RESUMEN
OBJECTIVE: IĸB protein B cell lymphoma 3-encoded protein (BCL3) is a regulator of the NF-κB family of transcription factors. NF-κB signaling fundamentally influences the fate of bone-forming osteoblasts and bone-resorbing osteoclasts, but the role of BCL3 in bone biology has not been investigated. The objective of this study was to evaluate BCL3 in skeletal development, maintenance, and osteoarthritic pathology. METHODS: To assess the contribution of BCL3 to skeletal homeostasis, neonatal mice (n = 6-14) lacking BCL3 (Bcl3-/- ) and wild-type (WT) controls were characterized for bone phenotype and density. To reveal the contribution to bone phenotype by the osteoblast compartment in Bcl3-/- mice, transcriptomic analysis of early osteogenic differentiation and cellular function (n = 3-7) were assessed. Osteoclast differentiation and function in Bcl3-/- mice (n = 3-5) was assessed. Adult 20-week Bcl3-/- and WT mice bone phenotype, strength, and turnover were assessed. A destabilization of the medial meniscus model of osteoarthritic osteophytogenesis was used to understand adult bone formation in Bcl3-/- mice (n = 11-13). RESULTS: Evaluation of Bcl3-/- mice revealed congenitally increased bone density, long bone dwarfism, increased bone biomechanical strength, and altered bone turnover. Molecular and cellular characterization of mesenchymal precursors showed that Bcl3-/- cells displayed an accelerated osteogenic transcriptional profile that led to enhanced differentiation into osteoblasts with increased functional activity, which could be reversed with a mimetic peptide. In a model of osteoarthritis-induced osteophytogenesis, Bcl3-/- mice exhibited decreased pathological osteophyte formation (P < 0.05). CONCLUSION: Cumulatively, these findings demonstrate that BCL3 controls developmental mineralization to enable appropriate bone formation, whereas in a pathological setting, it contributes to skeletal pathology.
Asunto(s)
Proteínas del Linfoma 3 de Células B , Huesos , Osteogénesis , Animales , Ratones , Huesos/metabolismo , Densidad Ósea , Diferenciación Celular , FN-kappa B/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Proteínas del Linfoma 3 de Células B/genéticaRESUMEN
Since its discovery over 30 years ago the NF-ĸB family of transcription factors has gained the status of master regulator of the immune response. Much of what we understand of the role of NF-ĸB in immune development, homeostasis and inflammation comes from studies of mice null for specific NF-ĸB subunit encoding genes. The role of inflammation in diseases that affect a majority of individuals with health problems globally further establishes NF-ĸB as an important pathogenic factor. More recently, genomic sequencing has revealed loss of function mutations in the NFKB1 gene as the most common monogenic cause of common variable immunodeficiencies in Europeans. NFKB1 encodes the p105 subunit of NF-ĸB which is processed to generate the NF-ĸB p50 subunit. NFKB1 is the most highly expressed transcription factor in macrophages, key cellular drivers of inflammation and immunity. Although a key role for NFKB1 in the control of the immune system is apparent from Nfkb1-/- mouse studies, we know relatively little of the role of NFKB1 in regulating human macrophage responses. In this study we use the THP1 monocyte cell line and CRISPR/Cas9 gene editing to generate a model of NFKB1-/- human macrophages. Transcriptomic analysis reveals that activated NFKB1-/- macrophages are more pro-inflammatory than wild type controls and express elevated levels of TNF, IL6, and IL1B, but also have reduced expression of co-stimulatory factors important for the activation of T cells and adaptive immune responses such as CD70, CD83 and CD209. NFKB1-/- THP1 macrophages recapitulate key observations in individuals with NFKB1 haploinsufficiency including decreased IL10 expression. These data supporting their utility as an in vitro model for understanding the role of NFKB1 in human monocytes and macrophages and indicate that of loss of function NFKB1 mutations in these cells is an important component in the associated pathology.
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Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Inflamación/genética , Macrófagos/metabolismo , Subunidad p50 de NF-kappa B/genética , Transcriptoma , Inmunidad Adaptativa , Sistemas CRISPR-Cas , Citocinas/genética , Citocinas/metabolismo , Humanos , Inmunidad Celular , Inflamación/inmunología , Inflamación/metabolismo , Activación de Macrófagos , Macrófagos/inmunología , Subunidad p50 de NF-kappa B/deficiencia , Fenotipo , RNA-Seq , Células THP-1 , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMEN
The NF-ĸB transcription factor is a critical regulator of immune homeostasis and inflammatory responses and is a critical factor in the pathogenesis of inflammatory disease. The pathways to NF-ĸB activation are paradigms for signal-induced ubiquitination and proteasomal degradation, control of transcription factor function by subcellular localisation, and the control of gene transcription and physiological processes by signal transduction mechanisms. Despite the importance of NF-ĸB in disease, the NF-ĸB pathway remains unexploited for the treatment of inflammatory disease. Our understanding of NF-ĸB comes mostly from studies of transgenic mice and cell lines where components of the pathway have been deleted or over expressed. Recent advances in quantitative proteomics offer new opportunities to understand the NF-ĸB pathway using the absolute abundance of individual pathway components. We have analysed available quantitative proteomic datasets to establish the structure of the NF-ĸB pathway in human immune cells under both steady state and activated conditions. This reveals a conserved NF-κB pathway structure across different immune cell lineages and identifies important differences to the current model of the NF-ĸB pathway. These include the findings that the IKK complex in most cells is likely to consist predominantly of IKKß homodimers, that the relative abundancies of IκB proteins show strong cell type variation, and that the components of the non-canonical NF-ĸB pathway are significantly increased in activated immune cells. These findings challenge aspects of our current view of the NF-κB pathway and identify outstanding questions important for defining the role of key components in regulating inflammation and immunity.
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FN-kappa B , Proteómica , Animales , Humanos , Quinasa I-kappa B/metabolismo , Ratones , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas , Transducción de SeñalRESUMEN
Chromosomal rearrangements of the mixed lineage leukaemia (MLL, also known as KMT2A) gene on chromosome 11q23 are amongst the most common genetic abnormalities observed in human acute leukaemias. MLL rearrangements (MLLr) are the most common cytogenetic abnormalities in infant and childhood acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) and do not normally acquire secondary mutations compared to other leukaemias. To model these leukaemias, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL-AF9 (MA9) chromosomal rearrangements in murine hematopoietic stem and progenitor cell lines and primary cells. By utilizing a dual-single guide RNA (sgRNA) approach targeting the breakpoint cluster region of murine Mll and Af9 equivalent to that in human MA9 rearrangements, we show efficient de novo generation of MA9 fusion product at the DNA and RNA levels in the bulk population. The leukaemic features of MA9-induced disease were observed including increased clonogenicity, enrichment of c-Kit-positive leukaemic stem cells and increased MA9 target gene expression. This approach provided a rapid and reliable means of de novo generation of Mll-Af9 genetic rearrangements in murine haematopoietic stem and progenitor cells (HSPCs), using CRISPR/Cas9 technology to produce a cellular model of MA9 leukaemias which faithfully reproduces many features of the human disease in vitro.
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Edición Génica/métodos , Células Madre Hematopoyéticas/citología , N-Metiltransferasa de Histona-Lisina/genética , Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-kit/genética , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Puntos de Rotura del Cromosoma , Modelos Animales de Enfermedad , Células HEK293 , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Modelos Biológicos , Células 3T3 NIHRESUMEN
The transcription factor NF-ĸB is a master regulator of the innate immune response and plays a central role in inflammatory diseases by mediating the expression of pro-inflammatory cytokines. Ubiquitination-triggered proteasomal degradation of DNA-bound NF-ĸB strongly limits the expression of its target genes. Conversely, USP7 (deubiquitinase ubiquitin-specific peptidase 7) opposes the activities of E3 ligases, stabilizes DNA-bound NF-ĸB, and thereby promotes NF-ĸB-mediated transcription. Using gene expression and synthetic peptide arrays on membrane support and overlay analyses, we found here that inhibiting USP7 increases NF-ĸB ubiquitination and degradation, prevents Toll-like receptor-induced pro-inflammatory cytokine expression, and represents an effective strategy for controlling inflammation. However, the broad regulatory roles of USP7 in cell death pathways, chromatin, and DNA damage responses limit the use of catalytic inhibitors of USP7 as anti-inflammatory agents. To this end, we identified an NF-ĸB-binding site in USP7, ubiquitin-like domain 2, that selectively mediates interactions of USP7 with NF-ĸB subunits but is dispensable for interactions with other proteins. Moreover, we found that the amino acids 757LDEL760 in USP7 critically contribute to the interaction with the p65 subunit of NF-ĸB. Our findings support the notion that USP7 activity could be potentially targeted in a substrate-selective manner through the development of noncatalytic inhibitors of this deubiquitinase to abrogate NF-ĸB activity.
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Factor de Transcripción ReIA/metabolismo , Peptidasa Específica de Ubiquitina 7/metabolismo , Ubiquitinación , Animales , Células Cultivadas , Femenino , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Moleculares , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Peptidasa Específica de Ubiquitina 7/químicaRESUMEN
Proinflammatory responses induced by Toll-like receptors (TLRs) are dependent on the activation of the NF-ĸB and mitogen-activated protein kinase (MAPK) pathways, which coordinate the transcription and synthesis of proinflammatory cytokines. We demonstrate that BCL-3, a nuclear IĸB protein that regulates NF-ĸB, also controls TLR-induced MAPK activity by regulating the stability of the TPL-2 kinase. TPL-2 is essential for MAPK activation by TLR ligands, and the rapid proteasomal degradation of active TPL-2 is a critical mechanism limiting TLR-induced MAPK activity. We reveal that TPL-2 is a nucleocytoplasmic shuttling protein and identify the nucleus as the primary site for TPL-2 degradation. BCL-3 interacts with TPL-2 and promotes its degradation by promoting its nuclear localization. As a consequence, Bcl3-/- macrophages have increased TPL-2 stability following TLR stimulation, leading to increased MAPK activity and MAPK-dependent responses. Moreover, BCL-3-mediated regulation of TPL-2 stability sets the MAPK activation threshold and determines the amount of TLR ligand required to initiate the production of inflammatory cytokines. Thus, the nucleus is a key site in the regulation of TLR-induced MAPK activity. BCL-3 links control of the MAPK and NF-ĸB pathways in the nucleus, and BCL-3-mediated TPL-2 regulation impacts on the cellular decision to initiate proinflammatory cytokine production in response to TLR activation.
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Proteínas del Linfoma 3 de Células B/metabolismo , Núcleo Celular/metabolismo , Proteínas I-kappa B/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Receptores Toll-Like/metabolismo , Animales , Proteínas del Linfoma 3 de Células B/genética , Citocinas/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Macrófagos/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Células RAW 264.7RESUMEN
Phosphorylation of the NF-κB transcription factor is an important regulatory mechanism for the control of transcription. Here we identify serine 80 (S80) as a phosphorylation site on the p50 subunit of NF-κB, and IKKß as a p50 kinase. Transcriptomic analysis of cells expressing a p50 S80A mutant reveals a critical role for S80 in selectively regulating the TNFα inducible expression of a subset of NF-κB target genes including pro-inflammatory cytokines and chemokines. S80 phosphorylation regulates the binding of p50 to NF-κB binding (κB) sites in a sequence specific manner. Specifically, phosphorylation of S80 reduces the binding of p50 at κB sites with an adenine at the -1 position. Our analyses demonstrate that p50 S80 phosphorylation predominantly regulates transcription through the p50:p65 heterodimer, where S80 phosphorylation acts in trans to limit the NF-κB mediated transcription of pro-inflammatory genes. The regulation of a functional class of pro-inflammatory genes by the interaction of S80 phosphorylated p50 with a specific κB sequence describes a novel mechanism for the control of cytokine-induced transcriptional responses.
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ADN/metabolismo , Quinasa I-kappa B/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , FN-kappa B/metabolismo , Serina/metabolismo , Transcripción Genética , Animales , Sitios de Unión/genética , Dominio Catalítico , Células Cultivadas , ADN/genética , Células HEK293 , Humanos , Ratones , FN-kappa B/química , Subunidad p50 de NF-kappa B/química , Fosforilación , Unión Proteica , Especificidad por Sustrato/genéticaRESUMEN
Objective: We have previously shown that increased circulating interleukin-6 (IL-6) results in enhanced CD4+ T cell signaling via signal transduction and activator of transcription-3 (STAT3) in early rheumatoid arthritis (RA). We tested the hypothesis that transcriptional "imprinting" of T-cells by this mechanism skews downstream effector responses, reinforcing immune dysregulation at a critical, but targetable, disease phase. Methods: We modeled naïve CD4+ T cell exposure to pathophysiological concentrations of IL-6 in vitro, assessing the dynamic transcriptional and functional consequences for downstream effector cells utilizing microarray and flow cytometry. Fresh blood from treatment-naïve early arthritis patients was phenotyped in parallel for comparison. Results: T cell sensitivity to IL-6 was most marked in the naïve subset, and related to gp130 rather than IL-6R expression. Exposure of healthy naïve CD4+ T cells to IL-6 induced the same STAT3 target genes as previously seen to discriminate RA patients from disease controls. After TCR stimulation IL-6 pre-exposed cells exhibited enhanced proliferative capacity, activation, and a propensity toward Th1 differentiation, compared to non-exposed cells. An entirely analogous phenotype was observed in early RA compared to control CD4+ T cells. Conclusions: Sustained IL-6 exposure at a critical point in the natural history of RA "primes" the adaptive immune system to respond aberrantly to TCR stimulation, potentiating disease induction with implications for the optimal timing of targeted therapy.
Asunto(s)
Artritis Reumatoide/inmunología , Linfocitos T CD4-Positivos/inmunología , Interleucina-6/inmunología , Modelos Inmunológicos , Transducción de Señal/inmunología , Transcripción Genética/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T/inmunología , Factor de Transcripción STAT3/inmunologíaRESUMEN
OBJECTIVES: Dysregulated signal transduction and activator of transcription-3 (STAT3) signalling in CD4+ T cells has been proposed as an early pathophysiological event in RA. We sought further evidence for this observation, and to determine its clinical relevance. METHODS: Microarray technology was used to measure gene expression in purified peripheral blood CD4+ T cells from treatment-naïve RA patients and disease controls newly recruited from an early arthritis clinic. Analysis focused on 12 previously proposed transcripts, and concurrent STAT3 pathway activation was determined in the same cells by flow cytometry. A pooled analysis of previous and current gene expression findings incorporated detailed clinical parameters and employed multivariate analysis. RESULTS: In an independent cohort of 161 patients, expression of 11 of 12 proposed signature genes differed significantly between RA patients and controls, robustly validating the earlier findings. Differential regulation was most pronounced for the STAT3 target genes PIM1, BCL3 and SOCS3 (>1.3-fold difference; P < 0.005), each of whose expression correlated strongly with paired intracellular phospho-STAT3. In a meta-analysis of 279 patients the same three genes accounted for the majority of the signature's ability to discriminate RA patients, which was found to be independent of age, joint involvement or acute phase response. CONCLUSION: The STAT3-mediated dysregulation of BCL3, SOCS3 and PIM1 in circulating CD4+ T cells is a discriminatory feature of early RA that occurs independently of acute phase response. The mechanistic and functional implications of this observation at a cellular level warrant clarification.
Asunto(s)
Artritis Reumatoide/diagnóstico , Linfocitos T CD4-Positivos/metabolismo , Regulación de la Expresión Génica/inmunología , Factor de Transcripción STAT3/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Artritis/diagnóstico , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Estudios de Casos y Controles , Análisis por Conglomerados , Diagnóstico Diferencial , Diagnóstico Precoz , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , Transducción de Señal/genética , Transducción de Señal/inmunología , Transcriptoma , Adulto JovenRESUMEN
Tolerance is a long-recognized property of macrophages that leads to an altered response to repeated or chronic exposure to endotoxin. The physiological role of tolerance is to limit the potential damage to host tissue that may otherwise result from prolonged production of pro-inflammatory cytokines. Tolerance is induced by all toll-like receptor (TLR) ligands tested to date, however, tolerance induced by the TLR4 ligand lipopolysaccharide (LPS) is by far the best studied. LPS tolerance involves a global transcriptional shift from a pro-inflammatory response toward one characterized by the expression of anti-inflammatory and pro-resolution factors. Although largely reversible, LPS-tolerance leads to a hybrid macrophage activation state that is pro-inflammatory in nature, but possesses distinct regulatory anti-inflammatory features. Remarkably, a comparative transcriptomic analysis of tolerance induced by different TLR ligands has not previously been reported. Here, we describe the transcriptomic profiles of mouse macrophages tolerized with ligands for TLR2, TLR3, TLR4 and TLR 9. While we identified TLR-specific transcriptional profiles in macrophages tolerized with each ligand, tolerance induced by TLR4 represented an archetype pattern, such that each gene tolerized by any of the TLRs tested was also found to be tolerized by TLR4. Pro-inflammatory cytokines are not universally suppressed in all tolerant cells, but distinct patterns of cytokine expression distinguished TLR-specific tolerance. Analysis of gene regulatory regions revealed specific DNA sequence motifs associated with distinct states of TLR tolerance, implicating previously identified as well as novel transcriptional regulators of tolerance in macrophages. These data provide a basis for the future exploitation of TLR-specific tolerant states to achieve therapeutic re-programming of the innate immune response.
Asunto(s)
Tolerancia Inmunológica , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Animales , Femenino , Perfilación de la Expresión Génica , Inmunidad Innata , Memoria Inmunológica , Ligandos , Activación de Macrófagos/genética , Ratones , Poli I-C/inmunología , Transducción de Señal , TranscriptomaRESUMEN
Trib2 pseudokinase is involved in the etiology of a number of cancers including leukaemia, melanoma, ovarian, lung and liver cancer. Both high and low Trib2 expression levels correlate with different types of cancer. Elevated Trib2 expression has oncogenic properties in both leukaemia and lung cancer dependent on interactions with proteasome machinery proteins and degradation of transcription factors. Here, we demonstrated that Trib2 deficiency conferred a growth and survival advantage both at steady state and in stress conditions in leukaemia cells. In response to stress, wild type leukaemia cells exited the cell cycle and underwent apoptosis. In contrast, Trib2 deficient leukaemia cells continued to enter mitosis and survive. We showed that Trib2 deficient leukaemia cells had defective MAPK p38 signalling, which associated with a reduced γ-H2Ax and Chk1 stress signalling response, and continued proliferation following stress, associated with inefficient activation of cell cycle inhibitors p21, p16 and p19. Furthermore, Trib2 deficient leukaemia cells were more resistant to chemotherapy than wild type leukaemia cells, having less apoptosis and continued propagation. Trib2 re-expression or pharmacological activation of p38 in Trib2 deficient leukaemia cells sensitised the cells to chemotherapy-induced apoptosis comparable with wild type leukaemia cells. Our data provide evidence for a tumour suppressor role of Trib2 in myeloid leukaemia via activation of p38 stress signalling. This newly identified role indicates that Trib2 may counteract the propagation and chemotherapy resistance of leukaemia cells.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide/metabolismo , Sistema de Señalización de MAP Quinasas , Mitosis , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Estrés Fisiológico , Proteínas Supresoras de Tumor/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Mieloide/genética , Leucemia Mieloide/patología , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
The NF-κB transcription factor was discovered 30 years ago and has since emerged as the master regulator of inflammation and immune homeostasis. It achieves this status by means of the large number of important pro- and antiinflammatory factors under its transcriptional control. NF-κB has a central role in inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and autoimmunity, as well as diseases comprising a significant inflammatory component such as cancer and atherosclerosis. Here, we provide an overview of the studies that form the basis of our understanding of the role of NF-κB subunits and their regulators in controlling inflammation. We also describe the emerging importance of posttranslational modifications of NF-κB in the regulation of inflammation, and highlight the future challenges faced by researchers who aim to target NF-κB transcriptional activity for therapeutic benefit in treating chronic inflammatory diseases.
Asunto(s)
Inflamación/genética , FN-kappa B/metabolismo , Transcripción Genética , Animales , Humanos , Modelos Biológicos , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
The t(15;17) translocation generates a PML-RARα fusion protein causative for acute promyelocytic leukemia (APL). Li et al. now identify the pseudokinase stress protein TRIB3 as an important factor in APL disease progression and therapy resistance. Targeting the interaction of TRIB3 and PML-RARα using peptide technology provides a novel therapeutic approach.
Asunto(s)
Proteínas de Neoplasias/genética , Tretinoina , Humanos , Leucemia Promielocítica Aguda , Proteínas de Fusión Oncogénica/genética , Translocación GenéticaRESUMEN
Promyelocytic Leukemia (PML) is a nuclear protein that forms sub-nuclear structures termed nuclear bodies associated with transcriptionally active genomic regions. PML is a tumour suppressor and regulator of cell differentiation. We demonstrate that PML promotes TNFα-induced transcriptional responses by promoting NF-κB activity. TNFα-treated PML-/- cells show normal IκBα degradation and NF-κB nuclear translocation but significantly reduced NF-κB DNA binding and phosphorylation of NF-κB p65. We also demonstrate that the PML retinoic acid receptor-α (PML-RARα) oncofusion protein, which causes acute promyelocytic leukemia, inhibits TNFα induced gene expression and phosphorylation of NF-κB. This study establishes PML as an important regulator of NF-κB and demonstrates that PML-RARα dysregulates NF-κB.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Proteína de la Leucemia Promielocítica/genética , Factor de Transcripción ReIA/genética , Animales , Embrión de Mamíferos , Fibroblastos/citología , Fibroblastos/metabolismo , Ontología de Genes , Genes Reporteros , Células HEK293 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Anotación de Secuencia Molecular , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Proteína de la Leucemia Promielocítica/metabolismo , Transducción de Señal , Factor de Transcripción ReIA/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Tribbles homolog 2 (TRIB2) is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1-3). Studies of TRIB2 indicate that many of the molecular interactions between the single Drosophila Tribbles (Trbl) protein and interacting partners are evolutionary conserved. In this study, we examined the relationship between TRIB2 and cell division cycle 25 (CDC25) family of dual-specificity protein phosphatases (mammalian homologues of Drosophila String), which are key physiological cell cycle regulators. Using co-immunoprecipitation we demonstrate that TRIB2 interacts with CDC25B and CDC25C selectively. Forced overexpression of TRIB2 caused a marked decrease in total CDC25C protein levels. Following inhibition of the proteasome, CDC25C was stabilized in the nuclear compartment. This implicates TRIB2 as a regulator of nuclear CDC25C turnover. In complementary ubiquitination assays, we show that TRIB2-mediated degradation of CDC25C is associated with lysine-48-linked CDC25C polyubiquitination driven by the TRIB2 kinase-like domain. A cell cycle associated role for TRIB2 is further supported by the cell cycle regulated expression of TRIB2 protein levels. Our findings reveal mitotic CDC25C as a new target of TRIB2 that is degraded via the ubiquitin proteasome system. Inappropriate CDC25C regulation could mechanistically underlie TRIB2 mediated regulation of cellular proliferation in neoplastic cells.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Fosfatasas de Especificidad Dual/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Fosfatasas cdc25/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Fosfatasas de Especificidad Dual/genética , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Unión Proteica/genética , Unión Proteica/fisiología , Ubiquitinación/genética , Ubiquitinación/fisiología , Fosfatasas cdc25/genéticaRESUMEN
Nuclear factor (NF)-κB has evolved as a latent, inducible family of transcription factors fundamental in the control of the inflammatory response. The transcription of hundreds of genes involved in inflammation and immune homeostasis require NF-κB, necessitating the need for its strict control. The inducible ubiquitination and proteasomal degradation of the cytoplasmic inhibitor of κB (IκB) proteins promotes the nuclear translocation and transcriptional activity of NF-κB. More recently, an additional role for ubiquitination in the regulation of NF-κB activity has been identified. In this case, the ubiquitination and degradation of the NF-κB subunits themselves plays a critical role in the termination of NF-κB activity and the associated transcriptional response. While there is still much to discover, a number of NF-κB ubiquitin ligases and deubiquitinases have now been identified which coordinate to regulate the NF-κB transcriptional response. This review will focus the regulation of NF-κB subunits by ubiquitination, the key regulatory components and their impact on NF-κB directed transcription.
RESUMEN
Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn's disease (CD), a chronic inflammatory bowel condition. It has been proposed that AIEC-infected macrophages produce high levels of pro-inflammatory cytokines thus contributing to the inflammation observed in CD. AIEC can replicate in macrophages and we wanted to determine if bacterial replication was linked to the high level of cytokine production associated with AIEC-infected macrophages. Therefore, we undertook a genetic analysis of the metabolic requirements for AIEC replication in the macrophage and we show that AIEC replication in this niche is dependent on bacterial glycolysis. In addition, our analyses indicate that AIEC have access to a wide range of nutrients in the macrophage, although the levels of purines and pyrimidines do appear to be limiting. Finally, we show that the macrophage response to AIEC infection is indistinguishable from the response to the non-replicating glycolysis mutant (ΔpfkAB) and a non-pathogenic strain of E. coli, MG1655. Therefore, AIEC does not appear to subvert the normal macrophage response to E. coli during infection.
Asunto(s)
Citocinas/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Glucólisis/genética , Macrófagos/microbiología , Pirimidinas/biosíntesis , Animales , Línea Celular , Enfermedad de Crohn/microbiología , Elementos Transponibles de ADN/genética , Escherichia coli/crecimiento & desarrollo , Biblioteca de Genes , Humanos , Metabolómica , RatonesRESUMEN
The NF-κB transcription factor is the master regulator of the inflammatory response and is essential for the homeostasis of the immune system. NF-κB regulates the transcription of genes that control inflammation, immune cell development, cell cycle, proliferation, and cell death. The fundamental role that NF-κB plays in key physiological processes makes it an important factor in determining health and disease. The importance of NF-κB in tissue homeostasis and immunity has frustrated therapeutic approaches aimed at inhibiting NF-κB activation. However, significant research efforts have revealed the crucial contribution of NF-κB phosphorylation to controlling NF-κB directed transactivation. Importantly, NF-κB phosphorylation controls transcription in a gene-specific manner, offering new opportunities to selectively target NF-κB for therapeutic benefit. This review will focus on the phosphorylation of the NF-κB subunits and the impact on NF-κB function.
RESUMEN
Autoimmune diseases arise from the loss of tolerance to endogenous self-antigens, resulting in a heterogeneous range of chronic conditions that cause considerable morbidity and mortality worldwide. In Western countries, over 5% of the population is affected by some form of autoimmune disease, with enhanced or inappropriate activation of nuclear factor (NF)-κB implicated in a number of these conditions. Although treatment strategies for autoimmunity have improved significantly in recent years, current therapeutics are still not capable of achieving satisfactory disease management in all patients, and as such, the therapeutic modulation of NF-κB is an attractive target in autoimmunity. To date, no NF-κB inhibitors have progressed to the clinic for the treatment of autoimmunity, but a variety of promising approaches targeting multiple stages of the NF-κB pathway are currently being explored. This review focuses on the current strategies being investigated for the inhibition of the NF-κB pathway in autoimmune diseases and considers potential future strategies for the therapeutic targeting of this crucial transcription factor.