RESUMEN
Intellectual disability in Down syndrome (DS) appears to be related to severe neurogenesis impairment during brain development. The molecular mechanisms underlying this defect are still largely unknown. Accumulating evidence has highlighted the importance of GSK3ß signaling for neuronal precursor proliferation/differentiation. In neural precursor cells (NPCs) from Ts65Dn mice and human fetuses with DS, we found reduced GSK3ß phosphorylation and, hence, increased GSK3ß activity. In cultures of trisomic subventricular-zone-derived adult NPCs (aNPCs) we found that deregulation of GSK3ß activity was due to higher levels of the AICD fragment of the trisomic gene APP that directly bound to GSK3ß. We restored GSK3ß phosphorylation in trisomic aNPCs using either lithium, a well-known GSK3ß inhibitor, or using a 5-HT receptor agonist or fluoxetine, which activated the serotonin receptor 5-HT1A. Importantly, this effect was accompanied by restoration of proliferation, cell fate specification and neuronal maturation. In agreement with results obtained in vitro, we found that early treatment with fluoxetine, which was previously shown to rescue neurogenesis and behavior in Ts65Dn mice, restored GSK3ß phosphorylation. These results provide a link between GSK3ß activity alteration, APP triplication and the defective neuronal production that characterizes the DS brain. Knowledge of the molecular mechanisms underlying neurogenesis alterations in DS may help to devise therapeutic strategies, potentially usable in humans. Results suggest that drugs that increase GSK3ß phosphorylation, such as lithium or fluoxetine, may represent useful tools for the improvement of neurogenesis in DS.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/enzimología , Síndrome de Down/enzimología , Glucógeno Sintasa Quinasa 3/metabolismo , Células-Madre Neurales/enzimología , Neurogénesis , Animales , Encéfalo/metabolismo , Proliferación Celular , Modelos Animales de Enfermedad , Síndrome de Down/metabolismo , Femenino , Feto , Glucógeno Sintasa Quinasa 3 beta , Humanos , Ratones , Ratones Transgénicos , Células-Madre Neurales/metabolismo , FosforilaciónRESUMEN
Mental retardation in Down syndrome (DS) appears to be related to severe neurogenesis impairment during critical phases of brain development. Recent lines of evidence in the cerebellum of a mouse model for DS (the Ts65Dn mouse) have shown a defective responsiveness to Sonic Hedgehog (Shh), a potent mitogen that controls cell division during brain development, suggesting involvement of the Shh pathway in the neurogenesis defects of DS. Based on these premises, we sought to identify the molecular mechanisms underlying derangement of the Shh pathway in neural precursor cells (NPCs) from Ts65Dn mice. By using an in vitro model of NPCs obtained from the subventricular zone and hippocampus, we found that trisomic NPCs had an increased expression of the Shh receptor Patched1 (Ptch1), a membrane protein that suppresses the action of a second receptor, Smoothened (Smo), thereby maintaining the pathway in a repressed state. Partial silencing of Ptch1 expression in trisomic NPCs restored cell proliferation, indicating that proliferation impairment was due to Ptch1 overexpression. The overexpression of Ptch1 in trisomic NPCs resulted from increased levels of AICD [a transcription-promoting fragment of amyloid precursor protein (APP)] and increased AICD binding to the Ptch1 promoter. Our data provide novel evidence that Ptch1 overexpression underlies derangement of the Shh pathway in trisomic NPCs with consequent proliferation impairment. The demonstration that Ptch1 overexpression in trisomic NPCs is due to an APP fragment provides a link between this trisomic gene and the defective neuronal production that characterizes the DS brain.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Síndrome de Down/genética , Células-Madre Neurales/fisiología , Neuronas/fisiología , Receptores de Superficie Celular/biosíntesis , Acetilación , Animales , Ciclo Celular/genética , Proliferación Celular , Ciclohexilaminas/farmacología , Metilación de ADN , Síndrome de Down/embriología , Síndrome de Down/metabolismo , Femenino , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/farmacología , Hipocampo/embriología , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ventrículos Laterales/embriología , Ventrículos Laterales/metabolismo , Ventrículos Laterales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Receptores Patched , Receptor Patched-1 , Complejo Represivo Polycomb 1 , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Proteínas Represoras/genética , Receptor Smoothened , Tiofenos/farmacología , Regulación hacia Arriba , Alcaloides de Veratrum/farmacología , Proteína con Dedos de Zinc GLI1 , Proteína Gli2 con Dedos de Zinc , Proteína Gli3 con Dedos de ZincRESUMEN
BACKGROUND: Basal-like carcinoma are aggressive breast cancers that frequently carry p53 inactivating mutations, lack estrogen receptor-α (ERα) and express the cancer stem cell markers CD133 and CD44. These tumors also over-express Interleukin 6 (IL-6), a pro-inflammatory cytokine that stimulates the growth of breast cancer stem/progenitor cells. RESULTS: Here we show that p53 deficiency in breast cancer cells induces a loss of methylation at IL-6 proximal promoter region, which is maintained by an IL-6 autocrine loop. IL-6 also elicits the loss of methylation at the CD133 promoter region 1 and of CD44 proximal promoter, enhancing CD133 and CD44 gene transcription. In parallel, IL-6 induces the methylation of estrogen receptor (ERα) promoter and the loss of ERα mRNA expression. Finally, IL-6 induces the methylation of IL-6 distal promoter and of CD133 promoter region 2, which harbour putative repressor regions. CONCLUSION: We conclude that IL-6, whose methylation-dependent autocrine loop is triggered by the inactivation of p53, induces an epigenetic reprogramming that drives breast carcinoma cells towards a basal-like/stem cell-like gene expression profile.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Interleucina-6/metabolismo , Antígeno AC133 , Antígenos CD/genética , Antígenos CD/metabolismo , Western Blotting , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Metilación de ADN/fisiología , Ensayo de Inmunoadsorción Enzimática , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Interleucina-6/genética , Interleucina-6/farmacología , Péptidos/genética , Péptidos/metabolismo , Interferencia de ARN , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The endocannabinoid system is involved in the regulation of many physiological effects in the central and peripheral nervous system. Recent findings have demonstrated the presence of a functional endocannabinoid system within neuronal progenitors located in the hippocampus and ventricular/subventricular zone that participates in the regulation of cell proliferation. It is presently unknown whether the endocannabinoid system exerts a widespread effect on neuronal precursors from different neurogenic regions, and very little is known about the signaling by which it regulates neuronal precursor proliferation. Herein, we demonstrate the presence of cannabinoid CB(1) receptors in granule cell precursors (GCPs) during early cerebellar development. Activation of CB(1) receptors by HU-210 promoted GCP proliferation in vitro, an effect that was prevented by a selective CB(1) antagonist. Accordingly, in vivo experiments showed that GCP proliferation was increased by chronic HU-210 treatment and that in CB(1)-deficient mice cell proliferation was significantly lower than in wild-type littermates, indicating that the endocannabinoid system is physiologically involved in regulation of GCP proliferation. The pro-proliferative effect of cannabinoids in GCPs was mediated through the CB(1)/AKT/glycogen synthase kinase-3beta/beta-catenin pathway. Involvement of this pathway was also observed in cultures of neuronal precursors from the subventricular zone, suggesting that this pathway may be a general mechanism by which endocannabinoids regulate proliferation of neuronal precursors. These observations suggest that endocannabinoids constitute a new family of lipid signaling cues that may exert a widespread effect on neuronal precursor proliferation during brain development.
