RESUMO
Runx2 is a member of the Runx family of transcription factors (Runx1-3) with a restricted expression pattern. It has so far been detected predominantly in skeletal tissues where, inter alia, it regulates the expression of the beta-galactoside-specific lectin galectin-3. Here we show that, in contrast to Runx3, Runx1 and Runx2 are expressed in a variety of human glioma cells. Runx2 expression pattern in these cells correlated completely with that of galectin-3, but not with that of other galectins. A similar correlation in the expression pattern of galectin-3 and Runx2 transcripts was detected in distinct types of 70 primary neural tumors, such as glioblastoma multiforme, but not in others, such as gangliocytomas. In glioma cells, Runx2 is directly involved in the regulation of galectin-3 expression, as shown by RNAi and transcription factor binding assays demonstrating that Runx2 interacts with a Runx2-binding motif present in the human galectin-3 promoter. Knockdown of Runx2 was thus accompanied by a reduction of both galectin-3 mRNA and protein levels by at least 50%, dependent on the glial tumor cell line tested. Reverse transcriptase-polymerase chain reaction analyses, aimed at finding other potential target genes of Runx2 in glial tumor cells, revealed the presence of bone sialoprotein, osteocalcin, osteopontin, and osteoprotegerin. However, their expression patterns only partially overlap with that of Runx2. These data suggest a functional contribution of Runx-2-regulated galectin-3 expression to glial tumor malignancy.
Assuntos
Neoplasias Encefálicas/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Galectina 3/biossíntese , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioma/metabolismo , Western Blotting , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Expressão Gênica , Glioma/genética , Humanos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Chondroitin sulfate proteoglycan (CS-PG) expression is increased in response to CNS injury and limits the capacity for axonal regeneration. Previously we have shown that neurocan is one of the CS-PGs that is upregulated (Asher et al., 2000). Here we show that another member of the aggrecan family, versican, is also upregulated in response to CNS injury. Labeling of frozen sections 7 d after a unilateral knife lesion to the cerebral cortex revealed a clear increase in versican immunoreactivity around the lesion. Western blot analysis of extracts prepared from injured and uninjured tissue also revealed considerably more versican in the injured tissue extract. In vitro studies revealed versican to be a product of oligodendrocyte lineage cells (OLCs). Labeling was seen between the late A2B5-positive stage and the O1-positive pre-oligodendrocyte stage. Neither immature, bipolar A2B5-positive cells, nor differentiated, myelin-forming oligodendrocytes were labeled. The amount of versican in conditioned medium increased as these cells differentiated. Versican and tenascin-R colocalized in OLCs, and coimmunoprecipitation indicated that the two exist as a complex in oligodendrocyte-conditioned medium. Treatment of pre-oligodendrocytes with hyaluronidase led to the release of versican, indicating that its retention at the cell surface is dependent on hyaluronate (HA). In rat brain, approximately half of the versican is bound to hyaluronate. We also provide evidence of a role for CS-PGs in the axon growth-inhibitory properties of oligodendrocytes. Because large numbers of OLCs are recruited to CNS lesions, these results suggest that OLC-derived versican contributes to the inhospitable environment of the injured CNS.
Assuntos
Lesões Encefálicas/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Oligodendroglia/metabolismo , Regulação para Cima/fisiologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Lesões Encefálicas/patologia , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Células Cultivadas , Córtex Cerebral/lesões , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Proteoglicanas de Sulfatos de Condroitina/farmacologia , Meios de Cultivo Condicionados/metabolismo , Modelos Animais de Doenças , Feminino , Ácido Hialurônico/metabolismo , Imuno-Histoquímica , Lectinas Tipo C , Oligodendroglia/citologia , Testes de Precipitina , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologia , Células-Tronco/metabolismo , Tenascina/metabolismo , VersicanasRESUMO
J1-160 and J1-180 are developmentally late appearing J1 extracellular matrix glycoproteins derived from oligodendrocytes. They prevent adhesion of neurons (but not of astrocytes or fibroblasts) when offered as a substrate in mixture with laminin (Pesheva et al., J. Cell Biol., 109, 1765 - 1778, 1989). In the present study we have examined the influence of divalent cations on the inhibitory substrate properties of J1-160/180 glycoproteins towards adhesion of neurons. By metal chelate affinity chromatography, we show that J1-180, but not J1-160, binds Ca2+, while both J1 components are capable of binding Zn2+ and other divalent metal ions. Divalent cation binding was observed by gel filtration, aggregation assays with coated latex beads and electron microscopic examination to elicit aggregation of the molecules. Divalent cation binding also affects their non-permissive substrate properties towards neurons from early postnatal mouse cerebellum. Without divalent cations, J1-160 and J1-180 are inhibitory for substrate adhesion of neurons independently of the adhesive substrate present (laminin or poly-l-lysine). This effect is neutralized when J1-180 is preincubated with Ca2+ or Zn2+ prior to coating as substrate. In contrast, preincubation with Ca2+ ions does not affect the inhibitory substrate properties of J1-160 under these conditions. These observations show that J1-160/180 molecules may undergo self-aggregation in a divalent cation-dependent mechanism, which correlates with the neutralization of their inhibitory effect on neuronal adhesion. The aggregation state of the molecules may thus influence the process of myelination by a homophilic binding mechanism and determine the effectiveness of neurite extension during central nervous system development and under traumatic conditions in the adult.
RESUMO
Tenascin-R is an extracellular matrix glycoprotein that is restricted to the central nervous system, where it acts as a multifunctional and versatile molecule. We report spatial and temporal distribution of tenascin-R in the developing human cerebral cortex for the first time. At 7.5 gestational weeks (GW), tenascin-R was expressed in a restricted area of the basal telencephalon. At 9.5 and 11 GW, it showed a unique double band expression pattern that delineated the boundaries of the future cortical plate. From 14 to 30 GW, tenascin-R labeling extended to the whole cortex from the deep layers toward the marginal zone with an inside-to-outside progression pattern reminiscent of neuronal migration. Moreover, tenascin-R labeling initially appeared in the form of thin, straight, or slightly tortuous intercellular processes directed toward the surface in parallel with the axis of neuronal migration. At the end of pregnancy and at adulthood, diffuse and homogeneous immunolabeling of the whole cortex thickness was observed. The striatum and thalamus were faintly positive for TNR as early as 14 GW, and this positivity intensified with brain maturation. At all developmental stages, the germinative zone, the corpus callosum, the anterior commissure, and the internal capsule appeared clearly negative for tenascin-R immunostaining whereas the adjacent parenchyma was immunopositive. Our results show that tenascin-R expression is tightly regulated in a spatiotemporal manner during brain development, especially cortical plate formation. Its pattern of expression suggests a role for tenascin-R in corticogenesis.
Assuntos
Movimento Celular/fisiologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Neurônios/fisiologia , Tenascina/metabolismo , Adulto , Córtex Cerebral/citologia , Feminino , Idade Gestacional , Humanos , Neurônios/citologia , Gravidez , Trimestres da GravidezRESUMO
It is currently unclear whether retinal ganglion cell (RGC) axon regeneration depends on down-regulation of axon growth-inhibitory proteins, and to what extent outgrowth-promoting substrates contribute to RGC axon regeneration in reptiles. We performed an immunohistochemical study of the regulation of the axon growth-inhibiting extracellular matrix molecules tenascin-R and chondroitin sulphate proteoglycan (CSPG), the axon outgrowth-promoting extracellular matrix proteins fibronectin and laminin, and the axonal tenascin-R receptor protein F3/contactin during RGC axon regeneration in the lizard, Gallotia galloti. Tenascin-R and CSPG were expressed in an extracellular matrix-, oligodendrocyte/myelin- and neuron-associated pattern and up-regulated in the regenerating optic pathway. The expression pattern of tenascin-R was not indicative of a role in channeling or restriction of re-growing RGC axons. Up-regulation of fibronectin, laminin, and F3/contactin occurred in spatiotemporal patterns corresponding to tenascin-R expression. Moreover, we analyzed the influence of substrates containing tenascin-R, fibronectin, and laminin on outgrowth of regenerating lizard RGC axons. In vitro regeneration of RGC axons was not inhibited by tenascin-R, and further improved on mixed substrates containing tenascin-R together with fibronectin or laminin. These results indicate that RGC axon regeneration in Gallotia galloti does not require down-regulation of tenascin-R or CSPG. Presence of tenascin-R is insufficient to prevent RGC axon growth, and concomitant up-regulation of axon growth-promoting molecules like fibronectin and laminin may override the effects of neurite growth inhibitors on RGC axon regeneration. Up-regulation of contactin in RGCs suggests that tenascin-R may have an instructive function during axon regeneration in the lizard optic pathway.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Regeneração Nervosa/fisiologia , Tenascina/metabolismo , Regulação para Cima/fisiologia , Vias Visuais/metabolismo , Vias Visuais/fisiopatologia , Fatores Etários , Animais , Animais Recém-Nascidos , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas do Olho/metabolismo , Lateralidade Funcional , Gânglios Espinais/citologia , Lagartos/anatomia & histologia , Lagartos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/transplante , Traumatismos do Nervo Óptico/fisiopatologia , Ratos , Retina/fisiologia , Retina/transplante , Tenascina/genética , Fatores de TempoRESUMO
Axon growth inhibitory CNS matrix proteins, such as tenascin-R (TN-R), have been supposed to contribute to the poor regenerative capacity of adult mammalian CNS. With regard to TN-R function in low vertebrates capable of CNS regeneration, questions of particular interest concern the (co)evolution of ligand-receptor pairs and cellular response mechanisms associated with axon growth inhibition and oligodendrocyte differentiation. We address here these questions in a series of comparative in vivo and in vitro analyses using TN-R proteins purified from different vertebrates (from fish to human). Our studies provide strong evidence that unlike TN-R of higher vertebrates, fish TN-R proteins are not repellent for fish and less repellent for mammalian neurons and do not interfere with F3/contactin- and fibronectin-mediated mammalian cell adhesion and axon growth. However, axonal repulsion is induced in fish neurons by mammalian TN-R proteins, suggesting that the intracellular inhibitory machinery induced by TN-R-F3 interactions is already present during early vertebrate evolution. In contrast to TN-R-F3, TN-R-sulfatide interactions, mediating oligodendrocyte adhesion and differentiation, are highly conserved during vertebrate evolution. Our findings thus indicate the necessity of being cautious about extrapolations of the function of ligand-receptor pairs beyond a species border and, therefore, about the phylogenetic conservation of a molecular function at the cellular/tissue level.
Assuntos
Sistema Nervoso Central/metabolismo , Evolução Molecular , Filogenia , Tenascina/metabolismo , Vertebrados/metabolismo , Animais , Axônios/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Moléculas de Adesão Celular Neuronais/farmacologia , Células Cultivadas , Sistema Nervoso Central/citologia , Contactinas , Ensaio de Imunoadsorção Enzimática/métodos , Fibronectinas/farmacologia , Humanos , Imuno-Histoquímica/métodos , Técnicas In Vitro , Indóis , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sulfoglicoesfingolipídeos/farmacologia , Vertebrados/classificaçãoRESUMO
Tenascin-R (TN-R) is a member of the tenascin family of multidomain matrix glycoproteins that is expressed exclusively in the central nervous system by oligodendrocytes and small neurons during postnatal development and in the adult. TN-R contributes to the regulation of axon extension and regeneration, neurite formation and synaptogenesis, and neuronal growth and migration. TN-R can be modified with three distinct sulfated oligosaccharide structures: HNK-1 (SO(4)-3-GlcUAbeta1,3Galbeta1,4GlcNAc), GalNAc-4-SO(4), and chondroitin sulfate. We have determined that TN-R expressed in dendrite-rich regions of the rat cerebellum, hippocampus, and cerebral cortex is one of the major matrix glycoproteins that bears N-linked carbohydrates terminating with beta1,4-linked GalNAc-4-SO(4). The syntheses of these unique sulfated structures on TN-R are differentially regulated. Levels of HNK-1 on TN-R rise and fall in parallel to the levels of TN-R during postnatal development of the cerebellum. In contrast, levels of GalNAc-4-SO(4) are regulated independently from those of TN-R, rising late in cerebellar development and continuing into adulthood. As a result, the pattern of TN-R modification with distinct sulfated carbohydrate structures changes dramatically over the course of postnatal cerebellar development in the rat. Because TN-R interacts with a number of different matrix components and, depending on the circumstances, can either activate or inhibit neurite outgrowth, the highly regulated addition of these unique sulfated structures may modulate the adhesive properties of TN-R over the course of development and during synapse maintenance. In addition, the 160-kDa form of TN-R is particularly enriched for terminal GalNAc-4-SO(4) later in development and in the adult, suggesting additional levels of regulation.
Assuntos
Neurônios/metabolismo , Tenascina/química , Animais , Western Blotting , Encéfalo/embriologia , Encéfalo/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Sulfatos de Condroitina/química , Cisteína/química , Eletroforese em Gel de Poliacrilamida , Glicosilação , Hipocampo/metabolismo , Humanos , Ligantes , Camundongos , Oligossacarídeos/química , Peptídeos/química , Testes de Precipitina , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Ratos , Ressonância de Plasmônio de Superfície , Sinapses/metabolismo , Tenascina/metabolismo , Fatores de Tempo , Tripsina/químicaRESUMO
Galectin-3 (gal-3) is a member of the galectin family of lectins whose expression strongly depends on the cellular state. Here we show that in PC12 cells the expression of gal-3 protein is regulated via Ras- and mitogen-activated protein kinase (MAPK)-dependent and independent signalling pathways and correlates with nerve growth factor (NGF)-mediated neuronal differentiation. Gal-3 expression, activation of the MAPK ERK1/2 and neurite outgrowth are induced by NGF and basic fibroblast growth factor (bFGF), but not by ciliary neurotrophic factor (CNTF), epidermal growth factor, insulin or interleukin-6 (IL-6). In addition, in NGF-treated PC12 cells, gal-3 expression, ERK1/2 activation and neurite outgrowth could be specifically inhibited at the level of TrkA, Ras and MAPK-kinase, whereas expression of an oncogenic form of Ras leads to gal-3 expression and neurite outgrowth in the absence of growth factors. In NGF-primed PC12 cells, subsequent treatment with CNTF or IL-6 induces ERK1/2 activation and neurite outgrowth, but not gal-3 expression. Treatment of PC12 cells with staurosporine induces gal-3 expression and neurite outgrowth without ERK1/2 activation. NGF- and staurosporine-induced gal-3-expression is also regulated at the transcriptional level. Our data suggest the presence of complex induction mechanisms of gal-3 expression in neuronally differentiating PC12 cells involving NGF-, but not CNTF- and IL-6-driven (in NGF-primed cells) Ras/MAPK-related signalling pathways. Staurosporine, in contrast, induces gal-3 expression by a Ras/MAPK-independent mechanism.
Assuntos
Galectina 3/biossíntese , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Proteínas ras/metabolismo , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Fator Neurotrófico Ciliar/farmacologia , Inibidores Enzimáticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Galectina 3/análise , Galectina 3/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Células PC12 , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/químicaRESUMO
Lipid rafts are microdomains of the plasma membrane which are enriched in glycosphingolipids and specific proteins. The reported interactions of several raft-associated proteins (such as, e.g., F3) with tenascin C and tenascin R prompted us to consider that these oligomeric multidomain glycoproteins of the extracellular matrix (ECM) could associate with rafts. Here, we show punctate immunocytochemical distributions of tenascin C (TN-C) and tenascin R (TN-R) at the membrane surface of neural cells resembling the pattern reported for raft-associated proteins. Moreover, cholesterol depletion with methyl-beta-cyclodextrin reduced the punctate surface staining of TN-C. Consistently, TN-C was associated with lipid rafts of neonatal mouse brain according to sucrose density gradient centrifugation experiments. Furthermore, TN-R was also found in rafts prepared from myelin of adult mice. Thus, brain-derived tenascins are able to associate with lipid rafts.
Assuntos
Microdomínios da Membrana/metabolismo , Neurônios/metabolismo , Tenascina/metabolismo , Animais , Química Encefálica , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Feminino , Camundongos , Neurônios/ultraestrutura , GravidezRESUMO
PURPOSE: A major reason for the poor functional recovery after peripheral nerve injury is the outgrowth of supernumerary axonal branches at the lesion site. Projecting within several nerve fascicles, the branches of one axon often re-innervate synchronously muscles with antagonis-tic functions and impair any coordinated activity. We hypothetized that accelerated axonal elongation through extracellular matrix proteins fos-tering neurite outgrowth might reduce axonal branching and improve recovery of function. METHODS: In a control group of rats, ramus zygomaticus, ramus buccalis, and ramus marginalis mandibulae of the facial nerve were transected and the stumps labeled with DiI, Fluoro-Gold (FG), and Fast Blue (FB). RESULTS: Neuron counts showed that the zygomatic ramus contained axons of 204 +/- 88 DiI-labeled motoneurons in the dorsal facial subnu-cleus. No perikarya were labeled by 2 or 3 tracers. After transection and suture of the facial nerve trunk, the zygomatic ramus contained axons of 328 +/- 50 motoneurons dispersed throughout the whole facial nucleus. The occurrence of double-labeled (DiI+FG and DiI+FB) motoneu-rons showed that about 30 % of all axons in the zygomatic ramus had a twin branch projecting within the buccal and/or mandibular ramus. CONCLUSIONS: Entubulation of transected facial nerve in a silicone tube containing phosphate buffered saline, collagen type I, laminin, fibronectin, or tenascin did not reduce the portion of double-labeled motoneurons. We conclude that (i) axonal branching follows a rather con-stant pattern regardless of changes in the local microenvironment; (ii) despite their known effect to support neurite outgrowth, all tested extra-cellular matrix proteins do not suppress axonal branching in the rat facial nerve model.