RESUMEN
We have previously shown that low concentrations of a specific proteasome inhibitor accelerate exit from the cell cycle and enhance oligodendroglial cell (OLGc) differentiation. To elucidate the mechanisms involved in this process, OLGcs of the N20.1 cell line, transfected with a reporter gene driven by the MBP promoter, were treated with proteasome inhibitors and/or inhibitors of different signaling pathways. Partial proteasome inhibition resulted in enhanced activation of the MBP promoter which involved the tyrosine kinase, PI3-Akt and PKC pathways, accompanied by an increase in the levels of p21(Cip1), p27(Kip1) and Sp1 and by a decrease in Nkx2.2. Binding of Sp1 to DNA was also increased. These results were not observed when the Sp1 binding site was mutated. We conclude that the enhanced activation of the MBP promoter induced by partial inhibition of the proteasome could be due, at least in part, to the stabilization of p27(Kip1) and Sp1.
Asunto(s)
Diferenciación Celular/genética , Proteína Básica de Mielina/genética , Oligodendroglía/citología , Oligodendroglía/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteína Homeobox Nkx-2.2 , Inmunoprecipitación , Ratones , Proteína Básica de Mielina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Factor de Transcripción Sp1/metabolismo , TransfecciónRESUMEN
In order to further characterize the still unknown mechanism of cuprizone-induced demyelination, we investigated its effect on rat primary oligodendroglial cell cultures. Cell viability was not significantly affected by this treatment. However, when concentrations of IFNgamma and/or TNFalpha having no deleterious effects per se on cell viability were added together with cuprizone, cell viability decreased significantly. In mitochondria isolated from cuprizone-treated glial cells, we observed a marked decrease in the activities of the various complexes of the respiratory chain, indicating a disruption of mitochondrial function. An enhancement in oxidant production was also observed in cuprizone and/or TNFalpha-treated oligodendroglial cells. In in vivo experiments, inhibition of microglial activation with minocycline prevented cuprizone-induced demyelination. Based on the above-mentioned results we suggest that these microglial cells appear to have a very active role in cuprizone-induced oligodendroglial cell death and demyelination, through the production and secretion of pro-inflammatory cytokines.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Cuprizona/farmacología , Interferón gamma/metabolismo , Microglía/metabolismo , Oligodendroglía/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Medios de Cultivo Condicionados , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/prevención & control , Inmunohistoquímica , Masculino , Ratones , Minociclina/uso terapéutico , Oligodendroglía/citología , RatasRESUMEN
The selective degradation of abnormal or short half-life proteins in eukaryotic cells proceeds through the ubiquitin-mediated proteolytic system (UbPS). The signals that tag the proteins for their ubiquitination are well known. In the present study, our aim was to investigate the relationship between the action of ceramide and the changes in the expression of certain mRNAs of the Ub pathway and in the activation of the UbPS in cultured astrocytes (ASTs). Changes in the expression of components that are known to be substrates of the UbPS and that participate in the regulation of the cell death process were also studied. Addition of different concentrations of C2 ceramide to cultured ASTs produced an increase in the expression of the Ub gene and in the gene that encodes E1, one of the enzymes involved in the ubiquitination process, without any changes on cell viability. Immunocytochemical studies showed an increase in the expression of Bcl-2 with no changes in cytochrome c. Also, there was an increase in the nuclear reactivity of NFkappaB, suggesting a translocation of this factor towards the nucleus. Western blots showed a decrease in IkappaB and its phosphorylated form as well as an increase in Bcl-2 with no changes in cytochrome c. All of these compounds appear to be acting as possible modulators of AST responses to C2 ceramide. Our results suggest that in AST primary cultures, C2 ceramide, at the concentrations used in this study, does not produce apoptosis. However, it induces an activation of the UbPS, probably as a consequence of an activation of phosphatases and kinases, or through the generation of reactive oxygen species, which act as triggering signals of the UbPS. The fundamental role of NFkappaB and Bcl-2 as antiapoptotic factors is discussed.