothesis. When functional
antagonism occurs following co-transfection of transcription factors,
it is usually thought to be due to "unphysiologic squelching"
caused by unnaturally high levels of transcription factors. However,
such unphysiologic squelching cannot explain the inhibitory effects of
liganded PPAR
on leptin gene expression, since negative regulation
by TZ in primary rat adipocytes and CV-1 cells occurred in the presence
of endogenous PPAR
and did not require the addition of exogenous
PPAR
.
In the present study, a canonical DR+1 site was observed at position
3951 to
3939 of the murine leptin promoter. This site binds
PPAR
/RXR heterodimers and is a positive peroxisome
proliferator-activated receptor response element when linked to a
heterologous promoter. Indeed, the murine leptin 5
-flanking region
bears resemblance to other adipocyte genes, such as aP2 and PEPCK, in
that it contains a functional C/EBP
binding site within the proximal
promoter as well as a functional PPAR
binding site further upstream.
Although inclusion of this site in leptin reporter gene constructs
attenuated the negative regulation by TZ, the dominant role of PPAR
on leptin expression appears to be inhibitory in both primary rat
adipocytes and heterologous cell lines. This contrasts with net
positive regulation of the aP2 or PEPCK promoter. The reason for this
is presently unknown but might be explained by the presence of two PPAR
binding sites within the aP2 and PEPCK 5
-flanking regions, while only one was found within the leptin 5
region. Alternatively, additional elements may be present within the leptin 5
region which
modify transactivation by PPAR
.
In light of recent observations demonstrating positive synergy between
PPAR and C/EBP
on fat cell differentiation (15), it is somewhat
paradoxical that PPAR
would functionally antagonize C/EBP
action
on the leptin promoter. However, closer examination indicates that
these two observations need not be mutually exclusive. The mechanisms
by which PPAR
and C/EBP
synergize to accelerate fat cell
differentiation are unknown but could be explained by a number of
possibilities. First, PPAR
and C/EBP
could synergistically cooperate on the promoters of adipocyte genes possessing binding sites
for both transcription factors (i.e. aP2 and PEPCK). While this is plausible, it should be noted that such synergistic effects on
promoter-reporter gene constructs have not been reported. Indeed, in
the present study, C/EBP
not only failed to synergize with PPAR
on the aP2 promoter but actually antagonized the effect of TZ (Fig.
6C). Alternatively, PPAR
and C/EBP
could each
separately induce additional, presently unknown, important
transcription factors which then might cooperate on 5
-flanking regions
of adipocyte genes. Finally, PPAR
and C/EBP
might each be
responsible for increasing expression of subsets of genes which
together are required for efficient fat cell differentiation. These
latter two possibilities are not at odds with functional antagonism of
PPAR
and C/EBP
on leptin gene expression.
In summary, the present study reports that TZ treatment of primary rat
adipocytes decrease leptin mRNA levels. This effect appears to be
due, at least in part, to functional antagonism of liganded PPAR on
C/EBP
transactivation of the leptin promoter. Identification of the
precise mechanism for this antagonism will be the subject of future
investigations.
We thank Dong-Er Zhang for the GST-C/EBP
expression construct, Alan Friedman for the MSV C/EBP expression
vector, and T. Nagaya for TK109 pA3Luc. The
9-cis-retinoic acid was a gift from Arthur Levin.