RESUMEN
Heparan sulfate (HS) chains, covalently linked to heparan sulfate proteoglycans (HSPG), promote synaptic development and functions by connecting various synaptic adhesion proteins (AP). HS binding to AP could vary according to modifications of HS chains by different sulfotransferases. 3-O-sulfotransferases (Hs3sts) produce rare 3-O-sulfated HSs (3S-HSs), of poorly known functions in the nervous system. Here, we showed that a peptide known to block herpes simplex virus by interfering with 3S-HSs in vitro and in vivo (i.e. G2 peptide), specifically inhibited neural activity, reduced evoked glutamate release, and impaired synaptic assembly in hippocampal cell cultures. A role for 3S-HSs in promoting synaptic assembly and neural activity is consistent with the synaptic interactome of G2 peptide, and with the detection of Hs3sts and their products in synapses of cultured neurons and in synaptosomes prepared from developing brains. Our study suggests that 3S-HSs acting as receptors for herpesviruses might be important regulators of neuronal and synaptic development in vertebrates.
Asunto(s)
Proteoglicanos de Heparán Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Hipocampo/metabolismo , Sulfotransferasas/metabolismo , Sinapsis/metabolismo , Animales , Células Cultivadas , Ratones , Neurogénesis/fisiología , Neuronas/metabolismoRESUMEN
BACKGROUND: Thioredoxin (TRX)-1, a ubiquitous 12-kDa protein, exerts antioxidant and anti-inflammatory effects. In contrast, the truncated form, called TRX80, produced by macrophages induces upregulation of proinflammatory cytokines. TRX80 also promotes the differentiation of mouse peritoneal and human macrophages toward a proinflammatory M1 phenotype. METHODS: TRX1 and TRX80 plasma levels were determined with a specific ELISA. A disintegrin and metalloproteinase domain-containing protein (ADAM)-10, ADAM-17, and ADAM-10 activities were measured with SensoLyte 520 ADAM10 Activity Assay Kit, Fluorimetric, and InnoZyme TACE Activity Kit, respectively. Western immunoblots were performed with specific antibodies to ADAM-10 or ADAM-17. Angiogenesis study was evaluated in vitro with human microvascular endothelial cells-1 and in vivo with the Matrigel plug angiogenesis assay in mice. The expression of macrophage phenotype markers was investigated with real-time polymerase chain reaction. Phosphorylation of Akt, mechanistic target of rapamycin, and 70S6K was determined with specific antibodies. The effect of TRX80 on NLRP3 inflammasome activity was evaluated by measuring the level of interleukin-1ß and -18 in the supernatants of activated macrophages with ELISA. Hearts were used for lesion surface evaluation and immunohistochemical studies, and whole descending aorta were stained with Oil Red O. For transgenic mice generation, the human scavenger receptor (SR-A) promoter/enhancer was used to drive macrophage-specific expression of human TRX80 in mice. RESULTS: In this study, we observed a significant increase of plasma levels of TRX80 in old subjects compared with healthy young subjects. In parallel, an increase in expression and activity of ADAM-10 and ADAM-17 in old peripheral blood mononuclear cells compared with those of young subjects was observed. Furthermore, TRX80 was found to colocalize with tumor necrosis factor-α, a macrophage M1 marker, in human atherosclerotic plaque. In addition, TRX80 induced the expression of murine M1 macrophage markers through Akt2/mechanistic target of rapamycin-C1/70S6K pathway and activated the inflammasome NLRP3, leading to the release of interleukin-1ß and -18, potent atherogenic cytokines. Moreover, TRX80 exerts a powerful angiogenic effect in both in vitro and in vivo mouse studies. Finally, transgenic mice that overexpress human TRX80 specifically in macrophages of apoE-/- mice have a significant increase of aortic atherosclerotic lesions. CONCLUSIONS: TRX80 showed an age-dependent increase in human plasma. In mouse models, TRX80 was associated with a proinflammatory status and increased atherosclerosis.
Asunto(s)
Envejecimiento , Aterosclerosis/patología , Fragmentos de Péptidos/sangre , Tiorredoxinas/sangre , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Adulto , Anciano , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Inflamación , Interleucina-18/sangre , Interleucina-1beta/sangre , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Tiorredoxinas/farmacologíaRESUMEN
The development of the central nervous system requires an appropriate micro-environment that is conditioned by a combination of various extracellular components. Most of the known signaling factors, such as neurotransmitters or neuropeptides, are soluble and diffuse into the extracellular matrix. However, other secreted molecules like proteoglycans or glycosaminoglycans anchor in the extracellular matrix to influence cerebral ontogenesis. As such, pleiotrophin (PTN), which binds the proteoglycans syndecan-3 (SDC3) and protein tyrosine phosphatase zeta (PTPζ), has been described as a pro-migratory and a pro-differentiating secreted cytokine on cortical neurons. In rat cerebellum, PTN is highly expressed during the first postnatal week, suggesting that this cytokine could participate to the development of the cerebellar cortex. According to this hypothesis, our spatio-temporal cartography of PTN, PTPζ and SDC3 indicated that, in mouse, the PTNergic system was present in the cerebellum at least from the first postnatal day (P0). Until P12, PTN was mainly expressed by granule cell precursors and located in the extracellular matrix, while SDC3 was expressed by Purkinje cells, Golgi cells and granule cell precursors, and PTPζ was present on Purkinje cells and Bergmann fibers. In vitro studies confirmed the presence of SDC3 on immature granule cells and demonstrated that PTN could stimulate directly their velocity in culture. In contrast, subarachnoidal injection of PTN in the cerebellum significantly reduced the rate of migration of granule cells, exacerbated their apoptosis and induced an atrophy of the Purkinje cell dendritic tree. Since differentiated granule cells did not express SDC3 or PTPζ, the PTN effect observed on migration and apoptosis may be indirectly mediated by Purkinje and/or Bergmann cells. From P21 to adulthood, the distribution of PTN, SDC3 and PTPζ changed and their expression dramatically decreased even if they were still detectable. PTN and SDC3 immunolabeling was restricted around Purkinje cell bodies and Golgi cells, whereas PTPζ was located around interneurons. These data suggested that, in the cerebellum of adult mice, PTN participates to the perineuronal nets that control neuronal plasticity. To conclude, the present work represents the first spatio-temporal characterization of the PTNergic system in the mouse cerebellum and indicates that PTN may contribute to cerebellum ontogenesis during the postnatal development as well as to neuronal plasticity at adulthood.
Asunto(s)
Proteínas Portadoras/metabolismo , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Citocinas/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores de Edad , Animales , Calbindinas/metabolismo , Proteínas Portadoras/farmacología , Caspasa 3/metabolismo , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Citocinas/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato Descarboxilasa/metabolismo , Técnicas In Vitro , Captura por Microdisección con Láser , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Estadísticas no Paramétricas , Sindecano-3/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
We hypothesized that kallikrein-related peptidase 14 (KLK14) is produced by colonic tumors and can promote tumorigenesis by activating proteinase-activated receptors (PARs). We found that KLK14 is expressed in human colon adenocarcinoma cells but not in adjacent cancer-free tissue; KLK14 mRNA, present in colon cancer, leads to KLK14 protein expression and secretion; and KLK14 signals viaPAR-2 in HT-29 cells to cause (1) receptor activation/internalization, (2) increases in intracellular calcium, (3) stimulation of ERK1/2/MAP kinase phosphorylation, and (4) cell proliferation. We suggest that KLK14, acting via PAR-2, represents an autocrine/paracrine regulator of colon tumorigenesis.