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1.
Int J Biol Macromol ; 153: 1262-1271, 2020 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-31770559

RESUMEN

The tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a key cytoplasm signaling adaptor that mediates signals activated by TNFR superfamily and the interleukin-1/Toll-like receptor (IL-1/TLR) superfamily. In the present research, a housefly Musca domestica TRAF6 (MdTRAF6) gene is identified and characterized, with a 51.7-kDa protein possessing a RING domain and a conserved C-terminal TRAF homology MATH domain encoded. MdTRAF6 is widely expressed in diverse tissues with high expression levels in gut and fat body, which is of the highest levels in adult in all growth stages. The expression of MdTRAF6 could be remarkably induced by bacterial challenge, and the silencing MdTRAF6 could alter the expressions of NF-κB-like genes (relish and dorsal) and antimicrobial peptide genes (cecropin, diptericin, attacin, muscin), thus leading elevated mortalities of larvae followed by bacterial infection. Inspiringly. MdTRAF6-depleted adult flies display higher mortality, lower fertility and reduced survival of offspring than the controls. Further investigation reveals that knockdown of MdTRAF6 disturbs the ovarian development and impaires the expressions of vitellogenin and vitellogenin receptor genes in the adult females. All these phenotypes show crucial roles of MdTRAF6 in innate immunity via positive regulation of the Toll pathway and negative regulation of the Imd pathway, and in reproduction by maintaining ovarian development.


Asunto(s)
Moscas Domésticas/crecimiento & desarrollo , Moscas Domésticas/inmunología , Proteínas de Insectos/metabolismo , Ovario/crecimiento & desarrollo , Factor 6 Asociado a Receptor de TNF/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Silenciador del Gen , Moscas Domésticas/genética , Moscas Domésticas/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/deficiencia , Proteínas de Insectos/genética , Factor 6 Asociado a Receptor de TNF/química , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética , Regulación hacia Arriba
2.
Commun Biol ; 2: 292, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31396572

RESUMEN

Receptor activator of nuclear factor (NF)-κB (RANK) signaling promotes pregnancy-dependent epithelial cell differentiation and expansion for mammary gland development, which requires NF-κB pathway-dependent Cyclin D1 induction and inhibitor of DNA binding 2 (Id2) pathway-dependent anti-apoptotic gene induction. However, the roles of tumor necrosis factor receptor-associated factor 6 (TRAF6) remain unclear despite its requirement in RANK signaling. Here we show that TRAF6 is crucial for both mammary stem cell maintenance and pregnancy-induced epithelial cell expansion. TRAF6 deficiency impairs phosphoinositide 3-kinase (PI3K)/AKT and canonical NF-κB pathways, whereas noncanonical NF-κB signaling remains functional. Therefore, we propose that TRAF6 promotes cell proliferation by activating PI3K/AKT signaling to induce retinoblastoma phosphorylation in concert with noncanonical NF-κB pathway-dependent Cyclin D1 induction. Furthermore, TRAF6 inhibits apoptosis by activating canonical NF-κB signaling to induce anti-apoptotic genes with the Id2 pathway. Therefore, proper orchestration of TRAF6-dependent and -independent RANK signals likely establishes mammary gland formation.


Asunto(s)
Proliferación Celular , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Células Madre/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo/trasplante , Animales , Apoptosis , Línea Celular , Ciclina D1/metabolismo , Femenino , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones Endogámicos BALB C , Ratones Noqueados , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación , Embarazo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética
3.
J Exp Med ; 215(2): 501-519, 2018 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-29339448

RESUMEN

M cells are located in the follicle-associated epithelium (FAE) that covers Peyer's patches (PPs) and are responsible for the uptake of intestinal antigens. The differentiation of M cells is initiated by receptor activator of NF-κB. However, the intracellular pathways involved in M cell differentiation are still elusive. In this study, we demonstrate that the NF-κB pathway activated by RANK is essential for M cell differentiation using in vitro organoid culture. Overexpression of NF-κB transcription factors enhances the expression of M cell-associated molecules but is not sufficient to complete M cell differentiation. Furthermore, we evaluated the requirement for tumor necrosis factor receptor-associated factor 6 (TRAF6). Conditional deletion of TRAF6 in the intestinal epithelium causes a complete loss of M cells in PPs, resulting in impaired antigen uptake into PPs. In addition, the expression of FAE-associated genes is almost silenced in TRAF6-deficient mice. This study thus demonstrates the crucial role of TRAF6-mediated NF-κB signaling in the development of M cells and FAE.


Asunto(s)
Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , FN-kappa B/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Humanos , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Organoides/citología , Organoides/inmunología , Organoides/metabolismo , Ganglios Linfáticos Agregados/citología , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/metabolismo , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética
4.
J Leukoc Biol ; 96(3): 427-36, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24812060

RESUMEN

TLRs act as sentinels in professional immune cells to detect and initiate the innate immune response to pathogen challenge. TLR4 is a widely expressed TLR, responsible for initiating potent immune responses to LPS. TRAM acts to bridge TLR4 with TRIF, orchestrating the inflammatory response to pathogen challenge. We have identified a putative TRAF6-binding motif in TRAM that could mediate a novel signaling function for TRAM in TLR4 signaling. TRAM and TRAF6 association was confirmed by immunoprecipitation of endogenous, ectopically expressed and recombinant proteins, which was ablated upon mutation of a key Glu residue in TRAM (TRAM E183A). TRAF6 and TRAM were observed colocalizing using confocal microscopy following ectopic expression in cells and the ability of TRAM and TRAM E183A to activate luciferase-linked reporter assays was determined in HEK293 and TRAF6-deficient cells. Importantly, TRAM-deficient macrophages reconstituted with TRAM E183A display significantly reduced inflammatory TNF-α, IL-6, and RANTES protein production compared with WT TRAM. These results demonstrate a novel role for TRAM in TLR4-mediated signaling in regulating inflammatory responses via its interaction with TRAF6, distinct from its role as a bridging adaptor between TLR4 and TRIF.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Inflamación/fisiopatología , Factor 6 Asociado a Receptor de TNF/fisiología , Receptor Toll-Like 4/fisiología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Proteínas Adaptadoras del Transporte Vesicular/genética , Secuencias de Aminoácidos , Animales , Células Cultivadas , Citocinas/biosíntesis , Fibroblastos , Genes Reporteros , Células HEK293 , Humanos , Inmunoprecipitación , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Mutación Missense , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Mapeo de Interacción de Proteínas , ARN Interferente Pequeño/farmacología , Receptores de Interleucina/deficiencia , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/química , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética
5.
PLoS One ; 8(9): e74639, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24058613

RESUMEN

Regulatory T cells (Tregs) maintain immune homeostasis by limiting inflammatory responses. TRAF6 plays a key role in the regulation of innate and adaptive immunity by mediating signals from various receptors including the T-cell receptor (TCR). T cell-specific deletion of TRAF6 has been shown to induce multiorgan inflammatory disease, but the role of TRAF6 in Tregs remains to be investigated. Here, we generated Treg-specific TRAF6-deficient mice using Foxp3-Cre and TRAF6-flox mice. Treg-specific TRAF6-deficient (cKO) mice developed allergic skin diseases, arthritis, lymphadenopathy and hyper IgE phenotypes. Although TRAF6-deficient Tregs possess similar in vitro suppression activity compared to wild-type Tregs, TRAF6-deficient Tregs did not suppress colitis in lymphopenic mice very efficiently due to reduced number of Foxp3-positive cells. In addition, the fraction of TRAF6-deficient Tregs was reduced compared with wild-type Tregs in female cKO mice without inflammation. Moreover, adoptive transfer of Foxp3 (+) Tregs into Rag2(-/-) mice revealed that TRAF6-deficient Tregs converted into Foxp3(-) cells more rapidly than WT Tregs under lymphopenic conditions. Fate-mapping analysis also revealed that conversion of Tregs from Foxp3(+) to Foxp3(-) (exFoxp3 cells) was accelerated in TRAF6-deficient Tregs. These data indicate that TRAF6 in Tregs plays important roles in the maintenance of Foxp3 in Tregs and in the suppression of pathogenic Th2 type conversion of Tregs.


Asunto(s)
Autoinmunidad/inmunología , Linfocitos T Reguladores/inmunología , Factor 6 Asociado a Receptor de TNF/metabolismo , Células Th2/inmunología , Animales , Proliferación Celular , Femenino , Factores de Transcripción Forkhead/metabolismo , Integrasas/metabolismo , Interleucina-2/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Fenotipo , Factor de Transcripción STAT5/metabolismo , Factor 6 Asociado a Receptor de TNF/deficiencia
6.
J Clin Invest ; 123(8): 3510-24, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23867620

RESUMEN

TRAF6, an E3 ubiquitin protein ligase, plays a critical role in T cell tolerance by regulating medullary thymic epithelial cell (mTEC) development. mTECs regulate T cell tolerance by ectopically expressing self-antigens and eliminating autoreactive T cells in the thymus. Here we show that mice with mTEC depletion due to conditional deletion of Traf6 expression in murine thymic epithelial cells (Traf6ΔTEC mice) showed a surprisingly narrow spectrum of autoimmunity affecting the liver. The liver inflammation in Traf6ΔTEC mice exhibited all the histological and immunological characteristics of human autoimmune hepatitis (AIH). The role of T cells in AIH establishment was supported by intrahepatic T cell population changes and AIH development after transfer of liver T cells into immunodeficient mice. Despite a 50% reduction in natural Treg thymic output, peripheral tolerance in Traf6ΔTEC mice was normal, whereas compensatory T regulatory mechanisms were evident in the liver of these animals. These data indicate that mTECs exert a cell-autonomous role in central T cell tolerance and organ-specific autoimmunity, but play a redundant role in peripheral tolerance. These findings also demonstrate that Traf6ΔTEC mice are a relevant model with which to study the pathophysiology of AIH, as well as autoantigen-specific T cell responses and regulatory mechanisms underlying this disease.


Asunto(s)
Células Epiteliales/patología , Hepatitis Autoinmune/patología , Timo/patología , Traslado Adoptivo , Animales , Anticuerpos Antinucleares/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Hepatitis Autoinmune/inmunología , Humanos , Tolerancia Inmunológica , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Plasmáticas/patología , Linfocitos T/inmunología , Linfocitos T/trasplante , Linfocitos T Reguladores/inmunología , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética , Timo/inmunología
7.
J Immunol ; 190(8): 4027-36, 2013 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-23514740

RESUMEN

TNFR-associated factor (TRAF)6 is an essential ubiquitin E3 ligase in immune responses, but its function in adaptive immunity is not well understood. In this study, we show that TRAF6 is recruited to the peripheral ring of the T cell immunological synapse in Jurkat T cells or human primary CD4(+) T cells conjugated with staphylococcal enterotoxin E-pulsed B cells. This recruitment depends on TRAF6 interacting with linker for activation of T cells (LAT) via its TRAF domain. Although LAT was indispensable for TCR/CD28-induced TRAF6 ubiquitination and its ligase activity, RNA interference-induced TRAF6 knockdown in T cells decreased TCR/CD28-induced LAT ubiquitination, tyrosine phosphorylation, and association with tyrosine kinase ZAP70. Overexpression of TRAF6 or its catalytically inactive form C70A promoted and decreased, respectively, LAT tyrosine phosphorylation upon stimulation. Moreover, LAT was ubiquitinated at Lys(88) by TRAF6 via K63-linked chain. In addition, TRAF6 was required for and synergized with LAT to promote the TCR/CD28-induced activation of NFAT. These results reveal a novel function and mechanism of TRAF6 action in the TCR-LAT signaling pathway distinct from its role in TCR-induced NF-κB activation, indicating that LAT also plays an adapter role in TCR/CD28-induced activation of TRAF6.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de la Membrana/metabolismo , Mapeo de Interacción de Proteínas/métodos , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/inmunología , Factor 6 Asociado a Receptor de TNF/fisiología , Antígenos CD28/fisiología , Células HEK293 , Humanos , Células Jurkat , Fosforilación/inmunología , Cultivo Primario de Células , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/metabolismo , Ubiquitinación/inmunología
9.
Circulation ; 126(14): 1739-51, 2012 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-22935568

RESUMEN

BACKGROUND: Previous studies implicated Toll-like receptor signaling as a critical pathogenic pathway in atherosclerosis, but the cell-specific mechanisms by which Toll-like receptors act to control atherosclerotic plaque development remain poorly understood. METHODS AND RESULTS: To study the cell-specific role of tumor necrosis factor receptor-associated factor 6 (TRAF6) in atherosclerosis, we generated ApoE(-/-) mice with endothelial cell- or myeloid cell-specific TRAF6 deficiency using Cre/LoxP-mediated gene targeting. Endothelial TRAF6 deficiency reduced atherosclerosis in female ApoE(-/-) mice by inhibiting nuclear factor-κB-dependent proinflammatory gene expression and monocyte adhesion to endothelial cells. In contrast, myeloid cell-specific TRAF6 deficiency caused exacerbated atherosclerosis, with larger plaques containing more necrotic areas in both male and female ApoE(-/-) mice. TRAF6-deficient macrophages showed impaired expression of the antiinflammatory and atheroprotective cytokine interleukin-10, elevated endoplasmic reticulum stress, increased sensitivity to oxidized low-density lipoprotein-induced apoptosis, and reduced capacity to clear apoptotic cells. Thus, the reduced antiinflammatory properties, coupled with increased sensitivity to apoptosis and impaired efferocytosis capacity of TRAF6-deficient macrophages, result in exacerbated atherosclerosis development in TRAF6(MYKO)/ApoE(-/-) mice. CONCLUSION: Toll-like receptor-mediated TRAF6 signaling acts in endothelial cells to promote atherosclerosis but displays atheroprotective, antiinflammatory and prosurvival functions in myeloid cells.


Asunto(s)
Aterosclerosis/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Mieloides/metabolismo , Factor 6 Asociado a Receptor de TNF/deficiencia , Receptores Toll-Like/fisiología , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Células Endoteliales/patología , Femenino , Marcación de Gen/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/patología , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/fisiología
10.
Nature ; 483(7391): 623-6, 2012 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-22407319

RESUMEN

Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-κB signalling pathway. We determined the 2.0 Å crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Shigella flexneri/enzimología , Shigella flexneri/inmunología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Amidohidrolasas/genética , Secuencia de Aminoácidos , Animales , Ácido Aspártico/metabolismo , Proteína 10 de la LLC-Linfoma de Células B , Biocatálisis , Caspasas/metabolismo , Dominio Catalítico/genética , Cristalografía por Rayos X , Cisteína/metabolismo , Análisis Mutacional de ADN , Diglicéridos/antagonistas & inhibidores , Diglicéridos/metabolismo , Disentería Bacilar/microbiología , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Células HEK293 , Células HeLa , Histidina/metabolismo , Humanos , Inmunidad Innata , Inflamación/enzimología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Shigella flexneri/genética , Shigella flexneri/patogenicidad , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Factores de Virulencia/metabolismo
11.
J Clin Invest ; 121(10): 4095-105, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21911935

RESUMEN

Somatic mutations and copy number alterations (as a result of deletion or amplification of large portions of a chromosome) are major drivers of human lung cancers. Detailed analysis of lung cancer-associated chromosomal amplifications could identify novel oncogenes. By performing an integrative cytogenetic and gene expression analysis of non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) cell lines and tumors, we report here the identification of a frequently recurring amplification at chromosome 11 band p13. Within this region, only TNF receptor-associated factor 6 (TRAF6) exhibited concomitant mRNA overexpression and gene amplification in lung cancers. Inhibition of TRAF6 in human lung cancer cell lines suppressed NF-κB activation, anchorage-independent growth, and tumor formation. In these lung cancer cell lines, RAS required TRAF6 for its oncogenic capabilities. Furthermore, TRAF6 overexpression in NIH3T3 cells resulted in NF-κB activation, anchorage-independent growth, and tumor formation. Our findings show that TRAF6 is an oncogene that is important for RAS-mediated oncogenesis and provide a mechanistic explanation for the previously apparent importance of constitutive NF-κB activation in RAS-driven lung cancers.


Asunto(s)
Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , FN-kappa B/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Proteínas ras/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/etiología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Pequeñas/etiología , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Pequeñas/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 11/genética , Amplificación de Genes , Humanos , Neoplasias Pulmonares/etiología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células 3T3 NIH , Oncogenes , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/deficiencia , Ensayo de Tumor de Célula Madre
12.
J Immunol ; 185(11): 6555-62, 2010 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-21041727

RESUMEN

CD40 is required for T cell-dependent humoral immunity, but it can also contribute to the pathogenesis of autoimmunity and B cell malignancy. The TNFR-associated factor (TRAF)2 and TRAF6 adaptor proteins are positive regulators of CD40 signaling required to activate downstream kinase cascades and transcription factors. In contrast, TRAF3 can serve as a negative regulator of CD40 signaling, and CD40 signals are amplified in TRAF3(-/-) B cells. We previously reported a gain-of-function polymorphism of the human CD40 receptor, hCD40-P227A, which signals in an amplified manner to B lymphocytes. In this study, we show that hCD40-P227A binds more TRAF3 and TRAF5, as well as certain associated proteins, than wild-type-CD40. Studies in TRAF-deficient B cell lines revealed that hCD40-P227A uses TRAF3 as a positive rather than negative regulator. Although located outside of any known TRAF binding sites, the P227A polymorphism can alter TRAF binding and dramatically changes the role played by TRAF3 in CD40 signaling.


Asunto(s)
Antígenos CD40/antagonistas & inhibidores , Antígenos CD40/metabolismo , Variación Genética/inmunología , Transducción de Señal/inmunología , Factor 3 Asociado a Receptor de TNF/metabolismo , Regulación hacia Arriba/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Animales , Sitios de Unión/genética , Sitios de Unión/inmunología , Antígenos CD40/genética , Línea Celular , Células Clonales , Humanos , Interleucina-6/biosíntesis , Ratones , Datos de Secuencia Molecular , Polimorfismo Genético/inmunología , Transducción de Señal/genética , Factor 3 Asociado a Receptor de TNF/deficiencia , Factor 3 Asociado a Receptor de TNF/fisiología , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética , Regulación hacia Arriba/genética
13.
J Immunol ; 185(3): 1606-15, 2010 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-20622119

RESUMEN

GM-CSF, IL-3, and IL-5 are proinflammatory cytokines that control the production and function of myeloid and lymphoid cells. Their receptors are composed of a ligand-specific alpha subunit and a shared common signal-transducing beta subunit (beta common receptor or GM-CSFR beta [beta(c)]). The pleiotropic nature of biologic outcomes mediated by beta(c) and the presence of large, uncharacterized regions of its cytoplasmic domain suggest that much remains to be learned about its downstream signaling pathways. Although some previous work has attempted to link beta(c) with NF-kappaB activation, a definitive mechanism that mediates this pathway has not been described and, to date, it has not been clear whether the receptor can directly activate NF-kappaB. We demonstrate that NF-kappaB activation by beta(c) is dependent on TNFR-associated factor 6 (TRAF6) and that association of TRAF6 with beta(c) requires a consensus-binding motif found in other molecules known to interact with TRAF6. Furthermore, point mutation of this motif abrogated the ability of beta(c) to mediate NF-kappaB activation and reduced the viability of an IL-3-dependent hematopoietic cell line. Because this receptor plays a key role in hematopoiesis and the beta(c) cytoplasmic domain identified in this work mediates hematopoietic cell viability, this new pathway is likely to contribute to immune cell biology. This work is significant because it is the first description of a TRAF6-dependent signaling pathway associated with a type I cytokine receptor. It also suggests that TRAF6, a mediator of TNFR and TLR signaling, may be a common signaling intermediate in diverse cytokine receptor systems.


Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/fisiología , FN-kappa B/fisiología , Factor 6 Asociado a Receptor de TNF/metabolismo , Transporte Activo de Núcleo Celular/inmunología , Animales , Células Cultivadas , Células Clonales , Secuencia de Consenso , Subunidad beta Común de los Receptores de Citocinas/antagonistas & inhibidores , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fibroblastos/inmunología , Fibroblastos/metabolismo , Humanos , Ratones , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Biosíntesis de Proteínas/inmunología , Estructura Terciaria de Proteína/genética , Transporte de Proteínas/inmunología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/fisiología
14.
J Virol ; 84(13): 6605-14, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20410275

RESUMEN

Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1) is required for EBV B-lymphocyte transformation, transforms rodent fibroblasts, and can induce lymphoma and epithelial hyperplasia in transgenic mice. Two domains have been identified within the intracellular carboxy terminus that can activate NF-kappaB, C-terminus-activating region 1 (CTAR1) and CTAR2, through interactions with tumor necrosis receptor-associated factors (TRAFs). CTAR1 can activate both the canonical and noncanonical NF-kappaB pathways and has unique effects on cellular gene expression. The epidermal growth factor receptor (EGFR) is highly induced by LMP1-CTAR1 in epithelial cells through activation of a novel NF-kappaB form containing p50 homodimers and Bcl-3. To further understand the regulation of NF-kappaB in CTAR1-induced EGFR expression, we evaluated the ability of CTAR1 to induce EGFR in mouse embryonic fibroblasts (MEFs) defective for different NF-kappaB effectors. CTAR1-mediated EGFR induction required the NF-kappaB-inducing kinase (NIK) but not the IkappaB kinase (IKK) complex components that regulate canonical or noncanonical NF-kappaB pathways. CTAR1-mediated induction of nuclear p50 occurred in IKKbeta-, IKKgamma-, and NIK-defective MEFs, indicating that this induction is not dependent on the canonical or noncanonical NF-kappaB pathways. EGFR and nuclear p50 were expressed at high levels in TRAF2(-/-) fibroblasts and were not induced by CTAR1. In TRAF3(-/-) MEFs, CTAR1 induced nuclear p50 but did not affect basal levels of STAT3 serine phosphorylation or induce EGFR expression. EGFR was induced by LMP1 in TRAF6(-/-) MEFs. These findings suggest that this novel NF-kappaB pathway is differentially regulated by TRAF2 and TRAF3, and that distinct interactions of LMP1 and its effectors regulate LMP1-mediated gene expression.


Asunto(s)
Receptores ErbB/biosíntesis , Herpesvirus Humano 4/fisiología , Subunidad p50 de NF-kappa B/metabolismo , Proteínas de la Matriz Viral/metabolismo , Acoplamiento Viral , Animales , Fibroblastos/virología , Quinasa I-kappa B/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor 2 Asociado a Receptor de TNF/deficiencia , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor 3 Asociado a Receptor de TNF/deficiencia , Factor 3 Asociado a Receptor de TNF/metabolismo , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/metabolismo , Quinasa de Factor Nuclear kappa B
15.
Sci Signal ; 2(93): ra66, 2009 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-19843958

RESUMEN

The cytokine interleukin-1 (IL-1) mediates immune and inflammatory responses by activating the transcription factor nuclear factor kappaB (NF-kappaB). Although transforming growth factor-beta-activated kinase 1 (TAK1) and mitogen-activated protein kinase (MAPK) kinase kinase 3 (MEKK3) are both crucial for IL-1-dependent activation of NF-kappaB, their potential functional and physical interactions remain unclear. Here, we showed that TAK1-mediated activation of NF-kappaB required the transient formation of a signaling complex that included tumor necrosis factor receptor-associated factor 6 (TRAF6), MEKK3, and TAK1. Site-specific, lysine 63-linked polyubiquitination of TAK1 at lysine 209, likely catalyzed by TRAF6 and Ubc13, was required for the formation of this complex. After TAK1-mediated activation of NF-kappaB, TRAF6 subsequently activated NF-kappaB through MEKK3 independently of TAK1, thereby establishing continuous activation of NF-kappaB, which was required for the production of sufficient cytokines. Therefore, we propose that the cooperative activation of NF-kappaB by two mechanistically and temporally distinct MEKK3-dependent pathways that diverge at TRAF6 critically contributes to immune and inflammatory systems.


Asunto(s)
Interleucina-1/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Animales , Línea Celular , Humanos , MAP Quinasa Quinasa Quinasa 3/deficiencia , MAP Quinasa Quinasa Quinasa 3/metabolismo , Quinasas Quinasa Quinasa PAM/deficiencia , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Unión Proteica , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación
17.
Nature ; 460(7251): 103-7, 2009 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-19494812

RESUMEN

CD8 T cells, which have a crucial role in immunity to infection and cancer, are maintained in constant numbers, but on antigen stimulation undergo a developmental program characterized by distinct phases encompassing the expansion and then contraction of antigen-specific effector (T(E)) populations, followed by the persistence of long-lived memory (T(M)) cells. Although this predictable pattern of CD8 T-cell responses is well established, the underlying cellular mechanisms regulating the transition to T(M) cells remain undefined. Here we show that tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), an adaptor protein in the TNF-receptor and interleukin-1R/Toll-like receptor superfamily, regulates CD8 T(M)-cell development after infection by modulating fatty acid metabolism. We show that mice with a T-cell-specific deletion of TRAF6 mount robust CD8 T(E)-cell responses, but have a profound defect in their ability to generate T(M) cells that is characterized by the disappearance of antigen-specific cells in the weeks after primary immunization. Microarray analyses revealed that TRAF6-deficient CD8 T cells exhibit altered expression of genes that regulate fatty acid metabolism. Consistent with this, activated CD8 T cells lacking TRAF6 display defective AMP-activated kinase activation and mitochondrial fatty acid oxidation (FAO) in response to growth factor withdrawal. Administration of the anti-diabetic drug metformin restored FAO and CD8 T(M)-cell generation in the absence of TRAF6. This treatment also increased CD8 T(M) cells in wild-type mice, and consequently was able to considerably improve the efficacy of an experimental anti-cancer vaccine.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Ácidos Grasos/metabolismo , Memoria Inmunológica/inmunología , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Hipoglucemiantes/farmacología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Listeriosis/metabolismo , Listeriosis/microbiología , Metformina/farmacología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-cbl/deficiencia , Proteínas Proto-Oncogénicas c-cbl/genética , Factor 6 Asociado a Receptor de TNF/genética
18.
J Immunol ; 182(3): 1660-6, 2009 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-19155515

RESUMEN

IL-17 alone is a relatively weak inducer of gene expression, but cooperates with other cytokines, including TNF-alpha, to generate a strong response in part via prolongation of mRNA t(1/2). Because TNFR-associated factor 6 (TRAF6) has been reported to be essential for signaling by IL-17, we examined its involvement in IL-17-mediated mRNA stabilization. Although overexpression of TRAF6 in HeLa cells activates NF-kappaB, it does not stabilize transfected KC mRNA. Furthermore, a dominant-negative TRAF6 abrogates NF-kappaB activation, but does not block IL-17-induced chemokine mRNA stabilization. IL-17 can stabilize KC and MIP-2 mRNAs comparably in TNF-alpha-treated mouse embryo fibroblasts from TRAF6(+/+) and TRAF6(-/-) mice. TRAF6 is known to couple upstream signals with activation of p38 MAPK and mitogen activated protein kinase activated protein kinase 2, both of which have been shown to be important for Toll/IL-1R-mediated mRNA stabilization in various cell types. Inhibition of p38 MAPK, however, does not block IL-17-induced KC mRNA stabilization, and IL-17 can stabilize KC mRNA equally in mouse embryo fibroblasts from both wild-type and mitogen activated protein kinase activated protein kinase 2/3 doubly-deficient mice. Finally, IL-17 can amplify the levels of multiple TNF-alpha-stimulated mRNAs in wild-type and TRAF6-deficient cells, but not in cells from Act1(-/-) mice. Collectively, these findings demonstrate the existence of a TRAF6/p38 MAPK-independent pathway that couples the IL-17R with enhanced mRNA stability. Because the most potent effects of IL-17 on gene expression are obtained in cooperation with other cytokines such as TNF-alpha, these findings suggest that this pathway is a major contributing mechanism for response to IL-17.


Asunto(s)
Interleucina-17/fisiología , Estabilidad del ARN/inmunología , ARN Mensajero/metabolismo , Transducción de Señal/inmunología , Factor 6 Asociado a Receptor de TNF/fisiología , Animales , Células Cultivadas , Quimiocinas/biosíntesis , Quimiocinas/genética , Regulación de la Expresión Génica/inmunología , Células HeLa , Humanos , Mediadores de Inflamación/fisiología , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , ARN Mensajero/biosíntesis , Transducción de Señal/genética , Factor 6 Asociado a Receptor de TNF/biosíntesis , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética , Factor de Necrosis Tumoral alfa/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología
19.
J Biol Chem ; 283(46): 32110-8, 2008 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-18772140

RESUMEN

TRAF6 (tumor necrosis factor-associated factor 6) is an essential adaptor downstream from the tumor necrosis factor (TNF) receptor and Toll-like receptor superfamily members. This molecule is critical for dendritic cell maturation and T cell homeostasis. Here we show that TRAF6 is important in high affinity IgE receptor, FcepsilonRI-mediated mast cell activation. In contrast to dendritic cells and T cells, TRAF6-deficient mast cells matured normally and showed normal IgE-dependent degranulation. Importantly, TRAF6-deficient mast cells showed impaired production of cytokine interleukin-6, CCL-9, interleukin-13, and TNF following FcepsilonRI aggregation. Chromatin immunoprecipitation assay showed decreased NF-kappaB p65 binding to CCL-9 and TNF promoters in TRAF6-deficient mast cells. Antigen and IgE-induced IkappaB phosphorylation and NF-kappaB p65 translocation to the nucleus were diminished in TRAF6-deficient mast cells. NF-kappaB luciferase activity in response to antigen and IgE stimulation was severely impaired in TRAF6-deficient mast cells. In addition, antigen and IgE-induced phosphorylation of mitogen-activated protein kinase p38 and JNK, but not ERK1/2, was significantly reduced in TRAF6-deficient mast cells. These results identified TRAF6 as an important signal transducer in FcepsilonRI-mediated signaling in mast cells. Our findings implicate TRAF6 as a new adaptor/regulator molecule for allergen-mediated inflammation in allergy.


Asunto(s)
Citocinas/biosíntesis , Mastocitos/metabolismo , Receptores de IgE/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Calcio/metabolismo , Degranulación de la Célula , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inmunoglobulina E/inmunología , Hígado/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Noqueados , Fosforilación , Receptores de IgE/inmunología , Especificidad por Sustrato , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
20.
Cell Signal ; 20(9): 1679-86, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18603409

RESUMEN

IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L. However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38, JNK and Nuclear factor-kappaB (NF-kappaB) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappaB in TRAF6 deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAF6 and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappaB activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity.


Asunto(s)
Interleucinas/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Carboxipeptidasas A/genética , Carboxipeptidasas A/metabolismo , Línea Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-33 , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucinas/farmacología , Ratones , Proteínas Mutantes/metabolismo , Transducción de Señal/efectos de los fármacos , Factor 6 Asociado a Receptor de TNF/deficiencia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacos
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