RESUMO
Complement-derived anaphylatoxins regulate immune and inflammatory responses through G-protein-coupled receptor (GPCR)-mediated signalling. C5L2 (also known as GPR77) is a relatively new GPCR thought to be a non-signalling receptor binding to C5a, on the basis of sequence information and experimental evidence. Here we show, using gene targeting, that C5L2 is required to facilitate C5a signalling in neutrophils, macrophages and fibroblasts in vitro. Deficiency of C5L2 results in reduced inflammatory cell infiltration, suggesting that C5L2 is critical for optimal C5a-mediated cell infiltration in certain in vivo settings. C5L2 is also involved in optimizing C3a-induced signals. Furthermore, like mice incapable of C3a/complement 3a receptor (C3aR) signalling, C5L2-deficient mice are hypersensitive to lipopolysaccharide (LPS)-induced septic shock, show reduced ovalbumin (OVA)-induced airway hyper-responsiveness and inflammation, and are mildly delayed in haematopoietic cell regeneration after gamma-irradiation. Our data indicate that C5L2 can function as a positive modulator for both C5a- and C3a-anaphylatoxin-induced responses.
Assuntos
Complemento C3a/metabolismo , Complemento C5a/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Bovinos , Células Cultivadas , Ativação do Complemento , Complemento C3a/imunologia , Complemento C5a/imunologia , Fibroblastos , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Inflamação , Pulmão/citologia , Neutrófilos/citologia , Neutrófilos/metabolismo , Receptor da Anafilatoxina C5a , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Transdução de SinaisRESUMO
TNFα stimulation triggers both cell death and survival programs. Since dysregulated apoptosis or cell growth can cause inflammatory diseases, cancer, or autoimmune disorders, it is important to understand the molecular mechanism of controlling cell death and survival by TNFR downstream signaling molecules. In this study, we used normal diploid cells, mouse embryonic fibroblasts (MEFs), to mimic the general TNFα-resistant phenomenon seen under physiological conditions.We elucidated the TNFα-induced death signaling complexes in TNF α-resistant WT MEFs and TNFα-sensitive MEFs that were cFLIP-, RelA-, TRAF2- or RIP1-deficient. Consistent with TNFα-mediated killing, we detected TNFα-induced high molecular weight complexes containing caspase-8 and FADD by gel filtration in the deficient MEFs, especially in those devoid of cFLIP. In addition to the presence of caspase-8-FADD in the TNFα-induced-death complex in the deficient MEFs, we also detected an intermediate protein complex containing RIP1, TRAF2 and caspase-8.Moreover, we demonstrated a correlation between TNFα-sensitivity and death-inducing complex ability in two transformed cell lines, E1A- and Ras- transformed MEFs and PDGF-B-transformed NIH-3T3 cells with PDGF-B signaling inhibited by the tyrosine kinase inhibitor STI571. Taken together, our results suggest the involvement of cFLIP-, RelA-, RIP1-, or TRAF2-related mechanisms for preventing FADD-caspase-8 interaction in wild-type MEFs.
Assuntos
Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/deficiência , Caspase 8/metabolismo , Morte Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas Ativadoras de GTPase/deficiência , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Células NIH 3T3 , Fator 2 Associado a Receptor de TNF/deficiência , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Fas-associated death domain (FADD) and caspase-8 are key signal transducers for death receptor-induced apoptosis, whereas cellular FLICE-inhibitory protein (cFLIP) antagonizes this process. Interestingly, FADD and caspase-8 also play a role in T cell development and T cell receptor (TCR)-mediated proliferative responses. To investigate the underlying mechanism, we generated cFLIP-deficient T cells by reconstituting Rag-/- blastocysts with cFLIP-deficient embryonic stem cells. These Rag chimeric mutant mice (rcFLIP-/-) had severely reduced numbers of T cells in the thymus, lymph nodes, and spleen, although mature T lymphocytes did develop. Similar to FADD- or caspase-8-deficient cells, rcFLIP-/- T cells were impaired in proliferation in response to TCR stimulation. Further investigation revealed that cFLIP is required for T cell survival, as well as T cell cycling in response to TCR stimulation. Interestingly, some signaling pathways from the TCR complex appeared competent, as CD3 plus CD28 cross-linking was capable of activating the ERK pathway in rcFLIP-/- T cells. We demonstrate an essential role for cFLIP in T cell function.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Linfócitos T/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Caspase 8 , Caspases/metabolismo , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína de Domínio de Morte Associada a Fas , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Signaling from tumor necrosis factor receptor type 1 (TNFR1) can elicit potent inflammatory and cytotoxic responses that need to be properly regulated. It was suggested that the silencer of death domains (SODD) protein constitutively associates intracellularly with TNFR1 and inhibits the recruitment of cytoplasmic signaling proteins to TNFR1 to prevent spontaneous aggregation of the cytoplasmic death domains of TNFR1 molecules that are juxtaposed in the absence of ligand stimulation. In this study, we demonstrate that mice lacking SODD produce larger amounts of cytokines in response to in vivo TNF challenge. SODD-deficient macrophages and embryonic fibroblasts also show altered responses to TNF. TNF-induced activation of NF-kappaB is accelerated in SODD-deficient cells, but TNF-induced c-Jun N-terminal kinase activity is slightly repressed. Interestingly, the apoptotic arm of TNF signaling is not hyperresponsive in the SODD-deficient cells. Together, these results suggest that SODD is critical for the regulation of TNF signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD/metabolismo , Proteínas de Transporte/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Proteínas de Transporte/genética , Morte Celular/fisiologia , Células Cultivadas , Cicloeximida/metabolismo , Proteína Ligante Fas , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Inibidores da Síntese de Proteínas/metabolismo , Receptores Tipo I de Fatores de Necrose TumoralRESUMO
IRAK-4 is a protein kinase that is pivotal in mediating signals for innate immune responses. Here, we report that IRAK-4 signaling is also essential for eliciting adaptive immune responses. Thus, in the absence of IRAK-4, in vivo T cell responses were significantly impaired. Upon T cell receptor stimulation, IRAK-4 is recruited to T cell lipid rafts, where it induces downstream signals, including protein kinase C activation through the association with Zap70. This signaling pathway was found to be required for optimal activation of nuclear factor kappaB. Our findings suggest that T cells use this critical regulator of innate immunity for the development of acquired immunity, suggesting that IRAK-4 may be involved in direct signal cross talk between the two systems.
Assuntos
Ativação Linfocitária , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Linfócitos T/imunologia , Animais , Ativação Enzimática , Imunidade Inata , Quinases Associadas a Receptores de Interleucina-1 , Isoenzimas/metabolismo , Microdomínios da Membrana/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Interleukin 1 receptor (IL-1R)-associated kinase-4 (IRAK-4) is required for various responses induced by IL-1R and Toll-like receptor signals. However, the molecular mechanism of IRAK-4 signaling and the role of its kinase activity have remained elusive. In this report, we demonstrate that IRAK-4 is recruited to the IL-1R complex upon IL-1 stimulation and is required for the recruitment of IRAK-1 and its subsequent activation/degradation. By reconstituting IRAK-4-deficient cells with wild type or kinase-inactive IRAK-4, we show that the kinase activity of IRAK-4 is required for the optimal transduction of IL-1-induced signals, including the activation of IRAK-1, NF-kappaB, and JNK, and the maximal induction of inflammatory cytokines. Interestingly, we also discover that the IRAK-4 kinase-inactive mutant is still capable of mediating some signals. These results suggest that IRAK-4 is an integral part of the IL-1R signaling cascade and is capable of transmitting signals both dependent on and independent of its kinase activity.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Transdução de Sinais , Animais , Western Blotting , Linhagem Celular , Clonagem Molecular , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Genes Reporter , Vetores Genéticos , Humanos , Interleucina-1/metabolismo , Quinases Associadas a Receptores de Interleucina-1 , Luciferases/metabolismo , MAP Quinase Quinase 4 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , NF-kappa B/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Testes de Precipitina , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Transfecção , TransgenesRESUMO
The immediate-early gene Nur77, which encodes an orphan nuclear receptor, is rapidly induced by various stress stimuli, including tumor necrosis factor (TNF). Nur77 has been implicated in mediating apoptosis, particularly in T cells and tumor cells. We report here that Nur77 can play a role in antagonizing apoptosis in TNF signaling. Nur77 expression is strongly induced by TNF. Interestingly, unlike most antiapoptotic molecules, this induced expression of Nur77 is largely independent of NF-kappa B. Ectopic expression of Nur77 can protect wild-type, TRAF2-/-, and RelA-/- cells from apoptosis induced by TNF, whereas expression of a dominant-negative form of Nur77 (DN-Nur77) accelerates TNF-mediated cell death in the mutant cells. In mouse embryonic fibroblasts, Nur77 remains in the nucleus in response to TNF and is not translocated to the mitochondria, where it was reported to mediate apoptosis. Our results suggest that Nur77 is a survival effector protein in the context of TNF-mediated signaling.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Caspase 8 , Caspase 9 , Caspases/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , NF-kappa B/deficiência , NF-kappa B/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Transporte Proteico , Proteínas/genética , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Fator 2 Associado a Receptor de TNF , Fator de Transcrição RelA , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, and members of the pro-inflammatory interleukin-1 receptor (IL-1R) family, share homologies in their cytoplasmic domains called Toll/IL-1R/plant R gene homology (TIR) domains. Intracellular signalling mechanisms mediated by TIRs are similar, with MyD88 (refs 5-8) and TRAF6 (refs 9, 10) having critical roles. Signal transduction between MyD88 and TRAF6 is known to involve the serine-threonine kinase IL-1 receptor-associated kinase 1 (IRAK-1) and two homologous proteins, IRAK-2 (ref. 12) and IRAK-M. However, the physiological functions of the IRAK molecules remain unclear, and gene-targeting studies have shown that IRAK-1 is only partially required for IL-1R and TLR signalling. Here we show by gene-targeting that IRAK-4, an IRAK molecule closely related to the Drosophila Pelle protein, is indispensable for the responses of animals and cultured cells to IL-1 and ligands that stimulate various TLRs. IRAK-4-deficient animals are completely resistant to a lethal dose of lipopolysaccharide (LPS). In addition, animals lacking IRAK-4 are severely impaired in their responses to viral and bacterial challenges. Our results indicate that IRAK-4 has an essential role in innate immunity.