RESUMO
The inflammasome components NLRP3 and ASC are cytosolic proteins, which upon sensing endotoxins or danger cues, form multimeric complexes to process interleukin (IL)-1ß for secretion. Here we found that antigen (Ag)-triggered degranulation of IgE-sensitized mast cells (MCs) was mediated by NLRP3 and ASC. IgE-Ag stimulated NEK7 and Pyk2 kinases in MCs to induce the deposition of NLRP3 and ASC on granules and form a distinct protein complex (granulosome) that chaperoned the granules to the cell surface. MCs deficient in NLRP3 or ASC did not form granulosomes, degranulated poorly in vitro and did not evoke systemic anaphylaxis in mice. IgE-Ag-triggered anaphylaxis was prevented by an NLRP3 inhibitor. In endotoxin-primed MCs, pro-IL-1ß was rapidly packaged into granules after IgE-Ag stimulation and processed within granule remnants by proteases after degranulation, causing lethal anaphylaxis in mice. During IgE-Ag-mediated degranulation of endotoxin-primed MCs, granulosomes promoted degranulation, combined with exteriorization and processing of IL-1ß, resulting in severe inflammation.
Assuntos
Anafilaxia , Inflamassomos , Camundongos , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Mastócitos , Anafilaxia/metabolismo , Imunoglobulina E/metabolismo , Endotoxinas/metabolismo , Degranulação CelularRESUMO
BACKGROUND: Polyoxyethylene hydrogenated castor oil (HCO ethoxylates) is a nonionic surfactant used as an excipient for ointments and injections in human and veterinary drugs. Several polyethylene glycol (PEG) derivatives can be obtained depending on the number of moles of ethylene oxide (EO). HCO ethoxylates have the potential to cause anaphylactoid reactions. There is little published information about these types of reactions in dogs. OBJECTIVE: To determine the potential for HCO-ethoxylate-containing drugs to cause anaphylactoid reactions in dogs, employing intradermal testing (IDT) with various concentrations of HCO ethoxylates (HCO-25, -40, -60 and -80). ANIMALS: Four healthy male laboratory dogs. MATERIALS AND METHODS: We performed IDT with drugs containing HCO ethoxylates and HCO ethoxylates alone to determine threshold concentrations. The IDT scores and threshold concentrations were compared. Analysis of skin biopsies from IDT sites was used to measure the percentage of degranulated mast cells. The effect of histamine at IDT sites was investigated by pre-treatment with an antihistamine. RESULTS: All HCO-ethoxylate-containing drugs caused a wheal-and-flare reaction. The threshold concentrations (0.001% and 0.00001%) of each HCO-ethoxylate depended on the number of moles of EO (p < 0.05). Mast cell degranulation was enhanced by all HCO ethoxylates. The HCO-60-induced reaction was suppressed by an oral antihistamine. CONCLUSIONS AND CLINICAL RELEVANCE: The threshold concentration can serve as a consideration for developing safe new drug formulations and for clinical decision-making around using drugs containing PEG derivatives. IDT is useful to predict the risk of adverse effects. Antihistamines could demonstrate a prophylactic effect.
Assuntos
Anafilaxia , Óleo de Rícino , Doenças do Cão , Animais , Cães , Óleo de Rícino/efeitos adversos , Masculino , Anafilaxia/induzido quimicamente , Anafilaxia/veterinária , Doenças do Cão/induzido quimicamente , Polietilenoglicóis/efeitos adversos , Testes Intradérmicos/veterinária , Excipientes/efeitos adversos , Excipientes/química , Pele/efeitos dos fármacos , Pele/patologiaRESUMO
Condensed-bicyclic 4,6-substituted1,2,4-triazolo-1,3,4-thiadiazole derivatives (CBTT) have been shown to possess a wide spectrum of pharmacological activities. In this study, several novel CBTT derivatives were synthesized and investigated for their possible role as anti-neoplastic agents. The anti-proliferative effect of various CBTT derivatives was analyzed against tumor cell lines by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay. One of the potential CBTT derivative, 5-(3-(2,3-dichlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)flurobenzonitrile (DTTF) was found to be the most potent against cervical cancer SiHa cells and exhibited minimal effect against normal cells. Molecular docking analysis indicated that transcription factor NF-κB was one of the potential molecular targets modulated by DTTF. Specifically, the drug blocked the TNFα-induced phosphorylation of upstream IκBα kinase in a time-dependent manner leading to the suppression of NF-κB activation and nuclear translocation. DTTF also potentiated the apoptotic effect of TNFα, as well as significantly inhibited migration and invasion of tumor cells. Overall, these findings indicate a potential novel role and mechanism(s) of action of DTTF as an anticancer agent against diverse malignancies.
Assuntos
Apoptose/efeitos dos fármacos , Quinase I-kappa B/genética , Fator de Necrose Tumoral alfa/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Humanos , Quinase I-kappa B/química , Simulação de Acoplamento Molecular , NF-kappa B/química , NF-kappa B/genética , Invasividade Neoplásica/genética , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Tiadiazóis/administração & dosagem , Tiadiazóis/química , Fator de Transcrição RelA/química , Fator de Transcrição RelA/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin-mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.
Assuntos
Infecções por Escherichia coli/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Infecções Urinárias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Acetilcisteína , Animais , Linhagem Celular , Escherichia coli/metabolismo , Feminino , Imunofluorescência , Humanos , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Reação em Cadeia da Polimerase , Transporte Proteico/fisiologia , Transfecção , Bexiga Urinária/microbiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/metabolismoRESUMO
The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-ß production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by ß-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.
Assuntos
Regulação da Expressão Gênica/imunologia , Interferon Tipo I/imunologia , Monoéster Fosfórico Hidrolases/imunologia , Receptores do Ácido Retinoico/imunologia , Transdução de Sinais/imunologia , Humanos , Imunidade Inata/imunologia , Fator Regulador 3 de Interferon/imunologia , RNA Interferente PequenoRESUMO
Wound healing is critical for normal development and pathological processes including cancer cell metastasis. MAPK, Rho-GTPases and NFκB are important regulators of wound healing, but mechanisms for their integration are incompletely understood. Annexin-A1 (ANXA1) is upregulated in invasive breast cancer cells resulting in constitutive activation of NFκB. We show here that silencing ANXA1 increases the formation of stress fibers and focal adhesions, which may inhibit wound healing. ANXA1 regulated wound healing is dependent on the activation of ERK1/2. ANXA1 increases the activation of RhoA, which is dependent on ERK activation. Furthermore, active RhoA is important in NF-κB activation, where constitutively active RhoA potentiates NFκB activation, while dominant negative RhoA inhibits NFκB activation in response to CXCL12 stimulation and active MEKK plasmids. These findings establish a central role for ANXA1 in the cell migration through the activation of NFκB, ERK1/2 and RhoA.
Assuntos
Anexina A1/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Cicatrização/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Movimento Celular , Feminino , Humanos , Células MCF-7 , Células Tumorais CultivadasRESUMO
TLRs play a pivotal role in the recognition of bacteria and viruses. Members of the family recognize specific pathogen sequences to trigger both MyD88 and TRIF-dependent pathways to stimulate a plethora of cells. Aberrant activation of these pathways is known to play a critical role in the development of autoimmunity and cancer. However, how these pathways are entirely regulated is not fully understood. In these studies, we have identified Annexin-A1 (ANXA1) as a novel regulator of TLR-induced IFN-ß and CXCL10 production. We demonstrate that in the absence of ANXA1, mice produce significantly less IFN-ß and CXCL10, and macrophages and plasmacytoid dendritic cells have a deficiency in activation following polyinosinic:polycytidylic acid administration in vivo. Furthermore, a deficiency in activation is observed in macrophages after LPS and polyinosinic:polycytidylic acid in vitro. In keeping with these findings, overexpression of ANXA1 resulted in enhanced IFN-ß and IFN-stimulated responsive element promoter activity, whereas silencing of ANXA1 impaired TLR3- and TLR4-induced IFN-ß and IFN-stimulated responsive element activation. In addition, we show that the C terminus of ANXA1 directly associates with TANK-binding kinase 1 to regulate IFN regulatory factor 3 translocation and phosphorylation. Our findings demonstrate that ANXA1 plays an important role in TLR activation, leading to an augmentation in the type 1 IFN antiviral cytokine response.
Assuntos
Anexina A1/metabolismo , Interferon beta/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Anexina A1/biossíntese , Anexina A1/genética , Linhagem Celular , Quimiocina CXCL10/biossíntese , Células Dendríticas/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/metabolismo , Lipopolissacarídeos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosforilação , Poli I-C/farmacologia , Transdução de Sinais/imunologiaRESUMO
Acinetobacter baumannii is a major extensively drug-resistant lethal human nosocomial bacterium. However, the host innate immune mechanisms controlling A. baumannii are not well understood. Although viewed as an extracellular pathogen, A. baumannii can also invade and survive intracellularly. However, whether host innate immune pathways sensing intracellular bacteria contribute to immunity against A. baumannii is not known. Here, we provide evidence for the first time that intracellular antibacterial innate immune receptors Nod1 and Nod2, and their adaptor Rip2, play critical roles in the sensing and clearance of A. baumannii by human airway epithelial cells in vitro. A. baumannii infection upregulated Rip2 expression. Silencing of Nod1, Nod2, and Rip2 expression profoundly increased intracellular invasion and prolonged the multiplication and survival of A. baumannii in lung epithelial cells. Notably, the Nod1/2-Rip2 axis did not contribute to the control of A. baumannii infection of human macrophages, indicating that they play cell type-specific roles. The Nod1/2-Rip2 axis was needed for A. baumannii infection-induced activation of NF-κB but not mitogen-activated protein kinases. Moreover, the Nod1/2-Rip2 axis was critical to induce optimal cytokine and chemokine responses to A. baumannii infection. Mechanistic studies showed that the Nod1/2 pathway contributed to the innate control of A. baumannii infection through the production of ß-defensin 2 by airway epithelial cells. This study revealed new insights into the immune control of A. baumannii and may contribute to the development of effective immune therapeutics and vaccines against A. baumannii.
Assuntos
Infecções por Acinetobacter/imunologia , Acinetobacter baumannii/imunologia , Imunidade Inata/imunologia , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Infecções por Acinetobacter/genética , Infecções por Acinetobacter/microbiologia , Linhagem Celular , Quimiocinas/genética , Quimiocinas/imunologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Células HEK293 , Humanos , Imunidade Inata/genética , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , NF-kappa B/genética , NF-kappa B/imunologia , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologia , beta-Defensinas/genética , beta-Defensinas/imunologiaRESUMO
The TSC1-TSC2 complex has recently been implicated in cell survival responses. We observed that NF-kappaB signaling is attenuated in TSC1- and TSC2-deficient MEFs concomitant with reduced survival following DNA damage or TNFalpha stimulation. Reconstitution of TSC2 expression in TSC2(-/-) MEFs rescued survival in an NF-kappaB activity-dependent manner. Furthermore, in TSC2(-/-) MEFs, the rapamycin-mediated inhibition of deregulated mTOR activity restored NF-kappaB activation and survival. This rapamycin-mediated effect was reversed by inhibition of NF-kappaB transcriptional activation or by inhibition of ERK1/2 MAP kinase or PI-3K pathways, which lie on signaling cascades that lead to NF-kappaB activation. These results provide evidence for a crosstalk between the TSC/Rheb/mTOR pathway and the NF-kappaB induction pathways and indicate that NF-kappaB functions as an important survival factor that regulates TSC2-dependent cell survival.
Assuntos
NF-kappa B/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Fibroblastos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transcrição Gênica/genética , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
According to WHO, dengue virus is classed among major threats for future pandemics and remains at large an unmet medical need as there are currently no relevant antiviral drugs whereas vaccine developments have met with safety concerns, mostly due to secondary infections caused by antibody-dependant-enhancement in cross infections among the four dengue serotypes. This adds extra complexity in dengue antiviral research and has impeded the progress in this field. Following through our previous effort which born the allosteric, dual-mode inhibitor SP-471P (a carbazole derivative, EC50 1.1 µM, CC50 100 µM) we performed further optimisation while preserving the two arylamidoxime arms and the bromoaryl domain present in SP-471P. Examination of the relative positions of these functionalities within this three-point pharmacophore ultimately led us to an indolazepinone scaffold and our lead compound SP-1769B. SP-1769B is among the most cell-efficacious against all serotypes (DENV2/3 EC50 100 nM, DENV1/4 EC50 0.95-1.25 µM) and safest (CC50 > 100 µM) anti-dengue compounds in the literature that also completely inhibits a secondary ADE-driven infection.
RESUMO
Dengue virus (DENV) nonstructural protein 5 (NS5), consisting of methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, is critical for viral RNA synthesis within endoplasmic reticulum-derived replication complexes in the cytoplasm. However, a significant proportion of NS5 is localized to the nucleus of infected cells for DENV2, 3, and 4, whereas DENV1 NS5 is localized diffusely in the cytoplasm. We still have an incomplete understanding of how the DENV NS5 subcellular localization is regulated. Within NS5, two putative nuclear localization signal (NLS) sequences have been identified: NLSCentral residing in the palm of the RdRp domain as well as the recently discovered NLSC-term residing in the flexible region at the C-terminal of the RdRp domain. We have previously shown that DENV2 NS5 nuclear localization can be significantly reduced by single-point mutations to the NLSC-term. Here, we present biochemical, virological, and structural data demonstrating that the relative importance of either NLS in NS5 nuclear localization is unique to each of the four DENV serotypes. DENV1 NS5's cytoplasmic localization appears to be due to a functionally weak interaction between its NLSCentral and importin-α (IMPα), while DENV2 NS5 is almost exclusively nuclear through its NLSC-term's strong interaction with IMPα. Both NLSs of DENV3 NS5 appear to contribute to directing its nuclear localization. Lastly, in the case of DENV4, the regulation of its NS5 nuclear localization remains an enigma but appears to be associated with its NLSC-term.
Assuntos
Núcleo Celular , Vírus da Dengue , Sinais de Localização Nuclear , Sorogrupo , Proteínas não Estruturais Virais , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/química , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Núcleo Celular/metabolismo , Humanos , Citoplasma/metabolismo , Replicação Viral , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/química , Animais , Dengue/virologia , Transporte ProteicoRESUMO
Urinary tract infections (UTIs) account for almost 25% of infections in women. Many are recurrent (rUTI), with patients frequently experiencing chronic pelvic pain and urinary frequency despite clearance of bacteriuria after antibiotics. To elucidate the basis for these bacteria-independent bladder symptoms, we examined the bladders of patients with rUTI. We noticed a notable increase in neuropeptide content in the lamina propria and indications of enhanced nociceptive activity. In mice subjected to rUTI, we observed sensory nerve sprouting that was associated with nerve growth factor (NGF) produced by recruited monocytes and tissue-resident mast cells. Treatment of rUTI mice with an NGF-neutralizing antibody prevented sprouting and alleviated pelvic sensitivity, whereas instillation of native NGF into naïve mice bladders mimicked nerve sprouting and pain behavior. Nerve activation, pain, and urinary frequency were each linked to the presence of proximal mast cells, because mast cell deficiency or treatment with antagonists against receptors of several direct or indirect mast cell products was each effective therapeutically. Thus, our findings suggest that NGF-driven sensory sprouting in the bladder coupled with chronic mast cell activation represents an underlying mechanism driving bacteria-independent pain and voiding defects experienced by patients with rUTI.
Assuntos
Mastócitos , Bexiga Urinária , Humanos , Camundongos , Feminino , Animais , Bexiga Urinária/inervação , Bexiga Urinária/metabolismo , Fator de Crescimento Neural/metabolismo , Reinfecção/complicações , Reinfecção/metabolismo , Dor/etiologia , Dor/metabolismo , Dor/prevenção & controleRESUMO
Scrub typhus is a major infectious threat in the Asia-Pacific region. We report an unusual case of scrub typhus in a patient in Singapore who presented with sepsis and acute respiratory distress syndrome but lacked the pathognomonic eschar. The patient recovered after appropriate diagnosis and doxycycline treatment. Rickettsial diseases should be included in the differential diagnosis of febrile illnesses in regions where the diseases are endemic, and absence of eschar should not be the criterion used to rule out scrub typhus.
Assuntos
Síndrome do Desconforto Respiratório/diagnóstico , Tifo por Ácaros/complicações , Tifo por Ácaros/diagnóstico , Sepse/complicações , Sepse/diagnóstico , Adulto , Antibacterianos/uso terapêutico , Anticorpos Antibacterianos/sangue , Ásia , Western Blotting , Doxiciclina/uso terapêutico , Humanos , Imunoglobulina A/sangue , Imunoglobulina M/sangue , Masculino , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/patologia , Tifo por Ácaros/tratamento farmacológico , Tifo por Ácaros/patologia , Sepse/tratamento farmacológico , Sepse/patologia , Singapura , Resultado do TratamentoRESUMO
BACKGROUND: Increasing evidence indicates that the interaction between the CXC chemokine receptor-4 (CXCR4) and its ligand CXCL12 is critical in the process of metastasis that accounts for more than 90% of cancer-related deaths. Thus, novel agents that can downregulate the CXCR4/CXCL12 axis have therapeutic potential in inhibiting cancer metastasis. METHODS: In this report, we investigated the potential of an agent, plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), for its ability to modulate CXCR4 expression and function in various tumor cells using Western blot analysis, DNA binding assay, transient transfection, real time PCR analysis, chromatin immunoprecipitation, and cellular migration and invasion assays. RESULTS: We found that plumbagin downregulated the expression of CXCR4 in breast cancer cells irrespective of their HER2 status. The decrease in CXCR4 expression induced by plumbagin was not cell type-specific as the inhibition also occurred in gastric, lung, renal, oral, and hepatocellular tumor cell lines. Neither proteasome inhibition nor lysosomal stabilization had any effect on plumbagin-induced decrease in CXCR4 expression. Detailed study of the underlying molecular mechanism(s) revealed that the regulation of the downregulation of CXCR4 was at the transcriptional level, as indicated by downregulation of mRNA expression, inhibition of NF-κB activation, and suppression of chromatin immunoprecipitation activity. In addition, using a virtual, predictive, functional proteomics-based tumor pathway platform, we tested the hypothesis that NF-κB inhibition by plumbagin causes the decrease in CXCR4 and other metastatic genes. Suppression of CXCR4 expression by plumbagin was found to correlate with the inhibition of CXCL12-induced migration and invasion of both breast and gastric cancer cells. CONCLUSIONS: Overall, our results indicate, for the first time, that plumbagin is a novel blocker of CXCR4 expression and thus has the potential to suppress metastasis of cancer.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Naftoquinonas/farmacologia , Receptores CXCR4/metabolismo , Neoplasias da Mama , Linhagem Celular Tumoral , Quimiocina CXCL12/farmacologia , Quimiocina CXCL12/fisiologia , Simulação por Computador , Regulação para Baixo , Feminino , Genes Reporter , Humanos , Luciferases/biossíntese , Luciferases/genética , Modelos Biológicos , NF-kappa B/genética , NF-kappa B/metabolismo , Invasividade Neoplásica , Ligação Proteica , Receptores CXCR4/genética , Neoplasias Gástricas , Transcrição Gênica/efeitos dos fármacosRESUMO
Increasing evidences indicate that CXCR4/CXCL12 signaling pathway plays a pivotal role in the process of distant site metastasis that accounts for more than 90% of prostate cancer related deaths in patients. Thus, novel drugs that can downregulate CXCR4/CXCL12 axis have a great potential in the treatment of metastatic prostate cancer. In this report, we tested an agent, ursolic acid (UA) for its ability to modulate CXCR4 expression in prostate cancer cell lines and inhibit metastasis in vivo in transgenic adenocarcinoma of mouse prostate (TRAMP) model. We observed that UA downregulated the expression of CXCR4 in prostate cancer cells irrespective of their HER2 status in a dose- and time-dependent manner. Neither proteasome inhibitor nor lysosomal stabilization had any effect on UA-induced decrease in CXCR4 expression. When investigated for the molecular mechanisms, it was observed that the downregulation of CXCR4 was due to transcriptional regulation as indicated by downregulation of mRNA expression, inhibition of NF-κB activation and modulation of chromatin immunoprecipitation activity. Suppression of CXCR4 expression by UA further correlated with the inhibition of CXCL12-induced migration and invasion in prostate cancer cells. Finally, we also found that UA treatment can inhibit metastasis of prostate cancer to distal organs, including lung and liver and suppress CXCR4 expression levels in the prostate tissues of TRAMP mice. Overall, our experimental findings suggest that UA exerts its antimetastatic effects through the suppression of CXCR4 expression in prostate cancer both in vitro and in vivo.
Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Quimiocina CXCL12/metabolismo , Neoplasias da Próstata/metabolismo , Receptores CXCR4/metabolismo , Triterpenos/farmacologia , Adenocarcinoma/patologia , Animais , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Metástase Neoplásica/prevenção & controle , Neoplasias da Próstata/patologia , Complexo de Endopeptidases do Proteassoma , Transdução de Sinais/efeitos dos fármacos , Ácido UrsólicoRESUMO
Recently a G-protein-coupled receptor, MAS Related GPR Family Member X2 (MRGPRX2), was identified as a specific receptor on human mast cells responsible for IgE independent adverse drug reactions (ADR). Although a murine homologue, Mrgprb2, has been identified for this receptor, its affinity for many ADR-causing drugs is poor making it difficult to undertake in vivo studies to examine mechanisms of ADR and to develop therapeutic strategies. Here, we have created humanized mice capable of generating MRGPRX2-expressing human MCs allowing for the study of MRGPRX2 MCs-mediated ADR in vitro as well as in vivo. Humanized mice were generated by hydrodynamic-injection of plasmids expressing human GM-CSF and IL-3 into NOD-scid IL2R-γ-/- strain of mice that had been transplanted with human hematopoietic stem cells. These GM/IL-3 humice expressed high numbers of tissue human MCs but the MRGPRX2 receptor expressed in MCs were limited to few body sites including the skin. Importantly, large numbers of MRGPRX2-expressing human MCs could be cultured from the bone marrow of GM/IL-3 humice revealing these mice to be an important source of human MCs for in vitro studies of MRGPRX2-related MCs activities. When GM/IL-3 humice were exposed to known ADR causing contrast agents (meglumine and gadobutrol), the humice were found to experience anaphylaxis analogous to the clinical situation. Thus, GM/IL-3 humice represent a valuable model for investigating in vivo interactions of ADR-causing drugs and human MCs and their sequelae, and these mice are also a source of human MRGPRX2-expressing MCs for in vitro studies.
Assuntos
Modelos Animais de Doenças , Toxidermias/imunologia , Mastócitos/imunologia , Proteínas do Tecido Nervoso/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores de Neuropeptídeos/imunologia , Animais , Meios de Contraste/toxicidade , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Interleucina-3/genética , Mastócitos/efeitos dos fármacos , Meglumina/toxicidade , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Compostos Organometálicos/toxicidadeRESUMO
Mast cells (MCs) are strategically distributed at barrier sites and prestore various immunocyte-recruiting cytokines, making them ideal targets for selective activation to treat peripheral infections. Here, we report that topical treatment with mastoparan, a peptide MC activator (MCA), enhances clearance of Staphylococcus aureus from infected mouse skins and accelerates healing of dermonecrotic lesions. Mastoparan functions by activating connective tissue MCs (CTMCs) via the MRGPRX2 (Mas-related G protein-coupled receptor member X2) receptor. Peripheral CTMC activation, in turn, enhances recruitment of bacteria-clearing neutrophils and wound-healing CD301b+ dendritic cells. Consistent with MCs playing a master coordinating role, MC activation also augmented migration of various antigen-presenting dendritic cells to draining lymph nodes, leading to stronger protection against a second infection challenge. MCAs therefore orchestrate both the innate and adaptive immune arms, which could potentially be applied to combat peripheral infections by a broad range of pathogens.
Assuntos
Mastócitos/imunologia , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/metabolismo , Imunidade Adaptativa/efeitos dos fármacos , Administração Tópica , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Masculino , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genética , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Infecções Cutâneas Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Transfecção , Venenos de Vespas/administração & dosagem , Venenos de Vespas/uso terapêutico , Cicatrização/efeitos dos fármacos , Cicatrização/imunologiaRESUMO
Rab small GTPases control membrane trafficking through effectors that recruit downstream mediators such as motor proteins. Subcellular trafficking typically involves multiple Rabs, with each specific step mediated by a distinct Rab protein. We describe a collaboration between two distinct Rab-protein-orchestrated trafficking circuits in bladder epithelial cells (BECs) that expels intracellular uropathogenic Escherichia coli (UPEC) from their intracellular niche. RAB11a and RAB27b and their trafficking circuitry are simultaneously involved in UPEC expulsion. While RAB11a recruits its effector RAB11FIP3 and cytoskeletal motor Dynein, RAB27b mobilizes the effector MyRIP and motor Myosin VIIa to mediate bacterial expulsion. This collaboration is coordinated by deposition of the exocyst complex on bacteria-containing vesicles, an event triggered by the innate receptor Toll-like receptor 4. Both RAB11a and RAB27b are recruited and activated by the exocyst complex components SEC6/SEC15. Thus, the cell autonomous defense system can mobilize and coalesce multiple subcellular trafficking circuitries to combat infections.
Assuntos
Infecções por Escherichia coli/enzimologia , Escherichia coli Uropatogênica/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Transporte Proteico , Bexiga Urinária/enzimologia , Bexiga Urinária/microbiologia , Escherichia coli Uropatogênica/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Transcription of type I interferon genes during RNA virus infection requires signal communication between several pattern recognition receptor (PRR)-adaptor complexes located at distinct subcellular membranous compartments and a central cytoplasmic TBK1-interferon regulatory factor 3 (IRF3) kinase-transcription factor module. However, how the cell integrates signal transduction through spatially distinct modules of antiviral signaling pathways is less defined. RIG-I is a major cytosolic PRR involved in the control of several RNA viruses. Here we identify ArfGAP domain-containing protein 2 (ADAP2) as a key novel scaffolding protein that integrates different modules of the RIG-I pathway, located at distinct subcellular locations, and mediates cellular antiviral type I interferon production. ADAP2 served to bridge the mitochondrial membrane-bound upstream RIG-I adaptor MAVS and the downstream cytosolic complex of NEMO (regulatory subunit of TBK1), TBK1, and IRF3, leading to IRF3 phosphorylation. Furthermore, independently, ADAP2 also functioned as a major orchestrator of the interaction of TBK1 with NEMO and IRF3. Mutational and in vitro cell-free reconstituted RIG-I signaling assay-based analyses identified that the ArfGAP domain of ADAP2 mediates the interferon response. TRAF3 acted as a trigger for ADAP2 to recruit RIG-I pathway component proteins into a single macromolecular complex. This study provides important novel insights into the assembly and integration of different modules of antiviral signaling cascades.
Assuntos
Proteína DEAD-box 58/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Interferon Tipo I/biossíntese , Transdução de Sinais , Sistema Livre de Células , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/genética , Modelos Biológicos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Receptores Imunológicos , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/metabolismo , Transcrição Gênica/efeitos dos fármacos , Vesiculovirus/fisiologiaRESUMO
Cyclic GMP-AMP synthetase (cGAS) is a DNA-specific cytosolic sensor, which detects and initiates host defense responses against microbial DNA. It is thus curious that a recent study identified cGAS as playing important roles in inhibiting positive-sense single-stranded RNA (+ssRNA) viral infection, especially since RNA is not known to activate cGAS. Using a dengue virus serotype 2 (DENV-2) vaccine strain (PDK53), we show that infection creates an endogenous source of cytosolic DNA in infected cells through the release of mitochondrial DNA (mtDNA) to drive the production of cGAMP by cGAS. Innate immune responses triggered by cGAMP contribute to limiting the spread of DENV to adjacent uninfected cells through contact dependent gap junctions. Our result thus supports the notion that RNA virus indirectly activates a DNA-specific innate immune signaling pathway and highlights the breadth of the cGAS-induced antiviral response.