RESUMEN
Nitric oxide (NO) has been shown to play a crucial role in bone formation in vivo. We sought to determine the temporal effect of NO on murine embryonic stem cells (ESCs) under culture conditions that promote osteogenesis. Expression profiles of NO pathway members and osteoblast-specific markers were analyzed using appropriate assays. We found that NO was supportive of osteogenesis specifically during an early phase of in vitro development (days 3-5). Furthermore, ESCs stably overexpressing the inducible NO synthase showed accelerated and enhanced osteogenesis in vitro and in bone explant cultures. To determine the role of NO in early lineage commitment, a stage in ESC differentiation equivalent to primitive streak formation in vivo, ESCs were transfected with a T-brachyury-GFP reporter. Expression levels of T-brachyury and one of its upstream regulators, ß-catenin, the major effector in the canonical Wnt pathway, were responsive to NO levels in differentiating primitive streak-like cells. Our results indicate that NO may be involved in early differentiation through regulation of ß-catenin and T-brachyury, controlling the specification of primitive-streak-like cells, which may continue through differentiation to later become osteoblasts.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Óxido Nítrico/metabolismo , Osteogénesis , Línea Primitiva/embriología , beta Catenina/metabolismo , Animales , Recuento de Células , Diferenciación Celular/efectos de los fármacos , GMP Cíclico/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas Fetales/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Cloruro de Litio/farmacología , Ratones , Ratones Endogámicos C57BL , Minerales/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Osteogénesis/efectos de los fármacos , Fosfatidilserinas/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Línea Primitiva/citología , Línea Primitiva/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas de Dominio T Box/metabolismo , Factores de Tiempo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Hippocampal CA1 pyramidal neurons undergo delayed neurodegeneration after transient forebrain ischemia, and the phenomenon is dependent upon hyperactivation of l-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of glutamate receptors, resulting in aberrant intracellular calcium influx. The GluR2 subunit of AMPA receptors is critical in limiting the influx of calcium. The CA1 pyramidal neurons are very sensitive to ischemic damage and attempts to achieve neuroprotection, mediated by drugs, have been unsuccessful. Moreover, receptor antagonism strategies in the past have failed to provide long-term protection against ischemic injury. Long-term protection against severe forebrain ischemia can be conferred by fimbria-fornix (FF) deafferentation, which interrupts the afferent input to CA1. Our study evaluated the long-term protective effect of FF deafferentation, 12 days prior to induction of ischemia, on vulnerable CA1 neurons. Our results indicate that at 7 and 28 days post-ischemia, prior FF deafferentation protected 60% of neurons against ischemic cell death. Furthermore, we sought to evaluate whether FF deafferentation also sustained GluR2 levels in these neurons. GluR2 protein and mRNA expression were sustained by deafferentation at 70% of control following ischemia. Correlation studies revealed a positive correlation between GluR2 protein and mRNA level. These results demonstrate that protection conferred by FF deafferentation was long-term and related to sustained GluR2 expression.