RESUMEN
To ensure proper progression through a cell cycle, checkpoints have evolved to play a surveillance role in maintaining genomic integrity. In this study, we demonstrate that loss of CDK2 activity activates an intra-S-phase checkpoint. CDK2 inhibition triggers a p53-p21 response via ATM- and ATR-dependent p53 phosphorylation at serine 15. Phosphorylation of other ATM and ATR downstream substrates, such as H2AX, NBS1, CHK1, and CHK2 is also increased. We show that during S phase when CDK2 activity is inhibited, there is an unexpected loading of the minichromosome maintenance complex onto chromatin. In addition, there is an increased number of cells with more than 4N DNA content, detected in the absence of p53, suggesting that rereplication can occur as a result of CDK2 disruption. Our findings identify an important role for CDK2 in the maintenance of genomic stability, acting via an ATM- and ATR-dependent pathway.
Asunto(s)
Quinasas CDC2-CDC28/antagonistas & inhibidores , Quinasas CDC2-CDC28/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Cafeína/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Replicación del ADN , Proteínas de Unión al ADN , Inhibidores Enzimáticos/metabolismo , Humanos , Fosforilación , ARN Interferente Pequeño/metabolismo , Serina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de TumorRESUMEN
Asthma is a syndrome of chronic bronchial inflammation and airway remodelling. Initially, asthma has been categorized into atopic and nonatopic types, based on antigen-specific IgE levels. Moreover, recently, asthma has been classified into different endotypes based on its pathophysiology, leading to the selection of the most optimal and effective therapies. Although T helper cell type 2 (Th2) cytokines were proven to play critical roles in atopic asthma, IL-17A has been reported to be involved in severe refractory asthma. In this study, we measured the levels of 24 cytokines/chemokines in the sera of healthy controls (HCs) (n = 34) and patients with asthma (n = 77), that were compared among patient groups with different disease activities and characteristics. The serum levels of nine cytokines were significantly higher in patients with asthma than in HCs, and the levels of IL-17A and SCF were significantly different between uncontrolled and well-controlled patient groups (p = 0.003). The IL-17A levels were significantly correlated with those of IL-4, IL-25, IL-10, and IFN-γ in patients with uncontrolled asthma, and the patients with the highest levels of all the above cytokines were refractory to high-dose of inhaled corticosteroid therapy and have a history of acute exacerbation within 1 year, requiring systemic steroid therapy. This study examines the profiles of upregulation and downregulation of various cytokines and chemokines in relation to asthmatic control status. IL-17A was significantly upregulated in patients with the uncontrolled and refractory status. Therefore, IL-17A may play important roles in asthmatic exacerbation, and its high level, in combination with upregulated Th2 and other cytokines, may indicate the refractory endotype of asthma.
Asunto(s)
Asma/sangre , Asma/inmunología , Citocinas/sangre , Interleucina-17/sangre , Células Th2/inmunología , Células Th2/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antiasmáticos/uso terapéutico , Asma/diagnóstico , Asma/terapia , Estudios de Casos y Controles , Quimiocinas/metabolismo , Análisis por Conglomerados , Resistencia a Medicamentos , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Mediadores de Inflamación/sangre , Masculino , Persona de Mediana Edad , Fenotipo , Pruebas de Función Respiratoria , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovial pannus formation, leukocyte infiltration, and angiogenesis. Adenoviral production of interleukin-13 (IL-13) reduces levels of proinflammatory mediators in an explant model of RA synovial tissue in vitro. To assess this approach in an animal model of arthritis, we compared intra-articular injections of an adenovirus producing rat IL-13 (AxCArIL-13), a control virus, and rat ankles receiving phosphate-buffered saline (PBS) in rat adjuvant-induced arthritis (AIA). We demonstrate that IL-13 levels are normally low in ankles throughout the course of rat AIA. We show that administration of AxCArIL-13 before arthritis onset significantly reduces ankle circumference, paw volume, bony destruction, the number of polymorphonuclear cells (PMNs), the quantity of blood vessels, and levels of monocyte chemoattractant protein (MCP)-1 in ankles. When administered as a treatment to inflamed ankles, AxCArIL-13 decreases articular index scores, paw volumes, bony destruction, vascularization, tumor necrosis factor-alpha (TNF-alpha) levels, and the quantity of monocytes, lymphocytes, and PMNs. Thus, increasing IL-13 levels significantly ameliorates the course of rat AIA, suggesting that similar strategies for the treatment of human RA are worthy of further study.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Terapia Genética , Interleucina-13/genética , Interleucina-13/uso terapéutico , Adenoviridae , Animales , Tobillo/patología , Tobillo/fisiopatología , Artritis Experimental/inmunología , Artritis Experimental/patología , Huesos/efectos de los fármacos , Huesos/patología , Modelos Animales de Enfermedad , Fibroblastos , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/inmunología , Interleucina-13/administración & dosificación , Mycobacterium , RatasRESUMEN
Interleukin 17-producing helper T (Th17) cells play pathogenic roles in chronic inflammatory and autoimmune diseases, including arthritis, colitis and multiple sclerosis. Th17 cells selectively express the transcription factor RORγt, as well as the cytokine receptors IL-23R and CCR6. Identification of novel Th17 cell-specific molecules may have potential value as diagnostic markers in the above-mentioned inflammatory diseases. To that aim, we carried out a comparative microarray analysis on in vitro differentiated Th1, Th2, Treg and Th17 cells from naïve CD4(+) cells of BALB/c mice. Among a total of one hundred and twenty Th17 cell-specific molecules, twenty-nine were novel cell-surface molecules. Then we revealed that thirteen of them were up-regulated in vivo in inflamed tissues from experimental autoimmune diseases, including spontaneous SKG arthritis, inflammatory bowel disease (IBD) and experimental autoimmune encephalomyelitis (EAE). Next, we analyzed the expression of four membranous molecules, and revealed that podoplanin was expressed highly in the in vitro differentiated Th17 cells. Moreover, at the inflamed synovium of the arthritic SKG mice, most of the accumulating Th17 cells were podoplanin-positive. These results indicate that podoplanin would be a useful Th17 cell marker for diagnosing pathological conditions of autoimmune diseases, including rheumatoid arthritis.
Asunto(s)
Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Articulaciones/inmunología , Glicoproteínas de Membrana/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Regulación hacia Arriba , Animales , Artritis Experimental/genética , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Diferenciación Celular , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inmunofenotipificación , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Articulaciones/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Células Th17/citologíaRESUMEN
Therapeutic angiogenesis can be induced by the implantation of bone marrow cells (BMCs). However, the mechanism of BMC-mediated neovascularization remains to be clarified. We investigated the differential activities of bone marrow subpopulations in angiogenesis and cytokine production. BMCs were separated into positive and negative fractions by surface expression of Mac-1, Gr-1, CD19, and c-kit, respectively. After 7 days of culture in the presence of vascular endothelial growth factor (VEGF), the cells produced adherent cells which incorporate acetylated low-density lipoprotein (acLDL). Mac-1(+) and Mac-1(-) cells produced almost equal numbers of acLDL(+) cells, but only Mac-1(-) cells expressed endothelial markers, including Flk-1, vWF, and CD31. Similarly, the expression of endothelial markers was detected in Gr-1(-), CD19(-), and c-kit(+) BMC fractions at 7-day cultures, but not in Gr-1(+), CD19(+), or c-kit(-) cells. In contrast, freshly isolated Mac-1(+) and Gr-1(+) BMCs expressed higher levels of mRNAs for angiogenic cytokines (including VEGF-A, FGF-2, and HGF) than Mac-1(-) and Gr-1(-) cells, respectively. Moreover, Mac-1(+)/c-kit(+) BMC subpopulation expressed higher levels of VEGF-A and SDF-1 mRNAs than other subpopulations. These data demonstrate that a relatively small proportion of VEGF-cultured adherent cells are true endothelial cells with a Flk-1(+)/vWF(+)/CD31(+) phenotype. Moreover, endothelial stem/progenitor cells (EPCs) are limited primarily to Mac-1(-), Gr-1(-), and c-kit(+) BMC populations. In contrast, angiogenic cytokine mRNAs were also produced by Mac-1(+), Gr-1(+), and c-kit(-) BMCs, suggesting the heterogeneity of effector cell types for neovasculatization therapy.
Asunto(s)
Células de la Médula Ósea/citología , Citocinas/genética , Endotelio Vascular/citología , Células Madre/citología , Animales , Células de la Médula Ósea/fisiología , Técnicas de Cultivo de Célula , Separación Celular , Endotelio Vascular/fisiología , Fémur , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The commitment of naive T cells to polarized Th cells requires specific changes in their transcription factors. Retrovirally overexpressed GATA-3 has been reported to induce the Th2 cytokine profile in developing Th1 cells. In this study, we examined the role of the N-terminal finger (Nf) of GATA-3 in Th2 cell development. The Nf, as well as the C-terminal finger and the transactivation domain, is critical for the induction of the Th2 phenotype. Using the GATA-3-Nf as a bait, our yeast two-hybrid screening identified friend of GATA (FOG) in the Th2 cell-specific library. Naive T cells express significant levels of FOG mRNA, which was rapidly down-regulated upon commitment to both Th1 and Th2 lineages. In reporter assays, FOG blocked the GATA-3-mediated activation of several cytokine promoters. Finally, retroviral expression of FOG in developing Th2 cells suppressed both IL-4 and IL-5 and allowed for IFN-gamma production, which was accompanied by a significant level of T-bet mRNA expression. Serial deletion mutation analysis indicated that the N-terminal region, but not the consensus C-terminal binding protein-binding motif, of FOG is critical for the effects. Our results clearly indicate that 1) FOG is a repressor of GATA-3 in naive T cells and 2) the down-regulation of FOG induces Th2 cell differentiation by releasing GATA-3 from its repression.