RESUMEN
Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci.
Asunto(s)
Cromatina/metabolismo , Metilación de ADN , Proteínas Proto-Oncogénicas c-ret/genética , Activación Transcripcional , Tretinoina/farmacología , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Histona Desacetilasa 1/metabolismo , Humanos , Proteína 2 de Unión a Metil-CpG/metabolismo , Neuroblastoma , Complejo Represivo Polycomb 2 , Regiones Promotoras Genéticas , Receptores de Ácido Retinoico/metabolismo , Proteínas Represoras/metabolismo , Elementos de Respuesta , Receptor alfa de Ácido Retinoico , Complejo Correpresor Histona Desacetilasa y Sin3 , Factores de Transcripción/metabolismoRESUMEN
The ARF protein functions as an important sensor of hyper-proliferative stimuli restricting cell proliferation through both p53-dependent and -independent pathways. Although to date the majority of studies on ARF have focused on its anti-proliferative role, few studies have addressed whether ARF may also have pro-survival functions. Here we show for the first time that during the process of adhesion and spreading ARF re-localizes to sites of active actin polymerization and to focal adhesion points where it interacts with the phosphorylated focal adhesion kinase. In line with its recruitment to focal adhesions, we observe that hampering ARF function in cancer cells leads to gross defects in cytoskeleton organization resulting in apoptosis through a mechanism dependent on the Death-Associated Protein Kinase. Our data uncover a novel function for p14ARF in protecting cells from anoikis that may reflect its role in anchorage independence, a hallmark of malignant tumor cells.
Asunto(s)
Anoicis/fisiología , Adhesión Celular/fisiología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Adhesiones Focales/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Apoptosis/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Citoesqueleto/metabolismo , Citoesqueleto/fisiología , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Adhesiones Focales/fisiología , Células HeLa , Humanos , Células MCF-7 , Fosforilación/fisiología , Transducción de Señal/fisiologíaRESUMEN
Increasing evidence points to a role for dysfunctional glutamate N-methyl-D-aspartate receptor (NMDAR) neurotransmission in schizophrenia. D-aspartate is an atypical amino acid that activates NMDARs through binding to the glutamate site on GluN2 subunits. D-aspartate is present in high amounts in the embryonic brain of mammals and rapidly decreases after birth, due to the activity of the enzyme D-aspartate oxidase (DDO). The agonistic activity exerted by D-aspartate on NMDARs and its neurodevelopmental occurrence make this D-amino acid a potential mediator for some of the NMDAR-related alterations observed in schizophrenia. Consistently, substantial reductions of D-aspartate and NMDA were recently observed in the postmortem prefrontal cortex of schizophrenic patients. Here we show that DDO mRNA expression is increased in prefrontal samples of schizophrenic patients, thus suggesting a plausible molecular event responsible for the D-aspartate imbalance previously described. To investigate whether altered D-aspartate levels can modulate schizophrenia-relevant circuits and behaviors, we also measured the psychotomimetic effects produced by the NMDAR antagonist, phencyclidine, in Ddo knockout mice (Ddo(-)(/-)), an animal model characterized by tonically increased D-aspartate levels since perinatal life. We show that Ddo(-/-) mice display a significant reduction in motor hyperactivity and prepulse inhibition deficit induced by phencyclidine, compared with controls. Furthermore, we reveal that increased levels of D-aspartate in Ddo(-/-) animals can significantly inhibit functional circuits activated by phencyclidine, and affect the development of cortico-hippocampal connectivity networks potentially involved in schizophrenia. Collectively, the present results suggest that altered D-aspartate levels can influence neurodevelopmental brain processes relevant to schizophrenia.
Asunto(s)
Conducta Animal/efectos de los fármacos , D-Aspartato Oxidasa/genética , Antagonistas de Aminoácidos Excitadores/farmacología , Fenciclidina/farmacología , Corteza Prefrontal/metabolismo , Adulto , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatología , Estudios de Casos y Controles , D-Aspartato Oxidasa/metabolismo , Metilación de ADN , Modelos Animales de Enfermedad , Femenino , Neuroimagen Funcional , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/fisiopatología , Inhibición Prepulso/efectos de los fármacos , Inhibición Prepulso/genética , EsquizofreniaRESUMEN
Retinoic acid (RA) treatment of embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) induces growth arrest and terminal differentiation along the neuronal pathway. In the present study, we provide a functional link between RA and p27 function in the control of neuronal differentiation in NT2/D1 cells. We report that RA enhances p27 expression, which results in increased association with cyclin E/cyclin-dependent kinase 2 complexes and suppression of their activity; however, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by lack of neurite outgrowth and by the failure to modify surface antigens. As to the mechanism involved in RA-dependent p27 upregulation, our data support the concept that RA reduces p27 protein degradation through the ubiquitin/proteasome-dependent pathway. Taken together, these findings demonstrate that in embryonal carcinoma cells, p27 expression is required for growth arrest and proper neuronal differentiation.