RESUMEN
Engineered silica nanoparticles (NPs) have attracted increasing interest in several applications, and particularly in the field of nanomedicine, thanks to the high biocompatibility of this material. For their optimal and controlled use, the understanding of the mechanisms elicited by their interaction with the biological target is a prerequisite, especially when dealing with cells particularly vulnerable to environmental stimuli like neurons. Here we have combined different electrophysiological approaches (both at the single cell and at the population level) with a genomic screening in order to analyze, in GT1-7 neuroendocrine cells, the impact of SiO2 NPs (50 ± 3 nm in diameter) on electrical activity and gene expression, providing a detailed analysis of the impact of a nanoparticle on neuronal excitability. We find that 20 µg mL-1 NPs induce depolarization of the membrane potential, with a modulation of the firing of action potentials. Recordings of electrical activity with multielectrode arrays provide further evidence that the NPs evoke a temporary increase in firing frequency, without affecting the functional behavior on a time scale of hours. Finally, NPs incubation up to 24 hours does not induce any change in gene expression.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Nanopartículas , Células Neuroendocrinas/efectos de los fármacos , Neuronas/metabolismo , Dióxido de Silicio/farmacología , Animales , Línea Celular , Expresión Génica/efectos de los fármacos , Hipotálamo/citología , Ratones , Células Neuroendocrinas/fisiología , Neuronas/efectos de los fármacosRESUMEN
The dystrophin gene transcription is up-regulated during muscle cell differentiation. Its expression in muscle cells is induced by the binding of the positive regulators serum response factor and dystrophin promoter bending factor (DPBF) on a regulatory CArG element present on the promoter. Here we show that the dystrophin CArG box is also recognized by the zinc finger nuclear factor YY1. Transient transfection experiments show that YY1 negatively regulates dystrophin transcription in C2C12 muscle cells. On the dystrophin CArG element YY1 competes with the structural factor DPBF. We further show that YY1 and DPBF binding to the CArG element induce opposite DNA bends suggesting that their binding induces alternative promoter structures. Along with C2C12 myotube formation YY1 is reduced and we observed that YY1, but not DPBF, is a substrate of m-calpain, a protease that is up-regulated in muscle cell differentiation. Thus, high levels of YY1 in non-differentiated muscle cells down-regulate the dystrophin promoter, at least in part, by interfering with the spatial organization of the promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Distrofina/genética , Regulación de la Expresión Génica , Músculos/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Secuencia de Bases , Calpaína/metabolismo , Línea Celular , Cartilla de ADN , Distrofina/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Hidrólisis , Músculos/citología , Unión Proteica , Transcripción Genética , Factor de Transcripción YY1RESUMEN
Protein kinase CK2 is an ubiquitous and pleiotropic Ser/Thr protein kinase composed of two catalytic (alpha and/or alpha') and two noncatalytic (beta) subunits forming a heterotetrameric holoenzyme involved in cell growth and differentiation. Here we report the identification, cloning, and oncogenic activity of the murine CK2alpha' subunit. Serum treatment of quiescent mouse fibroblasts induces CK2alpha' mRNA expression, which peaks at 4 h. The kinetics of CK2alpha' expression correlate with increased kinase activity toward a specific CK2 holoenzyme peptide substrate. The ectopic expression of CK2alpha' (or CK2alpha) cooperates with Ha-ras in foci formation of rat primary embryo fibroblasts. Moreover, we observed that BALB/c 3T3 fibroblasts transformed with Ha-ras and CK2alpha' show a faster growth rate than cells transformed with Ha-ras alone. In these cells the higher growth rate correlates with an increase in calmodulin phosphorylation, a protein substrate specifically affected by isolated CK2 catalytic subunits but not by CK2 holoenzyme, suggesting that unbalanced expression of a CK2 catalytic subunit synergizes with Ha-ras in cell transformation.