Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 241
Filtrar
1.
Fish Shellfish Immunol ; 89: 548-554, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30991146

RESUMO

In this study, a sea cucumber Fas-associated death domain (FADD) named HLFADD was first cloned from Holothuria leucospilota. The full-length cDNA of HLFADD is 2137 bp in size, containing a 116-bp 5'-untranslated region (UTR), a 1334-bp 3'-UTR and a 687-bp open reading frame (ORF) encoding a protein of 228 amino acids with a deduced molecular weight of 26.42 kDa. HLFADD protein contains a conserved death effector domain at its N-terminal and a conserved death domain at its C-terminal, structurally similar to its counterparts in vertebrates. The over-expressed HLFADD protein could induce apoptosis in HEK293 cells, suggesting a possible death receptor-mediated apoptosis pathway in echinoderms adapted with FADD. Moreover, HLFADD mRNA is ubiquitously expressed in all examined tissues, with the highest transcript level in the coelomocytes, followed by intestine. In vitro experiments performed in the H. leucospilota coelomocytes, the expression of HLFADD mRNA was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic acid [poly (I:C)] challenge, suggesting that HLFADD might play important roles in the innate immune defense of sea cucumber against the invasion of bacteria and viruses.


Assuntos
Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/imunologia , Regulação da Expressão Gênica/imunologia , Holothuria/genética , Holothuria/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Proteína de Domínio de Morte Associada a Fas/química , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência , Regulação para Cima
2.
Fish Shellfish Immunol ; 88: 556-566, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30885740

RESUMO

Fas-associated protein with death domain (FADD) is an essential element in cell death, and also implicates in cell cycle progression, inflammation and innate immunity. In the study, an FADD (designated as RpFADD) was identified and characterized from manila clam, Ruditapes philippinarum. Multiple alignments and phylogenetic analysis strongly suggested that RpFADD was a new member of the FADD family. The RpFADD transcripts were constitutively expressed in a wide range of tissues, and dominantly expressed in hemocytes. After challenged with Vibrio anguillarum or Micrococcus luteus, the expression level of RpFADD transcripts was significantly induced and reached the maximum level at 72 h and 48 h, respectively. Knockdown of RpFADD down-regulated the transcript levels of RpIKK, RpTAK1 and RpNF-κB with the exception of RpIκB. Moreover, RpFADD primarily localized in the cell cytoplasm, and its over-expression promoted the apoptosis of HeLa cells. These results revealed that RpFADD perhaps regulated the NF-κB signaling pathways positively, which provided a better understanding of RpFADD in innate immunity.


Assuntos
Bivalves/genética , Proteína de Domínio de Morte Associada a Fas/genética , Imunidade Inata , Transdução de Sinais , Animais , Apoptose , Bivalves/imunologia , Bivalves/microbiologia , Clonagem Molecular , Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Hemócitos/imunologia , Hemócitos/microbiologia , Humanos , Micrococcus luteus , NF-kappa B/metabolismo , Filogenia , Transcriptoma , Vibrio
3.
Proc Natl Acad Sci U S A ; 116(9): 3518-3523, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808745

RESUMO

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme ß1,3-galactosyltransferase V (ß3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of ß3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of ß3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.


Assuntos
Neoplasias da Mama/genética , Galactosiltransferases/genética , Glicoesfingolipídeos/genética , Microdomínios da Membrana/genética , Antígenos Glicosídicos Associados a Tumores/genética , Antígenos Glicosídicos Associados a Tumores/metabolismo , Apoptose/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caveolina 1/genética , Caveolina 1/metabolismo , Progressão da Doença , Proteína de Domínio de Morte Associada a Fas/genética , Feminino , Proteína-Tirosina Quinases de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica/genética , Glicoesfingolipídeos/metabolismo , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Microdomínios da Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt/genética , Saporinas/genética , Transdução de Sinais/genética , Antígenos Embrionários Estágio-Específicos/genética , Antígenos Embrionários Estágio-Específicos/metabolismo
4.
RNA Biol ; 16(2): 233-248, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30628514

RESUMO

The development of chemotherapeutic drugs resistance such as doxorubicin (DOX) and cisplatin (DDP) is the major barrier in gastric cancer therapy. Emerging evidences reveal that microRNAs (miRNAs) contribute to chemosensitivity. In this study, we investigated the role of miR-633, an oncogenic miRNA, in gastric cancer chemoresistance. In gastric cancer tissue and cell lines, miR-633 expression was highly increased and correlated with down regulation of Fas-associated protein with death domain (FADD). Inhibition of miR-633 significantly increased FADD protein level and enhanced DOX/DDP induced apoptosis in vitro. MiR-633 antagomir administration remarkably decreased tumor growth in combination with DOX in vivo, suggesting that miR-633 targets FADD to block gastric cancer cell death. We found that the promoter region of miR-633 contained putative binding sites for forkhead box O 3 (Foxo3a), which can directly repress miR-633 transcription. In addition, we observed that DOX-induced nuclear accumulation of Foxo3a leaded to the suppression of miR-633 transcription. Together, our study revealed that miR-633/FADD axis played a significant role in the chemoresistance and Foxo3a regulated this pathway in gastric cancer. Thus, miR-633 antagomir resensitized gastric cancer cells to chemotherapy drug and had potentially therapeutic implication.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteína de Domínio de Morte Associada a Fas/genética , Proteína Forkhead Box O3/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Adulto , Idoso , Animais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Interferência de RNA , Transcrição Genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Mol Cell ; 73(3): 413-428.e7, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30598363

RESUMO

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.


Assuntos
Caspase 8/metabolismo , Instabilidade Cromossômica , Neoplasias do Colo/enzimologia , Fibroblastos/enzimologia , Mitose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Aneuploidia , Animais , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibroblastos/patologia , Células HT29 , Humanos , Inflamação/enzimologia , Inflamação/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais
6.
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
7.
Int Endod J ; 52(1): 5-12, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29904933

RESUMO

AIM: To investigate the DNA methylation profiles of immune response-related genes in apical periodontitis (AP) lesions. METHODOLOGY: The methylation profiles on the cytosine-phosphate-guanine (CpG) regions of 22 gene promoters involved in inflammation and autoimmunity were assessed in 60 human AP lesions and 24 healthy periodontal ligaments (controls) using a pathway-specific real-time polymerase chain reaction array (EpiTect® Methyl Signature PCR Array Human Inflammatory Response). Differentially methylated genes were subsequently assessed for their mRNA expression. Data analyses (One-way anova, Tukey's multiple comparisons tests and Mann-Whitney tests) were performed using GraphPad Prism 6 software. P values ≤ 0.05 were considered statistically significant. RESULTS: Significant DNA hypermethylation was observed for CXCL3 and FADD gene promoters in AP lesions when compared to control tissues (P < 0.001) and among other genes (P < 0.05). In contrast, IL12B and IL4R were associated with significant hypomethylation in comparison to other genes (P < 0.05). IL12B, IL4R, CXCL3 and FADD had differential mRNA expression in AP lesions and controls (P < 0.001). CONCLUSIONS: Differential methylation profiles of immune response-related genes, such as FADD, CXCL3, IL12B and IL4R, may have an influence on individual AP susceptibility and patient treatment outcomes, through their potential contributions to altered expression of disease-relevant genes. Methylation and/or genetic variations in additional genes may also contribute to the dynamics of AP development and should be considered in future studies.


Assuntos
Metilação de DNA , Periodontite Periapical/genética , Periodontite Periapical/imunologia , Periodontite Periapical/metabolismo , Transcriptoma , Adolescente , Adulto , Idoso , Autoimunidade/genética , Brasil , Quimiocinas/genética , Quimiocinas CXC/genética , Citocinas/genética , Proteína de Domínio de Morte Associada a Fas/genética , Regulação da Expressão Gênica , Humanos , Inflamação , Subunidade p40 da Interleucina-12/genética , Subunidade alfa de Receptor de Interleucina-4/genética , Pessoa de Meia-Idade , Ligamento Periodontal , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Citocinas/genética , Adulto Jovem
8.
Front Immunol ; 9: 2038, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30250469

RESUMO

Intestinal intraepithelial lymphocytes (IELs) play a critical role in mucosal immune system, which differ from thymus-derived cells and develop locally in gut. Although the development of IELs has been studied in some detail, the molecular cues controlling their local development remain unclear. Here, we demonstrate that FADD, a classic adaptor protein required for death-receptor-induced apoptosis, is a critical regulator of the intestinal IEL development. The mice with a dominant negative mutant of FADD (FADD-DN) display an abnormal development of intestinal IELs with a marked reduction in the numbers of CD8αα+TCRγδ+ T cells. As a precursor for CD8αα+ development, lamina propria lymphocytes in lin-negative expression (lin- LPLs) were analyzed and the massive accumulation of IL-7R-lin- LPLs was observed in FADD-DN mice. As IL-7R is one of Notch1-target genes, we further observed that the level of Notch1 expression was lower in Lin- LPLs from FADD-DN mice compared with normal mice. The downregulation of Notch1 expression induced by FADD-DN overexpression was also confirmed in Jurkat T cells. Considering that IL-7 and its receptor IL7-R play a differentiation inducing role in the development of intestinal IELs, the influence of FADD via its DD domain on Notch1 expression might be a possible molecular signal involved in the early IELs development. In addition, loss of γδ T-IELs in FADD-DN mice aggravates DSS-induced colitis, suggesting that FADD is a relevant contribution to the field of mucosal immunology and intestinal homeostasis.


Assuntos
Colite/imunologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , Mucosa Intestinal/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD8/metabolismo , Diferenciação Celular , Colite/induzido quimicamente , Sulfato de Dextrana , Proteína de Domínio de Morte Associada a Fas/genética , Humanos , Imunidade nas Mucosas , Células Jurkat , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/metabolismo , Transdução de Sinais
10.
Molecules ; 23(5)2018 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-29734760

RESUMO

Kaempferol is a widely distributed dietary flavonoid. Epidemiological studies have demonstrated kaempferol consumption lowers the risk of ovarian cancer. Our previous research proved that kaempferol suppresses human ovarian cancer cells by inhibiting tumor angiogenesis. However, the effects of kaempferol on the cell cycle and extrinsic apoptosis of ovarian cancer cells have not yet been studied. In the present study, we demonstrated that kaempferol induced G2/M cell cycle arrest via the Chk2/Cdc25C/Cdc2 pathway and Chk2/p21/Cdc2 pathway in human ovarian cancer A2780/CP70 cells. Chk2 was not responsible for kaempferol-induced apoptosis and up-regulation of p53. Kaempferol stimulated extrinsic apoptosis via death receptors/FADD/Caspase-8 pathway. Our study suggested that Chk2 and death receptors played important roles in the anticancer activity of kaempferol in A2780/CP70 cells. These findings provide more evidence of the anti-ovarian cancer properties of kaempferol and suggest that kaempferol could be a potential candidate for ovarian cancer adjuvant therapy.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Quinase do Ponto de Checagem 2/genética , Células Epiteliais/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Quempferóis/farmacologia , Receptores de Morte Celular/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia , Receptores de Morte Celular/metabolismo , Transdução de Sinais , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo
11.
Cell Mol Biol (Noisy-le-grand) ; 64(6): 31-35, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29808797

RESUMO

The Wnt/ß-catenin signaling pathway plays important roles in cancers such as colorectal cancer. Colon cancer cells secrete and express high levels of ß-catenin, which may stimulate autocrine signaling and further enhance activities of the canonical Wnt signaling pathway. Free ß-catenin in the cytoplasm and nucleus leads to its association with T cell factor (TCF)/lymphocyte enhancing factor (Lef) transcription factors, and subsequent transcriptional activation of downstream target genes. FADD plays a key role in cellular apoptosis in many different types of cancer. Therefore, a recombinant adenovirus is constructed, in which an apoptosis gene FADD is placed under control of a promoter containing Tcf-responsive elements. It is observed that FADD overexpression can suppress cell growth and enhance apoptosis of SW480 cells in vitro. In addition, Ad-FADD can also suppress the growth of subcutaneous xenografts in the nude mice. Together, these results suggest that Ad-FADD has anti-proliferative and pro-apoptotic effects in colon cancer cells, which provides a novel strategy for treatment of colorectal cancer.


Assuntos
Adenocarcinoma/terapia , Adenoviridae/genética , Neoplasias Colorretais/terapia , Proteína de Domínio de Morte Associada a Fas/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Terapia Genética , Vetores Genéticos/uso terapêutico , Via de Sinalização Wnt/fisiologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteína de Domínio de Morte Associada a Fas/biossíntese , Proteína de Domínio de Morte Associada a Fas/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células NIH 3T3 , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição TCF/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cell Death Dis ; 9(3): 346, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29497034

RESUMO

Proteasome inhibitors have achieved clinical success because they trigger intrinsic and extrinsic cell death to eliminate susceptible human cancers. The ubiquitin-proteasome protein degradation system regulates signaling pathways by controlling levels of components such as cellular inhibitor of apoptosis (cIAP)1 and cIAP2 in TNF-mediated cell death. Here, we sought to evaluate the contribution of necroptosis to the cell death pattern induced by the specific proteasome inhibitor Carfilzomib (Cf). Proteasome inhibitor-sensitive multiple myeloma cell lines die in response to Cf by apoptosis in combination with serine protease-dependent death, without any contribution of RIPK3-dependent necroptosis. Proteasome inhibition leads to the induction of apoptotic markers such as activated caspase-3 rather than necroptotic markers such as phosphorylated-MLKL in all cell lines tested. In HT-29 cells, Cf attenuates the late RIPK1 interaction with TNFR1 during TNF-induced necroptosis without altering the sensitivity of cIAP antagonists. Cf treatment results in decreased translocation of death signaling components RIPK1, FADD, caspase-8, cFLIP, and RIPK3 to detergent insoluble fractions. Our results show that proteasome inhibition with Cf impairs necroptosis and favors apoptosis even in cells with intact necroptotic machinery. Following the induction of TNFR1-mediated necroptosis, proteasome activity stabilizes effective aggregation and activation of ripoptosome/necrosome complexes.


Assuntos
Morte Celular/efeitos dos fármacos , Oligopeptídeos/farmacologia , Inibidores de Proteassoma/farmacologia , Apoptose/efeitos dos fármacos , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Apoptosis ; 23(3-4): 226-236, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29468482

RESUMO

Epirubicin is an anthracycline and is widely used in tumor treatment, but has toxic and undesirable side effects on wide range of cells and hematopoietic stem cells (HSC). Osteoblasts play important roles in bone development and in supporting HSC differentiation and maturation. It remains unknown whether epirubicin-induced bone loss and hematological toxicity are associated with its effect on osteoblasts. In primary osteoblast cell cultures, epirubicin inhibited cell growth and decreased mineralization. Moreover, epirubicin arrested osteoblasts in the G2/M phase, and this arrest was followed by apoptosis in which both the extrinsic (death receptor-mediated) and intrinsic (mitochondrial-mediated) apoptotic pathways were evoked. The factors involved in the extrinsic apoptotic pathway were increased FasL and FADD as well as activated caspase-8. Those involved in the intrinsic apoptotic pathway were decreased Bcl-2; increased reactive oxygen species, Bax, cytochrome c; and activated caspase-9 and caspase-3. These results demonstrate that epirubicin induced osteoblast apoptosis through the extrinsic and intrinsic apoptotic pathways, leading to the destruction of osteoblasts and consequent lessening of their functions in maintaining bone density and supporting hematopoietic stem cell differentiation and maturation.


Assuntos
Apoptose/efeitos dos fármacos , Epirubicina/farmacologia , Mitocôndrias/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Caspases/genética , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Citocromos c/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Osteoblastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
14.
Fish Shellfish Immunol ; 74: 517-529, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29355760

RESUMO

Fas-associated protein with death domain (FADD) is the key adaptor protein that transmits apoptotic signals mediated by the main death receptors. Besides being an essential instrument in cell death, FADD is also implicated in proliferation, cell cycle progression, tumor development, inflammation, innate immunity, and autophagy. In the present study, a FADD homologue (EcFADD) from the orange-spotted grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcFADD contains 808 base pairs (bp), including a 573 bp open reading frame that encodes a 190 amino acid protein with a predicted molecular mass of 21.81 kDa. Quantitative real-time polymerase chain reaction analysis indicated that EcFADD was distributed in all examined tissues. The expression of EcFADD in the spleen of E. coioides was differentially up-regulated when challenged with Singapore grouper iridovirus (SGIV) or polyinosine-polycytidylic acid(poly[I:C]). EcFADD was abundantly distributed in both the cytoplasm and nucleus in grouper spleen (GS) and fathead minnow (FHM) epithelial cells. Over-expression of EcFADD inhibited SGIV infection and replication and SGIV-induced apoptosis. To achieve antiviral and anti-apoptosis activities, FADD promoted the activation of interferon-stimulated response element (ISRE) and type I interferon (IFN) genes in the antiviral IFN signaling pathway and inhibited activation of apoptosis-related transcription factors p53. Our results not only characterize FADD but also reveal new immune functions and the molecular mechanisms by which FADD responds to virus infection and virus-induced apoptosis.


Assuntos
Bass/genética , Bass/imunologia , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína de Domínio de Morte Associada a Fas/química , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Filogenia , Poli I-C/farmacologia , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária
15.
J Immunol ; 200(1): 271-285, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29167229

RESUMO

In mammalian cells, signaling pathways triggered by TNF can be switched from NF-κB activation to apoptosis and/or necroptosis. The in vivo mechanisms underlying the mutual regulation of these three signaling pathways are poorly understood. In this article, we report that the embryonic lethality of RelA-deficient mice is partially prevented by the deletion of Rip3 or Mlkl, but it is fully rescued by the combined ablation of Fadd and Rip3 or Mlkl or by blocking RIP1 kinase activity (RIP1K45A). RelA-/-Fadd-/-Rip3-/- triple-knockout (TKO) and RelA-/-Rip1K45A/K45A mice displayed bacterial pneumonia leading to death ∼2 wk after birth. Moreover, RelA-/-Rip1K45A/K45A mice, but not TKO mice, developed severe inflammation associated with inflammatory skin lesion. Antibiotic treatment improved bacterial pneumonia, extended the lifespan of TKO and RelA-/-Rip1K45A/K45A mice, and alleviated skin inflammation in RelA-/-Rip1K45A/K45A mice. These results show the mechanisms underlying the in vivo mutual regulation between NF-κB activation and the cell death pathway and provide new insights into this interplay in embryonic development and host immune homeostasis.


Assuntos
Perda do Embrião/genética , Pneumonia/imunologia , Fator de Transcrição RelA/genética , Animais , Apoptose/genética , Células Cultivadas , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Feminino , Imunidade , Inflamação , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Necrose/genética , Pneumonia/genética , Gravidez , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Ativação Transcricional
16.
Cell Death Dis ; 8(8): e3025, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-29048428

RESUMO

Besides its tumor-selective apoptotic activity, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) promotes pro-survival, proliferative or migratory signaling (NF-κB, PI3K/Akt, MAPK and JNK; referred to as 'non-apoptotic' cascades). Indeed, apoptosis and non-apoptotic signaling can be activated in clonal populations of cancer cells in response to treatment and, as a result, only a part of the initial cellular population dies while a fraction survives and develops resistance to TRAIL-induced apoptosis (referred to as 'fractional survival'). Notably, the molecular characterization of the protein platforms streaming into tumoricidal versus tumor-promoting cascades that control fractional survival remained elusive. Here we demonstrate that, in the context of DR4-DR5-DcR2 hetero-oligomeric complexes, a single death receptor (DR5) suffices to assemble composite plasma membrane-proximal pro-apoptotic/pro-survival platforms that propagate TRAIL signaling to both death and survival pathways in clonal populations of cancer cells. Moreover, we show that while all members of TRAIL-induced complexes support survival, none of them acted exclusively pro-apoptotic. Indeed, key apoptotic proteins as FADD and procaspase-8 were also involved in transducing non-apoptotic signaling in response to this cytokine. Collectively, this study reveals the Janus faces of DR5, and the contributions of other death complex components in fractional survival that foster the generation of resistance. Our data highlight a new level of complexity in TRAIL signaling and point to an improved therapeutic rationale in view of hitherto disappointing results.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Fibroblastos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/genética , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo
17.
Free Radic Biol Med ; 112: 567-577, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28888620

RESUMO

Ligation of the death receptors for TNF-α, FasL, and TRAIL triggers two common pathways, caspase-dependent intrinsic apoptosis and intracellular reactive oxygen species (ROS) generation. The apoptotic pathway is well characterized; however, a signaling linker between the death receptor and ROS production has not been clearly elucidated. Here, we found that death receptor-induced ROS generation was strongly inhibited by mitochondrial complex I and II inhibitors, but not by inhibitors of NADPH oxidase, lipoxygenase, cyclooxygenase or xanthine oxidase, indicating that ROS are mostly generated by the impairment of the mitochondrial respiratory chain. ROS generation was accompanied by caspase-8 activation, Bid cleavage, and cytochrome c release; it was blocked in FADD- and caspase-8-deficient cells, as well as by caspase-8 knockdown and inhibitor. Moreover, Bid knockdown abrogated TNF-α- or TRAIL-induced ROS generation, whereas overexpression of truncated Bid (tBid) or knockdown of cytochrome c spontaneously elevated ROS production. In addition, p53-overexpressing cells accumulated intracellular ROS via cytochrome c release mediated by the BH3-only protein Noxa induction. In a cell-free reconstitution system, caspase-8-mediated Bid cleavage and recombinant tBid induced mitochondrial cytochrome c release and ROS generation, which were blocked by Bcl-xL and antioxidant enzymes. These data suggest that anti-apoptotic Bcl-2 proteins play an important role in mitochondrial ROS generation by preventing cytochrome c release. These data provide evidence that the FADD/caspase-8/Bid/cytochrome c axis is a crucial linker between death receptors and mitochondria, where they play a role in ROS generation and apoptosis.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Caspase 8/genética , Citocromos c/genética , Mitocôndrias Hepáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Células Jurkat , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , 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 bcl-X/genética , Proteína bcl-X/metabolismo
18.
J Biol Chem ; 292(42): 17337-17350, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28860194

RESUMO

The inhibition of host innate immunity pathways is essential for the persistence of attaching and effacing pathogens such as enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium during mammalian infections. To subvert these pathways and suppress the antimicrobial response, attaching and effacing pathogens use type III secretion systems to introduce effectors targeting key signaling pathways in host cells. One such effector is the arginine glycosyltransferase NleB1 (NleBCR in C. rodentium) that modifies conserved arginine residues in death domain-containing host proteins with N-acetylglucosamine (GlcNAc), thereby blocking extrinsic apoptosis signaling. Ectopically expressed NleB1 modifies the host proteins Fas-associated via death domain (FADD), TNFRSF1A-associated via death domain (TRADD), and receptor-interacting serine/threonine protein kinase 1 (RIPK1). However, the full repertoire of arginine GlcNAcylation induced by pathogen-delivered NleB1 is unknown. Using an affinity proteomic approach for measuring arginine-GlcNAcylated glycopeptides, we assessed the global profile of arginine GlcNAcylation during ectopic expression of NleB1, EPEC infection in vitro, or C. rodentium infection in vivo NleB overexpression resulted in arginine GlcNAcylation of multiple host proteins. However, NleB delivery during EPEC and C. rodentium infection caused rapid and preferential modification of Arg117 in FADD. This FADD modification was extremely stable and insensitive to physiological temperatures, glycosidases, or host cell degradation. Despite its stability and effect on the inhibition of apoptosis, arginine GlcNAcylation did not elicit any proteomic changes, even in response to prolonged NleB1 expression. We conclude that, at normal levels of expression during bacterial infection, NleB1/NleBCR antagonizes death receptor-induced apoptosis of infected cells by modifying FADD in an irreversible manner.


Assuntos
Apoptose , Citrobacter rodentium/enzimologia , Escherichia coli Enteropatogênica/enzimologia , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Glicosiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Fatores de Virulência/metabolismo , Citrobacter rodentium/patogenicidade , Escherichia coli Enteropatogênica/patogenicidade , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/genética , Proteína de Domínio de Morte Associada a Fas/genética , Glicosiltransferases/genética , Células HeLa , Humanos , Estabilidade Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fatores de Virulência/genética
19.
Cell Physiol Biochem ; 42(6): 2364-2376, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28848149

RESUMO

BACKGROUND: Long noncoding RNA (lncRNA) H19 is emerging as a vital regulatory molecule in the progression of different types of cancer and miR-675 is reported to be embedded in H19's first exon. However, their function and specific mechanisms of action have not been fully elucidated. The aim of this study was to identify a novel lncRNA-microRNA-mRNA functional network in gastric cancer. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the relative expression of H19 and miR-675 in normal (GES-1) and gastric cancer cell lines (SGC-7901, SGC-7901/DDP) as well as in tumor tissues. Gain and loss of function approaches were carried out to investigate the potential roles of H19/miR-675 in cell proliferation and apoptosis. Moreover, Fas associated via death domain (FADD) was validated to be the target of miR-675 via luciferase reporter assay. Western blotting was used to evaluate the protein expression of related signaling pathway. RESULTS: In our study H19 and miR-675 were increased in gastric cancer cell lines and tissues. Overexpression of H19 and miR-675 promoted cell proliferation and inhibited cell apoptosis, whereas knockdown of H19 and miR-675 inhibited these effects. By further examining the underlying mechanism, we showed that H19/miR-675 axis inhibited expression of FADD. FADD downregulation subsequently inhibited the caspase cleavage cascades including caspase 8 and caspase 3. CONCLUSION: Taken together, our results point to a novel regulatory pathway H19/miR-675/ FADD/caspase 8/caspase 3 in gastric cancer which may be potential target for cancer therapy.


Assuntos
Caspase 3/metabolismo , Caspase 8/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/patologia , Animais , Antagomirs/metabolismo , Apoptose , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Regulação para Baixo , Proteína de Domínio de Morte Associada a Fas/antagonistas & inibidores , Proteína de Domínio de Morte Associada a Fas/genética , Humanos , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , Alinhamento de Sequência , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Transplante Heterólogo
20.
Cell Death Differ ; 24(8): 1459-1469, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28574501

RESUMO

RIP1 is an essential regulator of TNF-induced signaling complexes mediating NF-κB activation, apoptosis and necroptosis. Loss of Rip1 rescues the embryonic lethality of Fadd or Caspase-8-deficient mice, even though the double knockout mice die shortly after birth like Rip1-deficient mice. Recent studies demonstrated that mice expressing RIP1 kinase-dead mutants developed normally and resisted necroptotic stimuli in vitro and in vivo. However, the impact of RIP1 kinase activity on Fadd-/- embryonic development remains unknown. Here, we engineered two RIP1 kinase inactive mutant mouse lines, a Rip1K45A/K45A mouse line as previously reported and a novel Rip1Δ/Δ mouse line with an altered P-loop in the kinase domain. While RIP1K45A could not rescue the embryonic lethality of Fadd-deficient mice at E11.5, RIP1Δ rescued lethality of Fadd-/- mice at E11.5 and Fadd-/-Rip1Δ/Δ mice eventually died at E16.5 due to excessive death of fetal liver cells and unregulated inflammation. Under necropotosis-inducing conditions, comparing to Rip1K45A/K45A cells, Rip1Δ/Δcells displayed reduced phosphorylation and oligomerization of RIP3 and MLKL, which lead to increased cell viability. Thus, our study provides genetic evidence that different kinase inactive mutations have distinct impacts on the embryogenesis of Fadd-deficient mice, which might attribute to their extents of protection on necroptosis signaling.


Assuntos
Desenvolvimento Embrionário/genética , Proteína de Domínio de Morte Associada a Fas/genética , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Embrião de Mamíferos , Proteína de Domínio de Morte Associada a Fas/deficiência , Feminino , Proteínas Ativadoras de GTPase/deficiência , Genes Letais , Camundongos , Camundongos Knockout , Mutação , Fosforilação , Gravidez , Multimerização Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA