From the Laboratoire de Biologie Cellulaire, INSERM Unité 327, Faculté de Médecine Xavier Bichat, Université Paris 7 Denis Diderot, Paris 75018, France
Received for publication, July 6, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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Cross-talk between Smad and mitogen-activated
protein kinase pathways has been described recently, and evidence for
Smad cooperation with AP-1 is emerging. Here we report that epidermal
growth factor (EGF) potentializes transforming growth factor Transforming growth factor Emerging evidence indicates that TGF- In the present study we addressed the question of whether AP-1/Smad
cooperation occurs in normal rat hepatocytes following a combined
stimulation with EGF and TGF- Plasmids--
Smad2-Gal4 constructs, prepared in J. Massagué's laboratory, Smad3-Gal4 plasmid, prepared by R. Derynck, and 6-Myc-Smad3, from CH. Heldin's laboratory, were all
generously provided by P. Ten Dijke. The plasmids 4xSBE-Luc and
6xMBE-Luc were provided by B. Vogelstein. Ti5xGal4-Luc was a gift from
X.-F. Wang. TAM-67 (18, 19) was provided by M. J. Birrer. DNMKK4
and I Antisense Jun Expression Vectors--
The 5'-untranslated
regions (underlined) of rat junD
(5'-CTAGACGGTCTGTACGGGCAGCGGACTGGGGGGCA, nt 73 to 102, acc.n°D26307), junB
(5'-CTAGACCAGCTCCCGAGGACGCGCGACCG-3', nt 1325 to 1348, acc.n°X54686) and c-jun
(5'-CTAGAGAGCGCTCCGTGAGTGACCGCGACTTTTCAAAGCG-3', nt 76 to 111, acc.n°X17163) were used as antisense probes to take advantage of the diversity in the nucleotide sequence in this region
and to provide specificity. These sequences were inserted between
EcoRI and XbaI cloning sites within the
polylinker of pCI-neo (Promega). The constructs were checked by DNA sequencing.
Hepatocyte Culture--
Hepatocytes were obtained from adult
male Harlan Sprague-Dawley rats (Charles River) weighing 180-200 g.
Animals were maintained on commercial chow (UAR, Villemoisson-sur-Orge,
France) and water ad libitum. Hepatocytes were isolated by
collagenase perfusion (21), as modified by Balavoine et al.
(22). The hepatocytes were purified by Percoll gradient centrifugation
(23), and viability was found to be >85% by trypan blue exclusion.
Hepatocytes were suspended in William's E medium (Life Technologies,
Inc.) supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin,
and 10% fetal calf serum, and they were plated on collagen-coated
Petri dishes.
Electrophoretic Mobility Shift Assays (EMSA)--
Nuclear
extracts were prepared according to Andrews and Faller (24) with minor
modifications. Cells were suspended in lysis buffer (10 mM
Hepes/KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml
aprotinin, 0.5 mM spermidine) and centrifuged at 500 × g for 30 s at 4 °C. The nuclear pellet was
suspended in 20 mM Hepes/KOH, pH 7.9, containing 25%
glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 4 mM
dithiothreitol, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml
aprotinin and centrifuged at 18,000 × g for 2 min at
4 °C. The supernatants were stored at
Single-stranded oligonucleotides corresponding to the upper
strand of the Smad-binding element (SBE)
(5'-GGAGTATGTCTAGACTGACAATGTAC-3') (6) or the
TPA-responsive element (TRE) (5'-TAAAGCATGAGTCAGACACCTC-3') were used (the cis elements are shown by the
underlines). They were end-labeled with T4 polynucleotide
kinase in the presence of [
For supershift analyses, 2 µl of antibody specific to the Jun family
(anti-c-Jun, anti-JunB, and anti-JunD), the Fos family (c-Fos and
Fra-1), or Smad3 protein (Santa Cruz Biotechnology) were incubated with
the nuclear extracts for 2 h at 4 °C prior to incubation with
the 32P-radiolabeled probe.
Cell Transfection and Luciferase (Luc) Assays--
Immediately
after isolation, hepatocytes at a density of 20 × 106
cells/0.8 ml of PBS containing 5% fetal bovine serum (25) were electroporated (Gene Pulser, Bio-Rad) at 160 V and 960 microfarads in
the presence of 50 µg of reporter plasmid, 30 µg of expression vector, and 30 µg of RSV-CAT plasmid to correct for transfection efficiencies. The total amount of DNA was adjusted to 400 µg with salmon sperm DNA. After electroporation, the hepatocytes were plated in
the presence of 10% fetal bovine serum for 1 h and then deprived
of serum for 18 h. They were finally incubated for 24 h in
serum-free medium in the presence or absence of EGF (20 ng/ml), TGF- Coimmunoprecipitation--
Approximately 1 mg of proteins from
cells lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM PMSF, 4 mM dithiothreitol, 0.5 mM spermidine, 1 µg/ml
leupeptin, 1 µg/ml aprotinin) were incubated for 2 h at 4 °C
with protein A-Sepharose beads coated with anti-c-Jun or anti-JunB
antibodies (Santa Cruz Biotechnology). The beads were then collected
and washed four times with buffer A (PBS containing 0.05% deoxycholate sodium, 0.01% SDS, and 0.5% Triton X-100), 3 times with buffer B
(0.5% Triton X-100, 1 mM EDTA, 500 mM NaCl,
125 mM Tris-HCl), and once with 10 mM Tris-HCl,
pH 8.1. Bound proteins were eluted by boiling in 1× Laemmli buffer and
subjected to SDS-PAGE analysis (10% acrylamide) and then
electroblotted onto nitrocellulose membranes. The blots were incubated
for 1 h with a 1/1000 dilution of mouse anti-Myc antibody (Santa
Cruz Biotechnology) in PBS containing 0.1% Tween 20, followed by
incubation with 1/5000 dilution of horseradish peroxidase-conjugated
anti-mouse IgG (Amersham Pharmacia Biotech). Immune complexes were
revealed by the enhanced chemiluminescence kit (Amersham Pharmacia
Biotech) according to the manufacturer's instructions and exposed to
x-ray film for visualization.
Western Blot Analysis--
Nuclear extracts prepared as above
(50 µg) were separated by SDS-PAGE and electrophoretically
transferred to nitrocellulose membranes (Schleicher & Schuell).
Membranes were probed with either anti-phospho-c-Jun antibody (New
England Biolabs), which recognizes phosphorylated serine 63 of c-Jun,
or with anti-c-Jun antibody (Oncogene Research Products), which
recognizes both phosphorylated and nonphosphorylated forms of c-Jun.
Immunoreactive bands were visualized by enhanced chemiluminescence
(Amersham Pharmacia Biotech).
Statistical Tests--
We used a Student's t test
and the one-way analysis of variance followed by the least significant
difference test.
EGF Synergizes with TGF- AP-1 Is Involved in the Synergism between EGF and TGF- c-Jun and JunB Cooperate with Smad3--
AP-1 is a dimeric
transcriptional complex composed of Fos (c-Fos, FosB, Fra1, and Fra2)
and Jun (c-Jun, JunB and JunD) proteins. Interactions between Smad3 and
all Jun family members has been reported (11, 12). To determine which
Jun protein cooperates with Smad3 during EGF and TGF- Endogenous c-Jun Associates with Smad3 upon Costimulation with
TGF- Costimulation with TGF- Activations of PI3-Kinase and p38 Are Implicated in Stimulation of
Smad3 Transactivation--
Tyrosine phosphorylation of specific
residues of the EGF receptor activates several signaling pathways
(reviewed in Ref. 28), all of which are implicated in AP-1 activation.
An important pathway triggered by EGF is the PI3-kinase cascade, which
is directly activated by phosphorylation of the EGF receptor (29, 30). Accordingly, hepatocytes were cotransfected with Ti5xGal and Gal4-Smad3 plasmids and treated with LY294002, a specific inhibitor of the PI3-kinase pathway. LY294002 did not change Smad3 transcriptional activity induced by EGF or TGF-
To assess further the role of PI3-kinase activation on Smad3
transactivation, we evaluated the effect of the transfection of an
activated Ras expression vector carrying a point mutation enabling the
selective activation of the PI3-kinase pathway (RasV12C40). As shown on
Fig. 7A, basal Smad3
transactivation was elevated (about 3-fold) in RasV12C40-transfected
hepatocytes compared with pRSV-transfected unstimulated cells,
mimicking the EGF-induced effect on Smad3 transcriptional activity.
TGF- EGF Induces Phosphorylation of c-Jun by PI3-Kinase and
p38-dependent Pathways--
Our results indicated that the
synergistic effect of TGF- EGF/TGF- There is increasing evidence that several signaling pathways
interfere with Smads to regulate TGF- Binding of EGF to its receptor activates the PI3-kinase pathway
(28-30), a cascade involved in AP-1 activation (29, 33). We show that
this pathway is involved in the potentiation of Smad3 transactivation
by Jun proteins since inhibition of the PI3-kinase pathway by the
highly selective inhibitor LY294002 or by a PI3-kinase dominant
negative mutant efficiently blocked the EGF-induced Smad3 transactivation, whereas activation of the PI3-kinase pathway by
RasV12C40 reproduced the synergistic effect of EGF stimulation on Smad3
transactivation. The PI3-kinase-activating effect was blunted in cells
transfected with TAM-67 which are deficient in AP-1 transactivating
function, strongly suggesting that the effect of PI3-kinase is through
AP-1 activation rather than the result of direct
PI3-kinase-dependent phosphorylation of Smad3. The
involvement of PI3-kinase in Smad transactivation has never been
demonstrated before. That PI3-kinase activation may be involved in
TGF- Another important signaling pathway that originates from the EGF
receptor is the MAPK pathway, including the ERK1 and ERK2 pathways, the
JNK/SAPK, and the p38 MAPK pathways (Ref. 39 and for review see Ref.
40), all implicated in EGF-induced AP-1 activation (14). In our study,
neither inhibition of the JNK pathway by transfection of a dominant
negative mutant (DNMKK4) nor inhibition of the ERK pathway by the
specific inhibitor PD98059 decreased the EGF-induced potentialization
of Smad3 transactivation, whereas involvement of the p38 pathway was
suggested by the inhibitory effect of SB 202190 on EGF/TGF- In summary, we have described another novel mechanism allowing TGF- (TGF-
)
induced Smad3 transactivation in rat hepatocytes, an effect
abrogated by TAM-67, a dominant negative mutant of AP-1. Antisense
transfection experiments indicated that c-Jun and JunB were involved in
the synergistic effect, and endogenous c-Jun physically associated with
Smad3 during a combined EGF/TGF-
treatment. We next investigated which signaling pathway transduced by EGF was responsible for the
Jun-induced synergism. Whereas inhibition of JNK had no effect, inhibition of the phosphatidylinositol-3' kinase (PI3-kinase) pathway
by LY294002 or by expression of a dominant negative mutant of
PI3-kinase reduced EGF/TGF-
-induced Smad3 transcriptional activity.
Transfection of an activated Ras with a mutation enabling the
activation of the PI3-kinase pathway alone mimicked the EGF/TGF-
potentiation of Smad3 transactivation, and TAM-67 abolished this effect, suggesting that the PI3-kinase pathway stimulates Smad3 via
AP-1 stimulation. The EGF/TGF-
-induced activation of Smad3 correlated with PI3-kinase and p38-dependent but not
JNK-dependent phosphorylation of c-Jun. Since potentiation
of a Smad-binding element-driven gene was also induced by EGF/TGF-
treatment, this novel mechanism of Jun/Smad cooperation might be
crucial for diversifying TGF-
responses.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
)1 is a member of a
large family of cytokines that includes bone morphogenetic proteins, activins, and several more distantly related factors (1). TGF-
is
central in the regulation of many biological processes, including cell
differentiation, growth, adhesion, and apoptosis. TGF-
signals through a system of transmembrane serine/threonine kinase receptors composed of type I and type II receptors (TGF-
RI and TGF-
RII) (see Refs. 2-4 and reviewed in Ref. 5). Ligand binding to TGF-
RII
recruits and activates the TGF-
RI receptor, which phosphorylates Smad2 and Smad3 on their SSXS motif. Smad proteins encompass
a conserved amino-terminal domain that binds DNA and a conserved carboxyl-terminal domain that binds receptors and partner Smads. These
domains are separated by a less conserved linker region. Phosphorylated
Smad2 or Smad3 forms stable complexes with Smad4, which translocate
into the nucleus, where they bind the consensus GTCTAGAC sequence found
in the promoter of many TGF-
-responsive genes (6). Disruption of the
Smad pathway or Smad mutations have underscored the functional
importance of this signaling pathway in the transcriptional response of
target cells to TGF-
(reviewed in Ref. 7).
signaling may also cross-talk
with the mitogen-activated protein kinase (MAPK) family of
serine/threonine protein kinases. Antagonistic or synergistic interplay
between these kinases and Smad signaling has been described. Extracellular signal-regulated kinases (ERK), members of the MAPK, cause a rapid increase in the phosphorylation of Smad2 and Smad3 in
their linker region, preventing their translocation into the nucleus
and therefore providing a mechanism of repression of TGF-
signaling
(8, 9). At the opposite, a synergistic mechanism between Smads and MAPK
has been proposed, in a kinase downstream of the MAP kinase kinase
MEK1-induced Smad2 phosphorylation on the SSXS motif and its
nuclear translocation (10). Recent evidence also indicates that Smad
cooperates with AP-1 (11-13), a heterodimer of Fos and Jun family
members (14). Stimulation of AP-1-dependent transcription
can be achieved by phosphorylation of the c-Jun transactivation domain
by c-Jun NH2-terminal kinase (JNK)/stress-activated protein
kinase (SAPK), another member of the MAPK family. Smad and AP-1
response elements are juxtaposed in the promoters of several
TGF-
-inducible genes, such as plasminogen activator inhibitor-1 or
c-jun, and both sites appear to be critical in the
TGF-
response (15-17).
. We show that under these experimental
conditions AP-1 induces a strong activation of Smad3 transactivation
independent of AP-1 binding to its cognate cis-element. This
synergism was mediated by c-Jun and JunB, and a protein-protein
interaction between Smad3 and endogenous c-Jun was found during
EGF/TGF-
stimulation. Furthermore, we demonstrate that activation of
AP-1 via the phosphatidylinositol 3-kinase (PI3-kinase), but not the
JNK pathway, is implicated in this functional synergism. Finally, we
show that Jun/Smad3 cooperation induced by EGF is effective on a
SBE-driven reporter gene. These data suggest that Jun/Smad3 synergism
independent of binding to TRE elements might represent another
important mechanism of regulation of TGF-
-inducible genes in hepatocytes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
A32-36 were the generous gifts of A. Atfi and G. Cherqui.
The expression vectors for RasV12C40 and p85
iSH2-N (p85DN,
thereafter), kindly provided by J. Downward, have been described
(20).
80 °C. Protein
concentration was determined with the BCA protein assay reagent (Pierce).
32P]ATP (5,000 Ci/mmol,
Amersham Pharmacia Biotech). The labeled oligonucleotides were annealed
with their respective unlabeled lower strands. Nuclear extracts (5-20
µg of protein) were incubated in binding buffer (20 mM
Hepes, pH 7.9, 5 mM MgCl2, 4 mM
dithiothreitol, 20% glycerol, 0.1 mM PMSF, 5 mM benzamidine, 2 mM levamisole, 0.1 µg/ml
aprotinin, 0.1 µg/ml bestatin) containing 2 µg of poly(dI-dC) and
32P-labeled double-stranded probe (3 × 104 cpm) for 20 min at 4 °C. The reaction mixture was
then loaded onto a 6% polyacrylamide gel in 0.09 M Tris
borate, 2 mM EDTA, pH 8.0 buffer, and electrophoresed at 11 V/cm for 2 h at 20 °C. The gels were dried and exposed to x-ray
film for autoradiography.
(3 ng/ml), or EGF plus TGF-
. For Luc assays, cells were washed with
chilled PBS and lysed for 15 min on ice with lysis buffer (25 mM Tris/HPO4, pH 7.8, 8 mM
MgCl2, 1% Triton X-100, 1% bovine serum albumin, 15%
glycerol, 1 mM EDTA, and 1 mM dithiothreitol), and then the lysates were centrifuged 10 min at 13,000 rpm and stored
at
20 °C. Luc activities were determined with a luminometer (EGG
Instruments) and normalized to the amount of proteins in the extracts.
For the measurement of chloramphenicol acetyltransferase enzyme
activities, cell extracts were prepared as described in Nadori et
al. (26). Chloramphenicol acetyltransferase assays were performed
according to Seed and Sheen (27) and normalized relative to the amount
of protein in the extracts.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
for Smad 3 Transactivation--
To
determine whether EGF and TGF-
signalizations cross-talk in
hepatocytes, we investigated the transcriptional activity of Smad
proteins by using a single Gal-hybrid system, which makes use of an
expression vector for Smad3-Gal4 fusion protein and a reporter plasmid
containing five concatemerized Gal4-binding sites (Ti5xGal4). In
hepatocytes transiently transfected with the Tix5Gal4 reporter alone, a
very low level of Luc activity was detected (Fig.
1). In cells cotransfected with Ti5xGal4
and Smad3-Gal4 expression vector treatment with TGF-
induced a
10-fold increase in Luc activity. EGF treatment alone slightly
activated basal Smad3 activity, whereas simultaneous treatment with EGF and TGF-
led to a potent synergistic increase in Smad3
transcriptional activity, when compared with that upon TGF-
treatment alone (Fig. 1). The same results were observed in hepatocytes
transfected with a Smad2-Gal4 expression plasmid (data not shown).
These results indicated that whereas the EGF signaling pathway alone
weakly activates Smad3 transcriptional activity in hepatocytes, it
potently synergizes with Smads once they have been activated by
TGF-
.
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Fig. 1.
EGF synergizes with TGF-
for Smad3 transactivation. Hepatocytes were transiently
transfected with Ti5xGal-Luc and Smad3-Gal4 expression vector, and they
were left untreated or treated with EGF (20 ng/ml), TGF-
(3 ng/ml),
or a combination of both for 24 h. Luc activity was determined and
normalized to transfection efficiency. Values of samples from cells
transfected with Tix5Gal and Smad3-Gal4 and left untreated were
arbitrarily set to 1. The results are the mean ± S.E. of three
independent transfections.
on Smad3
Transactivation--
Since one of the major targets of EGF signaling
is activation of the AP-1 complex, we first determined whether
inhibition of AP-1 would interfere with Smad3 transcriptional activity.
Hepatocytes were cotransfected with Smad3-Gal4 and either TAM-67, a
dominant negative c-Jun expression vector which blocks the activity of all endogenous Jun and Fos proteins by forming nonfunctional
heterodimers (18, 19), or the empty pCMV plasmid, as a control. Fig.
2A shows that cotransfection
with TAM-67 did not significantly modify the transcriptional activity
of Smad3 in unstimulated hepatocytes or in hepatocytes treated with EGF
or TGF-
alone, whereas it induced an important decrease of the Smad3
transcriptional activity triggered by TGF-
/EGF costimulation, an
effect not observed in hepatocytes transfected with the control plasmid
(Fig. 2A). We have recently observed that EGF activates
NF-
B binding and transactivation in normal rat
hepatocytes.2 We thus
investigated whether inhibition of NF-
B activity could also
influence the synergism between EGF and TGF-
on Smad3
transactivation. Hepatocytes were cotransfected with Smad3-Gal4,
Ti5xGal, and either with the I
B
A32/36 expression vector encoding
for a mutated, nonphosphorylatable form of I
B protein which blocks
NF-
B nuclear translocation and activity or with the Rc/CMV control
plasmid. In these experiments no modification of the transcriptional
activity of Smad3 was observed after costimulation with EGF and TGF-
(Fig. 2B).
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Fig. 2.
Activation of AP-1 but not
NF- B is involved in
EGF/TGF-
-induced Smad3 transactivation.
Hepatocytes were cotransfected with Ti5xGal-Luc and Smad3-Gal4
expression vectors and TAM-67 or the control empty plasmid (pCMV) in
A or I
B
A32/A36 or the control empty plasmid (pRc/CMV)
in B. They were left untreated or treated with EGF (20 ng/ml), TGF-
(3 ng/ml), or a combination of both for 24 h.
Luciferase activity was determined and normalized for transfection
efficiency. Value of samples from cells transfected with Tix5Gal and
Smad3-Gal4 and left untreated was arbitrarily set to 1. The results are
the mean ± S.E. of three independent transfections in
A and represent the average of two independent experiments
in B. *, p < 0.01 by Student's
t test.
costimulation,
we transiently cotransfected the hepatocytes with Ti5xGal4, Smad3-Gal4,
and with either c-jun, junB, or junD
antisense expression vectors. The efficiency of the antisense strategy
was checked in separate experiments by cotransfecting hepatocytes with
these antisense constructs and with a TRE-driven gene, the 5xTRE-Luc
plasmid. After 32 h of transfection, c-jun and
junB antisense expression vectors inhibited Luc activity by
70 and 40%, respectively, compared with cells transfected with the
control pCI-neo vector (Fig.
3A). Cotransfection of the
junD antisense plasmid did not significantly inhibit Luc activity. Since the half-life of JunD is much longer than for the other Jun proteins, the absence of effect of this construct could
be related to the relatively short delay between transfection of the
antisense RNA and the Luc assay. Treatment with EGF or TGF-
of
hepatocytes transfected with the c-jun, the junB,
or the junD antisense plasmid did not significantly decrease
the transcriptional activity of Smad3 (Fig. 3B). By
contrast, a combined treatment with EGF and TGF-
of hepatocytes
expressing c-jun or junB antisense mRNA
decreased Smad3 transactivation by ~40%, whereas antisense
junD expression had no inhibitory effect. These data suggest
the involvement of c-Jun and JunB in Smad3 cooperation, but according
to the commentary above they do not allow us to completely rule out a
possible action of JunD.
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Fig. 3.
c-Jun cooperates with Smad3.
A, hepatocytes were transfected with a TRE-Luc reporter gene
and with antisense expression plasmid as indicated. Luc activity was
determined after 24 h and normalized for transfection efficiency.
B, hepatocytes were cotransfected with Ti5xGal-LUC and
Smad3-Gal4 expression vectors, and with c-jun,
junB, or junD antisense expression vectors. They
were left untreated or treated with EGF (20 ng/ml), TGF- (3 ng/ml),
or a combination of both for 24 h. Luc activity was determined and
normalized for transfection efficiency. Value of samples from cells
transfected with Tix5Gal and Smad3-Gal4 and left untreated was
arbitrarily set to 1. The results are the mean ± S.E. of three
independent transfections. *, p < 0.01, by one-way
analysis of variance.
and EGF--
Hepatocytes were transfected with an expression
vector for a 6xMyc-tagged Smad3 protein and left unstimulated or
treated with EGF, TGF-
, or a combination of both for 24 h.
Proteins immunoprecipitated with anti-c-Jun antibody were then
immunoblotted with an anti-Myc antibody. As shown in Fig.
4, association of c-Jun with Smad3 was
detectable only after costimulation with EGF and TGF-
.
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Fig. 4.
Endogenous c-Jun associates with Smad3 during
costimulation with TGF- and EGF.
Hepatocytes were transiently transfected with a 6xMyc-tagged Smad3
expression vector and left unstimulated or stimulated for 24 h
with EGF (20 ng/ml), TGF-
(3 ng/ml), or both as indicated. Lysates
were incubated with anti-c-Jun-coated protein A-Sepharose beads before
performing SDS-PAGE and a Western blot with anti-Myc antibody. Lysate
from nontransfected cells (NT) was also tested.
and EGF Does Not Increase TRE or SBE
Binding--
To determine whether the DNA binding activity of Jun or
Smad proteins was modified during costimulation with TGF-
and EGF, we performed EMSA with a TRE probe that binds AP-1 proteins or an SBE
probe containing the consensus GTCTAGAC sequence that binds Smad
proteins (6). Whereas TGF-
treatment alone did not modify the level
of AP-1 binding to the TRE cis-element, stimulation with EGF
induced a 2.5-fold increase, as expected (26) (Fig. 5A). EMSA performed with the
SBE probe and nuclear extracts from unstimulated cells generated two
retarded bands, whose intensity was not modified by treatment with EGF
or TGF-
. Treatment with TGF-
induced the formation of a third
slower band, visible as soon as 30 min after TGF-
stimulation (data
not shown) which was still visible at 2 h (Fig. 5B).
The intensity of this TGF-
-induced retarded band was not modified
after costimulation with EGF and TGF-
(Fig. 5B).
Supershift experiments with antibodies specific to Smad3 induced a
complete displacement of the slowly migrating retarded band induced by
TGF-
but did not modify the intensities of the other bands. No
supershifts were observed after addition of c-Jun, JunB, JunD, c-Fos,
or Fra-1 antibodies to EMSA performed with the SBE probe (data not
shown). Competition experiments indicated that the three retarded bands
were specific, since they were completely competed by a 100-fold molar
excess of unlabeled SBE probe, whereas they remained unchanged in the
presence of an excess of unlabeled NF-
B probe (Fig. 5C).
In addition, competition with a 100-fold molar excess of cold TRE probe
decreased the binding of the two faster migrating bands, indicating
that part of the proteins that bind to the SBE also interact with an
AP-1 cis-element (Fig. 5C).
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Fig. 5.
Costimulation with TGF-
and EGF does not increase TRE or SBE binding. A
and B, equal amounts (10 µg) of nuclear protein extracts
from hepatocytes nonstimulated or stimulated with EGF (20 ng/ml),
TGF-
(3 ng/ml), or both for 2 h were incubated with a
32P-labeled TRE probe in A or SBE probe in
B, and electrophoresed in a 6% polyacrylamide gel.
Competition for TRE binding was performed by adding a 100-fold molar
excess of unlabeled TRE probe to the incubation mixture (competitor).
B, anti-Smad3 antibody was added in the SBE gel retardation
assay. Arrowheads point to the retarded bands, and the
arrow indicates the supershifted band induced by anti-Smad3.
C, competition experiments for SBE binding. Increasing
concentrations of unlabeled SBE probe or a 100-fold molar excess of TRE
probe or NF-
B probe were added to the reaction mixture before
electrophoresis.
(data not shown), but it inhibited by 61% the Smad3 activity triggered by a combined treatment with EGF
and TGF-
(Fig. 6A). To
check the specificity of the LY294002 effect, we used another approach
to inhibit PI3-kinase activation. Hepatocytes were transfected with
p85DN, a plasmid that allows the expression of a mutated catalytic
PI3-kinase subunit exerting a dominant negative effect on PI3-kinase
activity (20). Transient expression of this plasmid induced a 40%
decrease of EGF/TGF-
-induced Smad3 transactivation (Fig.
6B). This result therefore is in agreement with the
involvement of the PI3-kinase pathway in EGF/TGF-
synergism. Another
well identified pathway triggered by the EGF receptor is the Ras-MAPK
pathway leading to ERK, JNK, or p38 activation. We therefore asked
whether inactivation of one of these pathways could also block
EGF/TGF-
-induced Smad3 transactivation. The possible involvement of
the p38 pathway was tested by treatment of hepatocytes cotransfected
with Smad3-Gal4 and Tix5Gal with SB202190, a selective inhibitor of p38
MAPK. In these transfected cells, SB202190 induced a 50% decrease of
Smad3 transactivation in the presence of EGF and TGF-
(Fig.
6A). Simultaneous inhibition of the p38 and PI3-kinase
pathways by treating hepatocytes with both LY294002 and SB202190 did
not synergize for the inhibition of EGF/TGF-
-induced Smad3
transactivation (Fig. 6A) suggesting that the two pathways
are interdependent. Involvement of the ERK pathway was tested by the
use of PD98059, a potent and specific inhibitor of MEK1 activation, a
kinase directly upstream of ERK. PD98059 treatment did not decrease the
Smad3-dependent transactivation induced by EGF, TGF-
(data not shown), or a combination of both (Fig. 3A).
Finally, to test the role of the JNK pathway, hepatocytes were
cotransfected with GAL4-Smad3, the Tix5xGAL4 reporter plasmid, and
DNMKK4, a dominant negative mutant of MKK4, a kinase predominantly involved in the activation of JNK (31). Coexpression of DNMKK4 did not
modify the level of Smad transactivation induced by EGF, TGF-
(data
not shown), or a combination of both (Fig. 3B), compared with hepatocytes cotransfected with the empty pcDNA3 vector.
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Fig. 6.
Effects of the inhibition of PI3-kinase and
MAPK pathways on EGF/TGF- synergism.
A, hepatocytes transfected with Ti5xGal and Smad3-Gal4
plasmids were treated with a combination of EGF (20 ng/ml) and TGF-
(3 ng/ml) for 24 h in the presence or in the absence of the
PI3-kinase inhibitor LY294002 (35 µM), the p38 inhibitor
SB202190 (10 µM), a mixture of both, or the ERK inhibitor
PD98059 (50 µM). Value of samples from cells transfected
with Tix5Gal and Smad3-Gal4 in the absence of the inhibitors was
arbitrarily set to 100. The results are the mean ± S.E. of three
independent transfections except for cells treated with PD98059 for
which the average of two independent experiments is shown.
B, hepatocytes were cotransfected with Ti5xGal and
Smad3-Gal4 plasmids and with p85DN or the empty plasmid pSG5, or DNMKK4
or the control plasmid pcDNA3. They were treated with a combination
of EGF (20 ng/ml) and TGF-
(3 ng/ml) for 24 h. Luciferase
activity was determined and normalized for transfection efficiency.
Value of samples from cells transfected with Tix5Gal and Smad3-Gal4
alone was arbitrarily set to 100. The results represent the average of
two independent transfections.
treatment further increased by 3-fold Smad3 transactivation, to
reach a level close to that detected in hepatocytes costimulated with
EGF and TGF-
but not expressing RasV12C40 (Fig. 7A). To
determine whether PI3-kinase activates Smad3 transcriptional activity
via a direct mechanism (i.e. Smad phosphorylation) or rather
via the induction of AP-1 activity, hepatocytes were transfected with
RasV12C40 in the presence of the AP-1 dominant negative TAM-67 or the
control empty plasmid pCMV. Inactivation of AP-1 blunted the basal
PI3-kinase-mediated activation of Smad3 transactivation induced by
RasV12C40 and inhibited by 70% Smad3 transactivation in the presence
of TGF-
(Fig. 7A). These data indicated that AP-1
activity is necessary for the stimulating effect of PI3-kinase on Smad3
transactivation. Treatment of RasV12C40-transfected hepatocytes with
the PI3-kinase inhibitor LY294002 led to a 40% decrease of Smad3
transactivation (Fig. 7B), consistent with the assumption
that the effect of this activated Ras on Smad3 transactivation depends,
at least in part, on its PI3-kinase activating function. Since p38 is a
well known downstream effector of PI3-kinase, we also investigated
whether inhibition of p38 by SB202190 would modify the effect of
PI3-kinase activation on Smad3 transactivation. Treatment of
RasV12C40-transfected hepatocytes with SB202190 inhibited Smad3
transactivation by 30% (Fig. 7B). Thus, at least part of the effect of PI3kinase on Jun-mediated Smad3 transactivation depends
on the p38 kinase pathway.
View larger version (38K):
[in a new window]
Fig. 7.
The PI3-kinase and p38 pathways are involved
in AP-1-mediated EGF/TGF- synergism on Smad3
transactivation. A, hepatocytes were cotransfected with
Ti5xGal and Smad3-Gal4 plasmids with or without RasV12C40 or its empty
plasmid pRSV, or TAM-67, or its empty plasmid pCMV, as indicated. They
were left untreated or treated with EGF (20 ng/ml), TGF-
(3 ng/ml),
or both, as indicated, for 24 h. Value of samples from cells
transfected with Tix5Gal and Gal4-Smad3 and left untreated was
arbitrarily set to 1. The results are the mean ± S.E. of three
independent experiments. B, hepatocytes were cotransfected
with Ti5xGal, Smad3-Gal4, and RasV12C40 and treated with EGF + TGF-
in the presence or in the absence of LY294002 (35 µM) or
SB202190 (10 µM). Value of samples from cells treated
with EGF + TGF-
in the absence of inhibitors was arbitrarily set to
100. The results are the mean ± S.E. of three independent
transfections.
+ EGF on Smad3 transactivation depended
on the activation of Jun proteins. Phosphorylation of the
transactivation domain of c-Jun on Ser-63 and Ser-73 by JNK is the best
described mechanism of c-Jun activation. Accordingly, we first looked
at the phosphorylation of c-Jun on Ser-63, by Western blotting using an
antibody directed against Ser-63-phosphorylated c-Jun. Stimulation with
TGF-
alone did not increase c-Jun phosphorylation, whereas, as
expected, a strong phosphorylation of c-Jun on Ser-63 was induced by
EGF stimulation. The intensity of the band specific for c-Jun Ser-63 phosphorylation was not modified by costimulation with EGF and TGF-
in comparison with cells treated with EGF alone, even in the presence
of LY294002 or SB202190 (Fig.
8A). This observation was
consistent with our demonstration that inhibition of JNK does not
suppress EGF/TGF-
synergism on Smad3 transactivation. To determine
whether phosphorylation of c-Jun by a pathway different from JNK was
triggered during EGF + TGF-
treatment, we next performed Western
blotting using a c-Jun antibody that recognizes both the nonphosphorylated and the phosphorylated forms of c-Jun. As shown on
Fig. 8B, stimulation with EGF, but not TGF-
, induced a
strong accumulation of c-Jun protein, with appearance of a slower band, indicating the presence of phosphorylated forms of the protein. This
retarded band was also detectable after TGF-
+ EGF treatment. In
extracts from hepatocytes treated with TGF-
+ EGF in the presence of
LY294002, the retarded band disappeared almost completely, whereas it
was slightly decreased upon treatment with SB202190. These data
indicated that besides the JNK-dependent phosphorylation of
Jun on serine 63, EGF/TGF-
induces the phosphorylation c-Jun on
other site(s) through both PI3-kinase and p38-dependent
pathways.
View larger version (25K):
[in a new window]
Fig. 8.
Inhibition of c-Jun phosphorylation by
PI3-kinase and p38-kinase inhibitors. Equal amounts (60 µg) of
nuclear protein extracts from hepatocytes nonstimulated or stimulated
with EGF (20 ng/ml), TGF- (3 ng/ml), or both in the presence or in
the absence of the PI3-kinase inhibitor LY294002 (35 µM)
or the p38-kinase inhibitor SB202190 (10 µM) for 1 h
were electrophoresed in a 10% acrylamide gel and transferred on
nitrocellulose membranes. The membranes were probed as follows:
A, with anti c-Jun-phospho-Ser-63 antibody; B,
with anti-c-Jun antibody.
Potentiation of Smad3 Transactivation Is Active on an
SBE-containing Promoter--
We next investigated whether the
EGF/TGF-
potentiation of Smad3 transactivation found in our Gal4
system was also operative in the context of an SBE-driven gene.
Hepatocytes were transfected with a construct that contains a Luc gene
under the control of four SBE repeats (4xSBE-Luc) or with a control
plasmid containing six mutated SBE repeats (6xMBE-Luc). As shown in
Fig. 9A, no Luc activity was
detected after transfection of the MBE plasmid. EGF treatment alone did
not stimulate the SBE promoter activity. Stimulation with TGF-
induced Luc activity by 10-fold, as expected, and a combined treatment
with EGF and TGF-
further increased (about 2-fold) Luc activity
(Fig. 9A). Thus, although of weaker magnitude, a synergistic
effect on an SBE-dependent transcription was also induced
by EGF/TGF-
stimulation. Cotransfection with TAM-67 completely prevented the synergistic effect of EGF/TGF-
on Luc expression (data
not shown). Finally, we also tested the effect of the activation of the
PI3-kinase pathway on SBE-mediated transactivation by cotransfection with the RasV12C40 plasmid. As shown in Fig. 9B, a high
level of transactivation was induced by TGF-
treatment alone,
mimicking the EGF synergistic effect, and inhibition of AP-1 by TAM-67
blunted this response, indicating that it depended on AP-1
activation.
View larger version (42K):
[in a new window]
Fig. 9.
AP-1-dependent
EGF/TGF- synergy is operative on an SBE-driven
gene. A, hepatocytes were transfected with 6xMBE-Luc or
4xSBE-Luc, in the absence or in the presence of TAM-67. They were
stimulated for 24 h with EGF (20 ng/ml), TGF-
(3 ng/ml), or
both, as indicated. Value of samples from cells transfected with
4xSBE-Luc and left untreated was arbitrarily set to 1. The results are
the mean ± S.E. of three independent transfections. B,
hepatocytes were transfected with 4xSBE-Luc alone or with RasV12C40, in
the absence or in the presence of TAM-67 or the empty plasmid pCMV.
They were treated with TGF-
(3 ng/ml) with or without EGF (20 ng/ml), as indicated. The bars represent the mean of two
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-responsive genes, resulting in
antagonistic as well as synergistic effects. In the present study, we
show that EGF alone induces a mild activation of Smad3 transcriptional
activity only detectable under conditions of Smad3 overexpression, in
agreement with previous reports (10). More importantly, we show that a
combined treatment with EGF and TGF-
strongly activates (about
30-fold) Smad3 transactivation. This synergistic effect is dependent on
the presence of AP-1 proteins, since it is prevented by transfection of
TAM-67, a dominant negative c-Jun truncated on its transactivating
domain acting as an inhibitor of AP-1 function (18, 19). Recently,
cooperation of Smad proteins with the AP-1 proteins Fos and Jun has
been documented in mink lung epithelial cells (11, 12). In
vitro binding of Smad3 and Smad4 to all three Jun family members
as well as in vivo association between Smads and a
TGF-
-phosphorylated form of endogenous c-Jun induced by JNK have
been demonstrated in HaCaT cells (12). Cooperation of AP-1 proteins
with Smad3 and Smad4 occurred via Jun proteins bound to their cognate
cis-element, the TRE, or to composite sites containing
juxtaposed AP-1 and SBE sites (11, 12, 17), and the interaction was
shown to involve 13 carboxyl-terminal amino acids conserved in the
three Jun proteins (12). The mechanism described herein differs from
these previous studies by two major features as follows: 1) by using a
Gal reporter system, we could demonstrate that the interaction of Smad3
with c-Jun induced a synergistic effect on Smad3 transactivation
independently of binding to the TRE; 2) overexpression of TAM-67, a
dominant negative c-Jun which is truncated in its transactivating
NH2-terminal domain but still possesses the COOH-terminal
domain implicated in the physical interaction with Smad proteins (11),
abrogated the EGF/TGF-
-induced stimulation of Smad3 transactivation,
implying that the functional cooperation with Smad3 requires the
amino-terminal transactivation domain of Jun proteins. In addition, we
also show that the EGF-induced cooperative effect of Jun proteins on
Smad3 transactivation was not paralleled by any modification of the binding of Smad proteins to their cognate cis-element, the
SBE site, indicating that the increased transcriptional activity of Smad3 proteins is probably due to their association with Jun proteins rather than to the recruitment of additional Smads to the SBE. Despite
the fact that a physical association between Jun proteins and Smads has
been reported to occur in vitro (11, 12) and the
demonstration that c-Jun coimmunoprecipitates with Smad3 during EGF/TGF-
stimulation (our present result), we failed in detecting c-Jun or other AP-1 proteins bound to the SBE by supershift
experiments. This could be due to the lack of sensitivity of the method
that is known to produce false negative results (32).
signaling had been previously suggested from the observation
that wortmannin, another PI3-kinase inhibitor, inhibits
TGF-
-stimulated chemotaxis of human neutrophil leukocytes (34). It
has also been demonstrated that TGF-
markedly enhanced EGF-induced
PI3-kinase activity in human airway smooth muscle cells (35). More
recently, involvement of PI3-kinase in the inhibitory effect of
insulin, EGF, or interleukin-6 on TGF-
-induced apoptosis has been
reported (36-38), but the relationship with AP-1 function or Smad
signaling was not investigated. We show here that one mechanism by
which PI3-kinase may cross-talk with Smad signaling is through
activation of Jun proteins, which themselves cooperate with Smad3 for transactivation.
-induced
Smad3 transactivation. These data confirm and extend the previous
demonstration that p38 increases the transcriptional activity of
TGF-
-inducible genes (41-44). In these studies, p38 activation was
shown to be induced by TGF-
-activated kinase, a MAP kinase kinase
kinase also involved in TGF-
signaling (45, 46). According to this model, TGF-
-activated kinase-induced p38 phosphorylation in response to TGF-
triggers the phosphorylation of activating transcription factor 2, a basic leucine zipper protein member of the activating transcription factor/cAMP-response element-binding protein family that
shares many structural characteristics with AP-1 proteins. Activated
transcription factor 2 forms a complex with Smad4 that is
transcriptionally active on Smad-regulated genes (41, 42). The
mechanism of p38 activation found in the present study is clearly
different from this model. It is very likely that the activating effect
of p38 on Smad3 transactivation detected in our study lies downstream
of PI3-kinase activation, since inhibition of the two pathways was not
clearly additive. That PI3-kinase can contribute to the activation of
protein kinases of the MAPK family, such as ERK, has been previously
shown (47), but a direct demonstration of p38 activation by the
PI3-kinase pathway has never been published. Finally, since our Western
blot experiments indicated that EGF induces c-Jun phosphorylation by
mechanisms dependent of PI3-kinase and p38, it is very likely, but not
proven, that these phosphorylation events are implicated in the
potentiation of Smad3 transactivation by EGF. In sharp contrast with
our results describing a stimulating effect of Jun/Smad3 cooperation
for transactivation in the context of PI3-kinase-induced Jun
activation, two recent examples of interplay between Jun proteins and
Smad3 leading to transcriptional repression of Smad3 have recently been
published (13, 48). In one model, Jun activated via a TGF-
-induced JNK binds to Smad3 on an SBE element and inhibits Smad3 transcriptional activity (13). In a second model, c-Jun activated by tumor necrosis factor
binds to Smad3 and prevents its binding to DNA, therefore acting again as a transcriptional repressor (48). Collectively, these
data and ours suggest that the functional interplay between Jun and
Smads is far more complicated than previously thought and might vary
according to the mechanisms of Jun activation.
to integrate with regulatory networks of the cell. Whether this pathway
is specific to the hepatocyte environment remains to be determined. We
show that such a mechanism operates on an SBE-driven gene, although
with a lower magnitude, and therefore represents a potential mechanism
of regulation of TGF-
-inducible genes. In normal hepatocytes,
simultaneous treatment with EGF and TGF-
induces a proliferation
arrest (49-51). Whether Jun/Smad3 synergism is at work on the promoter
of Smad-responsive gene inhibitors of the cell cycle will be the
subject of our next investigations.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Massagué, P. Ten Dijke, B. Vogelstein, X. F. Wang, A. Atfi, G. Cherqui, J. Downward, and M. J. Birrer for providing plasmids and A. Groyer and F. Daniel for critical evaluation of the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed. Tel.:
33-01-44-85-61-90; Fax: 33-01-44-85-92-79; E-mail:
bernuau@bichat.inserm.fr.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M005919200
2 M. Rahmani, P. Péron, J. Weitzmann, L. Bakiri, B. Lardeux, and D. Bernuau, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor
;
ERK, extracellular signal-regulated
kinase;
MAPK, mitogen-activated protein kinase;
JNK/SAPK, c-Jun
NH2-terminal kinase/stress-activated protein kinase;
EGF, epidermal growth factor;
PI3-kinase, phosphatidylinositol 3'-kinase;
Luc, luciferase;
Gal, galactosidase;
SBE, Smad-binding element;
TRE, TPA-responsive element;
PBS, phosphate-buffered saline;
EMSA, electrophoretic mobility shift assay;
PMSF, phenylmethylsulfonyl
fluoride;
MAP, mitogen-activated protein;
MAPK, MAP kinase;
PAGE, polyacrylamide gel electrophoresis.
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