From the Departments of Pharmacology and
§ Psychiatry and Behavioral Sciences, Duke University
Medical Center, Durham, North Carolina 27710 and the ¶ Department
of Biochemistry, Boston University School of Medicine,
Boston, Massachusetts 02118
Received for publication, September 14, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The The Two groups of transcription factors are known to be responsible for
initiating and maintaining adipocyte differentiation: the
CCAAT/enhancer-binding proteins (C/EBP) (11-17) and PPAR Materials--
Materials were obtained from the following
sources: dexamethasone, 3-isobutyl-1-methylxanthine, insulin,
puromycin, benzamidine, poly(dI/dC), and soybean trypsin inhibitor were
from Sigma; phenylmethylsulfonyl fluoride was from Life
Technologies, Inc.; Dulbecco's modified Eagle's medium (DMEM) was
from Cellgro; fetal bovine, calf, and cosmic calf sera from Hyclone;
[ Isolation and Sequencing of Genomic Clones and Construction of
Cell Culture and Transfections--
COS-7 and C3H10T1/2 (T1/2)
clone 8 fibroblasts (American Type Culture Collection) were maintained
in DMEM + 10% fetal bovine serum. 3T3-L1 and NIH-3T3 cells were
cultured in DMEM + 10% calf serum. The NIH-C/EBP Enzymatic Assays--
Luciferase activity was determined in a TD
20/20 luminometer (Promega) using the luciferase assay kit (Promega).
Oligonucleotides--
Oligonucleotides used in this study were
synthesized by Life Technologies, Inc. Nucleotide sequences are listed
in Table I.
Preparation of Nuclear Extracts and Electrophoretic
Mobility Shift Assays--
Nuclear extracts were prepared following
the method of Schreiber (31). Protein determinations were made by
the Bradford method (32). Double-stranded oligonucleotide probes were
annealed and end-labeled with [ Isolation and Analysis of RNA--
Total cellular RNA was
prepared using TRI reagent according to the manufacturer's
specifications (Molecular Research Center). RNA (40 µg) was denatured
by the glyoxal procedure, fractionated through 1.2% agarose gels, and
blotted onto Biotrans (ICN) membranes as detailed previously (33).
Radiolabeled probes were prepared by random primer synthesis (PrimeIT,
Stratagene) of the purified DNA fragments in the presence of
[ Adenylyl Cyclase Assay--
Cells were grown in 12-well plates
for cAMP assays according to published methods (37). Briefly, cells
were preincubated in serum-free DMEM, 25 mM HEPES, pH 7.5 (SFM) for 20 min, followed by fresh SFM containing 0.25 mM
isobutylmethylxanthine for 5 min. CL316,243 (5 µM final)
was added, and the plates were returned to the incubator for 20 min
(determined to be an optimal time in pilot experiments). Treatment was
terminated by rapidly aspirating the medium and adding cold 5%
trichloroacetic acid (100 µl/well). Two hundred µl of 50 mM KPO4, pH 7.4, was added to each well to partially neutralize the trichloroacetic acid. Cyclic AMP in the cell
lysate was determined by radioimmunoassay (38) using a polyclonal
antiserum to iodinated cAMP (39).
Differentiation-dependent Expression of the
C/EBP
In support of these mRNA expression studies, we assessed the
functional activity of the Structure and Activity of the Mouse
In experiments designed to evaluate the tissue-specificity of the
C/EBP
Finally, we wanted to determine whether binding of the The
Next we mutated the C/EBP site at In this study we have shown that the mouse Our observation that expression of the In addition to C/EBP Although we show a pivotal role for C/EBP3-adrenergic receptor
(
3AR) is expressed predominantly in adipocytes, and it
plays a major role in regulating lipolysis and adaptive thermogenesis.
Its expression in a variety of adipocyte cell models is preceded by the
appearance of CCAAT/enhancer-binding protein
(C/EBP
), which has
been shown to regulate a number of other adipocyte-specific genes.
Importantly, it has been demonstrated that several adipocyte cell lines
that fail to express C/EBP
exhibit reduced insulin sensitivity,
despite an apparent adipogenic phenotype. Here we show that
transcription and function of the
3AR correlates with
C/EBP
expression in these adipocyte models. A 5.13-kilobase pair
fragment of the mouse
3AR promoter was isolated and
sequenced. This fragment conferred a 50-fold increase in luciferase reporter gene expression in adipocytes. Two putative C/EBP binding sites exist at
3306 to
3298 and at
1462 to
1454, but only the
more distal site is functional. Oligonucleotides corresponding to both
the wild-type and mutated
3306 element were inserted upstream of a
thymidine kinase luciferase construct. When cotransfected in
fibroblasts with a C/EBP
expression vector, reporter gene expression
increased 3-fold only in the wild-type constructs. The same mutation,
when placed into the intact 5.13-kilobase pair promoter, reduced
promoter activity in adipocytes from 50-fold to <10-fold.
Electrophoretic mobility shift analysis demonstrated that the site at
3306 generated a specific protein-oligonucleotide complex that was
supershifted by C/EBP
antibody, while a probe corresponding to a
putative site at
1462 did not. These results define C/EBP
as a key
transcriptional regulator of the mouse
3AR gene during adipogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-adrenergic receptor
(
3AR)1 is a
unique member of the
AR family because, unlike the
1AR and
2AR, it is expressed predominantly in adipocytes and regulates both lipolysis and
nonshivering thermogenesis (reviewed in Ref. 1). In genetic and dietary models of obesity, progressive accumulation of adipose tissue is
associated with defects in the ability of catecholamines to mobilize
lipid stores (2-4). We have previously shown that the expression and
function of the adipocyte
ARs are blunted in most models of obesity
(5, 6). Nevertheless, a curious aspect of
3AR biology is
that, despite defects in
3AR expression and function,
selective agonists for this receptor have been shown to prevent or
reverse obesity (4, 7-10). The efficacy of these drugs is related to
increased brown adipose tissue thermogenesis and a restoration of
expression of the
3AR and
1AR in white adipose tissue depots (4). For these reasons it is important to
understand the tissue-specific and hormonal factors that regulate the
expression of this receptor.
(18-20). From a large body of work in model adipocyte cell lines, such as
3T3-L1, it has been shown that the C/EBPs are expressed in a
cascade-like fashion during the early stages of adipocyte
differentiation, with C/EBP
and C/EBP
preceding the appearance of
C/EBP
(15, 21). More recent studies indicate that the expression of
the adipogenic transcription factor PPAR
is partially under the
control of the C/EBP family of transcription factors and vice
versa (22, 23). Additionally, insulin-sensitive glucose uptake has
been shown to be impaired in adipogenic cells that lack C/EBP
, due to deficits in insulin receptor and insulin receptor substrate-1 (22, 24). Like other adipocyte-specific genes, the
3AR
is not expressed in preadipocytes, but appears late in the adipogenic program of both white and brown adipocytes (Refs. 25 and 26; this
report). Because of the pivotal role of C/EBP
in activating many
adipocyte-specific genes, the focus of our studies was to determine the
role of C/EBP
in initiating
3AR gene transcription during adipogenesis. Our results show that C/EBP
is required for the
adipocyte-dependent expression of the mouse
3AR gene, and we define the C/EBP binding site in the
3AR promoter that is responsible for this regulation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP and [
-32P]ATP from
PerkinElmer Life Sciences. CL316,243 was a gift from American
Cyanamid Co. (Pearl River, NY). GL237310x (rosiglitazone) and GF211835x
(LGD1069) were generous gifts from Drs. Steve Kliewer and Jurgenn
Lehmann (Glaxo/Wellcome).
3AR Promoter-Luciferase Plasmids--
A mouse genomic
DNA library constructed in
-DASH (Stratagene) was screened with a
mouse
3AR probe comprising the first 108 amino acids of
the protein (27). Eight independent positive clones were isolated and
analyzed by restriction enzyme mapping and Southern blot hybridization
with the 108 amino-terminal probe and a with second probe corresponding
to the first 600 nucleotides 5' to the initiator methionine. A
6641-nucleotide BamHI fragment was subcloned into the
pGEM-4Z plasmid, and two isolates containing the insert in opposite
orientations were sequenced along both strands by the Duke University
Automated DNA Sequencing Facility. The sequence of the 5283-nucleotide
BamHI-NarI fragment can be retrieved as
GenBankTM accession no. AF303739. This
BamHI-NarI fragment was subcloned into the
luciferase reporter construct pGL3Basic (Promega) at the
BglII/HindIII sites to generate
m
3-Luc. A series of deletion mutants was created by
digesting the m
3-Luc with the following enzymes to yield
the indicated fragments: NheI and SpeI (3288 base
pairs), KpnI (2079 base pairs), EcoRV and MluI (1107 base pairs), or MluI and
BglII (557 base pairs). A site-directed mutation of the
putative C/EBP response element at
3306 to
3298 was generated using
the GeneEditor kit (Promega) such that the sequence TGGAGCAAT was
changed to GACTAGCCT. The C/EBP
expression vector has been described
previously (28). Plasmids for transfections were purified using the
Promega Megaprep system.
, Swiss-PPAR
,
and NIH-PPAR
cell lines were maintained and differentiated as
described (13, 14, 24). T1/2 cells were seeded into six-well dishes and
differentiation proceeded as described (29). For most cell lines, 2 µg of DNA were transfected per well using 3 µl of FuGENE 6 as
outlined by the supplier (Roche Molecular Biochemicals). For
preparation of nuclear extracts enriched in C/EBP
, 20 µg of
C/EBP
expression vector were transfected into 10-cm dishes of COS-7
cells by calcium phosphate coprecipitation (30). The 3T3-L1 cells were
cotransfected with 4 µg of reporter vectors, 4 µg of either the
murine sarcoma virus-C/EBP
expression vector or the empty
murine sarcoma virus expression vector, and 2 µg of
cytomegalovirus-
-galactosidase using calcium phosphate
coprecipitation. T1/2 cells were harvested for luciferase activity
72 h after transfection, while all others were harvested after
48 h.
-Galactosidase activity was determined by a colorimetric assay
(absorbance at 570 nm) using chlorophenol
red-
-D-galactopyranoside as the substrate. Luciferase
data were normalized by dividing the light units by
-galactosidase activity.
Nucleotide sequences of oligonucleotides used in EMSAs
-32P]ATP (111TBq/mmol)
by T4 polynucleotide kinase. Nuclear extracts (10 µg) were incubated
with labeled oligonucleotide probes for 30 min on ice before loading on
pre-run polyacrylamide gels (Invitrogen) in 0.5× TBE (44.5 mM Tris borate, 1 mM EDTA, pH 8.0). For
competition studies, unlabeled nucleotides were incubated with nuclear
extracts on ice for 15 min prior to addition of the labeled
oligonucleotides. Rat liver nuclear extract was obtained from Geneka
Biotechnology (Montréal, Quebec, Canada) and gel shifts were
performed per manufacturer's instructions, using 2 µg of
extract/reaction. Reactions were resolved in 1× TGE (50 mM
Tris, 380 mM glycine, and 2 mM EDTA). Where
indicated, antibodies used in supershift analysis that are specific to
C/EBP
(sc-61x), C/EBP
(sc-150x), and C/EBP
(sc-151x) were
obtained from Santa Cruz Biotechnology.
-32P]deoxy CTP (111TBq/mmol) to a specific
activity > 2 × 109 dpm/µg DNA. The DNA
fragments that were used as probes were obtained from the following
sources. A fragment specific for mouse
3AR was prepared
as described previously (5). Mouse PPAR
cDNA was a gift from
Jurgenn Lehmann. The aP2 probe was a gift from Bruce Spiegelman (34). A
cDNA fragment for rat C/EBP
was described previously (13). A rat
cDNA probe for cyclophilin was used as an internal
hybridization/quantitation standard as described previously (35). Blots
were hybridized and washed as described previously (5, 36).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3AR Gene--
T1/2 fibroblasts have been shown to
differentiate into white adipocytes in response to PPAR
ligands, and
these cells can serve as a vehicle for examining the expression of
transfected genes (29), but the expression of
3AR in
these cells as a function of differentiation was unknown. Fig.
1 presents the pattern and time course of
expression of several adipogenic genes, including
3AR,
in these cells during differentiation. Total RNA was isolated every
other day from day 2 after ligand treatment until day 12 and analyzed
by Northern blotting (Fig. 1A). A progressive increase in
the expression of C/EBP
, PPAR
, and aP2 preceded detectable levels
of
3AR transcripts. The internal control transcript,
cyclophilin, was unchanged throughout this period. Fig. 1B
depicts the concomitant increase in the ability of a selective
3AR agonist to stimulate adenylyl cyclase activity
during differentiation of the T1/2 cells. By day 3 of differentiation,
there is a 2-fold increase in
3AR-stimulated adenylyl
cyclase activity. However, in subsequent days, there is a substantial
increase in cAMP production such that, by day 10,
3AR-stimulated cAMP production is enhanced 25-fold over
basal.
View larger version (29K):
[in a new window]
Fig. 1.
Differentiation-dependent
expression of 3AR-and agonist-stimulated adenylyl
cyclase activity in T1/2 adipocytes. A, Forty µg of total
cellular RNA from differentiating T1/2 adipocytes were fractionated
through 1.2% agarose gels and blotted as described under
"Experimental Procedures." The blot was hybridized with
-32P-labeled probes for
3AR, C/EBP
,
PPAR
, aP2, and cyclophilin. B, fully differentiated cells
were incubated with the
3AR-selective agonist CL316,243
(5 µM) for 20 min, and the cAMP produced was measured by
radioimmunoassay as described under "Experimental Procedures." The
data are expressed as picomoles of cAMP produced/well/min.
Is Necessary for
3AR Expression in
Adipocytes--
Previous studies have shown that ectopic expression of
either PPAR
or C/EBP
in nonadipogenic fibroblasts stimulates
adipogenesis (11, 20). Coexpression of the two proteins (22) results in
a synergism of adipogenesis that is comparable to that seen in the
well-studied 3T3-L1 model (20). Fig. 2
compares the expression of
3AR and other relevant
adipocyte genes in a collection of cell lines that either possess
de novo adipogenic capacity in response to hormonal
induction or were engineered to express various combinations of C/EBPs
and PPAR
to assess their contribution to the adipocyte phenotype.
/
39 cells are NIH-3T3 cells that were designed to express both
C/EBP
and C/EBP
under a tetracycline-responsive promoter to
ascertain the role of glucocorticoids and C/EBP
in the initiation of
adipogenesis (13, 14). When these cells are induced to differentiate,
they accumulate lipid droplets and they express both PPAR
and aP2
(Fig. 2). However, despite obvious adipocyte morphology, these cells
fail to express either C/EBP
or
3AR. In two other
adipocyte models, ectopic retroviral expression of PPAR
induces
C/EBP
and
3AR expression in the Swiss-3T3 cells (24),
but not in NIH-3T3 cells (20), again despite lipid accumulation and
expression of aP2. Finally, we examined
3AR expression
in another cell line, NIH-
, which ectopically express C/EBP
in the NIH-3T3 fibroblast background (24). As shown in the
right-hand side of Fig. 2, when these
cells are induced to differentiate they express
3AR
mRNA, in addition to PPAR
and aP2, as early as day 6.
View larger version (37K):
[in a new window]
Fig. 2.
Expression of adipogenic transcripts in
differentiating adipocyte cell line models. Forty µg of total
cellular RNA were isolated on the indicated days from either the
/
39, NIH-P
, or the Swiss-P
cells and were fractionated
through 1.2% agarose gels and blotted as described under
"Experimental Procedures." The blot was hybridized with
-32P-labeled
3AR, C/EBP
, PPAR
, aP2,
and cyclophilin probes.
3AR in these various
adipocyte cell lines by the ability of the
3AR-specific
agonist, CL316,243, to stimulate adenylyl cyclase activity. Table
II shows that there is no detectable
increase in
3AR stimulated cyclase activity in
undifferentiated versus differentiated NIH-PPAR
cells,
whereas there was a 5-fold increase in cAMP production in the NIH-
cells when comparing the differentiated and undifferentiated states. Together, these data strongly suggest that C/EBP
is necessary for
3AR expression and function in adipocytes.
CL316,243-stimulated adenylyl cyclase activity
3AR-specific agonist
CL316,243 (5 µM) for 20 min and subjected to RIA (see
"Experimental Procedures"). The experiments were performed in
triplicate.
3AR
Promoter.--
We sequenced a 5.28-kb BamHI-Nar I fragment
containing 150 nucleotides of exon 1 and 5.13 kb of sequence 5' to the
transcription start site (40) of the mouse
3AR gene.
Sequence analysis with SIGNALSCAN (GCG, University of Wisconsin,
Madison, WI) revealed a number of putative transcription factor binding
sites within the 5.13-kb promoter. As outlined in Fig.
3, two putative C/EBP protein binding
sites between
3306 and
3298 and between
1462 and
1354 were
identified. Several other putative regulatory elements, including
glucocorticoid and AP-1 binding sites, were also identified (see
GenBankTM accession no. AF303739).
View larger version (10K):
[in a new window]
Fig. 3.
Structure of the mouse
3AR promoter. A 5285-nucleotide
fragment of the
3AR gene, including 5135 nucleotides of
5'-flanking promoter sequence, was isolated and sequenced as described.
The location of restriction endonucleases for creating many of the
reporter constructs and the location and sequence of the two putative
C/EBP sites are shown.
3AR promoter, the promoter region was subcloned into the pGL3-Basic luciferase vector (Promega). This construct was then transfected into differentiating T1/2 adipocytes or proliferating NIH-3T3 cells. Relative to the promoter-less parent vector, the 5.13-kb
3AR fragment stimulated luciferase expression 50-fold in
the adipocytes (Fig. 4A). By
contrast, in NIH-3T3 cells, the same construct induced luciferase
expression <2-fold. These results are consistent with the observation
that
3AR mRNA is primarily observed in adipocytes.
To determine the importance of the putative C/EBP regions for the
activity of the mouse
3AR promoter, we evaluated the
activity of several 5'-deletion constructs (Fig. 4B). The
activity of the
3138 promoter truncation, which lacks the proposed
C/EBP site at
3306, is decreased to less than 50% of the
5.13 kb
promoter fragment. The
1929 bp deletion led to a further 30%
decrease of luciferase activity, while deletion of the second putative
C/EBP site at
1462, shown by the
957 bp truncation, had no further
effect. These data support the hypothesis that C/EBP
is necessary
for both the expression and function of the
3AR in these
adipocyte models.
View larger version (19K):
[in a new window]
Fig. 4.
Tissue-specific activity of the mouse
3AR promoter. A,
luciferase activity of the 5.13-kb mouse
3AR promoter
after transfection into differentiating T1/2 adipocytes and NIH-3T3
cells. Transfections were performed as described. Error
bars show the standard error of triplicate wells. The
results are representative of several experiments. B,
schematic representation of the different reporter constructs. Several
5'-deletion series of mouse
3AR promoter-luciferase
constructs were transfected into differentiating T1/2 adipocytes.
Error bars show the standard error of triplicate
wells.
Binds the
3AR
3306 Element--
To
establish whether either of the putative C/EBP binding sites was
capable of binding C/EBP proteins, we performed a series of gel shift
and antibody supershift assays. For these experiments, nuclear extracts
were prepared from COS-7 cells that had been transfected with a
C/EBP
expression vector, as described previously (41). The
oligonucleotides used in these experiments are shown in Table I. Using
a consensus C/EBP oligonucleotide as the probe resulted in a major
binding species (filled arrow) that was
supershifted (open arrow) in the presence of
antisera to C/EBP
(Fig. 5A,
lanes 2 and 3). Addition of a 100-fold
molar excess of the unlabeled
3AR
3306 eliminated this
band (lane 4). When the
3AR
3306 element was used as a probe (Fig. 5A, lanes
5-8), several binding species were detected, with one major
band that comigrated with the major C/EBP band in lane
2. This major band was completely supershifted
(open arrow) by anti-C/EBP
antibody
(lane 5 versus lane
6), but was eliminated when excess unlabeled oligonucleotide for a consensus C/EBP binding site (42) was included as a competitor (lane 7). In contrast, an oligonucleotide with
mutations in the
3306 C/EBP was unable to affect the gel shift
pattern of the labeled
3306 oligonucleotide (lane
8). In Fig. 5B, we again used the C/EBP consensus
sequence (lanes 1-4) and the
3306
3AR element (lanes 5-8) as probes
to further examine the specificity of binding. The major band
(filled arrow) was eliminated in the presence of 100-fold molar excess of the
3306
3AR C/EBP element,
but neither an excess of the
1462 C/EBP element nor of a Sp1
consensus affected binding. In lanes 5-9, the
3306 C/EBP element was used as a probe. A 100-fold molar excess of
the C/EBP consensus oligonucleotide inhibited binding of the major
band. The addition of the unlabeled C/EBP element at
1462 was able to
partially inhibit binding of the
3306 C/EBP element while the
addition of unlabeled Sp1 oligonucleotide or the mutant
3306
oligonucleotide had no effect. The relative affinity of C/EBP
for
the
3306 C/EBP element versus the consensus sequence was
assessed by including 10-, 50-, or 100-fold molar excess of unlabeled
oligonucleotides (Fig. 5C). The major band shown in
lane 1 (filled arrow) is
eliminated in the presence of increasing amounts of the
3306
oligonucleotide (lanes 2-4), while the mutant
3306 had no effect (lanes 5-7). As
anticipated, addition of the unlabeled consensus C/EBP sequence
completely blocked the appearance of the major band (lanes
8-10). Finally, as shown in Fig. 5D, the
oligonucleotide corresponding to the putative C/EBP site at
1462
produced gel shift bands with nuclear extracts, but these bands do not
appear to bind C/EBP
. First, the relative position of these faint
bands did not correspond to C/EBP binding as observed for the
3306
sequence and identical to the consensus, nor was the band pattern
affected when either the consensus or the
3306 C/EBP were used as
competitors. More importantly, the addition of anti-C/EBP
antibody
did not affect the abundance or position of these bands.
View larger version (48K):
[in a new window]
Fig. 5.
Electrophoretic mobility shift analysis with
probes corresponding to putative C/EBP sites within the mouse
3AR promoter. A and
B, 32P-labeled oligonucleotides corresponding to
residues
3306 to
3298 of the mouse
3AR promoter or a
consensus C/EBP were incubated with nuclear extract from COS-7 cells
transfected with a C/EBP
expression vector. Ten µg of nuclear
extract was used per sample. For competition assays, 100-fold molar
excess of unlabeled oligonucleotide was used. Supershifting was
performed by adding 1 µl of C/EBP
antibody. C, a
32P-labeled oligonucleotide corresponding to residues
3306 to
3298 was incubated with nuclear extract from COS-7 cells
transfected with a C/EBP
expression vector. For competition assays,
10-, 50-, or 100-fold molar excess of unlabeled oligonucleotide was
used. D, 32P-labeled oligonucleotides
corresponding to residues
1462 to
1454 or residues
3306 to
3298
of the mouse
3AR promoter were incubated with nuclear
extract from COS-7 cells transfected with a C/EBP
expression vector.
For competition assays, 100-fold molar excess of unlabeled
oligonucleotides were used; 1 µl of C/EBP
antibody was used for
supershift analysis.
3306 element
was specific for C/EBP
. To address this, we performed gel shifts
using rat liver nuclear extract, which contains multiple C/EBP
isoforms, and antibodies specific for C/EBP
, C/EBP
, and C/EBP
.
As shown in Fig. 6, the
3306 C/EBP
sequence produced strong gel shift bands when incubated with the rat
liver extract (lane 2), while a consensus C/EBP
oligonucleotide inhibited the appearance of these bands
(lane 3). A C/EBP mutant had no effect (lane 4) (for sequences see Table I). The major
band was nearly completely supershifted by antisera to C/EBP
(lanes 5, 8, and 9).
Addition of anti-C/EBP
supershifted a lower, weaker band (lanes 6, 8, and 10), and
anti-C/EBP
had no effect (lanes 7, 9, and 10). For comparison, the same consensus
C/EBP sequence used in Fig. 5 (see Table I) was used as the probe in
lanes 11 and 12. The major band was
supershifted by the addition of C/EBP
antibody. From these data, we
conclude that C/EBP
binds the
3306 C/EBP element within the mouse
3AR promoter.
View larger version (68K):
[in a new window]
Fig. 6.
The 3306 C/EBP site binds proteins present
in rat liver nuclear extract. 32P-Labeled
oligonucleotides corresponding to either the
3306 C/EBP in the mouse
3AR promoter or a C/EBP consensus were incubated with 2 µg of nuclear protein. For competition assays, 100-fold molar excess
of unlabeled oligonucleotide of a consensus C/EBP binding site or a
mutant C/EBP site were used. In supershift assays 2 µl of the
indicated antibodies were added; in the lanes where there are two
antibodies present, 1 µl of each antibody was used.
3AR C/EBP
Element Confers Activity to a
Heterologous Promoter--
Having identified a potential role for
C/EBP
in the regulation of the
3AR by correlative
expression and gel shift analysis, we sought to determine whether the
putative C/EBP at
3306 was functional. Two approaches were taken.
First, wild-type and mutated versions of the
3306 C/EBP
element
were transfected in the presence or absence of a C/EBP
expression
vector. Fig. 7 shows that the insertion
of one copy of the putative C/EBP
element results in a 3-fold
increase in luciferase activity, while the presence of two tandem C/EBP
elements results in a 9-fold enhancement. In contrast, the mutant
3306 C/EBP element abolished transactivation by C/EBP
.
View larger version (10K):
[in a new window]
Fig. 7.
The 3306 C/EBP site in the mouse
3AR promoter confers transcriptional
responsiveness to C/EBP
. Complementary
oligonucleotides corresponding to the C/EBP region at
3306 to
3298
(and a mutant version) were annealed and ligated into a thymidine
kinase-luciferase construct as described under "Experimental
Procedures." Four micrograms of the constructs were transfected into
proliferating 3T3-L1 cells with or without 4 µg of C/EBP
expression vector and 2 µg of
-galactosidase vector. Shown is a
representative experiment performed in triplicate.
3306 to
3298 from TGGAGCAAT to
GACTAGCCT within the context of the intact 5.13-kb
3AR promoter. This mutation is the same as the one used in the C/EBP
transactivation experiments in Fig. 7, as well as the gel shift assays
in Fig. 5. As shown in Fig. 8, when the
3AR promoter constructs containing wild-type and mutant
C/EBP sites were introduced into differentiating T1/2 cells, luciferase
activity from the mutant was reduced by more than half, to a level
equivalent to that observed in the
2852 deletion (Fig.
4B). These data demonstrate that this site mediates the
effect of C/EBP
on
3AR gene expression.
View larger version (11K):
[in a new window]
Fig. 8.
A mutation in the 3306 C/EBP-like region
inhibits mouse
3AR promoter
activity. Differentiating T1/2 cells were transfected with either
the pGL3 Basic vector, the
5.13 kb promoter, or the mut
5.13 kb.
The mut
5.13 kb contains a mutation in the C/EBP site at
3306 in
context of the original
5.13 kb construct. Shown is a representative
experiment performed in triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3AR gene
is specifically activated by C/EBP
and that this activation is
correlated with the binding of C/EBP
to an element residing between
3306 and
3298 bp upstream of the
3AR gene
transcription start site. By utilizing several cell lines that contain
various combinations of the C/EBPs and PPAR
, we showed that only
adipocytes expressing C/EBP
possess
3AR transcripts
and concomitant functional activity. A 5.13-kb promoter fragment of the
mouse
3AR gene containing two putative C/EBP binding
sites, at
3306 to
3298 and at
1462 to
1454, confers robust
expression of a luciferase reporter preferentially in adipocytes. This
transcriptional activity is significantly decreased upon deletion of
the more distal C/EBP element, while removal of the more proximal
element had no further effect. We also showed that this C/EBP element
at
3306 conveys transcriptional activity to C/EBP
in
vitro. Finally, electrophoretic mobility shift assays provided
evidence that C/EBP
interacts specifically with the element at
3306 and not the element at
1462 in the mouse
3AR
promoter. In every case, mutation of the
3306 C/EBP site eliminates
these responses.
3AR gene is
positively regulated by C/EBP
is consistent with numerous reports
showing the role of this transcription factor in adipogenesis. For
example, ectopic expression of C/EBP
is sufficient to induce
adipocyte differentiation in a number of cell lines, while the
expression of an antisense C/EBP
construct in 3T3-L1 adipocytes
blocks differentiation (43). Consistent with these in vitro
studies, C/EBP
null mice fail to develop white adipose tissue
(44).
, C/EBP
and C/EBP
have also been shown to
be critical regulators of adipocyte differentiation. C/EBP
and
C/EBP
are transiently expressed and precede the appearance of
C/EBP
(12). Interestingly, overexpression of C/EBP
, but not
C/EBP
, in preadipocytes converts them to adipocytes, suggesting that
C/EBP
can substitute for C/EBP
(15). However, the
/
39 cells, which constitutively express C/EBP
and C/EBP
, acquire an
adipocyte phenotype, as evidenced by the presence of PPAR
and aP2,
but fail to express either C/EBP
or
3AR. As shown in our gel shift assays, it appears that C/EBP
is capable of binding the C/EBP element at
3306 in the mouse
3AR promoter.
Perhaps this is not surprising because C/EBP isoforms have been shown to bind to a common DNA consensus sequence (21, 41). Despite this
interaction, it is clear that C/EBP
or C/EBP
alone is
insufficient to induce
3AR.
in transactivating the
3AR gene during adipogenesis, tissues containing high
levels of C/EBP
, such as liver and lung, do not contain appreciable amounts of
3AR. In addition, we show that the NIH-
cells do not express
3AR in the preadipocyte state,
despite the constitutive expression of C/EBP
. Therefore, it is clear
that some other transcription factor(s) are important in directing the
adipocyte-specific expression of the
3AR gene. Another
key regulator of adipogenesis is PPAR
. Constitutive expression of
PPAR
in fibroblasts can induce the conversion to the adipocyte
phenotype (20). PPAR
ligands, such as the thiazolidinediones, which
are a class of insulin-sensitizing agents, convert fibroblasts and
multipotential stem cells to adipocytes (13, 29). As well, PPAR
(
/
) animals completely lack adipose tissue (45-47). Previous
studies showed that, despite expression of PPAR
and development of
an adipocyte morphology, a lack of C/EBP
results in decreased
insulin-stimulated glucose transport (22, 24). Despite this apparent
cooperation between C/EBP
and PPAR
in adipocyte differentiation,
our initial attempts to locate a DR-1 PPAR
response element (PPRE)
within our mouse
3AR promoter fragment or to demonstrate
transactivation of the
3AR by PPAR
have been
unsuccessful. It is plausible that a PPRE lies outside of the promoter
region that we have isolated and studied. However, it is known that
PPREs in several PPAR
target genes deviate significantly from the
consensus DR-1 site (18). For this reason we are currently
investigating some regions that weakly resemble a PPRE half-site. To
fully understand the role of the
3AR in obesity, it will
be important for us to determine not only what other critical
transcription factor(s) is/are required for the regulation of the
3AR gene, but how their effects on
3AR
expression may contribute to obesity.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the following people for gifts of
plasmids and other reagents: Steve Kliewer, Jurgen Lehmann, Steve
McKnight, Pamela Mellon, Bruce Spiegelman, Ormand MacDougald, Donald
McDonnell, and Tom Gettys. We also thank Steven Kingsman and Michael
Seldin for the mouse genomic DNA library in the -DASH vector,
Putting Xu and Sheridan Snedden for help with library screening, and
other members of the Collins laboratory for advice and helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R01DK46793 (to S. C.) and R01DK53092 (to S. C.) and a National Institutes of Health minority predoctoral award (to T. M. D.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF303739.
To whom correspondence should be addressed: Duke University
Medical Center, Box 3557, Durham, NC 27710. Tel.: 919-684-8991; Fax:
919-684-3071; E-mail: colli008@mc.duke.edu.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M008440200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AR, adrenergic
receptor;
C/EBP, CCAAT/enhancer-binding protein;
PPAR, peroxisome
proliferator-activated receptor;
aP2, adipocyte fatty acid-binding
protein;
bp, base pair(s);
kb, kilobase pair(s);
DMEM, Dulbecco's
modified Eagle's medium;
SFM, serum-free medium;
PPRE, peroxisome
proliferator-activated receptor response element.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Collins, S., Cao, W., Soeder, K. J., and Snedden, S. K. (2000) in Adipocyte Biology and Hormone Signaling (Ntambi, J. M., ed), Vol. 37 , pp. 51-62, IOS Press, Washington, D. C. |
2. | Laudat, M. H., and Pairault, J. (1975) Eur. J. Biochem. 56, 583-589[Abstract] |
3. | Shepherd, R. E., Malbon, C. C., Smith, C. J., and Fain, J. N. (1977) J. Biol. Chem. 252, 7243-7248[Medline] [Order article via Infotrieve] |
4. |
Collins, S.,
Daniel, K. W.,
Petro, A. E.,
and Surwit, R. S.
(1997)
Endocrinology
138,
405-413 |
5. | Collins, S., Daniel, K. W., Rohlfs, E. M., Ramkumar, V., Taylor, I. L., and Gettys, T. W. (1994) Mol. Endocrinol. 8, 518-527[Abstract] |
6. | Collins, S., Daniel, K. W., and Rohlfs, E. M. (1999) Int. J. Obesity 23, 669-677[CrossRef] |
7. | Arch, J. R. S., Ainsworth, A. T., Cawthorne, M. A., Piercy, V., Sennitt, M. V., Thody, V. E., Wilson, C., and Wilson, S. (1984) Nature 309, 163-165[Medline] [Order article via Infotrieve] |
8. |
Himms-Hagen, J.,
Cui, J.,
Danforth, E., Jr.,
Taatjes, D. J.,
Lang, S. S.,
Waters, B. L.,
and Claus, T. H.
(1994)
Am. J. Physiol.
266,
R1371-R1382 |
9. | Largis, E. E., Burns, M. G., Muenkel, H. A., Dolan, J. A., and Claus, T. H. (1994) Drug Dev. Res. 32, 69-76 |
10. | Sasaki, N., Uchida, E., Niiyama, M., Yoshida, T., and Saito, M. (1998) J. Vet. Med. Sci. 60, 465-469[CrossRef][Medline] [Order article via Infotrieve] |
11. | Freytag, S. O., Paielli, D. L., and Gilbert, J. D. (1994) Genes Dev. 8, 1654-1663[Abstract] |
12. |
Darlington, G. J.,
Ross, S. E.,
and MacDougald, O. A.
(1998)
J. Biol. Chem.
273,
30057-30060 |
13. |
Wu, Z.,
Xie, Y.,
Morrison, R.,
Bucher, N.,
and Farmer, S.
(1998)
J. Clin. Invest.
101,
22-32 |
14. | Wu, Z., Xie, Y., Bucher, N. L. R., and Farmer, S. R. (1995) Genes Dev. 9, 2350-2363[Abstract] |
15. | Yeh, W. C., Cao, Z., Classon, M., and McKnight, S. L. (1995) Genes Dev. 9, 168-181[Abstract] |
16. | Schwarz, E. J., Reginato, M. J., Shao, D., Krakow, S. L., and Lazar, M. A. (1997) Mol. Cell. Biol. 17, 1552-1561[Abstract] |
17. | Lin, F., and Lane, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8757-8761[Abstract] |
18. | Brun, R. P., Tontonoz, P., Forman, B. M., Ellis, R., Chen, J., Evans, R. M., and Spiegelman, B. M. (1996) Genes Dev. 10, 974-984[Abstract] |
19. | Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I., and Spiegelman, B. M. (1994) Genes Dev. 8, 1224-1234[Abstract] |
20. | Tontonoz, P., Hu, E., and Spiegelman, B. M. (1994) Cell 79, 1147-1156[Medline] [Order article via Infotrieve] |
21. | Cao, Z., Umek, R. M., and McKnight, S. L. (1991) Genes Dev. 5, 1538-1552[Abstract] |
22. | Wu, Z., Rosen, E. D., Brun, R., Hauser, S., Adelmant, G., Troy, A. E., MeKeon, C., Darlington, G. J., and Spiegelman, B. M. (1999) Mol. Cell 3, 151-158[Medline] [Order article via Infotrieve] |
23. | Wu, Z., Puigserver, P., Andersson, U., Zhang, C., Adelmant, G., Mootha, V., Troy, A., Cinti, S., Lowell, B., Scarpulla, R. C., and Spiegelman, B. M. (1999) Cell 98, 115-124[Medline] [Order article via Infotrieve] |
24. |
El-Jack, A. K.,
Hamm, J. K.,
Pilch, P. F.,
and Farmer, S. R.
(1999)
J. Biol. Chem.
274,
7946-7951 |
25. |
Feve, B.,
Emorine, L.,
Lasnier, F.,
Strosberg, D.,
and Pairault, J.
(1991)
J. Biol. Chem.
266,
20329-20336 |
26. | Cannon, B., and Nedergaard, J. (1996) Biochem. Soc. Trans. 24, 40-45 |
27. |
Rohlfs, E. M.,
Daniel, K. W.,
Premont, R. T.,
Kozak, L. P.,
and Collins, S.
(1995)
J. Biol. Chem.
270,
10723-10732 |
28. | Friedman, A. D., Landschulz, W. H., and McKnight, S. L. (1989) Genes Dev. 3, 1314-1322[Abstract] |
29. | Kliewer, S. A., Lenhard, J. M., Wilson, T. M., Patel, I., Morris, D. C., and Lehmann, J. M. (1995) Cell 83, 813-819[Medline] [Order article via Infotrieve] |
30. | Gorman, C. (1984) Philos. Trans. R. Soc. Lond. B Biol. Sci. 307, 342-346 |
31. | Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Medline] [Order article via Infotrieve] |
32. | Bradford, M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Collins, S.,
Caron, M. G.,
and Lefkowitz, R. J.
(1988)
J. Biol. Chem.
263,
9067-9070 |
34. |
Spiegelman, B. M.,
Frank, M.,
and Green, H.
(1983)
J. Biol. Chem.
258,
10083-10089 |
35. |
Collins, S.,
and Surwit, R. S.
(1996)
J. Biol. Chem.
271,
9437-9440 |
36. | Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5201-5205[Abstract] |
37. |
Cotecchia, S.,
Kobila, B.,
Daniel, K.,
Nolan, R.,
Lapetina, E.,
Caron, L., R,
and Regan, J.
(1990)
J. Biol. Chem.
265,
63-69 |
38. | Harper, J. F., and Brooker, G. (1975) J. Cyclic Nucleotide Res. 1, 207-218[Medline] [Order article via Infotrieve] |
39. |
Gettys, T. W.,
Ramkumar, V.,
Uhing, R. J.,
Seger, L.,
and Taylor, I.
(1991)
J. Biol. Chem.
266,
15949-15955 |
40. | van Spronsen, A., Nahmias, C., Krief, S., Briend-Sutren, M.-M., Strosberg, A. D., and Emorine, L. J. (1993) Eur. J. Biochem. 213, 1117-1124[Abstract] |
41. |
Elberg, G.,
Gimble, J. M.,
and Tsai, S. Y.
(2000)
J. Biol. Chem.
275,
27815-27822 |
42. | Vinson, C. R., Sigler, P. B., and McKnight, S. L. (1989) Science 246, 911-916[Medline] [Order article via Infotrieve] |
43. | Lin, F.-T., and Lane, M. D. (1992) Genes Dev. 6, 533-544[Abstract] |
44. | Wang, N., Finegold, M., Bradley, A., Ou, C., Abdelsayed, S., Wilde, M., Taylor, L., Wilson, D., and Darlington, G. (1995) Science 269, 1108-1112[Medline] [Order article via Infotrieve] |
45. | Barak, Y., Nelson, M. C., Ong, E. S., Jones, Y. Z., Ruiz-Lozano, P., Chien, K. R., Koder, A., and Evans, R. M. (1999) Mol. Cell 4, 585-595[Medline] [Order article via Infotrieve] |
46. | Rosen, E. D., Sarraf, P., Troy, A. E., Bradwin, G., Moore, K., Milstone, D. S., Spiegelman, B. M., and Mortensen, R. M. (1999) Mol. Cell 4, 611-617[Medline] [Order article via Infotrieve] |
47. | Kubota, N., Terauchi, Y., Miki, H., Tamemoto, H., Yamauchi, T., Komeda, K., Satoh, S., Nakano, R., Ishii, C., Sugiyama, T., Eto, K., Tsubamoto, Y., Okuno, A., Murakami, K., Sekihara, H., Hasegawa, G., Naito, M., Toyoshima, Y., Tanaka, S., Shiota, K., Kitamura, T., Fujita, T., Ezaki, O., Aizawa, S., Nagai, R., Tobe, K., Kimura, S., and Kadowaki, T. (1999) Mol. Cell 4, 597-609[Medline] [Order article via Infotrieve] |