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1.
J Immunol ; 205(6): 1564-1579, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32747502

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human coronavirus causing severe disease and mortality. MERS-CoV infection failed to elicit robust IFN response, suggesting that the virus might have evolved strategies to evade host innate immune surveillance. In this study, we identified and characterized type I IFN antagonism of MERS-CoV open reading frame (ORF) 8b accessory protein. ORF8b was abundantly expressed in MERS-CoV-infected Huh-7 cells. When ectopically expressed, ORF8b inhibited IRF3-mediated IFN-ß expression induced by Sendai virus and poly(I:C). ORF8b was found to act at a step upstream of IRF3 to impede the interaction between IRF3 kinase IKKε and chaperone protein HSP70, which is required for the activation of IKKε and IRF3. An infection study using recombinant wild-type and ORF8b-deficient MERS-CoV further confirmed the suppressive role of ORF8b in type I IFN induction and its disruption of the colocalization of HSP70 with IKKε. Ectopic expression of HSP70 relieved suppression of IFN-ß expression by ORF8b in an IKKε-dependent manner. Enhancement of IFN-ß induction in cells infected with ORF8b-deficient virus was erased when HSP70 was depleted. Taken together, HSP70 chaperone is important for IKKε activation, and MERS-CoV ORF8b suppresses type I IFN expression by competing with IKKε for interaction with HSP70.


Assuntos
Ativação Enzimática/imunologia , Quinase I-kappa B/imunologia , Interferon Tipo I/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Proteínas Virais/imunologia , Betacoronavirus , Linhagem Celular , Infecções por Coronavirus , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Interferon Tipo I/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Pandemias , Pneumonia Viral , Proteínas Virais/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-32656094

RESUMO

As an emerging swine enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) not only causes serious diarrhea in suckling piglets but also possesses the potential for cross-species transmission, which has sparked growing interest when studying this emerging virus. We previously identified a novel accessory protein NS7a encoded by PDCoV; however, the function of NS7a was not resolved. In this study, we demonstrated that PDCoV NS7a is an interferon antagonist. Overexpression of NS7a notably inhibited Sendai virus (SeV)-induced interferon-ß (IFN-ß) production and the activation of IRF3 rather than NF-κB. NS7a also inhibited IFN-ß promoter activity induced by RIG-I, MDA5, MAVS, TBK1, and IKKε, which are key components of the RIG-I-like receptor (RLR) signaling pathway but not IRF3, the transcription factor downstream of TBK1/IKKε. Surprisingly, NS7a specifically interacts with IKKε but not with the closely related TBK1. Furthermore, NS7a interacts simultaneously with the kinase domain (KD) and the scaffold dimerization domain (SDD) of IKKε, competing with TRAF3, and IRF3 for binding to IKKε, leading to the reduction of RLR-mediated IFN-ß production. The interactions of TRAF3-IKKε and IKKε-IRF3 are also attenuated in PDCoV-infected cells. Taken together, our results demonstrate that PDCoV NS7a inhibits IFN-ß production by disrupting the association of IKKε with both TRAF3 and IRF3, revealing a new mechanism utilized by a PDCoV accessory protein to evade the host antiviral innate immune response.


Assuntos
Infecções por Coronavirus/metabolismo , Coronavirus/metabolismo , Quinase I-kappa B/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/antagonistas & inibidores , Fator 3 Associado a Receptor de TNF/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Coronavirus/genética , Coronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Células HEK293 , Humanos , Quinase I-kappa B/imunologia , Evasão da Resposta Imune , Fator Regulador 3 de Interferon/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/biossíntese , Interferon beta/imunologia , Receptores do Ácido Retinoico/metabolismo , Vírus Sendai/imunologia , Vírus Sendai/metabolismo , Transdução de Sinais , Suínos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia
3.
Mol Cell Biol ; 40(15)2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32393609

RESUMO

The diversified NF-κB transcription factor family has been extensively characterized in organisms ranging from flies to humans. However, homologs of NF-κB and many upstream signaling components have recently been characterized in basal phyla, including Cnidaria (sea anemones, corals, hydras, and jellyfish), Porifera (sponges), and single-celled protists, including Capsaspora owczarzaki and some choanoflagellates. Herein, we review what is known about basal NF-κBs and how that knowledge informs on the evolution and conservation of key sequences and domains in NF-κB, as well as the regulation of NF-κB activity. The structures and DNA-binding activities of basal NF-κB proteins resemble those of mammalian NF-κB p100 proteins, and their posttranslational activation appears to have aspects of both canonical and noncanonical pathways in mammals. Several studies suggest that the single NF-κB proteins found in some basal organisms have dual roles in development and immunity. Further research on NF-κB in invertebrates will reveal information about the evolutionary roots of this major signaling pathway, will shed light on the origins of regulated innate immunity, and may have relevance to our understanding of the responses of ecologically important organisms to changing environmental conditions and emerging pathogen-based diseases.


Assuntos
Regulação da Expressão Gênica/genética , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Regulação da Expressão Gênica/imunologia , Humanos , Quinase I-kappa B/imunologia , Imunidade Inata/imunologia , NF-kappa B/imunologia , Transdução de Sinais/fisiologia , Fator de Transcrição RelA/imunologia
4.
Nat Commun ; 11(1): 1786, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286300

RESUMO

Protein-protein interactions featuring intricate binding epitopes remain challenging targets for synthetic inhibitors. Interactions of NEMO, a scaffolding protein central to NF-κB signaling, exemplify this challenge. Various regulators are known to interact with different coiled coil regions of NEMO, but the topological complexity of this protein has limited inhibitor design. We undertook a comprehensive effort to block the interaction between vFLIP, a Kaposi's sarcoma herpesviral oncoprotein, and NEMO using small molecule screening and rational design. Our efforts reveal that a tertiary protein structure mimic of NEMO is necessary for potent inhibition. The rationally designed mimic engages vFLIP directly causing complex disruption, protein degradation and suppression of NF-κB signaling in primary effusion lymphoma (PEL). NEMO mimic treatment induces cell death and delays tumor growth in a PEL xenograft model. Our studies with this inhibitor reveal the critical nexus of signaling complex stability in the regulation of NF-κB by a viral oncoprotein.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma de Efusão Primária/metabolismo , NF-kappa B/metabolismo , Animais , Linhagem Celular , Dicroísmo Circular , Herpesvirus Humano 8/metabolismo , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linfoma de Efusão Primária/genética , Masculino , Camundongos , Microscopia Confocal , Modelos Biológicos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Mol Immunol ; 121: 195-203, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32298923

RESUMO

Cells recognize virus nucleic acid by pattern recognition receptors (PRRs), virus involve in the activation of signaling cascade of variable adaptor proteins, TANK-binding kinase1(TBK1)/ inhibitor of nuclear factor kappa-B kinase subunit epsilon(IKKi) complex, IκB kinase(IKKs) to trigger activation of transcription factor, interferon regulatory factor 3/7(IRF3/7), ultimately, leading to the production of type I interferon and exert anti-viral effects. In this study, E3 ubiquitin ligase ankyrin repeat and SOCS box-containing 8(ASB8) interacted with TBK1/IKKi and phosphorylation modification of ASB8 at site of Ser17 to further strengthen its ubiquitination activity were verified. Conversely, phosphorylated ASB8 accelerate K48-linked ubiquitination and degradation of TBK1/IKKi, which further reduces phosphorylation level of IRF3 and inhibits production of IFN-ß. At the same time, a new bridge molecule Leucine-rich repeat containing protein 10B(LRRC10B) upregulated after viral infection are involved in the formation and interaction with ASB8-TBK1/IKKi complex was reported. Our study reveals a new mechanism of ubiquitin ligase ASB8 modulating antiviral innate immunity by altering stability of TBK1/IKKi kinase complex.


Assuntos
Quinase I-kappa B/metabolismo , Interferon beta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/imunologia , Fosforilação/imunologia , Proteínas Serina-Treonina Quinases/imunologia , RNA Interferente Pequeno/metabolismo , Serina/metabolismo , Transdução de Sinais/imunologia , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação/imunologia
6.
J Virol ; 94(10)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132236

RESUMO

The nuclear factor kappa B (NF-κB) is a potent transcription factor, activation of which typically results in robust proinflammatory signaling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in the gradual and persistent activation of NF-κB, illustrated by proteasome-dependent degradation of the inhibitory NF-κB regulator IκB and nuclear translocation and phosphorylation of the NF-κB subunit p65. PRV-induced persistent activation of NF-κB does not result in expression of negative feedback loop genes, like the gene for IκBα or A20, and does not trigger expression of prototypical proinflammatory genes, like the gene for tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). In addition, PRV infection inhibits TNF-α-induced canonical NF-κB activation. Hence, PRV infection triggers persistent NF-κB activation in an unorthodox way and dramatically modulates the NF-κB signaling axis, preventing typical proinflammatory gene expression and the responsiveness of cells to canonical NF-κB signaling, which may aid the virus in modulating early proinflammatory responses in the infected host.IMPORTANCE The NF-κB transcription factor is activated via different key inflammatory pathways and typically results in the fast expression of several proinflammatory genes as well as negative feedback loop genes to prevent excessive inflammation. In the current report, we describe that infection of cells with the porcine alphaherpesvirus pseudorabies virus (PRV) triggers a gradual and persistent aberrant activation of NF-κB, which does not result in expression of hallmark proinflammatory or negative feedback loop genes. In addition, although PRV-induced NF-κB activation shares some mechanistic features with canonical NF-κB activation, it also shows remarkable differences; e.g., it is largely independent of the canonical IκB kinase (IKK) and even renders infected cells resistant to canonical NF-κB activation by the inflammatory cytokine TNF-α. Aberrant PRV-induced NF-κB activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-κB activation.


Assuntos
Células Epiteliais/virologia , Expressão Gênica , NF-kappa B/metabolismo , Pseudorraiva/virologia , Animais , Linhagem Celular , Citocinas/metabolismo , Técnicas de Silenciamento de Genes , Herpesvirus Suídeo 1/patogenicidade , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Evasão da Resposta Imune , NF-kappa B/genética , Fosforilação , Transdução de Sinais , Suínos , Fatores de Transcrição/metabolismo , Transcriptoma , Fator de Necrose Tumoral alfa/metabolismo
7.
Biochem Biophys Res Commun ; 525(3): 589-594, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32115150

RESUMO

Uveitis is a sight-threatening intraocular inflammatory disease that accounts for almost 10% of blindness worldwide. NF-κB signaling plays pivotal roles in inflammatory diseases. We have reported that IMD-0354, which inhibits NF-κB signaling via selective blockade of IKK-ß, suppresses inflammation in several ocular disease models. Here, we examined the therapeutic effect of IMD-0354 in an experimental autoimmune uveoretinitis (EAU) model, a well-established animal model for endogenous uveitis in humans. Systemic administration of IMD-0354 significantly suppressed the clinical and histological severity, inflammatory edema, and the translocation of NF-κB p65 into the nucleus of retinas in EAU mice. Furthermore, IMD-0354 treatment significantly inhibited the levels of several Th1/Th17-mediated pro-inflammatory cytokines in vitro. Our current data demonstrate that inhibition of IKKß with IMD-0354 ameliorates inflammatory responses in the mouse EAU model, suggesting that IMD-0354 may be a promising therapeutic agent for human endogenous uveitis.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Benzamidas/uso terapêutico , Quinase I-kappa B/antagonistas & inibidores , Retinite/tratamento farmacológico , Uveíte/tratamento farmacológico , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Benzamidas/administração & dosagem , Benzamidas/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citocinas/biossíntese , Edema/complicações , Edema/patologia , Quinase I-kappa B/metabolismo , Inflamação/complicações , Inflamação/patologia , Masculino , Camundongos , NF-kappa B/metabolismo , Retinite/imunologia , Retinite/patologia , Índice de Gravidade de Doença , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Uveíte/imunologia , Uveíte/patologia
8.
PLoS One ; 15(3): e0229395, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32130250

RESUMO

Inhibition of the key glycolytic activator 6-phosphofructokinase 2/fructose-2,6-bisphosphatase-3 (PFKFB3) by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) strongly attenuates pathological angiogenesis in cancer and inflammation. In addition to modulating endothelial proliferation and migration, 3PO also dampens proinflammatory activation of endothelial cells and experimental inflammation in vivo, suggesting a potential for 3PO in the treatment of chronic inflammation. The aim of our study was to explore if the anti-inflammatory action of 3PO in human endothelial cells was mediated by inhibition of PFKFB3 and glycolysis and assess if other means of PFKFB3 inhibition reduced inflammatory activation in a similar manner. We found that 3PO caused a rapid and transient reduction in IL-1ß- and TNF-induced phosphorylation of both IKKα/ß and JNK, thus inhibiting signaling through the NFκB and the stress-activated kinase pathways. However, in contrast to 3PO-treatment, neither shRNA-mediated silencing of PFKFB3 nor treatment with the alternative PFKFB3 inhibitor 7,8-dihydroxy-3-(4-hydroxy-phenyl)-chromen-4-one (YN1) prevented cytokine-induced NFκB signaling and upregulation of the adhesion molecules VCAM-1 and E-selectin, implying off target effects of 3PO. Collectively, our results suggest that the anti-inflammatory action of 3PO in human endothelial cells is not limited to inhibition of PFKFB3 and cellular glycolysis.


Assuntos
Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosfofrutoquinase-2/metabolismo , Piridinas/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
9.
J Virol ; 94(10)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32161168

RESUMO

Viruses commonly antagonize innate immune pathways that are primarily driven by nuclear factor kappa B (NF-κB), interferon regulatory factor (IRF), and the signal transducer and activator of transcription proteins (STAT) family of transcription factors. Such a strategy allows viruses to evade immune surveillance and maximize their replication. Using an unbiased transcriptome sequencing (RNA-seq)-based approach to measure gene expression induced by transfected viral genomic RNA (vgRNA) and reovirus infection, we discovered that mammalian reovirus inhibits host cell innate immune signaling. We found that, while vgRNA and reovirus infection both induce a similar IRF-dependent gene expression program, gene expression driven by the NF-κB family of transcription factors is lower in infected cells. Potent agonists of NF-κB such as tumor necrosis factor alpha (TNF-α) and vgRNA failed to induce NF-κB-dependent gene expression in infected cells. We demonstrate that NF-κB signaling is blocked due to loss of critical members of the inhibitor of kappa B kinase (IKK) complex, NF-κB essential modifier (NEMO), and IKKß. The loss of the IKK complex components prevents nuclear translocation and phosphorylation of NF-κB, thereby preventing gene expression. Our study demonstrates that reovirus infection selectively blocks NF-κB, likely to counteract its antiviral effects and promote efficient viral replication.IMPORTANCE Host cells mount a response to curb virus replication in infected cells and prevent spread of virus to neighboring, as yet uninfected, cells. The NF-κB family of proteins is important for the cell to mediate this response. In this study, we show that in cells infected with mammalian reovirus, NF-κB is inactive. Further, we demonstrate that NF-κB is rendered inactive because virus infection results in reduced levels of upstream intermediaries (called IKKs) that are needed for NF-κB function. Based on previous evidence that active NF-κB limits reovirus infection, we conclude that inactivating NF-κB is a viral strategy to produce a cellular environment that is favorable for virus replication.


Assuntos
Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Infecções por Reoviridae/metabolismo , Reoviridae/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Regulação da Expressão Gênica , Quinase I-kappa B/genética , Quinase I-kappa B/farmacologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , Reoviridae/genética , Reoviridae/fisiologia , Transcriptoma , Fator de Necrose Tumoral alfa/metabolismo
10.
J Immunol ; 204(7): 1881-1891, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32066597

RESUMO

Type I IFN mediates the innate immune system to provide defense against viral infections. NF-κB-inducing kinase (NIK) potentiates the basal activation of endogenous STING, which facilitates the recruitment of TBK1 with the ectopically expressed IRF3 to induce IFN production. Moreover, NIK phosphorylates IKKα and confers its ability to phosphorylate p100 (also known as NF-κB2) in mammals. Our study demonstrated that NIK plays a critical role in IFN production in teleost fish. It was found that NIK interacts with IKKα in the cytoplasm and that IKKα phosphorylates the NIK at the residue Thr432, which is different from the mammals. Overexpression of NIK caused the activation of IRF3 and NF-κB, which in turn led to the production of IFN and IFN-stimulated genes (ISGs). Furthermore, the ectopic expression of NIK was observed to be associated with a reduced replication of the fish virus, whereas silencing of endogenous NIK had an opposite effect in vitro. Furthermore, NIK knockdown significantly reduced the expression of IFN and key ISGs in zebrafish larvae after spring viremia of carp virus infection. Additionally, the replication of spring viremia of carp virus was enhanced in NIK knockdown zebrafish larvae, leading to a lower survival rate. In summary, our findings revealed a previously undescribed function of NIK in activating IFN and ISGs as a host antiviral response. These findings may facilitate the establishment of antiviral therapy to combat fish viruses.


Assuntos
Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Carpas/metabolismo , Carpas/virologia , Linhagem Celular , Quinase I-kappa B/metabolismo , Viremia/metabolismo , Viremia/virologia , Peixe-Zebra
11.
Cell Mol Biol Lett ; 25: 5, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32082390

RESUMO

Background: MicroRNAs (miRNAs or miRs) can participate in the development and progression of neuroblastoma. Many studies have indicated that miR-429 can participate in tumor development. However, the mechanism underlying miR-429-mediated progression of neuroblastoma remains largely unclear. Methods: Colony formation and apoptosis assays were used to determine the effect of miR-429 on cell proliferation. Its impact on cell migration was determined using the wound-healing and Transwell assays. The target gene of miR-429 was confirmed via western blotting and luciferase reporter assays. A nude mouse xenograft model with miR-429 overexpression was used to assess the effect on tumor growth. Results: Our findings indicate that miR-429 is downregulated in neuroblastoma cell lines. We also found that it can induce apoptosis and inhibit proliferation in cells of those lines. MiR-429 can bind to the 3'-UTR of IKKß mRNA and overexpression of IKKß can reverse cell proliferation, blocking the effect of miR-429. Furthermore, miR-429 overexpression inhibited neuroblastoma growth in our nude mouse xenograft model. Conclusion: We provide important insight into miR-429 as a tumor suppressor through interaction with IKKß, which is a catalytic subunit of the IKK complex that activates NF-κB nuclear transport. Our results demonstrate that miR-429 may be a new target for the treatment of neuroblastoma.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Genes Supressores de Tumor , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Neuroblastoma/metabolismo , Regiões 3' não Traduzidas , Animais , Apoptose/genética , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Neuroblastoma/genética , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Nat Commun ; 11(1): 685, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019925

RESUMO

IκB kinase 2 (IKK2) is well known for its pivotal role as a mediator of the canonical NF-κB pathway, which has important functions in inflammation and immunity, but also in cancer. Here we identify a novel and critical function of IKK2 and its co-factor NEMO in the activation of oncogenic c-Jun N-terminal kinase (JNK) signaling, induced by the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV). Independent of its kinase activity, the TGFß-activated kinase 1 (TAK1) mediates LMP1 signaling complex formation, NEMO ubiquitination and subsequent IKK2 activation. The tumor progression locus 2 (TPL2) kinase is induced by LMP1 via IKK2 and transmits JNK activation signals downstream of IKK2. The IKK2-TPL2-JNK axis is specific for LMP1 and differs from TNFα, Interleukin-1 and CD40 signaling. This pathway mediates essential LMP1 survival signals in EBV-transformed human B cells and post-transplant lymphoma, and thus qualifies as a target for treatment of EBV-induced cancer.


Assuntos
Linfócitos B/virologia , Herpesvirus Humano 4/fisiologia , Quinase I-kappa B/metabolismo , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Transformação Celular Viral , Herpesvirus Humano 4/genética , Humanos , Quinase I-kappa B/genética , Linfoma/enzimologia , Linfoma/genética , Linfoma/metabolismo , MAP Quinase Quinase 4/genética , MAP Quinase Quinase Quinases/genética , Camundongos , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais
13.
PLoS Genet ; 16(2): e1008626, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32059017

RESUMO

Neuronal pruning is a commonly observed phenomenon for the developing nervous systems to ensure precise wiring of neural circuits. The function of Ik2 kinase and its downstream mediator, Spindle-F (Spn-F), are essential for dendrite pruning of Drosophila sensory neurons during development. However, little is known about how Ik2/Spn-F signaling is transduced in neurons and ultimately results in dendrite pruning. Our genetic analyses and rescue experiments demonstrated that the small GTPase Rab11, especially the active GTP-bound form, is required for dendrite pruning. We also found that Rab11 shows genetic interactions with spn-F and ik2 on pruning. Live imaging of single neurons and antibody staining reveal normal Ik2 kinase activation in Rab11 mutant neurons, suggesting that Rab11 could have a functional connection downstream of and/or parallel to the Ik2 kinase signaling. Moreover, we provide biochemical evidence that both the Ik2 kinase activity and the formation of Ik2/Spn-F/Rab11 complexes are central to promote Rab11 activation in cells. Together, our studies reveal that a critical role of Ik2/Spn-F signaling in neuronal pruning is to promote Rab11 activation, which is crucial for dendrite pruning in neurons.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Quinase I-kappa B/metabolismo , Plasticidade Neuronal/genética , Células Receptoras Sensoriais/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Dendritos/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrião não Mamífero , Técnicas de Silenciamento de Genes , Microscopia Intravital , Proteínas Associadas aos Microtúbulos/metabolismo , Células Receptoras Sensoriais/citologia , Transdução de Sinais/fisiologia , Imagem com Lapso de Tempo , Proteínas rab de Ligação ao GTP/genética
14.
Clin Exp Metastasis ; 37(2): 283-292, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32020377

RESUMO

We explored the role of the transcription factor, NF-κB, and its upstream kinase IKKß in regulation of migration, invasion, and metastasis of cisplatin-resistant head and neck squamous cell carcinoma (HNSCC). We showed that cisplatin-resistant HNSCC cells have a stronger ability to migrate and invade, as well as display higher IKKß/NF-κB activity compared to their parental partners. Importantly, we found that knockdown of IKKß, but not NF-κB, dramatically impaired cell migration and invasion in these cells. Consistent with this, the IKKß inhibitor, CmpdA, also inhibited cell migration and invasion. Previous studies have already shown that N-Cadherin, an epithelial-mesenchymal transition (EMT) marker, and IL-6, a pro-inflammatory cytokine, play important roles in regulation of HNSCC migration, invasion, and metastasis. We found that cisplatin-resistant HNSCC expressed higher levels of N-Cadherin and IL-6, which were significantly inhibited by CmpdA. More importantly, we showed that CmpdA treatment dramatically abated cisplatin-resistant HNSCC cell metastasis to lungs in a mouse model. Our data demonstrated the crucial role of IKKß in control of migration, invasion, and metastasis, and implicated that targeting IKKß may be a potential therapy for cisplatin-resistant metastatic HNSCC.


Assuntos
Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Quinase I-kappa B/antagonistas & inibidores , Neoplasias Pulmonares/prevenção & controle , NF-kappa B/metabolismo , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Técnicas de Silenciamento de Genes , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica/genética , Metástase Neoplásica/prevenção & controle , Oxazinas/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/secundário , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Artigo em Inglês | MEDLINE | ID: mdl-32036038

RESUMO

Macrophages are the effector immune cells with plasticity to differentiate as M1 (classically activated) and M2 (alternatively activated) phenotypes. Prostaglandins (PGs) have various important roles and are involved in the regulation of macrophage activation. However, the role of PGF2α in macrophage activation remains unclear. We investigated the role of PGF2α receptor (FP)-mediated signaling in the M1 macrophage polarization using murine macrophage RAW264.7 cells. Stimulation with lipopolysaccharide (LPS) + interferon (IFN)-γ increased the mRNA expression of the M1 macrophage markers such as inducible nitric oxide synthase, tumor necrosis factor-α, and CD11c. Pre-treatment with AL8810, an FP receptor antagonist, further enhanced the expression of these genes. In contrast, treatment with fluprostenol, an FP receptor agonist, decreased the LPS + IFN-γ-induced expression of M1 markers. LPS-induced M1 macrophage polarization was dependent on the activation of NF-κB p65. Treatment with IκB kinase ß inhibitor reduced AL8810-induced mRNA expression of the M1 markers. Stimulation with LPS + IFN-γ increased the expression of IL-10. Pre-treatment with AL8810 lowered LPS + IFN-γ-induced IL-10 expression, and further enhanced LPS + IFN-γ-stimulated nuclear translocation of NF-κB p65. In contrast, co-treatment with IL-10 reversed AL8810-induced nuclear translocation of NF-κB p65. These results indicate that the FP receptor signaling was involved in the control of M1 polarization of macrophages via IL-10-regulated nuclear translocation of NF-κB p65.


Assuntos
Interleucina-10/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Receptores de Prostaglandina/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Núcleo Celular/metabolismo , Dinoprosta/análogos & derivados , Dinoprosta/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Interferon gama/imunologia , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Prostaglandinas F Sintéticas/farmacologia , Células RAW 264.7 , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Tiofenos/farmacologia
16.
Oxid Med Cell Longev ; 2020: 3602824, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32064021

RESUMO

Abdominal aortic aneurysm (AAA) is a vascular disorder that is considered a chronic inflammatory disease. However, the precise molecular mechanisms involved in AAA have not been fully elucidated. Recently, significant progress has been made in understanding the function and mechanism of action of inhibitor of kappa B kinase epsilon (IKKε) in inflammatory and metabolic diseases. The angiotensin II- (Ang II-) induced or pharmacological inhibitors were established to test the effects of IKKε on AAA in vivo. After mice were continuously stimulated with Ang II for 28 days, morphologically, we found that knockout of IKKε reduced AAA formation and drastically reduced maximal diameter and severity. We also observed a decrease in elastin degradation and medial destruction, which were independent of systolic blood pressure or plasma cholesterol concentrations. Western blot analyses and immunohistochemical staining were carried out to measure IKKε expression in AAA tissues and cell lines. AAA phenotype of mice was measured by ultrasound and biochemical indexes. In zymography, immunohistology staining, immunofluorescence staining, and reactive oxygen species (ROS) analysis, TUNEL assay was used to examine the effects of IKKε on AAA progression in AAA mice. IKKε deficiency significantly inhibited inflammatory macrophage infiltration, matrix metalloproteinase (MMP) activity, ROS production, and vascular smooth muscle cell (VSMC) apoptosis. We used primary mouse aortic VSMC isolated from apolipoprotein E (Apoe) -/- and Apoe-/-IKKε -/- mice. Mechanistically, IKKε deficiency blunted the activation of the ERK1/2 pathway. The IKKε inhibitor, amlexanox, has the same impact in AAA. Our results demonstrate a critical role of IKKε in AAA formation induced by Ang II in Apoe-/- mice. Targeting IKKε may constitute a novel therapeutic strategy to prevent AAA progression.


Assuntos
Angiotensina II/toxicidade , Aneurisma da Aorta Abdominal/metabolismo , Apoptose/genética , Quinase I-kappa B/deficiência , Inflamação/metabolismo , Estresse Oxidativo/genética , Idoso , Animais , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/fisiopatologia , Apolipoproteínas E/deficiência , Apoptose/efeitos dos fármacos , Elastina/metabolismo , Feminino , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Inflamação/genética , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/genética , Macrófagos/metabolismo , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Espécies Reativas de Oxigênio/metabolismo
17.
J Immunol ; 204(6): 1499-1507, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32024699

RESUMO

As an important effector in response to various intracellular or extracellular stimuli, the NF-κB family extensively participates in a wide spectrum of biological events, and its dysregulation may result in many pathological conditions, such as microbial infection, tumor progression, and neurodegenerative disorders. Previous investigations showed that multiple types of ubiquitination play critical roles in the modulation of the NF-κB signaling pathway, yet the molecular mechanisms are still poorly understood. In the current study, we identified TRIM25, an E3 ubiquitin ligase, as a novel positive regulator in mediating NF-κB activation in human embryonic kidney 293T (HEK293T), HeLa cells, THP-1 cells, and PBMCs. The expression of TRIM25 promoted TNF-α-induced NF-κB signaling, whereas the knockdown had the opposite effect. Furthermore, TRIM25 interacted with TRAF2 and enhanced the K63-linked polyubiquitin chains attached to TRAF2. Moreover, TRIM25 bridged the interaction of TRAF2 and TAK1 or IKKß. To our knowledge, our study has identified a previously unrecognized role for TRIM25 in the regulation of NF-κB activation by enhancing the K63-linked ubiquitination of TRAF2.


Assuntos
Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Leucócitos Mononucleares , Lisina/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Cultura Primária de Células , Células THP-1 , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Ubiquitinação/imunologia
18.
Arterioscler Thromb Vasc Biol ; 40(3): 638-655, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31893948

RESUMO

OBJECTIVE: Although often studied independently, little is known about how aortic valve endothelial cells and valve interstitial cells interact collaborate to maintain tissue homeostasis or drive valve calcific pathogenesis. Inflammatory signaling is a recognized initiator of valve calcification, but the cell-type-specific downstream mechanisms have not been elucidated. In this study, we test how inflammatory signaling via NFκB (nuclear factor κ-light-chain enhancer of activated B cells) activity coordinates unique and shared mechanisms of valve endothelial cells and valve interstitial cells differentiation during calcific progression. Approach and Results: Activated NFκB was present throughout the calcific aortic valve disease (CAVD) process in both endothelial and interstitial cell populations in an established mouse model of hypercholesterolemia-induced CAVD and in human CAVD. NFκB activity induces endothelial to mesenchymal transformation in 3-dimensional cultured aortic valve endothelial cells and subsequent osteogenic calcification of transformed cells. Similarly, 3-dimensional cultured valve interstitial cells calcified via NFκB-mediated osteogenic differentiation. NFκB-mediated endothelial to mesenchymal transformation was directly demonstrated in vivo during CAVD via genetic lineage tracking. Genetic deletion of NFκB in either whole valves or valve endothelium only was sufficient to prevent valve-specific molecular and cellular mechanisms of CAVD in vivo despite the persistence of a CAVD inducing environment. CONCLUSIONS: Our results identify NFκB signaling as an essential molecular regulator for both valve endothelial and interstitial participation in CAVD pathogenesis. Direct demonstration of valve endothelial cell endothelial to mesenchymal transformation transmigration in vivo during CAVD highlights a new cellular population for further investigation in CAVD morbidity. The efficacy of valve-specific NFκB modulation in inhibiting hypercholesterolemic CAVD suggests potential benefits of multicell type integrated investigation for biological therapeutic development and evaluation for CAVD.


Assuntos
Valva Aórtica/metabolismo , Calcinose/metabolismo , Diferenciação Celular , Células Endoteliais/metabolismo , Doenças das Valvas Cardíacas/metabolismo , NF-kappa B/metabolismo , Osteogênese , Animais , Valva Aórtica/patologia , Calcinose/etiologia , Calcinose/patologia , Células Cultivadas , Microambiente Celular , Modelos Animais de Doenças , Células Endoteliais/patologia , Doenças das Valvas Cardíacas/etiologia , Doenças das Valvas Cardíacas/genética , Doenças das Valvas Cardíacas/patologia , Humanos , Hipercolesterolemia/complicações , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/genética , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
19.
Int J Oral Sci ; 12(1): 1, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31900382

RESUMO

Odontogenic tumours are a heterogeneous group of lesions that develop in the oral cavity region and are characterized by the formation of tumoural structures that differentiate as teeth. Due to the diversity of their histopathological characteristics and clinical behaviour, the classification of these tumours is still under debate. Alterations in morphogenesis pathways such as the Hedgehog, MAPK and WNT/ß-catenin pathways are implicated in the formation of odontogenic lesions, but the molecular bases of many of these lesions are still unknown. In this study, we used genetically modified mice to study the role of IKKß (a fundamental regulator of NF-κB activity and many other proteins) in oral epithelial cells and odontogenic tissues. Transgenic mice overexpressing IKKß in oral epithelial cells show a significant increase in immune cells in both the oral epithelia and oral submucosa. They also show changes in the expression of several proteins and miRNAs that are important for cancer development. Interestingly, we found that overactivity of IKKß in oral epithelia and odontogenic tissues, in conjunction with the loss of tumour suppressor proteins (p53, or p16 and p19), leads to the appearance of odontogenic tumours that can be classified as ameloblastic odontomas, sometimes accompanied by foci of secondary ameloblastic carcinomas. These tumours show NF-κB activation and increased ß-catenin activity. These findings may help to elucidate the molecular determinants of odontogenic tumourigenesis and the role of IKKß in the homoeostasis and tumoural transformation of oral and odontogenic epithelia.


Assuntos
Células Epiteliais/metabolismo , Genes Supressores de Tumor , Quinase I-kappa B/biossíntese , Mucosa Bucal/patologia , Tumores Odontogênicos/patologia , Odontoma/patologia , RNA Mensageiro/genética , Animais , Western Blotting , Células Epiteliais/patologia , Citometria de Fluxo , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Transgênicos , Mucosa Bucal/metabolismo , Tumores Odontogênicos/metabolismo , Odontoma/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
20.
Cancer Chemother Pharmacol ; 85(2): 331-343, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31989218

RESUMO

One promising strategy for minimizing chemotherapeutic resistance in hepatocellular carcinoma (HCC) is the use of effective chemosensitizers. We studied the complementary multi-targeted molecular mechanisms of metformin and celastrol in mice with diethylnitrosamine-induced HCC to investigate whether metformin could augment the sensitivity of HCC tissue to the effect of celastrol. Simultaneous administration of celastrol (2 mg/kg) and metformin (200 mg/kg) improved liver function, enhanced the histological picture and prolonged survival. Additionally, combination therapy exerted anti-inflammatory activity, as indicated by the decreased levels of TNF-α and IL-6. This protective role could be attributed to inhibition of inflammasome activation. Herein, our data revealed downregulated NLRP3 gene expression, suppressed caspase-1 activity and reduced levels of the active forms of IL-1ß and IL-18. Under this condition, pyroptotic activity was suppressed. In contrast, in the celastrol and celastrol + metformin groups, the apoptotic potential was amplified, as revealed by the increase in the caspase-9 and caspase-3 levels and Bax:BCL-2 ratio. In addition to their repressive effect on the gene expression of NFκBp65, TNFR and TLR4, metformin and celastrol inhibited phosphorylation-induced activation of IκBκB and NFκBp65 and decreased IκBα degradation. Combination therapy with metformin and celastrol repressed markers of angiogenesis, metastasis and tumour proliferation, as revealed by the decreased hepatic levels of VEGF, MMP-2/9 and cyclin D1 mRNA, respectively. In conclusion, by inhibiting NLRP3 inflammasome and its prerequisite NFκB signalling, simultaneous administration of metformin and celastrol appears to have additive benefits in the treatment of HCC compared to cela monotherapy. This effect warrants further clinical investigation.


Assuntos
Apoptose/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Inflamassomos/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Metformina/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Triterpenos/farmacologia , Animais , Antineoplásicos/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Dietilnitrosamina/farmacologia , Inflamassomos/metabolismo , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos
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