A Central Role for the JNK Pathway in Mediating the Antagonistic Activity of Pro-inflammatory Cytokines against Transforming Growth Factor-beta -driven SMAD3/4-specific Gene Expression*

Franck VerrecchiaDagger , Charlotte TacheauDagger , Erwin F. Wagner§, and Alain MauvielDagger

From Dagger  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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have focused our attention on the molecular events underlying the antagonistic activities of pro-inflammatory cytokines against transforming growth factor-beta (TGF-beta )/SMAD signaling. Using jnk1/2-knockout (jnk-/-) and Ikappa B kinase-gamma /nemo-/- fibroblasts, we have determined the specific roles played by the JNK/AP-1 and NF-kappa 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-alpha (TNF-alpha ) did not inhibit the formation of SMAD-DNA complexes and the resulting SMAD-driven transcription in response to TGF-beta . On the other hand, lack of NF-kappa B activity in nemo-/- fibroblasts did not affect the antagonistic effect of pro-inflammatory cytokines against TGF-beta . 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-alpha , 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-beta /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-beta -induced SMAD signaling. In addition, we demonstrate that such a JNK-dependent regulatory mechanism underlies the antagonistic activity of TNF-alpha against TGF-beta -induced up-regulation of type I and III collagens in fibroblasts.

    INTRODUCTION
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INTRODUCTION
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Transforming growth factor-beta (TGF-beta )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-alpha (TNF-alpha ) 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-beta 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-beta (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-beta and pro-inflammatory cytokines, including TNF-alpha and IL-1 (7). More recently, it was shown that several signaling pathways, such as those for NF-kappa 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-beta receptors (8, 9). NF-kappa B has been suggested to be part of the signals responsible for SMAD7 gene activation by TNF-alpha (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-beta 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-beta /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-beta . Our study establishes a key role for JNK activation by TNF-alpha in blocking TGF-beta -induced SMAD signaling and SMAD-dependent transactivation of fibrillar collagen genes, whereas the NF-kappa 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-beta /SMAD signaling.

    MATERIALS AND METHODS
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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-beta 1 was purchased from R&D Systems (Minneapolis, MN); it is referred to as TGF-beta throughout the text. Human recombinant IL-1beta and TNF-alpha were purchased from Roche Diagnostic.

Plasmid Constructs-- (CAGA)9-Lux was used as a reporter construct specific for SMAD3/4-driven signaling (21). pNF-kappa B-Lux and pAP-1-TA-Lux (MercuryTM pathway profiling vectors, Clontech, Palo Alto, CA) were used to determine NF-kappa 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-alpha , 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-beta -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
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ABSTRACT
INTRODUCTION
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MKK4 and MKK7 Participate in the Antagonistic Activity of Pro-inflammatory Cytokines against TGF-beta /SMAD Signaling-- We previously provided evidence that inhibition of c-Jun expression prevents the antagonistic activity of TNF-alpha against TGF-beta /SMAD signaling (6). Conversely, c-Jun overexpression was shown to block TGF-beta /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-beta /SMAD signaling, respectively (12).

We first wanted to determine whether JNK activity is required for pro-inflammatory cytokines to antagonize TGF-beta /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-alpha on TGF-beta -driven (CAGA)9-Lux transactivation in human dermal fibroblasts. On the other hand, overexpression of a dominant-negative form of Ikappa B kinase-alpha , known to block NF-kappa B activation and subsequent nuclear translocation, was without effect on the inhibitory effect exerted by TNF-alpha . Similar results were obtained when IL-1beta (10 units/ml) was used instead of TNF-alpha (data not shown).


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Fig. 1.   MKK4 and MKK7 participate in the antagonistic activity of TNF-alpha against TGF-beta /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) Ikappa B kinase-alpha (IKK-alpha ), 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-beta and TNF-alpha (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.

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-alpha 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-alpha . A similar rescue mechanism was observed when IL-1beta was used instead of TNF-alpha (data not shown), indicating that both MKK4 and MKK7 may contribute to the signaling cascade activated by pro-inflammatory cytokines to counteract TGF-beta /SMAD signaling.

JNK Activity Is Required for TNF-alpha to Antagonize TGF-beta /SMAD Signaling-- To ascertain the role played by the JNK pathway and to definitely rule out the possible implication of NF-kappa B, the antagonistic activity of TNF-alpha against TGF-beta /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-kappa B-dependent gene transactivation in response to TNF-alpha was absent in nemo-/- fibroblasts, but normal in jnk-/- fibroblasts. Inversely, AP-1-dependent gene expression downstream of TNF-alpha 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-alpha 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-alpha . As shown in Fig. 2D, TNF-alpha 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-kappa 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-alpha . Total protein extracts from WT, jnk-/-, and nemo-/- fibroblasts treated with TNF-alpha 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-beta in jnk-/-, nemo-/-, and junAA immortalized fibroblasts. As shown in Fig. 3A, full SMAD3/4-dependent responsiveness downstream of TGF-beta was observed in all cell lines, indicating that neither JNK nor NF-kappa B activity nor the c-Jun phosphorylation state plays a role in TGF-beta -driven SMAD3/4 responses. Of note, using the Gal4-based transactivation assay system, we determined that TGF-beta 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-beta transcriptional antagonism in a cellular context exhibiting normal TGF-beta /SMAD responsiveness, but devoid of either JNK or NF-kappa B activity or in which c-Jun phosphorylation by JNK is impossible. As shown in Fig. 3A, TNF-alpha -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-beta .


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Fig. 3.   JNK activity is required for TNF-alpha to antagonize TGF-beta /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-beta was added without or with TNF-alpha , and incubations were continued for 24 h before luciferase activity was determined. Note that TNF-alpha exerted an antagonistic activity against TGF-beta 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-beta and/or TNF-alpha . The SMAD content of the TGF-beta -induced complex (arrow) was verified by supershift with an anti-SMAD3 antibody (right panel). Note that the TGF-beta -induced SMAD-DNA complex was not reduced by TNF-alpha in jnk-/- fibroblasts. C, EMSAs were performed using a consensus NF-kappa 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-kappa B binding from their respective DNA recognition sites.

To determine whether this cellular context-specific inhibitory activity of TNF-alpha correlates with the known ability of TNF-alpha 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-beta and/or TNF-alpha for 30 min, a time point previously shown to be ideal for the detection of SMAD-DNA complexes after TGF-beta 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-beta (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-alpha efficiently reduced TGF-beta -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-alpha 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-alpha was added concomitantly with TGF-beta (see above) are not due to reduced SMAD3 levels in response to TNF-alpha . Parallel EMSA with an NF-kappa B-specific probe (Fig. 3C) highlighted the lack of correlation between TNF-alpha -dependent reduction in SMAD-DNA complexes seen in Fig. 3B and the induction of NF-kappa B DNA-binding activity.


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Fig. 4.   c-Jun overexpression antagonizes TGF-beta /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-beta (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-beta , and whole cell lysates were prepared at various time points for Western blot analysis of phospho-JNK (P-JNK), JNK, and beta -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-alpha (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 beta -actin contents. Note the high amounts of phospho-c-Jun in c-Jun-transfected cells, even in the absence of TNF-alpha (lane 3).

Because c-Jun is a key effector of TNF-alpha inhibitory activity in SMAD signaling (6), we overexpressed c-Jun instead of adding exogenous TNF-alpha 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-beta -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-beta was examined. As shown in Fig. 4B (upper panel), no JNK phosphorylation in response to TGF-beta (detected by Western blot analysis of endogenous phospho-JNK proteins) was observed 15 min to 24 h after TGF-beta 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-beta 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-alpha (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-beta /SMAD signaling.

jnk1 Expression in jnk-/- Fibroblasts Restores the Inhibitory Effect of Pro-inflammatory Cytokines on TGF-beta /SMAD Signaling-- To verify that the lack of TNF-alpha effect against TGF-beta /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-alpha in jnk-/- fibroblasts, attesting that the lack of inhibitory effect of TNF-alpha 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-alpha on TGF-beta /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-beta was added without or with TNF-alpha , 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.

c-Jun and JunB (but Not ATF2) Inhibit TGF-beta /SMAD Signaling in a JNK-dependent Manner-- Our next aim was to determine which JNK substrate(s) may be able to interfere with TGF-beta /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-beta in wild-type cells, but not in jnk-/- fibroblasts. Similarly, in human dermal fibroblasts, c-Jun and JunB inhibitory activity against TGF-beta /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-beta /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-beta 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-beta 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-beta /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-beta -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-beta /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-beta /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-beta 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-alpha -induced inhibition of TGF-beta /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-beta and TNF-alpha 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-alpha was added, and luciferase activity was measured 24 h later. Data from a representative experiment are shown.

We have determined that TNF-alpha efficiently blocks TGF-beta signaling in junAA fibroblasts (Fig. 3). Together with our data indicating the critical role for c-Jun phosphorylation by JNK in mediating the TNF-alpha effect in human and mouse fibroblasts, these observations led us to investigate (a) whether JNK plays a role downstream of TNF-alpha 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-alpha and TGF-beta on (CAGA)9-Lux was determined. As shown in Fig. 7B, dominant-negative MKK4 efficiently blocked the effect of TNF-alpha against TGF-beta , 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-alpha effect, indicating that JunB substitutes for c-JunAA in mediating the inhibitory activity of TNF-alpha 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-alpha 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-alpha , 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-alpha Prevents TGF-beta -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-alpha against TGF-beta -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-beta in the three cell types (lanes 2, 6, and 10, respectively). TNF-alpha antagonized the TGF-beta 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-alpha antagonizes TGF-beta -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-beta -induced type I and type III collagen gene expression by TNF-alpha . Subconfluent WT, jnk-/-, and nemo-/- fibroblast cultures were treated with TGF-beta and TNF-alpha 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-beta effect on both COL1A1 and COL3A1 mRNA steady-state levels by TNF-alpha in jnk-/- fibroblasts.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta /SMAD signaling, whereas NF-kappa 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-beta /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-beta . 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-beta 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-beta -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-beta 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-beta is entirely consistent with our previous observation that TGF-beta 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-beta /SMAD Signaling, whereas NF-kappa B Activity Is Not-- As described above, in some cell types, JNK positively regulates some of the SMAD- and TGF-beta -mediated transcriptional responses, yet JNK activators only partially stimulate transcriptional responses characteristic of TGF-beta without coincident SMAD pathway activation. It has also been reported that, in some cell types, triggering of the SAPK/JNK pathway by TGF-beta itself could participate in a negative feedback loop controlling SMAD-driven TGF-beta responses (12). Although they are somewhat opposite with regard to JNK activation by TGF-beta itself, these results are in full agreement with the concept of an interdependent relationship between the JNK and SMAD pathways in TGF-beta -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-kappa 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-kappa B activity allowed us to definitely rule out the involvement of the latter in the antagonistic activity of TNF-alpha against SMAD signaling.

Both TNF-alpha and IL-1beta , prototypic inflammatory cytokines, inhibited TGF-beta -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-alpha 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-beta (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-alpha might block SMAD signaling is through NF-kappa B activation. The latter may, in turn, induce SMAD7 expression, a molecule that interferes with SMAD phosphorylation by TGF-beta receptor type I and subsequent translocation into the cell nucleus (10). Activation of SMAD7 expression through the NF-kappa B cascade appears to be restricted to certain subsets of mouse embryos fibroblasts, as (a) in human embryonic kidney 293 cells, NF-kappa B activation inhibits SMAD7 gene expression (11); (b) we did not previously observe any activation of SMAD7 expression by TNF-alpha 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 Ikappa B kinase-alpha , which blocks NF-kappa B activation, did not interfere with TNF-alpha blockade of SMAD signaling (data not shown); and (d) mouse embryo fibroblasts devoid of NF-kappa B activity, e.g. nemo-/-, allowed full inhibitory activity of TNF-alpha against SMAD signaling (this study). The latter data unequivocally eliminate the NF-kappa B pathway from playing a role in the interference exerted by pro-inflammatory cytokines with TGF-beta /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-alpha . The latter phenomenon is likely representative of JNK1 activation by TNF-alpha , 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-beta /SMAD Signaling-- In human dermal and WT mouse fibroblasts, most of the antagonistic activity of TNF-alpha against TGF-beta /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-alpha stimulation (see Fig. 2, B and C), we unveiled a novel function of JunB downstream of TNF-alpha signaling. Specifically, we determined that JunB could substitute for c-Jun in mediating the inhibitory activity of TNF-alpha 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-beta /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-beta 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-beta . We have also demonstrated that such a JNK-dependent mechanism underlies the inhibitory activity of TNF-alpha against TGF-beta -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-beta , transforming growth factor-beta ; IL-1, interleukin-1; TNF-alpha , tumor necrosis factor-alpha ; 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.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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