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1.
J Fish Biol ; 96(1): 185-193, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31721203

RESUMO

In this study, we cloned the complementary (c)DNA sequences of tumour necrosis factor receptor (TNFR)-associated factor 3 (traf3) in Nile tilapia, Oreochromis niloticus. The expression patterns of the traf3 gene were investigated and preliminary functional analyses were performed. In healthy fish, traf3 transcript was broadly expressed in all examined tissues, with the highest expression level in the blood and the lowest in the liver. The traf3 gene reached its highest expression at 8 days post-fertilisation (dpf) during embryonic development. Moreover, we found that expression of traf3 was clearly altered following stimulation with Streptococcus agalactiae in vivo and that traf3 could be induced by lipopolysaccharides (LPS), Poly I: C and S. agalactiae WC1535 in Nile tilapia macrophages. Overexpression in 293T cells showed that Traf3 protein was mainly distributed in the cytoplasm and could significantly increase nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Taken together, these results implied that traf3 could play important roles in the immune response to pathogen invasion.


Assuntos
Ciclídeos/imunologia , NF-kappa B/metabolismo , Fator 3 Associado a Receptor de TNF , Animais , Técnicas de Cultura de Células , Ciclídeos/genética , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Expressão Gênica , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , NF-kappa B/imunologia , Streptococcus agalactiae/imunologia , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/imunologia , Fator 3 Associado a Receptor de TNF/metabolismo
2.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801245

RESUMO

BACKGROUND: Ovaries are sensitive to chemotherapy, which may lead to early depletion of primordial follicle reserve. One strategy for gonadal function preservation is temporary ovarian suppression with Gonadotropin Releasing Hormone agonists (GnRHa) during chemotherapy. To date, GnRHa protective mechanism of action remains not fully elucidated. METHODS: We collected 260 immature cumulus cell-oocyte complexes (COC) from 111 women < 38 years old, with a normal ovarian reserve. The COC were randomly assigned to the following groups: a) control; culture with the addition of b) GnRHa; c) cyclophosphamide; d) cyclophosphamide plus GnRHa. After in vitro treatments, RNA and proteins were extracted from oocytes and cumulus cells (CC), separately. Potential effects of drugs were evaluated on GnRH receptors, apoptosis pathways, ceramide pathway, and glutathione synthesis by quantitative PCR and, whenever possible, by Western blot. RESULTS: Cyclophosphamide triggered activation of the extrinsic pathway of apoptosis mediated by BAX in CC. The co-administration of GnRHa inhibited the apoptosis pathway in CC. According to our model, the GnRHa does not directly act on oocytes, which do not express GnRH receptors. Moreover, glutathione synthesis was decreased after GnRHa treatment both in CC and oocytes. CONCLUSION: Our data suggest that the protective mechanisms induced by GnRHa is mediated by an anti-apoptotic effect on CC.


Assuntos
Apoptose/efeitos dos fármacos , Células do Cúmulo/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Receptores LHRH/genética , Proteína X Associada a bcl-2/genética , Adulto , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Ceramidas/metabolismo , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Ciclofosfamida/farmacologia , Feminino , Regulação da Expressão Gênica , Glutationa/metabolismo , Humanos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Reserva Ovariana/genética , Receptores LHRH/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Receptor fas/genética , Receptor fas/metabolismo
3.
Biochimie ; 167: 217-227, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31654668

RESUMO

RANKL induces osteoclastogenesis via JNK1 signal that exerts an anti-apoptotic effect during osteoclastogenesis. However, the classic downstream c-Jun/AP-1 pathway is not included in anti-apoptosis of JNK1. Thus, the detail mechanism underlying JNK1-resisted apoptosis remains unknown during RANKL-induced osteoclastogenesis. RANKL-induced autophagy results in the degradation of the osteoclastogenesis-inhibitor TRAF3, and TRAF3 is thought as a regulator of apoptosis. Given the key effect of JNK1 in mediating autophagy, our study aims to investigate the significance of TRAF3 in bridging JNK1-mediated autophagy and apoptotic resistance during osteoclastogenesis. In this study, by using Bone Marrow-derived macrophages (BMMs) as osteoclast precursors (OCPs), we found that RANKL-induced TRAF3 degradation was significantly suppressed by JNK inhibitor (SP600125), which was restored by overexpression of Beclin1 (key autophagic protein). Nevertheless, TRAF3 silencing partially reversed the reduced osteoclastogenesis under SP600125 intervention. Besides, OCP apoptosis was positively regulated by TRAF3 overexpression, regardless of the application of autophagy inhibitor or SP600125. Remarkably, the enhanced apoptosis caused by the pharmacological inhibition of Beclin1 was reversed by TRAF3 silencing. Together, these results suggest that JNK1-mediated autophagy could promote RANKL-induced osteoclastogenesis via enhancing TRAF3 degradation. Importantly, JNK1 could prevent OCP apoptosis through autophagy-TRAF3 signaling, which provides more potential targets for clinical treatment of pathological bone loss.


Assuntos
Macrófagos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Fator 3 Associado a Receptor de TNF/fisiologia , Animais , Apoptose , Autofagia , Proteína Beclina-1/metabolismo , Células Cultivadas , Macrófagos/citologia , Camundongos Endogâmicos C57BL , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Osteoclastos/citologia , Ligante RANK/metabolismo , Transdução de Sinais
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.
J Biol Chem ; 294(39): 14231-14240, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31375559

RESUMO

Innate immune detection of viral nucleic acids during viral infection activates a signaling cascade that induces type I and type III IFNs as well as other cytokines, to generate an antiviral response. This signaling is initiated by pattern recognition receptors, such as the RNA helicase retinoic acid-inducible gene I (RIG-I), that sense viral RNA. These sensors then interact with the adaptor protein mitochondrial antiviral signaling protein (MAVS), which recruits additional signaling proteins, including TNF receptor-associated factor 3 (TRAF3) and TANK-binding kinase 1 (TBK1), to form a signaling complex that activates IFN regulatory factor 3 (IRF3) for transcriptional induction of type I IFNs. Here, using several immunological and biochemical approaches in multiple human cell types, we show that the GTPase-trafficking protein RAB1B up-regulates RIG-I pathway signaling and thereby promotes IFN-ß induction and the antiviral response. We observed that RAB1B overexpression increases RIG-I-mediated signaling to IFN-ß and that RAB1B deletion reduces signaling of this pathway. Additionally, loss of RAB1B dampened the antiviral response, indicated by enhanced Zika virus infection of cells depleted of RAB1B. Importantly, we identified the mechanism of RAB1B action in the antiviral response, finding that it forms a protein complex with TRAF3 to facilitate the interaction of TRAF3 with mitochondrial antiviral signaling protein. We conclude that RAB1B regulates TRAF3 and promotes the formation of innate immune signaling complexes in response to nucleic acid sensing during RNA virus infection.


Assuntos
Imunidade Inata , Fator 3 Associado a Receptor de TNF/metabolismo , Infecção por Zika virus/imunologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Animais , Chlorocebus aethiops , Proteína DEAD-box 58/metabolismo , Células HEK293 , Humanos , Interferon beta/metabolismo , Ligação Proteica , Transdução de Sinais , Células Vero
6.
EMBO J ; 38(18): e102075, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31390091

RESUMO

RIG-I-MAVS antiviral signaling represents an important pathway to stimulate interferon production and confer innate immunity to the host. Upon binding to viral RNA and Riplet-mediated polyubiquitination, RIG-I promotes prion-like aggregation and activation of MAVS. MAVS subsequently induces interferon production by activating two signaling pathways mediated by TBK1-IRF3 and IKK-NF-κB respectively. However, the mechanism underlying the activation of MAVS downstream pathways remains elusive. Here, we demonstrated that activation of TBK1-IRF3 by MAVS-Region III depends on its multimerization state and identified TRAF3IP3 as a critical regulator for the downstream signaling. In response to virus infection, TRAF3IP3 is accumulated on mitochondria and thereby facilitates the recruitment of TRAF3 to MAVS for TBK1-IRF3 activation. Traf3ip3-deficient mice demonstrated a severely compromised potential to induce interferon production and were vulnerable to RNA virus infection. Our findings uncover that TRAF3IP3 is an important regulator for RIG-I-MAVS signaling, which bridges MAVS and TRAF3 for an effective antiviral innate immune response.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Viroses/imunologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Células HEK293 , Células HeLa , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Mitocôndrias/metabolismo , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Viroses/genética
7.
Mol Med Rep ; 20(3): 2843-2850, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322228

RESUMO

Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation and joint inflammation. A previous study showed that microRNA (miR)­671­3p is involved in the development of OA, however, its function and molecular target in chondrocytes during the pathogenesis of OA remain to be fully elucidated. In the present study, miR­671­3p was significantly downregulated in knee OA cartilage tissues compared with normal cartilage tissues. The expression levels of pro­inflammatory cytokines, including interleukin (IL)­1ß, IL­6, IL­8 and tumor necrosis factor (TNF)­α, in the knee OA cartilage tissues were significantly higher than those in the normal cartilage tissues. Through gain­of­function and loss­of­function experiments, miR­671­3p was shown to significantly affect matrix synthesis gene expression, cell proliferation, apoptosis and inflammation in chondrocytes from patients with OA. Subsequent bioinformatics analysis identified potential target sites of the miR­671­3p located in the 3'untranslated region of TNF receptor­associated factor (TRAF3). The results of a dual­luciferase reporter assay showed that TRAF3 is a target gene of miR­671­3p. Western blot analysis demonstrated that miR­671­3p inhibited the gene expression of TRAF3. Furthermore, the restoration of TRAF3 markedly abrogated the effect of miR­671­3p. Taken together, the present study suggests that miR­671­3p may be important in the pathogenesis of OA through targeting TRAF3 and regulating chondrocyte apoptosis and inflammation, which may be a potential molecular target for OA treatment.


Assuntos
Condrócitos/patologia , MicroRNAs/genética , Osteoartrite do Joelho/genética , Fator 3 Associado a Receptor de TNF/genética , Idoso , Apoptose , Proliferação de Células , Condrócitos/citologia , Condrócitos/metabolismo , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/patologia , Regulação para Cima
8.
Biol Res ; 52(1): 38, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31349873

RESUMO

BACKGROUND: Breast cancer is the second common malignant cancer among females worldwide. Accumulating studies have indicated that deregulation of miRNA expression in breast cancer will contribute to tumorigenesis and form different cancer subtypes. However, the reported studies on miR-29b-3p-regulated breast cancer are limited so far. Herein, we investigated the role and mechanism of miR-29b-3p in the triple negative breast cancer cell line MDA-MB-231. METHODS: The relative miR-29b-3p expression in different breast cancer cell lines were determined by qRT-PCR. CCK8 and colony formation assay were used to determine the influence of miR-29b-3p on cell proliferation. Migration assay and invasion assay were performed for cell migration and invasion respectively. To study the cell integrity immunofluorescence was performed. TUNEL assay, flow cytometry assay, hoechst staining and western blot were conducted to determine the influence of miR-29b-3p inhibitor on cell apoptosis. TRAF3 was found to be the target gene of miR-29b-3p using bioinformatics predictions. Dual-luciferase assay was performed to determine the relative luciferase activity in NC, miR-29b-3p mimic, miR-29b-3p inhibitor with TRAF3 3'-UTR wt or TRAF3 3'-UTR mt reporter plasmids. The proteins expression of NF-κB signaling pathway in MDA-MB-231 after transfection with NC, miR-29b-3p mimic, miR-29b-3p inhibitor were determined by western blot. RESULTS: The miR-29b-3p expression was significantly increased in MDA-MB-231 compare with MCF-10A. miR-29b-3p inhibitor reduced the cell viability of MDA-MB-231 and inhibited cell migration and invasion. Cell cytoskeleton integrity destroyed after miR-29b-3p inhibitor treatment. Furthermore, we identified the mechanism and found miR-29b-3p targets the TRAF3 and activates NF-κB signaling pathway. CONCLUSIONS: From the above studies, our results indicated that miR-29b-3p acts as a promoter for the development of MDA-MB-231.


Assuntos
Apoptose/efeitos dos fármacos , Regulação para Baixo/genética , MicroRNAs/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Luciferases/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Neoplasias de Mama Triplo Negativas/patologia
9.
Nat Commun ; 10(1): 2795, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243287

RESUMO

Inflammaging induces osteoporosis by promoting bone destruction and inhibiting bone formation. TRAF3 limits bone destruction by inhibiting RANKL-induced NF-κB signaling in osteoclast precursors. However, the role of TRAF3 in mesenchymal progenitor cells (MPCs) is unknown. Mice with TRAF3 deleted in MPCs develop early onset osteoporosis due to reduced bone formation and enhanced bone destruction. In young mice TRAF3 prevents ß-catenin degradation in MPCs and maintains osteoblast formation. However, TRAF3 protein levels decrease in murine and human bone samples during aging when TGFß1 is released from resorbing bone. TGFß1 induces degradation of TRAF3 in murine MPCs and inhibits osteoblast formation through GSK-3ß-mediated degradation of ß-catenin. Thus, TRAF3 positively regulates MPC differentiation into osteoblasts. TRAF3 deletion in MPCs activated NF-κB RelA and RelB to promote RANKL expression and enhance bone destruction. We conclude that pharmacologic stabilization of TRAF3 during aging could treat/prevent age-related osteoporosis by inhibiting bone destruction and promoting bone formation.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Envelhecimento , Animais , Diferenciação Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/fisiologia , Osteoporose , Fator 3 Associado a Receptor de TNF/genética
10.
Genome Med ; 11(1): 26, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039804

RESUMO

BACKGROUND: Genome wide association studies have identified > 200 susceptibility loci accounting for much of the heritability of multiple sclerosis (MS). Epstein-Barr virus (EBV), a memory B cell tropic virus, has been identified as necessary but not sufficient for development of MS. The molecular and immunological basis for this has not been established. Infected B cell proliferation is driven by signalling through the EBV produced cell surface protein LMP1, a homologue of the MS risk gene CD40. METHODS: We have investigated transcriptomes of B cells and EBV-infected B cells at Latency III (LCLs) and identified MS risk genes with altered expression on infection and with expression levels associated with the MS risk genotype (LCLeQTLs). The association of LCLeQTL genomic burden with EBV phenotypes in vitro and in vivo was examined. The risk genotype effect on LCL proliferation with CD40 stimulation was assessed. RESULTS: These LCLeQTL MS risk SNP:gene pairs (47 identified) were over-represented in genes dysregulated between B and LCLs (p < 1.53 × 10-4), and as target loci of the EBV transcription factor EBNA2 (p < 3.17 × 10-16). Overall genetic burden of LCLeQTLs was associated with some EBV phenotypes but not others. Stimulation of the CD40 pathway by CD40L reduced LCL proliferation (p < 0.001), dependent on CD40 and TRAF3 MS risk genotypes. Both CD40 and TRAF3 risk SNPs are in binding sites for the EBV transcription factor EBNA2, with expression of each correlated with EBNA2 expression dependent on genotype. CONCLUSIONS: These data indicate targeting EBV may be of therapeutic benefit in MS.


Assuntos
Linfócitos B/metabolismo , Antígenos CD4/genética , Herpesvirus Humano 4/fisiologia , Esclerose Múltipla/genética , Polimorfismo de Nucleotídeo Único , Fator 3 Associado a Receptor de TNF/genética , Linfócitos B/virologia , Células Cultivadas , Endonucleases/genética , Herpesvirus Humano 4/patogenicidade , Humanos , Locos de Características Quantitativas , Transcriptoma , Latência Viral , Replicação Viral
11.
Br Poult Sci ; 60(4): 357-365, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31046421

RESUMO

1. Tumour necrosis factor receptor-associated factor 3 (TRAF3) is a key regulator of innate immunity and acquired immunity, and has a salient anti-viral role. 2. In this experiment, the duck TRAF3 (DuTRAF3) gene was cloned according to the Anas platyrhynchos TRAF3 sequence to explore its function. The TRAF3 open reading frame contains 1704 bp that encode a protein of 567 amino acids, which contain a RING finger domain, two zinc finger motifs, a coiled-coil region, and a MATH domain. 3. Reverse transcription-polymerase chain reaction showed that DuTRAF3 was expressed in all the examined tissues, with a comparatively higher expression in the spleen and brain tissues. 4. In HEK293T cells, DuTRAF3 overexpression resulted in a significantly increased NF-κB activity and interferon (IFN)-ß promoter activation. 5. Following resiquimod (R848) and poly(I:C) stimulation of duck peripheral blood mononuclear cells (PBMCs), the expressions of TRAF3 and IFN-ß were significantly upregulated; in addition, following R848 stimulation, the mRNA levels of IL-6, IL-8 and IL-10 were also significantly upregulated. After infection with the Newcastle Disease Virus LaSota vaccine strain, the mRNA levels of IL-6 and IL-10 were significantly upregulated, while that of TRAF3 was downregulated. 6. These results suggest that DuTRAF3 has an important role to play in innate antiviral immune responses.


Assuntos
Proteínas Aviárias/genética , Patos/genética , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Doenças das Aves Domésticas/imunologia , Fator 3 Associado a Receptor de TNF/genética , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Clonagem Molecular , Patos/metabolismo , Perfilação da Expressão Gênica/veterinária , Filogenia , Alinhamento de Sequência/veterinária , Fator 3 Associado a Receptor de TNF/química , Fator 3 Associado a Receptor de TNF/metabolismo
12.
Int Immunopharmacol ; 71: 181-187, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30909133

RESUMO

Osteoarthritis (OA) is a degenerative joint disease with characteristics of reduced cartilage cellularity, subchondral sclerosis and synoviti. Ultrasonic diagnosis plays pivotal role in diagnosing OA in the clinical, while biological markers are equal important to the diagnosis of OA. This study aimed to identify and characterize a biomarker, the expression of microRNA-107 (miR-107) in normal and OA chondrocytes, and to explore its effect on OA pathogenesis. Transfection with miR-107 mimic or inhibitor was used to investigate the effect of miR-107 on OA chondrocytes and to identify miR-107 target. Activation of AKT, mTOR and P65 was evaluated by Western blot analysis. Chondrocyte apoptosis was detected by using flow cytometer. Our results showed that the expression level of miR-107 in OA chondrocytes was obviously lower than control chondrocytes. Overexpression of miR-107 inhibited apoptosis and promoted autophagy in OA chondrocytes. Additionally, overexpression of miR-107 inhibited the activation of AKT/mTOR and NF-κB pathway by targeting TRAF3 genes. In vivo analysis revealed that miR-107 was also lowly expressed in rats with OA, and its abnormal expression significantly affected cell apoptosis. In conclusion, miR-107 regulated apoptosis and autophagy of OA chondrocytes by targeting TRAF3, and it might be used as a potential target for OA therapy.


Assuntos
Apoptose/genética , Autofagia/genética , Condrócitos/fisiologia , MicroRNAs/genética , Osteoartrite/genética , Fator 3 Associado a Receptor de TNF/genética , Adulto , Idoso , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
13.
Brain Behav Immun ; 79: 174-185, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30711510

RESUMO

Neuroinflammation occurs after germinal matrix hemorrhage (GMH) and induces secondary brain injury. Interferon-α (IFN-α) has been shown to exert anti-inflammatory effects in infectious diseases via activating IFNAR and its downstream signaling. We aimed to investigate the anti-inflammatory effects of Recombinant human IFN-α (rh-IFN-α) and the underlying mechanisms in a rat GMH model. Two hundred and eighteen P7 rat pups of both sexes were subjected to GMH by an intraparenchymal injection of bacterial collagenase. Rh-IFN-α was administered intraperitoneally. Small interfering RNA (siRNA) of IFNAR, and siRNA of tumor necrosis factor receptor associated factor 3 (TRAF3) were administered through intracerebroventricular (i.c.v.) injections. JAK1 inhibitor ruxolitinib was given by oral lavage. Post-GMH evaluation included neurobehavioral function, Nissl staining, Western blot analysis, and immunofluorescence. Our results showed that endogenous IFN-α and phosphorylated IFNAR levels were increased after GMH. Administration of rh-IFN-α improved neurological functions, attenuated neuroinflammation, inhibited microglial activation, and ameliorated post-hemorrhagic hydrocephalus after GMH. These observations were concomitant with IFNAR activation, increased expression of phosphorylated JAK1, phosphorylated STAT1 and TRAF3, and decreased levels of phosphorylated NF-κB, IL-6 and TNF-α. Specifically, knockdown of IFNAR, JAK1 and TRAF3 abolished the protective effects of rh-IFN-α. In conclusion, our findings demonstrated that rh-IFN-α treatment attenuated neuroinflammation, neurological deficits and hydrocephalus formation through inhibiting microglial activation after GMH, which might be mediated by IFNAR/JAK1-STAT1/TRAF3/NF-κB signaling pathway. Rh-IFN-α may be a promising therapeutic agent to attenuate brain injury via its anti-inflammatory effect.


Assuntos
Hemorragia Cerebral Intraventricular/imunologia , Interferon-alfa/metabolismo , Neuroimunomodulação/fisiologia , Animais , Animais Recém-Nascidos , Lesões Encefálicas/metabolismo , Hemorragia Cerebral Intraventricular/induzido quimicamente , Hemorragia Cerebral Intraventricular/fisiopatologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Proteínas I-kappa B/metabolismo , Inflamação/metabolismo , Interferon-alfa/farmacologia , Interferon-alfa/fisiologia , Janus Quinase 1/metabolismo , Janus Quinase 1/fisiologia , Masculino , Microglia/metabolismo , NF-kappa B/imunologia , NF-kappa B/metabolismo , Neuroimunomodulação/imunologia , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fator 3 Associado a Receptor de TNF/metabolismo
14.
J Med Virol ; 91(3): 482-492, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30267576

RESUMO

The mitochondrial antiviral signal protein mitochondrial antiviral signaling protein, also known as virus-induced signaling adaptor (VISA), plays a key role in regulating host innate immune signaling pathways. This study identifies FK506 binding protein 8 (FKBP8) as a candidate interacting protein of VISA through the yeast two-hybrid technique. The interaction of FKBP8 with VISA, retinoic acid inducible protein 1 (RIG-I), and IFN regulatory factor 3 (IRF3) was confirmed during viral infection in mammalian cells by coimmunoprecipitation. Overexpression of FKBP8 using a eukaryotic expression plasmid significantly attenuated Sendai virus-induced activation of the promoter interferons ß (IFN-ß), and transcription factors nuclear factor κ-light chain enhancer of activated B cells (NF-κB) and IFN-stimulated response element (ISRE). Overexpression of FKBP8 also decreased dimer-IRF3 activity, but enhanced virus replication. Conversely, knockdown of FKBP8 expression by RNA interference showed opposite effects. Further studies indicated that FKBP8 acts as a negative interacting partner to regulate RLR-VISA signaling by acting on VISA and TANK binding kinase 1 (TBK1). Additionally, FKBP8 played a negative role on virus-induced signaling by inhibiting the formation of TBK1-IRF3 and VISA-TRAF3 complexes. Notably, FKBP8 also promoted the degradation of TBK1, RIG-I, and TRAF3 resulting from FKBP8 reinforced Sendai virus-induced endogenous polyubiquitination of RIG-I, TBK1, and TNF receptor-associated factor 3 (TRAF3). Therefore, a novel function of FKBP8 in innate immunity antiviral signaling regulation was revealed in this study.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Imunidade Inata , Vírus Sendai , Transdução de Sinais , Proteínas de Ligação a Tacrolimo/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/imunologia , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/imunologia , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinação
15.
J Cell Physiol ; 234(5): 7467-7474, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30367484

RESUMO

Exercise is an effective therapy for insulin resistance. However, the underlying mechanism remains to be elucidated. Previous research demonstrated that TGFß-activated kinase 1 (TAK1)-dependent signaling plays a crucial character in hepatic insulin resistance. Hepatic ubiquitin specific protease 4 (USP4), USP18, and dual-specificity phosphatases 14 (DUSP14) can suppress TAK1 phosphorylation, besides tumor necrosis factor receptor-associated factor 3 (TRAF3) and tripartite motif 8 (TRIM8) promote its phosphorylation. In this study, we tried to verify our hypothesis that exercise improves insulin resistance in high-fat diet (HFD)-induced obese (DIO) rats via regulating the TAK1 dependent signaling and TAK1 regulators in liver. Forty male Sprague-Dawley rats were randomized into four groups (n = 10): standard diet and sedentary as normal control; fed on HFD and DIO-sedentary; fed on HFD and DIO-chronic exercise; and fed on HFD and DIO-acute exercise. HFD feeding resulted in increased body weight, visceral fat mass, serum FFAs and hepatic lipid deposition, but decreased hepatic glycogen content and insulin sensitivity. Moreover, hepatic TRAF3 and TRIM8 protein levels increased, whereas USP4, USP18, and DUSP14 protein levels were decreased under obese status, which resulted in enhanced TAK1 phosphorylation and impaired insulin signaling. Exercise training, containing chronic and acute mode, both ameliorated insulin resistance. Meanwhile, decreased TAK1, c-Jun N-terminal kinase 1 (JNK1), and insulin receptor substrate 1 (IRS1) phosphorylation enhanced Akt phosphorylation in liver. Moreover, exercise enhanced USP4 and DUSP14 protein levels, whereas decreased TRIM8 protein levels in obese rats' liver. These results showed that exercise triggered a crucial modulation in TAK1-dependent signaling and its regulators in obese rats' liver, and distinct improvement in insulin sensitivity, which provide new insights into the mechanism by which physical exercise improves insulin resistance.


Assuntos
Resistência à Insulina/fisiologia , Insulina/metabolismo , Fígado/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Obesidade/metabolismo , Condicionamento Físico Animal/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteínas de Transporte/metabolismo , Dieta Hiperlipídica/efeitos adversos , Gordura Intra-Abdominal/metabolismo , Masculino , Obesidade/fisiopatologia , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Fator 3 Associado a Receptor de TNF/metabolismo , Proteases Específicas de Ubiquitina/metabolismo
16.
Cancer Prev Res (Phila) ; 12(1): 57-66, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30463990

RESUMO

Persistent high-risk HPV infection is considered as a major cause of cervical cancer. Nevertheless, only some infected individuals actually develop cervical cancer. The RIG-I pathway in innate immunity plays an important role in antivirus response. Here, we hypothesized that altered function of mitochondrial antiviral signaling protein (MAVS) and mitochondrial TNF receptor-associated factor 3(TRAF3), key molecules downstream of the viral sensors RIG-I, may impair their ability of clearing HPV and thereby influence the risk for cervical precancerous lesions. To investigate the effects of MAVS and TRAF3 polymorphisms on susceptibility to cervical precancerous lesions, 8 SNPs were analyzed in 164 cervical precancerous lesion cases and 428 controls. Gene-environment interactions were also calculated. We found that CA genotype of rs6052130 in MAVS gene were at 1.48 times higher risk of developing cervical precancerous lesion than individuals with CC genotype (CA vs. CC: ORadjusted = 1.48, 95% CI, 1.02-2.16). In addition, a significant synergetic interaction between high-risk HPV infection and rs6052130 was found on an additive scale. A significantly decreased risk of cervical precancerous lesions for the TC genotype of rs12435483 in the TRAF3 gene (ORadjusted = 0.67, 95% CI, 0.45-0.98) was also found. Moreover, MDR analysis identified a significant three-locus interaction model, involving high-risk HPV infection, TRAF3 rs12435483 and number of full-term pregnancies. Our results indicate that the MAVS rs6052130 and TRAF3 rs12435483 confer genetic susceptibility to cervical precancerous lesions. Moreover, MAVS rs6052130-mutant individuals have an increased vulnerability to high-risk HPV-induced cervical precancerous lesions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Predisposição Genética para Doença , Infecções por Papillomavirus/complicações , Polimorfismo de Nucleotídeo Único , Lesões Pré-Cancerosas/epidemiologia , Fator 3 Associado a Receptor de TNF/genética , Neoplasias do Colo do Útero/epidemiologia , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , China/epidemiologia , Feminino , Interação Gene-Ambiente , Genótipo , Humanos , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/virologia , Transdução de Sinais , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia
17.
FEBS J ; 286(3): 523-535, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30536547

RESUMO

Fas (CD95) signalling is best known for its role in apoptosis, however, recent reports have shown it to be involved in other cellular responses as well, including inflammation. Fas and its adaptor protein FADD are known to negatively regulate LPS-induced proinflammatory responses, but their role in LPS-induced type I interferon production is unknown. Here, we demonstrate that Fas engagement on macrophages, using an agonistic Fas antibody CH11, augments LPS-induced NF-κB responses, causing increased production of TNFα, IL-8, IL-6 and IL-12. Conversely, costimulation with both LPS and CH11 causes a significant reduction in the level of interferon-beta (IFNß) production. This differential effect involves the Fas adaptor FADD because while LPS-induced IL-6 production increased in FADD-/- murine embryonic fibroblasts, LPS-induced IFNß production was significantly reduced in these cells. Overexpression of a dominant negative form of FADD (FADD-DD) inhibits LPS-induced IFNß luciferase but not LPS-induced NF-κB luciferase. In contrast, overexpression of full-length FADD inhibited LPS-induced NF-κB luciferase activation but was seen to augment LPS-induced IFNß luciferase. Moreover, FADD-DD inhibits TRIF-, TRAM-, IKKε-, TBK-1- and TRAF3-induced IFNß luciferase production, with coimmunoprecipitation experiments demonstrating an interaction between FADD and TRIF. These data identify FADD as a novel component of the noncanonical Toll-like receptor 4/IFNß signalling pathway and demonstrate that both Fas and its adaptor FADD can differentially regulate the production of LPS-induced proinflammatory cytokines and type I interferons.


Assuntos
Proteína de Domínio de Morte Associada a Fas/genética , Interferon beta/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Receptor fas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Anticorpos/farmacologia , Proteína de Domínio de Morte Associada a Fas/imunologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Interferon beta/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Células Jurkat , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , NF-kappa B/genética , NF-kappa B/imunologia , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Células RAW 264.7 , Transdução de Sinais , Células THP-1 , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/imunologia , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Receptor fas/antagonistas & inibidores , Receptor fas/imunologia
18.
Biochem Biophys Res Commun ; 508(4): 1088-1092, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30553450

RESUMO

Osteoclasts play an important role in bone remodeling. The inflammatory cytokine IL-17A could modulate the RANKL-induced osteoclastogenesis by regulating the autophagic activity. It is well accepted that protective autophagy has an anti-apoptotic effect. It is necessary to elucidate whether IL-17A can influence the apoptosis of osteoclast precursors (OCPs) through autophagy responses during osteoclastogenesis. The results showed that apoptosis of RAW264.7-derived OCPs was promoted by high levels of IL-17A, but the opposite anti-apoptotic function was shown by low levels of IL-17A. Furthermore, the enhanced apoptosis by high levels of IL-17A was reversed by overexpression of autophagy protein Beclin1; conversely, the inhibited apoptosis by low levels of IL-17A was restored by knockdown of Beclin1. It was also found that Beclin1 suppression with Beclin1 inhibitor (spautin1) could block the reduced apoptosis by low levels of IL-17A, which was recovered by TRAF3 knockdown. Moreover, the enhanced apoptosis by high levels of IL-17A decreased following the downregulation of TRAF3. Importantly, overexpression of caspase3 further attenuated osteoclastogenesis treated by high levels of IL-17A, without significantly affecting osteoclastogenesis stimulated by low levels of IL-17A. In conclusion, IL-17A modulates apoptosis of OCPs through Beclin1-autophagy-TRAF3 signaling pathway, thereby influencing osteoclastogenesis. Therefore, our study sheds lights on the improvement of clinical strategies of dental implantation or orthodontic treatment by revealing the novel targets in the bone remodeling.


Assuntos
Apoptose , Autofagia , Interleucina-17/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/metabolismo , Animais , Proteína Beclina-1/metabolismo , Caspase 3/metabolismo , Camundongos , Osteoclastos/ultraestrutura , Células RAW 264.7
19.
Mol Immunol ; 106: 53-62, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30579117

RESUMO

The inhibition of tumor necrosis factor receptor-associated factor 3 (TRAF3) degradation induces endotoxin tolerance (ET) in macrophages. However, the mechanisms leading to TRAF3 inhibition by ET are largely unknown. Here, we found that ubiquitin-specific peptidase 25 (USP25), a deubiquitinating enzyme (DUB), interacted with TRAF3 and stabilized ET in Kupffer cells (KCs). Lentiviral knockdown of USP25 activated K48-linked ubiquitination of TRAF3 and the cytoplasmic translocation of the Myd88-associated multiprotein complex in tolerized KCs. This outcome led to a subsequent activation of Myd88-dependent c-Jun N-terminal kinase (JNK) and p38-mediated downregulation of inflammatory cytokines. The overexpression of TRAF3 attenuated the proinflammatory effects of USP25 knockdown in tolerized KCs. Thus, our findings reveal a novel mechanism of endotoxin-mediated TRAF3 degradation in KCs.


Assuntos
Endotoxinas/imunologia , Tolerância Imunológica , Macrófagos do Fígado/imunologia , Proteólise , Fator 3 Associado a Receptor de TNF/imunologia , Ubiquitina Tiolesterase/imunologia , Ubiquitinação/imunologia , Animais , Técnicas de Silenciamento de Genes , Lentivirus , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , Masculino , Camundongos , Fator 3 Associado a Receptor de TNF/genética , Ubiquitina Tiolesterase/genética , Ubiquitinação/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia
20.
Front Immunol ; 9: 2618, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30524423

RESUMO

CD137 (4-1BB, Tnsfr9) is a member of the TNF-receptor (TNFR) superfamily without known intrinsic enzymatic activity in its cytoplasmic domain. Hence, akin to other members of the TNFR family, it relies on the TNFR-Associated-Factor (TRAF) family of adaptor proteins to build the CD137 signalosome for transducing signals into the cell. Thus, upon CD137 activation by binding of CD137L trimers or by crosslinking with agonist monoclonal antibodies, TRAF1, TRAF2, and TRAF3 are readily recruited to the cytoplasmic domain of CD137, likely as homo- and/or heterotrimers with different configurations, initiating the construction of the CD137 signalosome. The formation of TRAF2-RING dimers between TRAF2 molecules from contiguous trimers would help to establish a multimeric structure of TRAF-trimers that is probably essential for CD137 signaling. In addition, available studies have identified a large number of proteins that are recruited to CD137:TRAF complexes including ubiquitin ligases and proteases, kinases, and modulatory proteins. Working in a coordinated fashion, these CD137-signalosomes will ultimately promote CD137-mediated T cell proliferation and survival and will endow T cells with stronger effector functions. Current evidence allows to envision the molecular events that might take place in the early stages of CD137-signalosome formation, underscoring the key roles of TRAFs and of K63 and K48-ubiquitination of target proteins in the signaling process. Understanding the composition and fine regulation of CD137-signalosomes assembly and disassembly will be key to improve the therapeutic activities of chimeric antigen receptors (CARs) encompassing the CD137 cytoplasmic domain and a new generation of CD137 agonists for the treatment of cancer.


Assuntos
Complexos Multiproteicos/metabolismo , Neoplasias/tratamento farmacológico , Fator 1 Associado a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Proliferação de Células , Humanos , Ativação Linfocitária , Terapia de Alvo Molecular , Transdução de Sinais , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação
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