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

ROLE OF PHOSPHATIDYLINOSITOL 3-KINASE-INDUCED AP-1 ACTIVATION*
Open AccessPublished:March 30, 2001DOI:https://doi.org/10.1074/jbc.M005919200
      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 β (TGF-β)−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-β 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-β-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-β 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-β-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-β treatment, this novel mechanism of Jun/Smad cooperation might be crucial for diversifying TGF-β responses.
      TGF-β
      transforming growth factor β
      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
      Transforming growth factor β (TGF-β)1 is a member of a large family of cytokines that includes bone morphogenetic proteins, activins, and several more distantly related factors (
      • Kingsley D.M.
      ). TGF-β is central in the regulation of many biological processes, including cell differentiation, growth, adhesion, and apoptosis. TGF-β signals through a system of transmembrane serine/threonine kinase receptors composed of type I and type II receptors (TGF-βRI and TGF-βRII) (see Refs.
      • Heldin C.-H.
      • Miyazono K.
      • ten Dijke P.
      ,
      • Attisano L.
      • Wrana J.L.
      ,
      • Chen Y.-G.
      • Hata A.
      • Lo R.S.
      • Wotton D.
      • Shi Y.
      • Pavletich N.
      • Massagué J.
      and reviewed in Ref.
      • Hu P.P.-C.
      • Datto M.B.
      • Wang X.-F.
      ). Ligand binding to TGF-βRII recruits and activates the TGF-βRI 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-β-responsive genes (
      • Zawel L.
      • Le Dai J.
      • Buckhaults P.
      • Zhou S.
      • Kinzler K.W.
      • Vogelstein B.
      • Kern S.E.
      ). 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-β (reviewed in Ref.
      • Massagué J.
      ).
      Emerging evidence indicates that TGF-β 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-β signaling (
      • Kretzschmar M.
      • Doody J.
      • Massagué J.
      ,
      • Kretzschmar M.
      • Doody J.
      • Timokhina I.
      • Massagué J.
      ). 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 (
      • De Caestecker M.P.
      • Parks W.T.
      • Frank C.J.
      • Castagnino P.
      • Bottaro D.P.
      • Roberts A.B.
      • Lechleider R.J.
      ). Recent evidence also indicates that Smad cooperates with AP-1 (
      • Zhang Y.
      • Feng X.-H.
      • Derynck R.
      ,
      • Liberati N.T.
      • Datto M.B.
      • Frederick J.P.
      • Shen X.
      • Wong C.
      • Rougier-Chapman E.M.
      • Wang X.-F.
      ,
      • Dennler S.
      • Prunier C.
      • Ferrand N.
      • Gauthier J.-M.
      • Atfi A.
      ), a heterodimer of Fos and Jun family members (
      • Karin M.
      ). 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-β-inducible genes, such as plasminogen activator inhibitor-1 or c-jun, and both sites appear to be critical in the TGF-β response (
      • Dennler S.
      • Itoh S.
      • Vivien D.
      • ten Dijke P.
      • Huet S.
      • Gauthier J.-M.
      ,
      • Yingling J.M.
      • Datto M.B.
      • Wong C.
      • Frederick J.P.
      • Liberati N.T.
      • Wang X.-F.
      ,
      • Wong C.
      • Rougier-Chapman E.M.
      • Frederick J.P.
      • Datto M.B.
      • Liberati N.T.
      • Li J.-M.
      • Wang X.-F.
      ).
      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-β. 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-β 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-β-inducible genes in hepatocytes.

      DISCUSSION

      There is increasing evidence that several signaling pathways interfere with Smads to regulate TGF-β-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 (
      • De Caestecker M.P.
      • Parks W.T.
      • Frank C.J.
      • Castagnino P.
      • Bottaro D.P.
      • Roberts A.B.
      • Lechleider R.J.
      ). More importantly, we show that a combined treatment with EGF and TGF-β 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 (
      • Brown P.H.
      • Alani R.
      • Preis L.H.
      • Szabo E.
      • Birrer M.J.
      ,
      • Brown P.H.
      • Chen T.K.
      • Birrer M.J.
      ). Recently, cooperation of Smad proteins with the AP-1 proteins Fos and Jun has been documented in mink lung epithelial cells (
      • Zhang Y.
      • Feng X.-H.
      • Derynck R.
      ,
      • Liberati N.T.
      • Datto M.B.
      • Frederick J.P.
      • Shen X.
      • Wong C.
      • Rougier-Chapman E.M.
      • Wang X.-F.
      ). In vitro binding of Smad3 and Smad4 to all three Jun family members as well as in vivo association between Smads and a TGF-β-phosphorylated form of endogenous c-Jun induced by JNK have been demonstrated in HaCaT cells (
      • Liberati N.T.
      • Datto M.B.
      • Frederick J.P.
      • Shen X.
      • Wong C.
      • Rougier-Chapman E.M.
      • Wang X.-F.
      ). Cooperation of AP-1 proteins with Smad3 and Smad4 occurred via Jun proteins bound to their cognatecis-element, the TRE, or to composite sites containing juxtaposed AP-1 and SBE sites (
      • Zhang Y.
      • Feng X.-H.
      • Derynck R.
      ,
      • Liberati N.T.
      • Datto M.B.
      • Frederick J.P.
      • Shen X.
      • Wong C.
      • Rougier-Chapman E.M.
      • Wang X.-F.
      ,
      • Wong C.
      • Rougier-Chapman E.M.
      • Frederick J.P.
      • Datto M.B.
      • Liberati N.T.
      • Li J.-M.
      • Wang X.-F.
      ), and the interaction was shown to involve 13 carboxyl-terminal amino acids conserved in the three Jun proteins (
      • Liberati N.T.
      • Datto M.B.
      • Frederick J.P.
      • Shen X.
      • Wong C.
      • Rougier-Chapman E.M.
      • Wang X.-F.
      ). 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 (
      • Zhang Y.
      • Feng X.-H.
      • Derynck R.
      ), abrogated the EGF/TGF-β-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 (
      • Zhang Y.
      • Feng X.-H.
      • Derynck R.
      ,
      • Liberati N.T.
      • Datto M.B.
      • Frederick J.P.
      • Shen X.
      • Wong C.
      • Rougier-Chapman E.M.
      • Wang X.-F.
      ) and the demonstration that c-Jun coimmunoprecipitates with Smad3 during EGF/TGF-β 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 (
      • Osborn M.T.
      • Herrin K.
      • Buzen F.G.
      • Hurlburt B.K.
      • Chambers T.C.
      ).
      Binding of EGF to its receptor activates the PI3-kinase pathway (
      • Hackel P.O.
      • Zwick E.
      • Prenzel N.
      • Ullrich A.
      ,
      • Huang C.
      • Ma W.-Y.
      • Dong Z.
      ,
      • Logan S.K.
      • Falasca M.
      • Hu P.
      • Schlessinger J.
      ), a cascade involved in AP-1 activation (
      • Huang C.
      • Ma W.-Y.
      • Dong Z.
      ,
      • Huang C.
      • Schmid P.C.
      • Ma W.-Y.
      • Schmid H.H.O.
      • Dong Z.
      ). 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-β signaling had been previously suggested from the observation that wortmannin, another PI3-kinase inhibitor, inhibits TGF-β-stimulated chemotaxis of human neutrophil leukocytes (
      • Thelen M.
      • Uguccioni M.
      • Bosiger J.
      ). It has also been demonstrated that TGF-β markedly enhanced EGF-induced PI3-kinase activity in human airway smooth muscle cells (
      • Krymskaya V.P.
      • Hoffman R.
      • Eszterhas A.
      • Ciocca V.
      • Panettieri Jr., R.A.
      ). More recently, involvement of PI3-kinase in the inhibitory effect of insulin, EGF, or interleukin-6 on TGF-β-induced apoptosis has been reported (
      • Roberts R.A.
      • James N.H.
      • Cosulich S.C.
      ,
      • Chen R.-H.
      • Su Y.-H.
      • Chuang R.L.C.
      • Chang T.-Y.
      ,
      • Chen R.-H.
      • Chang M.-C.
      • Su Y.-H.
      • Tsai S.-T.
      • Kuo M.-L.
      ), 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.
      • Kyriakis J.M.
      • Banerjee P.
      • Nikolakaki E.
      • Dai T.
      • Rubie E.A.
      • Ahmad M.F.
      • Avruch J.
      • Woodgett J.R.
      and for review see Ref.
      • Tibbles L.A.
      • Woodgett J.R.
      ), all implicated in EGF-induced AP-1 activation (
      • Karin M.
      ). 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-β-induced Smad3 transactivation. These data confirm and extend the previous demonstration that p38 increases the transcriptional activity of TGF-β-inducible genes (
      • Hanafusa H.
      • Ninomiya-Tsuji J.
      • Masuyama N.
      • Nishita M.
      • Fujisawa J.-I
      • Shibuya H.
      • Matsumoto K.
      • Nishida E.
      ,
      • Sano Y.
      • Harada J.
      • Tashiro S.
      • Gotoh-Mandeville R.
      • Maekawa T.
      • Ishii S.
      ,
      • Adachi-Yamada T.
      • Nakamura M.
      • Irie K.
      • Tomoyasu Y.
      • Sano Y.
      • Mori E.
      • Gotoh S.
      • Ueno N.
      • Nishida Y.
      • Matsumoto K.
      ,
      • Kimura N.
      • Matsuo R.
      • Nakashima K.
      • Taga T.
      ). In these studies, p38 activation was shown to be induced by TGF-β-activated kinase, a MAP kinase kinase kinase also involved in TGF-β signaling (
      • Yamaguchi K.
      • Shirakabe K.
      • Shibuya H.
      • Irie K.
      • Oishi I.
      • Ueno N.
      • Taniguchi T.
      • Nishida E.
      • Marsumoto K.
      ,
      • Shibuya H.
      • Yamaguchi K.
      • Shirakabe K.
      • Tonegawa A.
      • Gotoh Y.
      • Ueno N.
      • Irie K.
      • Nishida E.
      • Matsumoto K.
      ). According to this model, TGF-β-activated kinase-induced p38 phosphorylation in response to TGF-β 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 (
      • Hanafusa H.
      • Ninomiya-Tsuji J.
      • Masuyama N.
      • Nishita M.
      • Fujisawa J.-I
      • Shibuya H.
      • Matsumoto K.
      • Nishida E.
      ,
      • Sano Y.
      • Harada J.
      • Tashiro S.
      • Gotoh-Mandeville R.
      • Maekawa T.
      • Ishii S.
      ). 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 (
      • Bondeva T.
      • Pirola L.
      • Bulgarelli-Leva G.
      • Rubio I.
      • Wetzker R.
      • Wymann M.P.
      ), 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 (
      • Dennler S.
      • Prunier C.
      • Ferrand N.
      • Gauthier J.-M.
      • Atfi A.
      ,
      • Verrecchia F.
      • Pessah M.
      • Atfi A.
      • Mauviel A.
      ). In one model, Jun activated via a TGF-β-induced JNK binds to Smad3 on an SBE element and inhibits Smad3 transcriptional activity (
      • Dennler S.
      • Prunier C.
      • Ferrand N.
      • Gauthier J.-M.
      • Atfi A.
      ). In a second model, c-Jun activated by tumor necrosis factor α binds to Smad3 and prevents its binding to DNA, therefore acting again as a transcriptional repressor (
      • Verrecchia F.
      • Pessah M.
      • Atfi A.
      • Mauviel A.
      ). 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-β 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-β-inducible genes. In normal hepatocytes, simultaneous treatment with EGF and TGF-β induces a proliferation arrest (
      • Nakamura T.
      • Tomita Y.
      • Hirai R.
      • Yamaoka K.
      • Kaji K.
      • Ichihara A.
      ,
      • Carr B.I.
      • Hayashi I.
      • Branum E.L.
      • Moses H.L.
      ,
      • Mc Mahon J.B.
      • Richards W.L.
      • DelCampo A.A.
      • Song M.K.
      • Thorgeirsson S.S.
      ). 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.

      Acknowledgments

      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.

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