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Neuritogenesis Induced by Thyroid Hormone-treated Astrocytes Is Mediated by Epidermal Growth Factor/Mitogen-activated Protein Kinase-Phosphatidylinositol 3-Kinase Pathways and Involves Modulation of Extracellular Matrix Proteins*

  • Rodrigo Martinez
    Affiliations
    From the Instituto de Ciências Biomédicas, Departamento de Anatomia, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, RJ, Brazil
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  • Flávia Carvalho Alcantara Gomes
    Correspondence
    To whom correspondence should be addressed: Instituto de Ciências Biomédicas, Departamento de Anatomia, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco F, Ilha do Fundão 21941-590, Rio de Janeiro, RJ, Brazil. Tel.: 55-21-2562-6460;
    Affiliations
    From the Instituto de Ciências Biomédicas, Departamento de Anatomia, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, RJ, Brazil
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  • Author Footnotes
    * This work was supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior-Comité Francais d'Evaluation de la Coopération Universitaire avec le Brésil (CAPES-COFECUB), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho de Ensino para Graduados e Pesquisa-Universidade Federal do Rio de Janeiro (CEPG-UFRJ), and Programa de Apoio a Núcleos de Excelência2-Ministério de Ciência e Tecnologia (PRONEX2-MCT).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.
Open AccessPublished:September 27, 2002DOI:https://doi.org/10.1074/jbc.M209284200
      Thyroid hormone (T3) plays a crucial role in several steps of cerebellar ontogenesis. By using a neuron-astrocyte coculture model, we have investigated the effects of T3-treated astrocytes on cerebellar neuronal differentiation in vitro. Neurons plated onto T3-astrocytes presented a 40–60% increase on the total neurite length and an increment in the number of neurites. Treatment of astrocytes with epidermal growth factor (EGF) yielded similar results, suggesting that this growth factor might mediate T3-induced neuritogenesis. EGF and T3 treatment increased fibronectin and laminin expression by astrocytes, suggesting that astrocyte neurite permissiveness induced by these treatments is mostly due to modulation of extracellular matrix (ECM) components. Such increase in ECM protein expression as well as astrocyte permissiveness to neurite outgrowth was reversed by the specific EGF receptor tyrosine kinase inhibitor, tyrphostin. Moreover, studies using selective inhibitors of several transduction-signaling cascades indicated that modulation of ECM proteins by EGF is mainly through a synergistic activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. In this work, we provide evidence of a novel role of EGF as an intermediary factor of T3 action on cerebellar ontogenesis. By modulating the content of ECM proteins, EGF increases neurite outgrowth. Our data reveal an important role of astrocytes as mediators of T3-induced cerebellar development and partially elucidate the role of EGF and mitogen-activated protein kinase/phosphatidylinositol 3-kinase pathways on this process.
      Thyroid hormone (3,5,3′-triiodothyronine, T3)
      The abbreviations used are: T3, triiodothyronine; NS, nervous system; CNS, central nervous system; ECM, extracellular matrix protein; EGF, epidermal growth factor; EGFR, EGF receptor; EGL, external granular layer; MAPK, mitogen-activated protein (MAP) kinase; MEK, MAPK/extracellular signal regulated kinase kinase; PI3K, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; CM, conditioned medium
      1The abbreviations used are: T3, triiodothyronine; NS, nervous system; CNS, central nervous system; ECM, extracellular matrix protein; EGF, epidermal growth factor; EGFR, EGF receptor; EGL, external granular layer; MAPK, mitogen-activated protein (MAP) kinase; MEK, MAPK/extracellular signal regulated kinase kinase; PI3K, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; CM, conditioned medium
      is essential for normal development of the vertebrate nervous system (NS), influencing diverse processes of brain development such as neuronal migration, neurite outgrowth, synapse formation, myelination, and glial cell differentiation (
      • Bernal J.
      • Nunez J.
      ,
      • Lima F.R.S.
      • Gervais A.
      • Colin C.
      • Izembart M.
      • Moura Neto V.
      • Mallat M.
      ,
      • Gomes F.C.A.
      • Lima F.R.S.
      • Trentin A.G.
      • Moura Neto V.
      ,
      • Gomes F.C.A.
      • Spohr T.C.L.S.
      • Martinez R.
      • Moura Neto V.
      ,
      • Forrest D.
      • Reh T.A.
      • Rüsch A.
      ). Although the T3 role on central nervous system (CNS) morphogenesis is well documented, the precise mechanism of hormone action is not completely understood. To gain insights into T3 effects on CNS we have focused on the cerebellum ontogenesis, which is one of the most dramatically affected brain structures in hypothyroidism (
      • Nicholson J.L.
      • Altman J.
      ,
      • Morte B.
      • Manzano J.
      • Scanlan T.
      • Vennström B.
      • Bernal J.
      ).
      Most of the granular cells of the cerebellum arise from the external granular cell layer (EGL). Postnatally, these cells migrate from the premigratory zone of the EGL to the internal granular layer, leaving their axons behind to produce the molecular layer. These events are accompanied by a progressive morphological differentiation of Purkinje cells characterized by perisomatic extensions and dendritic trees (
      • Miale I.L.
      • Sidman R.L.
      ,
      • Komuro H.
      • Yacubova E.
      • Rakic P.
      ,
      • Anderson G.W.
      ). Although cerebellar histogenesis is well studied, the molecular mechanisms that control proliferation and differentiation of granular cells are still unknown. These processes have been shown to undergo dramatic modulation by thyroid hormone (
      • Nicholson J.L.
      • Altman J.
      ,
      • Anderson G.W.
      ,
      • Cayrou C.
      • Denver R.J.
      • Puymirat J.
      ). Besides a series of abnormalities found in the cerebellar cortex, hypothyroidism causes a decrease in EGL proliferation rate, increased neuronal death in the internal granular layer, impaired migration of granular cells, and a deficiency in the elaboration of Purkinje cell dendritic trees, spines, and synapses (
      • Nicholson J.L.
      • Altman J.
      ).
      Although a few genes have been shown to be directly modulated by T3 in the cerebellum, the molecular mechanism of T3 action on this brain region is still controversial (
      • Koibuchi N.
      • Fukuda H.
      • Chin W.W.
      ,
      • Anderson G.W.
      • Hagen S.G.
      • Larson R.J.
      • Strait K.A.
      • Schwartz H.L.
      • Mariash C.N.
      • Oppenheimer J.H.
      ,
      • Potter G.B.
      • Facchinetti F.
      • Beaudoin III, G.M.
      • Thompson C.C.
      ). It has been proposed that such endocrine regulation of cerebellar development might be the result of T3-dependent modulation of secretion of several growth factors such as neurotrophin 3, nerve growth factor, insulin growth factor, and brain-derived neurotrophic factor (
      • Beck K.D.
      • Knusel B.
      • Hefti F.
      ,
      • Figueiredo B.C.
      • Almazan G.
      • Ma Y.
      • Tetzlaff W.
      • Miller F.D.
      • Cuello A.C.
      ).
      Astrocytes have been pointed out as the major source of trophic factors in the CNS (
      • Garcia-Segura L.M.
      • Naftolin F.
      • Hutchison J.B.
      • Azcoitia I.
      • Chowen J.A.
      ,
      • Dong Y.
      • Benveniste E.M.
      ,
      • Gomes F.C.A.
      • Maia C.G.
      • Menezes J.R.L.
      • Moura Neto V.
      ). The fact that thyroid hormone treatment of astrocytes is associated in vitro with the secretion of several growth factors makes the astrocyte a putative candidate for mediating T3 action on neural histogenesis (
      • Gomes F.C.A.
      • Maia C.G.
      • Menezes J.R.L.
      • Moura Neto V.
      ,
      • Trentin A.G.
      • Alvarez-Silva M.
      • Moura Neto V.
      ). Recently, we described a novel mechanism for T3 action over granular neurons mediated by astrocytes. We demonstrated that cerebellar astrocytes treated by T3 secrete a combination of growth factors such as epidermal growth factor (EGF) and tumor necrosis factor-β, which induces proliferation of cerebellar granular neurons in vitro(
      • Gomes F.C.A.
      • Maia C.G.
      • Menezes J.R.L.
      • Moura Neto V.
      ).
      In the present work, we used an in vitro system of neuron-astrocyte coculture to assess the effects of T3 mediated by astrocytes on another step of cerebellar morphogenesis such as granule cell differentiation. We provide evidence that EGF secreted by astrocytes in response to T3 presents a binary role in cerebellar ontogenesis; acting directly on neurons, EGF promotes proliferation of granular cell precursors, and indirectly, EGF increases neuronal morphological differentiation by modulating the content of two astrocytic extracellular matrix (ECM) proteins, laminin and fibronectin. Furthermore, we suggest that EGF modulation of ECM proteins is mainly mediated by activation of MAPK and PI3K pathways. Together, our work gives glial cells a novel attribute as mediators of the endocrine-regulated cerebellar development and describes an additional role for EGF on brain morphogenesis.

      DISCUSSION

      In the present work, we provide the first evidence that EGF secreted by T3-treated astrocytes induces EGL neurons to undergo differentiation initiated by outgrowth of neurites. Such an event is mediated by EGF modulation of laminin and fibronectin astrocytic expression through MAPK and PI3K pathways. The present findings together with those previously described by us (
      • Gomes F.C.A.
      • Maia C.G.
      • Menezes J.R.L.
      • Moura Neto V.
      ) suggest a binary role for EGF on cerebellar ontogenesis, directly, on granular precursors proliferation and, indirectly, through ECM components in neurite outgrowth. Our data create a new scenario on the role of EGF and glial cells as mediators of T3 action on cerebellar development.
      Astrocytes have been well recognized as the major source of ECM components including fibronectin and laminin both in vivoand in vitro (
      • Garcia-Abreu J.
      • Cavalcante L.A.
      • Moura Neto V.
      ,
      • Menet V.
      • Giménez y Ribotta M.
      • Chauvet N.
      • Drian M.J.
      • Lannoy J.
      • Colucci-Guyon E.
      • Privat A.
      ,
      • Palu E.
      • Liesi P.
      ). The pattern of these ECM proteins on the astrocyte surface, which is highly modulated by thyroid hormone, provides directional cues to neurite outgrowth (
      • Garcia-Abreu J.
      • Cavalcante L.A.
      • Moura Neto V.
      ,
      • Freire E.
      • Gomes F.C.A.
      • Linden R.
      • Moura Neto V.
      • Coelho-Sampaio T.
      ,
      • Farwell A.P.
      • Dubord-Tomasetti S.A.
      ,
      • Calloni G.W.
      • Alvarez-Silva M.
      • Vituri C.
      • Trentin A.G.
      ,
      • Trentin A.G.
      • Moura Neto V.
      ).
      We now report that astrocytes treated by T3 or EGF greatly increased laminin and fibronectin fibrils in the extracellular space, thus providing a permissive substrate to neurite outgrowth. Our data contrast with those obtained from Farwell and Dubord-Tomasetti (
      • Farwell A.P.
      • Dubord-Tomasetti S.A.
      ), who demonstrated that T4 but not T3 increases laminin expression. We believe, however, that this apparent discrepancy between these two works most likely reflect fundamental differences in the technical approaches such as hormone treatment schedule, hormone concentration, and differences in culture conditions. Furthermore, those authors have cultured astrocytes derived from whole brain, whereas we have used in our study astrocytes derived from cerebellum. It has been speculated that spatial differences in the expression of T3 receptors account for the variety of T3 response elicited in brain structures (
      • Gomes F.C.A.
      • Lima F.R.S.
      • Trentin A.G.
      • Moura Neto V.
      ,
      • Lima F.R.S.
      • Gonçalves N.
      • Gomes F.C.A.
      • de Freitas M.S.
      • Moura Neto V.
      ).
      The addition of the EGFR tyrosine kinase inhibitor, tyrphostin, to T3-treated astrocytes greatly inhibited the ECM increment elicited by the hormone as well as impaired astrocyte permissivity to neurite extension. These data strongly suggest that T3-induced ECM augmentation in astrocytes is mediated by EGF. Furthermore, because no additive effects on neurite outgrowth were observed in astrocytes treated by EGF and T3 in combination, it seems likely that the two growth factors act probably through the same pathway, i.e. induction of ECM components. Because T3-astrocytes already produce EGF (
      • Gomes F.C.A.
      • Maia C.G.
      • Menezes J.R.L.
      • Moura Neto V.
      ), we assume that the addition of exogenous EGF raises the growth factor concentration beyond the saturation limit optimum for its effect.
      T3 has been proposed to directly modulate some ECM genes (
      • Alvarez-Dolado M.
      • González-Sancho J.M.
      • Bernal J.
      • Muñoz A.
      ,
      • Alvarez-Dolado M.
      • Ruiz M.
      • Del Rı́o J.A.
      • Alcántara S.
      • Burgaya F.
      • Sheldon M.
      • Nakajima K.
      • Bernal J.
      • Howell B.W.
      • Curran T.
      • Soriano E.
      • Muñoz A.
      ); however, a direct T3 regulation has not been undoubtedly reported for laminin and fibronectin. Our results do not completely rule out a T3 direct regulation of these proteins; however, they reveal an additional new mechanism for ECM protein modulation mediated by EGF in the NS. Together with ours, the recent finding that fibronectin mRNA is increased by activation of the EGFR in cardiac fibroblasts (
      • Matsubara H.
      • Moriguchi Y.
      • Mori Y.
      • Masaki H.
      • Tsutsumi Y.
      • Shibasaki Y.
      • Uchiyama-Tanaka Y.
      • Fujiyama S.
      • Koyama Y.
      • Nose-Fujiyama A.
      • Iba S.
      • Tateishi E.
      • Iwasaka T.
      ) and EGFR gene amplification is associated with laminin overexpression in tumor cell lines (
      • Ono Y.
      • Nakanishi Y.
      • Gotoh M.
      • Sakamoto M.
      • Hirotashi S.
      ) suggest that EGF modulation of laminin and fibronectin might be a more general process occurring in several tissues. We completely rule out a direct action of EGF on cerebellar neurite outgrowth since addition of EGF (data not shown) or T3CM (which contains EGF) directly on neuronal cultures does not increase neuritogenesis. This is the first time a T3 action on ECM protein expression and neuronal outgrowth mediated by an intermediary growth factor in NS is clearly described.
      EGF is implicated in widespread effects in CNS such as proliferation and differentiation of a variety of neuronal progenitors, postmitotic neurons, and glial cells (
      • Fricker-Gates R.A.
      • Winkler C.
      • Kirik D.
      • Rosenblad C.
      • Carpenter M.K.
      • Björklund A.
      ,
      • Yamada M.
      • Ikeuchi T.
      • Hatanaka H.
      ). EGF exerts most of its cellular actions through activation of the EGF receptor, which belongs to a family of structurally related tyrosine kinase receptors (
      • Carpenter G.
      ). Immunoreactivity for EGFR has been demonstrated in several regions of the embryonic and adult brains such as frontal cortex, hippocampus, cerebellum, and striatum (
      • Yamada M.
      • Ikeuchi T.
      • Hatanaka H.
      ,
      • Kornblum H.I.
      • Hussaim R.J.
      • Bronstein J.M.
      • Gall C.M.
      • Lee D.C.
      • Seroogy K.B.
      ), which support a role for EGF during brain development. Signaling through EGFR is triggered by ligand binding, receptor dimerization, and tyrosine phosphorylation and is classically associated with activation of the Raf-MEK-MAP/extracellular signal-regulated kinase pathway (
      • Yamada M.
      • Ikeuchi T.
      • Hatanaka H.
      ,
      • Danielsen A.J.
      • Maihle N.J.
      ). In our work, the specific inhibitor of MEK1/2 kinase, PD98059, greatly inhibited laminin and fibronectin overexpression induced by EGF. Similar results were yielded by administration of the specific EGFR inhibitor, tyrphostin, which suggested that MAPK pathway is activated downstream of EGFR tyrosine kinase (data not shown). Although the molecular mechanism of ECM modulation by EGF in NS has not been described yet, EGFR transactivation was found to up-regulate fibronectin in a MEK-extracellular signal-regulated kinase-dependent manner in other systems (
      • Kaiura T.L.
      • Itoh H.
      • Kent K.C.
      ,
      • Rescan C.
      • Coutant A.
      • Talarmin H.
      • Theret N.
      • Glaise D.
      • Guguen- Guillouzo C.
      • Baffet G.
      ,
      • Moriguchi Y.
      • Matsubara H.
      • Mori Y.
      • Murasawa S.
      • Masaki H.
      • Maruyama K.
      • Tsutsumi Y.
      • Shibasaki Y.
      • Tanaka Y.
      • Nakajima T.
      • Oda K.
      • Iwasaka T.
      ). Activation of EGFR is followed by induction of the Ras signaling pathway characterized by a kinase cascade, including Raf, MAPK kinase, and MAPK. It has been suggested that activated MAPK can translocate into the nucleus where it phosphorylates and activates several transcriptional factors (
      • Yamada M.
      • Ikeuchi T.
      • Hatanaka H.
      ). Recently, it has been demonstrated that the Ras-MAPK cascade described above is just one of the transcytoplasmic nuclear-signaling pathways activated by EGF (
      • Yart A.
      • Chap H.
      • Raynal P.
      ). This is the case of PI3K, the activity of which has been described to be stimulated by EGF. PI3Ks are a conserved family of lipid kinases that catalyze the phosphorylation of the 3′ position of the inositol ring of phosphoinositides (
      • Yart A.
      • Chap H.
      • Raynal P.
      ). They produce lipids implicated in several cellular processes. Although the mechanism involved in EGFR activation of MAP does not display an obvious role for PI3K, pharmacological inhibitors of PI3K were found to strongly interfere with MAPK pathways in several systems (
      • Yart A.
      • Chap H.
      • Raynal P.
      ,
      • Yart A.
      • Laffargue M.
      • Mayeux P.
      • Chretien S.
      • Peres C.
      • Tonks N.
      • Roche S.
      • Payrastre B.
      • Chap H.
      • Raynal P.
      ,
      • Carballada R.
      • Yasuo H.
      • Lemaire P.
      ,
      • Kim J.
      • Eckhart A.D.
      • Eguchi S.
      • Koch W.
      ). In agreement with these data, the addition of the PI3K pathway inhibitor, LY294002, completely abolished EGF-induced ECM overexpression. Recent evidence has been accumulated pointing a functional cross-talking between PI3K and MAP kinase pathways (
      • Yart A.
      • Chap H.
      • Raynal P.
      ,
      • Carballada R.
      • Yasuo H.
      • Lemaire P.
      ,
      • Kim J.
      • Eckhart A.D.
      • Eguchi S.
      • Koch W.
      ,
      • Fang X.
      • Yu S.
      • Eder A.
      • Mao M.
      • Bast Jr., R.C.
      • Boyd D.
      • Mills G.B.
      ,
      • Yu C.F.
      • Liu Z.-X.
      • Cantley L.G.
      ).
      Because we previously demonstrated that the effects of EGF on neuronal proliferation involved the protein kinase A-cAMP pathway, we sought to investigate the role of this pathway on EGF-induced neuritogenesis. The addition of the protein kinase A inhibitor KT5720 had no effect on EGF-induced ECM overexpression and neuritogenesis. Taken together, two models for the T3/EGF neuritogenesis induced by astrocytes might be proposed. Thyroid hormone induces cerebellar astrocytes to secrete EGF, which induces neuronal proliferation (Ref.
      • Gomes F.C.A.
      • Maia C.G.
      • Menezes J.R.L.
      • Moura Neto V.
      and Fig.9). By autocrine mechanism, EGF activates astrocytic EGFR. Transactivation of EGFR leads to 1) induction of PI3K followed by MAPK pathway activation, or 2) alternatively, EGFR may activate two separate cascades, a PI3K-dependent pathway and the classical MAPK pathway (Fig.9). The fact that the administration of LY294002 and PD98059 alone is sufficient to completely inhibit ECM overproduction, and concomitant addition does not yield additive inhibition (data not shown) call in favor of converging rather than independent pathways. Full elucidation of the molecular mechanisms implicating PI3K and MAPK pathways await further experiments.
      Figure thumbnail gr9
      Figure 9Schematic representation outlining the roles of MAPK and PI3K pathways in the EGF-induced cerebellar neuritogenesis. Thyroid hormone induces astrocytes to secrete EGF. Directly, EGF induces neuronal proliferation; indirectly, by modulating the content of astrocytic ECM proteins, EGF increases neurite outgrowth. EGF effects are triggered by EGFR tyrosine kinase signaling mediated by cooperation between MAPK and PI3K pathways. Two possibilities are depicted; 1) PI3K and MAPK cascades are independently activated, or 2) The two pathways cross-talk somewhere. The influences of general tyrosine kinase inhibition (genistein), EGFR inhibition (tyrphostin), classic MAPK pathway inhibition (PD98059), and PI3K pathway inhibition (LY294002) are shown.
      We reported a new attribute of EGF as mediator of thyroid hormone action on cerebellar development. Our results suggest that EGF might play a crucial role in distinct aspects of granular cell development in culture. How these in vitro results could account forin vivo cerebellar ontogenesis? Expression of the EGFR and T3 receptor does appear to be temporally uncoordinated in cerebellum. The early germinative zone of the EGL (E15–19) was not undoubtedly reported to express T3 receptor, which will be expressed later in the development in the postmitotic premigratory zone of EGL and in the internal granular layer (
      • Bradley D.J.
      • Towle H.C.
      • Young W.S.
      ), whereas EGFR mRNA is highly expressed in the EGL (
      • Seroogy K.B.
      • Gall C.M.
      • Lee D.C.
      • Kornblum H.I.
      ). These data highlight the importance of a mediator for T3 activity (possibly glia cells via EGF secretion) at least on these early events of cerebellum ontogenesis.
      Other factors also modulated by thyroid hormone such as the neurotrophins family have been also implicated in the regulation of several steps of cerebellar development (
      • Lindholm D.
      • Hammer S.
      • Zirrgiebel U.
      ). Our work points to EGF as an additional growth factor in the modulation of cerebellar granular cell ontogenesis, thus providing support for a multiple novel neurotrophic activity of growth factors in the development of cerebellar cortex. The fact that replacement of neurotrophin-3 or brain-derived neurotrophic factor results in some rescue of cerebellar development in hypothyroid animals (
      • Neveu I.
      • Arenas E.
      ) points to the possibility of using glia-derived growth factors as putative therapy to congenital hypothyroidism. Understanding the molecular relationship of thyroid hormones and neuron-astrocyte interactions could open in the future a new avenue to explore and rescue the abnormalities exhibited by the hypothyroid brain. Our work provides the first evidence that EGF secreted by astrocytes mediates thyroid hormone neuritogenesis in the cerebellum. The complexity of the processes underlying axonal growth suggests the existence of multiple sites of possible regulation. Therefore, it is likely that modulation of ECM proteins by EGF reported here in this paper might provide a potential mechanism by which this morphogenetic hormone exerts its effects on neurite outgrowth and establishment of neuronal connections.

      Acknowledgments

      We thank Adiel Batista do Nascimento for care and breeding of the animals. We are also in debt with Ângela Langer for technical assistance and João R. L. de Menezes for critically reading the manuscript. We specially thank Vivaldo Moura Neto for helpful discussion during the work.

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