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1.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445397

RESUMO

Tumor necrosis factor-alpha (TNFα) can bind two distinct receptors (TNFR1/2). The transmembrane form (tmTNFα) preferentially binds to TNFR2. Upon tmTNFα cleavage by the TNF-alpha-converting enzyme (TACE), its soluble (sTNFα) form is released with higher affinity for TNFR1. This assortment empowers TNFα with a plethora of opposing roles in the processes of tumor cell survival (and apoptosis) and anti-tumor immune stimulation (and suppression), in addition to angiogenesis and metastases. Its functions and biomarker potential to predict cancer progression and response to immunotherapy are reviewed here, with a focus on lung cancer. By mining existing sequencing data, we further demonstrate that the expression levels of TNF and TACE are significantly decreased in lung adenocarcinoma patients, while the TNFR1/TNFR2 balance are increased. We conclude that the biomarker potential of TNFα alone will most likely not provide conclusive findings, but that TACE could have a key role along with the delicate balance of sTNFα/tmTNFα as well as TNFR1/TNFR2, hence stressing the importance of more research into the potential of rationalized treatments that combine TNFα pathway modulators with immunotherapy for lung cancer patients.


Assuntos
Regulação para Baixo , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/genética , Fator de Necrose Tumoral alfa/genética , Proteína ADAM17/genética , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
2.
Nat Commun ; 12(1): 4405, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285232

RESUMO

Stimulator of interferon genes (STING) promotes anti-tumour immunity by linking innate and adaptive immunity, but it remains unclear how intratumoural treatment with STING agonists yields anti-tumour effects. Here we demonstrate that intratumoural injection of the STING agonist cGAMP induces strong, rapid, and selective apoptosis of tumour endothelial cells (ECs) in implanted LLC tumour, melanoma and breast tumour, but not in spontaneous breast cancer and melanoma. In both implanted and spontaneous tumours, cGAMP greatly increases TNFα from tumour-associated myeloid cells. However, compared to spontaneous tumour ECs, implanted tumour ECs are more vulnerable to TNFα-TNFR1 signalling-mediated apoptosis, which promotes effective anti-tumour activity. The spontaneous tumour's refractoriness to cGAMP is abolished by co-treatment with AKT 1/2 inhibitor (AKTi). Combined treatment with cGAMP and AKTi induces extensive tumour EC apoptosis, leading to extensive tumour apoptosis and marked growth suppression of the spontaneous tumour. These findings propose an advanced avenue for treating primary tumours that are refractory to single STING agonist therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Membrana/agonistas , Neoplasias/tratamento farmacológico , Nucleotídeos Cíclicos/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunidade Inata/efeitos dos fármacos , Injeções Intralesionais , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasias/irrigação sanguínea , Neoplasias/imunologia , Neoplasias/patologia , Nucleotídeos Cíclicos/uso terapêutico , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/metabolismo
3.
Front Immunol ; 12: 622471, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34163464

RESUMO

Search for novel regulatory protein fragments with potential functional roles is required both for understanding the immune response mechanisms and the development of targeted immunotherapy. Earlier we demonstrated that the PGLYRP1/Tag7 innate immunity protein can be regarded as an inhibitor of TNFα cytotoxic activity via the interaction with its TNF receptor 1 (TNFR1). A C-terminal peptide fragment 17.1 of the molecule is responsible for this function. In this study we have identified a minimal 8-mer region of this peptide (hereinafter - 17.1A) capable to bind to TNFR1. As a result of such interaction, the cytotoxic signals induced by this receptor are blocked. Also, this peptide demonstrates an anti-inflammatory activity in vivo in the complete Freund's adjuvant (CFA)-induced arthritis model in laboratory mice. Peptide 17.1A is capable to reduce periarticular inflammation, inhibit the development of synovitis and exhibit a protective effect on cartilage and bone tissues. This peptide can turn out to be a promising medicinal agent for autoimmune arthritis and other diseases.


Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Citocinas/metabolismo , Fibroblastos/imunologia , Inflamação/imunologia , Peptídeos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Animais , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Linhagem Celular , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Humanos , Imunidade Inata , Camundongos , Ligação Proteica , Fator de Necrose Tumoral alfa/metabolismo
4.
Int J Mol Sci ; 22(11)2021 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067256

RESUMO

Tumor necrosis factor (TNF) is one of the main cytokines regulating a pro-inflammatory environment. It has been related to several cell functions, for instance, phagocytosis, apoptosis, proliferation, mitochondrial dynamic. Moreover, during mycobacterial infections, TNF plays an essential role to maintain granuloma formation. Several effector mechanisms have been implicated according to the interactions of the two active forms, soluble TNF (solTNF) and transmembrane TNF (tmTNF), with their receptors TNFR1 and TNFR2. We review the impact of these interactions in the context of mycobacterial infections. TNF is tightly regulated by binding to receptors, however, during mycobacterial infections, upstream activation signalling pathways may be influenced by key regulatory factors either at the membrane or cytosol level. Detailing the structure and activation pathways used by TNF and its receptors, such as its interaction with solTNF/TNFRs versus tmTNF/TNFRs, may bring a better understanding of the molecular mechanisms involved in activation pathways which can be helpful for the development of new therapies aimed at being more efficient against mycobacterial infections.


Assuntos
Proteínas de Membrana/metabolismo , Infecções por Mycobacterium/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Membrana Celular/metabolismo , Humanos , Transdução de Sinais/fisiologia
5.
Mol Med Rep ; 24(1)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33955507

RESUMO

The protein extracted from red algae Pyropia yezoensis has various biological activities, including anti­inflammatory, anticancer, antioxidant, and antiobesity properties. However, the effects of P. yezoensis protein (PYCP) on tumor necrosis factor­α (TNF­α)­induced muscle atrophy are unknown. Therefore, the present study investigated the protective effects and related mechanisms of PYCP against TNF­α­induced myotube atrophy in C2C12 myotubes. Treatment with TNF­α (20 ng/ml) for 48 h significantly reduced myotube viability and diameter and increased intracellular reactive oxygen species levels; these effects were significantly reversed in a dose­dependent manner following treatment with 25­100 µg/ml PYCP. PYCP inhibited the expression of TNF receptor­1 in TNF­α­induced myotubes. In addition, PYCP markedly downregulated the nuclear translocation of nuclear factor­κB (NF­κB) by inhibiting the phosphorylation of inhibitor of κB. Furthermore, PYCP treatment suppressed 20S proteasome activity, IL­6 production, and the expression of the E3 ubiquitin ligases, atrogin­1/muscle atrophy F­box and muscle RING­finger protein­1. Finally, PYCP treatment increased the protein expression levels of myoblast determination protein 1 and myogenin in TNF­α­induced myotubes. The present findings indicate that PYCP may protect against TNF­α­induced myotube atrophy by inhibiting the proinflammatory NF­κB pathway.


Assuntos
Proteínas de Algas/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Atrofia Muscular/tratamento farmacológico , Substâncias Protetoras/farmacologia , Rodófitas/química , Transdução de Sinais/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Interleucina-6/metabolismo , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/metabolismo , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/metabolismo , Proteína MyoD/metabolismo , Miogenina/metabolismo , NF-kappa B/metabolismo , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
6.
Life Sci Alliance ; 4(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33858959

RESUMO

Tumor necrosis factor receptor 1 (TNFR1) activates NF-κB-dependent pro-inflammatory gene expression, but also induces cell death by triggering apoptosis and necroptosis. Inhibition of inhibitor of NF-κB kinase (IKK)/NF-κB signaling in keratinocytes paradoxically unleashed spontaneous TNFR1-mediated skin inflammation in mice, but the underlying mechanisms remain poorly understood. Here, we show that TNFR1 causes skin inflammation in mice with epidermis-specific knockout of IKK2 by inducing receptor interacting protein kinase 1 (RIPK1)-dependent necroptosis, and to a lesser extent also apoptosis, of keratinocytes. Combined epidermis-specific ablation of the NF-κB subunits RelA and c-Rel also caused skin inflammation by inducing TNFR1-mediated keratinocyte necroptosis. Contrary to the currently established model that inhibition of NF-κB-dependent gene transcription causes RIPK1-independent cell death, keratinocyte necroptosis, and skin inflammation in mice with epidermis-specific RelA and c-Rel deficiency also depended on RIPK1 kinase activity. These results advance our understanding of the mechanisms regulating TNFR1-induced cell death and identify RIPK1-mediated necroptosis as a potent driver of skin inflammation.


Assuntos
Queratinócitos/metabolismo , Necroptose/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose/fisiologia , Feminino , Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , NF-kappa B/fisiologia , Necroptose/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Pele/metabolismo , Pele/patologia , Fator de Necrose Tumoral alfa/farmacologia
7.
Cell Death Dis ; 12(4): 360, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824270

RESUMO

In the early 1990s, it has been described that LTα and LTß form LTα2ß and LTαß2 heterotrimers, which bind to TNFR1 and LTßR, respectively. Afterwards, the LTαß2-LTßR system has been intensively studied while the LTα2ß-TNFR1 interaction has been ignored to date, presumably due to the fact that at the time of identification of the LTα2ß-TNFR1 interaction one knew already two ligands for TNFR1, namely TNF and LTα. Here, we show that LTα2ß interacts not only with TNFR1 but also with TNFR2. We furthermore demonstrate that membrane-bound LTα2ß (memLTα2ß), despite its asymmetric structure, stimulates TNFR1 and TNFR2 signaling. Not surprising in view of its ability to interact with TNFR2, LTα2ß is inhibited by Etanercept, which is approved for the treatment of rheumatoid arthritis and also inhibits TNF and LTα.


Assuntos
Linfotoxina-alfa/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/agonistas , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Humanos , Membranas/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/agonistas , Receptores Tipo I de Fatores de Necrose Tumoral/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/imunologia
8.
J Cell Biol ; 220(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33914027

RESUMO

Activation of inflammation by lipopolysaccharide (LPS) is an important innate immune response. Here we investigated the contribution of caspases to the LPS-mediated inflammatory response and discovered distinctive temporal roles of RIPK1 in mediating proinflammatory cytokine production when caspases are inhibited. We propose a biphasic model that differentiates the role of RIPK1 in early versus late phase. The early production of proinflammation cytokines stimulated by LPS with caspase inhibition is mediated by the NF-κB pathway that requires the scaffold function of RIPK1 but is kinase independent. Autocrine production of TNFα in the late phase promotes the formation of a novel TNFR1-associated complex with activated RIPK1, FADD, caspase-8, and key mediators of NF-κB signaling. The production of proinflammatory cytokines in the late phase can be blocked by RIPK1 kinase inhibitor Nec-1s. Our study demonstrates a mechanism by which the activation of RIPK1 promotes its own scaffold function to regulate the NF-κB-mediated proinflammatory cytokine production that is negatively regulated by caspases to restrain inflammatory signaling.


Assuntos
Caspase 8/química , Inibidores de Caspase/farmacologia , Citocinas/metabolismo , Imunidade Inata/efeitos dos fármacos , Inflamação/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Caspase 8/genética , Caspase 8/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
9.
Immunity ; 54(3): 468-483.e5, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33484643

RESUMO

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.


Assuntos
Vasos Sanguíneos/imunologia , Dermatite de Contato/imunologia , Inflamação/imunologia , Mastócitos/imunologia , Neutrófilos/imunologia , Pele/patologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Circulação Sanguínea , Degranulação Celular , Células Cultivadas , Doenças do Sistema Imunitário , Transtornos Leucocíticos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação de Neutrófilo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Vesículas Secretórias/metabolismo , Fator de Necrose Tumoral alfa/genética
10.
Nat Commun ; 12(1): 86, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397971

RESUMO

Inflammation and cell death are closely linked arms of the host immune response to infection, which when carefully balanced ensure host survival. One example of this balance is the tightly regulated transition from TNFR1-associated pro-inflammatory complex I to pro-death complex II. By contrast, here we show that a TRIF-dependent complex containing FADD, RIPK1 and caspase-8 (that we have termed the TRIFosome) mediates cell death in response to Yersinia pseudotuberculosis and LPS. Furthermore, we show that constitutive binding between ZBP1 and RIPK1 is essential for the initiation of TRIFosome interactions, caspase-8-mediated cell death and inflammasome activation, thus positioning ZBP1 as an effector of cell death in the context of bacterial blockade of pro-inflammatory signaling. Additionally, our findings offer an alternative to the TNFR1-dependent model of complex II assembly, by demonstrating pro-death complex formation reliant on TRIF signaling.


Assuntos
Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Caspase 8/metabolismo , Morte Celular/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Ligação Proteica/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Yersinia
11.
Nat Commun ; 12(1): 582, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495441

RESUMO

Tumour necrosis factor (TNF) is a trimeric protein which signals through two membrane receptors, TNFR1 and TNFR2. Previously, we identified small molecules that inhibit human TNF by stabilising a distorted trimer and reduce the number of receptors bound to TNF from three to two. Here we present a biochemical and structural characterisation of the small molecule-stabilised TNF-TNFR1 complex, providing insights into how a distorted TNF trimer can alter signalling function. We demonstrate that the inhibitors reduce the binding affinity of TNF to the third TNFR1 molecule. In support of this, we show by X-ray crystallography that the inhibitor-bound, distorted, TNF trimer forms a complex with a dimer of TNFR1 molecules. This observation, along with data from a solution-based network assembly assay, leads us to suggest a model for TNF signalling based on TNF-TNFR1 clusters, which are disrupted by small molecule inhibitors.


Assuntos
Multimerização Proteica/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/química , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Necrose Tumoral alfa/química , Algoritmos , Animais , Ligação Competitiva/efeitos dos fármacos , Humanos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Bibliotecas de Moléculas Pequenas/química , Fator de Necrose Tumoral alfa/metabolismo
12.
Nat Commun ; 12(1): 583, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495445

RESUMO

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.


Assuntos
Anticorpos Monoclonais/metabolismo , Complexos Multiproteicos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Anticorpos Monoclonais/farmacologia , Células Cultivadas , Cristalografia por Raios X , Epitopos/química , Epitopos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/química , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Necrose Tumoral alfa/química
13.
J Leukoc Biol ; 109(2): 363-371, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32401398

RESUMO

TNFR1 is a crucial regulator of NF-ĸB-mediated proinflammatory cell survival responses and programmed cell death (PCD). Deregulation of TNFα- and TNFR1-controlled NF-ĸB signaling underlies major diseases, like cancer, inflammation, and autoimmune diseases. Therefore, although being routinely used, antagonists of TNFα might also affect TNFR2-mediated processes, so that alternative approaches to directly antagonize TNFR1 are beneficial. Here, we apply quantitative single-molecule localization microscopy (SMLM) of TNFR1 in physiologic cellular settings to validate and characterize TNFR1 inhibitory substances, exemplified by the recently described TNFR1 antagonist zafirlukast. Treatment of TNFR1-mEos2 reconstituted TNFR1/2 knockout mouse embryonic fibroblasts (MEFs) with zafirlukast inhibited both ligand-independent preligand assembly domain (PLAD)-mediated TNFR1 dimerization as well as TNFα-induced TNFR1 oligomerization. In addition, zafirlukast-mediated inhibition of TNFR1 clustering was accompanied by deregulation of acute and prolonged NF-ĸB signaling in reconstituted TNFR1-mEos2 MEFs and human cervical carcinoma cells. These findings reveal the necessity of PLAD-mediated, ligand-independent TNFR1 dimerization for NF-ĸB activation, highlight the PLAD as central regulator of TNFα-induced TNFR1 oligomerization, and demonstrate that TNFR1-mEos2 MEFs can be used to investigate TNFR1-antagonizing compounds employing single-molecule quantification and functional NF-ĸB assays at physiologic conditions.


Assuntos
NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Transdução de Sinais , Imagem Individual de Molécula , Compostos de Tosil/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular , Citocinas/biossíntese , Células HeLa , Humanos , Camundongos , Multimerização Proteica/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
14.
Food Chem Toxicol ; 147: 111856, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33246054

RESUMO

CIL-102 (1-[4-(furo [2,3-b]quinolin-4-ylamino)phenyl]ethanone) is a major active agent and an alkaloid derivative of Camptotheca acuminata, which has valuable biological properties, including anti-tumorigenic activity. However, the molecular mechanisms of CIL-102 related to inductive apoptosis in human gastric cancer remain unclear. By using diphenyltetrazolium bromide (MTT), annexin-V-fluorescein-isothiocyanate (FITC)/propidium iodide staining and a 2',7' -dichlorofluorescin diacetate (DCFDA), a Fluo-3 fluorescence staining assay, the cell death and cell viability in gastric cancer cells and an in vivo xenograft mouse model, with or without the addition of CIL-102, were measured, respectively. Furthermore, signaling pathways and apoptotic molecules were also detected by western blots and an immunohistochemical assay. Our results demonstrated that CIL-102 treatment significantly induced the cell apoptosis of gastric cancer cells, along with increased ROS production and increased intracellular Ca2+ levels. In addition, through the inactivation of CDK1/cyclin B1, CIL-102 treatment induced the cell cycle G2/M arrest of gastric cancer cells. Moreover, our data revealed that multiple signaling pathways were involved in the H3K4 trimethylation of TNFR1 and TRAIL proteins by CIL-102, including ROS-derived and JNK/mTOR/p300 pathways in gastric cancer AGS cells. The CIL-102 treatment also consistently inhibited tumor growth and increased tumor apoptosis, as measured by TUNEL assay in an in vivo xenograft mouse model. An immunohistochemical analysis revealed that the upregulation of the TNFR1 and TRAIL proteins and the downregulation of PCNA and CDK1 proteins were found in the CIL-102-treated gastric cancer xenograft mouse model, compared to that of the saline control. Together, this study sheds light on the novel mechanism associated with CIL-102 for inducing gastric cancer apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Quinolinas/farmacologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Neoplasias Gástricas/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Humanos , Metilação , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Quinolinas/administração & dosagem , Quinolinas/química , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Neoplasias Gástricas/patologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Regulação para Cima/efeitos dos fármacos
15.
J Neurosci ; 41(6): 1349-1362, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33303682

RESUMO

There are significant neurogenic and inflammatory influences on blood pressure, yet the role played by each of these processes in the development of hypertension is unclear. Tumor necrosis factor α (TNFα) has emerged as a critical modulator of blood pressure and neural plasticity; however, the mechanism by which TNFα signaling contributes to the development of hypertension is uncertain. We present evidence that following angiotensin II (AngII) infusion the TNFα type 1 receptor (TNFR1) plays a key role in heightened glutamate signaling in the hypothalamic paraventricular nucleus (PVN), a key central coordinator of blood pressure control. Fourteen day administration of a slow-pressor dose of AngII in male mice was associated with transcriptional and post-transcriptional (increased plasma membrane affiliation) regulation of TNFR1 in the PVN. Further, TNFR1 was shown to be critical for elevated NMDA-mediated excitatory currents in sympathoexcitatory PVN neurons following AngII infusion. Finally, silencing PVN TNFR1 prevented the increase in systolic blood pressure induced by AngII. These findings indicate that TNFR1 modulates a cellular pathway involving an increase in NMDA-mediated currents in the PVN following AngII infusion, suggesting a mechanism whereby TNFR1 activation contributes to hypertension via heightened hypothalamic glutamate-dependent signaling.SIGNIFICANCE STATEMENT Inflammation is critical for the emergence of hypertension, yet the mechanisms by which inflammatory mediators contribute to this dysfunction are not clearly defined. We show that tumor necrosis factor α receptor 1 (TNFR1) in the paraventricular hypothalamic nucleus (PVN), a critical neuroregulator of cardiovascular function, plays an important role in the development of hypertension in mice. In the PVN, TNFR1 expression and plasma membrane localization are upregulated during hypertension induced by angiotensin II (AngII). Further, TNFR1 activation was essential for NMDA signaling and the heightening NMDA currents during hypertension. Finally, TNFR1 silencing in the PVN inhibits elevated blood pressure induced by AngII. These results point to a critical role for hypothalamic TNFR1 signaling in hypertension.


Assuntos
Angiotensina II/toxicidade , Ácido Glutâmico/metabolismo , Hipertensão/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Animais , Hipertensão/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Metilaspartato/metabolismo , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos
16.
Angew Chem Int Ed Engl ; 60(12): 6733-6743, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33331089

RESUMO

Selective modulation of ligand-receptor interaction is essential in targeted therapy. In this study, we design an intelligent "scan and unlock" DNA automaton (SUDA) system to equip a native protein-ligand with cell-identity recognition and receptor-mediated signaling in a cell-type-specific manner. Using embedded DNA-based chemical reaction networks (CRNs) on the cell surface, SUDA scans and evaluates molecular profiles of cell-surface proteins via Boolean logic circuits. Therefore, it achieves cell-specific signal modulation by quickly unlocking the protein-ligand in proximity to the target cell-surface to activate its cognate receptor. As a proof of concept, we non-genetically engineered hepatic growth factor (HGF) with distinct logic SUDAs to elicit target cell-specific HGF signaling and wound healing behaviors in multiple heterogeneous cell types. Furthermore, the versatility of the SUDA strategy was shown by engineering tumor necrotic factor-α (TNFα) to induce programmed cell death of target cell subpopulations through cell-specific modulation of TNFR1 signaling.


Assuntos
DNA/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , DNA/química , Fator de Crescimento de Hepatócito/química , Humanos , Ligantes , Modelos Moleculares , Receptores Tipo I de Fatores de Necrose Tumoral/química , Transdução de Sinais
17.
J Pharmacol Exp Ther ; 377(1): 86-99, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33310816

RESUMO

Intracerebral hemorrhage (ICH) is a devastating disease with the high mortality. The poor outcome of ICH is partially due to a combination of various secondary insults, including in the ischemic area. Xuemaitong capsule (XMT), a kind of traditional Chinese medicine, has been applied to clinic practice. The purpose of this study is to explore the mechanism of XMT in alleviating secondary damage in the ischemic area after ICH. We screened XMT target, compound components, and ICH-related targets using network pharmacology, cluster analysis, and enrichment analysis. We found that the tumor necrosis factor (TNF) signaling pathway might be the key signaling pathway for XMT treatment of ICH. An ICH rat model was established, as demonstrated by poor neurologic score. In the ICH rats, Western blot analysis and immunofluorescence indicated the upregulated expression of TNF receptor 1 (TNFR1), mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and caspase-3 (CASP3). Importantly, administration of XMT alleviated inflammation, edema, and increased perfusion in the ischemic area, whereas the expression of TNFR1, MAPK, NF-κB, and CASP3 was decreased. Furthermore, Fluoro-Jade B and terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling staining revealed that XMT application also inhibited apoptosis and degradation of ischemic area neurons. In conclusion, this evidence elucidates that XMT alleviates neuron apoptosis, ischemic area inflammation, edema, and perfusion through the TNFR1-mediated CASP3/NF-κB/MAPK axis. SIGNIFICANCE STATEMENT: Tumor necrosis factor (TNF) is the key signaling pathway of Xuemaitong (XMT) to intervention during intracerebral hemorrhage. Fourteen key targets [intercellular adhesion molecule 1, interleukin (IL) 6, TNF, C-C motif chemokine ligand 2, prostaglandin-endoperoxide synthase 2, v-rel reticuloendotheliosis viral oncogene homolog A, matrix metalloproteinase 9, endothelin-1 (EDN1), mitogen-activated protein kinase (MAPK) 1, fos proto-oncogene protein, caspase-3 (CASP3), jun proto-oncogene, IL1B, MAPK8] are retrieved from the data base. XMT can inhibit neuron apoptosis in the ischemic area via regulating TNF receptor 1 (TNFR1)/CASP3. XMT alleviates inflammation and edema through regulating TNFR1/nuclear factor-κB and TNFR1/MAPK signaling pathways. XMT alleviates hypoperfusion in the cerebral ischemic area through mediating TNFR1/MAPK/EDN1.


Assuntos
Anti-Inflamatórios/farmacologia , Isquemia Encefálica/tratamento farmacológico , Hemorragia Cerebral/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Caspase 3/metabolismo , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/uso terapêutico , Endotelina-1/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/uso terapêutico , Ratos , Ratos Sprague-Dawley , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
18.
Methods Mol Biol ; 2248: 121-137, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33185872

RESUMO

Inhibition of tumor necrosis factor receptor 1 (TNFR1) is a billion-dollar industry for treatment of autoimmune and inflammatory diseases. As current therapeutics of anti-TNF leads to dangerous side effects due to global inhibition of the ligand, receptor-specific inhibition of TNFR1 signaling is an intensely pursued strategy. To monitor directly the structural changes of the receptor in living cells, we engineered a fluorescence resonance energy transfer (FRET) biosensor by fusing green and red fluorescent proteins to TNFR1. Expression of the FRET biosensor in living cells allows for detection of receptor-receptor interactions and receptor structural dynamics. Using the TNFR1 FRET biosensor, in conjunction with a high-precision and high-throughput fluorescence lifetime detection technology, we developed a time-resolved FRET-based high-throughput screening platform to discover small molecules that directly target and modulate TNFR1 functions. Using this method in screening multiple pharmaceutical libraries, we have discovered a competitive inhibitor that disrupts receptor-receptor interactions, and allosteric modulators that alter the structural states of the receptor. This enables scientists to conduct high-throughput screening through a biophysical approach, with relevance to compound perturbation of receptor structure, for the discovery of novel lead compounds with high specificity for modulation of TNFR1 signaling.


Assuntos
Técnicas Biossensoriais , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Receptores Tipo I de Fatores de Necrose Tumoral/química , Linhagem Celular , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Expressão Gênica , Genes Reporter , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligantes , Microscopia de Fluorescência , Ligação Proteica , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Bibliotecas de Moléculas Pequenas , Software , Relação Estrutura-Atividade
19.
Int J Biol Macromol ; 169: 371-383, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33347929

RESUMO

Anti-TNF inhibitors are efficacious in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA), Crohn's disease (CD), juvenile idiopathic arthritis (JIA), and ankylosing spondylitis (AS). However, more and more clinical case reports revealed that anti-TNF inhibitors could increase the risk of viral, fungal, and bacterial (especially intracellular) infection. In this study, based on Immune Epitope Database (IEDB) online B cell epitope prediction and the knowledge of TNF three dimensional (3D) structure we developed a novel vaccine (DTNF114-TNF114) that targeting TNF epitope 1-14, which produced antibodies only partially binding to trans-membrane TNF (tmTNF), therefore partially sparing tmTNF-TNFR1/2 interaction. Immunization with DTNF114-TNF114 significantly protected and prolonged the survival rate of mice challenged with lipopolysaccharide (LPS); and in the mCherry expressing Mycobacterium bovis Bacillus Calmette-Guérin (mCherry-BCG) infection model, DTNF114-TNF114 immunization significantly decreased soluble TNF (solTNF) level in serum, meanwhile did not suppress host immunity against infection. Thus, this novel and infection concern-free vaccine provides a potential alternative or supplement to currently clinically used anti-TNF inhibitors.


Assuntos
Epitopos/imunologia , Mycobacterium bovis/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Vacina BCG/farmacologia , Linhagem Celular , Bases de Dados Factuais , Feminino , Imunização , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Tuberculose/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo
20.
PLoS Biol ; 18(12): e3000967, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33270628

RESUMO

Tumor necrosis factor-alpha (TNF-α) plays an important pathogenic role in cardiac hypertrophy and heart failure (HF); however, anti-TNF is paradoxically negative in clinical trials and even worsens HF, indicating a possible protective role of TNF-α in HF. TNF-α exists in transmembrane (tmTNF-α) and soluble (sTNF-α) forms. Herein, we found that TNF receptor 1 (TNFR1) knockout (KO) or knockdown (KD) by short hairpin RNA or small interfering RNA (siRNA) significantly alleviated cardiac hypertrophy, heart dysfunction, fibrosis, and inflammation with increased tmTNF-α expression, whereas TNFR2 KO or KD exacerbated the pathological phenomena with increased sTNF-α secretion in transverse aortic constriction (TAC)- and isoproterenol (ISO)-induced cardiac hypertrophy in vivo and in vitro, respectively, indicating the beneficial effects of TNFR2 associated with tmTNF-α. Suppressing TNF-α converting enzyme by TNF-α Protease Inhibitor-1 (TAPI-1) to increase endogenous tmTNF-α expression significantly alleviated TAC-induced cardiac hypertrophy. Importantly, direct addition of exogenous tmTNF-α into cardiomyocytes in vitro significantly reduced ISO-induced cardiac hypertrophy and transcription of the pro-inflammatory cytokines and induced proliferation. The beneficial effects of tmTNF-α were completely blocked by TNFR2 KD in H9C2 cells and TNFR2 KO in primary myocardial cells. Furthermore, we demonstrated that tmTNF-α displayed antihypertrophic and anti-inflammatory effects by activating the AKT pathway and inhibiting the nuclear factor (NF)-κB pathway via TNFR2. Our data suggest that tmTNF-α exerts cardioprotective effects via TNFR2. Specific targeting of tmTNF-α processing, rather than anti-TNF therapy, may be more useful for the treatment of hypertrophy and HF.


Assuntos
Cardiomegalia/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/efeitos dos fármacos , Cardiomegalia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/fisiologia
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