RESUMO
The divergence of the common dendritic cell progenitor1-3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages4,5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors-such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer4,6-but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis7 suggested that Nfil3 acts upstream of Id2, Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the -165 kb Zeb2 enhancer8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPß. In vivo mutational analysis using CRISPR-Cas9 targeting showed that these NFIL3-C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3-C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (TH2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths9-11. Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the -165 kb Zeb2 enhancer.
Assuntos
Diferenciação Celular , Células Dendríticas , Elementos Facilitadores Genéticos , Mutação , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Células Dendríticas/classificação , Células Dendríticas/citologia , Células Dendríticas/patologia , Elementos Facilitadores Genéticos/genética , Epistasia Genética , Proteína 2 Inibidora de Diferenciação , Linfócitos/citologia , Camundongos , Células Mieloides/citologia , Nematospiroides dubius/imunologia , Proteínas Repressoras , Células Th2/citologia , Células Th2/imunologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genéticaRESUMO
Senescent cells are beneficial for repairing acute tissue damage, but they are harmful when they accumulate in tissues, as occurs with advancing age. Senescence-associated extracellular vesicles (S-EVs) can mediate cell-to-cell communication and export intracellular content to the microenvironment of aging tissues. Here, we studied the uptake of EVs from senescent cells (S-EVs) and proliferating cells (P-EVs) and found that P-EVs were readily taken up by proliferating cells (fibroblasts and cervical cancer cells) while S-EVs were not. We thus investigated the surface proteome (surfaceome) of P-EVs relative to S-EVs derived from cells that had reached senescence via replicative exhaustion, exposure to ionizing radiation, or treatment with etoposide. We found that relative to P-EVs, S-EVs from all senescence models were enriched in proteins DPP4, ANXA1, ANXA6, S10AB, AT1A1, and EPHB2. Among them, DPP4 was found to selectively prevent uptake by proliferating cells, as ectopic overexpression of DPP4 in HeLa cells rendered DPP4-expressing EVs that were no longer taken up by other proliferating cells. We propose that DPP4 on the surface of S-EVs makes these EVs refractory to internalization by proliferating cells, advancing our knowledge of the impact of senescent cells in aging-associated processes.
Assuntos
Senescência Celular , Vesículas Extracelulares , Humanos , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Células HeLa , Vesículas Extracelulares/metabolismo , EnvelhecimentoRESUMO
Senescent cell accumulation in aging tissues is linked to age-associated diseases and declining function, prompting efforts to eliminate them. Mass spectrometry analysis revealed that DPP4 (dipeptidyl peptidase 4) was selectively expressed on the surface of senescent, but not proliferating, human diploid fibroblasts. Importantly, the differential presence of DPP4 allowed flow cytometry-mediated isolation of senescent cells using anti-DPP4 antibodies. Moreover, antibody-dependent cell-mediated cytotoxicity (ADCC) assays revealed that the cell surface DPP4 preferentially sensitized senescent, but not dividing, fibroblasts to cytotoxicity by natural killer cells. In sum, the selective expression of DPP4 on the surface of senescent cells enables their preferential elimination.
Assuntos
Senescência Celular/fisiologia , Dipeptidil Peptidase 4/metabolismo , Proteínas de Membrana/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Citotoxicidade Celular Dependente de Anticorpos , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Diploide , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/enzimologia , Espectrometria de Massas , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismoRESUMO
Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are two distinct and predominant types of human lung cancer. IκB kinase α (IKKα) has been shown to suppress lung SCC development, but its role in ADC is unknown. We found inactivating mutations and homologous or hemizygous deletions in the CHUK locus, which encodes IKKα, in human lung ADCs. The CHUK deletions significantly reduced the survival time of patients with lung ADCs harboring KRAS mutations. In mice, lung-specific Ikkα ablation (IkkαΔLu ) induces spontaneous ADCs and promotes KrasG12D-initiated ADC development, accompanied by increased cell proliferation, decreased cell senescence, and reactive oxygen species (ROS) accumulation. IKKα deletion up-regulates NOX2 and down-regulates NRF2, leading to ROS accumulation and blockade of cell senescence induction, which together accelerate ADC development. Pharmacologic inhibition of NADPH oxidase or ROS impairs KrasG12D-mediated ADC development in IkkαΔLu mice. Therefore, IKKα modulates lung ADC development by controlling redox regulatory pathways. This study demonstrates that IKKα functions as a suppressor of lung ADC in human and mice through a unique mechanism that regulates tumor cell-associated ROS metabolism.
Assuntos
Adenocarcinoma/genética , Quinase I-kappa B/fisiologia , Neoplasias Pulmonares/genética , Acetofenonas , Acetilcisteína , Adenocarcinoma/metabolismo , Animais , Proliferação de Células , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Epigênese Genética , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , NADPH Oxidase 2/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
RNA is a versatile biomaterial that can be used to engineer nanoassemblies for personalized treatment of various diseases. Despite promising advancements, the design of RNA nanoassemblies with minimal recognition by the immune system remains a major challenge. Here, an approach is reported to engineer RNA fibrous structures to operate as a customizable platform for efficient coordination of siRNAs and for maintaining low immunostimulation. Functional RNA fibers are studied in silico and their formation is confirmed by various experimental techniques and visualized by atomic force microscopy (AFM). It is demonstrated that the RNA fibers offer multiple advantages among which are: i) programmability and modular design that allow for simultaneous controlled delivery of multiple siRNAs and fluorophores, ii) reduced immunostimulation when compared to other programmable RNA nanoassemblies, and iii) simple production protocol for endotoxin-free fibers with the option of their cotranscriptional assembly. Furthermore, it is shown that functional RNA fibers can be efficiently delivered with various organic and inorganic carriers while retaining their structural integrity in cells. Specific gene silencing triggered by RNA fibers is assessed in human breast cancer and melanoma cell lines, with the confirmed ability of functional fibers to selectively target single nucleotide mutations.
RESUMO
Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1ß and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation. Some effectors, like Y. pestis YopM, may block inflammasome activation. Here we show that YopM prevents Y. pestis induced activation of the Pyrin inflammasome induced by the RhoA-inhibiting effector YopE, which is a GTPase activating protein. YopM blocks YopE-induced Pyrin-mediated caspase-1 dependent IL-1ß/IL-18 production and cell death. We also detected YopM in a complex with Pyrin and kinases RSK1 and PKN1, putative negative regulators of Pyrin. In contrast to wild-type mice, Pyrin deficient mice were also highly susceptible to an attenuated Y. pestis strain lacking YopM, emphasizing the importance of inhibition of Pyrin in vivo. A complex interplay between the Y. pestis T3SS and IL-1ß/IL-18 production is evident, involving at least four inflammasome pathways. The secreted effector YopJ triggers caspase-8- dependent IL-1ß activation, even when YopM is present. Additionally, the presence of the T3SS needle/translocon activates NLRP3 and NLRC4-dependent IL-1ß generation, which is blocked by YopK, but not by YopM. Taken together, the data suggest YopM specificity for obstructing the Pyrin pathway, as the effector does not appear to block Y. pestis-induced NLRP3, NLRC4 or caspase-8 dependent caspase-1 processing. Thus, we identify Y. pestis YopM as a microbial inhibitor of the Pyrin inflammasome. The fact that so many of the Y. pestis T3SS components are participating in regulation of IL-1ß/IL-18 release suggests that these effects are essential for maximal control of innate immunity during plague.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Inflamassomos/imunologia , Peste/imunologia , Pirina/imunologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Yersinia pestis/imunologiaRESUMO
C/EBPß is an auto-repressed protein that becomes post-translationally activated by Ras-MEK-ERK signalling. C/EBPß is required for oncogene-induced senescence (OIS) of primary fibroblasts, but also displays pro-oncogenic functions in many tumour cells. Here, we show that C/EBPß activation by H-Ras(V12) is suppressed in immortalized/transformed cells, but not in primary cells, by its 3' untranslated region (3'UTR). 3'UTR sequences inhibited Ras-induced cytostatic activity of C/EBPß, DNA binding, transactivation, phosphorylation, and homodimerization, without significantly affecting protein expression. The 3'UTR suppressed induction of senescence-associated C/EBPß target genes, while promoting expression of genes linked to cancers and TGFß signalling. An AU-rich element (ARE) and its cognate RNA-binding protein, HuR, were required for 3'UTR inhibition. These components also excluded the Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPß translation controls de-repression by Ras signalling. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras-induced C/EBPß activation and OIS to proceed. Our findings reveal a novel mechanism whereby non-coding mRNA sequences selectively regulate C/EBPß activity and suppress its anti-oncogenic functions.
Assuntos
Regiões 3' não Traduzidas , Envelhecimento , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica , Proteína Oncogênica p21(ras)/metabolismo , Linhagem Celular , Humanos , Modelos Biológicos , RNA Mensageiro/metabolismoRESUMO
Members of the C/EBP family of transcription factors bind to the Taz2 domain of p300/CBP and mediate its phosphorylation through the recruitment of specific kinases. Short sequence motifs termed homology boxes A and B, which comprise their minimal transactivation domains (TADs), are conserved between C/EBP activators and are necessary for specific p300/CBP binding. A possible mode of interaction between C/EBP TADs and the p300 Taz2 domain was implied by the crystal structure of a chimeric protein composed of residues 1723-1818 of p300 Taz2 and residues 37-61 of C/EBPâ. The segment corresponding to the C/EBPâ TAD forms two orthogonally disposed helices connected by a short linker and interacts with the core structure of Taz2 from a symmetry-related molecule. It is proposed that other members of the C/EBP family interact with the Taz2 domain in the same manner. The position of the C/EBPâ peptide on the Taz2 protein interaction surface suggests that the N-termini of C/EBP proteins are unbound in the C/EBP-p300 Taz2 complex. This observation is in agreement with the known location of the docking site of protein kinase HIPK2 in the C/EBPß N-terminus, which associates with the C/EBPß-p300 complex.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Fatores de Transcrição de p300-CBP/química , Sequência de Aminoácidos , Proteínas Estimuladoras de Ligação a CCAAT/química , Cristalografia por Raios X , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Although inflammation plays a central role in the pathogenesis of acute lung injury, the molecular mechanisms underlying inflammatory responses in acute lung injury are poorly understood, and therapeutic options remain limited. CCAAT/enhancer-binding proteins, C/EBPß and C/EBPδ, are expressed in the lung and have been implicated in the regulation of inflammatory mediators. However, their functions in lung pathobiological characteristics are not well characterized. Herein, we show that C/EBPß and C/EBPδ are activated in mouse lung after intrapulmonary deposition of lipopolysaccharide (LPS). Mice carrying a targeted deletion of the C/EBPδ gene displayed significant attenuation of the lung permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), and neutrophils in bronchial alveolar lavage fluids compared with wild-type mice. These phenotypes were consistent with morphological evaluation of lung, which showed reduced inflammatory cell influx and minimal intra-alveolar hemorrhage. Moreover, mutant mice expressed considerably less tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 in bronchial alveolar lavage fluids in LPS-injured lung compared with wild-type mice. In contrast, C/EBPß deficiency had no effect on LPS-induced lung injury. By using small-interfering RNA-mediated knockdown for C/EBPδ, we demonstrate, for the first time to our knowledge, that C/EBPδ plays a critical role for the tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 production in LPS-stimulated alveolar macrophages. These findings demonstrate that C/EBPδ, but not C/EBPß, plays an important role in LPS-induced lung inflammatory responses and injury.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Animais , Líquido da Lavagem Broncoalveolar , Proteína delta de Ligação ao Facilitador CCAAT/deficiência , Linhagem Celular , Quimiocinas/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Interleucina-6/metabolismo , Lipopolissacarídeos , Luciferases/metabolismo , Pulmão/enzimologia , Pulmão/patologia , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/patologia , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Psoriasis is a relapsing skin disease characterized by abnormal keratinocyte proliferation and differentiation and by an influx of inflammatory immune cells. Recently, IL-17 cytokines have been strongly implicated as critical for the pathogenesis of this disease. IL-17A (also known as IL-17) and IL-17F are the signature cytokines of Th17 cells, but are also produced by innate cells, including γδ T cells present in skin, whereas epithelial cells, including keratinocytes, may produce IL-17C. IL-17 cytokines signal via the adaptor protein connection to IκB kinase and stress-activated protein kinases (CIKS)/Act1. Psoriasis is a disease with a strong genetic predisposition, and the gene encoding CIKS has recently been identified as a susceptibility locus. Unexpectedly, one predisposing gene variant features a mutation that impairs rather than enhances CIKS-mediated IL-17 cytokine signaling, counter to the predicted role for IL-17 cytokines in psoriatic inflammation. In this study, we demonstrate, however, that this mutant adaptor does not impair the IL-17-specific contributions to the genetic response when combined with TNF-α, a cytokine also prominent in psoriatic inflammation. Interestingly, TNF-α signals compensate IL-17 signaling defects imposed by this mutant adaptor even for genes that are not induced by TNF-α alone, including the transcription factors CCAAT/enhancer binding protein δ and IκBζ, which help regulate secondary gene expression in response to IL-17. Based on these findings we discuss a scenario in which the mutant adaptor may interfere with homeostatic maintenance of epithelial barriers, thereby potentially enabling the initiation of inflammatory responses to insults, whereas this same mutant adaptor would still be able to mediate IL-17-specific contributions to inflammation once TNF-α is present.
Assuntos
Predisposição Genética para Doença/genética , Interleucina-17/imunologia , Psoríase/genética , Transdução de Sinais/imunologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Citocinas/imunologia , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Interleucina-17/metabolismo , Camundongos , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Psoríase/imunologia , Psoríase/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/imunologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
C/EBPs, particularly C/EBPß and C/EBPδ, are known to participate in the regulation of many genes associated with inflammation. However, very little is known regarding the activation and functions of C/EBPß and C/EBPδ in acute lung inflammation and injury. In this study, we show that both C/EBPß and C/EBPδ activation are triggered in lungs and in alveolar macrophages following intrapulmonary deposition of IgG immune complexes. We further show that mice carrying a targeted deletion of the C/EBPß gene displayed significant attenuation of the permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), total number of WBCs, and neutrophils in bronchoalveolar lavage fluids compared with wild-type mice. Moreover, the mutant mice expressed considerably less TNF-α, IL-6, and CXC/CC chemokine and soluble ICAM-1 proteins in bronchoalveolar lavage fluids, and corresponding mRNAs in the IgG immune complex-injured lung, compared with wild-type mice. These phenotypes were associated with a significant reduction in morphological lung injury. In contrast, C/EBPδ deficiency had no effect on IgG immune complex-induced lung injury. IgG immune complex-stimulated C/EBPß-deficient alveolar macrophages released significantly less TNF-α, IL-6, MIP-2, keratinocyte cell-derived chemokine, and MIP-1α compared with wild-type cells. Similar decreases in IgG immune complex-induced inflammatory mediator production were observed following small interfering RNA ablation of C/EBPß in a murine alveolar macrophage cell line. These findings implicate C/EBPß as a critical regulator of IgG immune complex-induced inflammatory responses and injury in the lung.
Assuntos
Lesão Pulmonar Aguda/imunologia , Complexo Antígeno-Anticorpo/administração & dosagem , Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Animais , Complexo Antígeno-Anticorpo/efeitos adversos , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/deficiência , Proteína delta de Ligação ao Facilitador CCAAT/deficiência , Proteína delta de Ligação ao Facilitador CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/fisiologia , Linhagem Celular , Modelos Animais de Doenças , Imunoglobulina G/administração & dosagem , Imunoglobulina G/efeitos adversos , Mediadores da Inflamação/administração & dosagem , Mediadores da Inflamação/efeitos adversos , Mediadores da Inflamação/fisiologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
CCAAT/enhancer-binding protein ß (C/EBPß) and C/EBPδ are known to participate in the regulation of many genes associated with inflammation. However, little is known about the activation and function of C/EBPß and -δ in inflammatory responses elicited by Fcγ receptor (FcγR) activation. Here we show that C/EBPß and -δ activation are induced in IgG immune complex (IC)-treated macrophages. The increased expression of C/EBPß and -δ occurred at both mRNA and protein levels. Furthermore, induction of C/EBPß and -δ was mediated, to a large extent, by activating FcγRs. Using siRNA-mediated knockdown as well as macrophages deficient for C/EBPß and/or -δ, we demonstrate that C/EBPß and -δ play a critical role in the production of TNF-α, MIP-2, and MIP-1α in IgG IC-stimulated macrophages. Moreover, both ERK1/2 and p38 MAPK are involved in C/EBP induction and TNF-α, MIP-2, and MIP-1α production induced by IgG IC. We provide the evidence that C5a regulates IgG IC-induced inflammatory responses by enhancing ERK1/2 and p38 MAPK activities as well as C/EBPß and -δ activities. Collectively, these data suggest that C/EBPß and -δ are key regulators for FcγR-mediated induction of cytokines and chemokines in macrophages. Furthermore, C/EBPs may play an important regulatory role in IC-associated inflammatory responses.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Quimiocinas/biossíntese , Complemento C5a/metabolismo , Receptores de IgG/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/genética , Linhagem Celular , Complemento C5a/genética , Inflamação/genética , Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de IgG/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Senescent cells release a variety of cytokines, proteases, and growth factors collectively known as the senescence-associated secretory phenotype (SASP). Sustained SASP contributes to a pattern of chronic inflammation associated with aging and implicated in many age-related diseases. Here, we investigated the expression and function of the immunomodulatory cytokine BAFF (B-cell activating factor; encoded by the TNFSF13B gene), a SASP protein, in multiple senescence models. We first characterized BAFF production across different senescence paradigms, including senescent human diploid fibroblasts (WI-38, IMR-90) and monocytic leukemia cells (THP-1), and tissues of mice induced to undergo senescence. We then identified IRF1 (interferon regulatory factor 1) as a transcription factor required for promoting TNFSF13B mRNA transcription in senescence. We discovered that suppressing BAFF production decreased the senescent phenotype of both fibroblasts and monocyte-like cells, reducing IL6 secretion and SA-ß-Gal staining. Importantly, however, the influence of BAFF on the senescence program was cell type-specific: in monocytes, BAFF promoted the early activation of NF-κB and general SASP secretion, while in fibroblasts, BAFF contributed to the production and function of TP53 (p53). We propose that BAFF is elevated across senescence models and is a potential target for senotherapy.
Assuntos
Fator Ativador de Células B , Senescência Celular , Humanos , Animais , Camundongos , Senescência Celular/genética , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Fator Ativador de Células B/farmacologia , Secretoma , Envelhecimento/genética , Citocinas/metabolismoRESUMO
Deregulated IL-12 and IL-23 production from activated myeloid lineage cells is a key driver of numerous T cell-dependent autoimmune and inflammatory diseases. IL-12 and IL-23 share a common p40 subunit encoded by Il12b, which is negatively regulated at the transcriptional level by the STAT3 (signal transducer and activator of transcription 3)-activating anti-inflammatory cytokine IL-10. We found that IL-10 targets an enhancer 10 kb upstream of the Il12b transcriptional start site. Within the enhancer, a single 10-bp site is required for the inhibitory effects of IL-10 and is bound by NFIL3 (nuclear factor, interleukin 3-regulated), a B-ZIP transcription factor. Myeloid cells lacking NFIL3 produce excessive IL-12p40 and increased IL-12p70. Thus, the STAT3-dependent expression of NFIL3 is a key component of a negative feedback pathway in myeloid cells that suppresses proinflammatory responses.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica/fisiologia , Subunidade p40 da Interleucina-12/biossíntese , Fator de Transcrição STAT3/metabolismo , Transcrição Gênica/fisiologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Células Cultivadas , Interleucina-10/genética , Interleucina-10/metabolismo , Subunidade p40 da Interleucina-12/genética , Camundongos , Camundongos Knockout , Células Mieloides/citologia , Células Mieloides/metabolismo , Fator de Transcrição STAT3/genéticaRESUMO
Signaling through Ras GTPases controls the activity of many transcription factors including CCAAT/enhancer-binding protein (C/EBPbeta), which regulates oncogenic H-Ras(V12)-induced senescence and growth arrest. Here we report that C/EBPbeta (LAP) DNA binding is inhibited by N-terminal sequences and derepressed by oncogenic Ras signaling. Sequence and mutational analyses showed that auto-repression involves two LXXLF (phiXXphiphi)-like motifs (LX1 and LX2) and a third element, auto-inhibitory domain (AID), located within conserved region CR5. LX1 is a critical component of the transactivation domain and has been shown to mediate C/EBPbeta binding to the TAZ2 region of p300/CREB-binding protein coactivators. C/EBPbeta auto-repression also involves a C-terminal regulatory domain (CRD) adjacent to the leucine zipper. CRD contains a third phiXXphiphi motif (LX3) and a short sequence, KQL, which has similarity to a region in the protein-binding site of TAZ2. The C/EBPbeta N- and C-terminal domains physically associate in a manner that requires the basic region and CRD. We propose a model in which the regulatory sequences form a hydrophobic core that reciprocally inhibits DNA binding and transactivation. We also suggest a mechanism for C/EBPbeta derepression involving several recently identified modifications within AID and CRD. Finally, we show that association of activated C/EBPbeta with p300/CREB-binding protein requires the LX2 and AID auto-inhibitory elements. Thus, the N-terminal regulatory elements have dual roles in auto-inhibition and coactivator binding.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , DNA/química , Elementos Facilitadores Genéticos , Fatores de Transcrição de p300-CBP/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Dados de Sequência Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Ratos , Transdução de Sinais , Transcrição GênicaRESUMO
CD1d is a nonclassical Ag-presenting molecule that presents glycolipid Ags to NKT cells that are involved in immune defense and tumor rejection. It also plays a role in immunoregulatory functions in the epidermis. The mechanisms controlling the expression of CD1d are not well understood. Therefore, we cloned the CD1d gene promoter and characterized its activities in primary human keratinocytes and other cell lines of epithelial origin. We found that a CCAAT box in the CD1d promoter is required for its expression in keratinocytes. We show here that transcription factor C/EBP-beta binds to the CCAAT box in the CD1d promoter in vitro and in vivo. Consistent with these observations, deletion of the gene encoding for C/EBP-beta caused a loss of CD1d expression. The in vivo regulation of CD1d has significant implications for the pathologic mechanisms of certain immunologic skin diseases in which NKT cells play a role, such as allergic contact dermatitis and psoriasis. Together, these data show a central role for C/EBP-beta in regulating CD1d transcription.
Assuntos
Antígenos CD1d/genética , Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Regulação da Expressão Gênica , Queratinócitos/metabolismo , Transcrição Gênica , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Células Epiteliais , Humanos , Células T Matadoras Naturais , Regiões Promotoras Genéticas , Dermatopatias/etiologia , Dermatopatias/imunologiaRESUMO
The lamin B receptor (LBR) is an integral nuclear envelope protein that interacts with chromatin and has homology to sterol reductases. Mutations in LBR result in Pelger-Huët anomaly and HEM-Greenberg skeletal dysplasia, whereas in mice Lbr mutations result in ichthyosis. To further understand the function of the LBR and its role in disease, we derived a novel mouse model with a gene-trap insertion into the Lbr locus (Lbr(GT/GT)). Phenotypically, the Lbr(GT/GT) mice are similar to ichthyosis mice. The Lbr(GT/GT) granulocytes lack a mature segmented nucleus and have a block in late maturation. Despite these changes in nuclear morphology, the innate granulocyte immune function in the killing of Staphylococcus aureus bacteria appears to be intact. Granulocyte differentiation requires the transcription factor C/EBPepsilon. We identified C/EBPepsilon binding sites within the Lbr promoter and used EMSAs and luciferase assays to show that Lbr is transcriptionally regulated by C/EBPepsilon. Our findings indicate that the Lbr(GT/GT) mice are a model for Pelger-Huët anomaly and that Lbr, under transcriptional regulation of C/EBPepsilon, is necessary for morphological but not necessarily functional granulocyte maturation.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Neutrófilos/citologia , Anomalia de Pelger-Huët/genética , Anomalia de Pelger-Huët/fisiopatologia , Receptores Citoplasmáticos e Nucleares/genética , Transcrição Gênica , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular , Forma do Núcleo Celular , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Insercional , Ativação de Neutrófilo , Neutrófilos/fisiologia , Anomalia de Pelger-Huët/embriologia , Anomalia de Pelger-Huët/metabolismo , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/metabolismo , Staphylococcus aureus/fisiologia , Receptor de Lamina BRESUMO
Small interfering RNAs (siRNAs) are potential therapeutic substances due to their gene silencing capability as exemplified by the recent approval by the US Food and Drug Administration (FDA) of the first siRNA therapeutic agent (patisiran). However, the delivery of naked siRNAs is challenging because of their short plasma half-lives and poor cell penetrability. In this study, we used vesicles made from bolaamphiphiles (bolas), GLH-19 and GLH-20, to investigate their ability to protect siRNA from degradation by nucleases while delivering it to target cells, including cells in the brain. Based on computational and experimental studies, we found that GLH-19 vesicles have better delivery characteristics than do GLH-20 vesicles in terms of stability, binding affinity, protection against nucleases, and transfection efficiency, while GLH-20 vesicles contribute to efficient release of the delivered siRNAs, which become available for silencing. Our studies with vesicles made from a mixture of the two bolas (GLH-19 and GLH-20) show that they were able to deliver siRNAs into cultured cancer cells, into a flank tumor and into the brain. The vesicles penetrate cell membranes and the blood-brain barrier (BBB) by endocytosis and transcytosis, respectively, mainly through the caveolae-dependent pathway. These results suggest that GLH-19 strengthens vesicle stability, provides protection against nucleases, and enhances transfection efficiency, while GLH-20 makes the siRNA available for gene silencing.
RESUMO
The prevalence of obesity, an established epidemiologic risk factor for many chronic diseases including cancer, has been steadily increasing in the US over several decades. The mechanisms used to regulate energy balance and adiposity and the relationship of these factors to cancer are not completely understood. Here we have used knockout mice to examine the roles of the transcription factors CCAAT/enhancer-binding protein (C/EBP) beta and C/EBPdelta in regulating body composition and systemic levels of hormones such as insulin-like growth factor-1 (IGF-1), leptin and insulin that mediate energy balance. Dual-energy X-ray absorptiometry showed that C/EBPbeta, either directly or indirectly, modulated body weight, fat content and bone density in both males and females, while the effect of C/EBPdelta was minor and only affected adiposity and body weight in female animals. Levels of IGF-1, leptin and insulin in the serum were decreased in both male and female C/EBPbeta(-/-) mice, and C/EBPbeta was associated with their promoters in vivo. Moreover, colon adenocarcinoma cells displayed reduced tumorigenic potential when transplanted into C/EBPbeta-deficient animals, especially males. Thus, C/EBPbeta contributes to endocrine expression of IGF-1, leptin and insulin, which modulate energy balance and can contribute to cancer progression by creating a favorable environment for tumor cell proliferation and survival.