RESUMO
Antiviral responses must rapidly defend against infection while minimizing inflammatory damage, but the mechanisms that regulate the magnitude of response within an infected cell are not well understood. miRNAs are small non-coding RNAs that suppress protein levels by binding target sequences on their cognate mRNA. Here, we identify miR-144 as a negative regulator of the host antiviral response. Ectopic expression of miR-144 resulted in increased replication of three RNA viruses in primary mouse lung epithelial cells: influenza virus, EMCV, and VSV. We identified the transcriptional network regulated by miR-144 and demonstrate that miR-144 post-transcriptionally suppresses TRAF6 levels. In vivo ablation of miR-144 reduced influenza virus replication in the lung and disease severity. These data suggest that miR-144 reduces the antiviral response by attenuating the TRAF6-IRF7 pathway to alter the cellular antiviral transcriptional landscape.
Assuntos
Influenza Humana/imunologia , MicroRNAs/metabolismo , Orthomyxoviridae/genética , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Animais , Linhagem Celular , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Influenza Humana/virologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Orthomyxoviridae/imunologia , Orthomyxoviridae/fisiologia , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Carga Viral , Replicação ViralRESUMO
The regulation of host-pathogen interactions during Mycobacterium tuberculosis (Mtb) infection remains unresolved. MicroRNAs (miRNAs) are important regulators of the immune system, and so we used a systems biology approach to construct an miRNA regulatory network activated in macrophages during Mtb infection. Our network comprises 77 putative miRNAs that are associated with temporal gene expression signatures in macrophages early after Mtb infection. In this study, we demonstrate a dual role for one of these regulators, miR-155. On the one hand, miR-155 maintains the survival of Mtb-infected macrophages, thereby providing a niche favoring bacterial replication; on the other hand, miR-155 promotes the survival and function of Mtb-specific T cells, enabling an effective adaptive immune response. MiR-155-induced cell survival is mediated through the SH2 domain-containing inositol 5-phosphatase 1 (SHIP1)/protein kinase B (Akt) pathway. Thus, dual regulation of the same cell survival pathway in innate and adaptive immune cells leads to vastly different outcomes with respect to bacterial containment.
Assuntos
Imunidade Adaptativa/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , MicroRNAs/genética , Mycobacterium tuberculosis/imunologia , Tuberculose/genética , Tuberculose/imunologia , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citocinas/biossíntese , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Ativação Linfocitária , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Transcriptoma , Tuberculose/metabolismoRESUMO
Type I alveolar epithelial cells are a replicative niche for influenza in vivo, yet their response to infection is not fully understood. To better characterize their cellular responses, we have created an immortalized murine lung epithelial type I cell line (LET1). These cells support spreading influenza virus infection in the absence of exogenous protease and thus permit simultaneous analysis of viral replication dynamics and host cell responses. LET1 cells can be productively infected with human, swine and mouse-adapted strains of influenza virus and exhibit expression of an antiviral transcriptional programme and robust cytokine secretion. We characterized influenza virus replication dynamics and host responses of lung type I epithelial cells and identified the capacity of epithelial cell-derived type I IFN to regulate specific modules of antiviral effectors to establish an effective antiviral state. Together, our results indicate that the type I epithelial cell can play a major role in restricting influenza virus infection without contribution from the haematopoietic compartment.
Assuntos
Células Epiteliais/imunologia , Células Epiteliais/virologia , Imunidade Inata , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Replicação Viral , Animais , Linhagem Celular , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Cellular fusion of macrophages into multinucleated giant cells is a distinguishing feature of the granulomatous response to inflammation, infection, and foreign bodies (Kawai and Akira. 2011. Immunity 34: 637-650). We observed a marked increase in fusion of macrophages genetically deficient in Dicer, an enzyme required for canonical microRNA (miRNA) biogenesis. Gene expression profiling of miRNA-deficient macrophages revealed an upregulation of the IL-4-responsive fusion protein Tm7sf4, and analyses identified miR-7a-1 as a negative regulator of macrophage fusion, functioning by directly targeting Tm7sf4 mRNA. miR-7a-1 is itself an IL-4-responsive gene in macrophages, suggesting feedback control of cellular fusion. Collectively, these data indicate that miR-7a-1 functions to regulate IL-4-directed multinucleated giant cell formation.
Assuntos
Diferenciação Celular/imunologia , Células Gigantes de Langhans/imunologia , Macrófagos/citologia , Macrófagos/imunologia , MicroRNAs/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Fusão Celular/métodos , Células Cultivadas , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Células Gigantes de Langhans/citologia , Células Gigantes de Langhans/metabolismo , Células HEK293 , Humanos , Interleucina-4/fisiologia , Macrófagos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/genética , Ribonuclease III/deficiência , Ribonuclease III/genética , Transcrição Gênica/imunologiaRESUMO
Precise control of the innate immune response is essential to ensure host defense against infection while avoiding inflammatory disease. Systems-level analyses of Toll-like receptor (TLR)-stimulated macrophages suggested that SHANK-associated RH domain-interacting protein (SHARPIN) might play a role in the TLR pathway. This hypothesis was supported by the observation that macrophages derived from chronic proliferative dermatitis mutation (cpdm) mice, which harbor a spontaneous null mutation in the Sharpin gene, exhibited impaired IL-12 production in response to TLR activation. Systems biology approaches were used to define the SHARPIN-regulated networks. Promoter analysis identified NF-κB and AP-1 as candidate transcription factors downstream of SHARPIN, and network analysis suggested selective attenuation of these pathways. We found that the effects of SHARPIN deficiency on the TLR2-induced transcriptome were strikingly correlated with the effects of the recently described hypomorphic L153P/panr2 point mutation in Ikbkg [NF-κB Essential Modulator (NEMO)], suggesting that SHARPIN and NEMO interact. We confirmed this interaction by co-immunoprecipitation analysis and furthermore found it to be abrogated by panr2. NEMO-dependent signaling was affected by SHARPIN deficiency in a manner similar to the panr2 mutation, including impaired p105 and ERK phosphorylation and p65 nuclear localization. Interestingly, SHARPIN deficiency had no effect on IκBα degradation and on p38 and JNK phosphorylation. Taken together, these results demonstrate that SHARPIN is an essential adaptor downstream of the branch point defined by the panr2 mutation in NEMO.
Assuntos
Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Animais , Sequência de Bases , Proteínas de Transporte/genética , Primers do DNA/genética , Imunidade Inata/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , NF-kappa B/metabolismo , Mapeamento de Interação de Proteínas , Transdução de Sinais , Análise de Sistemas , Biologia de Sistemas , Receptor 2 Toll-Like/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that exhibits specific tumoricidal activity against a variety of tumors. It is expressed on different cells of the immune system and plays a role in natural killer cell-mediated tumor surveillance. In allogeneic hematopoietic-cell transplantation, the reactivity of the donor T cell against malignant cells is essential for the graft-versus-tumor (GVT) effect. Cytolytic activity of T cells is primarily mediated through the Fas-Fas ligand and perforin-granzyme pathways. However, T cells deficient for both Fas ligand and perforin can still exert GVT activity in vivo in mouse models. To uncover a potential role for TRAIL in donor T cell-mediated GVT activity, we compared donor T cells from TRAIL-deficient and wild-type mice in clinically relevant mouse bone-marrow transplantation models. We found that alloreactive T cells can express TRAIL, but the absence of TRAIL had no effect on their proliferative and cytokine response to alloantigens. TRAIL-deficient T cells showed significantly lower GVT activity than did TRAIL-expressing T cells, but no important differences in graft-versus-host disease, a major complication of allogeneic hematopoietic cell transplantation, were observed. These data suggest that strategies to enhance TRAIL-mediated GVT activity could decrease relapse rates of malignancies after hematopoietic cell transplantation without exacerbation of graft-versus-host disease.
Assuntos
Efeito Enxerto vs Tumor/imunologia , Glicoproteínas de Membrana/fisiologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Ligante Indutor de Apoptose Relacionado a TNF , Transplante Homólogo , Células Tumorais CultivadasRESUMO
Extensive studies of mice deficient in one or several cytokine receptors have failed to support an indispensable role of cytokines in development of multiple blood cell lineages. Whereas B1 B cells and Igs are sustained at normal levels throughout life of mice deficient in IL-7, IL-7Ralpha, common cytokine receptor gamma chain, or flt3 ligand (FL), we report here that adult mice double deficient in IL-7Ralpha and FL completely lack visible LNs, conventional IgM+ B cells, IgA+ plasma cells, and B1 cells, and consequently produce no Igs. All stages of committed B cell progenitors are undetectable in FL-/- x IL-7Ralpha-/- BM that also lacks expression of the B cell commitment factor Pax5 and its direct target genes. Furthermore, in contrast to IL-7Ralpha-/- mice, FL-/- x IL-7Ralpha-/- mice also lack mature B cells and detectable committed B cell progenitors during fetal development. Thus, signaling through the cytokine tyrosine kinase receptor flt3 and IL-7Ralpha are indispensable for fetal and adult B cell development.
Assuntos
Linfócitos B/fisiologia , Diferenciação Celular/fisiologia , Proteínas de Membrana/metabolismo , Receptores de Interleucina-7/metabolismo , Animais , Linfócitos B/citologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Nódulos Linfáticos Agregados/metabolismo , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Transdução de Sinais/imunologia , Transdução de Sinais/fisiologiaRESUMO
Th17 cells, a subset of T cells involved in autoimmunity and host defense against extracellular Gram-negative infection, express both IL-17A and IL-17F. Both IL-17A and IL-17F can signal via the IL-17RA; however, IL-17F does so at a 1- to 2-log higher concentration than IL-17A. In this study, we show that the IL-17F homodimer via IL-17RA is a negative regulator of IL-17 production in T cells and suggest a mechanism whereby IL-17RA on T cells serves as an autocrine/paracrine regulator of IL-17 synthesis in T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Concanavalina A/toxicidade , Interleucina-17/imunologia , Mitógenos/toxicidade , Receptores de Interleucina-17/imunologia , Animais , Comunicação Autócrina/genética , Comunicação Autócrina/imunologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/imunologia , Interleucina-17/genética , Camundongos , Camundongos Knockout , Comunicação Parácrina/genética , Comunicação Parácrina/imunologia , Receptores de Interleucina-17/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Striking parallels exist between immune and nervous system cellular signalling mechanisms. Molecules originally shown to be critical for immune responses also serve neuronal functions, and similarly neural guidance cues can modulate immune function. We show here that semaphorin 7A (Sema7A), a membrane-anchored member of the semaphorin family of guidance proteins previously known for its immunomodulatory effects, can also mediate neuronal functions. Unlike many other semaphorins, which act as repulsive guidance cues, Sema7A enhances central and peripheral axon growth and is required for proper axon tract formation during embryonic development. Unexpectedly, Sema7A enhancement of axon outgrowth requires integrin receptors and activation of MAPK signalling pathways. These findings define a previously unknown biological function for semaphorins, identify an unexpected role for integrins and integrin-dependent intracellular signalling in mediating semaphorin responses, and provide a framework for understanding and interfering with Sema7A function in both immune and nervous systems.
Assuntos
Antígenos CD/metabolismo , Axônios/metabolismo , Integrinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Semaforinas/metabolismo , Animais , Antígenos CD/genética , Linhagem Celular , Ativação Enzimática , Feminino , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Proteínas Ligadas por GPI , Deleção de Genes , Humanos , Integrinas/química , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroglia/citologia , Neuroglia/metabolismo , Subunidades Proteicas , Proteínas Tirosina Quinases/metabolismo , Ratos , Receptores Virais/genética , Receptores Virais/metabolismo , Semaforinas/genéticaRESUMO
All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor alpha, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17-/- knockout mice corroborated the essential role of adam17-/- in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.
Assuntos
Endopeptidases/metabolismo , Receptores ErbB/metabolismo , Metaloendopeptidases/metabolismo , Fenilalanina/análogos & derivados , Proteínas ADAM , Proteína ADAM12 , Proteína ADAM17 , Anfirregulina , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Betacelulina , Células Cultivadas , Desintegrinas/genética , Desintegrinas/metabolismo , Família de Proteínas EGF , Embrião de Mamíferos/anatomia & histologia , Endopeptidases/genética , Fator de Crescimento Epidérmico/metabolismo , Epirregulina , Fibroblastos/citologia , Fibroblastos/metabolismo , Genótipo , Glicoproteínas/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloendopeptidases/genética , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Fenilalanina/metabolismo , Inibidores de Proteases/metabolismo , Estrutura Terciária de Proteína , Acetato de Tetradecanoilforbol/metabolismo , Tiofenos/metabolismo , Fator de Crescimento Transformador alfa/metabolismoRESUMO
The epidermis is a stratified, continually renewing epithelium dependent on a balance among cell proliferation, differentiation, and death for homeostasis. In normal epidermis, a mitotically active basal layer gives rise to terminally differentiating keratinocytes that migrate outward and are ultimately sloughed from the skin surface as enucleated squames. Although many proteins are known to function in maintaining epidermal homeostasis, the molecular coordination of these events is poorly understood. RIP4 is a novel RIP (receptor-interacting protein) family kinase with ankyrin repeats cloned from a keratinocyte cDNA library. RIP4 deficiency in mice results in perinatal lethality associated with abnormal epidermal differentiation. The phenotype of RIP4(-/-) mice in part resembles that of mice lacking IKKalpha, a component of a complex that regulates NF-kappaB. Despite the similar keratinocyte defects in RIP4- and IKKalpha-deficient mice, these kinases function in distinct pathways. RIP4 functions cell autonomously within the keratinocyte lineage. Unlike IKKalpha, RIP4-deficient skin fails to fully differentiate when grafted onto a normal host. Instead, abnormal hair follicle development and epidermal dysplasia, indicative of progression into a more pathologic state, are observed. Thus, RIP4 is a critical component of a novel pathway that controls keratinocyte differentiation.
Assuntos
Diferenciação Celular/fisiologia , Queratinócitos/fisiologia , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Animais , Células Epidérmicas , Epiderme/crescimento & desenvolvimento , Epiderme/patologia , Epiderme/fisiologia , Feminino , Homeostase , Queratinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas/genética , Proteína Serina-Treonina Quinases de Interação com ReceptoresRESUMO
Endothelial CD39 metabolizes ADP released from activated platelets. Recombinant soluble human CD39 (solCD39) potently inhibited ex vivo platelet aggregation in response to ADP and reduced cerebral infarct volumes in mice following transient middle cerebral artery occlusion, even when given 3 hours after stroke. Postischemic platelet and fibrin deposition were decreased and perfusion increased without increasing intracerebral hemorrhage. In contrast, aspirin did not increase postischemic blood flow or reduce infarction volume, but did increase intracerebral hemorrhage. Mice lacking the enzymatically active extracellular portion of the CD39 molecule were generated by replacement of exons 4-6 (apyrase-conserved regions 2-4) with a PGKneo cassette. Although CD39 mRNA 3' of the neomycin cassette insertion site was detected, brains from these mice lacked both apyrase activity and CD39 immunoreactivity. Although their baseline phenotype, hematological profiles, and bleeding times were normal, cd39(-/-) mice exhibited increased cerebral infarct volumes and reduced postischemic perfusion. solCD39 reconstituted these mice, restoring postischemic cerebral perfusion and rescuing them from cerebral injury. These data demonstrate that CD39 exerts a protective thromboregulatory function in stroke.
Assuntos
Adenosina Trifosfatases/fisiologia , Antígenos CD/fisiologia , Apirase/fisiologia , Isquemia Encefálica/sangue , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/farmacologia , Animais , Antígenos CD/genética , Antígenos CD/farmacologia , Apirase/deficiência , Apirase/genética , Apirase/farmacologia , Aspirina/farmacologia , Isquemia Encefálica/fisiopatologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Agregação Plaquetária/efeitos dos fármacos , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/prevenção & controle , Trombose/sangue , Trombose/fisiopatologia , Trombose/prevenção & controleRESUMO
Protein kinase C-associated kinase (PKK; DIK/RIP4) is an ankyrin-repeat containing serine/threonine receptor-interacting protein (RIP)-family kinase that can activate NFkappaB, and is required for keratinocyte development. In earlier studies, the expression of a catalytically inactive mutant of PKK in the B cell lineage resulted in a marked decrease in peripheral B cells in the spleen and a severe reduction of B-1 B cells. Here we explore the consequences of a null mutation in PKK with respect to the generation of peripheral B cell lineages and the activation of NFkappaB. We show that PKK is not required for the production of B cells in the bone marrow or for the development and maintenance of all mature B lymphocyte populations. We also show that PKK is not required for the activation of NFkappaB downstream of the BCR, CD40, or TLR-4 in B cells. Taken together, these data demonstrate that the loss of this RIP-family kinase does not compromise B lymphocyte development and maintenance, but leaves open the possibility that PKK may have a redundant role in these processes.
Assuntos
Linfócitos B/citologia , Linfócitos B/enzimologia , Proteínas Quinases/fisiologia , Animais , Medula Óssea/imunologia , Antígenos CD40/metabolismo , Linhagem da Célula , Ativação Enzimática , Camundongos , Camundongos Mutantes , Mutação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptor 4 Toll-Like/metabolismo , Quinase Induzida por NF-kappaBRESUMO
Currently, the best clinical predictor for inflammatory bowel disease (IBD) is family history. Over 163 sequence variants have been associated with IBD in genome-wide association studies, but they have weak effects and explain only a fraction of the observed heritability. It is expected that additional variants contribute to the genomic architecture of IBD, possibly including rare variants with effect sizes larger than the identified common variants. Here we applied a family study design and sequenced 38 individuals from five families, under the hypothesis that families with multiple IBD-affected individuals harbor one or more risk variants that (i) are shared among affected family members, (ii) are rare and (iii) have substantial effect on disease development. Our analysis revealed not only novel candidate risk variants but also high polygenic risk scores for common known risk variants in four out of the five families. Functional analysis of our top novel variant in the remaining family, a rare missense mutation in the ubiquitin ligase TRIM11, suggests that it leads to increased nuclear factor of kappa light chain enhancer in B-cells (NF-κB) signaling. We conclude that an accumulation of common weak-effect variants accounts for the high incidence of IBD in most, but not all families we analyzed and that a family study design can identify novel rare variants conferring risk for IBD with potentially large effect size, such as the TRIM11 p.H414Y mutation.
RESUMO
ADAMs (A Disintegrin And Metalloprotease domain) are metalloprotease-disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion. Since such events are critical for bone resorption and osteoclast recruitment, we investigated whether they require ADAMs. We report here which ADAMs we have identified in bone cells, as well as our analysis of the generation, migration and resorptive activity of osteoclasts in developing metatarsals of mouse embryos lacking catalytically active ADAM 17 [TNFalpha converting enzyme (TACE)]. The absence of TACE activity still allowed the generation of cells showing an osteoclastic phenotype, but prevented their migration into the core of the diaphysis and the subsequent formation of marrow cavity. This suggests a role of TACE in the recruitment of osteoclasts to future resorption sites.
Assuntos
Desenvolvimento Ósseo/fisiologia , Medula Óssea/metabolismo , Metaloendopeptidases/metabolismo , Ossos do Metatarso/fisiologia , Osteoclastos/fisiologia , Proteínas ADAM , Proteína ADAM17 , Animais , Medula Óssea/enzimologia , Reabsorção Óssea/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular/fisiologia , Primers do DNA/genética , Diáfises/citologia , Diáfises/crescimento & desenvolvimento , Desintegrinas/química , Imuno-Histoquímica , Glicoproteínas de Membrana/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/genética , Ossos do Metatarso/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos DBA , Osteoclastos/citologia , Osteoclastos/enzimologia , Fenótipo , Estrutura Terciária de Proteína , Ligante RANK , Coelhos , Receptor Ativador de Fator Nuclear kappa-B , Fator de Necrose Tumoral alfaRESUMO
Tumour necrosis factor alpha (TNF alpha)-converting enzyme (TACE/ADAM-17, where ADAM stands for a disintegrin and metalloproteinase) releases from the cell surface the extracellular domains of TNF and several other proteins. Previous studies have found that, while purified TACE preferentially cleaves peptides representing the processing sites in TNF and transforming growth factor alpha, the cellular enzyme nonetheless also sheds proteins with divergent cleavage sites very efficiently. More recent work, identifying the cleavage site in the p75 TNF receptor, quantifying the susceptibility of additional peptides to cleavage by TACE and identifying additional protein substrates, underlines the complexity of TACE-substrate interactions. In addition to substrate specificity, the mechanism underlying the increased rate of shedding caused by agents that activate cells remains poorly understood. Recent work in this area, utilizing a peptide substrate as a probe for cellular TACE activity, indicates that the intrinsic activity of the enzyme is somehow increased.
Assuntos
Alanina/metabolismo , Metaloendopeptidases/metabolismo , Valina/metabolismo , Proteínas ADAM , Proteína ADAM17 , Indução Enzimática , Metaloendopeptidases/biossíntese , Metaloendopeptidases/química , Especificidade por SubstratoRESUMO
The receptor-interacting protein (RIP) family kinase RIP4 interacts with protein kinase C (PKC) isoforms and is implicated in PKC-dependent signaling pathways. RIP4(-/-) mice die at birth with epidermal differentiation defects, causing fusions of all external orifices and loss of the esophageal lumen. To further understand RIP4 function in the skin, we generated transgenic mice with epidermal-specific expression of RIP4 using the human keratin-14 promoter (K14-RIP4). The K14-RIP4 transgene rescued the epidermal phenotype of RIP4(-/-) mice, showing that RIP4 acts autonomously in the epidermis to regulate differentiation. Although RIP4(-/-) mice share many phenotypic similarities with inhibitor kappaB kinase (IKK)alpha(-/-) mice and stratifin repeated epilation (Sfn(Er/Er)) mice, the K14-RIP4 transgene failed to promote epidermal differentiation in these mutant backgrounds. Unexpectedly, topical treatment of K14-RIP4 mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced dramatic, neutrophilic inflammation, an effect that was independent of tumor necrosis factor type 1 receptor (TNFR1/p55) function. Despite their enhanced sensitivity to TPA, K14-RIP4 mice did not have an altered frequency of tumor formation in TPA-promoted skin cancer initiated with 7,12-dimethylbenz[a]anthracene (DMBA). These data suggest that RIP4 functions in the epidermis through PKC-specific signaling pathways to regulate differentiation and inflammation.
Assuntos
Dermatite de Contato/imunologia , Dermatite de Contato/fisiopatologia , Epiderme/imunologia , Epiderme/patologia , Proteínas Quinases , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Carcinógenos/toxicidade , Diferenciação Celular/fisiologia , Dermatite de Contato/patologia , Feminino , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Queratina-14/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Regiões Promotoras Genéticas/fisiologia , Proteína Quinase C/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/imunologia , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
The interleukin (IL)-1 family members IL-1alpha, -1beta, and -18 are potent inflammatory cytokines whose activities are dependent on heterodimeric receptors of the IL-1R superfamily, and which are regulated by soluble antagonists. Recently, several new IL-1 family members have been identified. To determine the role of one of these family members in the skin, transgenic mice expressing IL1F6 in basal keratinocytes were generated. IL1F6 transgenic mice exhibit skin abnormalities that are dependent on IL-1Rrp2 and IL-1RAcP, which are two members of the IL-1R family. The skin phenotype is characterized by acanthosis, hyperkeratosis, the presence of a mixed inflammatory cell infiltrate, and increased cytokine and chemokine expression. Strikingly, the combination of the IL-1F6 transgene with an IL1F5 deficiency results in exacerbation of the skin phenotype, demonstrating that IL-1F5 has antagonistic activity in vivo. Skin from IL1F6 transgenic, IL1F5(-/-) pups contains intracorneal and intraepithelial pustules, nucleated corneocytes, and dilated superficial dermal blood vessels. Additionally, expression of IL1RL2, -1F5, and -1F6 is increased in human psoriatic skin. In summary, dysregulated expression of novel agonistic and antagonistic IL-1 family member ligands can promote cutaneous inflammation, revealing potential novel targets for the treatment of inflammatory skin disorders.
Assuntos
Inflamação/fisiopatologia , Interleucina-1/fisiologia , Dermatopatias/fisiopatologia , Animais , Cápsulas Bacterianas , Humanos , Interleucina-1/genética , Interleucina-1/imunologia , Ligantes , Camundongos , Camundongos Transgênicos , Polissacarídeos Bacterianos/genética , Regiões Promotoras Genéticas , Pele/patologiaRESUMO
IL-17 is an inflammatory cytokine produced primarily by a unique lineage of CD4 T cells that plays critical roles in the pathogenesis of multiple autoimmune diseases. IL-17RA is a ubiquitously expressed receptor that is essential for IL-17 biologic activity. Despite widespread receptor expression, the activity of IL-17 is most classically defined by its ability to induce the expression of inflammatory cytokines, chemokines, and other mediators by stromal cells. The lack of IL-17 responsiveness in mouse stromal cells genetically deficient in IL-17RA is poorly complemented by human IL-17RA, suggesting the presence of an obligate ancillary component whose activity is species specific. This component is IL-17RC, a distinct member of the IL-17R family. Thus, the biologic activity of IL-17 is dependent on a complex composed of IL-17RA and IL-17RC, suggesting a new paradigm for understanding the interactions between the expanded family of IL-17 ligands and their receptors.
Assuntos
Interleucina-17/fisiologia , Receptores de Interleucina/química , Receptores de Interleucina/fisiologia , Transdução de Sinais/imunologia , Animais , Linhagem Celular , Linhagem Celular Transformada , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Interleucina-17/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subunidades Proteicas/química , Subunidades Proteicas/fisiologia , Receptores de Interleucina/deficiência , Receptores de Interleucina/genética , Receptores de Interleucina-17RESUMO
Brain inflammatory reactions have been described in various neurological disorders, including epilepsy. Although there is clear evidence that cytokines affect neuroglial functions and blood-brain barrier permeability, scarce information is available on the functional consequences of brain inflammation on seizures. We studied the role of tumor necrosis factor-alpha (TNF)-alpha and its p55 and p75 receptors in seizure modulation. We found that intrahippocampal injection of murine recombinant TNF-alpha potently inhibits seizure in mice while human recombinant TNF-alpha, which shows strong specificity for mouse p55 receptors, was ineffective. p75 receptors were detected in mouse hippocampal neurons, whereas p55 receptors were absent. Transgenic mice with a perturbed TNF-alpha system showed profound alterations in seizure susceptibility: astrocytic overexpression of TNF-alpha was associated with reduced seizures, whereas mice lacking TNF-alpha p75 or both p55 and p75, receptors showed prolonged seizures. Mice deficient in p55 receptor only showed reduced seizures; and both p75 and TNF receptor-associated factor 2 protein levels were upregulated in their hippocampi. Our findings show that increased brain levels of TNF-alpha result in significant inhibition of seizures in mice, and this action is mediated by neuronal p75 receptors. This evidence highlights a novel function of TNF-alpha in brain and indicates a new system for anticonvulsive intervention.