A Central Role for the JNK Pathway in Mediating the
Antagonistic Activity of Pro-inflammatory Cytokines against
Transforming Growth Factor-
-driven SMAD3/4-specific Gene
Expression*
Franck
Verrecchia
,
Charlotte
Tacheau
,
Erwin F.
Wagner§, and
Alain
Mauviel
¶
From
INSERM U532, Institut de Recherche sur la Peau
Hôpital Saint-Louis, 75475 Paris Cedex 10, France and the
§ Research Institute for Molecular Pathology, University of
Vienna, A-1030 Vienna, Austria
Received for publication, July 11, 2002, and in revised form, October 17, 2002
 |
ABSTRACT |
We have focused our attention on the molecular
events underlying the antagonistic activities of pro-inflammatory
cytokines against transforming growth factor-
(TGF-
)/SMAD
signaling. Using jnk1/2-knockout
(jnk
/
) and I
B
kinase-
/nemo
/
fibroblasts, we have
determined the specific roles played by the JNK/AP-1 and NF-
B/Rel
pathways in this phenomenon. We demonstrate that, in a cellular context
devoid of JNK activity (i.e.
jnk
/
fibroblasts), interleukin-1 and tumor
necrosis factor-
(TNF-
) did not inhibit the formation of SMAD-DNA
complexes and the resulting SMAD-driven transcription in response to
TGF-
. On the other hand, lack of NF-
B activity in
nemo
/
fibroblasts did not affect the
antagonistic effect of pro-inflammatory cytokines against TGF-
. In
the latter cell type, overexpression of antisense c-jun
mRNA or of a dominant-negative form of MKK4 blocked the
inhibitory activity of TNF-
, similar to what was observed in normal
human dermal fibroblasts. Among JNK substrates, c-Jun and JunB (but not
activating transcription factor-2) antagonized TGF-
/SMAD signaling
in a JNK-dependent manner. Overexpression of JNK1 in
jnk
/
fibroblasts restored the ability of
cytokines and Jun proteins to interfere with SMAD signaling. In
junAA mouse embryo fibroblasts, in which c-Jun can no
longer be phosphorylated by JNK, JunB substituted for c-Jun in
mediating the cytokine effect against SMAD-driven transcription in a
JNK-dependent manner. These results suggest a critical role
for JNK-mediated c-Jun and JunB phosphorylation in transmitting
the inhibitory effect of pro-inflammatory cytokines against
TGF-
-induced SMAD signaling. In addition, we demonstrate that such a
JNK-dependent regulatory mechanism underlies the
antagonistic activity of TNF-
against TGF-
-induced up-regulation
of type I and III collagens in fibroblasts.
 |
INTRODUCTION |
Transforming growth factor-
(TGF-
)1 is a potent
anabolic factor for fibroblasts, stimulating extracellular matrix
component synthesis and inhibiting the activity of matrix
metalloproteinases both by diminishing their expression and by
enhancing the expression of their natural inhibitors, the TIMPs
(tissue inhibitors of
metalloproteases) (1). On the other hand,
pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumor
necrosis factor-
(TNF-
) have potent catabolic activities,
inhibiting the expression of several extracellular matrix components
and activating that of matrix metalloproteinases (2). Antagonistic
activities of pro-inflammatory cytokines against TGF-
are thus
believed to play an essential role in maintaining tissue homeostasis
and extracellular matrix deposition (3, 4).
Several studies have addressed the molecular mechanisms underlying the
antagonistic activities exerted by pro-inflammatory cytokines and
TGF-
(3, 5, 6). For example, differential induction of c-Jun and
JunB, transcription factors of the AP-1 family that exhibit
antagonistic transcriptional activities, underlies the differential
regulation of matrix metalloproteinase-1 gene expression by TGF-
and
pro-inflammatory cytokines, including TNF-
and IL-1 (7). More
recently, it was shown that several signaling pathways, such as those
for NF-
B and JAK (Janus kinase)/STAT (signal transducer and activator of
transcription), activated in response to various stimuli,
such as cytokines, shear stress, and UV light, lead to an increased
expression of inhibitory SMAD7, which, in turn, prevents
signaling from the TGF-
receptors (8, 9). NF-
B has been suggested
to be part of the signals responsible for SMAD7 gene
activation by TNF-
(10), although it is clearly a cell-type specific
mechanism (6, 11). Alternatively, induction of c-Jun by cytokines has
been shown to directly interfere with the SMAD pathway either by
preventing SMAD3 binding to cognate DNA sequences or by sequestering
the transcriptional coactivator p300 (6, 12, 13). In this respect,
activation of JunB expression in response to SMAD signaling downstream
of the TGF-
receptors was recently shown to be part of a negative
autoregulatory loop attenuating SMAD-specific transcriptional responses
(14).
The JNK MAPKs, also called SAPKs, are activated upon exposure of cells
to cytokines, growth factors, and environmental stress such as UV
irradiation and heat shock (15). Three distinct genes encode JNKs,
jnk1, jnk2, and jnk3; the first two
are ubiquitously expressed, whereas the latter one is selectively
expressed in the heart, testis, and brain. Dual Thr and Tyr
phosphorylation of JNK by the two MAPK kinases MKK4 and MKK7 results in
its activation and nuclear translocation, after which it
phosphorylates several transcription factors such as c-Jun and ATF2
(16). Phosphorylation of c-Jun by JNK is thought to be critical for its
maximal transcriptional activity (17). On the other hand,
overexpression of constitutively active MEKK1
(MAPK/ERK kinase
kinase-1), which, in turn, activates several
MAPKs including JNK, has been shown to inhibit TGF-
/SMAD signaling
through stabilization of SMAD-Jun interactions (12).
In this study, using genetically modified
jnk1/2
/
,
nemo
/
, and junAA mouse embryo
fibroblasts, we have further refined the understanding of the molecular
mechanisms underlying the antagonistic activities of pro-inflammatory
cytokines against TGF-
. Our study establishes a key role for JNK
activation by TNF-
in blocking TGF-
-induced SMAD signaling and
SMAD-dependent transactivation of fibrillar collagen genes,
whereas the NF-
B pathway, also activated by these cytokines, plays
little role (if any) in this regulation. In addition, we provide novel
evidence that JunB is a substrate for JNK that is able to substitute
for c-Jun in mediating the JNK-dependent inhibitory effect
of pro-inflammatory cytokines against TGF-
/SMAD signaling.
 |
MATERIALS AND METHODS |
Cell Cultures--
Immortalized fibroblast cell lines were
derived from wild-type (WT),
jnk1/2
/
(referred to as
jnk
/
; targeted disruption of the
jnk1 and jnk2 genes) (18), and nemo
/
(targeted disruption of the
nemo gene) (19) mouse embryos. junAA immortalized
fibroblasts were derived from mouse embryos carrying a mutant
c-jun allele in which the JNK phosphoacceptor Ser63 and Ser73 residues are mutated to
alanines (20). Human dermal fibroblasts were established by explanting
neonatal foreskins. Cells were grown in Dulbecco's modified Eagle's
medium supplemented with 10% heat-inactivated fetal calf serum (FCS),
2 mM glutamine, 100 units/ml penicillin, 50 µg/ml
streptomycin G, and 0.25 µg/ml FungizoneTM. Human
recombinant TGF-
1 was purchased from R&D Systems (Minneapolis, MN);
it is referred to as TGF-
throughout the text. Human recombinant IL-1
and TNF-
were purchased from Roche Diagnostic.
Plasmid Constructs--
(CAGA)9-Lux was used as a
reporter construct specific for SMAD3/4-driven signaling (21).
pNF-
B-Lux and pAP-1-TA-Lux (MercuryTM pathway profiling
vectors, Clontech, Palo Alto, CA) were used to
determine NF-
B- and AP-1-driven transcription, respectively. For
c-Jun, JunB, and ATF2 expression, we used full-length human cDNAs
cloned into the pRSVe expression vector (3, 22). Dominant-negative MKK4
(22) and MKK7 and JNK1 (23) expression vectors were used to modulate
JNK activity. Antisense vectors pRSV/AS-c-jun and pRSV/AS-junB (3, 7) and fusion protein expression vectors VP16AD-JunB and Gal4BD-SMAD3 (13) have been described
previously. To determine JNK activation by TNF-
, we used a reporter
system derived from the mammalian one-hybrid system, consisting of a reporter plasmid (Gal4-Lux) and a transactivator plasmid encoding a
chimeric transactivator protein (Gal4BD-c-Jun) consisting of the
DNA-binding domain of Gal4 (Gal4BD) and the transactivation domain of
c-Jun that requires phosphorylation by JNK to fully transactivate
Gal4-Lux (Stratagene, La Jolla, CA).
Transient Cell Transfections and Reporter
Assays--
Transfections were performed using the calcium
phosphate/DNA coprecipitation procedure with a commercial assay kit
(Promega, Madison, WI). pRSV-
-galactosidase was cotransfected in
every experiment to monitor transfection efficiencies. In this context, it should be noted that basal promoter activities did not vary significantly between the various cell lines used in this study (<15%), with the exception of the junAA-transformed
fibroblasts, which exhibited a 50-60% reduction in transfection
efficiency compared with all other cell lines. Interestingly,
endogenous levels of c-Jun, JNK, and SMAD3/4-DNA complexes and mRNA
steady-state levels for type I and III collagens were very similar
between cell lines (see corresponding data description under
"Results"). Luciferase activity was determined with a commercial
kit (Promega). For high transfection efficiency of the pRSV-c-Jun
expression vector, cells were electroporated with a
NucleofectorTM (Amaxa GmbH, Köln, Germany) according
to the manufacturer's protocol. Transfection efficiency was estimated
to be ~80% by fluorescence-activated cell sorter analysis of a
cotransfected green fluorescent protein expression vector (data not shown).
Northern Blotting--
Total RNA was obtained using an RNeasy
kit (QIAGEN GmbH, Hilden, Germany) and analyzed by Northern
hybridization (20 µg/lane) with 32P-labeled cDNA
probes for COL1A1 (24), COL3A1 (25), and
glyceraldehyde-3-phosphate dehydrogenase (26). Hybridization signal was
revealed with a PhosphorImager (Storm 840, Amersham Biosciences).
Electrophoresis Mobility Shift Assays (EMSAs)--
A
(CAGA)3 probe (21) was used to determine specific
SMAD3/4-DNA interactions. Nuclear extracts were isolated using a
small-scale preparation (27).
Western Blot Analyses--
Whole cell lysates from fibroblast
cultures were prepared by scraping into Laemmli buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 0.5 mM phenylmethylsulfonyl fluoride) after two washes in cold
phosphate-buffered saline. The protein concentration of lysates was
assayed with a one-step colorimetric method (Bio-Rad protein reagent),
and 25 µg of protein was denatured by heating at 95 °C for 3 min
prior to resolution by SDS-PAGE. After electrophoresis, proteins were
transferred to Hybond ECL nitrocellulose filters (Amersham
Biosciences). The filters were placed in blocking solution (1×
phosphate-buffered saline and 5% nonfat milk) for 1 h, followed by incubation with rabbit anti-phospho-c-Jun polyclonal antiserum (Upstate Biotechnology, Inc., Lake Placid, NY) or anti-c-Jun or anti-SMAD3 polyclonal antiserum (Santa Cruz Biotechnology, Santa Cruz,
CA) at a dilution of 1:1000 in blocking solution for 1 h. After
incubation, the filters were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Santa Cruz
Biotechnology) for 1 h. The filters were then washed, developed according to ECL protocols (Amersham Biosciences), and revealed with
the Storm 840 PhosphorImager.
 |
RESULTS |
MKK4 and MKK7 Participate in the Antagonistic Activity of
Pro-inflammatory Cytokines against TGF-
/SMAD
Signaling--
We previously provided evidence that inhibition of
c-Jun expression prevents the antagonistic activity of TNF-
against
TGF-
/SMAD signaling (6). Conversely, c-Jun overexpression was shown
to block TGF-
/SMAD signaling. Possible mechanisms underlying c-Jun inhibitory effects were identified: (a) interference with
SMAD-DNA complex formation and (b) sequestration of the
shared transcriptional coactivator p300. Also, it was previously
reported that inhibition or activation of JNK activity in HepG2 cells
could activate or inhibit TGF-
/SMAD signaling, respectively
(12).
We first wanted to determine whether JNK activity is required for
pro-inflammatory cytokines to antagonize TGF-
/SMAD signaling. As
shown in Fig. 1A, expression
of both an antisense vector against c-Jun and a dominant-negative MKK4
vector prevented the inhibitory effect of TNF-
on
TGF-
-driven (CAGA)9-Lux transactivation in human dermal
fibroblasts. On the other hand, overexpression of a dominant-negative
form of I
B kinase-
, known to block NF-
B activation and
subsequent nuclear translocation, was without effect on the inhibitory
effect exerted by TNF-
. Similar results were obtained when IL-1
(10 units/ml) was used instead of TNF-
(data not shown).

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Fig. 1.
MKK4 and MKK7 participate in the antagonistic
activity of TNF- against
TGF- /SMAD signaling. A,
subconfluent human dermal fibroblasts were cotransfected with 2 µg of
(CAGA)9-Lux together with 4 µg of dominant-negative
(D/N) I B kinase- (IKK- ),
pRSV/AS-c-Jun, or dominant-negative MKK4 expression vector.
Empty pCMV was used to maintain equivalent amounts of transfected DNA
in each plate. After glycerol shock, the cells were placed in medium
supplemented with 1% FCS. Six hours later, TGF- and TNF- (10 ng/ml) were added, and incubations were continued for 24 h before
luciferase activity was determined. B, subconfluent human
dermal fibroblasts were cotransfected with 2 µg of
(CAGA)9-Lux together with 4 µg of dominant-negative MKK4
and/or MKK7 expression vector. Empty pCMV was used to maintain
equivalent amounts of transfected DNA in each plate. After glycerol
shock, the cells were placed in medium supplemented with 1% FCS. Six
hours later, growth factors and cytokines were added, and incubations
were continued for 24 h before luciferase activity was determined.
Bars indicate means ± S.D. of three independent
experiments, each performed with duplicate samples.
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|
To further understand the mechanisms by which pro-inflammatory
cytokines (known inducers of JNK activity) are capable of interfering with the SMAD pathway, we next investigated the potential implication of another MAPK kinase, MKK7 (known to activate JNK), in mediating TNF-
inhibition of SMAD signaling. As shown in Fig. 1B,
when MKK4 activity was blocked by expression of its dominant-negative mutant form, MKK7 overexpression was able to significantly rescue the
inhibitory activity of TNF-
. A similar rescue mechanism was observed
when IL-1
was used instead of TNF-
(data not shown), indicating
that both MKK4 and MKK7 may contribute to the signaling cascade
activated by pro-inflammatory cytokines to counteract TGF-
/SMAD signaling.
JNK Activity Is Required for TNF-
to Antagonize
TGF-
/SMAD Signaling--
To ascertain the role played by
the JNK pathway and to definitely rule out the possible implication of
NF-
B, the antagonistic activity of TNF-
against TGF-
/SMAD
signaling was examined in immortalized fibroblast lines derived from
jnk
/
and nemo
/
mouse embryos. To fully exploit the analytical power of such a cellular
system, it was important to determine how the various signaling
pathways to be investigated were regulated by cytokines in the
different cell lines. First, as shown in Fig.
2A and consistent with our
expectations from the literature, NF-
B-dependent gene transactivation in response to TNF-
was absent in
nemo
/
fibroblasts, but normal in
jnk
/
fibroblasts. Inversely,
AP-1-dependent gene expression downstream of TNF-
was
normal in nemo
/
fibroblasts, but absent in
jnk
/
fibroblasts (Fig. 2B).
Second, we examined the capacity of pro-inflammatory cytokines to
activate JNK in the various cell lines. As shown in Fig. 2C,
TNF-
efficiently transactivated the Gal4-Lux construct in the
presence of Gal4BD-c-Jun in WT and nemo
/
fibroblasts, but not in jnk
/
fibroblasts,
suggesting that simultaneous targeting of jnk1 and jnk2 is sufficient to completely eliminate JNK activity.
This is likely representative of an absence of JNK3 expression in these cells, which would be consistent with its known strict tissue-specific expression (15). The specificity of JNK activity in the various cell
types was further confirmed by Western blot analysis of phospho-c-Jun content in response to TNF-
. As shown in Fig. 2D, TNF-
stimulation of both WT and nemo
/
fibroblasts
resulted in a dramatic induction of c-Jun phosphorylation (lanes
2 and 6 versus lanes 1 and 5),
the latter not being detected in the jnk
/
fibroblasts (lane 4 versus lane 3).

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Fig. 2.
Cytokine and growth factor responses in
jnk /
and
nemo /
fibroblasts. Subconfluent WT, jnk / , and
nemo / fibroblast cultures were transfected
with 2 µg of pNF- B-Lux (A), pAP-1-Lux (B),
or Gal4-Lux together with the Gal4BD-c-Jun fusion protein expression
vector (C) prior to stimulation with TNF- . Total protein
extracts from WT, jnk / , and
nemo / fibroblasts treated with TNF- for
1 h using anti-phospho-c-Jun (P-cJun) and anti-c-Jun
antibodies were analyzed Western blotting (D).
|
|
To complete our characterization of these cell lines in terms of growth
factor response, we next examined the transactivation of
(CAGA)9-Lux by TGF-
in jnk
/
,
nemo
/
, and junAA immortalized
fibroblasts. As shown in Fig.
3A, full SMAD3/4-dependent responsiveness downstream of TGF-
was
observed in all cell lines, indicating that neither JNK nor NF-
B
activity nor the c-Jun phosphorylation state plays a role in
TGF-
-driven SMAD3/4 responses. Of note, using the Gal4-based
transactivation assay system, we determined that TGF-
did not
activate JNK in any cell type (data not shown). Together, these results
validate our model system for further investigation of the functional
aspects of cytokine/TGF-
transcriptional antagonism in a cellular
context exhibiting normal TGF-
/SMAD responsiveness, but devoid of
either JNK or NF-
B activity or in which c-Jun phosphorylation by JNK is impossible. As shown in Fig. 3A, TNF-
-mediated
inhibition of SMAD signaling, as measured using the
(CAGA)9-Lux vector as a reporter system, was consistently
observed in immortalized WT fibroblasts, as well as in
nemo
/
and junAA fibroblasts, but
not in jnk
/
fibroblasts, suggesting
(a) a critical role for JNK and (b) the existence
of alternative mechanisms not requiring c-Jun phosphorylation to allow
the inhibitory activity of pro-inflammatory cytokines against
TGF-
.

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Fig. 3.
JNK activity is required for
TNF- to antagonize
TGF- /SMAD signaling. A,
subconfluent WT, jnk / ,
nemo / , and junAA fibroblast
cultures were transfected with (CAGA)9-Lux. After glycerol
shock, the cells were placed in medium supplemented with 1% FCS. Six
hours later, TGF- was added without or with TNF- , and incubations
were continued for 24 h before luciferase activity was determined.
Note that TNF- exerted an antagonistic activity against TGF- in
all but jnk / fibroblasts. B,
EMSAs were performed using the SMAD3/4-specific (CAGA)3
oligonucleotide (21) as a probe together with nuclear extracts from WT,
jnk / , and nemo /
fibroblast cultures treated for 30 min with TGF- and/or TNF- . The
SMAD content of the TGF- -induced complex (arrow) was
verified by supershift with an anti-SMAD3 antibody (right
panel). Note that the TGF- -induced SMAD-DNA complex was not
reduced by TNF- in jnk / fibroblasts.
C, EMSAs were performed using a consensus NF- B
oligonucleotide as a probe together with nuclear extracts from WT,
jnk / , and nemo /
fibroblast cultures as described for B. Note the complete
dissociation of SMAD and NF- B binding from their respective DNA
recognition sites.
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|
To determine whether this cellular context-specific inhibitory activity
of TNF-
correlates with the known ability of TNF-
to interfere
with SMAD-DNA complex formation in normal human dermal fibroblasts (6),
EMSA experiments were carried out using nuclear extracts from WT,
jnk
/
, and nemo
/
fibroblasts treated with TGF-
and/or TNF-
for 30 min, a time point previously shown to be ideal for the detection of SMAD-DNA complexes after TGF-
stimulation (28, 29). As shown in Fig. 3B (left panel), in
the three cell types, a unique protein-DNA complex was equally
induced by TGF-
(second, sixth, and
tenth bars). Supershift experiments with an anti-SMAD3
antibody confirmed that this complex is indeed a SMAD-DNA complex
(Fig. 3B, right panel). TNF-
efficiently reduced TGF-
-induced SMAD-DNA complex formation in both
WT (lane 4 versus lane 2) and
nemo
/
(lane 12 versus lane
10) mouse fibroblast extracts, whereas no reduction in SMAD-DNA
complex formation could be observed in jnk
/
fibroblasts (lane 8 versus lane 6). No change in
total SMAD3 content (measured by Western blot analysis of whole cell
extracts) was observed in WT fibroblasts treated with TNF-
for 30 min to 24 h (data not shown), indicating that the reduced amount
of SMAD-DNA complexes observed in EMSAs and the reduced
SMAD-dependent gene transactivation when TNF-
was added
concomitantly with TGF-
(see above) are not due to reduced SMAD3
levels in response to TNF-
. Parallel EMSA with an NF-
B-specific
probe (Fig. 3C) highlighted the lack of correlation
between TNF-
-dependent reduction in SMAD-DNA complexes
seen in Fig. 3B and the induction of NF-
B
DNA-binding activity.

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Fig. 4.
c-Jun overexpression antagonizes
TGF- /SMAD signaling: a role for basal JNK
activity. A, subconfluent WT,
jnk / , and nemo /
fibroblast cultures were transfected with (CAGA)9-Lux
without or with the pRSV-c-Jun expression vector. After glycerol shock,
the cells were placed in medium supplemented with 1% FCS. Six hours
later, TGF- (10 ng/ml) was added, and incubations were continued for
24 h before luciferase activity was determined. Bars
indicate means ± S.D. of three independent experiments, each
performed with duplicate samples. B, WT fibroblast cultures
were treated with TGF- , and whole cell lysates were prepared at
various time points for Western blot analysis of phospho-JNK
(P-JNK), JNK, and -actin contents. C, WT
fibroblast cultures were cotransfected with the Gal4-Lux reporter
construct and Gal4BD-c-Jun fusion protein expression vector in the
presence of either empty pRSVe or the dominant-negative MKK4
expression vector. Results from three separate experiments are shown as
means ± S.D. D, WT fibroblasts were electroporated
with empty pRSVe or the c-Jun expression vector using a
NucleofectorTM. Twenty-four hours later, TNF- (10 ng/ml)
was added for 30 min. Whole cell extracts were then analyzed by Western
blotting for phospho-c-Jun (P-cJun), c-Jun, and -actin
contents. Note the high amounts of phospho-c-Jun in c-Jun-transfected
cells, even in the absence of TNF- (lane 3).
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|
Because c-Jun is a key effector of TNF-
inhibitory activity in SMAD
signaling (6), we overexpressed c-Jun instead of adding exogenous
TNF-
and examined its inhibitory potential on SMAD signaling in WT,
jnk
/
, and nemo
/
fibroblasts. As shown in Fig. 4A, c-Jun inhibitory activity
against SMAD-driven TGF-
-induced (CAGA)9-Lux
transactivation was readily observed in WT and
nemo
/
fibroblasts, but absent in
jnk
/
fibroblasts, suggesting a critical role
for c-Jun N-terminal phosphorylation in the inhibitory activity of
c-Jun against the SMAD pathway. To determine the origin of such JNK
activity in WT cells, several approaches were taken. First, potential
activation of JNK by TGF-
was examined. As shown in Fig.
4B (upper panel), no JNK phosphorylation in
response to TGF-
(detected by Western blot analysis of endogenous
phospho-JNK proteins) was observed 15 min to 24 h after TGF-
addition. An antibody directed against JNK verified that the total JNK
content was identical in each sample (middle panel). These
data are consistent with our previous demonstration that TGF-
is
able to transactivate AP-1-dependent genes only in
epithelial cells, but not in fibroblasts (7). Second, to determine the
possibility of basal JNK activity in unstimulated cultured fibroblasts,
we used a Gal4-based transactivation system. Specifically, a
Gal4BD-c-Jun fusion protein expression vector was cotransfected
together with the Gal4-Lux reporter vector in WT fibroblasts in the
absence or presence of a dominant-negative MKK4 expression vector. As
shown in Fig. 4C, expression of Gal4BD-c-Jun led to a
2.5-fold elevation of Gal4-Lux activity. Overexpression of
dominant-negative MKK4 fully repressed Gal4BD-c-Jun-induced luciferase
activity, consistent with the idea that transactivation of Gal4-Lux by
Gal4BD-c-Jun is a JNK-dependent mechanism. Third, we
determined whether, upon c-Jun overexpression, a significant fraction
of c-Jun would be phosphorylated. Significantly higher amounts of c-Jun
protein were detected in extracts from fibroblasts transfected with a
c-Jun expression vector with NucleofectorTM compared with
extracts from mock-transfected cells (Fig. 4D, middle
panel, lane 3 versus lane 1). Interestingly,
such high expression of c-Jun was accompanied by strongly elevated
levels of phospho-c-Jun (upper panel, lane 3 versus lane 1), very similar to the levels obtained in
mock-transfected cells treated with TNF-
(lane 2).
Together, these data suggest the existence of detectable levels of JNK
activity in unstimulated cultured fibroblasts, sufficient to convey
both Gal4-Lux transactivation by Gal4BD-c-Jun and the inhibitory
effect of overexpressed c-Jun against TGF-
/SMAD signaling.
jnk1 Expression in jnk
/
Fibroblasts Restores the
Inhibitory Effect of Pro-inflammatory Cytokines on
TGF-
/SMAD Signaling--
To verify that the lack of
TNF-
effect against TGF-
/SMAD signaling in
jnk
/
fibroblasts is effectively due to the
knockout of JNK activity, the effect of jnk1 expression
rescue was examined. As shown in Fig. 5,
ectopic expression of jnk1, which had no significant effect on SMAD signaling per se in WT fibroblasts, entirely rescued
the inhibitory effect of TNF-
in jnk
/
fibroblasts, attesting that the lack of inhibitory effect of TNF-
is
strictly due to the absence of JNK activity.

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Fig. 5.
jnk1 expression in
jnk /
fibroblasts restores the inhibitory effect of TNF-
on TGF- /SMAD signaling. Subconfluent WT and
jnk / fibroblast cultures were transfected
with (CAGA)9-Lux in the absence or presence of the JNK1
expression vector. After glycerol shock, the cells were placed in
medium supplemented with 1% FCS. Six hours later, TGF- was added
without or with TNF- , and incubations were continued for 24 h
before luciferase activity was determined. Bars indicate
means ± S.D. of three independent experiments, each performed
with duplicate samples.
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c-Jun and JunB (but Not ATF2) Inhibit TGF-
/SMAD
Signaling in a JNK-dependent Manner--
Our next aim was
to determine which JNK substrate(s) may be able to interfere with
TGF-
/SMAD signaling. For this purpose, the effect of overexpression
of c-Jun, JunB, and ATF2, three known JNK substrates (16), on
(CAGA)9-Lux transactivation was examined in WT and
jnk
/
fibroblasts. The results presented in
Fig. 6A indicate that c-Jun and JunB (but not ATF2) exerted an inhibitory activity against TGF-
in wild-type cells, but not in jnk
/
fibroblasts. Similarly, in human dermal fibroblasts, c-Jun and JunB
inhibitory activity against TGF-
/SMAD signaling was strongly limited
by overexpression of dominant-negative MKK4 (Fig. 6B), implying that, in this cell type, JNK activity is also critical for the
effect of both c-Jun and JunB.

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Fig. 6.
c-Jun and JunB (but not ATF2) inhibit
TGF- /SMAD signaling in a JNK-dependent
manner. A, subconfluent WT and
jnk / fibroblasts were transfected in
parallel with (CAGA)9-Lux together with c-Jun, JunB, and
ATF2 expression vectors. After glycerol shock, the cells were placed in
medium supplemented with 1% FCS. Eighteen hours later, TGF- was
added, and incubations were continued for 24 h before luciferase
activity was determined. Bars indicate means ± S.D. of
three independent experiments, each performed with duplicate samples.
B, subconfluent human dermal fibroblast cultures were
transfected in parallel with (CAGA)9-Lux together with
either pRSV-c-Jun or pRSV-JunB without or with the dominant-negative
(D/N) MKK4 expression vector. After glycerol
shock, the cells were placed in medium supplemented with 1% FCS.
Eighteen hours later, TGF- was added, and incubations were continued
for 24 h before luciferase activity was determined.
Bars indicate means ± S.D. of three independent
experiments, each performed with duplicate samples.
|
|
Further Evidence That JunB Function Is Dependent on JNK--
From
the results presented above, it appears that JunB may be a substrate
for JNK, an issue that has been somewhat controversial (30-32). To
confirm this hypothesis, we compared the effect of JunB on TGF-
/SMAD
signaling in WT and jnk
/
fibroblasts in the
absence or presence of exogenously added jnk1 expression vector. As shown in Fig.
7A, JunB
overexpression efficiently blocked TGF-
-driven
SMAD-dependent transactivation in WT fibroblasts. The
inhibitory effect of JunB was lost in jnk
/
fibroblasts, but rescued by ectopic jnk1 expression in the
latter cell type, attesting for a direct role of JNK activity in
controlling the JunB effect against TGF-
/SMAD signaling. Of note,
the same results were obtained when c-Jun was used instead of JunB in
the same experimental setting (data not shown).

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|
Fig. 7.
JNK-dependent JunB
functions. A, JNK1 overexpression restores JunB
inhibitory activities against TGF- /SMAD signaling in
jnk / fibroblasts. Subconfluent WT and
jnk / fibroblast cultures were transfected in
parallel with (CAGA)9-Lux together with either empty pRSV
or pRSV-JunB without or with the JNK1 expression vector. After glycerol
shock, the cells were placed in medium supplemented with 1% FCS.
Eighteen hours later, TGF- was added, and incubations were continued
for 24 h before luciferase activity was determined. B,
in junAA fibroblasts, JunB substitutes for c-JunAA and
mediates TNF- -induced inhibition of TGF- /SMAD signaling in a
JNK-dependent manner. Subconfluent junAA
fibroblasts were cotransfected with (CAGA)9-Lux together
with either the pRSV/AS-junB or dominant-negative
(D/N) MKK4 expression vector. Empty pCMV was used
to maintain equivalent amounts of transfected DNA in each plate. After
glycerol shock, the cells were placed in medium supplemented with 1%
FCS. Six hours later, TGF- and TNF- were added, and incubations
were continued for 24 h before luciferase activity was determined.
In A and B, bars indicate means ± S.D. of at least three experiments, each performed with duplicate
samples. C, JNK activity increases SMAD3-JunB interactions
in the mammalian two-hybrid system. Subconfluent WT and
jnk / fibroblast cultures were transfected
with Gal4-Lux, Gal4BD-SMAD3, and/or VP16AD-JunB together with
either the empty pCMVe or JNK1 expression vector. Six hours following
glycerol shock, TNF- was added, and luciferase activity was measured
24 h later. Data from a representative experiment are shown.
|
|
We have determined that TNF-
efficiently blocks TGF-
signaling in
junAA fibroblasts (Fig. 3). Together with our data
indicating the critical role for c-Jun phosphorylation by JNK in
mediating the TNF-
effect in human and mouse fibroblasts, these
observations led us to investigate (a) whether JNK plays a
role downstream of TNF-
in junAA cells and (b)
whether JunB may substitute for c-Jun in the latter cell type. To this
end, junAA fibroblasts were transfected with either a
dominant-negative MKK4 or an antisense junB expression
vector, and the antagonism between TNF-
and TGF-
on
(CAGA)9-Lux was determined. As shown in Fig. 7B,
dominant-negative MKK4 efficiently blocked the effect of TNF-
against TGF-
, indicating that this inhibitory mechanism in
junAA fibroblasts is also dependent on JNK function.
Furthermore, overexpression of the antisense junB vector
resulted in almost complete abolishment of the TNF-
effect,
indicating that JunB substitutes for c-JunAA in mediating the
inhibitory activity of TNF-
against SMAD signaling in a
JNK-dependent manner.
One of the mechanisms by which Jun proteins interfere with the SMAD
pathway involves direct SMAD-Jun interaction, not compatible with
SMAD-DNA complex formation (13, 33, 34). To determine whether JNK
activity may play a role in controlling SMAD3-JunB interactions, we
adapted the mammalian one-hybrid Gal4-based transactivation assay in
jnk
/
fibroblasts. As shown in Fig.
7C, in the absence of the JNK1 expression vector,
expression of VP16AD-JunB only slightly enhanced Gal4BD-SMAD3-mediated transactivation, representative of weak interactions between SMAD3 and JunB. No effect of TNF-
on this interaction could be observed. On the other hand, when jnk1
was coexpressed, VP16AD-JunB expression resulted in a dramatic
enhancement of the Gal4BD-SMAD3 effect, representative of
JNK-dependent SMAD3-JunB interactions, which were further
enhanced by exogenous TNF-
, reflecting activation of the MEK1
(MAPK/ERK
kinase-1)/MKK4/JNK cascade by the latter.
Together, these results provide strong evidence for a role of JNK in
enhancing SMAD-Jun direct interactions that are not compatible with
SMAD-DNA complex formation (i.e. no transactivation).
TNF-
Prevents TGF-
-induced COL1A1 and COL3A1 Gene Expression
in a JNK-dependent Manner--
The data presented above
demonstrate that JNK plays a essential role downstream of
pro-inflammatory cytokines in interfering with the SMAD pathway. To
determine the role of JNK in a physiologically relevant gene context,
we examined the antagonistic activity of TNF-
against
TGF-
-induced up-regulation of the endogenous extracellular matrix
genes COL1A1 and COL3A1. These fibrillar collagen
genes were previously identified as SMAD3/4 targets (35). As shown in
Fig. 8, strong enhancement of both
COL1A1 and COL3A1 mRNA steady-state levels
(6-8-fold) was observed in response to TGF-
in the three cell types
(lanes 2, 6, and 10, respectively).
TNF-
antagonized the TGF-
effect on type I and type III collagen
gene expression in both WT and nemo
/
fibroblasts (lanes 4 versus lanes 2 and 12 versus lane 10, respectively), but not in
jnk
/
fibroblasts (lane 8 versus lane 6). Glyceraldehyde-3-phosphate dehydrogenase mRNA steady-state levels showed no modulation by cytokines in any of the cell types (lower panel). These data
are in agreement with our observation that TNF-
antagonizes
TGF-
-induced COL1A2 expression and promoter
transactivation in a c-Jun/JNK-dependent manner (36,
37).

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|
Fig. 8.
Role of JNK in the down-regulation of
TGF- -induced type I and type III collagen gene
expression by TNF- . Subconfluent WT,
jnk / , and nemo /
fibroblast cultures were treated with TGF- and TNF- for 24 h
in medium containing 1% serum. Shown are representative Northern
hybridization signals of total RNA with COL1A1,
COL3A1, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) probes. Note the absence of down-regulation of
TGF- effect on both COL1A1 and COL3A1 mRNA
steady-state levels by TNF- in jnk /
fibroblasts.
|
|
 |
DISCUSSION |
Using both normal human dermal fibroblasts and genetically
modified immortalized mouse fibroblast lines established from
jnk
/
, nemo
/
, and
junAA embryos, we have provided definite evidence for a central role for JNK in allowing the inhibitory activity of
pro-inflammatory cytokines against TGF-
/SMAD signaling, whereas
NF-
B activity plays no role in the phenomenon. We have identified
c-Jun and JunB (but not ATF2) as JNK substrates responsible for this
effect and determined that JNK activity allows for strong
protein-protein interactions between JunB and SMAD3, not compatible
with SMAD-DNA complex formation (33) and leading to reduced
SMAD-dependent gene transcription (13).
Lack of JNK Activity Does Not Alter the TGF-
/SMAD
Response--
The use of cell lines genetically devoid of JNK
activity allowed us to rule out any implication of the latter MAPK in
the activation of the SMAD cascade by TGF-
. Our results, obtained mainly by transfecting the artificial constructs
(CAGA)9-Lux and (SMAD binding
element)4-Lux (38) (data not shown), both of them highly
specific for the SMAD3/4 pathway, differ from those of Engel et
al. (39), who demonstrated that JNK phosphorylation of SMAD3
facilitates its activation by TGF-
receptor type I and subsequent nuclear translocation. Several explanations can be found for
these discrepancies. The latter results were obtained using indirect
approaches to interfere with Rho and Rac GTPase-driven TGF-
-induced
JNK activity. A second reason that may explain the discrepancies is
that, in several experiments, Engel et al. utilized the
construct 3TP-Lux as a SMAD reporter, and it is now well established, as was somewhat suggested by the authors themselves, that its transactivation is highly AP-1-dependent, in other words,
JNK-dependent. It is also worth noting that, in all mouse
fibroblast lines we tested, TGF-
itself did not activate JNK, as
measured either by the modified one-hybrid transactivation system based
on c-Jun phosphorylation (data not shown) or by Western blotting (Fig. 4B), therefore contrasting with the biphasic activation of
JNK observed in Mv1Lu and MDA-MB-468 cells (39). This cell
type-specific activation of JNK by TGF-
is entirely consistent with
our previous observation that TGF-
induces
AP-1-dependent gene transactivation in epithelial cells,
but not in fibroblasts (7).
JNK Activity Is Central to the Inhibitory Activity of
Pro-inflammatory Cytokines against TGF-
/SMAD Signaling, whereas
NF-
B Activity Is Not--
As described above, in some cell types,
JNK positively regulates some of the SMAD- and TGF-
-mediated
transcriptional responses, yet JNK activators only partially stimulate
transcriptional responses characteristic of TGF-
without coincident
SMAD pathway activation. It has also been reported that, in some cell
types, triggering of the SAPK/JNK pathway by TGF-
itself could
participate in a negative feedback loop controlling SMAD-driven TGF-
responses (12). Although they are somewhat opposite with regard to JNK activation by TGF-
itself, these results are in full agreement with
the concept of an interdependent relationship between the JNK and SMAD
pathways in TGF-
-mediated transcription.
Another interesting and novel observation from our work is that JNK
activity is a prerequisite for pro-inflammatory cytokines to interfere
with the SMAD pathway, whereas the NF-
B pathway, although critical
for numerous inflammatory responses, plays little role (if any) in this
phenomenon. Again, the use of genetically altered cell lines devoid of
any NF-
B activity allowed us to definitely rule out the involvement
of the latter in the antagonistic activity of TNF-
against SMAD signaling.
Both TNF-
and IL-1
, prototypic inflammatory cytokines, inhibited
TGF-
-induced SMAD signaling in human dermal fibroblasts and in the
various mouse lines tested, except in the
jnk
/
fibroblasts. Their inhibitory activity
was restored in the latter cell type upon overexpression of
jnk1. In human dermal fibroblasts, their inhibitory activity
was prevented by a dominant-negative form of MKK4 that blocks JNK
activation (17). Concomitant expression of MKK7 together with
dominant-negative MKK4 restored most of the inhibitory activity of
TNF-
against SMAD signaling, suggesting that both MKK4 and MKK7 are
capable of mediating this cytokine effect. Searching for JNK substrates
involved in the inhibitory mechanism, we identified c-Jun and JunB as
potential candidates, both of which are known to be up-regulated in
fibroblasts by several pro-inflammatory cytokines, whether in the
presence or absence of TGF-
(3). These two Jun family members are
able to interfere with SMAD signaling, but this is the first
demonstration that their inhibitory activity can be exerted only
through JNK activation downstream of pro-inflammatory cytokines. ATF2,
another known JNK substrate, did not interfere with the SMAD pathway.
Another mechanism by which TNF-
might block SMAD signaling is
through NF-
B activation. The latter may, in turn, induce SMAD7 expression, a molecule that interferes with SMAD phosphorylation by
TGF-
receptor type I and subsequent translocation into the cell
nucleus (10). Activation of SMAD7 expression through the NF-
B
cascade appears to be restricted to certain subsets of mouse embryos
fibroblasts, as (a) in human embryonic kidney 293 cells, NF-
B activation inhibits SMAD7 gene expression (11);
(b) we did not previously observe any activation of SMAD7
expression by TNF-
in human dermal fibroblasts (6); (c)
in several primary and immortalized mouse cell lines tested during this
study, transfection of a dominant-negative mutant of I
B kinase-
,
which blocks NF-
B activation, did not interfere with TNF-
blockade of SMAD signaling (data not shown); and (d) mouse
embryo fibroblasts devoid of NF-
B activity, e.g.
nemo
/
, allowed full inhibitory activity of
TNF-
against SMAD signaling (this study). The latter data
unequivocally eliminate the NF-
B pathway from playing a role in the
interference exerted by pro-inflammatory cytokines with TGF-
/SMAD signaling.
JunB Function Depends on JNK Activity--
Several of the
experiments described in this study reinforce the idea that JunB is a
JNK substrate. First, we found that, in human dermal fibroblasts, JunB
inhibitory activity against SMAD signaling was blocked by expression of
a dominant-negative mutant form of MKK4 blocking the JNK signal
transduction pathway (17). Second, we observed that JunB did not exert
its inhibitory activity in jnk
/
fibroblasts
unless episomal expression of jnk1 was allowed by means of a
transfected expression vector. Finally, using the mammalian two-hybrid
system, we determined that protein-protein interactions between SMAD3
and JunB were very weak in jnk
/
fibroblasts,
potentiated by ectopic jnk1 expression and further enhanced
by TNF-
. The latter phenomenon is likely representative of JNK1
activation by TNF-
, resulting in enhanced functionality of JunB upon
phosphorylation. Together, these data provide ample evidence for JunB
as a JNK1 substrate. They also indicate that JNK activity promotes
SMAD3-JunB interactions, which result in decreased
SMAD-dependent gene transcription, as this association is
not compatible with SMAD binding to its cognate DNA sequences (6,
13).
JunB May Substitute for c-Jun in Mediating the Inhibitory Activity
of Pro-inflammatory Cytokines against TGF-
/SMAD
Signaling--
In human dermal and WT mouse fibroblasts, most of the
antagonistic activity of TNF-
against TGF-
/SMAD signaling is
dependent on c-Jun, as evidenced using antisense c-Jun approaches (Ref. 6 and this study). Earlier studies have shown that c-Jun needs to be
phosphorylated at Ser63 and Ser73 to become
transcriptionally active (40, 41) and for
c-Jun-dependent apoptosis (20, 42). Our data indicate that
c-Jun phosphorylation by JNK is also critical for inhibitory activities
that do not depend on c-Jun binding to specific DNA sequences. JunAA, a
protein in which the phosphoacceptor Ser63 and
Ser73 residues have been mutated to alanines, fails to
transactivate from AP-1 elements and to cotransform (20).
Moreover, a dominant-negative JNK1 mutant was shown to interfere with
c-Jun-dependent transformation (20, 42), further
emphasizing the central role of Jun phosphorylation in
Jun-dependent biological responses.
junAA fibroblasts allowed us to evaluate the role of JNK in
a cellular context in which its main substrate, c-Jun, is not functional as such. In this cellular context, in which JNK was activated upon TNF-
stimulation (see Fig. 2, B and
C), we unveiled a novel function of JunB downstream of
TNF-
signaling. Specifically, we determined that JunB could
substitute for c-Jun in mediating the inhibitory activity of TNF-
against SMAD signaling in a JNK-dependent manner. This is
an important result, as it is too often considered that JNK
inactivation and c-Jun targeting are somewhat similar. Furthermore, in
several instances, JunB has been shown to exert antagonistic activities
against c-Jun (3, 7, 43, 44).
Substitutions of some of the functions of AP-1 family members with JunB
have also been described. For example, defects in the placentation of
fra-1-knockout embryos can be rescued by a junB transgene, although with a low efficiency (45).
Knock-in mice in which c-jun has been replaced by
junB, although dying a few hours after birth because of
malformed cardiac outflow tracts, develop normal livers, clearly
indicating that JunB is able to complement for c-Jun in hepatic
development (46).
Conclusion--
We have reported a critical role for JNK in the
mechanism of suppression of TGF-
/SMAD3 signaling by pro-inflammatory
cytokines, involving the transcription factors c-Jun and JunB. These
AP-1 components are key factors in the transmission of signals from various pro-inflammatory cytokines known to antagonize TGF-
in the
context of tissue repair and maintenance of tissue homeostasis. Our
experiments demonstrate that JNK, which is critical in conferring transcriptional activity to Jun proteins, is also instrumental in
allowing these proteins to exert inhibitory activities independent of
their DNA-binding ability and associated transactivating capabilities, such as interfering with SMAD-dependent gene transcription
downstream of TGF-
. We have also demonstrated that such a
JNK-dependent mechanism underlies the inhibitory activity
of TNF-
against TGF-
-induced SMAD-dependent fibrillar
collagen gene expression.
 |
ACKNOWLEDGEMENTS |
We are indebted to Drs. A. Atfi (INSERM U482,
Paris, France); A. Israel and G. Courtois (Pasteur Institute,
Paris, France); S. Dennler and J.-M. Gauthier (GlaxoSmithKline, Les
Ulis, France); R. Derynck (University of California, San
Francisco, San Francisco, CA); A. B. Roberts (NCI, National
Institutes of Health, Bethesda, MD); R. J. Lechleider
(Georgetown University, Washington, D. C.); M. Pasparakis
(European Microbiology Laboratory (EMBL), Monterotondo, Italy);
and A. Moustakas and C. H. Heldin (Ludwig Institute for Cancer Research (LICR), Uppsala, Sweden) for providing the cellular and
molecular reagents essential for these studies.
 |
FOOTNOTES |
*
This work was supported by INSERM, the Ligue Nationale
contre le Cancer (Comité de Paris), the Association pour la
Recherche contre le Cancer (France), and the Electricité de
France (Service de Radioprotection).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 and reprint requests should be
addressed: INSERM U532, Institut de Recherche sur la Peau, Pavillon Bazin, Hôpital Saint-Louis, 1, avenue Claude Vellefaux, 75475 Paris Cedex 10, France. Tel.: 33-1-5372-2069; Fax: 33-1-5372-2051; E-mail: mauviel@chu-stlouis.fr.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M206927200
 |
ABBREVIATIONS |
The abbreviations used are:
TGF-
, transforming growth factor-
;
IL-1, interleukin-1;
TNF-
, tumor
necrosis factor-
;
JNK, c-Jun N-terminal kinase;
MAPK, mitogen-activated protein kinase;
SAPK, stress-activated protein
kinase;
MKK, mitogen-activated protein kinase kinase;
ERK, extracellular signal-regulated kinase;
ATF2, activating transcription
factor-2;
WT, wild-type;
FCS, fetal calf serum;
Lux, luciferase;
VP16AD, VP16 activation domain;
Gal4BD, Gal4
DNA-binding domain;
EMSA, electrophoretic mobility shift assay.
 |
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