Potentiation of Smad Transactivation by Jun Proteins during a Combined Treatment with Epidermal Growth Factor and Transforming Growth Factor-beta in Rat Hepatocytes

ROLE OF PHOSPHATIDYLINOSITOL 3-KINASE-INDUCED AP-1 ACTIVATION*

Philippe Péron, Mohamed Rahmani, Yvrick Zagar, Anne-Marie Durand-Schneider, Bernard Lardeux, and Dominique BernuauDagger

From the Laboratoire de Biologie Cellulaire, INSERM Unité 327, Faculté de Médecine Xavier Bichat, Université Paris 7 Denis Diderot, Paris 75018, France

Received for publication, July 6, 2000, and in revised form, November 26, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cross-talk between Smad and mitogen-activated protein kinase pathways has been described recently, and evidence for Smad cooperation with AP-1 is emerging. Here we report that epidermal growth factor (EGF) potentializes transforming growth factor beta  (TGF-beta )-induced Smad3 transactivation in rat hepatocytes, an effect abrogated by TAM-67, a dominant negative mutant of AP-1. Antisense transfection experiments indicated that c-Jun and JunB were involved in the synergistic effect, and endogenous c-Jun physically associated with Smad3 during a combined EGF/TGF-beta treatment. We next investigated which signaling pathway transduced by EGF was responsible for the Jun-induced synergism. Whereas inhibition of JNK had no effect, inhibition of the phosphatidylinositol-3' kinase (PI3-kinase) pathway by LY294002 or by expression of a dominant negative mutant of PI3-kinase reduced EGF/TGF-beta -induced Smad3 transcriptional activity. Transfection of an activated Ras with a mutation enabling the activation of the PI3-kinase pathway alone mimicked the EGF/TGF-beta potentiation of Smad3 transactivation, and TAM-67 abolished this effect, suggesting that the PI3-kinase pathway stimulates Smad3 via AP-1 stimulation. The EGF/TGF-beta -induced activation of Smad3 correlated with PI3-kinase and p38-dependent but not JNK-dependent phosphorylation of c-Jun. Since potentiation of a Smad-binding element-driven gene was also induced by EGF/TGF-beta treatment, this novel mechanism of Jun/Smad cooperation might be crucial for diversifying TGF-beta responses.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor beta  (TGF-beta )1 is a member of a large family of cytokines that includes bone morphogenetic proteins, activins, and several more distantly related factors (1). TGF-beta is central in the regulation of many biological processes, including cell differentiation, growth, adhesion, and apoptosis. TGF-beta signals through a system of transmembrane serine/threonine kinase receptors composed of type I and type II receptors (TGF-beta RI and TGF-beta RII) (see Refs. 2-4 and reviewed in Ref. 5). Ligand binding to TGF-beta RII recruits and activates the TGF-beta RI receptor, which phosphorylates Smad2 and Smad3 on their SSXS motif. Smad proteins encompass a conserved amino-terminal domain that binds DNA and a conserved carboxyl-terminal domain that binds receptors and partner Smads. These domains are separated by a less conserved linker region. Phosphorylated Smad2 or Smad3 forms stable complexes with Smad4, which translocate into the nucleus, where they bind the consensus GTCTAGAC sequence found in the promoter of many TGF-beta -responsive genes (6). Disruption of the Smad pathway or Smad mutations have underscored the functional importance of this signaling pathway in the transcriptional response of target cells to TGF-beta (reviewed in Ref. 7).

Emerging evidence indicates that TGF-beta signaling may also cross-talk with the mitogen-activated protein kinase (MAPK) family of serine/threonine protein kinases. Antagonistic or synergistic interplay between these kinases and Smad signaling has been described. Extracellular signal-regulated kinases (ERK), members of the MAPK, cause a rapid increase in the phosphorylation of Smad2 and Smad3 in their linker region, preventing their translocation into the nucleus and therefore providing a mechanism of repression of TGF-beta signaling (8, 9). At the opposite, a synergistic mechanism between Smads and MAPK has been proposed, in a kinase downstream of the MAP kinase kinase MEK1-induced Smad2 phosphorylation on the SSXS motif and its nuclear translocation (10). Recent evidence also indicates that Smad cooperates with AP-1 (11-13), a heterodimer of Fos and Jun family members (14). Stimulation of AP-1-dependent transcription can be achieved by phosphorylation of the c-Jun transactivation domain by c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK), another member of the MAPK family. Smad and AP-1 response elements are juxtaposed in the promoters of several TGF-beta -inducible genes, such as plasminogen activator inhibitor-1 or c-jun, and both sites appear to be critical in the TGF-beta response (15-17).

In the present study we addressed the question of whether AP-1/Smad cooperation occurs in normal rat hepatocytes following a combined stimulation with EGF and TGF-beta . We show that under these experimental conditions AP-1 induces a strong activation of Smad3 transactivation independent of AP-1 binding to its cognate cis-element. This synergism was mediated by c-Jun and JunB, and a protein-protein interaction between Smad3 and endogenous c-Jun was found during EGF/TGF-beta stimulation. Furthermore, we demonstrate that activation of AP-1 via the phosphatidylinositol 3-kinase (PI3-kinase), but not the JNK pathway, is implicated in this functional synergism. Finally, we show that Jun/Smad3 cooperation induced by EGF is effective on a SBE-driven reporter gene. These data suggest that Jun/Smad3 synergism independent of binding to TRE elements might represent another important mechanism of regulation of TGF-beta -inducible genes in hepatocytes.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- Smad2-Gal4 constructs, prepared in J. Massagué's laboratory, Smad3-Gal4 plasmid, prepared by R. Derynck, and 6-Myc-Smad3, from CH. Heldin's laboratory, were all generously provided by P. Ten Dijke. The plasmids 4xSBE-Luc and 6xMBE-Luc were provided by B. Vogelstein. Ti5xGal4-Luc was a gift from X.-F. Wang. TAM-67 (18, 19) was provided by M. J. Birrer. DNMKK4 and Ikappa Balpha A32-36 were the generous gifts of A. Atfi and G. Cherqui. The expression vectors for RasV12C40 and p85alpha Delta iSH2-N (p85DN, thereafter), kindly provided by J. Downward, have been described (20).

Antisense Jun Expression Vectors-- The 5'-untranslated regions (underlined) of rat junD (5'-CTAGACGGTCTGTACGGGCAGCGGACTGGGGGGCA, nt 73 to 102, acc.n°D26307), junB (5'-CTAGACCAGCTCCCGAGGACGCGCGACCG-3', nt 1325 to 1348, acc.n°X54686) and c-jun (5'-CTAGAGAGCGCTCCGTGAGTGACCGCGACTTTTCAAAGCG-3', nt 76 to 111, acc.n°X17163) were used as antisense probes to take advantage of the diversity in the nucleotide sequence in this region and to provide specificity. These sequences were inserted between EcoRI and XbaI cloning sites within the polylinker of pCI-neo (Promega). The constructs were checked by DNA sequencing.

Hepatocyte Culture-- Hepatocytes were obtained from adult male Harlan Sprague-Dawley rats (Charles River) weighing 180-200 g. Animals were maintained on commercial chow (UAR, Villemoisson-sur-Orge, France) and water ad libitum. Hepatocytes were isolated by collagenase perfusion (21), as modified by Balavoine et al. (22). The hepatocytes were purified by Percoll gradient centrifugation (23), and viability was found to be >85% by trypan blue exclusion. Hepatocytes were suspended in William's E medium (Life Technologies, Inc.) supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum, and they were plated on collagen-coated Petri dishes.

Electrophoretic Mobility Shift Assays (EMSA)-- Nuclear extracts were prepared according to Andrews and Faller (24) with minor modifications. Cells were suspended in lysis buffer (10 mM Hepes/KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 0.5 mM spermidine) and centrifuged at 500 × g for 30 s at 4 °C. The nuclear pellet was suspended in 20 mM Hepes/KOH, pH 7.9, containing 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 4 mM dithiothreitol, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin and centrifuged at 18,000 × g for 2 min at 4 °C. The supernatants were stored at -80 °C. Protein concentration was determined with the BCA protein assay reagent (Pierce).

Single-stranded oligonucleotides corresponding to the upper strand of the Smad-binding element (SBE) (5'-GGAGTATGTCTAGACTGACAATGTAC-3') (6) or the TPA-responsive element (TRE) (5'-TAAAGCATGAGTCAGACACCTC-3') were used (the cis elements are shown by the underlines). They were end-labeled with T4 polynucleotide kinase in the presence of [lambda -32P]ATP (5,000 Ci/mmol, Amersham Pharmacia Biotech). The labeled oligonucleotides were annealed with their respective unlabeled lower strands. Nuclear extracts (5-20 µg of protein) were incubated in binding buffer (20 mM Hepes, pH 7.9, 5 mM MgCl2, 4 mM dithiothreitol, 20% glycerol, 0.1 mM PMSF, 5 mM benzamidine, 2 mM levamisole, 0.1 µg/ml aprotinin, 0.1 µg/ml bestatin) containing 2 µg of poly(dI-dC) and 32P-labeled double-stranded probe (3 × 104 cpm) for 20 min at 4 °C. The reaction mixture was then loaded onto a 6% polyacrylamide gel in 0.09 M Tris borate, 2 mM EDTA, pH 8.0 buffer, and electrophoresed at 11 V/cm for 2 h at 20 °C. The gels were dried and exposed to x-ray film for autoradiography.

For supershift analyses, 2 µl of antibody specific to the Jun family (anti-c-Jun, anti-JunB, and anti-JunD), the Fos family (c-Fos and Fra-1), or Smad3 protein (Santa Cruz Biotechnology) were incubated with the nuclear extracts for 2 h at 4 °C prior to incubation with the 32P-radiolabeled probe.

Cell Transfection and Luciferase (Luc) Assays-- Immediately after isolation, hepatocytes at a density of 20 × 106 cells/0.8 ml of PBS containing 5% fetal bovine serum (25) were electroporated (Gene Pulser, Bio-Rad) at 160 V and 960 microfarads in the presence of 50 µg of reporter plasmid, 30 µg of expression vector, and 30 µg of RSV-CAT plasmid to correct for transfection efficiencies. The total amount of DNA was adjusted to 400 µg with salmon sperm DNA. After electroporation, the hepatocytes were plated in the presence of 10% fetal bovine serum for 1 h and then deprived of serum for 18 h. They were finally incubated for 24 h in serum-free medium in the presence or absence of EGF (20 ng/ml), TGF-beta (3 ng/ml), or EGF plus TGF-beta . For Luc assays, cells were washed with chilled PBS and lysed for 15 min on ice with lysis buffer (25 mM Tris/HPO4, pH 7.8, 8 mM MgCl2, 1% Triton X-100, 1% bovine serum albumin, 15% glycerol, 1 mM EDTA, and 1 mM dithiothreitol), and then the lysates were centrifuged 10 min at 13,000 rpm and stored at -20 °C. Luc activities were determined with a luminometer (EGG Instruments) and normalized to the amount of proteins in the extracts. For the measurement of chloramphenicol acetyltransferase enzyme activities, cell extracts were prepared as described in Nadori et al. (26). Chloramphenicol acetyltransferase assays were performed according to Seed and Sheen (27) and normalized relative to the amount of protein in the extracts.

Coimmunoprecipitation-- Approximately 1 mg of proteins from cells lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM PMSF, 4 mM dithiothreitol, 0.5 mM spermidine, 1 µg/ml leupeptin, 1 µg/ml aprotinin) were incubated for 2 h at 4 °C with protein A-Sepharose beads coated with anti-c-Jun or anti-JunB antibodies (Santa Cruz Biotechnology). The beads were then collected and washed four times with buffer A (PBS containing 0.05% deoxycholate sodium, 0.01% SDS, and 0.5% Triton X-100), 3 times with buffer B (0.5% Triton X-100, 1 mM EDTA, 500 mM NaCl, 125 mM Tris-HCl), and once with 10 mM Tris-HCl, pH 8.1. Bound proteins were eluted by boiling in 1× Laemmli buffer and subjected to SDS-PAGE analysis (10% acrylamide) and then electroblotted onto nitrocellulose membranes. The blots were incubated for 1 h with a 1/1000 dilution of mouse anti-Myc antibody (Santa Cruz Biotechnology) in PBS containing 0.1% Tween 20, followed by incubation with 1/5000 dilution of horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia Biotech). Immune complexes were revealed by the enhanced chemiluminescence kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions and exposed to x-ray film for visualization.

Western Blot Analysis-- Nuclear extracts prepared as above (50 µg) were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell). Membranes were probed with either anti-phospho-c-Jun antibody (New England Biolabs), which recognizes phosphorylated serine 63 of c-Jun, or with anti-c-Jun antibody (Oncogene Research Products), which recognizes both phosphorylated and nonphosphorylated forms of c-Jun. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Statistical Tests-- We used a Student's t test and the one-way analysis of variance followed by the least significant difference test.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

EGF Synergizes with TGF-beta for Smad 3 Transactivation-- To determine whether EGF and TGF-beta signalizations cross-talk in hepatocytes, we investigated the transcriptional activity of Smad proteins by using a single Gal-hybrid system, which makes use of an expression vector for Smad3-Gal4 fusion protein and a reporter plasmid containing five concatemerized Gal4-binding sites (Ti5xGal4). In hepatocytes transiently transfected with the Tix5Gal4 reporter alone, a very low level of Luc activity was detected (Fig. 1). In cells cotransfected with Ti5xGal4 and Smad3-Gal4 expression vector treatment with TGF-beta induced a 10-fold increase in Luc activity. EGF treatment alone slightly activated basal Smad3 activity, whereas simultaneous treatment with EGF and TGF-beta led to a potent synergistic increase in Smad3 transcriptional activity, when compared with that upon TGF-beta treatment alone (Fig. 1). The same results were observed in hepatocytes transfected with a Smad2-Gal4 expression plasmid (data not shown). These results indicated that whereas the EGF signaling pathway alone weakly activates Smad3 transcriptional activity in hepatocytes, it potently synergizes with Smads once they have been activated by TGF-beta .


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Fig. 1.   EGF synergizes with TGF-beta for Smad3 transactivation. Hepatocytes were transiently transfected with Ti5xGal-Luc and Smad3-Gal4 expression vector, and they were left untreated or treated with EGF (20 ng/ml), TGF-beta (3 ng/ml), or a combination of both for 24 h. Luc activity was determined and normalized to transfection efficiency. Values of samples from cells transfected with Tix5Gal and Smad3-Gal4 and left untreated were arbitrarily set to 1. The results are the mean ± S.E. of three independent transfections.

AP-1 Is Involved in the Synergism between EGF and TGF-beta on Smad3 Transactivation-- Since one of the major targets of EGF signaling is activation of the AP-1 complex, we first determined whether inhibition of AP-1 would interfere with Smad3 transcriptional activity. Hepatocytes were cotransfected with Smad3-Gal4 and either TAM-67, a dominant negative c-Jun expression vector which blocks the activity of all endogenous Jun and Fos proteins by forming nonfunctional heterodimers (18, 19), or the empty pCMV plasmid, as a control. Fig. 2A shows that cotransfection with TAM-67 did not significantly modify the transcriptional activity of Smad3 in unstimulated hepatocytes or in hepatocytes treated with EGF or TGF-beta alone, whereas it induced an important decrease of the Smad3 transcriptional activity triggered by TGF-beta /EGF costimulation, an effect not observed in hepatocytes transfected with the control plasmid (Fig. 2A). We have recently observed that EGF activates NF-kappa B binding and transactivation in normal rat hepatocytes.2 We thus investigated whether inhibition of NF-kappa B activity could also influence the synergism between EGF and TGF-beta on Smad3 transactivation. Hepatocytes were cotransfected with Smad3-Gal4, Ti5xGal, and either with the Ikappa Balpha A32/36 expression vector encoding for a mutated, nonphosphorylatable form of Ikappa B protein which blocks NF-kappa B nuclear translocation and activity or with the Rc/CMV control plasmid. In these experiments no modification of the transcriptional activity of Smad3 was observed after costimulation with EGF and TGF-beta (Fig. 2B).


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Fig. 2.   Activation of AP-1 but not NF-kappa B is involved in EGF/TGF-beta -induced Smad3 transactivation. Hepatocytes were cotransfected with Ti5xGal-Luc and Smad3-Gal4 expression vectors and TAM-67 or the control empty plasmid (pCMV) in A or Ikappa Balpha A32/A36 or the control empty plasmid (pRc/CMV) in B. They were left untreated or treated with EGF (20 ng/ml), TGF-beta (3 ng/ml), or a combination of both for 24 h. Luciferase activity was determined and normalized for transfection efficiency. Value of samples from cells transfected with Tix5Gal and Smad3-Gal4 and left untreated was arbitrarily set to 1. The results are the mean ± S.E. of three independent transfections in A and represent the average of two independent experiments in B. *, p < 0.01 by Student's t test.

c-Jun and JunB Cooperate with Smad3-- AP-1 is a dimeric transcriptional complex composed of Fos (c-Fos, FosB, Fra1, and Fra2) and Jun (c-Jun, JunB and JunD) proteins. Interactions between Smad3 and all Jun family members has been reported (11, 12). To determine which Jun protein cooperates with Smad3 during EGF and TGF-beta costimulation, we transiently cotransfected the hepatocytes with Ti5xGal4, Smad3-Gal4, and with either c-jun, junB, or junD antisense expression vectors. The efficiency of the antisense strategy was checked in separate experiments by cotransfecting hepatocytes with these antisense constructs and with a TRE-driven gene, the 5xTRE-Luc plasmid. After 32 h of transfection, c-jun and junB antisense expression vectors inhibited Luc activity by 70 and 40%, respectively, compared with cells transfected with the control pCI-neo vector (Fig. 3A). Cotransfection of the junD antisense plasmid did not significantly inhibit Luc activity. Since the half-life of JunD is much longer than for the other Jun proteins, the absence of effect of this construct could be related to the relatively short delay between transfection of the antisense RNA and the Luc assay. Treatment with EGF or TGF-beta of hepatocytes transfected with the c-jun, the junB, or the junD antisense plasmid did not significantly decrease the transcriptional activity of Smad3 (Fig. 3B). By contrast, a combined treatment with EGF and TGF-beta of hepatocytes expressing c-jun or junB antisense mRNA decreased Smad3 transactivation by ~40%, whereas antisense junD expression had no inhibitory effect. These data suggest the involvement of c-Jun and JunB in Smad3 cooperation, but according to the commentary above they do not allow us to completely rule out a possible action of JunD.


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Fig. 3.   c-Jun cooperates with Smad3. A, hepatocytes were transfected with a TRE-Luc reporter gene and with antisense expression plasmid as indicated. Luc activity was determined after 24 h and normalized for transfection efficiency. B, hepatocytes were cotransfected with Ti5xGal-LUC and Smad3-Gal4 expression vectors, and with c-jun, junB, or junD antisense expression vectors. They were left untreated or treated with EGF (20 ng/ml), TGF-beta (3 ng/ml), or a combination of both for 24 h. Luc activity was determined and normalized for transfection efficiency. Value of samples from cells transfected with Tix5Gal and Smad3-Gal4 and left untreated was arbitrarily set to 1. The results are the mean ± S.E. of three independent transfections. *, p < 0.01, by one-way analysis of variance.

Endogenous c-Jun Associates with Smad3 upon Costimulation with TGF-beta and EGF-- Hepatocytes were transfected with an expression vector for a 6xMyc-tagged Smad3 protein and left unstimulated or treated with EGF, TGF-beta , or a combination of both for 24 h. Proteins immunoprecipitated with anti-c-Jun antibody were then immunoblotted with an anti-Myc antibody. As shown in Fig. 4, association of c-Jun with Smad3 was detectable only after costimulation with EGF and TGF-beta .


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Fig. 4.   Endogenous c-Jun associates with Smad3 during costimulation with TGF-beta and EGF. Hepatocytes were transiently transfected with a 6xMyc-tagged Smad3 expression vector and left unstimulated or stimulated for 24 h with EGF (20 ng/ml), TGF-beta (3 ng/ml), or both as indicated. Lysates were incubated with anti-c-Jun-coated protein A-Sepharose beads before performing SDS-PAGE and a Western blot with anti-Myc antibody. Lysate from nontransfected cells (NT) was also tested.

Costimulation with TGF-beta and EGF Does Not Increase TRE or SBE Binding-- To determine whether the DNA binding activity of Jun or Smad proteins was modified during costimulation with TGF-beta and EGF, we performed EMSA with a TRE probe that binds AP-1 proteins or an SBE probe containing the consensus GTCTAGAC sequence that binds Smad proteins (6). Whereas TGF-beta treatment alone did not modify the level of AP-1 binding to the TRE cis-element, stimulation with EGF induced a 2.5-fold increase, as expected (26) (Fig. 5A). EMSA performed with the SBE probe and nuclear extracts from unstimulated cells generated two retarded bands, whose intensity was not modified by treatment with EGF or TGF-beta . Treatment with TGF-beta induced the formation of a third slower band, visible as soon as 30 min after TGF-beta stimulation (data not shown) which was still visible at 2 h (Fig. 5B). The intensity of this TGF-beta -induced retarded band was not modified after costimulation with EGF and TGF-beta (Fig. 5B). Supershift experiments with antibodies specific to Smad3 induced a complete displacement of the slowly migrating retarded band induced by TGF-beta but did not modify the intensities of the other bands. No supershifts were observed after addition of c-Jun, JunB, JunD, c-Fos, or Fra-1 antibodies to EMSA performed with the SBE probe (data not shown). Competition experiments indicated that the three retarded bands were specific, since they were completely competed by a 100-fold molar excess of unlabeled SBE probe, whereas they remained unchanged in the presence of an excess of unlabeled NF-kappa B probe (Fig. 5C). In addition, competition with a 100-fold molar excess of cold TRE probe decreased the binding of the two faster migrating bands, indicating that part of the proteins that bind to the SBE also interact with an AP-1 cis-element (Fig. 5C).


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Fig. 5.   Costimulation with TGF-beta and EGF does not increase TRE or SBE binding. A and B, equal amounts (10 µg) of nuclear protein extracts from hepatocytes nonstimulated or stimulated with EGF (20 ng/ml), TGF-beta (3 ng/ml), or both for 2 h were incubated with a 32P-labeled TRE probe in A or SBE probe in B, and electrophoresed in a 6% polyacrylamide gel. Competition for TRE binding was performed by adding a 100-fold molar excess of unlabeled TRE probe to the incubation mixture (competitor). B, anti-Smad3 antibody was added in the SBE gel retardation assay. Arrowheads point to the retarded bands, and the arrow indicates the supershifted band induced by anti-Smad3. C, competition experiments for SBE binding. Increasing concentrations of unlabeled SBE probe or a 100-fold molar excess of TRE probe or NF-kappa B probe were added to the reaction mixture before electrophoresis.

Activations of PI3-Kinase and p38 Are Implicated in Stimulation of Smad3 Transactivation-- Tyrosine phosphorylation of specific residues of the EGF receptor activates several signaling pathways (reviewed in Ref. 28), all of which are implicated in AP-1 activation. An important pathway triggered by EGF is the PI3-kinase cascade, which is directly activated by phosphorylation of the EGF receptor (29, 30). Accordingly, hepatocytes were cotransfected with Ti5xGal and Gal4-Smad3 plasmids and treated with LY294002, a specific inhibitor of the PI3-kinase pathway. LY294002 did not change Smad3 transcriptional activity induced by EGF or TGF-beta (data not shown), but it inhibited by 61% the Smad3 activity triggered by a combined treatment with EGF and TGF-beta (Fig. 6A). To check the specificity of the LY294002 effect, we used another approach to inhibit PI3-kinase activation. Hepatocytes were transfected with p85DN, a plasmid that allows the expression of a mutated catalytic PI3-kinase subunit exerting a dominant negative effect on PI3-kinase activity (20). Transient expression of this plasmid induced a 40% decrease of EGF/TGF-beta -induced Smad3 transactivation (Fig. 6B). This result therefore is in agreement with the involvement of the PI3-kinase pathway in EGF/TGF-beta synergism. Another well identified pathway triggered by the EGF receptor is the Ras-MAPK pathway leading to ERK, JNK, or p38 activation. We therefore asked whether inactivation of one of these pathways could also block EGF/TGF-beta -induced Smad3 transactivation. The possible involvement of the p38 pathway was tested by treatment of hepatocytes cotransfected with Smad3-Gal4 and Tix5Gal with SB202190, a selective inhibitor of p38 MAPK. In these transfected cells, SB202190 induced a 50% decrease of Smad3 transactivation in the presence of EGF and TGF-beta (Fig. 6A). Simultaneous inhibition of the p38 and PI3-kinase pathways by treating hepatocytes with both LY294002 and SB202190 did not synergize for the inhibition of EGF/TGF-beta -induced Smad3 transactivation (Fig. 6A) suggesting that the two pathways are interdependent. Involvement of the ERK pathway was tested by the use of PD98059, a potent and specific inhibitor of MEK1 activation, a kinase directly upstream of ERK. PD98059 treatment did not decrease the Smad3-dependent transactivation induced by EGF, TGF-beta (data not shown), or a combination of both (Fig. 3A). Finally, to test the role of the JNK pathway, hepatocytes were cotransfected with GAL4-Smad3, the Tix5xGAL4 reporter plasmid, and DNMKK4, a dominant negative mutant of MKK4, a kinase predominantly involved in the activation of JNK (31). Coexpression of DNMKK4 did not modify the level of Smad transactivation induced by EGF, TGF-beta (data not shown), or a combination of both (Fig. 3B), compared with hepatocytes cotransfected with the empty pcDNA3 vector.


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Fig. 6.   Effects of the inhibition of PI3-kinase and MAPK pathways on EGF/TGF-beta synergism. A, hepatocytes transfected with Ti5xGal and Smad3-Gal4 plasmids were treated with a combination of EGF (20 ng/ml) and TGF-beta (3 ng/ml) for 24 h in the presence or in the absence of the PI3-kinase inhibitor LY294002 (35 µM), the p38 inhibitor SB202190 (10 µM), a mixture of both, or the ERK inhibitor PD98059 (50 µM). Value of samples from cells transfected with Tix5Gal and Smad3-Gal4 in the absence of the inhibitors was arbitrarily set to 100. The results are the mean ± S.E. of three independent transfections except for cells treated with PD98059 for which the average of two independent experiments is shown. B, hepatocytes were cotransfected with Ti5xGal and Smad3-Gal4 plasmids and with p85DN or the empty plasmid pSG5, or DNMKK4 or the control plasmid pcDNA3. They were treated with a combination of EGF (20 ng/ml) and TGF-beta (3 ng/ml) for 24 h. Luciferase activity was determined and normalized for transfection efficiency. Value of samples from cells transfected with Tix5Gal and Smad3-Gal4 alone was arbitrarily set to 100. The results represent the average of two independent transfections.

To assess further the role of PI3-kinase activation on Smad3 transactivation, we evaluated the effect of the transfection of an activated Ras expression vector carrying a point mutation enabling the selective activation of the PI3-kinase pathway (RasV12C40). As shown on Fig. 7A, basal Smad3 transactivation was elevated (about 3-fold) in RasV12C40-transfected hepatocytes compared with pRSV-transfected unstimulated cells, mimicking the EGF-induced effect on Smad3 transcriptional activity. TGF-beta treatment further increased by 3-fold Smad3 transactivation, to reach a level close to that detected in hepatocytes costimulated with EGF and TGF-beta but not expressing RasV12C40 (Fig. 7A). To determine whether PI3-kinase activates Smad3 transcriptional activity via a direct mechanism (i.e. Smad phosphorylation) or rather via the induction of AP-1 activity, hepatocytes were transfected with RasV12C40 in the presence of the AP-1 dominant negative TAM-67 or the control empty plasmid pCMV. Inactivation of AP-1 blunted the basal PI3-kinase-mediated activation of Smad3 transactivation induced by RasV12C40 and inhibited by 70% Smad3 transactivation in the presence of TGF-beta (Fig. 7A). These data indicated that AP-1 activity is necessary for the stimulating effect of PI3-kinase on Smad3 transactivation. Treatment of RasV12C40-transfected hepatocytes with the PI3-kinase inhibitor LY294002 led to a 40% decrease of Smad3 transactivation (Fig. 7B), consistent with the assumption that the effect of this activated Ras on Smad3 transactivation depends, at least in part, on its PI3-kinase activating function. Since p38 is a well known downstream effector of PI3-kinase, we also investigated whether inhibition of p38 by SB202190 would modify the effect of PI3-kinase activation on Smad3 transactivation. Treatment of RasV12C40-transfected hepatocytes with SB202190 inhibited Smad3 transactivation by 30% (Fig. 7B). Thus, at least part of the effect of PI3kinase on Jun-mediated Smad3 transactivation depends on the p38 kinase pathway.


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Fig. 7.   The PI3-kinase and p38 pathways are involved in AP-1-mediated EGF/TGF-beta synergism on Smad3 transactivation. A, hepatocytes were cotransfected with Ti5xGal and Smad3-Gal4 plasmids with or without RasV12C40 or its empty plasmid pRSV, or TAM-67, or its empty plasmid pCMV, as indicated. They were left untreated or treated with EGF (20 ng/ml), TGF-beta (3 ng/ml), or both, as indicated, for 24 h. Value of samples from cells transfected with Tix5Gal and Gal4-Smad3 and left untreated was arbitrarily set to 1. The results are the mean ± S.E. of three independent experiments. B, hepatocytes were cotransfected with Ti5xGal, Smad3-Gal4, and RasV12C40 and treated with EGF + TGF-beta in the presence or in the absence of LY294002 (35 µM) or SB202190 (10 µM). Value of samples from cells treated with EGF + TGF-beta in the absence of inhibitors was arbitrarily set to 100. The results are the mean ± S.E. of three independent transfections.

EGF Induces Phosphorylation of c-Jun by PI3-Kinase and p38-dependent Pathways-- Our results indicated that the synergistic effect of TGF-beta  + EGF on Smad3 transactivation depended on the activation of Jun proteins. Phosphorylation of the transactivation domain of c-Jun on Ser-63 and Ser-73 by JNK is the best described mechanism of c-Jun activation. Accordingly, we first looked at the phosphorylation of c-Jun on Ser-63, by Western blotting using an antibody directed against Ser-63-phosphorylated c-Jun. Stimulation with TGF-beta alone did not increase c-Jun phosphorylation, whereas, as expected, a strong phosphorylation of c-Jun on Ser-63 was induced by EGF stimulation. The intensity of the band specific for c-Jun Ser-63 phosphorylation was not modified by costimulation with EGF and TGF-beta in comparison with cells treated with EGF alone, even in the presence of LY294002 or SB202190 (Fig. 8A). This observation was consistent with our demonstration that inhibition of JNK does not suppress EGF/TGF-beta synergism on Smad3 transactivation. To determine whether phosphorylation of c-Jun by a pathway different from JNK was triggered during EGF + TGF-beta treatment, we next performed Western blotting using a c-Jun antibody that recognizes both the nonphosphorylated and the phosphorylated forms of c-Jun. As shown on Fig. 8B, stimulation with EGF, but not TGF-beta , induced a strong accumulation of c-Jun protein, with appearance of a slower band, indicating the presence of phosphorylated forms of the protein. This retarded band was also detectable after TGF-beta  + EGF treatment. In extracts from hepatocytes treated with TGF-beta  + EGF in the presence of LY294002, the retarded band disappeared almost completely, whereas it was slightly decreased upon treatment with SB202190. These data indicated that besides the JNK-dependent phosphorylation of Jun on serine 63, EGF/TGF-beta induces the phosphorylation c-Jun on other site(s) through both PI3-kinase and p38-dependent pathways.


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Fig. 8.   Inhibition of c-Jun phosphorylation by PI3-kinase and p38-kinase inhibitors. Equal amounts (60 µg) of nuclear protein extracts from hepatocytes nonstimulated or stimulated with EGF (20 ng/ml), TGF-beta (3 ng/ml), or both in the presence or in the absence of the PI3-kinase inhibitor LY294002 (35 µM) or the p38-kinase inhibitor SB202190 (10 µM) for 1 h were electrophoresed in a 10% acrylamide gel and transferred on nitrocellulose membranes. The membranes were probed as follows: A, with anti c-Jun-phospho-Ser-63 antibody; B, with anti-c-Jun antibody.

EGF/TGF-beta Potentiation of Smad3 Transactivation Is Active on an SBE-containing Promoter-- We next investigated whether the EGF/TGF-beta potentiation of Smad3 transactivation found in our Gal4 system was also operative in the context of an SBE-driven gene. Hepatocytes were transfected with a construct that contains a Luc gene under the control of four SBE repeats (4xSBE-Luc) or with a control plasmid containing six mutated SBE repeats (6xMBE-Luc). As shown in Fig. 9A, no Luc activity was detected after transfection of the MBE plasmid. EGF treatment alone did not stimulate the SBE promoter activity. Stimulation with TGF-beta induced Luc activity by 10-fold, as expected, and a combined treatment with EGF and TGF-beta further increased (about 2-fold) Luc activity (Fig. 9A). Thus, although of weaker magnitude, a synergistic effect on an SBE-dependent transcription was also induced by EGF/TGF-beta stimulation. Cotransfection with TAM-67 completely prevented the synergistic effect of EGF/TGF-beta on Luc expression (data not shown). Finally, we also tested the effect of the activation of the PI3-kinase pathway on SBE-mediated transactivation by cotransfection with the RasV12C40 plasmid. As shown in Fig. 9B, a high level of transactivation was induced by TGF-beta treatment alone, mimicking the EGF synergistic effect, and inhibition of AP-1 by TAM-67 blunted this response, indicating that it depended on AP-1 activation.


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Fig. 9.   AP-1-dependent EGF/TGF-beta synergy is operative on an SBE-driven gene. A, hepatocytes were transfected with 6xMBE-Luc or 4xSBE-Luc, in the absence or in the presence of TAM-67. They were stimulated for 24 h with EGF (20 ng/ml), TGF-beta (3 ng/ml), or both, as indicated. Value of samples from cells transfected with 4xSBE-Luc and left untreated was arbitrarily set to 1. The results are the mean ± S.E. of three independent transfections. B, hepatocytes were transfected with 4xSBE-Luc alone or with RasV12C40, in the absence or in the presence of TAM-67 or the empty plasmid pCMV. They were treated with TGF-beta (3 ng/ml) with or without EGF (20 ng/ml), as indicated. The bars represent the mean of two independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is increasing evidence that several signaling pathways interfere with Smads to regulate TGF-beta -responsive genes, resulting in antagonistic as well as synergistic effects. In the present study, we show that EGF alone induces a mild activation of Smad3 transcriptional activity only detectable under conditions of Smad3 overexpression, in agreement with previous reports (10). More importantly, we show that a combined treatment with EGF and TGF-beta strongly activates (about 30-fold) Smad3 transactivation. This synergistic effect is dependent on the presence of AP-1 proteins, since it is prevented by transfection of TAM-67, a dominant negative c-Jun truncated on its transactivating domain acting as an inhibitor of AP-1 function (18, 19). Recently, cooperation of Smad proteins with the AP-1 proteins Fos and Jun has been documented in mink lung epithelial cells (11, 12). In vitro binding of Smad3 and Smad4 to all three Jun family members as well as in vivo association between Smads and a TGF-beta -phosphorylated form of endogenous c-Jun induced by JNK have been demonstrated in HaCaT cells (12). Cooperation of AP-1 proteins with Smad3 and Smad4 occurred via Jun proteins bound to their cognate cis-element, the TRE, or to composite sites containing juxtaposed AP-1 and SBE sites (11, 12, 17), and the interaction was shown to involve 13 carboxyl-terminal amino acids conserved in the three Jun proteins (12). The mechanism described herein differs from these previous studies by two major features as follows: 1) by using a Gal reporter system, we could demonstrate that the interaction of Smad3 with c-Jun induced a synergistic effect on Smad3 transactivation independently of binding to the TRE; 2) overexpression of TAM-67, a dominant negative c-Jun which is truncated in its transactivating NH2-terminal domain but still possesses the COOH-terminal domain implicated in the physical interaction with Smad proteins (11), abrogated the EGF/TGF-beta -induced stimulation of Smad3 transactivation, implying that the functional cooperation with Smad3 requires the amino-terminal transactivation domain of Jun proteins. In addition, we also show that the EGF-induced cooperative effect of Jun proteins on Smad3 transactivation was not paralleled by any modification of the binding of Smad proteins to their cognate cis-element, the SBE site, indicating that the increased transcriptional activity of Smad3 proteins is probably due to their association with Jun proteins rather than to the recruitment of additional Smads to the SBE. Despite the fact that a physical association between Jun proteins and Smads has been reported to occur in vitro (11, 12) and the demonstration that c-Jun coimmunoprecipitates with Smad3 during EGF/TGF-beta stimulation (our present result), we failed in detecting c-Jun or other AP-1 proteins bound to the SBE by supershift experiments. This could be due to the lack of sensitivity of the method that is known to produce false negative results (32).

Binding of EGF to its receptor activates the PI3-kinase pathway (28-30), a cascade involved in AP-1 activation (29, 33). We show that this pathway is involved in the potentiation of Smad3 transactivation by Jun proteins since inhibition of the PI3-kinase pathway by the highly selective inhibitor LY294002 or by a PI3-kinase dominant negative mutant efficiently blocked the EGF-induced Smad3 transactivation, whereas activation of the PI3-kinase pathway by RasV12C40 reproduced the synergistic effect of EGF stimulation on Smad3 transactivation. The PI3-kinase-activating effect was blunted in cells transfected with TAM-67 which are deficient in AP-1 transactivating function, strongly suggesting that the effect of PI3-kinase is through AP-1 activation rather than the result of direct PI3-kinase-dependent phosphorylation of Smad3. The involvement of PI3-kinase in Smad transactivation has never been demonstrated before. That PI3-kinase activation may be involved in TGF-beta signaling had been previously suggested from the observation that wortmannin, another PI3-kinase inhibitor, inhibits TGF-beta -stimulated chemotaxis of human neutrophil leukocytes (34). It has also been demonstrated that TGF-beta markedly enhanced EGF-induced PI3-kinase activity in human airway smooth muscle cells (35). More recently, involvement of PI3-kinase in the inhibitory effect of insulin, EGF, or interleukin-6 on TGF-beta -induced apoptosis has been reported (36-38), but the relationship with AP-1 function or Smad signaling was not investigated. We show here that one mechanism by which PI3-kinase may cross-talk with Smad signaling is through activation of Jun proteins, which themselves cooperate with Smad3 for transactivation.

Another important signaling pathway that originates from the EGF receptor is the MAPK pathway, including the ERK1 and ERK2 pathways, the JNK/SAPK, and the p38 MAPK pathways (Ref. 39 and for review see Ref. 40), all implicated in EGF-induced AP-1 activation (14). In our study, neither inhibition of the JNK pathway by transfection of a dominant negative mutant (DNMKK4) nor inhibition of the ERK pathway by the specific inhibitor PD98059 decreased the EGF-induced potentialization of Smad3 transactivation, whereas involvement of the p38 pathway was suggested by the inhibitory effect of SB 202190 on EGF/TGF-beta -induced Smad3 transactivation. These data confirm and extend the previous demonstration that p38 increases the transcriptional activity of TGF-beta -inducible genes (41-44). In these studies, p38 activation was shown to be induced by TGF-beta -activated kinase, a MAP kinase kinase kinase also involved in TGF-beta signaling (45, 46). According to this model, TGF-beta -activated kinase-induced p38 phosphorylation in response to TGF-beta triggers the phosphorylation of activating transcription factor 2, a basic leucine zipper protein member of the activating transcription factor/cAMP-response element-binding protein family that shares many structural characteristics with AP-1 proteins. Activated transcription factor 2 forms a complex with Smad4 that is transcriptionally active on Smad-regulated genes (41, 42). The mechanism of p38 activation found in the present study is clearly different from this model. It is very likely that the activating effect of p38 on Smad3 transactivation detected in our study lies downstream of PI3-kinase activation, since inhibition of the two pathways was not clearly additive. That PI3-kinase can contribute to the activation of protein kinases of the MAPK family, such as ERK, has been previously shown (47), but a direct demonstration of p38 activation by the PI3-kinase pathway has never been published. Finally, since our Western blot experiments indicated that EGF induces c-Jun phosphorylation by mechanisms dependent of PI3-kinase and p38, it is very likely, but not proven, that these phosphorylation events are implicated in the potentiation of Smad3 transactivation by EGF. In sharp contrast with our results describing a stimulating effect of Jun/Smad3 cooperation for transactivation in the context of PI3-kinase-induced Jun activation, two recent examples of interplay between Jun proteins and Smad3 leading to transcriptional repression of Smad3 have recently been published (13, 48). In one model, Jun activated via a TGF-beta -induced JNK binds to Smad3 on an SBE element and inhibits Smad3 transcriptional activity (13). In a second model, c-Jun activated by tumor necrosis factor alpha  binds to Smad3 and prevents its binding to DNA, therefore acting again as a transcriptional repressor (48). Collectively, these data and ours suggest that the functional interplay between Jun and Smads is far more complicated than previously thought and might vary according to the mechanisms of Jun activation.

In summary, we have described another novel mechanism allowing TGF-beta to integrate with regulatory networks of the cell. Whether this pathway is specific to the hepatocyte environment remains to be determined. We show that such a mechanism operates on an SBE-driven gene, although with a lower magnitude, and therefore represents a potential mechanism of regulation of TGF-beta -inducible genes. In normal hepatocytes, simultaneous treatment with EGF and TGF-beta induces a proliferation arrest (49-51). Whether Jun/Smad3 synergism is at work on the promoter of Smad-responsive gene inhibitors of the cell cycle will be the subject of our next investigations.

    ACKNOWLEDGEMENTS

We thank J. Massagué, P. Ten Dijke, B. Vogelstein, X. F. Wang, A. Atfi, G. Cherqui, J. Downward, and M. J. Birrer for providing plasmids and A. Groyer and F. Daniel for critical evaluation of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 33-01-44-85-61-90; Fax: 33-01-44-85-92-79; E-mail: bernuau@bichat.inserm.fr.

Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M005919200

2 M. Rahmani, P. Péron, J. Weitzmann, L. Bakiri, B. Lardeux, and D. Bernuau, unpublished data.

    ABBREVIATIONS

The abbreviations used are: TGF-beta , transforming growth factor beta ; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; JNK/SAPK, c-Jun NH2-terminal kinase/stress-activated protein kinase; EGF, epidermal growth factor; PI3-kinase, phosphatidylinositol 3'-kinase; Luc, luciferase; Gal, galactosidase; SBE, Smad-binding element; TRE, TPA-responsive element; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; PMSF, phenylmethylsulfonyl fluoride; MAP, mitogen-activated protein; MAPK, MAP kinase; PAGE, polyacrylamide gel electrophoresis.

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