RESUMEN
T cell differentiation requires Notch1 signaling. In the present study, we show that an enhancer upstream of Notch1 active in double-negative (DN) mouse thymocytes is responsible for raising Notch1 signaling intrathymically. This enhancer is required to expand multipotent progenitors intrathymically while delaying early differentiation until lineage restrictions have been established. Early thymic progenitors lacking the enhancer show accelerated differentiation through the DN stages and increased frequency of B, innate lymphoid (IL) and natural killer (NK) cell differentiation. Transcription regulators for T cell lineage restriction and commitment are expressed normally, but IL and NK cell gene expression persists after T cell lineage commitment and T cell receptor ß VDJ recombination, Cd3 expression and ß-selection have been impaired. This Notch1 enhancer is inactive in double-positive (DP) thymocytes. Its aberrant reactivation at this stage in Ikaros mutants is required for leukemogenesis. Thus, the DN-specific Notch1 enhancer harnesses the regulatory architecture of DN and DP thymocytes to achieve carefully orchestrated changes in Notch1 signaling required for early lineage restrictions and normal T cell differentiation.
Asunto(s)
Inmunidad Innata , Timocitos , Ratones , Animales , Timocitos/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Linfocitos/metabolismo , Timo , Diferenciación Celular/genética , Linaje de la Célula/genéticaRESUMEN
Signaling through Notch receptors intrinsically regulates tumor cell development and growth. Here, we studied the role of the Notch ligand Jagged2 on immune evasion in non-small cell lung cancer (NSCLC). Higher expression of JAG2 in NSCLC negatively correlated with survival. In NSCLC pre-clinical models, deletion of Jag2, but not Jag1, in cancer cells attenuated tumor growth and activated protective anti-tumor T cell responses. Jag2-/- lung tumors exhibited higher frequencies of macrophages that expressed immunostimulatory mediators and triggered T cell-dependent anti-tumor immunity. Mechanistically, Jag2 ablation promoted Nr4a-mediated induction of Notch ligands DLL1/4 on cancer cells. DLL1/4-initiated Notch1/2 signaling in macrophages induced the expression of transcription factor IRF4 and macrophage immunostimulatory functionality. IRF4 expression was required for the anti-tumor effects of Jag2 deletion in lung tumors. Antibody targeting of Jagged2 inhibited tumor growth and activated IRF4-driven macrophage-mediated anti-tumor immunity. Thus, Jagged2 orchestrates immunosuppressive systems in NSCLC that can be overcome to incite macrophage-mediated anti-tumor immunity.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Factores Reguladores del Interferón , Proteína Jagged-2 , Neoplasias Pulmonares , Ratones Noqueados , Macrófagos Asociados a Tumores , Animales , Humanos , Ratones , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Factores Reguladores del Interferón/metabolismo , Factores Reguladores del Interferón/genética , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-2/metabolismo , Proteína Jagged-2/genética , Proteína Jagged-2/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Receptor Notch1/metabolismo , Receptor Notch1/genética , Receptores Notch/metabolismo , Transducción de Señal , Escape del Tumor/inmunología , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismoRESUMEN
The Notch signaling pathway comprises multiple ligands that are used in distinct biological contexts. In principle, different ligands could activate distinct target programs in signal-receiving cells, but it is unclear how such ligand discrimination could occur. Here, we show that cells use dynamics to discriminate signaling by the ligands Dll1 and Dll4 through the Notch1 receptor. Quantitative single-cell imaging revealed that Dll1 activates Notch1 in discrete, frequency-modulated pulses that specifically upregulate the Notch target gene Hes1. By contrast, Dll4 activates Notch1 in a sustained, amplitude-modulated manner that predominantly upregulates Hey1 and HeyL. Ectopic expression of Dll1 or Dll4 in chick neural crest produced opposite effects on myogenic differentiation, showing that ligand discrimination can occur in vivo. Finally, analysis of chimeric ligands suggests that ligand-receptor clustering underlies dynamic encoding of ligand identity. The ability of the pathway to utilize ligands as distinct communication channels has implications for diverse Notch-dependent processes.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células CHO , Proteínas de Unión al Calcio , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Embrión de Pollo , Cricetulus , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ligandos , Proteínas de la Membrana/genética , Ratones , Receptor Notch1/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Regulación hacia ArribaRESUMEN
The mechanisms by which transcription factor haploinsufficiency alters the epigenetic and transcriptional landscape in human cells to cause disease are unknown. Here, we utilized human induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) to show that heterozygous nonsense mutations in NOTCH1 that cause aortic valve calcification disrupt the epigenetic architecture, resulting in derepression of latent pro-osteogenic and -inflammatory gene networks. Hemodynamic shear stress, which protects valves from calcification in vivo, activated anti-osteogenic and anti-inflammatory networks in NOTCH1(+/+), but not NOTCH1(+/-), iPSC-derived ECs. NOTCH1 haploinsufficiency altered H3K27ac at NOTCH1-bound enhancers, dysregulating downstream transcription of more than 1,000 genes involved in osteogenesis, inflammation, and oxidative stress. Computational predictions of the disrupted NOTCH1-dependent gene network revealed regulatory nodes that, when modulated, restored the network toward the NOTCH1(+/+) state. Our results highlight how alterations in transcription factor dosage affect gene networks leading to human disease and reveal nodes for potential therapeutic intervention.
Asunto(s)
Epigénesis Genética , Redes Reguladoras de Genes , Receptor Notch1/genética , Células Endoteliales/metabolismo , Femenino , Haploinsuficiencia , Código de Histonas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/metabolismo , Masculino , Osteogénesis , Linaje , Receptor Notch1/metabolismo , Estrés Mecánico , Transcripción GenéticaRESUMEN
Gain-of-function NOTCH1 mutations drive oncogenic MYC expression in T-ALL cells. Zhou et al. (2022) reveal that Notch-targeted therapy-resistant T-ALL cells activate EBF1, which promotes a T-to-B lineage shift and maintains oncogenic MYC expression in the absence of Notch signaling.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Receptor Notch1/genética , Receptor Notch1/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de SeñalRESUMEN
Notch signaling is a key developmental pathway that is subject to frequent genetic and epigenetic perturbations in many different human tumors. Here we investigate whether long noncoding RNA (lncRNA) genes, in addition to mRNAs, are key downstream targets of oncogenic Notch1 in human T cell acute lymphoblastic leukemia (T-ALL). By integrating transcriptome profiles with chromatin state maps, we have uncovered many previously unreported T-ALL-specific lncRNA genes, a fraction of which are directly controlled by the Notch1/Rpbjκ activator complex. Finally we have shown that one specific Notch-regulated lncRNA, LUNAR1, is required for efficient T-ALL growth in vitro and in vivo due to its ability to enhance IGF1R mRNA expression and sustain IGF1 signaling. These results confirm that lncRNAs are important downstream targets of the Notch signaling pathway, and additionally they are key regulators of the oncogenic state in T-ALL.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , ARN Largo no Codificante/análisis , Receptor Notch1/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , ARN Largo no Codificante/genética , Transducción de Señal , Timo/patologíaRESUMEN
Tissue-resident memory T cells (TRM cells) in the airways mediate protection against respiratory infection. We characterized TRM cells expressing integrin αE (CD103) that reside within the epithelial barrier of human lungs. These cells had specialized profiles of chemokine receptors and adhesion molecules, consistent with their unique localization. Lung TRM cells were poised for rapid responsiveness by constitutive expression of deployment-ready mRNA encoding effector molecules, but they also expressed many inhibitory regulators, suggestive of programmed restraint. A distinct set of transcription factors was active in CD103+ TRM cells, including Notch. Genetic and pharmacological experiments with mice revealed that Notch activity was required for the maintenance of CD103+ TRM cells. We have thus identified specialized programs underlying the residence, persistence, vigilance and tight control of human lung TRM cells.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Memoria Inmunológica , Subtipo H3N2 del Virus de la Influenza A/inmunología , Pulmón/inmunología , Infecciones por Orthomyxoviridae/inmunología , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Infecciones del Sistema Respiratorio/inmunología , Animales , Antígenos CD/metabolismo , Células Cultivadas , Femenino , Humanos , Cadenas alfa de Integrinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Receptor Notch1/genética , Receptor Notch2/genéticaRESUMEN
Sequencing efforts led to the identification of somatic mutations that could affect the self-renewal and differentiation of cancer-initiating cells. One such recurrent mutation targets the binding pocket of the ubiquitin ligase Fbxw7. Missense FBXW7 mutations are prevalent in various tumors, including T cell acute lymphoblastic leukemia (T-ALL). To study the effects of such lesions, we generated animals carrying regulatable Fbxw7 mutant alleles. Here, we show that these mutations specifically bolster cancer-initiating cell activity in collaboration with Notch1 oncogenes but spare normal hematopoietic stem cell function. We were also able to show that FBXW7 mutations specifically affect the ubiquitylation and half-life of c-Myc protein, a key T-ALL oncogene. Using animals carrying c-Myc fusion alleles, we connected Fbxw7 function to c-Myc abundance and correlated c-Myc expression to leukemia-initiating activity. Finally, we demonstrated that small-molecule-mediated suppression of MYC activity leads to T-ALL remission, suggesting an effective therapeutic strategy.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Modelos Animales de Enfermedad , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Noqueados , Mutación Missense , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Receptor Notch1/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.
Asunto(s)
Calcineurina/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Biotinilación , Centrosoma/metabolismo , Simulación por Computador , Células HEK293 , Células HeLa , Humanos , Espectrometría de Masas , Monoéster Fosfórico Hidrolasas/química , Fosforilación , Mapas de Interacción de Proteínas , Proteoma/metabolismo , Receptor Notch1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Notch signaling pathway activity, particularly fluctuations in the biologically active effector fragment NICD, is required for rapid and efficient dynamic regulation of proper fate decisions in stem cells. In this study, we identified NEDD4-binding protein 1 (N4BP1), which is highly expressed in the developing mouse cerebral cortex, as a negative modulator of Notch signaling dynamics in neural progenitor cells. Intriguingly, N4BP1 regulated NICD stability specifically after Notch1 S3 cleavage through ubiquitin-mediated degradation that depended on its RAM domain, not its PEST domain, as had been extensively and previously described. The CoCUN domain in N4BP1, particularly the "Phe-Pro" motif (862/863 amino acid), was indispensable for mediating NICD degradation. The Ring family E3 ligase Trim21 was, in contrast to other NEDD4 family members, required for N4BP1-regulated NICD degradation. Overexpression of N4BP1 in cortical neural progenitors promoted neural stem cell differentiation, whereas neural progenitor cells lacking N4BP1 were sensitized to Notch signaling, resulting in the maintenance of stem-like properties in neural progenitor cells and lower production of cortical neurons.
Asunto(s)
Neocórtex , Células-Madre Neurales , Animales , Ratones , Diferenciación Celular/fisiología , Neocórtex/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Receptor Notch1/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal/fisiologíaRESUMEN
It is currently unclear whether tissue changes surrounding multifocal epithelial tumors are a cause or consequence of cancer. Here, we provide evidence that loss of mesenchymal Notch/CSL signaling causes tissue alterations, including stromal atrophy and inflammation, which precede and are potent triggers for epithelial tumors. Mice carrying a mesenchymal-specific deletion of CSL/RBP-Jκ, a key Notch effector, exhibit spontaneous multifocal keratinocyte tumors that develop after dermal atrophy and inflammation. CSL-deficient dermal fibroblasts promote increased tumor cell proliferation through upregulation of c-Jun and c-Fos expression and consequently higher levels of diffusible growth factors, inflammatory cytokines, and matrix-remodeling enzymes. In human skin samples, stromal fields adjacent to multifocal premalignant actinic keratosis lesions exhibit decreased Notch/CSL signaling and associated molecular changes. Importantly, these changes in gene expression are also induced by UVA, a known environmental cause of cutaneous field cancerization and skin cancer.
Asunto(s)
Proteínas Musculares/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Animales , Atrofia/metabolismo , Atrofia/patología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Células Cultivadas , Dermatitis/metabolismo , Dermatitis/patología , Eliminación de Gen , Técnicas de Silenciamiento del Gen , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Queratinocitos/patología , Queratosis/metabolismo , Queratosis/patología , Mesodermo/metabolismo , Mesodermo/patología , Ratones , Proteínas Musculares/genética , Receptor Notch1/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
ABSTRACT: Notch signaling regulates cell-fate decisions in several developmental processes and cell functions. However, the role of Notch in hepatic thrombopoietin (TPO) production remains unclear. We noted thrombocytopenia in mice with hepatic Notch1 deficiency and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO messenger RNA levels, concomitant with lower numbers of platelets and impaired megakaryocyte differentiation and maturation, which were rescued by addition of exogenous TPO. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes, consistent with the RNA-sequencing analysis. JAK2/STAT3 phosphorylation and TPO production was also impaired in cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Consistently, hepatocyte-specific Notch1 deletion inhibited JAK2/STAT3 phosphorylation and hepatic TPO production induced by administration of desialylated platelets in vivo. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation in hepatocytes. Hepatocyte Ashwell-Morell receptor (AMR), a heterodimer of asialoglycoprotein receptor 1 [ASGR1] and ASGR2, physically associates with Notch1, and inhibition of AMR impaired Notch1 signaling activation and hepatic TPO production. Furthermore, blockage of Delta-like 4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation, and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.
Asunto(s)
Hepatocitos , Janus Quinasa 2 , Hígado , Receptor Notch1 , Trombopoyetina , Animales , Receptor Notch1/metabolismo , Receptor Notch1/genética , Trombopoyetina/metabolismo , Trombopoyetina/genética , Ratones , Hígado/metabolismo , Hepatocitos/metabolismo , Janus Quinasa 2/metabolismo , Janus Quinasa 2/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Ratones Noqueados , Transducción de Señal , Fosforilación , Plaquetas/metabolismo , Ratones Endogámicos C57BL , Trombocitopenia/metabolismo , Trombocitopenia/genética , Trombocitopenia/patologíaRESUMEN
Notch has been implicated in human cancers and is a putative therapeutic target. However, the regulation of Notch activation in the nucleus remains largely uncharacterized. Therefore, characterizing the detailed mechanisms governing Notch degradation will identify attractive strategies for treating Notch-activated cancers. Here, we report that the long noncoding RNA (lncRNA) BREA2 drives breast cancer metastasis by stabilizing the Notch1 intracellular domain (NICD1). Moreover, we reveal WW domain containing E3 ubiquitin protein ligase 2 (WWP2) as an E3 ligase for NICD1 at K1821 and a suppressor of breast cancer metastasis. Mechanistically, BREA2 impairs WWP2-NICD1 complex formation and in turn stabilizes NICD1, leading to Notch signaling activation and lung metastasis. BREA2 loss sensitizes breast cancer cells to inhibition of Notch signaling and suppresses the growth of breast cancer patient-derived xenograft tumors, highlighting its therapeutic potential in breast cancer. Taken together, these results reveal the lncRNA BREA2 as a putative regulator of Notch signaling and an oncogenic player driving breast cancer metastasis.
Asunto(s)
Neoplasias de la Mama , Neoplasias Pulmonares , ARN Largo no Codificante , Humanos , Femenino , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias de la Mama/genética , Receptor Notch1/genética , Receptor Notch1/metabolismoRESUMEN
Notch signaling plays a critical role in cell fate decisions in all cell types. Furthermore, gain-of-function mutations in NOTCH1 have been uncovered in many human cancers. Disruption of Notch signaling has recently emerged as an attractive disease treatment strategy. However, the nuclear interaction landscape of the oncoprotein NOTCH1 remains largely unexplored. We therefore employed here a proximity-dependent biotin identification approach to identify in vivo protein associations with the nuclear Notch1 intracellular domain in live cells. We identified a large set of previously reported and unreported proteins that associate with NOTCH1, including general transcription and elongation factors, DNA repair and replication factors, coactivators, corepressors, and components of the NuRD and SWI/SNF chromatin remodeling complexes. We also found that Notch1 intracellular domain associates with protein modifiers and components of other signaling pathways that may influence Notch signal transduction and protein stability such as USP7. We further validated the interaction of NOTCH1 with histone deacetylase 1 or GATAD2B using protein network analysis, proximity-based ligation, in vivo cross-linking and coimmunoprecipitation assays in several Notch-addicted cancer cell lines. Through data mining, we also revealed potential drug targets for the inhibition of Notch signaling. Collectively, these results provide a valuable resource to uncover the mechanisms that fine-tune Notch signaling in tumorigenesis and inform therapeutic targets for Notch-addicted tumors.
Asunto(s)
Carcinogénesis , Neoplasias , Proteínas Oncogénicas , Receptor Notch1 , Humanos , Diferenciación Celular , Línea Celular , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Peptidasa Específica de Ubiquitina 7/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
The importance of macrophages in adipose tissue (AT) homeostasis and inflammation is well established. However, the potential cues that regulate their function remain incompletely understood. To bridge this important gap, we sought to characterize novel pathways involved using a mouse model of diet-induced obesity. By performing transcriptomics analysis of AT macrophages (ATMs), we found that late-stage ATMs from high-fat diet mice presented with perturbed Notch signaling accompanied by robust proinflammatory and metabolic changes. To explore the hypothesis that the deregulated Notch pathway contributes to the development of AT inflammation and diet-induced obesity, we employed a genetic approach to abrogate myeloid Notch1 and Notch2 receptors. Our results revealed that the combined loss of Notch1 and Notch2 worsened obesity-related metabolic dysregulation. Body and AT weight gain was higher, blood glucose levels increased and metabolic parameters were substantially worsened in deficient mice fed high-fat diet. Moreover, serum insulin and leptin were elevated as were triglycerides. Molecular analysis of ATMs showed that deletion of Notch receptors escalated inflammation through the induction of an M1-like pro-inflammatory phenotype. Our findings thus support a protective role of myeloid Notch signaling in adipose tissue inflammation and metabolic dysregulation.
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Tejido Adiposo , Dieta Alta en Grasa , Inflamación , Macrófagos , Obesidad , Receptor Notch1 , Receptor Notch2 , Transducción de Señal , Animales , Macrófagos/inmunología , Macrófagos/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo/inmunología , Ratones , Dieta Alta en Grasa/efectos adversos , Inflamación/inmunología , Inflamación/metabolismo , Transducción de Señal/inmunología , Obesidad/metabolismo , Obesidad/inmunología , Receptor Notch1/metabolismo , Receptor Notch1/genética , Receptor Notch2/metabolismo , Receptor Notch2/genética , Ratones Noqueados , Ratones Endogámicos C57BL , MasculinoRESUMEN
The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.
Asunto(s)
Complejo CD3/inmunología , Citocinas/biosíntesis , Motivo de Activación del Inmunorreceptor Basado en Tirosina/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Complejo CD3/metabolismo , Línea Celular , Proliferación Celular , Células HEK293 , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/metabolismoRESUMEN
Notch signaling induces gene expression of the T cell lineage and discourages alternative fate outcomes. Hematopoietic deficiency in the Notch target Hes1 results in severe T cell lineage defects; however, the underlying mechanism is unknown. We found here that Hes1 constrained myeloid gene-expression programs in T cell progenitor cells, as deletion of the myeloid regulator C/EBP-α restored the development of T cells from Hes1-deficient progenitor cells. Repression of Cebpa by Hes1 required its DNA-binding and Groucho-recruitment domains. Hes1-deficient multipotent progenitor cells showed a developmental bias toward myeloid cells and dendritic cells after Notch signaling, whereas Hes1-deficient lymphoid progenitor cells required additional cytokine signaling for diversion into the myeloid lineage. Our findings establish the importance of constraining developmental programs of the myeloid lineage early in T cell development.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Proteína alfa Potenciadora de Unión a CCAAT/inmunología , Proteínas de Homeodominio/inmunología , Receptor Notch1/inmunología , Linfocitos T/inmunología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Línea Celular , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Citometría de Flujo , Expresión Génica/inmunología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Linfopoyesis/genética , Linfopoyesis/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Células Mieloides/inmunología , Células Mieloides/metabolismo , Unión Proteica/inmunología , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/inmunología , Células Madre/inmunología , Células Madre/metabolismo , Linfocitos T/metabolismo , Factor de Transcripción HES-1RESUMEN
ABSTRACT: Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL), but pan-Notch inhibitors showed excessive toxicity in clinical trials. To find alternative ways to target Notch signals, we investigated cell division cycle 73 (Cdc73), which is a Notch cofactor and key component of the RNA polymerase-associated transcriptional machinery, an emerging target in T-ALL. Although we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, chromatin and nascent gene expression profiling showed that Cdc73 intersects with Ets1 and Notch at chromatin within enhancers to activate expression of known T-ALL oncogenes through its enhancer functions. Cdc73 also intersects with these factors within promoters to activate transcription of genes that are important for DNA repair and oxidative phosphorylation through its gene body functions. Consistently, Cdc73 deletion induced DNA damage and apoptosis and impaired mitochondrial function. The CDC73-induced DNA repair expression program co-opted by NOTCH1 is more highly expressed in T-ALL than in any other cancer. These data suggest that Cdc73 might induce a gene expression program that was eventually intersected and hijacked by oncogenic Notch to augment proliferation and mitigate the genotoxic and metabolic stresses of elevated Notch signaling. Our report supports studying factors such as CDC73 that intersect with Notch to derive a basic scientific understanding on how to combat Notch-dependent cancers without directly targeting the Notch complex.
Asunto(s)
5'-Nucleotidasa , Leucemia de Células T , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animales , Ratones , Línea Celular Tumoral , Cromatina , Daño del ADN/genética , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Factores de Transcripción/genética , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismoRESUMEN
Osteoarthritis (OA) is the main cause of cartilage damage and disability. This study explored the biological function of S-phase kinase-associated protein 2 (SKP2) and Kruppel-like factor 11 (KLF11) in OA progression and its underlying mechanisms. C28/I2 chondrocytes were stimulated with IL-1ß to mimic OA in vitro. We found that SKP2, Jumonji domain-containing protein D3 (JMJD3), and Notch receptor 1 (NOTCH1) were upregulated, while KLF11 was downregulated in IL-1ß-stimulated chondrocytes. SKP2/JMJD3 silencing or KLF11 overexpression repressed apoptosis and extracellular matrix (ECM) degradation in chondrocytes. Mechanistically, SKP2 triggered the ubiquitination and degradation of KLF11 to transcriptionally activate JMJD3, which resulted in activation of NOTCH1 through inhibiting H3K27me3. What's more, the in vivo study found that KLF11 overexpression delayed OA development in rats via restraining apoptosis and maintaining the balance of ECM metabolism. Taken together, ubiquitination and degradation of KLF11 regulated by SKP2 contributed to OA progression by activation of JMJD3/NOTCH1 pathway. Our findings provide promising therapeutic targets for OA.
Asunto(s)
Condrocitos , Histona Demetilasas con Dominio de Jumonji , Osteoartritis , Receptor Notch1 , Proteínas Quinasas Asociadas a Fase-S , Ubiquitinación , Receptor Notch1/metabolismo , Receptor Notch1/genética , Animales , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoartritis/genética , Ratas , Condrocitos/metabolismo , Condrocitos/patología , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Masculino , Transducción de Señal , Ratas Sprague-Dawley , Humanos , Apoptosis , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genéticaRESUMEN
BACKGROUND: NOTCH1 pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how NOTCH1 regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which NOTCH1 pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive. METHODS: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 genome editing was utilized to delete NOTCH1 in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and NOTCH1 knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq. RESULTS: Human NOTCH1 knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. NOTCH1 deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in NOTCH1 knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in NOTCH1 knockout cell populations. CONCLUSIONS: NOTCH1 is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic NOTCH1 variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.