 |
INTRODUCTION |
Cell differentiation requires changes in protein expression
patterns to allow manifestation of a specialized phenotype from a
precursor state. Defined transcription factors, often referred to as
"master regulators," are necessary and often sufficient to activate
the differentiation process. In mesenchymal differentiation, MyoD,
PPAR
,1 and CBFA1 represent
master regulators that drive muscle, fat, and bone cell
differentiation, respectively. The signaling pathways that regulate
their function are not well understood.
Several transcription factors play key roles in adipocyte
differentiation. C/EBP transcription factors, belonging to the basic region-leucine zipper superfamily, were the first to be recognized as
critical for adipogenesis. C/EBP
expression induces growth arrest
and adipocyte differentiation of mesenchymal cells (1-3), but the time
course of its expression is delayed relative to the earliest adipocyte
marker genes. The
and
isoforms of C/EBP were subsequently
identified as essential initiators of adipocyte differentiation (4, 5),
and mice with both genes deleted have grossly defective adipose tissue
development (6). Analysis of the gene for aP2, an early adipocyte
marker, led to the identification of PPAR
, a nuclear hormone
receptor (7). Like C/EBPs, PPAR
can drive adipogenesis in
mesenchymal cells (8), and deletion of PPAR
blocks adipocyte
differentiation in vivo (9). ADD-1/SREBP1c, a basic
helix-loop-helix transcription factor, may be involved in the
generation of the ligand for transcriptional activation of PPAR
(10).
Specification of adipocyte differentiation involves cooperation of
C/EBPs with PPAR
2. C/EBP
and C/EBP
are induced in response to
hormonal stimuli and, together, directly activate transcription of the
PPAR
2 gene (11, 12) and other genes linked to adipogenesis (13).
Whereas many adipocyte gene promoters contain binding sites for C/EBP
and PPAR
, no PPAR
binding site has been found in the PPAR
promoter (14). Activation of PPAR
2 transcription and transcriptional
activation by ligand results in further activation of adipocyte marker
genes. C/EBP
and -
are down-regulated as differentiation
proceeds, and their transcription functions are thought to be replaced
by C/EBP
(4). C/EBP
activates many of the same genes as C/EBP
and -
, and also triggers growth arrest that accompanies full
differentiation (1, 15). C/EBP
cooperates with PPAR
2 to activate
adipocyte gene expression, and both factors are required for adipocyte
differentiation (16). Thus, any of these transcription factors could
represent targets for regulation by signaling pathways that affect adipogenesis.
One signaling pathway that affects adipocyte differentiation is
initiated by TGF-
. TGF-
regulates mesenchymal differentiation, inhibiting osteoblast (17), myoblast (18), and adipocyte
differentiation (19, 20). TGF-
blocks adipocyte differentiation
in vitro (19, 21), and transgenic overexpression of TGF-
in adipose tissue inhibits differentiation in vivo (22).
However, TGF-
is expressed endogenously in adipose tissue in
vivo (23), and in cultured preadipocytes and adipocytes (24-26).
In animal models of obesity (23) and humans with obesity, TGF-
1
expression is increased, correlating directly with body mass index and
increased expression of PAI-1 (plasminogen activator
inhibitor-1), which in turn are closely related to insulin
resistance (27). These observations contrast with the ability of
TGF-
to strongly inhibit adipocyte differentiation. However,
increased TGF-
expression in obese adipose tissue is believed to be
related to increased tumor necrosis factor-
expression in obese
adipose tissue (28). Like TGF-
, tumor necrosis factor-
strongly
inhibits adipocyte differentiation in culture (29). Whereas there is
already an extensive body of literature on the role of tumor necrosis
factor-
in adipocyte tissue physiology and insulin resistance,
little is as yet known about the role of TGF-
in adipose tissue.
TGF-
signals through two types of transmembrane serine-threonine
kinase receptors. Ligand binding to the type II TGF-
receptor stabilizes complex formation with the type I TGF-
receptor and induces activation of the type I receptor (T
RI) by the type II receptor (T
RII) kinase (30). Smads then act as signaling effectors (31, 32). C-terminal phosphorylation of Smad2 or Smad3 by T
RI
results in a conformational change that promotes heteromerization with
Smad4, and stimulates nuclear translocation of Smad complexes. In the
nucleus, Smad proteins regulate transcription by binding to DNA and
interacting with other transcription factors.
We have shown that Smad3, and not Smad2, mediates inhibition of
adipocyte differentiation by TGF-
(20). Smad3 also acts as an
effector of TGF-
inhibition of osteoblast (33) and myoblast differentiation (34). In the latter case, Smad3 physically interacts with MyoD and disrupts its binding to DNA, thus reducing activation of
muscle-specific gene expression. In TGF-
-mediated inhibition of
osteoblast differentiation, Smad3 represses CBFA1 function without
disrupting its DNA binding, although the mechanism of Smad3-mediated
repression of CBFA1 remains to be characterized (33).
In this report, we examined the mechanism by which Smad3 and TGF-
inhibit adipocyte differentiation. We found that adipogenesis, driven
by either C/EBP
or C/EBP
, could be inhibited by TGF-
without a
decrease in C/EBP protein levels. C/EBPs physically interacted with
both Smad3 and Smad4, whereas PPAR
2 interacted weakly or not at all
with Smads. This interaction correlated with repression of
C/EBP-mediated transcription by Smad3 and Smad4 at adipocyte
differentiation-dependent promoters and multimerized C/EBP
binding sites. In contrast, Smad3 and Smad4 did not affect transcription by PPAR
2. Smad3 and Smad4 cooperated to repress the
transcription function of C/EBPs without inhibition of target DNA
sequence binding. These data represent the first example of direct
inhibition of the transactivation function of a transcription factor by Smads.
 |
MATERIALS AND METHODS |
Expression and Reporter Plasmids--
C/EBP
-pSV-SPORT and
PPAR
2-pBabepuro (8) were from B. Spiegelman. pCMX-RXR
and
PPRE-3X-TK-luc (35) were from R. Evans, pCMV-LAP, encoding C/EBP
starting from the second methionine (36), was from U. Schibler,
MSV-C/EBP
was from S. McKnight, and GAL4-C/EBP
(37) was from W. Roesler. The reporter
159leptin-luc, containing the mouse leptin
promoter from
159 to +9 bp relative to the transcription start (38),
was from B. Lowell, and the PPAR
2 promoter plasmid pGL3-
2p1000
(14) was from J. Auwerx.
For transient transfections, the coding regions for RXR
, PPAR
2,
C/EBP
, C/EBP
, and C/EBP
were cloned into pRK5 (39). To make
stable NIH3T3 cells expressing an adipogenic transcription factor, the
C/EBP
, C/EBP
, and C/EBP
coding sequences were cloned into
pBabepuro (40). VP16-C/EBP
and GAL4-C/EBP
were constructed using PCR to generate full-length C/EBP
fused to the VP16
transactivation domain in pXFVP16, or the GAL4 DNA-binding domain in
pXFGAL4 (41). The 3× C/EBP-luc reporter was made by cloning a
double-stranded oligonucleotide
(5'-AGATCTGTTGCGCAAGTGGAGGTTGCGCAAGTGGCAGGTTGCGCAAGCTCGAG-3'), containing 3 C/EBP binding sites, into pTA-luc (42). The PPAR
2 promoter construct
190PPAR
2-luc was made by PCR, using
pGL3-
2p1000 as a template, to generate a PPAR
2 promoter fragment
from
190 to +3 relative to the transcription start, which was cloned
into pTA-luc.
Cell Culture, Generation of Stable Cell
Lines--
3T3-F442A cells (43) were purchased from H. Green and
maintained as described (20). 3T3-L1 and NIH3T3 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium with 10%
calf serum, 10 units/ml penicillin, 10 µg/ml streptomycin, and
passaged prior to reaching confluence. Phoenix E cells (G. Nolan)
and COS-1 cells (ATCC) were cultured as described (34).
Retrovirally infected NIH3T3 cells were made as described (20).
Selection with 2 µg/ml puromycin was started 48 h postinfection. For differentiation, confluent cells were treated for 48 h with 1 µM dexamethasone and 0.5 mM
isobutylmethylxanthine in Dulbecco's modified Eagle's medium
containing 5 µg/ml insulin and 10% fetal bovine serum. Cells
expressing PPAR
2 were also treated with, and maintained in, 5 µM troglitizone (Parke-Davis). As needed, 10 ng/ml
TGF-
was added at the same time as the differentiation inducers and
readded when the medium was changed. All cells were maintained in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum plus
5 µg/ml insulin after the dexamethasone-isobutylmethylxanthine treatment.
Analysis of Lipid Accumulation, RNA, and Protein--
NIH3T3
cells, expressing the transcription factor of interest, were analyzed 8 days after initiation of differentiation treatment for lipid by Oil Red
O staining (20). Parallel cultures were harvested for RNA or protein
extraction. RNA was extracted using the SV total RNA isolation kit
(Promega) and processed for Northern analysis (20). For protein
extraction, cells were lysed in 300 µl/well with FLAG lysis buffer
(300 mM NaCl, 20 mM Tris, pH 7.5, 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin). 50 µg of clarified lysate was run
on SDS-PAGE gels and transferred to polyvinylidene difluoride. Western
blotting and development with ECL+ was performed as directed (Amersham
Biosciences), using anti-C/EBP
, anti-C/EBP
, and
anti-PPAR
primary antibodies from Santa Cruz Biotechnology.
Transient Transfections and Reporter Assays--
3T3-F442A or
3T3-L1 cells, plated 18 h prior to transfection at 9 × 104 cells per well of 6-well dishes, and COS cells, plated
at 1 × 106 per 10-cm plate, were transfected using
LipofectAMINE (Invitrogen). For 3T3-F442A and 3T3-L1 cells, 1 µg of DNA was used per well, and for COS cells, 5 µg of DNA was
used per plate, and the total amount of DNA was constant by addition of
pRK5. For luciferase assay, cells were lysed in 300 µl/well with 1×
reporter lysis buffer (Promega), and assayed using reagents from BD
Pharmingen. Assays were performed at least 3 times in duplicate or
triplicate. All values are expressed as the -fold induction relative to
the basal activity.
GST Interaction Assays and Immunoprecipitations--
GST-Smad
proteins (44) were prepared and purified on glutathione-Sepharose beads
(Amersham Biosciences). 35S-Labeled C/EBP
, -
,
-
, or PPAR
2 was generated by in vitro translation
using the TNT quick-coupled transcription/translation kit
(Promega) and [35S]methionine. 5 µl of translation
mixture, adjusted to 1 ml with GST pull-down buffer, was incubated with
2 µg of GST or GST-Smads, and adsorbed proteins were analyzed, as
described (33).
For analysis of Smad-C/EBP interactions in vivo, transfected
COS cells were metabolically labeled with [35S]methionine
and cysteine and processed for immunoprecipitation as described (45),
except that cells were lysed in FLAG lysis buffer. Precipitates were
washed twice with HSA (12.5 mM potassium phosphate buffer,
pH 7.4, 600 mM NaCl), once with MDB (0.1% SDS, 0.05%
Nonidet P-40, 300 mM NaCl, 10 mM Tris, pH 8.3),
once with HSA, and once with SA (12.5 mM potassium
phosphate buffer, pH 7.4, 300 mM NaCl). For sequential
immunoprecipitation of C/EBP immunoprecipitates, 50 µl of a buffer
containing 1% SDS, 20 mM Tris, pH 7.5, 50 mM
NaCl, and 1 mM dithiothreitol was added to the washed
beads, and the samples were heated at 95 °C for 4 min. The eluted
samples were then immunoprecipitated using anti-FLAG antibody.
Immunoprecipitated proteins were run on SDS-PAGE, and gels were soaked
in Amplify (Amersham Biosciences) prior to autoradiography.
Electrophoretic Mobility Shift Assays and Biotinylated
Oligonucleotide Interactions--
For electrophoretic mobility shift
assay, the following oligonucleotides were synthesized: 2× wild-type
C/EBP (top strand only), 5'-CTTGGCATATTGCGCAATATGCTTGGCATATTGCGCAAT
ATGC-3'; 2× mutant C/EBP,
5'-CTAGCGATAaaGCGCttTATGCTTGCGATAaaGCGCttTATGC-3'. The mutations,
indicated by lowercase letters, are in the residues that are critical
for C/EBP binding (46). For biotinylated oligonucleotide binding
reactions, the identical oligonucleotides (top strand) were modified by
5' addition of biotin.
To generate nuclear extracts, 3T3-F442A cells were plated at 5 × 104 cells per 10-cm dish in Dulbecco's modified Eagle's
medium with 10% fetal bovine serum, 5 µg/ml insulin. Five days
later, 10 ng/ml TGF-
was added to some dishes. After 1 h,
nuclear extracts were prepared as described (47). The double-stranded,
wild-type 2× C/EBP oligonucleotide was 5' labeled with
[
-32P]ATP and 1 µl of labeled probe (20,000-50,000
cpm) was incubated with 5 µg of 3T3-F442A nuclear extract, in the
presence of 4 µg of bovine serum albumin, 1 µg of poly(dI-dC), 12 mM HEPES, pH 7.9, 12% glycerol, 0.12 mM EDTA,
0.9 mM MgCl2, 0.6 mM
dithiothreitol, and 120 mM KCl, in a volume of 10 µl, for
15-20 min at room temperature. For supershift analysis, 250 ng of
anti-C/EBP
, 2 µg of anti-Smad2/3 (N-19; Santa Cruz), or 1 µg of
anti-Smad4 (Upstate Biotechnology, Inc.) were added prior to probe
addition, and incubated 45 min at 4 °C.
For each biotinylated oligonucleotide reaction, 30 µl of
streptavidin magnetic beads (Promega) were washed twice with 2× B&W buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 2 M NaCl), then 100 ng of wild-type or mutant oligonucleotide
was bound to the beads in 1× B&W buffer. Beads were washed twice with
2× B&W buffer, once with binding buffer (5% glycerol, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM
dithiothreitol, 0.15% Triton X-100, 100 mM NaCl, 4 mM MgCl2), blocked for 30 min using 1% bovine
serum albumin in binding buffer, and resuspended in 50 µl of binding
buffer. Transfected COS cells were lysed in 1 ml of FLAG lysis buffer,
and 100 µl of clarified lysate was used in a 0.5-ml reaction with 150 µl of 3× binding buffer, 10 µg of poly(dI-dC), and 50 µl of
DNA-bound streptavidin magnetic beads. Incubations proceeded for 1 h at 4 °C with gentle mixing, followed by 5 washes in 1× binding
buffer. Bound proteins were analyzed by SDS-PAGE followed by Western
blot analysis.
 |
RESULTS |
TGF-
Inhibits C/EBP
- and
C/EBP
-mediated Adipocyte Differentiation--
We have
previously shown that TGF-
signaling inhibits adipocyte
differentiation through TGF-
-activated Smad3 (20). To address
whether TGF-
inhibits the function of individual adipocyte transcription factors, we generated NIH3T3 cell lines stably infected with retroviruses encoding C/EBP
, C/EBP
, C/EBP
, or PPAR
.
NIH3T3 cells, while unable to differentiate into adipocytes, support adipogenesis when any one of these transcription factors is expressed (4, 8). Furthermore, these cells have a functional TGF-
signaling
system (48, 49). Expression of the respective transcription factors was
confirmed by Western blot (Fig.
1A). Whereas TGF-
treatment
did not affect C/EBP expression, PPAR
expression in the stable
PPAR
-infected cells consistently decreased upon treatment with
TGF-
.

View larger version (103K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of TGF- on
adipogenesis in NIH3T3 cells, expressing
C/EBP , C/EBP , or
PPAR . A, stably infected NIH3T3 cells
overexpress adipocyte transcription factors in the presence or absence
of TGF- , as shown by Western blots. Equal amounts of protein were
loaded. B, morphology and lipid accumulation in NIH3T3 cells
overexpressing C/EBP or - or PPAR , in the absence or presence
of TGF- . Cells were subjected to differentiation treatments in the
absence or presence of 10 ng/ml TGF- . Neutral lipid accumulation was
visualized by Oil Red O staining 8 days after initiation of
treatment.
|
|
Adipocyte differentiation of these cells was induced, and cells
infected with empty vector were subjected to the same treatments. Consistent with previous reports (4, 8), all three transcription factors caused characteristic adipocyte rounding and lipid
accumulation, as assessed by microscopic examination and Oil Red O
staining of lipid accumulation (Fig. 1B). However, in the
presence of 10 ng/ml TGF-
, the differentiation of C/EBP
cells was
prevented. TGF-
also completely inhibited adipogenesis in NIH3T3
cells that stably expressed C/EBP
(data not shown). Cells expressing
C/EBP
were also inhibited, but to a lesser extent (Fig.
1B). We also observed a low level decrease in
differentiation of PPAR
expressing cells in response to TGF-
(Fig. 1B), which is likely related to the decreased PPAR
levels in TGF-
-treated cells (Fig. 1A).
TGF-
Represses Adipocyte Marker Gene Expression Activated by
C/EBP
or C/EBP
--
We assessed the effect
of TGF-
on the expression of several adipocyte differentiation
genes. Ectopic expression of any of the three transcription factors
induced expression of PPAR
, aP2, and adipsin mRNAs, but none
induced C/EBP
expression, consistent with previous reports (4, 50).
Expression of C/EBP
led to the highest levels of adipocyte marker
gene expression (Fig. 2). TGF-
treatment of these cells moderately decreased the level of PPAR
expression and strongly inhibited aP2 and adipsin expression (Fig. 2).
NIH3T3 cells expressing C/EBP
had lower levels of PPAR
, aP2, and
adipsin than C/EBP
-expressing cells, consistent with the lower
number of lipid-filled cells in cultures expressing C/EBP
versus C/EBP
(Fig. 1B). Treatment of the
C/EBP
-expressing cells with TGF-
blocked the expression of
PPAR
, aP2, and adipsin (Fig. 2). C/EBPs can directly activate the
PPAR
and aP2 promoters (11, 12, 51) and are critical for induction
of adipsin expression (52). Because the levels of C/EBP
and -
were unaffected by TGF-
, these results suggest that TGF-
inhibits
the transcriptional activity of C/EBP
and -
, and that this
repression may result in decreased adipocyte marker mRNA levels and
reduced adipogenesis.

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of TGF- on
adipocyte-specific mRNA expression in NIH3T3 cells expressing
adipocyte transcription factors. NIH3T3 cells, infected with empty
vector or expressing C/EBP , C/EBP , or PPAR were treated to
induce adipogenesis in the presence or absence of TGF- . Northern
blotting was performed using the indicated probes. In the case of
C/EBP , C/EBP , and PPAR , the endogenous and retroviral
transcripts are indicated. Ethidium bromide staining (EtBr,
bottom panel) demonstrates the RNA loading. The appearance
of a C/EBP -hybridizing band of the size of the viral transcript in
C/EBP overexpressing cells is likely because of cross-hybridization
of C/EBP RNA.
|
|
TGF-
treatment of PPAR
expressing cells decreased the levels of
both viral and endogenous PPAR
mRNA (Fig. 2) and protein (Fig.
1A). Expression of aP2 mRNA, which is directly activated by PPAR
(7), was only modestly decreased by TGF-
. This decrease was consistent with the decreased PPAR
mRNA and protein levels. In contrast, adipsin and PPAR
mRNAs, which require C/EBP
transcription factors for full induction, and are not directly induced
by PPAR
2 (14, 52), were strongly down-regulated. These data support the notion that TGF-
primarily inhibits the activity of C/EBPs, i.e. C/EBP
and C/EBP
, whereas inhibition of PPAR
may occur at the level of PPAR
protein and mRNA accumulation,
rather than its transcriptional activity.
Physical Interaction of Smad3 with C/EBPs--
To
evaluate a direct role of TGF-
in inhibiting the function of the
adipogenic transcription factors, we examined their ability to
physically interact with Smads. In vitro translated
C/EBP
, C/EBP
, C/EBP
, and PPAR
were tested for interaction
with GST-fused Smad1, -2, -3, or -4 (Fig.
3A). The three C/EBPs had an
identical interaction profile, having strong interaction with both
GST-Smad3 and GST-Smad4, very weak interaction with GST-Smad1, and no
interaction with Smad2. PPAR
exhibited a marginal interaction with
GST-Smad3 only. Neither the addition of PPAR
ligand nor
cotranslating PPAR
with its partner RXR
improved the ability of
PPAR
to interact with GST-Smads (data not shown).

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 3.
C/EBPs physically interact with Smad3 and
Smad4. A, in vitro interaction of
35S-labeled C/EBP , - , or - , or PPAR with
GST-Smad proteins. The complexes were subjected to SDS-PAGE, Coomassie
staining, and autoradiography. Because the amounts of GST proteins were
the same in the different experiments, only one Coomassie-stained gel
is shown (bottom panel). B, C/EBP interacts
with Smad3 and Smad4 in transfected COS cells. Lysates were subjected
to immunoprecipitation with anti-FLAG or anti-C/EBP antibodies, or
sequential immunoprecipitation with C/EBP , then anti-FLAG. The
different proteins are indicated (arrows).
|
|
The interaction of C/EBP
with Smad3 and Smad4 was also
observed in vivo (Fig. 3B). For this purpose, we
expressed C/EBP
, FLAG-tagged Smad3 or Smad4, and the constitutively
active T
RII/RI chimera (53) in various combinations in COS cells.
The high transfection efficiency of COS cells that allows this type of analysis stands in contrast with 3T3-F442A cells, which were not amenable for protein interaction experiments (data not shown). The
minimal sensitivity of COS cells to TGF-
, coincident with their very
low levels of TGF-
receptors, requires coexpression of an activated
TGF-
receptor rather than TGF-
treatment, to activate transfected
Smads. Transfected cells were 35S-labeled, and subjected to
immunoprecipitation with anti-FLAG or anti-C/EBP
, or sequential
immunoprecipitation with anti-C/EBP
followed by anti-FLAG (Fig.
3B). Smad3 or Smad4 were detected in anti-C/EBP
immunoprecipitations, but only when C/EBP
was expressed.
T
RII/RI expression did not stimulate this interaction, as has been observed for interactions of Smads with transcription factors expressed in COS cells (33). Identical results were obtained
when C/EBP
was expressed instead of C/EBP
(data not shown). The
inability of the anti-Smad or anti-C/EBP antibodies to
immunoprecipitate denatured proteins generated in sequential immunoprecipitation precluded a similar analysis of endogenous protein
interactions in 3T3-F442A cells (data not shown).
We next made truncation mutants of C/EBP
consisting of its
transcription activation domain or basic region plus leucine zipper, required for DNA binding and dimerization, and assessed their abilities
to interact with GST-fused Smad3 and -4. The interactions of Smad3 and
Smad4 with the basic region plus leucine zipper segment were strong,
but we also detected a weak, yet specific interaction with the
transcription activation domain (Fig.
4A). Conversely, C/EBP
primarily interacted with the MH1 (N) domain of Smad3, although there
was also a very weak interaction with the MH2 (C) domain (Fig.
4B). In transfected cells, C/EBP
interacted with the MH2
domain as well as, or perhaps better than, the MH1 domain of Smad3
(Fig. 4C). This stronger interaction of the MH2 domain in vivo versus in vitro has also been
observed with the interaction of Smad3 with MyoD (34), and may reflect
the stabilizing participation of additional proteins in the
transcription machinery. Neither truncated Smad3 protein could interact
as strongly with C/EBP
as full-length Smad3, consistent with an
interaction of both Smad3 domains with C/EBP
.

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 4.
Analysis of interaction domains of
C/EBP and Smad3. A, in
vitro interactions of C/EBP domains with Smad3 and Smad4.
Interactions of 35S-labeled C/EBP segments, generated
in vitro, with GST-Smad3 or -Smad4 were assessed as in Fig.
3A. The top and bottom panels show the
autoradiogram and Coomassie staining, respectively. TAD,
C/EBP transactivation domain; BLZ, C/EBP basic region
and leucine zipper. B, in vitro interactions of
35S-labeled, in vitro generated C/EBP with
GST-fused domains of Smad3. N, MH1 domain; L,
linker region; C, MH2 domain. C, interaction of
Smad3 domains with C/EBP in vivo. FLAG-tagged Smad3,
Smad3 NL, or Smad3 C were expressed in COS cells, with or without
C/EBP and T RII/RI chimeric receptor. 35S-Labeled cell
lysates were subjected to single or sequential immunoprecipitations, as
shown.
|
|
Smad3 Inhibits Transcriptional Activation of Adipocyte Marker Genes
by C/EBPs--
The adipogenic function of C/EBP
has been
shown to depend on the transcription activation domain of C/EBP
(4).
The association of Smad3 and Smad4 with C/EBPs (Figs. 3 and 4) and the
inhibition of differentiation of cells expressing C/EBPs by TGF-
(Figs. 1 and 2) suggests that Smad3, in cooperation with Smad4, may
decrease the ability of C/EBP
or C/EBP
to activate transcription.
We tested this hypothesis using promoters from the PPAR
2 and leptin genes, two adipocyte differentiation-induced promoters known to be
activated by C/EBPs.
As shown in Fig. 5A, C/EBP
activated transcription of a promoter segment of PPAR
2
containing 190 bp upstream from the transcription start. Smad3
inhibited C/EBP
-mediated transcription, and this inhibition was
enhanced in the presence of activated TGF-
receptor. TGF-
itself
exerted only a minimal decrease (data not shown), consistent with the
high numbers of reporter plasmids and high expression levels of
transcription factors in these transient transfection/reporter assays
(e.g. Refs. 54 and 55). Smad4 enhanced the ability of Smad3
to down-regulate C/EBP-mediated transcription of the PPAR-
promoter
segment. Similar results were seen using a promoter segment containing
159 base pairs upstream from the transcription start site of the leptin
gene (Fig. 5B). C/EBP
-induced transcription was inhibited
by Smad3 and by TGF-
signaling. This inhibition was more dramatic
when Smad4 was coexpressed with Smad3.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Smad3 or Smad3/4 repress
transcription from adipocyte differentiation-induced promoters by
C/EBP. 3T3-F442A cells were transfected with the indicated
plasmids and luciferase activities were measured. A,
activation of the PPAR 2 promoter by C/EBP is inhibited by Smad3
with Smad4 and TGF- receptor signaling. C/EBP was expressed with
increasing amounts of Smad3, with or without Smad4. B,
activation of the leptin promoter by C/EBP is inhibited by Smad3
with Smad4 and TGF- receptor signaling. Relative luciferase
activities are shown.
|
|
TGF-
Signaling and Smad3 Inhibit Transcription at
C/EBP Binding Sites--
The down-regulation of C/EBP
-
or
-activated transcription from the PPAR
2 and leptin promoters
by TGF-
and Smads did not exclude functional interactions with other
DNA binding transcription factors, besides C/EBP
and -
. We
therefore tested if Smad3 repressed transcription from an artificial
promoter that is specifically activated by C/EBP, i.e. an
artificial promoter with three tandem C/EBP binding sequences.
Luciferase reporter assays were again carried out in 3T3-F442A preadipocytes.
As shown in Fig. 6,
A-C, C/EBP
, -
, and -
activated
transcription from this 3× C/EBP promoter, and their activities were mildly inhibited by an activated TGF-
receptor. C/EBP-induced transcription was inhibited by Smad3, and strongly inhibited by Smad3
and Smad4, but not Smad4 alone, and their inhibition was enhanced when
the activated TGF-
receptor was coexpressed. Smad3 and Smad4 also
decreased the basal transcription in the absence of cotransfected
C/EBPs, presumably because of inhibition of endogenous C/EBP activity.
This repression was not because of decreased C/EBP expression (Fig. 6,
A and B, bottom panels). The
inhibition of the C/EBPs by Smad3/4 was not restricted to 3T3-F442A
preadipocytes, but was also observed in 3T3-L1 preadipocytes (Fig.
6D).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 6.
Smad3 or Smad3/4 repress
transcriptional activation of a C/EBP reporter gene by
C/EBP , - , or
- . Cells were transfected with the indicated
plasmids and a luciferase reporter driven by three C/EBP binding sites
(3XC/EBP-luc). A, Smad3 and Smad3/4 repress
activation of 3× C/EBP-luc by C/EBP in 3T3-F442A cells. Lower
panel shows a Western blot for C/EBP expression in parallel
transfected cell lysates. B, Smad3 and Smad3/4 repress
activation of 3× C/EBP-luc by C/EBP in 3T3-F442A cells. Lower
panel shows a Western blot for C/EBP expression. C,
Smad3 and Smad3/4 repress activation of 3× C/EBP-luc by C/EBP in
3T3-F442A cells. D, Smad3 and Smad3/4 repress C/EBP ,
- , and - activation of 3× C/EBP-luc in 3T3-L1 cells.
E, comparison of the effects of Smad3 and Smad4 on
PPAR 2/RXR -mediated transcription versus
C/EBP -mediated transcription. 3T3-F442A cells were transfected with
a luciferase reporter driven by a promoter with three PPAR binding
sites (3× PPRE-luc) or the 3× C/EBP-luc reporter. Transfected cells
expressed PPAR 2 and RXR to activate 3× PPRE-luc, or C/EBP to
activate 3× C/EBP-luc, with or without coexpressed Smad3/4 and
T RII/RI. Relative luciferase activities are shown.
|
|
We also tested the effects of Smad3 and Smad4 on transcription by
PPAR
2. We used a luciferase reporter containing 3 PPAR
binding
sites, similar to the 3× C/EBP luciferase reporter, and tested the
ability of Smad3 and Smad4 to repress transcription activated by
PPAR
2 in 3T3-F442A cells. Smad3 and Smad4 did not inhibit the
activation of the 3× PPRE reporter by PPAR
2 and RXR
(Fig.
6E), despite their ability to inhibit C/EBP
activation of
the 3× C/EBP luciferase reporter. These data support the idea that
TGF-
inhibits adipocyte differentiation by inhibiting the abilities
of C/EBPs, but not PPAR
2, to activate target gene expression.
DNA Binding of Smad3 Is Not Required for Repression of
C/EBP Activity--
Transcriptional activation by Smad3
involves Smad3 binding to DNA. However, Smad3 DNA binding is not
required in the one example of Smad3-mediated repression where this
requirement was tested (33). To determine whether Smad3 needs to bind
DNA to inhibit transcription by C/EBP, we tested the ability of the
R47D mutant of Smad3, which is unable to bind to DNA (56), to inhibit
C/EBP
-mediated transcription from 3× C/EBP-luc. As shown in Fig.
7A, Smad3 R47D repressed
transcriptional activation by C/EBP
, similarly to the wild-type
Smad3. However, Smad4 did not potentiate the inhibition by Smad3 R47D,
as compared with wild-type Smad3. Similar results (data not shown) were
obtained using the Smad3
LG mutant, which likewise does not bind DNA
(56).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 7.
Analysis of Smad3 repression of
C/EBP -activated transcription.
A, repression of C/EBP transcription does not require
Smad3 DNA binding. 3T3-F442A cells were transfected with 3× C/EBP-luc,
and combinations of Smad3 or its DNA binding-defective R47D mutant,
Smad4, C/EBP , and T RII/RI. B, the MH2 domain of Smad3
is necessary and sufficient for repression of C/EBP transcription.
3T3-F442A cells were transfected with Smad3, or Smad3 lacking the MH2
domain (Smad3NL), or the Smad3 MH2 domain (Smad3C), and combinations of
C/EBP , Smad4, and T RII/RI. Relative luciferase activities are
shown.
|
|
We also assessed the effects of Smad3NL, i.e. the Smad3 MH1
domain with the linker segment, and Smad3C, consisting of the MH2
domain, to inhibit activation of the C/EBP reporter by C/EBP. Smad3C
exhibited a stronger inhibition than Smad3, and Smad3NL, which can also
interact with C/EBP, did not inhibit C/EBP transcriptional activation,
and tended to slightly enhance it (Fig. 7B). These results
are consistent with the inability of DNA binding mutations in the MH1
domain to impair the repression of C/EBP by Smad3 (Fig. 7A).
Smad3 Does Not Inhibit DNA Binding of C/EBP--
Smad3
could repress C/EBP transcription through interference with the DNA
binding of C/EBP, similarly to the inhibition of MyoD activity by Smad3
(34). Alternatively, Smad3 could target the C/EBP transactivation
domain, which mediates transcription once C/EBP is bound to DNA.
Because Smad3 interacted strongly with the basic region plus leucine
zipper segment of C/EBP that binds DNA, we examined the abilities of
Smad3 and -4 to block DNA binding of C/EBP
.
Transcription from the transactivation domain of VP16 is not influenced
by TGF-
/Smad3 signaling (34). Thus, when fused to a DNA binding
segment, alterations in transcription from the DNA binding site are
likely to correlate with changes in DNA binding. A chimera of VP16 and
the basic region plus leucine zipper segment of C/EBP
did not
activate transcription from the 3× C/EBP promoter (data not shown). We
therefore fused the VP16 transactivation domain to full-size C/EBP
,
and this chimera activated transcription from C/EBP binding sites to a
higher level than C/EBP
itself (Fig.
8A). Coexpression of an
activated TGF-
receptor, Smad3 or -4, or Smad3/4, had little or no
effect on transcription by VP16-C/EBP
at the 3× C/EBP promoter; the
minor repression may have been because of repression of the C/EBP
activation domain in VP16-C/EBP
(see below). This is in contrast
with the strong repression of C/EBP
by Smad3/4 in the same assay
(Fig. 8A). These data suggest that Smad3 and/or Smad4 has
little or no effect on DNA binding of C/EBP
.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 8.
Smad3/4 and
TGF- do not inhibit C/EBP binding to DNA.
A, Smad3 or Smad3/4 do not inhibit transcription by
VP16-C/EBP . 3T3-F442A cells were transfected with VP16-C/EBP or
C/EBP , and combinations of Smad3, Smad4, or T RII/RI, and the
activation of the 3× C/EBP-luc reporter was measured. B,
Smad3 or Smad4 do not interfere with C/EBP binding to DNA. COS cells
were transfected with plasmids for C/EBP , FLAG-tagged Smad3, and/or
Smad4, using a 4-fold excess of Smad plasmid DNA relative to C/EBP
DNA. Biotinylated oligonucleotides with two consensus C/EBP binding
sequences (W), or a mutated sequence that does not bind
C/EBP (M), were incubated with cell lysates. Bound proteins
were precipitated by streptavidin-linked magnetic beads and subjected
to Western analysis. Equal amounts of cell lysates were blotted
directly to show the levels of Smads and C/EBP . C,
TGF- does not decrease C/EBP binding to DNA in gel-shift analysis
(electrophoretic mobility shift assay). 3T3-F442A cells, grown under
differentiation conditions, were untreated or treated for 1 h with
10 ng/ml TGF- . Nuclear extracts were incubated with a
32P-labeled 2× C/EBP binding sequence oligonucleotide.
wt 2× C/EBP, unlabeled 2× C/EBP
binding sequence oligonucleotide at indicated molar excess; mut
2× C/EBP, unlabeled mutant 2× C/EBP
oligonucleotide at the indicated molar excess;
-C/EBP , incubation with anti-C/EBP to
disrupt the C/EBP complex and generate a supershift (ss);
-Smad3, -Smad4, complexes incubated with these
antibodies.
|
|
We also used a biotinylated oligonucleotide binding assay to assess
whether excess Smad3 or -4 affected C/EBP
binding to its DNA
sequence. C/EBP
and/or Smad3 or -4 were expressed in COS cells and
their binding to an oligonucleotide comprising a wild-type or mutant
C/EBP binding sequence was assessed by Western blotting (Fig.
8B). C/EBP
specifically bound the wild-type but not the
mutant oligonucleotide. Smad3 or Smad4, expressed at high levels (as
high as 10-fold excess, data not shown), did not reduce C/EBP
binding to a single (not shown) or double C/EBP binding sequence; if
anything, Smad3 slightly increased binding. A small amount of Smad3
could be detected specifically bound to C/EBP at the DNA, but not in
the absence of C/EBP or to the mutant oligonucleotide in the presence
of C/EBP. The low level binding of Smad3 through C/EBP to the C/EBP
binding sequence is similar to the interaction of Smad3 through CBFA1
to the CBFA1-binding oligonucleotide (33). This weak, yet specific
protein interaction is likely stabilized by the many protein
interactions in the multiprotein transcription machinery (57). Smad4
binding to C/EBP at the DNA was barely detectable and similar to background.
Finally, we assessed whether 10 ng/ml TGF-
affected the binding of
endogenous C/EBP in 3T3-F442A cells to a 32P-labeled
oligonucleotide containing two C/EBP binding sites, in an
electrophoretic mobility shift assay. A complex was detected that was
specifically competed by excess C/EBP oligonucleotide, but not by
mutant C/EBP oligonucleotide, and could be displaced and supershifted
by a C/EBP
antibody (Fig. 8C). TGF-
treatment did not
affect the formation of this complex. These nuclear extracts were able
to form a TGF-
-inducible complex on a Smad-binding element
oligonucleotide, which could be supershifted with Smad3 or Smad4
antibody (data not shown), indicating that the cells were responsive to
TGF-
. However, anti-Smad3 or anti-Smad4 did not supershift the C/EBP
complex (Fig. 8C), suggesting an unstable interaction or
a conformation unfavorable for antibody-Smad interaction. This result is consistent with the low amount of interaction detected in the oligonucleotide pull-down assay (Fig. 8B), and is
similar to what has been seen for Smad3-Cbfa1 interaction (33).
Smad3 Represses the Transactivation Function of
C/EBPs--
Because Smad3 and Smad4 did not inhibit C/EBP
binding to DNA, we assessed whether Smad3 and -4 inhibited the
transcription function of C/EBPs. We fused C/EBP
to the DNA binding
domain of GAL4 and measured the activation of transcription of the
FR-luc reporter by this fusion protein. TGF-
/Smad3 signaling does
not affect the binding of the Gal4 DNA binding domain to the Gal4 binding sites in FR-luc (34); so this reporter system allows measurements of transactivation function under conditions of constant DNA binding.
As shown in Fig. 9, A and
B, TGF-
signaling repressed the transactivation function
of GAL4-fused C/EBP
. Smad3 also repressed GAL4-C/EBP
-activated
transcription of FR-luc, and this repression was stronger with an
activated TGF-
receptor. Smad4 only weakly suppressed transcription
by GAL4-C/EBP
(Fig. 9A). The repression was
dose-responsive (Fig. 9B), and greatest when Smad3 and -4 were coexpressed (Fig. 9, A and B). This profile
of repression by TGF-
/Smad3 signaling was similar to the repression
of C/EBP
function at the C/EBP binding site (Fig. 6). GAL4-C/EBP
was also repressed by Smad3 or Smad3/4 (Fig. 9C), similarly
to GAL4-C/EBP
. For unknown reasons, GAL4-C/EBP
was unable to
activate transcription of FR-luc in 3T3-F442A cells (data not
shown). Together, the data show that Smad3 cooperates with Smad4
to repress the transactivation function of C/EBPs, without affecting
C/EBP binding to the promoter DNA sequences of target genes, and that
this may represent a mechanism by which TGF-
blocks
adipogenesis.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 9.
Smad3 or Smad3/4 inhibit
the transcription function of C/EBP and
C/EBP . 3T3-F442A cells were transfected with the
indicated plasmids and assayed for luciferase activity. A,
Smad3 or Smad3/4 repress activation of the GAL4 reporter FR-luc by
GAL4-C/EBP . B, dose-response of repression of
GAL4-C/EBP by increasing amounts of Smad3/4. C,
Smad3 or Smad3/4 inhibit FR-luc activation by GAL4-C/EBP . Relative
luciferase activities are shown.
|
|
 |
DISCUSSION |
TGF-
/Smad3 Signaling Inhibits Adipogenic
Differentiation Primarily through Functional Repression of
C/EBP
and C/EBP
--
We previously
observed that inhibition of adipogenesis by TGF-
was accompanied by
reduced mRNA levels for PPAR
, C/EBP
, and ADD1/SREBP1c, but
not of C/EBP
and -
(20). These data suggested that TGF-
targets the transcription factor cascade upstream of PPAR
, possibly
by repressing the functions of C/EBP
and -
. We therefore
expressed the adipogenic transcription factors individually in NIH3T3
cells, and evaluated the effect of TGF-
on the adipocyte
differentiation program activated by each transcription factor. This
analysis revealed that TGF-
repressed adipogenesis directed by
C/EBP
or -
(or C/EBP
; data not shown), without decreasing
their levels. This indicates that TGF-
signaling represses the
function of C/EBP
and -
, a conclusion confirmed by the
TGF-
/Smad3-mediated repression of transcription by C/EBPs at
synthetic and natural promoters, including the PPAR
2 promoter. Taken
together, these observations let us conclude that adipogenic
differentiation is inhibited by TGF-
at the level of C/EBP
and
-
function, upstream from PPAR
expression.
The differentiation of NIH3T3 cells driven by ectopic PPAR
expression was also mildly inhibited by TGF-
. However, TGF-
also
decreased PPAR
mRNA and protein levels. This decrease in PPAR
2 expression may occur post-transcriptionally, because C/EBP
or -
expression from the same retroviral promoter was not affected by TGF-
. Also, the induction of aP2 expression was relatively unaffected by TGF-
, when considering the reduced level of PPAR
expression. The differentiation-dependent expression of aP2
depends primarily on PPAR
, with C/EBP playing a contributory role
(58), whereas C/EBP is critical for PPAR
and adipsin expression (14, 52). The relative insensitivity of aP2 expression to TGF-
, in
contrast to the strong repression of PPAR
and adipsin, in NIH3T3
cells expressing PPAR
, suggests that the transcription function of
PPAR
is less affected by TGF-
than that of C/EBP. Accordingly,
TGF-
/Smad3 signaling did not repress the transcription activity of
PPAR
. Together, our data suggest that the repression of C/EBP
and
-
by TGF-
/Smad3 signaling prevents induction of PPAR
expression, and that the function of PPAR
itself is not a direct
target of repression by TGF-
.
Mechanism of Smad3-mediated Repression of
C/EBPs--
The mechanism of TGF-
-induced
transcriptional activation through cooperation of Smads with
sequence-specific transcription factors, and the role of Smad
corepressors in reducing activation, are well documented. In contrast,
little is known about mechanisms of TGF-
-mediated repression of
transcription. In epithelial cells, TGF-
/Smad3 signaling can repress
transcription by the androgen receptor (59), and activin and Smad3
repress C/EBP
-induced transcription from the haptoglobin promoter
(60). In mesenchymal cells, TGF-
/Smad3 signaling represses the
functions of CBFA1 in osteoblastic differentiation (33), and of
myogenic basic helix-loop-helix transcription factors (34). A mechanism
of TGF-
/Smad-mediated repression was clearly demonstrated only in the latter case. In response to TGF-
, Smad3 represses MyoD function through physical interaction with the helix-loop-helix domain of MyoD,
interfering with its dimerization with E12/47, thus impairing MyoD
binding to DNA and blocking transcriptional activation. We now show
that in mesenchymal cells, TGF-
represses the functions of C/EBP
and -
through Smad3, resulting in inhibition of adipogenic differentiation by TGF-
. The physical association of Smad3 and -4 with C/EBPs provides the basis for this functional repression. In
contrast, Smad3 can only marginally, if at all, associate with PPAR
and did not reduce its transcriptional activity. Physical association
per se is not predictive of Smad-mediated repression versus activation. Indeed, Smads interact with various
transcription factors to activate transcription (31, 32); and
interaction of Smads with AMLs (acute myeloid leukemia
transcription factors) results in coactivation or repression of
transcription, depending on the cell and promoter sequence contexts
(33, 42). There is as yet no evidence for differential physical
interactions that mediate transcriptional activation versus repression.
Because Smad3 interacts with the DNA binding domain of C/EBP (Fig.
4A), we evaluated whether Smad3 or -4 impaired DNA binding of C/EBP
or -
. In contrast to MyoD (34), Smad3 did not decrease DNA binding of C/EBPs, suggesting a different mechanism of repression. Similarly, repression of CBFA1 transcription by Smad3 was not accompanied by decreased DNA binding of CBFA1 (33). The physical interactions of Smad3 and -4 with C/EBP are reminiscent of c-Jun (44,
56), another bZIP transcription factor. Smad3 interacts in
vitro with the DNA binding domains of both C/EBP or c-Jun. Smad3
did not disrupt and, instead, increased the DNA binding of c-Jun.
However, in contrast to the transcriptional cooperativity of Smad3 with
c-Jun, Smad3 repressed the C/EBP activity.
We also showed that direct DNA binding of Smad3 is not required for
repression of C/EBP function by Smad3. DNA binding-defective Smad3
mutants repressed C/EBP transcription (Fig. 7), repression occurred at
C/EBP binding sites without an adjacent Smad binding DNA sequence
(Figs. 5-7), and Smad3 only interacted with the C/EBP binding site
through C/EBP (Fig. 8). Similarly, Smad3 represses transcription by
CBFA1 without the need for DNA binding (33). These observations stand
in contrast with the required DNA binding of Smad3 in
TGF-
/Smad3-mediated transcriptional activation, e.g. in
the cooperativity of Smad3 with c-Jun (56). It remains to be explored
whether this lack of requirement of Smad3 binding to DNA is a general
aspect of Smad3-mediated repression.
In contrast to repression of MyoD, Smad3 represses C/EBP transcription
by repressing its transactivation function (Fig. 9). This is the first
evidence for Smad-mediated repression of the transactivation function
of a transcription factor. How Smad3 represses the transactivation
function is as yet unclear. One possibility would be that Smad3
recruits histone deacetylases, because its MH1 domain is able to
recruit deacetylase activity (61). However, the MH1 domain was
dispensable for repression of C/EBP, and the MH2 domain by itself
potently repressed C/EBP transcription. Furthermore, trichostatin A,
which inhibits class I and II histone deacetylase activities, did not
reverse Smad3-mediated repression of transcription by C/EBP (data not shown).
Another possibility would be that Smad3 interferes with the function of
CBP/p300 as coactivator for C/EBP. Indeed, C/EBP
interacts with, and
is transcriptionally activated by, p300 (62), and Smad3 also interacts
with CBP/p300 as coactivators (41, 63). However, p300 did not reverse
Smad3 repression of C/EBP (data not shown), in contrast to the partial
reversion observed on the haptoglobin promoter in hepatoma cells (60).
Instead, increased p300 levels repressed, rather than enhanced,
C/EBP-mediated transcription from the PPAR
promoter (data not
shown). Furthermore, a Smad3-binding, dominant-negative segment of p300
(64) did not inhibit the repression of C/EBP by Smad3 (data not shown). Perhaps CBP or p300 contribute to Smad3-mediated repression of C/EBP
function in our system, and/or as yet unidentified cofactors may be
involved. Further research will address the mechanism and co-factor(s)
involved in mediating this repression.