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1.
Scand J Immunol ; 91(1): e12840, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31630418

RESUMO

IL-17 participates in the development of many autoimmune diseases by promoting the expression of some chemokines. Chemokine C-C motif ligand 2 (CCL2) is an important factor at the infiltration of mononuclear cells in the myocardial tissue of viral myocarditis (VMC). It was found that IL-17 could aggravate myocardial injury by upregulating CCL2. But the underlying mechanism involved in CCL2 secretion induced by IL-17 in cardiac myocytes remains unclear. This study investigated the role of transcription factor AP-1 in IL-17 induced CCL2 expression. The results showed that IL-17 mediated the activation of Act1, TRAF6, p38MAPK and c-Jun/AP-1 not Wnt or PI3K signalling pathway to upregulate CCL2 expression in cardiac myocytes. After blocking Act1/TRAF6/p38MAPK cascade and interfering AP-1 with Curcumin or c-Jun siRNA, CCL2 expression induced by IL-17 was significantly attenuated at both mRNA and protein levels. Furthermore, the phosphorylation of c-Jun was suppressed when cardiac myocytes were treated with Act1 siRNA, TRAF6 siRNA, SB203580 (p38MAPK inhibitor) or SP600125 (JNK inhibitor) in cardiac myocytes. In conclusion, IL-17 could stimulate the expression of CCL2 in cardiac myocytes via Act1/TRAF6/p38MAPK-dependent AP-1 activation, which may provide a new target for the diagnosis and treatment of VMC.


Assuntos
Quimiocina CCL2/genética , Regulação da Expressão Gênica , Interleucina-17/metabolismo , Miócitos Cardíacos/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Animais Recém-Nascidos , Quimiocina CCL2/metabolismo , Interleucina-17/farmacologia , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Fosforilação , Transdução de Sinais/efeitos dos fármacos
2.
World J Microbiol Biotechnol ; 35(11): 168, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31654206

RESUMO

DNA methylation has been introduced as a promising biomarker for different diseases. Alterations in macrophage DNA methylation status have been documented during Mycobacterium tuberculosis (Mtb) infection. We conducted this study using a human methylation PCR array kit, which comprised a panel of 22 genes in TLR2 signaling pathway, in order to gain insights into epigenetic interactions between drug-susceptible and -resistant Mtb strains and THP-1-derived macrophages (one of the main host immunity cells during TB infection). We also evaluated the expression of Rv1988 gene in the studied isolates. It was found that the methylation level of all of the studied inflammatory genes, except Irak-2 and Tbk-1, increased in THP-1 macrophages, which were infected by extensively drug-resistant (XDR) Mtb strains, compared with the mock cells (P < 0.05). In susceptible strains, we only found hypomethylation in Irak-2 gene, in addition to a slight increase in the methylation levels of Ubev, Ube2n, and Traf6 genes. The present findings provide new insights into the potential role of resistant and susceptible Mtb strains in promoting aberrant epigenetic modifications in macrophages. Further investigations on the host epigenomes, infected with different Mtb isolates, are needed to elucidate their functions in immunological responses and to introduce new effective tools against Mtb infection.


Assuntos
Metilação de DNA , Epigênese Genética , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose/genética , Tuberculose/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Tuberculose Extensivamente Resistente a Medicamentos , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Macrófagos/microbiologia , Metiltransferases/genética , Mycobacterium tuberculosis/patogenicidade , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Células THP-1 , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 2 Toll-Like/genética , Tuberculose/microbiologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
3.
Life Sci ; 235: 116831, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31487530

RESUMO

AIMS: TRAF6 is an intracellular signal adapter molecule plays a significant role in tumor development. However, the specific mechanism causes and promotes of colorectal cancer keep largely unknown. Therefore, we sought to investigate the roles and the molecular mechanisms of TRAF6 in regulation colorectal cancer. MATERIAL AND METHODS: The immunohistochemistry analyzed the expression of TRAF6 in colorectal cancer samples and analyzed the effects of expression of TRAF6 on the prognosis in colorectal cancer. The roles of TRAF6 in regulating colorectal cancer cell proliferation, colony formation, cell migration, cell wound healing and cell invasion were evaluated in vitro. Animal studies were performed to investigate the effects of TRAF6 on tumor growth. mRNA abundance of key genes was analyzed via qPCR. Protein level of TRAF6 and NF-κB/AP-1 signaling pathways was examined by Western blot. Luciferase reporter and Immunofluorescence assays were used to identify the activities NF-κB/AP-1 signaling pathways. KEY FINDINGS: TRAF6 high expression in colorectal cancer tissues. And colorectal cancer patients with high expression of TRAF6 had a poor survival rate. TRAF6 knockdown can inhibit proliferation, migration, and invasion of colorectal cancer cells in vitro and in vivo experiments. TRAF6 activates the TRAF6-NF-κB/AP-1 signaling pathway by entering the nucleus, causing biobehavioral changes in colorectal cancer cells. SIGNIFICANCE: TRAF6 plays a vital role in the progression of colorectal cancer. What's more, research elucidating the biological mechanisms of TRAF6 can treated as potential therapeutic target for colorectal cancer.


Assuntos
Neoplasias Colorretais/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Neoplasias Colorretais/fisiopatologia , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica/fisiopatologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/fisiologia , Ensaio Tumoral de Célula-Tronco , Cicatrização/fisiologia
4.
PLoS Pathog ; 15(8): e1008002, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31404116

RESUMO

The galectin 3 binding protein (LGALS3BP, also known as 90K) is a ubiquitous multifunctional secreted glycoprotein originally identified in cancer progression. It remains unclear how 90K functions in innate immunity during viral infections. In this study, we found that viral infections resulted in elevated levels of 90K. Further studies demonstrated that 90K expression suppressed virus replication by inducing IFN and pro-inflammatory cytokine production. Upon investigating the mechanisms behind this event, we found that 90K functions as a scaffold/adaptor protein to interact with TRAF6, TRAF3, TAK1 and TBK1. Furthermore, 90K enhanced TRAF6 and TRAF3 ubiquitination and served as a specific ubiquitination substrate of TRAF6, leading to transcription factor NF-κB, IRF3 and IRF7 translocation from the cytoplasm to the nucleus. Conclusions: 90K is a virus-induced protein capable of binding with the TRAF6 and TRAF3 complex, leading to IFN and pro-inflammatory production.


Assuntos
Antígenos de Neoplasias/fisiologia , Biomarcadores Tumorais/fisiologia , Glicoproteínas/fisiologia , Fator 3 Associado a Receptor de TNF/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Viroses/imunologia , Replicação Viral , Vírus/imunologia , Animais , Células Cultivadas , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Viroses/metabolismo , Viroses/virologia
5.
Nucleic Acids Res ; 47(14): 7564-7579, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31216032

RESUMO

The multifunctional human DNA2 (hDNA2) nuclease/helicase is required to process DNA ends for homology-directed recombination repair (HDR) and to counteract replication stress. To participate in these processes, hDNA2 must localize to the nucleus and be recruited to the replication or repair sites. However, because hDNA2 lacks the nuclear localization signal that is found in its yeast homolog, it is unclear how its migration into the nucleus is regulated during replication or in response to DNA damage. Here, we report that the E3 ligase TRAF6 binds to and mediates the K63-linked polyubiquitination of hDNA2, increasing the stability of hDNA2 and promoting its nuclear localization. Inhibiting TRAF6-mediated polyubiquitination abolishes the nuclear localization of hDNA2, consequently impairing DNA end resection and HDR. Thus, the current study reveals a mechanism for the regulation of hDNA2 localization and establishes that TRAF6-mediated hDNA2 ubiquitination activates DNA repair pathways to maintain nuclear genome integrity.


Assuntos
Núcleo Celular/metabolismo , DNA Helicases/metabolismo , Genoma Humano/genética , Instabilidade Genômica , Poliubiquitina/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Helicases/genética , Reparo do DNA , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Interferência de RNA , Fator 6 Associado a Receptor de TNF/genética , Ubiquitinação
6.
Artigo em Chinês | MEDLINE | ID: mdl-31177708

RESUMO

Objective: To investigate the effects of silica dust on the expression of Myeloid differentiation factor 88 (MyD88) mRNA and tumor necrosis factor receptor-associated factor (TRAF6) mRNA of lung macrophages in rats. Methods: Selecting 40 SPF-class Wistar rats with average weight (200±20) g randomly divided into control group and 30 d, 60 d, 120 d experimental groups with 10 rats in each group according to body weight. The experimental groups rats were injected with 1 ml of SiO(2) (100 mg/ml) suspension through the trachea into lung only once, then they were respectively killed after 30, 60, 120 days. The control group rats were injected with 1 ml of saline into lung, and killed after 120 days. The lungs of the rats were taken for pathological observation. Lung macrophages were extracted and counted, and their activity was detected by MTT. RT-qPCR was used to assess the relative contents of MyD88 mRNA and TRAF6 mRNA. Results: Silica dust inhalation led to infiltration of lung tissue cells, thickening the alveolar wall and destruction of alveolar structure. The longer the exposure to dust, the more obvious the results were. The number of macrophages in all experimental groups and activity in the 30 d, 60 d groups were significantly higher than that in the control group (P<0.05) . Among them, 30 d group had the largest number and the highest activity. Compared with the control group, the expression of MyD88 mRNA and TRAF6 mRNA of lung macrophages in rats increased in the experimental groups (P<0.05) , especially in the 60 d group. Conclusion: Silica dust inhalation can increase the expression of MyD88 and TRAF6 in macrophages, suggesting that silica dust can induce silicosis fibrosis by activating TLR/NF-κB signal pathway.


Assuntos
Fator 88 de Diferenciação Mieloide , Dióxido de Silício , Fator 6 Associado a Receptor de TNF , Animais , Poeira , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Fator 88 de Diferenciação Mieloide/efeitos dos fármacos , Fator 88 de Diferenciação Mieloide/metabolismo , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Dióxido de Silício/toxicidade , Fator 6 Associado a Receptor de TNF/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/metabolismo
7.
Int J Mol Sci ; 20(12)2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216684

RESUMO

Acteoside, an active phenylethanoid glycoside compound isolated from herbs of Cistanche, was chosen for the investigation of anti-osteoporotic effect on postmenopausal osteoporosis by using an ovariectomized (OVX) mice model. The results from in vivo experiments showed that after daily oral administration of acteoside (20, 40, and 80 mg/kg body weight/day) for 12 weeks, bone mineral density and bone biomechanical properties of OVX mice were greatly enhanced, with significant improvement in bone microarchitecture. Furthermore, biochemical parameters of bone resorption markers as well as bone formation index, including tartrate-resistant acid phosphatase, cathepsin K, deoxypyridinoline, alkaline phosphatase, and bone gla-protein, were ameliorated by acteoside treatment, whereas the body, uterus, and vagina wet weights were seemingly not impacted by acteoside administration. Acteoside significantly affected osteoclastogenesis by attenuating nuclear factor kappa B (NF-κB) and stimulating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signal pathways through down-regulated levels of tumor-necrosis factor receptor-associated factor 6 (TRAF6), receptor activator of nuclear factor kappa B ligand (RANKL), RANK, NFKBIA, IκB kinase ß, nuclear factor of activated T-cells c2 (NFAT2), and up-regulated expressions of PI3K, AKT, and c-Fos. Accordingly, the current research validated our hypothesis that acteoside possesses potent anti-osteoporotic properties and may be a promising agent for the prevention of osteoporosis in the future.


Assuntos
Glucosídeos/farmacologia , Osteoporose/etiologia , Osteoporose/metabolismo , Ovariectomia/efeitos adversos , Fenóis/farmacologia , Substâncias Protetoras/farmacologia , Animais , Biomarcadores , Peso Corporal , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/genética , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Modelos Biológicos , Tamanho do Órgão , Osteoporose/tratamento farmacológico , Osteoporose/patologia , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo
8.
Biol Pharm Bull ; 42(5): 744-750, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061316

RESUMO

Increasing evidence supports that the efflux transporters, especially P-glycoprotein (P-gp), have vital roles on drug resistance in epilepsy. Overexpression of P-gp in the brain could reduce the anti-epileptic drugs (AEDs) concentration in the epileptogenic zone, resulting in drug resistance. Studies have demonstrated that recurrent seizures induce the expression of P-gp and status epilepticus (SE) could upregulate the expression of P-gp, resulting in drug resistance. MicroRNAs (miRNAs), as endogenous regulators, represent small regulatory RNA molecules that have been shown to act as negative regulators of gene expression in different biological processes. We investigated the impact of miR-146a-5p on the expression of P-gp in status epilepticus rat model. The expression of miR-146a-5p in rat cortex and hippocampus was measured by quantitative RT-PCR at 2 weeks after induction of SE. Meanwhile, we detected the expression of P-gp in the brain of SE rats using Western blotting and immunohistochemistry. Upregulation of miR-146a-5p and overexpression of P-gp were evident at 2 weeks after SE. Moreover, the expression of P-gp was downregulated by injection of miR-146a mimic into the hippocampus. We also detected the expression of interleukin-1 receptor-associated protein kinases-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) and nuclear factor-kappaB (NF-κB) p65 using Western blotting and immunohistochemistry, which indicated the expression of IRAK1, TRAF6 and NF-κB p-p65/p65 increased in the brain of SE rats, and overexpression of miR-146a-5p could downregulate the expression of IRAK1, TRAF6, NF-κB p-p65/p65 and P-gp. Our study indicated that miR-146a-5p may decrease the expression of P-gp in status epilepticus rats via NF-κB signaling pathway.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Córtex Cerebral/metabolismo , Hipocampo/metabolismo , MicroRNAs , Estado Epiléptico/metabolismo , Animais , Regulação para Baixo , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Cloreto de Lítio , Masculino , Pilocarpina , Ratos Sprague-Dawley , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelA/metabolismo
9.
Int J Mol Sci ; 20(9)2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083375

RESUMO

Geranylgeraniol (GGOH), a natural isoprenoid found in plants, has anti-inflammatory effects via inhibiting the activation of nuclear factor-kappa B (NFκB). However, its detailed mechanism has not yet been elucidated. Recent studies have revealed that isoprenoids can modulate signaling molecules in innate immune responses. We found that GGOH decreased the expression of lipopolysaccharide (LPS)-induced inflammatory genes in human macrophage-like THP-1 cells. Furthermore, we observed that the suppression of NFκB signaling proteins, in particular interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), occurred in GGOH-treated cells prior to LPS stimulation, suggesting an immunomodulatory effect. These results indicate that GGOH may modulate and help prevent excessive NFκB activation that can lead to numerous diseases.


Assuntos
Diterpenos/farmacologia , Inflamação/patologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , NF-kappa B/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Linhagem Celular , Humanos , Inflamação/genética , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células THP-1
10.
Biosci Biotechnol Biochem ; 83(6): 1011-1026, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31074699

RESUMO

TRAF6 is highly expressed in many tumors and plays an important role in the immune system. The aim of this study is to confirm anti-tumor activities of all naturally occurring Cinchona alkaloids that have been screened using computational docking program, and to validate the accuracy and specificity of the RING domain of TRAF6 as a potential anti-tumor target, and to explore their effect on the immune system. Results reported herein would demonstrate that Cinchona alkaloids could induce apoptosis in HeLa cells, inhibit the ubiquitination and phosphorylation of both AKT and TAK1, and up-regulate the ratio of Bax/Bcl-2. In addition, these compounds could induce apoptosis in vivo, and increase the secretion of TNF-α, IFN-γ, and IgG, while not significantly impacting the ratio of CD4+T/CD8+T. These investigations suggest that the RING domain of TRAF6 could serve as a de novo biological target for therapeutic treatment in cancers.


Assuntos
Apoptose/efeitos dos fármacos , Alcaloides de Cinchona/metabolismo , Alcaloides de Cinchona/farmacologia , Domínios Proteicos , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Ligação Competitiva , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática , Células HeLa , Humanos , Imunoglobulina G/sangue , Marcação In Situ das Extremidades Cortadas , Interferon gama/sangue , Contagem de Linfócitos , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfócitos T/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/química , Fator de Necrose Tumoral alfa/sangue , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/metabolismo
11.
Bioengineered ; 10(1): 197-206, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31117883

RESUMO

Dental pulp inflammation is a common bacterially driven inflammation characterized by the local accumulation of inflammatory mediators in human dental pulp. DNA methylation is a crucial epigenetic modification that that plays a fundamental role in gene transcription, and its role in inflammation-related diseases has recently attracted attention. However, its role in dental pulp inflammation is poorly understood. This study is aimed to elucidate the role of DNA methylation in lipopolysaccharide (LPS)-induced inflammatory reaction in human dental pulp cells (hDPCs). hDPCs were pretreated with DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and a cytokine antibody array was used to detect LPS-induced cytokine expression. The results indicated that 5-Aza-CdR significantly increased the expression of several pro-inflammatory cytokines in LPS-treated cells, including IL-6, IL-8, GM-CSF, MCP-2 and RANTES. The increased expression levels of IL-6 and IL-8 were further verified by qRT-PCR and ELISA. Furthermore, pretreatment with 5-Aza-CdR resulted in upregulation of p-IKKα/ß, p-IκBα, p-p65 and p-ERK in the NK-κB and MAPK pathways. In addition, the 5mC level of the TRAF6 promoter was significantly decreased following 5-Aza-CdR pretreatment in the LPS-stimulated hDPCs. The findings indicate that 5-Aza-CdR significantly enhances the expression of proinflammatory cytokines and activates the NF-κB and MAPK signaling pathways by eliciting a decline in the 5mc level in the TRAF6 promoter in hDPCs, suggesting that DNA methylation may play an important role in dental pulp inflammation. This study highlights the important role of DNA methylation in the immunity defense of dental pulp infection.


Assuntos
Decitabina/farmacologia , Polpa Dentária/citologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Fator 6 Associado a Receptor de TNF/metabolismo , Células Cultivadas , Citocinas/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator 6 Associado a Receptor de TNF/genética
12.
Cell Mol Biol Lett ; 24: 29, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31123462

RESUMO

Background: In its RING domain, tumor necrosis factor receptor-associated factor 6 (TRAF6) has ubiquitin E3 ligase activity that facilitates the formation of lysine 63-linked polyubiquitin chains. This activity is required to activate nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and plays an important role in the IκB kinase (IKK) complex. Methods: An in vitro ubiquitination assay was used to establish whether c-Cbl could promote TRAF6 ubiquitination. We assessed direct binding and performed fine mapping between c-Cbl and TRAF6 based on the results of an immunoprecipitation assay with cultured 293 T cells. The luciferase reporter assay was applied to establish if c-Cbl-mediated ubiquitination affected NF-κB activation after stimulus from various TRAF-mediated signals: tumor necrosis factor-α (TNF-α), receptor activator of NF-κB ligand (RANKL), and interleukin-1ß (IL-1ß). An in vivo ubiquitination assay was performed using endogenous immunoprecipitation of TRAF6 in bone marrow macrophages (BMMs) and osteoclasts. Results: Here, we report on a form of TRAF6 ubiquitination that is mediated by c-Cbl, leading to the formation of lysine 48-linked polyubiquitin chains. The NF-κB activity induced by RANKL and IL-1ß treatment is inhibited when c-Cbl is overexpressed, while the NF-κB activity induced by TNFα treatment is not. c-Cbl inhibits NF-κB activity mediated by TRAF6, but not by TRAF2. These findings show that c-Cbl ubiquitin ligase activity is essential for TRAF6 ubiquitination and negative regulation of NF-κB activity. Fine mapping revealed that the proline-rich domain of c-Cbl is critical for interaction with TRAF6. Stimulation with RANKL or interferon-γ (IFN-γ) caused c-Cbl to bind to polyubiquitinated TRAF6. Conclusions: These findings indicate that the interaction of TRAF6 with c-Cbl causes lysine 48-linked polyubiquitination for both negative feedback regulation and signaling cross-talk between RANKL and IFN-γ.


Assuntos
Lisina/metabolismo , NF-kappa B/metabolismo , Poliubiquitina/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Células HEK293 , Humanos , Interferon gama/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-cbl/química , Ligante RANK/farmacologia , Domínios RING Finger , Fator 6 Associado a Receptor de TNF/química , Ubiquitinação/efeitos dos fármacos
13.
Int Immunopharmacol ; 73: 23-40, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31078923

RESUMO

The incidence and mortality of type 2 diabetes mellitus (T2DM) rank among the top ten worldwide. Emerging studies indicate pathological roles for the immune system in inflammation, insulin resistance and islet ß-cell damage in subjects with T2DM. Methionine enkephalin (MENK) is present in endocrine cells of the pancreas and has been suggested to be an important mediator between the immune and neuroendocrine systems. Therefore, it may play a role in modulating insulin secretion from islet cells. Since little is known about the effect of MENK on T2DM, therefore it was the aim of this study to characterize the role and possible mechanism of action of MENK on plasma glucose and serum insulin levels in T2DM rats and INS-1 cells in vivo and in vitro. MENK significantly decreased the plasma glucose level and increased the serum insulin concentration in T2DM rats. It also increased the serum levels of the cytokines IL-5 and IL-10, while decreased TNF-α and IL-2 levels. We further confirmed that MENK regulated glucose metabolism by upregulating opioid receptor expression and modulating the IL-33/ST2 and MyD88-TRAF6-NF-κB p65 signaling pathways. Based on these results, an intraperitoneal injection of MENK represents a potentially new approach for T2DM.


Assuntos
Citocinas/imunologia , Diabetes Mellitus Tipo 2/imunologia , Encefalina Metionina/farmacologia , Receptores de Interleucina-1/imunologia , Animais , Glicemia/análise , Linhagem Celular Tumoral , Citocinas/sangue , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Masculino , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/imunologia , Pâncreas/metabolismo , Ratos Sprague-Dawley , Receptores Opioides/genética , Receptores Opioides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo
14.
Nat Commun ; 10(1): 1801, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996248

RESUMO

Macrophage-orchestrated, low-grade chronic inflammation plays a pivotal role in obesity and atherogenesis. However, the underlying regulatory mechanisms remain incompletely understood. Here, we identify major vault protein (MVP), the main component of unique cellular ribonucleoprotein particles, as a suppressor for NF-κB signaling in macrophages. Both global and myeloid-specific MVP gene knockout aggravates high-fat diet induced obesity, insulin resistance, hepatic steatosis and atherosclerosis in mice. The exacerbated metabolic disorders caused by MVP deficiency are accompanied with increased macrophage infiltration and heightened inflammatory responses in the microenvironments. In vitro studies reveal that MVP interacts with TRAF6 preventing its recruitment to IRAK1 and subsequent oligomerization and ubiquitination. Overexpression of MVP and its α-helical domain inhibits the activity of TRAF6 and suppresses macrophage inflammation. Our results demonstrate that macrophage MVP constitutes a key constraint of NF-κB signaling thereby suppressing metabolic diseases.


Assuntos
Aterosclerose/imunologia , Fígado Gorduroso/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Obesidade/imunologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Tecido Adiposo/patologia , Animais , Aterosclerose/etiologia , Aterosclerose/metabolismo , Biópsia , Células da Medula Óssea , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Quinase I-kappa B/metabolismo , Inflamação/etiologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , NF-kappa B/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Cultura Primária de Células , Transdução de Sinais/imunologia , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/imunologia
15.
EMBO J ; 38(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30979779

RESUMO

TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquitinated caspase-8 to TRAF6 for further polyubiquitination of caspase-8. Mutations of key TIM residues in TP53INP2 abrogate its interaction with TRAF6 and caspase-8, and subsequently reduce levels of death receptor-induced apoptosis. A screen of cancer cell lines showed that those with higher protein levels of TP53INP2 are more prone to TRAIL-induced apoptosis, making TP53INP2 a potential predictive marker of cancer cell responsiveness to TRAIL treatment. These findings uncover a novel mechanism for the regulation of caspase-8 ubiquitination and reveal TP53INP2 as an important regulator of the death receptor pathway.


Assuntos
Autofagia/genética , Proteínas Nucleares/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Caspase 8/metabolismo , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Receptores de Morte Celular/genética , Receptores de Morte Celular/metabolismo , Transdução de Sinais/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética
16.
Biomed Pharmacother ; 112: 108709, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30970514

RESUMO

OBJECTIVE: Poria cocos polysaccharide (PCP) is the major active ingredients of P. cocos and possesses various pharmacological effects, including anti-oxidative and anti-apoptosis effects and activity against cancer. This study investigated the immunomodulatory mechanism by which PCP acts on RAW 264.7 macrophages and LLC tumors in mice. METHODS: The concentrations of nitric oxide, and Th1, Th2, and Th17 cytokines were examined by Griess reaction and using a bead-based cytokine assessment kit. qRT-PCR and western blotting were used to investigate relevant signaling molecule expression. RESULTS: Levels of nitric oxide, IL-2, IL-6, IL-17 A, TNF, and IFN-γ were increased by PCP while levels of IL-4 and IL-10 were unaffected. The addition of TAK-242 (TLR4 inhibitor) or assessment in C57BL/10ScNJ (TLR4-deficient) mice markedly reduced this effect. In C57BL/10 J (TLR4+/+wild-type) mice, the indices of organ immune activity were all elevated, and oral PCP delivery resulted in a significant reduction in tumor volume over a 25 day period. Relative to controls, TLR4, MyD88, TRAF-6, p-NF-κB and p-c-JUN expression significantly increased, while TRAM expression did not change. Nevertheless, there was no PCP-dependent activation of MyD88, TRAF-6, TRAM, p-NF-κB or p-c-JUN in TLR4-deficient mice. CONCLUSION: These results support the concept that PCP may exhibit immunomodulatory activity through TLR4/TRAF6/NF-κB signaling both in vitro and in vivo.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , NF-kappa B/metabolismo , Polissacarídeos/uso terapêutico , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/metabolismo , Wolfiporia/química , Animais , Antineoplásicos/isolamento & purificação , Carcinoma Pulmonar de Lewis/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Fatores Imunológicos/isolamento & purificação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Polissacarídeos/isolamento & purificação , Células RAW 264.7 , Transdução de Sinais , Baço/efeitos dos fármacos , Baço/imunologia , Timo/efeitos dos fármacos , Timo/imunologia
17.
Cancer Sci ; 110(6): 1909-1920, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30945383

RESUMO

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been found to be involved in carcinogenesis in multiple cancers. However, the precise role of TRAF6 in cancer has not been extensively investigated and remains largely unknown. In this study, we aimed to investigate the biological function of TRAF6 and its underlying molecular mechanisms in cancer. A positive correlation between poor tumor differentiation and TRAF6 expression status was observed in both oral cancer and breast cancer. Overexpression of TRAF6 promoted proliferation, migration, and G0 /G1 to S phase transition in tumor cells. Tumor necrosis factor receptor-associated factor 6-mediated AKT ubiquitination and subsequent phosphorylation played an essential role in the control of tumor cell malignant behavior. In vivo treatment with TRAF6, but not the E3 ligase deficient TRAF6 mutant, facilitated tumor growth. Our findings indicate that TRAF6 contributes to malignant behavior of human cancers through promoting AKT ubiquitination and phosphorylation. Therefore, TRAF6 could serve as a therapeutic target in cancers.


Assuntos
Neoplasias da Mama/genética , Neoplasias Bucais/genética , Proteínas Proto-Oncogênicas c-akt/genética , Fator 6 Associado a Receptor de TNF/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Neoplasias Bucais/terapia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Terapêutica com RNAi/métodos , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
18.
Dig Dis Sci ; 64(7): 1844-1856, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30949903

RESUMO

OBJECTIVE: The role of TL1A in the intestinal mucosa barrier in inflammatory bowel disease (IBD) is still unclear. This study was aimed to investigate the expression levels of tight junction protein (TJ), myosin light chain kinase (MLCK), MyD88 and tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6) and how TL1A influences the intestinal barrier in IBD. METHODS: The mouse models of IBD were built using FMS-TL1A-GFP-transgenic mice and wild-type mice. The morphological and histopathological changes, bacterial translocation, permeability of colonic mucosa, and LPS level were assessed. Caco-2 cells were used to further investigate the association between TL1A and TNF-α and LPS. The protein level and mRNA changes of TJ proteins including ZO-1, occluding, JAMA, claudin-1, claudin-2, and claudin-3 were investigated using Western blot and real-time PCR. Protein changes of MLCK, MyD88 and TNF receptor-associated factor-6 (TRAF6), and TNF-α mRNA in the mouse colon were further assessed. RESULTS: The IBD models were successfully built. Cooper HS score and histopathological score of the colon were higher in DSS/WT group than in control/WT group (P < 0.05), higher in DSS/Tg group than in control/Tg group (P < 0.05), and higher in DSS/Tg group than in DSS/WT group. PAS, colonic permeability of the colon, and FITC-D examination showed the similar results and trends. Compared with control/WT group, the levels of TL1A and claudin-2 were higher and the levels of ZO-1, occludin, JAMA, claudin-1, and claudin-3 were lower in DSS/WT group (P < 0.05). Compared with control/Tg group, the levels of TL1A and claudin-2 were higher and the levels of ZO-1, occludin, JAMA, claudin-1, and claudin-3 were lower in DSS/Tg group. Compared with Caco-2 + TNF-α group, the expression level of occludin and claudin-1 in Caco-2 + LV-TNFSF15 + TNF-α group was significantly lower (P < 0.05); p-MLC level was significantly higher. Compared with Caco-2 + LPS group, the expression level of occludin and claudin-1 significantly decreased in Caco-2 + LV-TNFSF15 + LPS group; MyD88 and TRAF6 expression level significantly increased. CONCLUSION: The results suggested that TL1A could impair intestinal epithelial barrier in the mouse model of IBD and might regulate TJ expression via MLCK/p-MLC pathway and LPS-mediated MyD88/TRAF6 pathway.


Assuntos
Translocação Bacteriana , Colite/metabolismo , Colo/metabolismo , Sulfato de Dextrana , Microbioma Gastrointestinal , Mucosa Intestinal/metabolismo , Junções Íntimas/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Animais , Células CACO-2 , Colite/induzido quimicamente , Colite/microbiologia , Colite/patologia , Colo/microbiologia , Colo/ultraestrutura , Modelos Animais de Doenças , Feminino , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/ultraestrutura , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/metabolismo , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Permeabilidade , Fosforilação , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/microbiologia , Junções Íntimas/ultraestrutura , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética
19.
Arch Oral Biol ; 101: 100-107, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30913450

RESUMO

OBJECTIVE: To explore the gene mutation in an incontinentia pigmenti (IP) patient with syndromic tooth agenesis. METHODS: Long-range polymerase chain reaction (PCR) and Sanger sequencing were used to detect inhibitor of nuclear factor kappa-B kinase subunit gamma (IKBKG) mutation in the IP patient. We used the nuclear factor kappa B (NF-κB) reporter gene to assess activation of NF-κB, after transfecting an empty vector, wild-type, or mutant NF-κB essential modulator (NEMO) plasmid into IKBKG-deficient HEK293T cells, respectively. Furthermore, we performed immunoprecipitation and immunoblotting to describe the polyubiquitination of NEMO. Lastly, we detected the interactions between mutant NEMO and I kappa B kinase alpha (IKKα), I kappa B kinase beta (IKKß), TNF receptor associated factor 6 (TRAF6), HOIL-1-interacting protein (HOIP), hemo-oxidized iron regulatory protein 2 ligase 1 (HOIL-1), and SHANK-associated RH domain interactor (SHARPIN). RESULTS: A de novo nonsense mutation in IKBKG (c.924C > G; p.Tyr308*) was observed. The Tyr308* mutation inhibited activation of the NF-κB pathway by reducing K63-linked polyubiquitination and linear polyubiquitination. The mutant NEMO was not able to interact with TRAF6, HOIL-1, or SHARPIN. CONCLUSIONS: We identified a novel nonsense IKBKG mutation (c.924C > G; p.Tyr308*) in an IP patient with syndromic tooth agenesis. This research enriches the mutation spectrum of the IKBKG gene.


Assuntos
Anodontia/genética , Quinase I-kappa B/genética , Incontinência Pigmentar/genética , Mutação , Genes Reporter , Células HEK293 , Humanos , NF-kappa B/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo
20.
J Biol Chem ; 294(17): 6888-6898, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30872404

RESUMO

Virus-inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) is a radical SAM enzyme that plays a multifaceted role in the cellular antiviral response. Viperin has recently been shown to catalyze the SAM-dependent formation of 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), which inhibits some viral RNA polymerases. Viperin is also implicated in regulating Lys-63-linked polyubiquitination of interleukin-1 receptor-associated kinase-1 (IRAK1) by the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) as part of the Toll-like receptor-7 and -9 (TLR7/9) innate immune signaling pathways. In these pathways, the poly-ubiquitination of IRAK1 by TRAF6 is necessary to activate IRAK1, which then phosphorylates downstream targets and ultimately leads to the production of type I interferons. That viperin is a component of these pathways suggested that its enzymatic activity might be regulated by interactions with partner proteins. To test this idea, we have reconstituted the interactions between viperin, IRAK1, and TRAF6 by transiently expressing these enzymes in HEK 293T cells. We show that IRAK1 and TRAF6 increase viperin activity ∼10-fold to efficiently catalyze the radical-mediated dehydration of CTP to ddhCTP. Furthermore, we found that TRAF6-mediated ubiquitination of IRAK1 requires the association of viperin with both IRAK1 and TRAF6. Ubiquitination appears to depend on structural changes in viperin induced by SAM binding, but, significantly, does not require catalytically active viperin. We conclude that the synergistic activation of viperin and IRAK1 provides a mechanism that couples innate immune signaling with the production of the antiviral nucleotide ddhCTP.


Assuntos
Antivirais/metabolismo , Citidina Trifosfato/biossíntese , Imunidade Inata , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Adenosina/administração & dosagem , Adenosina/análogos & derivados , Células HEK293 , Meia-Vida , Humanos , Fosforilação , Ligação Proteica , S-Adenosilmetionina/metabolismo , Ubiquitinação
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