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Tumor Necrosis Factor-α Regulates Transforming Growth Factor-β-dependent Epithelial-Mesenchymal Transition by Promoting Hyaluronan-CD44-Moesin Interaction2

  • Eri Takahashi
    Affiliations
    From the Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo 160-8582

    the Department of Ophthalmology and Visual Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556
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  • Osamu Nagano
    Affiliations
    From the Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo 160-8582

    the Japan Science and Technology Agency, CREST, Tokyo 102-0075
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  • Takatsugu Ishimoto
    Affiliations
    From the Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo 160-8582
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  • Toshifumi Yae
    Affiliations
    From the Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo 160-8582
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  • Yoshimi Suzuki
    Affiliations
    the Department of Biomedical Research and Development, Link Genomics Inc., Tokyo 103-0024, and
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  • Takeshi Shinoda
    Affiliations
    the Department of Biomedical Research and Development, Link Genomics Inc., Tokyo 103-0024, and
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  • Satoshi Nakamura
    Affiliations
    the Department of Biomedical Research and Development, Link Genomics Inc., Tokyo 103-0024, and
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  • Shinichiro Niwa
    Affiliations
    the Department of Biomedical Research and Development, Link Genomics Inc., Tokyo 103-0024, and
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  • Shun Ikeda
    Affiliations
    the Laboratory of Medical Genomics, Department of Human Genome Research, Kazusa DNA Research Institute, Chiba 292-0818, Japan
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  • Hisashi Koga
    Affiliations
    the Laboratory of Medical Genomics, Department of Human Genome Research, Kazusa DNA Research Institute, Chiba 292-0818, Japan
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  • Hidenobu Tanihara
    Affiliations
    the Department of Ophthalmology and Visual Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556
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  • Hideyuki Saya
    Correspondence
    To whom correspondence should be addressed: Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: 81-3-5363-3981; Fax: 81-3-5363-3982
    Affiliations
    From the Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo 160-8582

    the Japan Science and Technology Agency, CREST, Tokyo 102-0075
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to O. N. and H. S.) and by a grant from the National Institute of Biomedical Innovation, Japan (to H. S.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Movies 1–5.
Open AccessPublished:December 04, 2009DOI:https://doi.org/10.1074/jbc.M109.056523
      Aberrant epithelial-mesenchymal transition (EMT) is involved in development of fibrotic disorders and cancer invasion. Alterations of cell-extracellular matrix interaction also contribute to those pathological conditions. However, the functional interplay between EMT and cell-extracellular matrix interactions remains poorly understood. We now show that the inflammatory mediator tumor necrosis factor-α (TNF-α) induces the formation of fibrotic foci by cultured retinal pigment epithelial cells through activation of transforming growth factor-β (TGF-β) signaling in a manner dependent on hyaluronan-CD44-moesin interaction. TNF-α promoted CD44 expression and moesin phosphorylation by protein kinase C, leading to the pericellular interaction of hyaluronan and CD44. Formation of the hyaluronan-CD44-moesin complex resulted in both cell-cell dissociation and increased cellular motility through actin remodeling. Furthermore, this complex was found to be associated with TGF-β receptor II and clathrin at actin microdomains, leading to activation of TGF-β signaling. We established an in vivo model of TNF-α-induced fibrosis in the mouse eye, and such ocular fibrosis was attenuated in CD44-null mice. The production of hyaluronan and its interaction with CD44, thus, play an essential role in TNF-α-induced EMT and are potential therapeutic targets in fibrotic disorders.

      Introduction

      The epithelial-mesenchymal transition (EMT)
      The abbreviations used are: EMT
      epithelial-mesenchymal transition
      ECM
      extracellular matrix
      TNF
      tumor necrosis factor
      TGF
      transforming growth factor
      HA
      hyaluronic acid
      HAS
      hyaluronic acid synthase
      ERM
      ezrin-radixin-moesin
      pERM
      phosphorylated ERM
      RPE cells
      retinal pigment epithelial cells
      α-SMA
      α-smooth muscle actin
      EAFD
      EMT-associated fibrotic deposit
      4-MU
      4-methylumbelliferone
      PKC
      protein kinase C
      MAPK
      mitogen-activated protein kinase
      siRNA
      small interfering RNA
      RT
      reverse transcription
      PVR
      proliferative vitreoretinopathy
      PBS
      phosphate-buffered saline
      DIC
      differential interference contrast.
      of epithelial cells is characterized by the loss of epithelial characteristics and the gain of mesenchymal attributes. During this transition, epithelial cells down-regulate cell-cell adhesion systems, lose their polarity, and acquire a mesenchymal phenotype associated with increased interaction with the extracellular matrix (ECM) and enhanced migratory capacity. The EMT is considered a critical event in metazoan embryogenesis as well as in physiological processes such as wound healing. However, it also plays an important role in pathological settings such as fibrotic disorders in various organs as well as cancer invasion and metastasis.
      The EMT associated with physiological processes is triggered by members of the transforming growth factor-β (TGF-β) family of proteins that function as morphogens (
      • Lee J.M.
      • Dedhar S.
      • Kalluri R.
      • Thompson E.W.
      ). In vitro studies have also shown that TGF-β is the major inducer of the EMT in epithelial cells (
      • Thiery J.P.
      ). Fibrotic disorders associated with pathological EMT result from a series of events including inflammation, leukocyte infiltration, and the production of cytokines and growth factors. TGF-β is one of the cytokines produced during inflammation and is, therefore, thought to heavily contribute to EMT-associated fibrosis (
      • Kalluri R.
      • Neilson E.G.
      ). However, given that TGF-β also possesses anti-inflammatory properties, the mechanism of pathological EMT induced by the inflammatory response may be multifactorial and differ from that of physiological EMT.
      In addition to growth factors, changes in the ECM microenvironment contribute to the EMT. Epithelial cells cultured in a type I collagen gel were found to undergo the EMT (
      • Zuk A.
      • Matlin K.S.
      • Hay E.D.
      ). Furthermore, collagen-induced changes in cadherin expression and cell morphology in epithelial cells were shown to be dependent on activation of intracellular signaling by collagen (
      • Shintani Y.
      • Wheelock M.J.
      • Johnson K.R.
      ). These observations implicated signaling pathways activated by cell adhesion to the ECM in acquisition of the mesenchymal phenotype. Hyaluronic acid (HA), or hyaluronan, is a major component of the ECM and plays a key role in tissue homeostasis as well as in pathological tissue remodeling (
      • Laurent T.C.
      • Fraser J.R.
      ). HA is synthesized by hyaluronic acid synthases (HASs) located at the plasma membrane. Three isoforms of mammalian HAS catalyze the synthesis of HA of distinct molecular sizes. HAS1 and HAS2 synthesize high molecular mass HA (200–2000 kDa), whereas HAS3 synthesizes low molecular mass HA (100–1000 kDa) (
      • Itano N.
      • Sawai T.
      • Yoshida M.
      • Lenas P.
      • Yamada Y.
      • Imagawa M.
      • Shinomura T.
      • Hamaguchi M.
      • Yoshida Y.
      • Ohnuki Y.
      • Miyauchi S.
      • Spicer A.P.
      • McDonald J.A.
      • Kimata K.
      ). HAS2-deficient mice fail to manifest the characteristic transformation of cardiac endothelial cells into mesenchyme (
      • Camenisch T.D.
      • Spicer A.P.
      • Brehm-Gibson T.
      • Biesterfeldt J.
      • Augustine M.L.
      • Calabro Jr., A.
      • Kubalak S.
      • Klewer S.E.
      • McDonald J.A.
      ). In addition, oligosaccharide forms of HA, which inhibit binding of endogenous HA to the HA receptor CD44, attenuate the EMT associated with cardiac development (
      • Zeng C.
      • Toole B.P.
      • Kinney S.D.
      • Kuo J.W.
      • Stamenkovic I.
      ,
      • Ghatak S.
      • Misra S.
      • Toole B.P.
      ,
      • Rodgers L.S.
      • Lalani S.
      • Hardy K.M.
      • Xiang X.
      • Broka D.
      • Antin P.B.
      • Camenisch T.D.
      ). These findings implicate HA-dependent changes in the tissue microenvironment in induction of the EMT.
      CD44 is the principal transmembrane adhesion receptor for HA and plays a central role in the remodeling and degradation of HA that lead to cell migration as well as to cancer invasion and metastasis (
      • Ponta H.
      • Sherman L.
      • Herrlich P.A.
      ,
      • Nagano O.
      • Saya H.
      ) The cytoplasmic tail of CD44 recruits ezrin-radixin-moesin (ERM) proteins that are linked to the actin cytoskeleton and thereby promote cell motility. Expression of CD44 is up-regulated not only in cancer cells but also in cells associated with inflammatory diseases (
      • Haynes B.F.
      • Hale L.P.
      • Patton K.L.
      • Martin M.E.
      • McCallum R.M.
      ,
      • Kuppner M.C.
      • Liversidge J.
      • McKillop-Smith S.
      • Lumsden L.
      • Forrester J.V.
      ,
      • Florquin S.
      • Nunziata R.
      • Claessen N.
      • van den Berg F.M.
      • Pals S.T.
      • Weening J.J.
      ), and inflammation-mediated fibrosis in the lung and kidney was shown to be attenuated in CD44-deficient mice (
      • Svee K.
      • White J.
      • Vaillant P.
      • Jessurun J.
      • Roongta U.
      • Krumwiede M.
      • Johnson D.
      • Henke C.
      ,
      • Teder P.
      • Vandivier R.W.
      • Jiang D.
      • Liang J.
      • Cohn L.
      • Puré E.
      • Henson P.M.
      • Noble P.W.
      ,
      • Rouschop K.M.
      • Sewnath M.E.
      • Claessen N.
      • Roelofs J.J.
      • Hoedemaeker I.
      • van der Neut R.
      • Aten J.
      • Pals S.T.
      • Weening J.J.
      • Florquin S.
      ,
      • Noble P.W.
      • Jiang D.
      ). However, the molecular mechanism by which the HA-CD44 interaction leads to the development of fibrotic disorders remains largely unknown.
      Proliferative vitreoretinopathy (PVR) is a disorder characterized by the formation of membranes on the surfaces of the retina and within the vitreous cavity after retinal detachment surgery, and intraocular inflammation and EMT are thought to be the pathogenesis of this disease (
      • Limb G.A.
      • Chignell A.H.
      • Woon H.
      • Green W.
      • Cole C.J.
      • Dumonde D.C.
      ,
      • Casaroli-Marano R.P.
      • Pagan R.
      • Vilaró S.
      ). The PVR membrane consists of extracellular matrix, retinal pigment epithelium (RPE), retinal glial cells, fibroblasts, and inflammatory macrophages (
      • Campochiaro P.A.
      ,
      • Lei H.
      • Hovland P.
      • Velez G.
      • Haran A.
      • Gilbertson D.
      • Hirose T.
      • Kazlauskas A.
      ). Intravitreal cell injection models of PVR show that not only fibroblasts, but also RPE cells, are associated with the formation of intraocular membrane (
      • Agrawal R.N.
      • He S.
      • Spee C.
      • Cui J.Z.
      • Ryan S.J.
      • Hinton D.R.
      ). Various growth factors and cytokines, which are inflammatory products of cell activation, were increased in vitreous aspirates from the eyes with PVR. One of the most prominent of the inflammatory cytokines is tumor necrosis factor-α (TNF-α), whose mRNA and proteins are widely expressed in PVR membranes (
      • Limb G.A.
      • Chignell A.H.
      • Woon H.
      • Green W.
      • Cole C.J.
      • Dumonde D.C.
      ,
      • Armstrong D.
      • Augustin A.J.
      • Spengler R.
      • Al-Jada A.
      • Nickola T.
      • Grus F.
      • Koch F.
      ) TNF-α is mainly derived from activated macrophages, and RPE and glial cells in PVR membranes also releases it (
      • Wiedemann P.
      ). Although TNF-α is thought to play a causative role in PVR, the underlying mechanism is unknown.
      We have now developed an in vitro model of EMT-associated fibrosis based on human RPE cells. With the use of this model, we identified TNF-α as an important inducer of EMT-associated fibrotic focus formation. In addition, we clarified that the HA-CD44-moesin interaction triggered by TNF-α is required for activation of TGF-β signaling that leads to induction of the mesenchymal phenotype in RPE cells. Furthermore, fibrosis induced by injection of TNF-α into the mouse retina was found to be markedly suppressed in CD44 knock-out mice. These findings indicate that the HA-CD44 interaction plays a key role in EMT-associated fibrotic disorders.

      DISCUSSION

      During embryonic development and normal wound healing, the EMT is tightly regulated by “on” and “off” switches. However, the balance between the EMT and the reverse transition is thought to become deregulated in pathological conditions such as chronic inflammation, favoring the EMT. Cells that have undergone the EMT under such conditions produce excess amounts of ECM and aggregate at sites of inflammation, resulting in the development of fibrosis (
      • Thiery J.P.
      ,
      • Noble P.W.
      • Jiang D.
      ,
      • Toole B.P.
      • Zoltan-Jones A.
      • Misra S.
      • Ghatak S.
      ).
      We have now provided evidence from both in vitro and in vivo models that the proinflammatory cytokine TNF-α plays a key role in the induction of fibrosis associated with the mesenchymal change of RPE cells. EMT-associated fibrosis was, thus, found to be induced by activation of TGF-β signaling as a result of HA-CD44-ERM interaction promoted by TNF-α (Fig. 8). Our findings provide both insight into the mechanism underlying the relation between chronic inflammation and the EMT as well as a basis for the development of new therapeutic strategies to avert fibrotic disorders.
      Figure thumbnail gr8
      FIGURE 8Model of the signaling pathways underlying TNF-α-induced EMT. TNF-α induces the expression of CD44 and the phosphorylation of ERM in a manner dependent on PKC activation and thereby promotes formation of the HA·CD44·pERM complex. This complex then triggers remodeling of the actin cytoskeleton and the CD44-TGF-β receptor interaction, leading to the activation of Smad signaling through TGF-β receptor clustering and EMT induction. Persistent activation of EMT results in fibrotic disorder.

      TNF-α Activates TGF-β Signaling for Induction of EMT in RPE Cells

      We studied EAFD formation as an indicator of EMT-associated fibrotic reactions in RPE cells. The formation of EAFDs appeared to result from the combination of ECM overproduction and migration of the fibroblast-like cells within the ECM deposit. EAFD formation, thus, provides an in vitro model of fibrotic disorders. We found that TNF-α or the combination of TNF-α and TGF-β2 induced EAFD formation. Whereas TGF-β2 alone was able to increase the expression of certain mesenchymal markers such as fibronectin as well as to induce small morphologic changes in RPE cells, it was not sufficient to induce EAFD formation. However, we found that TNF-α induced TGF2 gene expression and that the activation of TGF-β receptors was essential for TNF-α-induced EMT and EAFD formation. Persistent stimulation with TNF-α, which mimics inflammatory conditions, thus appears to activate TGF-β signaling, leading to fibrotic reactions as a result of deviation from the balance between the EMT and the reverse transition.

      TNF-α Promotes the Formation of HA·CD44·pERM Complex through PKC Activation

      The ECM component HA and its biosynthetic enzymes (HASs) have been implicated in the EMT associated with cardiac development (
      • Camenisch T.D.
      • Spicer A.P.
      • Brehm-Gibson T.
      • Biesterfeldt J.
      • Augustine M.L.
      • Calabro Jr., A.
      • Kubalak S.
      • Klewer S.E.
      • McDonald J.A.
      ) and various fibrotic disorders. Indeed, our present results show that pericellular interaction between HA and CD44 is necessary for TNF-α-induced EAFD formation and wound healing in vitro. However, the mechanism by which HA contributes to the EMT has remained unknown. We have now shown that CD44 expression is increased by TNF-α and that subsequent formation of the membrane-spanning HA·CD44·pERM complex is required for induction and maintenance of the EMT.
      Activation of PKC was found to be a key step in formation of the HA·CD44·pERM complex. Previous studies have shown that TNF-α activates PKC (
      • Wyatt T.A.
      • Ito H.
      • Veys T.J.
      • Spurzem J.R.
      ) and that PKC activation promotes HA synthesis by HAS (
      • Anggiansah C.L.
      • Scott D.
      • Poli A.
      • Coleman P.J.
      • Badrick E.
      • Mason R.M.
      • Levick J.R.
      ). We now show that TNF-α stimulation promotes association of HA with CD44 at the cell periphery and that this association is accompanied by the interaction of CD44 with pERM, in particular with phosphorylated moesin. Formation of the HA·CD44·pERM complex appears to promote a switch from cell-cell contact to cell-HA interaction through remodeling of the actin cytoskeleton. The phosphorylation of ERM proteins was mediated by PKC in response to TNF-α stimulation. Our results, thus, indicate that activation of PKC by TNF-α promotes the phosphorylation of ERM proteins, facilitating formation of the HA·CD44·pERM complex at the plasma membrane.

      HA·CD44·pERM Complex Is Required for Activation of TGF-β Signaling

      Several lines of evidence provide support for the notion that TNF-α activates TGF-β signaling by promoting formation of the HA·CD44·pERM complex. Clathrin-dependent internalization of TGF-β receptors into EEA1-positive endosomes, in which the Smad2 anchor SARA is enriched, has previously been shown to activate TGF-β signaling (
      • Di Guglielmo G.M.
      • Le Roy C.
      • Goodfellow A.F.
      • Wrana J.L.
      ). In contrast, the caveolin-dependent internalization of TGF-β promotes rapid receptor turnover, leading to inactivation of TGF-β signaling. Our results indicate that TNF-α-induced formation of the membrane-spanning HA·CD44·pERM complex resulted in reorganization of the actin cytoskeleton and promoted the formation of actin microdomains, which spatially organize signaling molecules at the membrane. Such CD44-positive microdomains were associated with TGF-β receptor II and clathrin but not with caveolin-1. Therefore, the clustering of TGF-β receptors at actin microdomains may result in activation of TGF-β signaling. It is, thus, possible that cells with preexisting HA·CD44·pERM complexes may be induced to undergo EMT-associated fibrotic reactions by TGF-β stimulation alone (in the absence of TNF-α).
      TGF-β is usually released from cells in a latent form that must be proteolytically processed to yield the biologically active form. This activation of TGF-β was recently shown to be dependent on CD44 and to be mediated by a matrix metalloproteinase (
      • Acharya P.S.
      • Majumdar S.
      • Jacob M.
      • Hayden J.
      • Mrass P.
      • Weninger W.
      • Assoian R.K.
      • Puré E.
      ). Such a mechanism cannot explain our findings with TGF-β, however, because we used the active form of TGF-β2 in our experiments. CD44 may, thus, play dual roles in the activation of TGF-β signaling by promoting the extracellular conversion of the latent form of TGF-β to the active form and by inducing the intracellular activation of the Smad-dependent signaling pathway.
      In summary, we have shown that TNF-α and TGF-β cooperatively induce the EMT and that formation of the HA·CD44·pERM complex in a manner dependent on PKC activation is an important step in TNF-α action, leading to loss of cell-cell contact, changes in cell morphology, and ECM overproduction. Both our RPE organ culture and subretinal injection experiments also showed that TNF-α induces EMT-associated fibrosis in the RPE in a manner dependent on CD44. The interactions among HA, CD44, and ERM proteins, in particular moesin, thus represent potential targets for the development of new therapeutic agents for the treatment of EMT-associated pathological states such as fibrosis and inflammation as well as tumor invasion and metastasis.

      Acknowledgments

      We thank I. Ishimatsu and N. Suzuki for technical assistance as well as K. Arai for secretarial assistance. We thank O. Sampetrean and S. H. Lee for critical discussions.

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