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1.
Artif Cells Nanomed Biotechnol ; 47(1): 3786-3792, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31549850

RESUMO

Phospholipase Cγ2 (PLCG2) has been implicated in the regulation of cell proliferation, transformation, and tumor growth. In this study, we investigate the mechanism of PLCG2 action using a short interference RNA (siRNA) method. The effects of PLCG2 on rat liver BRL-3A cells treated siRNA were studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT assay), bromodeoxyuridine (BrdU) labelling assay, flow cytometry method (FCM), quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The results showed when PLCG2 was reduced, cell vitality and proliferation rate were significantly decreased (p < .05 vs. control). FCM analysis showed that the number of cell division phase (G2 + M) was declined (p < .05 vs. control). RT-PCR and western blot revealed that the expression of signalling related genes NF-κB, FOS, JUN and ELK, target genes BCL2, CCNB1 and CCND1 were remarkably down-regulated in cells treated with PLCG2 siRNAs. Based on these results, we conclude PLCG2 plays an important role in rat liver cell proliferation via ERK and NF-κB pathway by regulating the expression of BCl2, MYC and CCND1.


Assuntos
Ciclina D1/metabolismo , Hepatócitos/citologia , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Fosfolipase C gama/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Proliferação de Células , Fosfolipase C gama/deficiência , Fosfolipase C gama/genética , RNA Interferente Pequeno/genética , Ratos
2.
Invest Ophthalmol Vis Sci ; 60(12): 4041-4051, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31560769

RESUMO

Purpose: Age-related macular degeneration (AMD) is the worldwide leading cause of blindness among the elderly. Although genome-wide association studies (GWAS) have identified AMD risk variants, their roles in disease etiology are not well-characterized, and they only explain a portion of AMD heritability. Methods: We performed pathway analyses using summary statistics from the International AMD Genomics Consortium's 2016 GWAS and multiple pathway databases to identify biological pathways wherein genetic association signals for AMD may be aggregating. We determined which genes contributed most to significant pathway signals across the databases. We characterized these genes by constructing protein-protein interaction networks and performing motif analysis. Results: We determined that eight genes (C2, C3, LIPC, MICA, NOTCH4, PLCG2, PPARA, and RAD51B) "drive" the statistical signals observed across pathways curated in the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and Gene Ontology (GO) databases. We further refined our definition of statistical driver gene to identify PLCG2 as a candidate gene for AMD due to its significant gene-level signals (P < 0.0001) across KEGG, Reactome, GO, and NetPath pathways. Conclusions: We performed pathway analyses on the largest available collection of advanced AMD cases and controls in the world. Eight genes strongly contributed to significant pathways from the three larger databases, and one gene (PLCG2) was central to significant pathways from all four databases. This is, to our knowledge, the first study to identify PLCG2 as a candidate gene for AMD based solely on genetic burden. Our findings reinforce the utility of integrating in silico genetic and biological pathway data to investigate the genetic architecture of AMD.


Assuntos
Estudo de Associação Genômica Ampla , Degeneração Macular/genética , Fosfolipase C gama/genética , Idoso , Estudos de Casos e Controles , Bases de Dados Factuais , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Domínios e Motivos de Interação entre Proteínas
3.
Blood ; 134(7): 641-644, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31243043

RESUMO

Mutational analyses performed following acquired ibrutinib resistance have suggested that chronic lymphocytic leukemia (CLL) progression on ibrutinib is linked to mutations in Bruton tyrosine kinase (BTK) and/or phospholipase Cγ2 (PLCG2) genes. Mutational information for patients still on ibrutinib is limited. We report a study aimed to provide a "snapshot" of the prevalence of mutations in a real-life CLL cohort still on ibrutinib after at least 3 years of treatment. Of 204 patients who initiated ibrutinib via an early-access program at 29 French Innovative Leukemia Organization (FILO) centers, 63 (31%) were still on ibrutinib after 3 years and 57 provided a fresh blood sample. Thirty patients had a CLL clone ≥0.5 × 109/L, enabling next-generation sequencing (NGS); BTK and PLCG2 mutations were detected in 57% and 13% of the NGS samples, respectively. After median follow-up of 8.5 months from sample collection, the presence of a BTK mutation was significantly associated with subsequent CLL progression (P = .0005 vs no BTK mutation). Our findings support that mutational analysis should be considered in patients receiving ibrutinib who have residual clonal lymphocytosis, and that clinical trials are needed to evaluate whether patients with a BTK mutation may benefit from an early switch to another treatment.


Assuntos
Tirosina Quinase da Agamaglobulinemia/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Fosfolipase C gama/genética , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Feminino , Seguimentos , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Mutação
4.
J Agric Food Chem ; 67(14): 3909-3918, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30892883

RESUMO

Stilbenes are phenolic compounds present in different higher plant families that have shown different biological activities, such as antioxidant properties and antitumoral and anti-atherosclerotic effects, among others. Angiogenesis is a key process involved in both cancer and cardiovascular diseases, the vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 being the main triggers. Certain polyphenol compounds, such as flavonoids, have shown a potent capacity to inhibit VEGF and, consequently, angiogenesis. The present work, therefore, aims to evaluate the potential effect of stilbenes on inhibiting VEGF and their subsequent effect on the downstream signaling pathway (PLCγ1, Akt, and eNOS). VEGFR-2 activation was studied through an ELISA assay in the HUVEC line, while the phosphorylation of intracellular downstream proteins PLCγ1, Akt, and eNOS was tested by Western blot. Student's t test was used to determine significant differences between samples. On the one hand, astringin, pallidol, and ω-viniferin showed the lowest IC50 values (2.90 ± 0.27, 4.42 ± 0.67, and 6.10 ± 1.29 µM, respectively) against VEGFR-2 activation. Additionally, VEGF-induced PLCγ1 phosphorylation was significantly inhibited by ε-viniferin, astringin, and ω-viniferin. However, ε-viniferin and pallidol simultaneously enhanced eNOS activation, proving to be via Akt activation in the case of ε-viniferin. For the first time, these data suggest that stilbenes such as astringin, pallidol, ω-viniferin, and ε-viniferin have a potential anti-angiogenic effect and they could be further considered as anti-VEGF ingredients in food and beverages. In addition, ε-viniferin and pallidol significantly allowed eNOS activation and could likely prevent the side effects caused by anti-VEGF hypertension drugs.


Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Extratos Vegetais/farmacologia , Estilbenos/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Vitis/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
5.
Oncol Rep ; 41(4): 2117-2125, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30720116

RESUMO

Pancreatic cancer (PC), a malignancy of the digestive system, has one of the highest rates of metastasis and mortality. It is characterized by the detachment, migration, implantation and infiltration of tumor cells to form metastases or recurrent foci. Tetraspanin 1 (TSPAN1), a novel member of the TSPAN superfamily, is highly expressed in many types of cancer, including gastric, colon, liver and esophageal cancer. It has also been associated with lymph node metastasis, tumor recurrence and metastasis. However, the role of TSPAN1 in PC has not been fully elucidated. The aim of the present study was to determine the expression of TSPAN1 in human PC tissue samples and cell lines. Additionally, the functions of TSPAN1 in PC cell migration and invasion were assessed. The protein and gene expression of TSPAN1 was analyzed in clinical PC tissue samples and human PC cell lines (SW1990, BxPC3, Capan1 and PANC­1) via immunohistochemistry, reverse transcription­quantitative polymerase chain reaction and western blotting. The effect of TSPAN1 downregulation and overexpression in PC cells, via transfection with siRNA and pLNCX­TSPAN1­cDNA recombinant plasmid, respectively, on cell invasion and migration were assessed. Additionally, the mRNA expression of matrix metalloproteinase (MMP2) and MMP9 were determined. In clinical PC tissue samples, the expression of TSPAN1 was markedly increased when compared with normal pancreatic tissue samples. TSPAN1 was also highly expressed in PC cell lines compared with HPDE, a normal pancreatic cell line. Transfection with siRNA targeting TSPAN1 in PC cell lines significantly suppressed PC cell migration and invasion, and downregulated the expression of MMP2. However, there was no effect on MMP9. Consistently, PC cell migration and invasion together with MMP2 mRNA expression were markedly increased following TSPAN1 ectopic overexpression. The present study utilized small interfering RNAs (siRNA) targeted to phospholipase Cγ (PLCγ) to demonstrate that TSPAN1 siRNA suppressed PC cell migration and invasion, and MMP2 mRNA expression by blocking the translocation and phosphorylation of PLCγ. The results of the present study revealed that TSPAN1 has an important function in human PC cell migration and invasion by modulating MMP2 expression via PLCγ. Thus, the results indicate that the silencing of TSPAN1 may be a potential therapeutic target for the treatment of PC.


Assuntos
Metaloproteinase 2 da Matriz/metabolismo , Neoplasias Pancreáticas/patologia , Fosfolipase C gama/metabolismo , Tetraspaninas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Movimento Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Pâncreas/patologia , Fosfolipase C gama/genética , Fosforilação , RNA Interferente Pequeno/metabolismo , Tetraspaninas/genética , Regulação para Cima
6.
Transl Psychiatry ; 9(1): 55, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30705288

RESUMO

Rare coding variants in TREM2, PLCG2, and ABI3 were recently associated with the susceptibility to Alzheimer's disease (AD) in Caucasians. Frequencies and AD-associated effects of variants differ across ethnicities. To start filling the gap on AD genetics in South America and assess the impact of these variants across ethnicity, we studied these variants in Argentinian population in association with ancestry. TREM2 (rs143332484 and rs75932628), PLCG2 (rs72824905), and ABI3 (rs616338) were genotyped in 419 AD cases and 486 controls. Meta-analysis with European population was performed. Ancestry was estimated from genome-wide genotyping results. All variants show similar frequencies and odds ratios to those previously reported. Their association with AD reach statistical significance by meta-analysis. Although the Argentinian population is an admixture, variant carriers presented mainly Caucasian ancestry. Rare coding variants in TREM2, PLCG2, and ABI3 also modulate susceptibility to AD in populations from Argentina, and they may have a European heritage.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Alzheimer/etnologia , Doença de Alzheimer/genética , Glicoproteínas de Membrana/genética , Fosfolipase C gama/genética , Receptores Imunológicos/genética , Grupo com Ancestrais do Continente Africano/genética , Idoso , Idoso de 80 Anos ou mais , Argentina/etnologia , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Predisposição Genética para Doença , Variação Genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Índios Norte-Americanos/genética , Masculino , Pessoa de Meia-Idade
7.
Int J Mol Sci ; 19(12)2018 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-30567287

RESUMO

Alisol B 23-acetate (AB23A), a natural triterpenoid, has been reported to exert hepatoprotective and antitumor activities. Aiming to investigate the anti-inflammatory activity, this study examined the effect of AB23A on mast cells and allergic reaction. AB23A inhibited the degranulation of mast cells stimulated by immunoglobulin E/antigen (IgE/Ag), and also decreased the synthesis of leukotriene C4 (LTC4), production of interlukin-6 (IL-6), and expression of cyclooxygenase-2 (COX-2) in a concentration-dependent manner with no significant cytotoxicity in bone marrow-derived mast cells (BMMCs). AB23A inhibited spleen tyrosine kinase (Syk) and the downstream signaling molecules including phospholipase Cγ (PLCγ), serine-threonine protein kinase/inhibitor of nuclear factor kappa-B kinase/nuclear factor kappa-B (Akt/IKK/NF-κB), and mitogen-activated protein kinases/cytosolic phospholipase A2 (MAPK/cPLA2). Furthermore, AB23A blocked mobilization of Ca2+. Similar results were obtained in other mast cell lines Rat basophilic leukemia (RBL)-2H3 cells and a human mast cell line (HMC-1). In addition, AB23A attenuated allergic responses in an acute allergy animal model, passive cutaneous anaphylaxis (PCA). Taken together, this study suggests that AB23A inhibits the activation of mast cells and ameliorates allergic reaction, and may become a lead compound for the treatment of mast cell-mediated allergic diseases.


Assuntos
Antialérgicos/farmacologia , Colestenonas/farmacologia , Hipersensibilidade/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Degranulação Celular/efeitos dos fármacos , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Imunoglobulina E/imunologia , Leucotrieno C4/biossíntese , Mastócitos/imunologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Fosfolipase C gama/genética , Proteínas Serina-Treonina Quinases/genética , Ratos , Baço/efeitos dos fármacos , Baço/enzimologia , Quinase Syk
8.
Nat Chem Biol ; 14(12): 1079-1089, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30429604

RESUMO

CD95L is a transmembrane ligand (m-CD95L) that is cleaved by metalloproteases to release a soluble ligand (s-CD95L). Unlike m-CD95L, interaction between s-CD95L and CD95 fails to recruit caspase-8 and FADD to trigger apoptosis and instead induces a Ca2+ response via docking of PLCγ1 to the calcium-inducing domain (CID) within CD95. This signaling pathway induces accumulation of inflammatory Th17 cells in damaged organs of lupus patients, thereby aggravating disease pathology. A large-scale screen revealed that the HIV protease inhibitor ritonavir is a potent disruptor of the CD95-PLCγ1 interaction. A structure-activity relationship approach highlighted that ritonavir is a peptidomimetic that shares structural characteristics with CID with respect to docking to PLCγ1. Thus, we synthesized CID peptidomimetics abrogating both the CD95-driven Ca2+ response and transmigration of Th17 cells. Injection of ritonavir and the CID peptidomimetic into lupus mice alleviated clinical symptoms, opening a new avenue for the generation of drugs for lupus patients.


Assuntos
Inflamação/prevenção & controle , Peptidomiméticos/farmacologia , Fosfolipase C gama/metabolismo , Células Th17/efeitos dos fármacos , Receptor fas/metabolismo , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/etiologia , Masculino , Camundongos Mutantes , Simulação de Acoplamento Molecular , Peptidomiméticos/química , Fosfolipase C gama/genética , Domínios Proteicos , Ritonavir/química , Ritonavir/farmacologia , Relação Estrutura-Atividade , Células Th17/metabolismo , Células Th17/patologia , Tiazóis/química , Tiazóis/farmacologia , Receptor fas/genética
9.
Mol Neurodegener ; 13(1): 53, 2018 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-30326945

RESUMO

BACKGROUND: Rare coding variants ABI3_rs616338-T and PLCG2_rs72824905-G were identified as risk or protective factors, respectively, for Alzheimer's disease (AD). METHODS: We tested the association of these variants with five neurodegenerative diseases in Caucasian case-control cohorts: 2742 AD, 231 progressive supranuclear palsy (PSP), 838 Parkinson's disease (PD), 306 dementia with Lewy bodies (DLB) and 150 multiple system atrophy (MSA) vs. 3351 controls; and in an African-American AD case-control cohort (181 AD, 331 controls). 1479 AD and 1491 controls were non-overlapping with a prior report. RESULTS: Using Fisher's exact test, there was significant association of both ABI3_rs616338-T (OR = 1.41, p = 0.044) and PLCG2_rs72824905-G (OR = 0.56, p = 0.008) with AD. These OR estimates were maintained in the non-overlapping replication AD-control analysis, albeit at reduced significance (ABI3_rs616338-T OR = 1.44, p = 0.12; PLCG2_rs72824905-G OR = 0.66, p = 0.19). None of the other cohorts showed significant associations that were concordant with those for AD, although the DLB cohort had suggestive findings (Fisher's test: ABI3_rs616338-T OR = 1.79, p = 0.097; PLCG2_rs72824905-G OR = 0.32, p = 0.124). PLCG2_rs72824905-G showed suggestive association with pathologically-confirmed MSA (OR = 2.39, p = 0.050) and PSP (OR = 1.97, p = 0.061), although in the opposite direction of that for AD. We assessed RNA sequencing data from 238 temporal cortex (TCX) and 224 cerebellum (CER) samples from AD, PSP and control patients and identified co-expression networks, enriched in microglial genes and immune response GO terms, and which harbor PLCG2 and/or ABI3. These networks had higher expression in AD, but not in PSP TCX, compared to controls. This expression association did not survive adjustment for brain cell type population changes. CONCLUSIONS: We validated the associations previously reported with ABI3_rs616338-T and PLCG2_rs72824905-G in a Caucasian AD case-control cohort, and observed a similar direction of effect in DLB. Conversely, PLCG2_rs72824905-G showed suggestive associations with PSP and MSA in the opposite direction. We identified microglial gene-enriched co-expression networks with significantly higher levels in AD TCX, but not in PSP, a primary tauopathy. This co-expression network association appears to be driven by microglial cell population changes in a brain region affected by AD pathology. Although these findings require replication in larger cohorts, they suggest distinct effects of the microglial genes, ABI3 and PLCG2 in neurodegenerative diseases that harbor significant vs. low/no amyloid ß pathology.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Fosfolipase C gama/genética , Afro-Americanos/genética , Idoso , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Humanos , Masculino , Microglia/metabolismo , Fatores de Risco
10.
Cell Death Dis ; 9(11): 1064, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30337526

RESUMO

Many genes of the human genome display pleiotropic activity, playing an important role in two or more unrelated pathways. Surprisingly, some of these functions can even be antagonistic, often letting to divergent functional outcomes depending on microenviromental cues and tissue/cell type-dependent parameters. Lately, the Bruton's tyrosine kinase (BTK) has emerged as one of such pleiotropic genes, with opposing effects in cancer pathways. While it has long been considered oncogenic in the context of B cell malignancies, recent data shows that BTK can also act as a tumour suppressor in other cells, as an essential member of the p53 and p73 responses to damage. Since BTK inhibitors are already being used clinically, it is important to carefully review these new findings in order to fully understand the consequences of blocking BTK activity in all the cells of the organism.


Assuntos
Tirosina Quinase da Agamaglobulinemia/genética , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Linfócitos B/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
11.
EMBO Rep ; 19(11)2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30224412

RESUMO

Coordinated expression of guidance molecules and their signal transduction are critical for correct brain wiring. Previous studies have shown that phospholipase C gamma1 (PLCγ1), a signal transducer of receptor tyrosine kinases, plays a specific role in the regulation of neuronal cell morphology and motility in vitro However, several questions remain regarding the extracellular stimulus that triggers PLCγ1 signaling and the exact role PLCγ1 plays in nervous system development. Here, we demonstrate that PLCγ1 mediates axonal guidance through a netrin-1/deleted in colorectal cancer (DCC) complex. Netrin-1/DCC activates PLCγ1 through Src kinase to induce actin cytoskeleton rearrangement. Neuronal progenitor-specific knockout of Plcg1 in mice causes axon guidance defects in the dorsal part of the mesencephalon during embryogenesis. Adult Plcg1-deficient mice exhibit structural alterations in the corpus callosum, substantia innominata, and olfactory tubercle. These results suggest that PLCγ1 plays an important role in the correct development of white matter structure by mediating netrin-1/DCC signaling.


Assuntos
Axônios/fisiologia , Encéfalo/embriologia , Netrina-1/metabolismo , Fosfolipase C gama/metabolismo , Animais , Axônios/patologia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Receptor DCC/metabolismo , Feminino , Masculino , Mesencéfalo/embriologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Netrina-1/genética , Fosfolipase C gama/genética , Fosforilação , Gravidez , Quinases da Família src/metabolismo
12.
Cell Physiol Biochem ; 48(5): 2011-2034, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30099438

RESUMO

BACKGROUND/AIMS: Eggs of all animal species display intense cytoplasmic Ca2+ increases at fertilization. Previously, we reported that unfertilized eggs of Astropecten aranciacus exposed to an actin drug latrunculin A (LAT-A) exhibit similar Ca2+ waves and cortical flashes after 5-10 min time lag. Here, we have explored the molecular mechanisms underlying this unique phenomenon. METHODS: Starfish eggs were pretreated with various agents such as other actin drugs or inhibitors of phospholipase C (PLC), and the changes of the intracellular Ca2+ levels were monitored by use of Calcium Green in the presence or absence of LAT-A. The concomitant changes of the actin cytoskeleton were visualized with fluorescent F-actin probes in confocal microscopy. RESULTS: We have shown that the LAT-A-induced Ca2+ increases are related to the disassembly of actin flaments: i) not only LAT-A but also other agents depolymerizing F-actin (i.e. cytochalasin B and mycalolide B) induced similar Ca2+ increases, albeit with slightly lower efficiency; ii) drugs stabilizing F-actin (i.e. phalloidin and jasplakinolide) either blocked or significantly delayed the LAT-A-induced Ca2+ increases. Further studies utilizing pharmacological inhibitors of PLC (U-73122 and neomycin), dominant negative mutant of PLC-É£, specific sequestration of PIP2 (RFP-PH), InsP3 uncaging, and quantitation of endogenous InsP3 all indicated that LAT-A induces Ca2+ increases by stimulating PLC rather than sensitizing InsP3 receptors. In support of the idea, it bears emphasis that LAT-A timely increased intracellular contents of InsP3 with concomitant decrease of PIP2 levels in the plasma membrane. CONCLUSION: Taken together, our results suggest that suboolemmal actin filaments may serve as a scaffold for cell signaling and modulate the activity of the key enzyme involved in intracellular Ca2+ signaling.


Assuntos
Citoesqueleto de Actina/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Estrelas-do-Mar/metabolismo , Citoesqueleto de Actina/química , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citocalasina B/farmacologia , Estrenos/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Microscopia Confocal , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Pirrolidinonas/farmacologia , Estrelas-do-Mar/crescimento & desenvolvimento , Tiazolidinas/farmacologia , Domínios de Homologia de src/genética
13.
J Autoimmun ; 94: 110-121, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30061013

RESUMO

NF-κB inducing kinase (NIK) is the key protein of the non-canonical NF-κB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIKΔT) mice. Despite showing normal development of lymphoid organs, NIKΔT mice were resistant to induction of CNS autoimmunity. T cells from NIKΔT mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK-/- T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dynamics. In line with this we found that NIK-deficient T cells were hampered in phosphorylation of Zap70, LAT, AKT, ERK1/2 and PLCγ upon TCR engagement. Hence, our data disclose a hitherto unknown function of NIK in T-cell priming and differentiation.


Assuntos
Actinas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Ativação Linfocitária , Proteínas Serina-Treonina Quinases/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Linfonodos/imunologia , Linfonodos/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Fosfolipase C gama/genética , Fosfolipase C gama/imunologia , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Baço/imunologia , Baço/patologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologia , Linfócitos T/patologia , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/imunologia
14.
BMC Ophthalmol ; 18(1): 170, 2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-30005593

RESUMO

BACKGROUND: Fungal keratitis (FK) is a sight-threatening disease, accounting for a significant portion with its complex presentation, suboptimal efficacy of the existing therapies and uncontrollable excessive innate inflammation. Phospholipase C-γ2 (PLCγ2) is a non-receptor tyrosine kinase that plays an important role at the early period of innate immunity. This study aimed to identify the role of PLCγ2 in Dectin-1-mediated Ca2+ Flux and its effect on the expression of proinflammatory mediators at the exposure to Aspergillus fumigatus (A. fumigatus) hyphae antigens in human corneal epithelial cells (HCECs). METHODS: The HCECs were preincubated with or without different inhibitors respectively before A. fumigatus hyphae stimulation. Intracellular calcium flux in HCECs and levels of PLCγ2 and spleen-tyrosine kinase (Syk) were detected by fluorescence imaging and Western Blotting. The expression of proinflammatory mediators was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: We demonstrated that an intracellular Ca2+ flux in HCECs was triggered by A. fumigatus hyphae and could be reduced by pre-treatment with PLCγ2-inhibitor U73122. A. fumigatus hyphae induced PLCγ2 phosphorylation was regulated by Dectin-1 via Syk. Furthermore, PLCγ2-deficient HCECs showed a drastic impairment in the Ca2+ signaling and the secretion of IL-6, CXCL1 and TNF-α. CONCLUSIONS: PLCγ2 plays a critical role for Ca2+ Flux in HCECs stimulated by A. fumigatus hyphae. Syk acts upstream of PLCγ2 in the Dectin-1 signaling pathway. The expressions of proinflammatory mediators induced by A. fumigatus are regulated by the activation of Dectin-1-mediated PLCγ2 signaling pathway in HCECs.


Assuntos
Aspergillus fumigatus/imunologia , Cálcio/metabolismo , Citocinas/biossíntese , Epitélio Anterior/metabolismo , Regulação da Expressão Gênica , Imunidade Inata/genética , Fosfolipase C gama/genética , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Epitélio Anterior/patologia , Infecções Oculares Fúngicas/genética , Infecções Oculares Fúngicas/imunologia , Infecções Oculares Fúngicas/metabolismo , Humanos , Fosfolipase C gama/biossíntese , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
15.
Biophys J ; 115(1): 31-45, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29972810

RESUMO

Phosphatidylinositol phospholipase Cγ (PLCγ) is an intracellular membrane-associated second-messenger signaling protein activated by tyrosine kinases such as fibroblast growth factor receptor 1. PLCγ contains the regulatory γ-specific array (γSA) comprising a tandem Src homology 2 (SH2) pair, an SH3 domain, and a split pleckstrin homology domain. Binding of an activated growth factor receptor to γSA leads to Tyr783 phosphorylation and consequent PLCγ activation. Several disease-relevant mutations in γSA have been identified; all lead to elevated phospholipase activity. In this work, we describe an allosteric mechanism that connects the Tyr783 phosphorylation site to the nSH2-cSH2 junction and involves dynamic interactions between the cSH2-SH3 linker and cSH2. Molecular dynamics simulations of the tandem SH2 protein suggest that Tyr783 phosphorylation is communicated to the nSH2-cSH2 junction by modulating cSH2 binding to sections of the cSH2-SH3 linker. NMR chemical shift perturbation analyses for designed tandem SH2 constructs reveal combined fast and slow dynamic processes that can be attributed to allosteric communication involving these regions of the protein, establishing an example in which complex N-site exchange can be directly inferred from 1H,15N-HSQC spectra. Furthermore, in tandem SH2 and γSA constructs, molecular dynamics and NMR results show that the Arg687Trp mutant in PLCγ1 (equivalent to the cancer mutation Arg665Trp in PLCγ2) perturbs the dynamic allosteric pathway. This combined experimental and computational study reveals a rare example of multistate kinetics involved in a dynamic allosteric process that is modulated in the context of a disease-relevant mutation. The allosteric influences and the weakened binding of the cSH2-SH3 linker to cSH2 should be taken into account in any more holistic investigation of PLCγ regulation.


Assuntos
Simulação de Dinâmica Molecular , Mutação , Neoplasias/genética , Ressonância Magnética Nuclear Biomolecular , Fosfolipase C gama/química , Fosfolipase C gama/metabolismo , Regulação Alostérica , Fosfolipase C gama/genética , Fosforilação , Domínios de Homologia de src
16.
Sci Signal ; 11(533)2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29871912

RESUMO

Members of the transient receptor potential (TRP) family of ion channels are cellular sensors involved in numerous physiological and pathological processes. We identified the TRP subfamily M member 7 (TRPM7) channel-kinase as a previously uncharacterized regulator of B cell activation. We showed that TRPM7 played a critical role in the early events of B cell activation through both its ion channel and kinase functions. DT40 B cells deficient in TRPM7 or expressing a kinase-deficient mutant of TRPM7 showed defective gathering of antigen and prolonged B cell receptor (BCR) signaling. We showed that lipid metabolism was altered in TRPM7-deficient cells and in cells expressing a kinase-deficient mutant of TRPM7 and suggest that PLC-γ2 may be a target of the kinase activity of TRPM7. Primary B cells that expressed less TRPM7 or were treated with a pharmacological inhibitor of TRPM7 also displayed defective antigen gathering and increased BCR signaling. Finally, we demonstrated that blocking TRPM7 function compromised antigen internalization and presentation to T cells. These data suggest that TRPM7 controls an essential process required for B cell affinity maturation and the production of high-affinity antibodies.


Assuntos
Apresentação do Antígeno , Linfócitos B/metabolismo , Canais de Cátion TRPM/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Linfócitos B/citologia , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação , Transdução de Sinais
17.
Cell Death Differ ; 25(10): 1796-1807, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29899383

RESUMO

The endoplasmic reticulum stress sensor, the unfolded protein response (UPR), regulates intracellular protein homeostasis. While transient activation of the reactive UPR by unfolded protein is protective, prolonged and sustained activation of the reactive UPR triggers CHOP-mediated apoptosis. In the recently characterized, evolutionarily conserved anticipatory UPR, mitogenic hormones and other effectors pre-activate the UPR; how strong and sustained activation of the anticipatory UPR induces cell death was unknown. To characterize this cell death pathway, we used BHPI, a small molecule that activates the anticipatory UPR through estrogen receptor α (ERα) and induces death of ERα+ cancer cells. We show that sustained activation of the anticipatory UPR by BHPI kills cells by inducing depletion of intracellular ATP, resulting in classical necrosis phenotypes, including plasma membrane disruption and leakage of intracellular contents. Unlike reactive UPR activation, BHPI-induced hyperactivation of the anticipatory UPR does not induce apoptosis or sustained autophagy. BHPI does not induce CHOP protein or PARP cleavage, and two pan-caspase inhibitors, or Bcl2 overexpression, have no effect on BHPI-induced cell death. Moreover, BHPI does not increase expression of autophagy markers, or work through recently identified programmed-necrosis pathways, such as necroptosis. Opening of endoplasmic reticulum IP3R calcium channels stimulates cell swelling, cPLA2 activation, and arachidonic acid release. Notably, cPLA2 activation requires ATP depletion. Importantly, blocking rapid cell swelling or production of arachidonic acid does not prevent necrotic cell death. Rapid cell death is upstream of PERK activation and protein synthesis inhibition, and results from strong and sustained activation of early steps in the anticipatory UPR. Supporting a central role for ATP depletion, reversing ATP depletion blocks rapid cell death, and the onset of necrotic cell death is correlated with ATP depletion. Necrotic cell death initiated by strong and sustained activation of the anticipatory UPR is a newly discovered role of the UPR.


Assuntos
Apoptose , Resposta a Proteínas não Dobradas , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácido Araquidônico/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Humanos , Necrose , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismo
18.
Int J Mol Sci ; 19(6)2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29912163

RESUMO

Platelet-derived growth factor (PDGF) has mitogenic and chemotactic effects on fibroblasts. An increase in intracellular Ca2+ is one of the first events that occurs following the stimulation of PDGF receptors (PDGFRs). PDGF activates Ca2+ elevation by activating the phospholipase C gamma (PLCγ)-signaling pathway, resulting in ER Ca2+ release. Store-operated Ca2+ entry (SOCE) is the major form of extracellular Ca2+ influx following depletion of ER Ca2+ stores and stromal interaction molecule 1 (STIM1) is a key molecule in the regulation of SOCE. In this study, wild-type and STIM1 knockout mouse embryonic fibroblasts (MEF) cells were used to investigate the role of STIM1 in PDGF-induced Ca2+ oscillation and its functions in MEF cells. The unexpected findings suggest that STIM1 knockout enhances PDGFR⁻PLCγ­STIM2 signaling, which in turn increases PDGF-BB-induced Ca2+ elevation. Enhanced expressions of PDGFRs and PLCγ in STIM1 knockout cells induce Ca2+ release from the ER store through PLCγ­IP3 signaling. Moreover, STIM2 replaces STIM1 to act as the major ER Ca2+ sensor in activating SOCE. However, activation of PDGFRs also activate Akt, ERK, and JNK to regulate cellular functions, such as cell migration. These results suggest that alternative switchable pathways can be observed in cells, which act downstream of the growth factors that regulate Ca2+ signaling.


Assuntos
Sinalização do Cálcio , Fator de Crescimento Derivado de Plaquetas/metabolismo , Molécula 1 de Interação Estromal/genética , Animais , Linhagem Celular , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Regulação para Cima
19.
Sci Rep ; 8(1): 5336, 2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29593227

RESUMO

Cish, participates within a multi-molecular E3 ubiquitin ligase complex, which ubiquitinates target proteins. It has an inhibitory effect on T cell activation mediated by PLC-γ1 regulation, and it functions as a potent checkpoint in CD8+ T cell tumor immunotherapy. To study the structural and functional relationships between Cish and PLC-γ1 during CD8+ T cell activation, we tested mutants of the Cish-SH2 (R107K) and D/BC (L222Q, C226Q) domains. We confirmed that Cish-SH2-specific binding was essential for PLC-γ1 ubiquitination and degradation. This domain was essential for the Cish-mediated inhibition of Ca2+ release upon TCR stimulation. No effect on inhibition of cytokine release was observed with SH2 or D/BC mutants, although the absence of Cish led to an increased release of IFN-γ and TNF-α. Using imaging we showed that Cish was expressed mostly in the cytoplasm and we did not see any Cish clustering at the plasma membrane upon stimulation. We conclude that the Cish-SH2 domain is essential for PLC-γ1 regulation in TCR-stimulated CD8+ T cells.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Fosfolipase C gama/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Domínios de Homologia de src , Animais , Linfócitos T CD8-Positivos/imunologia , Cálcio/metabolismo , Linhagem Celular , Citocinas/metabolismo , Expressão Gênica , Humanos , Ativação Linfocitária , Camundongos , Camundongos Knockout , Fosfolipase C gama/química , Fosfolipase C gama/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/genética
20.
Int Immunol ; 30(5): 205-213, 2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29538758

RESUMO

The intersection of granulomatosis and autoinflammatory disease is a rare occurrence that can be generally subdivided into purely granulomatous phenotypes and disease spectra that are inclusive of granulomatous features. NOD2 (nucleotide-binding oligomerization domain-containing protein 2)-related disease, which includes Blau syndrome and early-onset sarcoidosis, is the prototypic example of granulomatous inflammation in the context of monogenic autoinflammation. Granulomatous inflammation has also been observed in two related autoinflammatory diseases caused by mutations in PLCG2 (phospholipase Cγ2). More recently, mutations in LACC1 (laccase domain-containing protein 1) have been identified as the cause of a monogenic form of systemic juvenile idiopathic arthritis, which does not itself manifest granulomatous inflammation, but the same LACC1 mutations have also been shown to cause an early-onset, familial form of a well-known granulomatous condition, Crohn's disease (CD). Rare genetic variants of PLCG2 have also been shown to cause a monogenic form of CD, and moreover common variants of all three of these genes have been implicated in polygenic forms of CD. Additionally, common variants of NOD2 and LACC1 have been implicated in susceptibility to leprosy, a granulomatous infection. Although no specific mechanistic link exists between these three genes, they form an intriguing web of susceptibility to both monogenic and polygenic autoinflammatory and granulomatous phenotypes.


Assuntos
Artrite Juvenil/genética , Artrite/genética , Doença de Crohn/genética , Mutação/genética , Proteína Adaptadora de Sinalização NOD2/genética , Fosfolipase C gama/genética , Proteínas/genética , Sinovite/genética , Uveíte/genética , Animais , Autoimunidade , Interação Gene-Ambiente , Granuloma , Camundongos
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