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Amlexanox Reversibly Inhibits Cell Migration and Proliferation and Induces the Src-dependent Disassembly of Actin Stress Fibers in Vitro *

  • Matteo Landriscina
    Footnotes
    Affiliations
    Center for Molecular Medicine, Maine Medical Center Research Institute, South Portland, Maine 04106
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  • Igor Prudovsky
    Affiliations
    Center for Molecular Medicine, Maine Medical Center Research Institute, South Portland, Maine 04106
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  • Carla Mouta Carreira
    Footnotes
    Affiliations
    Center for Molecular Medicine, Maine Medical Center Research Institute, South Portland, Maine 04106
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  • Raffaella Soldi
    Affiliations
    Center for Molecular Medicine, Maine Medical Center Research Institute, South Portland, Maine 04106
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  • Francesca Tarantini
    Footnotes
    Affiliations
    Center for Molecular Medicine, Maine Medical Center Research Institute, South Portland, Maine 04106
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  • Thomas Maciag
    Correspondence
    To whom correspondence should be addressed: Center for Molecular Medicine, Maine Medical Center Research Institute, 125 John Roberts Rd., South Portland, Maine 04106. Tel.: 207-761-9783; Fax: 207-828-8071
    Affiliations
    Center for Molecular Medicine, Maine Medical Center Research Institute, South Portland, Maine 04106
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grants HL35627, HL32348, and AG07450 (to T. M.).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.
    ‡ Supported by a fellowship from the Catholic University of Rome.
    § Present address: Massachusetts General Hospital, Harvard Medical School, 100 Blossom St., Boston, MA 02114.
    ¶ Present address: Dept. of Geriatric Medicine, University of Florence, School of Medicine, Florence, Italy.
Open AccessPublished:October 20, 2000DOI:https://doi.org/10.1074/jbc.M002336200
      Amlexanox binds S100A13 and inhibits the release of fibroblast growth factor 1 (FGF1). Because members of the S100 gene family are known to be involved with the function of the cytoskeleton, we examined the ability of amlexanox to modify the cytoskeleton and report that amlexanox induces a dramatic reduction in the presence of actin stress fibers and the appearance of a random, non-oriented distribution of focal adhesion sites. Correspondingly, amlexanox induces the complete and reversible non-apoptotic inhibition of cell migration and proliferation, and although amlexanox does not induce either the down-regulation of F-actin levels or the depolymerization of actin filaments, it does induce the tyrosine phosphorylation of cortactin, a Src substrate known to regulate actin bundling. In addition, a dominant negative form of Src is able to partially rescue cells from the effect of amlexanox on both the actin cytoskeleton and cell migration. In contrast, the inhibition of cell proliferation by amlexanox correlates with the inhibition of cyclin D1 expression without interference of the receptor tyrosine kinase/mitogen-activated protein kinase signaling pathway. Last, the ability of amlexanox to inhibit FGF1 release is reversible and correlates with the restoration of the actin cytoskeleton, suggesting a role for the actin cytoskeleton in the FGF1 release pathway.
      FGF
      fibroblast growth factor
      BCS
      bovine calf serum
      DMI
      defined medium with insulin
      dn
      dominant negative
      FAK
      focal adhesion kinase
      Syt
      synaptotagmin
      NBD
      12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))
      HUVEC
      human umbilical vein endothelial cells
      DMEM
      Dulbecco's modified Eagle's medium
      Pipes
      1,4-piperazinediethanesulfonic acid
      ERK
      extracellular signal-regulated kinase
      PDGF
      platelet-derived growth factor
      PDGFR
      PDGF receptor
      PAGE
      polyacrylamide gel electrophoresis
      Amlexanox, an anti-allergic drug that binds S100A13, a relatively new member of the S100 gene family, is able to inhibit the heat shock-induced release of fibroblast growth factor (FGF1)1 (
      • Carreira C.
      • LaVallee T.
      • Tarantini F.
      • Jackson A.
      • Lathrop J.
      • Hampton B.
      • Burgess W.H.
      • Maciag T.
      ). FGF1 and FGF2 are the prototype members of a large family of heparin binding growth factor genes that regulate numerous biological processes, including mesoderm formation, neurogenesis, and angiogenesis in vivo(
      • Burgess W.H.
      • Maciag T.
      ). Since the FGF prototypes are characterized by the lack of a classical signal peptide sequence to provide access to the conventional endoplasmic reticulum-Golgi secretion pathway, it has been suggested that the release of both FGF1 and FGF2 may proceed through a novel release pathway (
      • Friesel R.E.
      • Maciag T.
      ). Our laboratory recently demonstrated that FGF1 is released as a reducing agent- and denaturant-sensitive complex, containing the p40 extravesicular domain of p65 Synaptotagmin (Syt) 1in vitro (
      • LaVallee T.
      • Tarantini F.
      • Gamble S.
      • Carreira C.
      • Jackson A.
      • Maciag T.
      ,
      • Tarantini F.
      • LaVallee T.
      • Jackson A.
      • Gamble S.
      • Carreira C.
      • Garfinkel S.
      • Burgess W.H.
      • Maciag T.
      ) and that FGF1, p40 Syt1, and S100A13 are components of a heparin binding complex in vivo (
      • Carreira C.
      • LaVallee T.
      • Tarantini F.
      • Jackson A.
      • Lathrop J.
      • Hampton B.
      • Burgess W.H.
      • Maciag T.
      ).
      It is well established that several members of the S100 gene family are associated with the cytoskeleton (
      • Mandinova A.
      • Atar D.
      • Schafer B.W.
      • Spiess M.
      • Aebi U.
      • Heizmann C.W.
      ,
      • Osterloh D.
      • Ivanenkov V.V.
      • Gerke V.
      ) and that the actin cytoskeleton is essential in transmembrane signaling, endocytosis, and secretion (
      • Schmidt A.
      • Hall M.
      ). There is also increasing evidence that actin microfilaments and the subplasmalemmal cytoskeleton are involved in several aspects of vesicle transport (
      • Nelson W.J.
      ). Indeed, in yeast, actin cytoskeleton mutants accumulate large secretory vesicles and exhibit defects in endocytosis that correlate with changes in actin-polarized organization (
      • Mulholland J.
      • Wesp A.
      • Riezman H.
      • Botstein D.
      ). The actin cytoskeleton is also required by mammalian cells for cell proliferation, motility, and morphological changes (
      • Schmidt A.
      • Hall M.
      ).
      The Src pathway also plays a central role in the modulation of the organization of the actin cytoskeleton in response to extracellular stimuli through the phosphorylation of several actin-binding proteins including cortactin (
      • Felsenfeld D.
      • Schwartzberg P.
      • Venegas A.
      • Tse R.
      • Sheetz M.
      ,
      • Zhan X.
      • Hu X.
      • Hampton B.
      • Burgess W.H.
      • Friesel R.
      • Maciag T.
      ). Indeed, the activation of the Src pathway correlates with the induction of cell migration and the redistribution of cortactin and F-actin, in response to FGF1 (
      • LaVallee T.M.
      • Prudovsky I.A.
      • McMahon G.A.
      • Hu X.
      • Maciag T.
      ).
      Since (i) amlexanox is an inhibitor of FGF1 and p40 Syt1 releasein vitro (
      • Carreira C.
      • LaVallee T.
      • Tarantini F.
      • Jackson A.
      • Lathrop J.
      • Hampton B.
      • Burgess W.H.
      • Maciag T.
      ), (ii) this reagent binds S100A13, a member of the heparin binding complex containing p40 Syt1 and FGF1 (
      • Carreira C.
      • LaVallee T.
      • Tarantini F.
      • Jackson A.
      • Lathrop J.
      • Hampton B.
      • Burgess W.H.
      • Maciag T.
      ,
      • Oyama Y.
      • Shishibori T.
      • Yamashita K.
      • Naya T.
      • Nakagiri S.
      • Maeta H.
      • Kobayashi R.
      ), and (iii) members of the S100 gene family are involved in the regulation of cytoskeletal function (
      • Mandinova A.
      • Atar D.
      • Schafer B.W.
      • Spiess M.
      • Aebi U.
      • Heizmann C.W.
      ,
      • Osterloh D.
      • Ivanenkov V.V.
      • Gerke V.
      ), we examined the effect of amlexanox on cell morphology, migration, proliferation, and cytoskeletal organization. We report that amlexanox induces a Src-dependent phosphorylation of cortactin that may be responsible for the reversible inhibition of cell migration and organization of actin stress fibers and induces a Src-independent reversible inhibition of cell proliferation that correlates with cyclin D1 down-regulation.

      DISCUSSION

      Amlexanox is a particularly efficient inhibitor of mammalian cell migration and proliferation in vitro. Cells exposed to amlexanox exhibited a dose-dependent inhibition of cell motility and growth without an increase in apoptotic cell death. Consistent with the inability of amlexanox to induce apoptosis, at least in concentrations used in these experiments, was the observation that the inhibition of cell migration and proliferation by amlexanox was reversible.
      In association with the suppression of cell migration and proliferation, prominent changes in cell morphology were also observed. Indeed, in most situations these morphologic changes were rapid and were readily visible after approximately 8 to 12 h. These morphologic alterations included an increase in cell size, the formation of a more flattened appearance, and the generation of long dendrite-like processes. Interestingly, the changes correlated with a strong attenuation of the presence of actin stress fibers in cells treated with amlexanox at concentrations that not only inhibit cell migration and proliferation but also alter and disorient the distribution of focal adhesion sites. In addition, we also observed more rapid and prominent morphological changes induced by amlexanox with a disappearance of actin stress fibers and subplasmalemmal cortex under serum-free conditions.
      It is clear that the effects of amlexanox on the cytoskeleton are not due to the down-regulation of intracellular levels of actin or to the depolymerization of actin filaments. Also, using an in vitrosystem of actin polymerization, we observed that amlexanox was unable to inhibit this process.
      A. Mandinova, U. Aebi, M. Landriscina, I. Prudovsky, and T. Maciag, unpublished observation.
      We therefore suggest that the process of actin microfilament bundling may be involved in mediating the effects of amlexanox. Supportive of this interpretation is the observation that jasplakinolide, an agent known to stabilize actin stress fibers (
      • Cramer L.
      ), is able to prevent the effects of amlexanox on cell morphology and actin cytoskeleton.
      The mechanism utilized by amlexanox appears to involve the function of cortactin and Src. Cortactin is a Src substrate and an F-actin-binding protein whose tyrosine phosphorylation results in a dramatic reduction in F-actin cross-linking activity and actin bundling (
      • Huang C.
      • Ni Y.
      • Wang T.
      • Gao Y.
      • Haudenschild C.C.
      • Zhan X.
      ). Indeed, amlexanox is able to rapidly induce and sustain the tyrosine phosphorylation of cortactin, and in response to amlexanox, cortactin is able to associate with a variety of phosphotyrosine-containing proteins including a p60 polypeptide. Since it has been demonstrated that cortactin is phosphorylated by Src in response to FGF1 and other growth factors as a relatively late event in the G1 phase of cell cycle (
      • Zhan X.
      • Hu X.
      • Hampton B.
      • Burgess W.H.
      • Friesel R.
      • Maciag T.
      ,
      • Zhan X.
      • Plourde C.
      • Hu X.
      • Friesel R.
      • Maciag T.
      ), we evaluated the role of Src pathway in mediating the effect of amlexanox. The overexpression of a dominant negative form of Src, known to act as an inhibitor of endogenous Src activity (
      • Thomas J.
      • Soriano P.
      • Brugge J.S.
      ), attenuated the effect of amlexanox on both cell morphology and actin stress fibers. In addition, amlexanox was also able to significantly reduce the levels of cortactin tyrosine phosphorylation in the dnSrc NIH 3T3 cell transfectants. The dnSrc NIH 3T3 cell transfectants were also less sensitive to amlexanox-induced inhibition of cell motility and to the redistribution of focal adhesion sites, suggesting that these effects of amlexanox may also be mediated, in part, by Src.
      Since it is well established that FAK is localized at focal adhesion sites (
      • Schaller M.
      • Hildebrand J.
      • Parsons J.
      ) and is involved in the regulation of cell migration in response to extracellular stimuli (
      • Sheetz M.
      • Felsenfeld D.
      • Galbraith C.
      ), we evaluated whether amlexanox was able to affect FAK phosphorylation. Interestingly, amlexanox was only able to produce a moderate down-regulation of FAK phosphorylation in both NIH 3T3 and the dnSrc NIH 3T3 cell transfectants, and this effect required a prolonged exposure (3–6 h) to amlexanox. These results suggest that the redistribution of focal adhesion sites in amlexanox-treated cells may be a consequence of the collapse of the actin cytoskeleton. This interpretation is consistent with the observation that a 1-h treatment with amlexanox under conditions of serum deprivation was able to affect the intensity and the distribution of vinculin-positive focal adhesion sites in NIH 3T3 cells but not dramatically alter the tyrosine phosphorylation of FAK (data not shown).
      Because the treatment of cells with amlexanox was able to prevent the FGF1-induced up-regulation of cyclin D1 in both quiescent and proliferating populations of Swiss 3T3 cells, we evaluated the capacity of FGF1, PDGF-BB, and insulin to activate their cell surface receptors in presence of amlexanox. Interestingly, the exposure of cells to amlexanox for 6 h did not prevent the ability of FGF1, PDGF-BB, and insulin to induce the auto-phosphorylation of their receptors. In addition, amlexanox treatment did not alter the ability of FGF1 and PDGF-BB to induce ERK-1 and ERK-2 phosphorylation in quiescent Swiss 3T3 cells and did not prevent the FGF1-induced migration of ERK-1 and ERK-2 to the nucleus (data not shown). We also evaluated the capacity of amlexanox to interfere with FGFR1 activation as a function of exposure time to amlexanox using quiescent populations of Swiss 3T3 cells, and we observed a decrease in the ability of FGF1 to induce the autophosphorylation of FGFR1 only after long term exposure (18 h) of the cells to amlexanox. Shorter time periods of exposure to amlexanox, which were able to induce the disaggregation of actin cytoskeleton and prevent the up-regulation of cyclin D1, failed to interfere with receptor signaling. Since amlexanox was able to inhibit the proliferation of dnSrc NIH 3T3 cells and the induction of cyclin D1 in dnSrc NIH 3T3 cells (data not shown), we suggest that the anti-proliferative effect of amlexanox is dissociated from its effect on the Src pathway. This could be explained by the ability of amlexanox to interfere with cyclin D1 induction and the molecular events that are responsible for the G1 transition, yet these appear to be independent of the induction of the polypeptide growth factor/receptor/Ras/mitogen-activated protein kinase signaling pathway. Our data may also suggest the importance of the actin cytoskeleton during pre-replicative events of the G1phase.
      Other biochemical agents such as the cytochalasines and latrunculins also induce the disorganization of actin cytoskeleton as well as the inhibition of cell proliferation (
      • Spector I.
      • Shochet N.R.
      • Blasberger D.
      • Kashman Y.
      ). However, unlike amlexanox, these agents induce apoptosis, and their effects on the actin cytoskeleton are more rapid and include the attenuation of stress fibers, a prominent decrease in F-actin protein levels, and a reduction in the subplasmalemmal F-actin cortex. In addition, the latrunculins and cytochalasines induce prominent cell rounding and the formation of cytoplasmic blebs, and these effects were not observed with amlexanox in the presence of serum (
      • Spector I.
      • Shochet N.R.
      • Blasberger D.
      • Kashman Y.
      ). Indeed, to our knowledge, this is the first study to identify a reagent that is able to induce the reversible disassembly of actin bundles without influencing actin polymerization and inducing apoptosis. The biological activities of amlexanox may also offer novel opportunities for its use as a reagent for studies in the fields of cell biology and experimental medicine. The reversible suppression of cell growth may potentially be used to synchronize cells, and its effect on actin stress fibers may prove useful for studies of the organization and functions of the actin cytoskeleton. In addition, amlexanox may also prove useful as a reagent to assess the role of actin stress fibers in the cellular trafficking of organelles and macromolecules. Lastly, the ability of amlexanox to reversibly inhibit human endothelial cell migration and proliferation suggest that its anti-inflammatory activities in vivo may possess an anti-angiogenic component. However, the poor solubility of amlexanox will require the development of novel methods for the efficientin vivo delivery of this rather interesting biological antagonist.
      The reason for studying the mechanism of amlexanox activity was the observation that amlexanox is able to bind S100A13 (
      • Oyama Y.
      • Shishibori T.
      • Yamashita K.
      • Naya T.
      • Nakagiri S.
      • Maeta H.
      • Kobayashi R.
      ), a protein that together with Syt1 is a component of a heparin binding multiprotein complex involved in the mechanism of FGF1 release (
      • Carreira C.
      • LaVallee T.
      • Tarantini F.
      • Jackson A.
      • Lathrop J.
      • Hampton B.
      • Burgess W.H.
      • Maciag T.
      ). Little is known about the biological functions and intracellular localization of S100A13, and it is a relatively novel member of the S100 gene family of calcium-binding proteins (
      • Wicki R.
      • Schafer B.W.
      • Erne P.
      • Heizmann C.W.
      ). However, it is well described that S100 gene family members, S100A1 and S100A4 (
      • Mandinova A.
      • Atar D.
      • Schafer B.W.
      • Spiess M.
      • Aebi U.
      • Heizmann C.W.
      ,
      • Osterloh D.
      • Ivanenkov V.V.
      • Gerke V.
      ) co-localize with actin stress fibers. In addition, S100B is able to associate with the actin-capping protein, CapZ (
      • Kilby P.M.
      • Van Eldik L.J.
      • Roberts G.C.
      ), and S100A2 is interactive with tropomyosins (
      • Gimona M.
      • Lando Z.
      • Dolginov Y.
      • Vandekerckhove J.
      • Kobayashi R.
      • Sobieszek A.
      • Helfman D.M.
      ), which are known to regulate the bundling of actin filaments (
      • Ishikawa R.
      • Yamashiro S.
      • Kohoma K.
      • Matsumura F.
      ). Thus, the ability of amlexanox to induce cortactin tyrosine phosphorylation and to modify actin stress fibers in vitro may reflect either the potential co-localization of S100A13 with the actin filaments or a potential role of S100A13 to cooperate in the organization of actin cytoskeleton.
      The effect of amlexanox on the Src pathway and actin stress fiber disassembly could underlie the mechanism used by this agent to interfere with the function of proteins involved in the release of FGF1 as well as its ability to suppress the release of histamine by mast cells, an important allergenic mediator (
      • Makino H.
      • Saijo T.
      • Ashida H.
      • Kuriki H.
      • Maki Y.
      ). We observed that the effect of amlexanox on FGF1 release is reversible and correlates with the restoration of the actin cytoskeleton. Moreover, latrunculin, another reagent that affects the integrity of the actin cytoskeleton through a different mechanism that involves the depolymerization of F-actin (
      • Spector I.
      • Shochet N.R.
      • Blasberger D.
      • Kashman Y.
      ), is also able to inhibit the release of FGF1 and p40 Syt1 in response to temperature stress. Thus it is possible that the function of the Src pathway and the actin cytoskeleton may be involved in the regulation of the intracellular trafficking responsible for FGF1 and Syt1 release. Since (i) p65 Syt1, a transmembrane component of intracellular vesicles (
      • Perin M.S.
      • Brose N.
      • Jahn R.
      • Sudhof T.C.
      ), is involved in the regulation of exocytotic and endocytotic organelle trafficking (
      • Zhang J.Z.
      • Davletov B.A.
      • Sudhof T.C.
      • Anderson R.G.W.
      ), including the docking of synaptic vesicles to the plasma membrane (
      • Sollner T.
      • Whiteheart S.W.
      • Brunner M.
      • Erdjument-Bromage H.
      • Geromanos S.
      • Tempst P.
      • Rothman J.
      ), and (ii) the function of intracellular p65 Syt1 is required for FGF1 release (
      • LaVallee T.
      • Tarantini F.
      • Gamble S.
      • Carreira C.
      • Jackson A.
      • Maciag T.
      ), we suggest that Syt1-positive intracellular vesicles containing the extravesicular FGF1 homodimer may traffic along actin stress fibers and that the function of S100A13 or other members of the S100 gene family may be involved in the regulation of this trafficking.

      Acknowledgments

      We thank the officers of Takeda Pharmaceuticals, Ltd. for their generosity in supplying amlexanox, A. Mandinova and U. Aebi (University of Basel), for their assessment of the ability of amlexanox to block actin polymerization in vitro, and R. Friesel (Maine Medical Center) for the dnSrc mutant.

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