RESUMEN
T-bet is important for differentiation of cytotoxic CD8 and Th1 CD4 T cells. We have discovered that Egr2 and 3 are potent inhibitors of T-bet function in CD4 and CD8 effector T cells. Egr2 and 3 were essential to suppress Th1 differentiation in Th2 and Th17 conditions in vitro and also to control IFN-γ-producing CD4 and CD8 T cells in response to virus infection. Together with Egr2 and 3, T-bet is induced in naive T cells by Ag stimulation, but Egr2 and 3 expression was inhibited by Th1-inducing cytokines. We found that Egr2 and 3 physically interact with the T-box domain of T-bet, blocking T-bet DNA binding and inhibiting T-bet-mediated production of IFN-γ. Thus, Egr2 and 3 are antagonists of T-bet function in effector T cells and are important for the control of inflammatory responses of T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 3 de la Respuesta de Crecimiento Precoz/metabolismo , Interferón gamma/biosíntesis , Proteínas de Dominio T Box/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Citocinas/farmacología , Proteína 2 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteína 3 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 3 de la Respuesta de Crecimiento Precoz/genética , Interferón gamma/inmunología , Ratones , Células TH1/inmunología , Células TH1/fisiología , Células Th17/inmunología , Células Th17/metabolismoAsunto(s)
Capecitabina/uso terapéutico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Capecitabina/efectos adversos , Cisplatino/efectos adversos , Cisplatino/uso terapéutico , Supervivencia sin Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Fluorouracilo/efectos adversos , Fluorouracilo/uso terapéutico , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , HumanosRESUMEN
MicroRNAs (miRNAs) are crucial in cancer development. However, the underlying mechanisms of miRNAs in osteosarcoma (OS) remain largely uncharacterized. The present study investigated the role of miR20a in OS cell proliferation. It was determined that miR20a expression is markedly upregulated in OS tissues and cells compared with the matched adjacent normal tissues and hFOB human osteoblast cell lines. Ectopic expression of miR20a promoted the proliferation and anchorageindependent growth of OS cells, whereas inhibition of miR20a reduced this effect. Bioinformatics analysis further revealed early growth response 2 (EGR2), as a potential target of miR20a. Data from luciferase reporter assays showed that miR20a directly binds to the 3'untranslated region (3'UTR) of EGR2 mRNA and represses expression at the transcriptional and translational levels. In functional assays, miR20a promoted OS cell proliferation and the cell cycle, which could be suppressed by an inhibitor of miR20a. In conclusion, the data provide compelling evidence that miR20a functions as an oncomiRNA, which is important in promoting cell proliferation in OS, and its oncogenic effect is mediated primarily through direct suppression of EGR2 expression.
Asunto(s)
Neoplasias Óseas/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Osteoblastos/metabolismo , Osteosarcoma/genética , Regiones no Traducidas 3' , Secuencia de Bases , Sitios de Unión , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Femenino , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Osteoblastos/patología , Osteosarcoma/metabolismo , Osteosarcoma/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
CNS/PNS interfaces constitute cell boundaries, since they delimit territories with different neuronal and glial contents. Despite their potential interest in regenerative medicine, the mechanisms restricting oligodendrocytes and astrocytes to the CNS, and Schwann cells to the PNS in mammals are not known. To investigate the involvement of peripheral glia and myelin in the maintenance of the CNS/PNS boundary, we have first made use of different mouse mutants. We show that inactivation of Krox20/Egr2, a master regulatory gene for myelination in Schwann cells, results in transgression of the CNS/PNS boundary by astrocytes and oligodendrocytes and in myelination of nerve root axons by oligodendrocytes. In contrast, such migration does not occur with the Trembler(J) mutation, which prevents PNS myelination without affecting Krox20 expression. Altogether these data suggest that maintenance of the CNS/PNS boundary requires a new Krox20 function separable from myelination control. Finally, we have analyzed a human patient affected by a congenital amyelinating neuropathy, associated with the absence of the KROX20 protein in Schwann cells. In this case, the nerve roots were also invaded by oligodendrocytes and astrocytes. This indicates that transgression of the CNS/PNS boundary by central glia can occur in pathological situations in humans and suggests that the underlying mechanisms are common with the mouse.
Asunto(s)
Proteína 2 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 2 de la Respuesta de Crecimiento Precoz/deficiencia , Neuroglía/fisiología , Raíces Nerviosas Espinales/patología , Animales , Astrocitos/fisiología , Pollos , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/fisiología , Humanos , Lactante , Ratones , Ratones Mutantes Neurológicos , Mutación Missense , Vaina de Mielina/fisiología , Oligodendroglía/fisiología , Enfermedades del Sistema Nervioso Periférico/congénito , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/patología , Células de Schwann/patología , Pez Cebra/genéticaRESUMEN
Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. In recent years, many genes have been identified as p53-regulated genes; however, no single target gene has been shown to be required for the apoptotic effect. Using microarray analysis, we have identified the transcription factor early growth response 2 (EGR2) as a target of the p53 family, specifically p53, p63 and p73. EGR2 expression was up-regulated by DNA damage-induced p53 activity, as well as by overexpression of p53 family genes. Furthermore, we identified a responsive element to p53, TAp63, and TAp73 within the EGR2 gene. This response element is highly conserved between human and rodents. We also found that overexpression of EGR2 induced apoptosis when combined with anticancer agents. Conversely, inactivation of EGR2 attenuated p53-mediated apoptosis. The results presented here suggest that EGR2 is a direct transcriptional target of p53 family that can in part mediate the p53-dependent apoptotic pathway.
Asunto(s)
Apoptosis/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Regulación de la Expresión Génica , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Células Cultivadas , Proteína 2 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HeLa , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , ARN Interferente Pequeño/farmacología , Ratas , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc/genéticaRESUMEN
Expansion of autoreactive T cells and their resistance to anergy was demonstrated in systemic lupus erythematosus (SLE). A pair of transcription factors, early growth response 2 (Egr-2) and 3 (Egr-3), are negative regulators of T cell activation that were shown to be important in anergy. A peptide (designated hCDR1 for human CDR1) based on the CDR-1 of an anti-DNA Ab ameliorated SLE in both induced and spontaneous lupus models. Our objectives were to determine the expression levels of Egr-2 and Egr-3 in autoreactive T cells following immunization with the lupus-inducing anti-DNA Ab that bears a common Id designated 16/6Id and also in a full-blown SLE and to determine the effect of hCDR1 on these transcription factors. We demonstrated diminished expression levels of Egr-2 and Egr-3 mRNA both early after immunization with the 16/6Id and in SLE-afflicted (NZB x NZW)F1 (New Zealand Black and New Zealand White) mice. Furthermore, by down-regulating Akt phosphorylation and up-regulating TGFbeta secretion, treatment with hCDR1 significantly up-regulated Egr-2 and Egr-3 expression. This was associated with an increased expression of the E3 ligase Cbl-b. Inhibition of Akt in T cells of immunized mice decreased, whereas silencing of the Egr-2 and Egr-3 in T cells of hCDR1-treated mice increased IFN-gamma secretion. Thus, hCDR1 down-regulates Akt phosphorylation, which leads to up-regulated expression of T cell Egr-2 and Egr-3, resulting in the inhibition of IFN-gamma secretion that is required for the maintenance of SLE.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anergia Clonal/efectos de los fármacos , Proteína 2 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 3 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Lupus Eritematoso Sistémico/inmunología , Fragmentos de Péptidos/farmacología , Animales , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 3 de la Respuesta de Crecimiento Precoz/genética , Proteína 3 de la Respuesta de Crecimiento Precoz/metabolismo , Humanos , Interferón gamma/metabolismo , Interleucina-2/farmacología , Ratones , Ratones Endogámicos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia ArribaRESUMEN
Proliferation of Schwann cells in vitro, unlike most mammalian cells, is not induced by serum alone but additionally requires cAMP elevation and mitogenic stimulation. How these agents cooperate to promote progression through the G1 phase of the cell cycle is unclear. We studied the integrative effects of these compounds on receptor-mediated signaling pathways and regulators of G1 progression. We show that serum alone induces strong cyclical expression of cyclin D1 and E1, 6 and 12 h after addition, respectively. Serum also promotes strong but transient erbB2, ERK, and Akt phosphorylation, but Schwann cells remain arrested in G1 due to high levels of the inhibitor, p27(Kip). Forskolin with serum promotes G1 progression in 22% of Schwann cells between 18 and 24 h by inducing a steady decline in p27(Kip) levels that reaches a nadir at 12 h coinciding with peak cyclin E1 expression. Forskolin also delays neuregulin-induced loss of erbB2 receptors allowing strong acute activation of PI3K, sustained erbB2 phosphorylation and G1 progression in 31% of Schwann cells. We find that the ability of forskolin to decrease p27(Kip) is associated with its ability to decrease Krox-20 expression that is induced by serum and further increased by neuregulin. Our results explain why serum is required but insufficient to stimulate proliferation and identify two routes by which forskolin promotes proliferation in the presence of serum and neuregulin. These findings provide insights into how G1 progression and, cell cycle arrest leading to myelination are regulated in Schwann cells.