RESUMEN
BACKGROUND: Thymic stromal lymphopoietin (TSLP) is released from the airway epithelium in response to various environmental triggers, inducing a type-2 inflammatory response, and is associated with airway inflammation, airway hyperresponsiveness (AHR), and exacerbations. TSLP may also induce AHR via a direct effect on airway smooth muscle and mast cells, independently of type-2 inflammation, although association between airway TSLP and AHR across asthma phenotypes has been described sparsely. OBJECTIVES: This study sought to investigate the association between AHR and levels of TSLP in serum, sputum, and bronchoalveolar lavage in patients with asthma with and without type-2 inflammation. METHODS: A novel ultrasensitive assay was used to measure levels of TSLP in patients with asthma (serum, n = 182; sputum, n = 81; bronchoalveolar lavage, n = 85) and healthy controls (serum, n = 47). The distribution and association among airway and systemic TSLP, measures of AHR, type-2 inflammation, and severity of disease were assessed. RESULTS: TSLP in sputum was associated with AHR independently of levels of eosinophils and fractional exhaled nitric oxide (ρ = 0.49, P = .005). Serum TSLP was higher in both eosinophil-high and eosinophil-low asthma compared to healthy controls: geometric mean: 1600 fg/mL (95% CI: 1468-1744 fg/mL) and 1294 fg/mL (95% CI: 1167-1435 fg/mL) versus 846 fg/mL (95% CI: 661-1082 fg/mL), but did not correlate with the level of AHR. Increasing age, male sex, and eosinophils in blood were associated with higher levels of TSLP in serum, whereas lung function, inhaled corticosteroid dose, and symptom score were not. CONCLUSIONS: The association between TSLP in sputum and AHR to mannitol irrespective of markers of type-2 inflammation further supports a role of TSLP in AHR that is partially independent of eosinophilic inflammation.
Asunto(s)
Asma , Eosinofilia , Inflamación , Linfopoyetina del Estroma Tímico , Humanos , Masculino , Asma/diagnóstico , Asma/metabolismo , Citocinas , Eosinofilia/diagnóstico , Eosinofilia/metabolismo , Eosinófilos , Inflamación/diagnóstico , Inflamación/metabolismo , Esputo , Linfopoyetina del Estroma Tímico/metabolismoRESUMEN
Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein cyclin F with suppression of the B-Myb/cyclin A pathway to ensure a DNA damage-induced checkpoint response in G2.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Ciclinas/metabolismo , Reparación del ADN/fisiología , Transactivadores/metabolismo , Línea Celular Tumoral , Cartilla de ADN/genética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Immunoblotting , Inmunoprecipitación , Luciferasas , Mutagénesis Sitio-Dirigida , Interferencia de ARN , ARN Interferente Pequeño/genética , UbiquitinaciónRESUMEN
To identify key connections between DNA-damage repair and checkpoint pathways, we performed RNA interference screens for regulators of the ionizing radiation-induced G2 checkpoint, and we identified the breast cancer gene BRCA2. The checkpoint was also abrogated following depletion of PALB2, an interaction partner of BRCA2. BRCA2 and PALB2 depletion led to premature checkpoint abrogation and earlier activation of the AURORA A-PLK1 checkpoint-recovery pathway. These results indicate that the breast cancer tumour suppressors and homologous recombination repair proteins BRCA2 and PALB2 are main regulators of G2 checkpoint maintenance following DNA-damage.
Asunto(s)
Proteína BRCA2/metabolismo , Fase G2/fisiología , Ensayos Analíticos de Alto Rendimiento , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína BRCA2/genética , Línea Celular , Daño del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Fase G2/genética , Biblioteca de Genes , Células HCT116 , Células HeLa , Humanos , Proteínas Nucleares/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Recombinación Genética , Transducción de Señal/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
The DNA damage induced G(2)/M checkpoint is an important guardian of the genome that prevents cell division when DNA lesions are present. The checkpoint prevents cells from entering mitosis by degrading CDC25A, a key CDK activator. CDC25A proteolysis is controlled by direct phosphorylation events that lead to its recognition by the ubiquitin ligase beta-TrCP. Recently we have identified NEK11, a member of NIMA-related kinase family, as the critical kinase triggering CDC25A degradation. NEK11 controls degradation of CDC25A by directly phosphorylating CDC25A on residues whose phosphorylation is required for beta-TrCP mediated CDC25A polyubiquitylation and degradation. The activity of NEK11 is in turn controlled by CHK1 that activates NEK11 via phosphorylation on serine 273. Since inhibition of NEK11 activity forces checkpoint-arrested cells into mitosis and cell death, NEK11 is, like CHK1, a strong candidate target for the development of novel anticancer drugs. Here we further support this notion by showing results suggesting that NEK11 expression increases during colon cancer development.
Asunto(s)
Ciclo Celular , Daño del ADN , Proteínas Quinasas/metabolismo , Transducción de Señal , Fosfatasas cdc25/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Fase G2 , Humanos , Mitosis , Modelos Biológicos , Quinasas Relacionadas con NIMA , Neoplasias/enzimología , Neoplasias/patología , Neoplasias/terapia , Lesiones Precancerosas/enzimología , Lesiones Precancerosas/patologíaRESUMEN
DNA damage-induced cell-cycle checkpoints have a critical role in maintaining genomic stability. A key target of the checkpoints is the CDC25A (cell division cycle 25 homologue A) phosphatase, which is essential for the activation of cyclin-dependent kinases and cell-cycle progression. To identify new genes involved in the G2/M checkpoint we performed a large-scale short hairpin RNA (shRNA) library screen. We show that NIMA (never in mitosis gene A)-related kinase 11 (NEK11) is required for DNA damage-induced G2/M arrest. Depletion of NEK11 prevents proteasome-dependent degradation of CDC25A, both in unperturbed and DNA-damaged cells. We show that NEK11 directly phosphorylates CDC25A on residues whose phosphorylation is required for beta-TrCP (beta-transducin repeat-containing protein)-mediated polyubiquitylation and degradation of CDC25A. Furthermore, we demonstrate that CHK1 (checkpoint kinase 1) directly activates NEK11 by phosphorylating it on Ser 273, indicating that CHK1 and NEK11 operate in a single pathway that controls proteolysis of CDC25A. Taken together, these results demonstrate that NEK11 is an important component of the pathway enforcing the G2/M checkpoint, suggesting that genetic mutations in NEK11 may contribute to the development of human cancer.