Activation of Extracellular Signal-Regulated Kinases and CREB/ATF-1 Mediate the Expression of CCAAT/Enhancer Binding Proteins ß and -
in Preadipocytes
Nathalie Belmonte,
Blaine W. Phillips,
Florence Massiera,
Phi Villageois,
Brigitte Wdziekonski,
Perla Saint-Marc,
Jennifer Nichols,
Jérôme Aubert,
Kumiko Saeki,
Akira Yuo,
Shuh Narumiya,
Gérard Ailhaud and
Christian Dani
Institute of Signaling, Development Biology and Cancer Research
(N.B., B.W.P., F.M., P.V., B.W., P.S.-M., J.A., G.A., C.D.), UMR 6543
Centre Nationale de la Recherche Scientifique, Centre de Biochimie
06108 Nice Cedex 2, France; Centre for Genome Research (J.N.),
University of Edinburgh, United Kingdom; Department of Hematology
(K.S., A.Y.), Research Institute, International Medical Centre of
Japan, Tokyo 162-8655, Japan; and Department of Pharmacology (S.N.),
Kyoto University, Kyoto 606-8501, Japan
Address all correspondence and requests for reprints to: Dr. Christian Dani, Centre de Biochimie (UMR 6543 CNRS), UNSA, Faculté des Sciences, Parc Valrose, 06108 Nice cedex 2, France. E-mail:
dani{at}unice.fr
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ABSTRACT
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The essential role of CCAAT/enhancer binding proteins (C/EBPs) ß
and
for adipocyte differentiation has been clearly established. In
preadipocytes, their expression is up-regulated by the activation of
leukemia inhibitory factor receptor (LIF-R) and prostacyclin receptor
(IP-R) via the extracellular signal-regulated kinase (ERK) pathway and
cAMP production, respectively. However, the molecular mechanisms by
which LIF and prostacyclin-induced signals are propagated to the
nucleus and the transcription factors mediating ERK and cAMP-induced
C/EBP gene expression were unknown. Here we report that both pathways
share cAMP responsive element binding protein/activation transcription
factor 1 (CREB/ATF-1) as common downstream effectors. LIF-R and
IP-R activation induced binding of CREB and/or ATF-1 to C/EBP promoters
and CREB-dependent transcription. Expression of dominant negative forms
of CREB dramatically reduced the LIF- and prostacyclin-stimulated C/EBP
ß and C/EBP
expression. Upon stimulation of the IP-R, the ERK
pathway was activated in a PKA-dependent manner. ERK activation by the
PKA pathway was not required for CREB/ATF-1 phosphorylation but
rather was necessary for CREB-dependent up-regulation of C/EBPs
expression. Our findings suggest that ERK activation is required for
CREB transcriptional activity, possibly by recruitment of a
coactivator.
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INTRODUCTION
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SEVERE OBESITY IS the result of an increase
in fat cell size, i.e. adipocyte hypertrophy, in combination
with increased fat cell number, i.e. adipose tissue
hyperplasia (1). New fat cells arise from a preexisting
pool of adipose precursor cells that are present irrespective of age
(2, 3). Therefore, the formation of new fat cells remains
an important issue from a physiological view point, and their enhanced
number in obesity emphasizes the critical role of the proliferation of
preadipose cells and their differentiation into adipose cells. The
characterization of the molecular events regulating the adipose
differentiation program (adipogenesis) has been the subject of numerous
investigations in the past two decades. A wealth of information has
been obtained both on the nature of the adipogenic factors, their
cognate signaling pathways, and the identification of key
transcriptional factors (4). So far, three families of
transcriptional factors have been identified to be important in mature
adipocyte development. Among members of the first family, PPAR
(=PPARß=NUC-1) appears rapidly in confluent cells that express early
markers of the adipogenic differentiation program but are still
triacylglycerol free, termed preadipocytes (5). The fatty
acid activation of PPAR
ectopically expressing fibroblasts leads to
the expression of PPAR
, which is critically required for terminal
differentiation of preadipocytes to triacylglycerol-filled adipocytes
(6). Both PPAR
and PPAR
function as heterodimers
with RXR
in a ligand-dependent fashion (7, 8). The
second group of adipogenic factors is the basic-helix-loop-helix
leucine zipper transcription factor family in which the /sterol
regulatory element binding protein-c appears quite early during
differentiation. Adipocyte determination and differentiation
factor-1 stimulates the expression of fatty acid synthetase and
lipoprotein lipase, which are responsible for fatty acid supply
(9, 10). CCAAT/enhancer binding proteins (C/EBPs) are the
third family of adipocyte-promoting transcription factors. They possess
both leucine zipper and basic and acidic domains and are active as
unliganded homodimers. It is now clear that transcription factors of
all three families play a sequential role in the program of adipocyte
differentiation and that the expression of C/EBPß and C/EBP
is the
earliest event to occur in preadipose cells. C/EBPß and C/EBP
both
promote adipogenesis of fibroblasts when they are ectopically expressed
(11, 12) and up-regulate the subsequent expression of
C/EBP
and PPAR
2. The essential role of these two C/EBPs for the
differentiation of adipocytes both in vitro and in
vivo has been clearly established using C/EBPß
-/-. C/EBP
-/- mice
(13).
We have shown that activation of two cell surface receptors,
i.e. the leukemia inhibitory factor-receptor (LIF-R) and the
prostacyclin-receptor (IP-R), stimulates the expression of both
C/EBPs and promotes adipogenesis. Preadipocytes but not adipocytes
secrete functional LIF and simultaneously express LIF-R. An antagonist
of LIF-R abolishes adipogenesis, strongly suggesting that secreted LIF
acts via a paracrine/autocrine mechanism. The reduced capacity of
lif-r-/- embryonic stem cells to
undergo adipocyte differentiation demonstrates that this receptor plays
a critical role in this process (14). Selective inhibitors
of the extracellular signal-regulated kinase (ERK) cascade inhibit
LIF-induced C/EBPß and C/EBP
gene expression and prevent
LIF-induced adipogenesis (14). Recently, we have
delineated a second pathway, which up-regulates C/EBPß and C/EBP
expression. Prostacyclin (PGI2), also secreted by
preadipocytes, acts via IP-R, which is expressed in preadipocytes only.
This pathway then up-regulates the expression of both C/EBPs by
triggering cAMP production and also promotes adipogenesis
(15). However, the molecular mechanisms by which LIF- and
prostacyclin-induced signals are propagated to the nucleus and the
transcriptional factor(s) mediating ERK- and cAMP-induced C/EBP gene
expression in preadipocyte cells are not known. This issue has been
addressed in this work.
In recent years, it has become clear that members of the leucine zipper
class of transcriptional factor cAMP-responsive element binding
proteins/activation transcription factor (CREB/ATF), respond to a
variety of external signals and play roles in cell proliferation and
differentiation (16, 17). CREB is the most extensively
studied cAMP-responsive element (CRE)-binding protein. Phosphorylation
of serine-133 is a critical event in CREB activation, leading to an
increase in its trans-activation potential by allowing the
recruitment and binding of coactivators such as CREB-binding protein
(CBP). ATF-1, another CRE-binding protein, has significant sequence
similarity to CREB, including in its phosphorylation domain. Initial
studies identified PKA as the major kinase responsible for Ser-133
phosphorylation, but subsequent studies have identified additional
pathways leading to CREB phosphorylation such as those that are
regulated by ERKs. Recently CREB, which is expressed before the
emergence of preadipocyte and adipocyte markers, has been shown to be
required for adipocyte differentiation. Expression of a constitutively
active form of CREB enhanced adipogenesis of 3T3-L1 cells while
expression of a dominant-negative form of CREB blocked adipogenesis
(18).
In this study, we used an antibody that specifically recognizes both
phosphorylated CREB and phosphorylated ATF-1. Our results demonstrate
that the regulation of C/EBPß and C/EBP
gene expression via the
LIF/LIF-R pathway is initially distinct from that of the
prostacyclin/IP-R pathway but that both pathways converge on the
activation of CREB/ATF-1. CREB/ATF-1 not only binds to a putative CRE
in the promoters of C/EBPß and C/EBP
genes but is also involved in
the regulation of their transcription. Stably transfected 3T3-F442A and
Ob1771 preadipose cells expressing dominant-negative forms of CREB
exhibit a potent attenuation of C/EBPß and C/EBP
gene expression
upon stimulation by LIF-R and IP-R agonists. Activation of the ERK
pathway upon LIF-R stimulation is sufficient for CREB phosphorylation
and induction of C/EBPß and C/EBP
transcription. While activation
of the PKA pathway upon stimulation of the IP-R is sufficient to induce
CREB phosphorylation, a PKA-dependent ERK activation is also required
to induce CREB-dependent transcription and stimulation of C/EBP gene
expression.
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RESULTS
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Stimulation of C/EBPß and C/EBP
Gene Expression by LIF and
Prostacyclin in ip-r-/-
or gp130-/- Mouse Embryo
Fibroblasts
It has been reported that exposure of Ob1771 and 3T3-F442A
preadipocytes to LIF stimulated C/EBPß and C/EBP
expression and
promoted adipogenesis. This response was transmitted via the cell
surface LIF-R/gp130 receptor complex and the ERK pathway
(14). Similarly, prostacyclin as well as its stable analog
(carba)prostacyclin (cPGI2) also induced C/EBPß and C/EBP
expression and appeared to act as a potent adipogenic hormone by
signaling through its cell surface IP-R and the cAMP-dependent pathway
in preadipocytes (15). As shown in Fig. 1
(upper panel), C/EBP
gene
expression was stimulated 5.7 ± 0.2-fold by LIF and 4.8 ±
0.3-fold by cPGI2 whereas addition of both factors simultaneously led
to an increase of 11.2 ± 1.6-fold. Similar data were obtained
with induction of C/EBPß gene expression. These results suggest that
the responses of the C/EBP genes to LIF and cPGI2 were additive.
Because preadipocytes secrete basal levels of both LIF and
prostacyclin, we were interested in determining whether each stimulus
could independently trigger C/EBP gene expression. Stimulation by LIF
and prostacyclin was examined in
ip-r-/- mouse embryonic fibroblasts
(MEFs) and gp130-/- MEFs. The gp130
subunit is known to be the common transducer of LIF and cytokines of
the IL-6 family. Thus gp130-/- MEFs
were deliberately used as both LIF and LIF-related cytokines appear to
be secreted from MEFs (14). As shown in Fig. 1
(left
panel), ip-r-/- MEFs were
unresponsive to the IP-R agonist with respect to the expression of
C/EBP genes; however, the cells clearly remained responsive to LIF.
Conversely, gp130-/- MEFs became
unresponsive to LIF, as expected, but remained responsive to cPGI2
(Fig. 1
, right panel). Taken together, these results
indicate that each of the LIF-R/LIF and IP-R/prostacyclin systems can
individually trigger the expression of C/EBPß and C/EBP, emphasizing
a redundancy at this early stage of adipogenesis. The transcriptional
factor(s) mediating ERK-and cAMP- induced C/EBPß and C/EBP
gene expression and a possible convergence between these two pathways
were then investigated.

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Figure 1. Stimulation of C/EBPß and C/EBP Expression in
Preadipose Cells and in ip-r-/- and
gp130-/- Mouse Embryo Fibroblasts
Upper panel, Ob1771 preadipose cells were maintained for
12 h in 1% FBS and then treated either with LIF (10 ng/ml) or
cPGI2 (1 µM) or with LIF plus
cPGI2 (10 ng/ml and 1 µM, respectively) for
1 h. Total RNA was extracted and 20 µg were subjected to
Northern blot analysis. Quantification of the hybridization signal was
performed as described in Materials and Methods,
standardized to ß-actin RNA signal, and results are expressed setting
the signal obtained in the absence of stimulation as 1. Results are the
means of two independent experiments. Lower panel, MEF
were maintained for 2 h in 1% FBS medium (C) and then treated
with LIF (10 ng/ml) or cPGI2) (1 µM) for
1 h. Total RNA was extracted and 20 µg were subjected to
Northern blot analysis.
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Binding of Agonists to LIF-R and IP-R Induces CREB/ATF-1
Transcriptional Activity
Various lines of evidence now support the notion that CREB family
members can be phosphorylated by several different kinases, which in
turn are activated by a variety of exogenous signals. Therefore, the
presence of Ser133 phosphorylated CREB and total CREB in preadipocytes
was examined by Western blot analysis. Using a (Ser133)phospho-CREB
(P-CREB) antibody which also detects the phosphorylated form of
CREB-related proteins, we showed that CREB and ATF-1 were rapidly
phosphorylated upon addition of LIF or cPGI2 to confluent Ob1771 and
3T3-F442A preadipocytes (Fig. 2A
). In
both cases, phosphorylated CREB/ATF-1 levels increased dramatically
within 10 min of treatment (>30-fold) and remained elevated for 30 min
while total CREB remained unchanged. Similar phosphorylation patterns
have been reported in 3T3-L1 preadipocytes treated with cAMP-elevating
agents (19). The ability of LIF and cPGI2 to regulate
CREB/ATF-1 transcriptional activity was subsequently investigated. For
this purpose, 3T3-F442A cells were transiently cotransfected with a
firefly luciferase reporter gene under the control of 16 repeated CREs
and a Renilla luciferase expression vector for normalization. As shown
in Fig. 2B
, addition of LIF or cPGI2 led to an increase in
CRE-dependent transcription. These observations were extended by
examining the capacity of nuclear extracts from Ob1771 preadipocytes
stimulated by LIF, cPGI2, or both to recognize the putative CRE present
in the C/EBPß and C/EBP
promoters through the use of EMSAs.
Potential CREs present in C/EBPß and C/EBP
promoters and probes
are shown in Fig. 3A
. It has been
previously reported that purified recombinant CREB was able to bind to
these promoter sequences (18). The results of the EMSA in
Fig. 3B
show a strong enhancement of binding activity to C/EBPß and
C/EBP
probes in LIF- or cPGI2- treated nuclear extracts compared
with that from unstimulated cells. These data suggest that
phosphorylation of CREB/ATF-1 promotes binding to CRE sequences. This
binding appeared specific as it was abolished by an excess of
competitor consensus CRE but not by mutated CRE (Fig. 3C
). Finally,
supershift experiments performed with nuclear extracts from
LIF-stimulated cells and a C/EBP
probe demonstrated that the complex
contained activated CREB/ATF1 (Fig. 3D
). At present, the ratio of CREB
and ATF-1 bound to the CRE has not been further analyzed.

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Figure 2. LIF-R and IP-R Activation Induces CREB/ATF-1
Phosphorylation and Transcriptional Activity
A, Ob1771 and 3T3-F442A cells were maintained for 15 h in 1% FBS
and then treated with 10 ng/ml LIF or 1 µM
cPGI2 for the indicated times. Protein lysates (25 µg)
were used for Western blot analysis using antibodies against
phospho-CREB and CREB. A representative result from three independent
experiments is shown. B, 3T3-F442A cells were cotransfected with
p MC16-firefly Luc and pEF-renilla Luc constructs and were untreated
(C) or treated for 16 h with LIF (10 ng/ml) or cPGI2
(1 µM). The figure plots normalized firefly luciferase
activity. The values represent the mean ± SEM of
three independent experiments performed in triplicate.
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Induction of C/EBPß and C/EBP
in Stably Transfected
Preadipocytes Expressing Dominant-Negative Forms of CREB and ATF-1
To directly assess the involvement of CREB/ATF-1 in the
up-regulation of the C/EBPß and C/EBP
genes by LIF or cPGI2,
stable retroviral transfection was used to generate Ob1771 and
3T3-F442A preadipocytes expressing dominant-negative forms of CREB.
Both dominant-negative forms, KCREB and ProCREB block gene activation
by titrating endogenous CREB and ATF-1 and preventing their interaction
with the CRE. KCREB has an Arg-to-Leu amino acid substitution at
position 301 in the DNA-binding domain and can not bind to DNA while it
still able to heterodimerize with endogenous CREB and ATF-1
(20). ProCREB contains a substitution of Arg at the same
position by Pro, resulting in comparable dominant negative activity as
KCREB (20). As shown in Fig. 4
, treatment of control-Ob1771
preadipocytes by LIF or cPGI2 led to an increase in the levels of
C/EBPß and C/EBP
mRNA as expected. LIF- or cPGI2-induced C/EBP
and C/EBPß gene expression was substantially reduced in
KCREB-expressing preadipocytes. Similar results were obtained by
stimulating the cells with the bisoxazole BMY45778, a potent agonist of
the IP-R (21). These observations were extended to stably
transfected 3T3-F442A preadipocytes expressing ProCREB. Similar to the
results obtained with KCREB, expression of ProCREB in preadipocytes
reduced C/EBP
stimulation by LIF or cPGI2. Interestingly, 3T3-F442A
preadipocytes did not respond to BMY45778, which will be discussed
later. Altogether, these data indicate that activation of CREB/ATF-1 is
necessary for the induction of C/EBPß and C/EBP
genes, via the
LIF-R and IP-R. Since our previous results demonstrated that ERK and
cAMP-related events were implicated in the up-regulation of C/EBP
genes, their involvement in CREB/ATF-1 activity was next
investigated.

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Figure 4. Expression of Dominant-Negative Forms of CREB in
Ob1771 and 3T3-F442A Cells Inhibits the LIF-R and IP-R Mediated
Induction of C/EBPß and C/EBP
AKV- or AKV-KCREB-expressing Ob1771 cells (A) and AKV- or
AKV-proCREB-expressing 3T3-F442A cells (B) were maintained for 2 h
in 1% FBS and stimulated with LIF (10 ng/ml), cPGI2 (1
µM), or BMY45778 (1 µM). After 1 h,
total RNA was prepared for Northern blot analysis. Blots were exposed
overnight. Quantification of the hybridization signal was performed as
described in Materials and Methods, standardized to
ß-actin RNA signal, and results are expressed by setting at 1 the
signal obtained in the absence of stimulation. Results are the means of
two independent experiments.
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A Pathway Linking PKA to ERK Is Required for the Activation of
C/EBP Gene Expression by IP-R
We have previously reported that the selective MAPK kinase (MEK)
inhibitor UO126 inhibits LIF-induced expression of C/EBP
and
C/EBPß, suggesting that the ERK pathway is involved. Stimulation of
IP-R by BMY45778 or cPGI2 resulted in the activation of C/EBP
(Fig. 5A
). This activation was PKA dependent as
pretreatment with 710 µM of the PKA inhibitor H89 led
to the complete inhibition of IP-R agonist-induced C/EBP
expression.
Surprisingly, complete inhibition was also observed by prior treatment
with 10 µM of the MEK inhibitor U0126 (Fig. 5A
).
This observation strongly suggested that IP-R induced the activation of
ERK. As shown in Fig. 5B
, both IP-R agonists induced ERK
phosphorylation. This phosphorylation was inhibited by the
MEK-inhibitor U0126, and the PKA inhibitor H89 (Fig. 5B
). Altogether,
these results demonstrated that activation of ERK by PKA was required
for C/EBP gene expression.

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Figure 5. ERK Activation Induced by IP-R Is Required for
C/EBP Gene Induction
A, Ob1771 cells were maintained for 2 h in 1% FBS, pretreated for
20 min with U0126 or H89 at the indicated concentrations, and then
stimulated with BMY45778 (1 µM) or cPGI2 (1
µM) for 1 h. Total RNA was prepared and C/EBP
expression was analyzed by Northern blot. B, Left panel,
Ob1771 cells were stimulated with BMY45778 (1 µM), cPGI2
(1 µM), or LIF (10 ng/ml) for the indicated times. DMSO,
the vehicle of cPGI2 and BMY45778, was also used. Right
panel, Ob1771 cells were pretreated with the inhibitors U0126
(10 µM) or H89 (7 µM) for 20 min before
stimulation by BMY45778 (1 µM) for the indicated times.
Cells were lysed and analyzed by Western blot for ERKs expression and
phosphorylation. The results are representative of three independent
experiments.
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Numerous studies have demonstrated that CREB phosphorylation and
transcriptional activity can occur through either PKA- or ERK-dependent
pathways (17). Therefore, we examined the contribution of
both pathways for CREB activation via IP-R. As shown, the PKA-inhibitor
H89 blocked CREB phosphorylation and transcriptional activity (Fig. 6
, A and B, respectively). In contrast,
the MEK-inhibitor U0126 did not affect CREB phosphorylation (Fig. 6A
),
but still inhibited the transcriptional activity of CREB induced by
IP-R activation (Fig. 6B
). This implied that an ERK-induced event was
required in addition to CREB phosphorylation for CREB-dependent
transcription of C/EBP. This requirement of ERK activation was
confirmed by another approach. In contrast to Ob1771 cells, which
respond to BMY45778 and cPGI2 through IP-R by up-regulating C/EBPß
and C/EBP
genes, 3T3-F442A cells appeared fully responsive to cPGI2
but unresponsive to BMY45778 (not shown). This lack of responsiveness
was not due to the lack of coupling of IP-R as phosphorylation of
CREB/ATF-1 was clearly observed under these conditions (Fig. 7A
). One possible explanation is that the
pathway linking PKA to ERK may not be functional in these cells when
exposed to BMY45778. To gain insight into the difference(s) between the
two cell lines and into the critical role of the ERK pathway, the
status of ERKs and their phosphorylated forms was examined after
exposure to different concentrations of BMY45778. Under these
conditions, ERK1 and ERK2 phosphorylation occurred in Ob1771
preadipocytes at levels as low as 20 nM, but remained
undetectable in 3T3-F442A preadipocytes even at 500 nM
(Fig. 7B
). The expression of B-Raf has been shown to be required for
ERK activation by the PKA pathway in a number of cell lines (22, 23). As shown in Fig. 7C
, B-Raf and Raf-1, the kinase upstream
of MEK, were expressed in both preadipose cell lines. Altogether, these
results strongly suggested that the lack of induction of C/EBP gene
expression in 3T3-F442A cells upon stimulation with BMY45778 was due to
the absence of ERK activation. These results confirmed that an
ERK-induced event was required, in addition to CREB/ATF-1
phosphorylation, for CREB/ATF-1-mediated transcription of C/EBP
genes.

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Figure 6. PKA and ERK Signaling Pathways Mediate CREB
Transcriptional Activity Induced by IP-R Activation
A, Ob1771 cells were pretreated for 20 min with U0126 (10
µM) or H89 (7 µM) and then stimulated with
cPGI2 (1 µM) for the indicated times. Phosphorylation of
CREB/ATF1 was observed by Western blot analysis and normalized by total
CREB expression. The results are representative of four independent
experiments. B, 3T3-F442A cells stably transfected with p MC16-Luc
reporter gene were pretreated for 20 min with U0126 (10
µM) or H89 (7 µM) before stimulation by
cPGI2 (1 µM). Luciferase activity was measured after
24 h.
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Figure 7. BMY45778 Induced Phosphorylation of CREB/ATF1 but
not ERK in 3T3-F442A Preadipocytes
A, 3T3-F442A preadipocytes were stimulated with BMY45778 (1
µM) for the indicated times. Cells were lysed and
analyzed by Western blot for CREB/ATF1 phosphorylation. B, Ob1771 or
3T3-F442A cells were stimulated either for 5 min with cPGI2
(1 µM) or BMY 45778 at the indicated concentrations or
for 10 min with LIF (10 ng/ml). Cells were lysed and analyzed by
Western blot for ERK phosphorylation. C, Unstimulated 3T3-F442A and
Ob1771 preadipose cells were lysed and analyzed by Western blot for
B-Raf and Raf-1 expression. Molecular weights are indicated. The
presence of B-Raf isoforms have been previously reported
(50 ).
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DISCUSSION
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We have previously shown that the C/EBPß and C/EBP
genes are up-regulated by both LIF/LIF-R and prostacyclin/IP-R, which
activate the ERK and the PKA signaling pathways, respectively. However,
the specific signaling molecules and transcription factors linking the
receptor-mediated events to increased gene expression had not been yet
identified. Our findings indicate that the LIF and
prostacyclin-activated pathways share CREB/ATF1 as a common downstream
effector, which in turn increases CRE-mediated expression of C/EBPß
and C/EBP
genes.
Several previous studies have demonstrated that the transcription
factors CREB and ATF1 can be activated by both the ERK and PKA
signaling pathways. This led us to examine whether CREB/ATF1 is
involved in LIF or prostacyclin-induced up-regulation of C/EBPß and
C/EBP
genes. We observed for the first time that LIF stimulation
results in the phosphorylation of CREB/ATF1. Similarly, cPGI2 also
induced the phosphorylation of CREB/ATF1. The phosphorylation of CREB
by both stimuli was rapid, occurring within 10 min, and preceded the
up-regulation of C/EBPß and C/EBP
, which takes place between 30
and 60 min. We next wanted to demonstrate that the CREB/ATF1
phosphorylation resulted in an active transcription factor, which could
affect C/EBP ß and C/EBP
induction. Stimulation of preadipocyte
cells with either LIF or cPGI2 resulted in the interaction between
CREB/ATF1 and the putative CRE present on the C/EBP genes as determined
by EMSA. In some systems, a constitutive binding of CREB to CRE
sequences in the absence of stimulus has been reported
(24), where phosphorylation of CREB results in the
enhancement of transcriptional activation by the recruitment of
additional coactivators (24, 25, 26). Alternatively,
phosphorylation of CREB might promote binding to the CRE (27, 28). The ability of LIF and cPGI2 to enhance its binding
activity, without inducing any change in overall CREB levels, suggested
that binding of CREB to C/EBP promoters could be regulated by
phosphorylation in preadipocytes.
The use of dominant-negative forms of CREB and ATF-1 (KCREB or
ProCREB) reduced the LIF and cPGI2-stimulated transcription of
C/EBPß and C/EBP
genes. These data collectively demonstrate that
LIF and cPGI2-induced signaling pathways converge upon CREB/ATF1, and
that the activation of this factor is associated with an
increased expression of the C/EBPß and C/EBP
genes.
This conclusion is strongly supported by a previous study which
demonstrates a role for CREB in adipogenesis (18). Reush
et al. (18) showed that cAMP elevating agents
induced CREB phosphorylation and subsequently adipogenesis in 3T3-L1
preadipocytes. However, this study did not examine the effect of
phospho-CREB on the expression of the C/EBP genes. Taken together,
these results highlight the importance of CREB in adipogenesis,
possibly by up-regulating the C/EBPß and C/EBP
genes.
After demonstrating the importance of CREB activation in the
up-regulation of the C/EBP genes, we studied the mechanisms by which
CREB is activated. Using a specific MEK inhibitor (UO126), we found
that the ERK pathway is required for CREB phosphorylation and C/EBP
expression induced by LIF. Similarly, the inhibition of the PKA pathway
using a PKA inhibitor abolished CREB/ATF1 phosphorylation and C/EBP
gene up-regulation in response to BMY45778 and cPGI2. These results
were expected, as LIF and cPGI2 are known to signal through the ERK and
PKA pathways, respectively. We were surprised, however, when the
addition of the MEK inhibitor resulted in the suppression of C/EBP
up-regulation after IP-R activation, as IP-R typically signals through
PKA and does not directly require the ERK signaling cascade. This
result demonstrated that ERK activation is an additional requirement
for C/EBP gene up-regulation.
Previous results showed that preadipocytes secrete basal levels of LIF,
which might then activate basal levels of ERK in an autocrine manner.
We thus studied C/EBP gene activation using MEFs with targeted
deletions in the gp130 and ip-r genes, enabling
us to study the two signaling pathways in isolation. We clearly
demonstrate that either ligand-receptor system alone is capable of
inducing the C/EBPs in the absence of the other. Therefore,
LIF-mediated ERK activation is not required for C/EBP expression
induced via IP-R. It thus appears that the ERK pathway is directly
activated by the IP-R ligands. A direct activation of the ERK cascade
by the IP-R ligands was confirmed by the observation that there is a
rapid phosphorylation of ERK after BMY45778 or cPGI2 treatment of
Ob1771 preadipocytes. This phosphorylation was inhibited by the
MEK-specific inhibitor, UO126, as well as by the PKA inhibitor, H89.
This led us to propose a pathway linking PKA to ERK. While PKA is
sufficient to phosphorylate CREB/ATF1, the activation of the ERK
pathway is critical for the final transactivation of the C/EBP genes by
IP-R activation.
We were able to confirm this result using another approach. Both cPGI2
and BMY45778 are considered to be high-affinity ligands for IP-R. The
clonal preadipocyte cell line Ob1771 showed an up-regulation of C/EBP
ß and C/EBP
after stimulation by both ligands. However, while
3T3-F442A preadipocytes and mouse embryonic fibroblasts show a response
to cPGI2, there is no change in C/EBP gene expression after BMY45778
stimulation. In 3T3-F442A cells we found that CREB is still
phosphorylated, indicating that the PKA is still activated by BMY45778
but ERK phosphorylation was undetectable. Using this cell model, we
confirmed that PKA-induced CREB phosphorylation is not sufficient for
C/EBP expression and that ERK activation is additionally required.
A weaker effect of the agonist BMY45778 compared with cPGI2 has been
previously described in the rat colon (29), which led the
authors to propose that BMY45778 interacts with a novel subtype of
IP-R. This proposal was supported by studies in the rat brain showing
different binding properties of several prostacyclin analogs
(30). Our results obtained in 3T3-F442A preadipocytes
could thus be explained if BMY45778 induced a weak activation of IP-R
leading to a PKA activation that was sufficient for CREB
phosphorylation but not for ERK pathway activation. Future studies will
address this hypothesis.
To date, the cAMP-induced stimulation of ERK activity has been reported
in neuronal and PC12 cells (22, 23, 31, 32), lymphoma
cells (33, 34), and melanocytes (35, 36). In
preadipocytes the step in which PKA activity is required to promote MEK
activity remains unclear. Because a MEK inhibitor blocked the
IP-R-mediated activation of ERK, we can envision various regulatory
points upstream of MEK linking PKA activity to the ERK signaling
pathways. The different isotypes of Raf kinase, which is the major MEK
activator, are known to be regulated by PKA (37). The
expression of B-Raf is generally required for ERK activation by the PKA
pathway (23). Raf-1 and B-Raf are expressed in 3T3-F442A
and Ob1771 cell lines (see Fig. 7C
). However, their role in
PKA-dependent activation of ERK in preadipose cells remains to be
investigated.
Our results show that to obtain CREB transcriptional activity, ERK
activation is required in addition to CREB phosphorylation. We suggest
that ERK activation is required for the regulation of CREB activity
itself and does not activate other transcription factors for C/EBP
induction as we show that the MEK inhibitor (UO126) blocked the cPGI2
up-regulation of a 16xCRE-luciferase reporter construct. This suggests
that the ERK activation is required for CREB activity possibly by
recruitment of a coactivator. The ability of MEK inhibitors to block
CREB-mediated transcription downstream of CREB phosphorylation has been
previously reported in PC12 cells (38) and in NIH 3T3
fibroblasts (39). Several transcriptional coactivators
leading to CREB- mediated transcription have been identified (40, 41). The transcriptional coactivator CREB binding protein (CBP)
is a putative candidate as it can associate with ERK (42),
an ERK-mediated phosphorylation of CBP stimulates its histone acetyl
transferase activity (43), and it has been proposed that
the ERK pathway increases CBP activity (38).
 |
MATERIALS AND METHODS
|
---|
Animals
Embryos from C57 BL/6J mice deficient for the prostacyclin
receptor gene
(ip-r-/-) at 14.5 d
post coitus were used to prepare fibroblasts after removing head,
heart, and legs. Embryos of strain (CD1) mice deficient for the
gp130 gene (gp130-/-)
were prepared similarly. Mouse embryo fibroblasts (MEF) thus obtained
were termed ip-r-/-
(44) and gp130-/- MEF,
respectively.
Cell Cultures
ip-r-/- and
gp130-/- MEF were used at passage 2
or 3 and grown to confluence at 37 C in H16 medium supplemented with
1x MEM nonessential amino acids, 2 mM glutamine,
1 mM pyruvate, and 10% heat-inactivated FBS
(Dutcher, S. A., France). Cells from Ob1771
(45) and 3T3-F442A (46) preadipocyte clonal
lines were grown to confluence in H16 medium containing 8% FBS. BOSC23
cells were grown in a 100-mm diameter culture dish in the presence of
10 ml of H16 medium containing 8% FBS.
Plasmids and Plasmid Constructions
The pCG expression vector containing KCREB or proCREB, two
dominant-negative forms of CREB (20), was digested with
BamHI and XbaI, and the resulting insert was
ligated into the BlueScript vector. The resulting vector was then
digested with SacI and HincII, and the insert was
ligated in the retroviral AKV bipe 2 vector. KCREB was obtained as
described previously (20), and ProCREB was obtained by
substituting Arg by Pro on position 301. The p
M-C16-Luc expression
vector (47) was a kind gift of J. C. Chambard (Nice,
France).
Luciferase Assays
3T3-F442A Preadipose cells were seeded at 5 x
104 cells per 35-mm diameter well. After 24
h, transient transfections were performed using the FuGene 6
transfection system (Roche Molecular Biochemicals,
Indianapolis, IN). Transfection mixes for each well contained 3 µg of
p
M-C16-firefly Luc construct (47) and 60 ng of
pEF-renilla Luc as internal control for normalizing firefly luciferase
activities. The p
M-C16-firefly Luc construct contained 16 repeated
CREs. Two days after transfection, cells reached confluence and were
stimulated for 16 h with the appropriate effectors. Cells were
lysed and both luciferase activities were determined using the
Dual-Luciferase reporter Assay System following the manufacturers
recommendations (Promega Corp., Madison, WI).
Retroviral Infection
For retroviral infection experiments, 8 x
105 BOSC23 cells were plated in a 100-mm diameter
culture dish. Twenty four hours later, exponentially growing cells were
transfected with 6 µg of AKV or AKV-KCREB/proCREB plasmids. The
virus-containing media (10 ml) were collected and centrifuged for 5 min
at 1,000 x g, and the supernatant was filtered through
0.45-µm pore size filters. 3T3-F442A and Ob1771 preadipose cells in
100-mm diameter culture dishes were infected at 50% confluence. After
24 h, infection media were removed and replaced every other day by
fresh H16 medium containing 8% FBS and 400 µg/ml of G418. One week
later, clones of cells were pooled and used for Northern blot
analysis.
Nuclear Extracts and Gel Retardation Assays
Probes were generated by adding 60 ng of double-stranded
oligonucleotide to T4-polynucleotide kinase buffer [0.5 M
Tris-HCl, pH 7.6, 0.1 M MgCl2, 10
mM dithiothreitol (DTT), 10 mM EDTA, 25%
polyethylene glycol (wt/vol)] with 1 µl
-32P-ATP and 5 U T4-polynucleotide kinase. The
EMSA was performed by incubation of the radiolabeled probe (70,000 cpm)
with 4 µg of nuclear extract in 1x binding buffer [10
mM Tris-HCl, pH 7.5, containing 50 mM NaCl, 0.5
mM DTT, 5 mM MgCl2, and
5% (vol/vol) glycerol] in the presence of a nonspecific competitor
[0.5 µg poly(dI-dC)(dI-dC) (Sigma, St. Louis, MO)], in
a final reaction volume of 20 µl. The binding reaction was
electrophoresed on a 6% polyacrylamide gel in 1x TBE buffer (90
mM Tris-HCl, pH 8.0, 90 mM boric acid, 2
mM EDTA). The gels were run for 1.5 h at 10
V/cm2 at 4 C to retain the stability of the
protein-DNA complexes.
RNA Analysis and DNA Probes
RNA was prepared and analyzed as previously described
(48). Quantification of the hybridization signal was
performed using a PhosphorImager screen (x-Bas1000, Fuji Photo Film Co., Ltd., Stamford, CT) coupled to the MacBas ver2.x
bio-imaging analyzer. The C/EBPß cDNA and the C/EBP
cDNA were
isolated from MSV/C/EBPß and MSV/C/EBP
plasmids (kindly provided
by S. L. McKnight, Tularik Inc., San Francisco, CA).
Western Blot Analysis
Confluent Ob1771 and 3T3-F442A cells were first maintained for
12 h in H16 medium containing 1% FBS, and then treated with
various effectors as indicated. Subsequently, cells were lysed with a
mixture containing 50 mM Tris-HCl buffer, pH 7.5, 1%
Triton X-100, 100 mM NaCl, 50 mM NaF, 5
mM EDTA, 40 mM ß-glycerophosphate, 2
µM sodium orthovanadate, 1 mM DTT, and 1x
protease inhibitor cocktail (Roche, Paris, France).
Proteins were separated on 10% SDS-polyacrylamide gels (30 µg of
protein per lane) and transferred on polyvinylidene difluoride (PVDF)
membranes (Amersham Pharmacia Biotech, Copenhagen,
Denmark). Membranes were soaked for 1 h in Tris-HCl buffer, pH
7.5, containing 150 mM NaCl, 0.1% Tween-20, and 5%
defatted milk. Total CREB and phosphorylated CREB were detected with
primary antibodies raised in rabbits and a secondary
peroxidase-conjugated antirabbit antibody according to the
manufacturers instructions (New England Biolabs, Inc.,
Beverly, MA). Monoclonal antibody directed against the dually
phosphorylated form of ERK1 and ERK2 was used according to the
manufacturers procedure (Sigma). Polyclonal antibody
against total ERKs and their use have been described (49).
Polyclonal antibodies directed against B-Raf and Raf-1 were purchased
from Santa Cruz Biotechnology, Inc. and used according to
the manufacturers instructions.
Materials
Leukemia Inhibitory Factor (LIF) was obtained from Euromedex
(France). cPGI2 was from Cayman Chemicals (Spibio, France). BMY45778
was a kind gift from N. Meanwell (Bristol-Myers Squibb Co., Princeton, NJ). The MEK inhibitor UO126 was purchased from
Promega Corp. and the PKA inhibitor H89 was from
FranceBiochem (France). Oligonucleotides were synthesized by
Amersham Pharmacia Biotech (Denmark). All other chemicals
were from Sigma-Aldrich (France).
 |
ACKNOWLEDGMENTS
|
---|
The secretarial assistance of G. Oillaux is gratefully
acknowledged. We are indebted to Drs. P. Lenormand (CNRS UMR 6543,
Nice) and R. Busca (INSERM U385, Nice) for helpful discussion and to
Drs. K. Yeow and R. Arkowitz for critical reading of the
manuscript.
 |
FOOTNOTES
|
---|
This work was supported by a special grant of the Bristol-Myers Squibb Co. Foundation to G. Ailhaud and a grant from Association
pour la Recherche sur le Cancer (ARC) (to C.D.) (Grant 9982). B.W.P.
was a recipient of an ARC fellowship.
Abbreviations: ATF-1, Activation transcription factor 1; CBP,
CREB-binding protein; C/EBP, CCAAT/enhancer binding protein; cPGI2,
(carba)prostacyclin; CRE, cAMP-responsive element; CREB,
cAMP-responsive element binding protein; DTT, dithiothreitol; ERK,
extracellular signal-regulated kinase; IP-R, prostacyclin receptor;
LIF, leukemia-inhibitory factor; LIF-R, LIF receptor; MEFS, mouse
embryonic fibroblasts; P-CREB, (Ser133)-phospho-CREB; PGI2,
prostacyclin.
Received for publication March 2, 2001.
Accepted for publication July 10, 2001.
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