RESUMO
Dulaglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, is widely used to treat diabetes. However, its effects on muscle wasting due to aging are poorly understood. In the current study, we investigated the therapeutic potential and underlying mechanism of dulaglutide in muscle wasting in aged mice. Dulaglutide improved muscle mass and strength in aged mice. Histological analysis revealed that the cross-sectional area of the tibialis anterior (TA) in the dulaglutide-treated group was thicker than that in the vehicle group. Moreover, dulaglutide increased the shift toward middle and large-sized fibers in both young and aged mice compared to the vehicle. Dulaglutide increased myofiber type I and type IIa in young (18.5% and 8.2%) and aged (1.8% and 19.7%) mice, respectively, compared to the vehicle group. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, decreased but increased by dulaglutide in aged mice. The expression of atrophic factors such as myostatin, atrogin-1, and muscle RING-finger protein-1 was decreased in aged mice, whereas that of the myogenic factor, MyoD, was increased in both young and aged mice following dulaglutide treatment. In aged mice, optic atrophy-1 (OPA-1) protein was decreased, whereas Toll-like receptor-9 (TLR-9) and its targeting inflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) were elevated in the TA and quadriceps (QD) muscles. In contrast, dulaglutide administration reversed this expression pattern, thereby significantly attenuating the expression of inflammatory cytokines in aged mice. These data suggest that dulaglutide may exert beneficial effects in the treatment of muscle wasting due to aging.
Assuntos
Envelhecimento/metabolismo , Peptídeos Semelhantes ao Glucagon/análogos & derivados , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Músculo Esquelético/fisiopatologia , Proteínas Recombinantes de Fusão/administração & dosagem , Sarcopenia/tratamento farmacológico , Sarcopenia/imunologia , Receptor Toll-Like 9/imunologia , Envelhecimento/efeitos dos fármacos , Envelhecimento/genética , Animais , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/imunologia , Peptídeos Semelhantes ao Glucagon/administração & dosagem , Humanos , Hipoglicemiantes/administração & dosagem , Interleucina-6/genética , Interleucina-6/imunologia , Masculino , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/imunologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/imunologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/imunologia , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/imunologia , Sarcopenia/etiologia , Sarcopenia/genética , Transdução de Sinais/efeitos dos fármacos , Receptor Toll-Like 9/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The evolutionarily conserved F-box family of proteins are well known for their role as the key component of SKP1-Cullin1-F-box (SCF) E3 ligase in controlling cell cycle, cell proliferation and cell death, carcinogenesis, and cancer metastasis. However, thus far, there is only limited investigation on their involvement in antiviral immunity. In contrast to the canonical function of FBXO6 associated with SCF E3 ligase complex, we report, in this study, that FBXO6 can also potently regulate the activation of IFN-I signaling during host response to viral infection by targeting the key transcription factor IFN-regulatory factor 3 (IRF3) for accelerated degradation independent of SCF in human embryonic kidney cells (HEK293T) and human lung cancer epithelial cells (A549). Structure and function delineation has further revealed that FBXO6 interacts with IAD domain of IRF3 through its FBA region to induce ubiquitination and degradation of IRF3 without the involvement of SCF. Thus, our studies have identified a general but, to our knowledge, previously unrecognized role and a novel noncanonical mechanism of FBXO6 in modulating IFN-I-mediated antiviral immune responses, which may protect the host from immunopathology of overreactive and harmful IFN-I production.
Assuntos
Proteínas Ligases SKP Culina F-Box/imunologia , Viroses/imunologia , Linhagem Celular , Humanos , Interferon Tipo I/imunologiaRESUMO
The inflammasome is a multiprotein complex that augments the proinflammatory response by increasing the generation and cellular release of key cytokines. Specifically, the NALP3 inflammasome requires two-step signaling, priming and activation, to be functional to release the proinflammatory cytokines IL-1ß and IL-18. The priming process, through unknown mechanisms, increases the protein levels of NALP3 and pro-IL-1ß in cells. Here we show that LPS increases the NALP3 protein lifespan without significantly altering steady-state mRNA in human cells. LPS exposure reduces the ubiquitin-mediated proteasomal processing of NALP3 by inducing levels of an E3 ligase component, FBXO3, which targets FBXL2. The latter is an endogenous mediator of NALP3 degradation. FBXL2 recognizes Trp-73 within NALP3 for interaction and targets Lys-689 within NALP3 for ubiquitin ligation and degradation. A unique small molecule inhibitor of FBXO3 restores FBXL2 levels, resulting in decreased NALP3 protein levels in cells and, thereby, reducing the release of IL-1ß and IL-18 in human inflammatory cells after NALP3 activation. Our findings uncover NALP3 as a molecular target for FBXL2 and suggest that therapeutic targeting of the inflammasome may serve as a platform for preclinical intervention.
Assuntos
Proteínas de Transporte/imunologia , Proteínas F-Box/imunologia , Inflamassomos/imunologia , Lipopolissacarídeos/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas F-Box/metabolismo , Humanos , Imunidade Inata , Inflamassomos/metabolismo , Interleucina-18/imunologia , Interleucina-1beta/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteólise , Proteínas Ligases SKP Culina F-Box/imunologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
OBJECTIVE: To construct eukaryotic expression vector of F-box protein 6 (FBXO6) gene. METHODS: The full-length FBXO6 cDNA containing two restriction sites (BglII and EcoRI) was synthesized by PCR. The pEGFP-C1-FBXO6 and pEGFP-C1-anti-FBXO6 vectors were constructed respectively. The size and sequence of cDNA fragments were confirmed by bacterial colony PCR and BglII and EcoRI digestion and sequencing. HEK293T cells were transfected with the pEGFP-C1-FBXO6 and pEGFP-C1-anti-FBXO6 vectors respectively, and then the expression of FBXO6 protein was detected using Western blotting. RESULTS: The pEGFP-C1-FBXO6 and pEGFP-C1-anti-FBXO6 contained FBXO6 fragments of proper size and sequence. FBXO6 protein was expressed efficiently in pEGFP-C1-FBXO6 transfected HEK293T cells, but it was down-regulated in pEGFP-C1-anti-FBXO6 transfected 293T cells. CONCLUSION: Both pEGFP-C1-FBXO6 and pEGFP-C1-anti-FBXO6 were successfully constructed.
Assuntos
Proteínas Recombinantes/biossíntese , Proteínas Ligases SKP Culina F-Box/genética , Anticorpos/genética , Clonagem Molecular , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Proteínas Ligases SKP Culina F-Box/imunologia , TransfecçãoRESUMO
Tolerance to endotoxins that is triggered by prior exposure to Toll-like receptor (TLR) ligands provides a mechanism with which to dampen inflammatory cytokines. The receptor-interacting protein RIP140 interacts with the transcription factor NF-κB to regulate the expression of genes encoding proinflammatory cytokines. Here we found lipopolysaccharide stimulation of kinase Syk-mediated tyrosine phosphorylation of RIP140 and interaction of the NF-κB subunit RelA with RIP140. These events resulted in more recruitment of the E3 ligase SCF to tyrosine-phosphorylated RIP140, which degraded RIP140 to inactivate genes encoding inflammatory cytokines. Macrophages expressing nondegradable RIP140 were resistant to the establishment of endotoxin tolerance for specific 'tolerizable' genes. Our results identify RelA as an adaptor with which SCF fine tunes NF-κB target genes by targeting the coactivator RIP140 and show an unexpected role for RIP140 degradation in resolving inflammation and endotoxin tolerance.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endotoxinas/imunologia , Tolerância Imunológica/imunologia , Inflamação/imunologia , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Imunoprecipitação da Cromatina , Técnicas de Silenciamento de Genes , Immunoblotting , Inflamação/metabolismo , Camundongos , Camundongos Transgênicos , NF-kappa B/imunologia , Proteínas Nucleares/imunologia , Proteína 1 de Interação com Receptor Nuclear , Proteínas Ligases SKP Culina F-Box/imunologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
MG with anti-MuSK antibodies (MuSK+) is often characterized with muscle atrophy and excellent response to plasma exchanges. To elucidate some MuSK+ MG features, we analyzed the functional effects of anti-MuSK Abs in human TE 671 muscle cells. We found that some MuSK+ sera induced a striking inhibition of proliferation, accompanied by: 1) cell cycle arrest, 2) atrogin-1 overexpression, 3) AChR subunits, rapsyn, Rho A and cdc42 downregulation. These effects correlated to disease severity and to anti-MuSK Abs titer and vanished following PE. Altogether, these results indicate that anti-MuSK Abs could be pathogenic by contributing to the muscle atrophy in MuSK+ MG patients.