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1.
Artigo em Inglês | MEDLINE | ID: mdl-32159362

RESUMO

Danger sensing is one of the most fundamental evolutionary features enabling multicellular organisms to perceive potential threats, escape from risky situations, fight actual intruders, and repair damage. Several endogenous molecules are used to "signal damage," currently referred to as "alarmins" or "damage-associated molecular patterns" (DAMPs), most being already present within all cells (preformed DAMPs), and thus ready to be released, and others neosynthesized following injury. Over recent years it has become overwhelmingly clear that adenosine 5'-triphosphate (ATP) is a ubiquitous and extremely efficient DAMP (thus promoting inflammation), and its main metabolite, adenosine, is a strong immunosuppressant (thus dampening inflammation). Extracellular ATP ligates and activates the P2 purinergic receptors (P2Rs) and is then degraded by soluble and plasma membrane ecto-nucleotidases to generate adenosine acting at P1 purinergic receptors (P1Rs). Extracellular ATP, P2Rs, ecto-nucleotidases, adenosine, and P1Rs are basic elements of the purinergic signaling network and fundamental pillars of inflammation.


Assuntos
Alarminas/genética , Inflamação/metabolismo , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P2/genética , Adenosina/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Alarminas/metabolismo , Animais , Membrana Celular/metabolismo , Humanos , Imunossupressores/metabolismo , Inflamação/fisiopatologia , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais/genética
2.
Biochem Biophys Res Commun ; 524(4): 798-802, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32037085

RESUMO

Inflammatory bowel disease (IBD) is a risk factor for the development of colorectal cancer (CRC) for which mutation to p53 is an early event leading to dysplasia. Interestingly, P2RY6 mRNA increases in both pathologies. In this study, we investigated if p53 and p53R273H mutant, commonly found in CRC and IBD, were involved in the transcriptional regulation of P2RY6. First, the P2RY6 promoter was defined as a region corresponding to -1600 to +273 nucleotides relative to the putative TATA-less transcriptional starting site found at position 73,264,505 of NCBI reference sequence NC_000010.11. We cloned this promoter region along with 5'-deletion constructs in the pGL4.10[luc2] vector for luciferase assays to delineate the minimal promoter region. We observed that p53 wt and p53R273H differentially regulated the transcription of the P2RY6 gene. In fact, increasing quantity of p53R273H enhanced the capacity of p53 wt to stimulate the transactivation of the P2RY6 promoter but this cooperative effect was lost when p53R273H was present in a ratio of 3:1. In accordance with the luciferase assays, ChIP analysis revealed that endogenous p53 wt was significantly associated with the P2RY6 proximal promoter, whereas the interaction of the p53R273H with the P2RY6 promoter was not significant. Although further studies are required to fully elucidate the molecular determinant controlling P2Y6 expression in diseases, we propose, for the first time, a molecular mechanism involving a collaboration between p53 wt and p53R273H to regulate the expression of this receptor.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Receptores Purinérgicos P2/genética , Transcrição Genética , Proteína Supressora de Tumor p53/genética , Células A549 , Substituição de Aminoácidos , Células CACO-2 , Proliferação de Células , Imunoprecipitação da Cromatina , Genes Reporter , Células HCT116 , Células HT29 , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
3.
Int J Mol Med ; 44(6): 2145-2160, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31638262

RESUMO

Endometriosis is associated with changes in long non­coding RNA (lncRNA) and mRNA expression, but the exact changes during the implantation window are unknown. Therefore, this study aimed to explore the lncRNA and mRNA expression profiles in the uterus of rats with endometriosis during the implantation window. A total of 35 non­pregnant female rats were randomized to the endometriosis (n=13), adipose tissue control (n=8) and blank control (n=14) groups. On the 5th day of pregnancy, the rats were sacrificed to obtain uterine tissues. lncRNA and mRNA were analyzed using gene chips. A total of five differentially expressed lncRNA and four mRNA were validated by reverse transcription­quantitative (RT­q)PCR. Immunohistochemistry and western blotting were used to determine the expression of the ADAM metallopeptidase with thrombospondin type 1 motif 7 (Adamts7), tumor protein p53 (Tp53), distal­less homeobox 3 (Dlx3) and pyrimidinergic receptor P2Y6 (P2ry6) proteins. There were 115 upregulated lncRNAs, 51 downregulated lncRNAs, 97 upregulated mRNAs and 85 downregulated mRNAs in the endometriosis group. RT­qPCR confirmed the trends for five lncRNAs and four mRNAs (Adamts7, Tp53, Dlx3 and P2ry6). The relative protein expression levels of Adamts7, P2ry6, Dlx3 and TP53 were significantly different in the endometriosis group (P<0.05 vs. controls). Bioinformatics predicted the co­expression relationship of the selected five lncRNA and four mRNA. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes predicted that Adamts7, P2ry6, Dlx3 and TP53 were involved in endometriosis­related inflammation and reproductive pathways. In conclusion, the changes in the expression of lncRNAs, mRNAs and proteins (Adamts7, P2ry6, Dlx3 and TP53) may possibly affect endometrial receptivity in rats with endometriosis during the implantation window, probably resulting in implantation failure of the embryo.


Assuntos
Proteína ADAMTS7/genética , Endometriose/genética , Proteínas de Homeodomínio/genética , Receptores Purinérgicos P2/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Animais , Implantação do Embrião/genética , Endometriose/patologia , Endométrio/crescimento & desenvolvimento , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Gravidez , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Ratos , Útero/crescimento & desenvolvimento , Útero/patologia
4.
Pharmacol Rep ; 71(5): 926-928, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31450027

RESUMO

BACKGROUND: Narcolepsy with cataplexy is a neurological sleep disorder, which is believed to arise from the autoimmune destruction of hypocretin-producing neurons. The purinergic receptor P2Y11 is associated with narcolepsy in genome-wide association studies, and P2RY11 sequencing has further revealed eight rare missense mutations associated with the disease. Some of these mutations alter the signaling properties of P2Y11, but for some, no functional effects have been discovered so far. METHODS: This study examined the surface expression of the eight narcolepsy-associated P2Y11 mutations using an in vitro surface expression assay. RESULTS: The assay showed excellent discrimination between cells transfected with tagged wild type and the untagged P2Y11 receptor, proving complete specificity towards the 3HA-N-tag used for the assay. Our results show a decreased surface expression of the R307W P2Y11 mutant and a surface expression similar to wild type for the other seven mutants. CONCLUSION: Based on the present findings, alteration in surface expression is not likely to play a role in how P2Y11 influences narcolepsy pathogenesis. This is important because intact surface expression increases the usefulness of P2Y11 as a future drug target.


Assuntos
Expressão Gênica , Narcolepsia/genética , Receptores Purinérgicos P2/genética , Variação Genética , Células HEK293 , Humanos , Mutação , Neurônios/metabolismo , Orexinas/metabolismo , Transfecção
5.
IUBMB Life ; 71(10): 1552-1560, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31301116

RESUMO

Rheumatoid arthritis is a common chronic inflammatory joint disease. Fibroblast-like synoviocytes-mediated inflammation is closely associated with the development of rheumatoid arthritis. In this study, we report that P2Y11 receptor activity is required for cytokine-induced inflammation in primary fibroblast-like synoviocytes (FLS). P2Y11R is fairly expressed in primary FLS isolated from healthy subjects and is elevated to around three- to four-fold in rheumatoid arthritis-derived FLS. The expression of P2Y11R is inducible upon IL-1ß treatment. Blockage of P2Y11R by its antagonist suppresses IL-1ß-induced TNF-α and IL-6 induction and ameliorates oxidative stress as determined by levels of cellular ROS and the oxidative byproduct 4-HNE. Moreover, blockage of P2Y11R by NF340 inhibits IL-1ß-induced matrix metalloproteinase protein expression as indicated by the levels of MMP-1, MMP-3, and MMP-13. Mechanistically, blockage of P2Y11R mitigates IL-1ß-activated NFκB signaling, which was revealed by reduced IκBα phosphorylation, nuclear p65 accumulation, and NFκB promoter activity. Our study provides evidence of a protective mechanism of P2Y11R antagonist NF340 against cytokine-induced inflammation. Therefore, targeting P2Y11R could have potential therapeutic implication in the treatment of RA.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Inflamação/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2/genética , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/patologia , Mediadores da Inflamação/farmacologia , Interleucina-1beta/genética , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Inibidor de NF-kappaB alfa/genética , NF-kappa B/genética , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores Purinérgicos P2/efeitos dos fármacos , Sinoviócitos/efeitos dos fármacos
6.
Biochim Biophys Acta Mol Basis Dis ; 1865(10): 2595-2605, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31271845

RESUMO

Extracellular nucleotides are released as constitutive danger signals by various cell types and activate nucleotide (P2) receptors such as P2Y6 receptor. P2Y6 activation on monocytes induces the secretion of the chemokine CXCL8 which may propagate intestinal inflammation. Also, P2Y6 expression is increased in infiltrating T cells of Crohn's disease patients. As inflammatory bowel disease (IBD) is associated with immune cell recruitment, we hypothesised that P2Y6 would participate to the establishment of inflammation in this disease. To address this, we used P2Y6 deficient (P2ry6--/-) mice in the dextran sodium sulfate (DSS) murine model of IBD. In disagreement with our hypothesis, P2Y6 deficient mice were more susceptible to inflammation induced by DSS than WT mice. DSS treated-P2ry6-/- mice showed increased histological damage and increased neutrophil and macrophage infiltration that correlated with increased mRNA levels of the chemokines KC and MCP-1. DSS treated-P2ry6-/- mice exhibited also higher levels of Th17/Th1 lymphocytes in their colon which correlated with increased levels of IFN-γ and IL-17A in the sera as well as increased mRNA levels of IFN-γ, IL-17A, IL-6, IL-23 and IL-1ß in P2ry6-/- colons. This inflammation was also accompanied by a decreased cell proliferation and goblet cell number. Importantly, injection of anti-IL-17 intraperitoneally partially protected P2ry6-/- mice from DSS-induced colitis. Taken together, in the absence of P2Y6, an exacerbated intestinal inflammation to DSS was observed which correlated with increased recruitment of Th17/Th1 lymphocytes. These data suggest a protective role of P2Y6 expressed on leukocytes in intestinal inflammation.


Assuntos
Inflamação/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Células Th17/metabolismo , Animais , Proliferação de Células , Quimiocina CCL2/metabolismo , Quimiocinas/metabolismo , Colite/induzido quimicamente , Colite/metabolismo , Colo/metabolismo , Citocinas/sangue , Citocinas/metabolismo , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Feminino , Inflamação/induzido quimicamente , Inflamação/patologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Interleucina-8/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th1/metabolismo , Células Th17/imunologia , Transcriptoma
7.
Biomed Res Int ; 2019: 3065818, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236404

RESUMO

Hepatocellular carcinoma (HCC) is a primary cause of cancer-related death in the world. Despite the fact that there are many methods to treat HCC, the 5-year survival rate of HCC is still at a low level. Emodin can inhibit the growth of HCC cells in vitro and in vivo. However, the gene regulation of emodin in HCC has not been well studied. In our research, RNA sequencing technology was used to identify the differentially expressed genes (DEGs) in HepG2 cells induced by emodin. A total of 859 DEGs were identified, including 712 downregulated genes and 147 upregulated genes in HepG2 cells treated with emodin. We used DAVID for function and pathway enrichment analysis. The protein-protein interaction (PPI) network was constructed using STRING, and Cytoscape was used for module analysis. The enriched functions and pathways of the DEGs include positive regulation of apoptotic process, structural molecule activity and lipopolysaccharide binding, protein digestion and absorption, ECM-receptor interaction, complement and coagulation cascades, and MAPK signaling pathway. 25 hub genes were identified and pathway analysis revealed that these genes were mainly enriched in neuropeptide signaling pathway, inflammatory response, and positive regulation of cytosolic calcium ion concentration. Survival analysis showed that LPAR6, C5, SSTR5, GPR68, and P2RY4 may be involved in the molecular mechanisms of emodin therapy for HCC. A quantitative real-time PCR (qRT-PCR) assay showed that the mRNA levels of LPAR6, C5, SSTR5, GPR68, and P2RY4 were significantly decreased in HepG2 cells treated with emodin. In conclusion, the identified DEGs and hub genes in the present study provide new clues for further researches on the molecular mechanisms of emodin.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Emodina/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Transcriptoma/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Mapeamento de Interação de Proteínas , Receptores Acoplados a Proteínas-G/genética , Receptores de Ácidos Lisofosfatídicos/genética , Receptores Purinérgicos P2/genética , Receptores de Somatostatina/genética , Transdução de Sinais/efeitos dos fármacos , Software
8.
J Exp Clin Cancer Res ; 38(1): 233, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159832

RESUMO

BACKGROUND: Previous study demonstrated that extracellular ATP could promote cell migration and invasion in multiple human cancers. Till now, the pro-invasive mechanisms of ATP and P2RX6, a preferred receptor for ATP, are still poorly studied in RCC. METHODS: Bioinformatics analysis was performed to identify the differentially expressed genes during RCC different stages. Tissue microarray, IHC staining and survival analysis was respectively used to evaluate potential clinical function. In vitro and in vivo assays were performed to explore the P2RX6 biological effects in RCC progression. RESULTS: We found that ATP might increase RCC cells migration and invasion through P2RX6. Mechanism dissection revealed that ATP-P2RX6 might modulate the Ca2+-mediated p-ERK1/2/MMP9 signaling to increase the RCC cells migration and invasion. Furthermore, METTL14 implicated m6A modification in RCC and down-regulated P2RX6 protein translation. In addition, human clinical survey also indicated the positive correlation of this newly identified signaling in RCC progression and prognosis. CONCLUSIONS: Our findings revealed that the newly identified ATP-P2RX6-Ca2+-p-ERK1/2-MMP9 signaling facilitates RCC cell invasion and metastasis. Targeting this novel signaling pathway with small molecules might help us to develop a new approach to better suppress RCC progression.


Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Metilação de DNA , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores Purinérgicos P2/genética , Animais , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Biologia Computacional/métodos , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Neoplasias Renais/metabolismo , Camundongos , Modelos Biológicos , Metástase Neoplásica , Estadiamento de Neoplasias , Fosforilação , Prognóstico , Transdução de Sinais
9.
Int J Dermatol ; 58(8): 946-952, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31077348

RESUMO

BACKGROUND: Autosomal recessive wooly hair/hypotrichosis is an inherited disorder of hair characterized by less dense, short, and tightly curled hair on the scalp and sometimes less dense to complete absence of eyebrows and eyelashes. Autosomal recessive wooly hair/hypotrichosis phenotypes are mostly associated with pathogenic sequence variants in LIPH and LPAR6 genes. METHODS: To find out the molecular basis of the disease, five families with autosomal recessive wooly hair/hypotrichosis were recruited for genetic analysis. Direct Sanger sequencing of LIPH and LPAR6 genes was carried out using BigDye chain termination chemistry. P2RY5 protein homology models were developed to study the effect of mutation on protein structure in a family having novel mutation. RESULTS: Sanger sequencing revealed a novel homozygous missense mutation (c.47A>T) in the LPAR6 gene in family A, while recurrent mutation (c.436G>A) was detected in the rest of the four families (B-E). Protein homology models for both native and mutant P2RY5 protein were developed to study the difference in subtle structural features because of Lys16Met (K16M) mutation. We observed that P2RY5K16M mutation results decrease in the number of ionic interactions detrimental to the protein stability. Protein modeling studies revealed that the novel mutation identified here decreased the number of ionic interactions by affecting physicochemical parameters of the protein, leading to an overall decrease in protein stability with no major secondary structural changes. CONCLUSION: The molecular analysis further confirms the frequent involvement of LPAR6 in autosomal recessive wooly hair/hypotrichosis, while the bioinformatic study revealed that the missense mutation destabilizes the overall structure of P2RY5 protein.


Assuntos
Genes Recessivos/genética , Doenças do Cabelo/genética , Cabelo/anormalidades , Hipotricose/genética , Receptores de Ácidos Lisofosfatídicos/genética , Biologia Computacional , Consanguinidade , Feminino , Humanos , Masculino , Mutação de Sentido Incorreto , Paquistão , Linhagem , Fenótipo , Estrutura Secundária de Proteína/genética , Receptores de Ácidos Lisofosfatídicos/química , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Homologia de Sequência de Aminoácidos
10.
J Neuroinflammation ; 16(1): 71, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30947729

RESUMO

BACKGROUND: HIV-associated neurocognitive disorders (HANDs) afflict more than half of HIV-1-positive individuals. The transactivator of transcription (Tat) produced by HIV virus elicits inflammatory process and is a major neurotoxic mediator that induce neuron damage during HAND pathogenesis. Activated astrocytes are important cells involved in neuroinflammation and neuronal damage. Purinergic receptors expressed in astrocytes participate in a positive feedback loop in virus-induced neurotoxicity. Here, we investigated that whether P2Y4R, a P2Y receptor subtype, that expressed in astrocyte participates in Tat-induced neuronal death in vitro and in vivo. METHODS: Soluble Tat protein was performed to determine the expression of P2Y4R and proinflammatory cytokines in astrocytes using siRNA technique via real-time PCR, Western blot, and immunofluorescence assays. Cytometric bead array was used to measure proinflammatory cytokine release. The TUNEL staining and MTT cell viability assay were analyzed for HT22 cell apoptosis and viability, and the ApopTag® peroxidase in situ apoptosis detection kit and cresyl violet staining for apoptosis and death of hippocampal neuron in vivo. RESULTS: We found that Tat challenge increased the expression of P2Y4R in astrocytes. P2Y4R signaling in astrocytes was involved in Tat-induced inflammatory cytokine production via PI3K/Akt- and ERK1/2-dependent pathways. Knockdown of P2Y4R expression significantly reduced inflammatory cytokine production and relieved Tat-mediated neuronal apoptosis in vitro. Furthermore, in vivo challenged with Tat, P2Y4R knockdown mice showed decreased inflammation and neuronal damage, especially in hippocampal CA1 region. CONCLUSIONS: Our data provide novel insights into astrocyte-mediated neuron damage during HIV-1 infection and suggest a potential therapeutic target for HANDs.


Assuntos
Astrócitos/efeitos dos fármacos , Citocinas/metabolismo , Neurônios/efeitos dos fármacos , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais/fisiologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glioma/patologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/patologia , Proteína Oncogênica v-akt , Fosfatidilinositol 3-Quinases , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/genética , Transdução de Sinais/genética , Transdução Genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
11.
Front Neural Circuits ; 13: 14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30894803

RESUMO

Little is known about the molecular and cellular mechanisms involved in the formation of the cranial peripheral sensory system in vertebrates. To identify genes involved in the formation of these circuits, we performed a forward genetic screen utilizing a transgenic zebrafish line (p2rx3.2:gfp sl1) that expresses green fluorescent protein (gfp) in sensory neurons of the Vth, VIIth, IXth and Xth cranial ganglia. Here, we describe a novel zebrafish mutant in which a missense mutation in the adam19b gene selectively affects the epibranchial sensory circuits.


Assuntos
Proteínas ADAM/metabolismo , Orientação de Axônios/fisiologia , Rombencéfalo/citologia , Rombencéfalo/fisiologia , Células Receptoras Sensoriais/metabolismo , Proteínas ADAM/genética , Animais , Animais Geneticamente Modificados , Orientação de Axônios/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Análise Mutacional de DNA , Embrião não Mamífero , Gânglios dos Invertebrados/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva , Mutação/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
12.
Int J Mol Sci ; 20(6)2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30909368

RESUMO

BACKGROUND: Vascular endothelial injury during ischemia generates apoptotic cell death and precedes apoptosis of underlying tissues. We aimed at studying the role of extracellular adenosine triphosphate (ATP) on endothelial cells protection against hypoxia injury. METHODS: In a hypoxic model on endothelial cells, we quantified the extracellular concentration of ATP and adenosine. The expression of mRNA (ectonucleotidases, adenosine, and P2 receptors) was measured. Apoptosis was evaluated by the expression of cleaved caspase 3. The involvement of P2 and adenosine receptors and signaling pathways was investigated using selective inhibitors. RESULTS: Hypoxic stress induced a significant increase in extracellular ATP and adenosine. After a 2-h hypoxic injury, an increase of cleaved caspase 3 was observed. ATP anti-apoptotic effect was prevented by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and CGS15943, as well as by selective A2A, A2B, and A3 receptor antagonists. P2 receptor-mediated anti-apoptotic effect of ATP involved phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), mitoKATP, and nitric oxide synthase (NOS) pathways whereas adenosine receptor-mediated anti-apoptotic effect involved ERK1/2, protein kinase A (PKA), and NOS. CONCLUSIONS: These results suggest a complementary role of P2 and adenosine receptors in ATP-induced protective effects against hypoxia injury of endothelial. This could be considered therapeutic targets to limit the development of ischemic injury of organs such as heart, brain, and kidney.


Assuntos
Trifosfato de Adenosina/metabolismo , Apoptose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Hipóxia/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina/metabolismo , Apoptose/genética , Biomarcadores , Espaço Extracelular/metabolismo , Expressão Gênica , Humanos , Hipóxia/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/genética , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P2/genética , Transdução de Sinais , Estresse Fisiológico/genética
13.
Cell Death Dis ; 10(3): 165, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30778044

RESUMO

Peripheral nerve injury causes neuropathic pain and microglia activation. P2Y12 receptors on microglia are thought to be a key player in the surveillance of the local environment, but whether or how these receptors are engaged in the cross-talk between microglia and neurons of the dorsal horn remain ambiguous. Using a rodent model of nerve injury-induced pain, we investigated the roles of P2Y12 in microglia activation, excitatory synaptic transmission, and nociceptive allodynia. We found that spinal nerve ligation (SNL) significantly increased the level of P2Y12 receptors specifically in the microglia of the ipsilateral dorsal horn. Injections of P2Y12 antagonists (MRS2395 or clopidogrel) attenuated microglia activation and increased the paw withdrawal latency in response to thermal stimuli on the ipsilateral side without affecting the basal threshold on the contralateral side. These effects on pain behaviors were replicated in P2Y12 knockout mice. Patch-clamp recordings further revealed that partial sciatic nerve ligation (PSNL)-induced excessive miniature excitatory postsynaptic currents (mEPSCs) were significantly attenuated in P2Y12 knockout mice. Moreover, we found that SNL activates the GTP-RhoA/ROCK2 signaling pathway and elevates the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), which was inhibited by the P2Y12 antagonist. The phosphorylation of p38 MAPK was inhibited by a ROCK inhibitor, but not vice versa, suggesting that p38 MAPK is downstream of ROCK activation. Our findings suggest that nerve injury engages the P2Y12 receptor-dependent GTP-RhoA/ROCK2 signaling pathway to upregulate excitatory synaptic transmission in the dorsal horn. This cross-talk ultimately participates in the manifestation of nociceptive allodynia, implicating P2Y12 receptor as a potential target for alleviating neuropathic pain.


Assuntos
Microglia/metabolismo , Neuralgia/fisiopatologia , Receptores Purinérgicos P2/fisiologia , Medula Espinal/metabolismo , Nervos Espinhais/fisiologia , Transmissão Sináptica/fisiologia , Adenina/análogos & derivados , Adenina/uso terapêutico , Animais , Clopidogrel/uso terapêutico , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuralgia/metabolismo , Neuralgia/terapia , Neurônios/fisiologia , Fosforilação/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2/uso terapêutico , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Transdução de Sinais/efeitos dos fármacos , Corno Dorsal da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Nervos Espinhais/cirurgia , Valeratos/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
14.
Arch Cardiovasc Dis ; 112(2): 124-134, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30600215

RESUMO

BACKGROUND: The P2Y13 purinergic receptor regulates hepatic high-density lipoprotein uptake and biliary sterol secretion; it acts downstream of the membrane ecto-F1-adenosine triphosphatase, which generates extracellular adenosine diphosphate that selectively activates P2Y13, resulting in high-density lipoprotein endocytosis. Previous studies have shown that the serum concentration of the F1-adenosine triphosphatase inhibitor inhibitory factor 1 is negatively associated with cardiovascular risk. AIM: To evaluate whether p2y13 genetic variants affect cardiovascular risk. METHODS: Direct sequencing of the p2y13 coding and flanking regions was performed in a subcohort of 168 men aged 45-74 years with stable coronary artery disease and 173 control subjects from the GENES study. The two most frequent mutations, rs3732757 and rs1466684, were genotyped in 767 patients with coronary artery disease and 789 control subjects, and their association with cardiovascular risk markers was analysed. RESULTS: Carriers of the rs3732757 261T and rs1466684 557G alleles represented 9% and 27.5% of the entire population, respectively. The allele frequencies were identical in patients with coronary artery disease and control subjects. The presence of 261T was associated with higher concentrations of plasma lipoprotein A-I and inhibitory factor 1, increased fat mass and a lower heart rate. Moreover, the proportion of patients with coronary artery disease with a pejorative systolic ankle-brachial index was lower in carriers of the 261T allele. In both populations, the 557G allele was associated with increased concentrations of lipoprotein(a), and an allele dose effect was observed. CONCLUSIONS: Two frequent p2y13 variants are associated with specific bioclinical markers of cardiovascular risk. Although neither one of these variants appears to be related to the development of atherosclerotic disease, they may modulate the risk of additional cardiovascular complications.


Assuntos
Adiposidade/genética , Doença da Artéria Coronariana/genética , Frequência Cardíaca/genética , Lipoproteína(a)/sangue , Polimorfismo de Nucleotídeo Único , Proteínas/análise , Receptores Purinérgicos P2/genética , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/fisiopatologia , França , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Fatores de Risco
15.
Food Chem Toxicol ; 123: 298-313, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30291944

RESUMO

Coffee is a drink prepared from roasted coffee beans and is lauded for its aroma and flavour. It is the third most popular beverage in the world. This beverage is known by its stimulant effect associated with the presence of methylxanthines. Caffeine, a purine-like molecule (1,3,7 trymetylxantine), is the most important bioactive compound in coffee, among others such as chlorogenic acid (CGA), diterpenes, and trigonelline. CGA is a phenolic acid with biological properties as antioxidant, anti-inflammatory, neuroprotector, hypolipidemic, and hypoglicemic. Purinergic system plays a key role inneuromodulation and homeostasis. Extracellular ATP, other nucleotides and adenosine are signalling molecules that act through their specific receptors, namely purinoceptors, P1 for nucleosides and P2 for nucleotides. They regulate many pathological processes, since adenosine, for instance, can limit the damage caused by ATP in the excitotoxicity from the neuronal cells. The primary purpose of this review is to discuss the effects of coffee, caffeine, and CGA on the purinergic system. This review focuses on the relationship/interplay between coffee, caffeine, CGA, and adenosine, and their effects on ectonucleotidases activities as well as on the modulation of P1 and P2 receptors from central nervous system and also in peripheral tissue.


Assuntos
Cafeína/metabolismo , Ácido Clorogênico/metabolismo , Extratos Vegetais/metabolismo , Purinas/metabolismo , Animais , Cafeína/química , Ácido Clorogênico/química , Coffea/química , Café/química , Café/metabolismo , Humanos , Extratos Vegetais/química , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais
16.
J Mol Cell Cardiol ; 121: 212-222, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30031814

RESUMO

Cardiac fibroblasts are important regulators of myocardial structure and function. Their implications in pathological processes such as Ischemia/Reperfusion are well characterized. Cardiac fibroblasts respond to stress by excessive proliferation and secretion of pro-inflammatory cytokines and other factors, e.g. ATP, leading to purinergic receptors activation. P2Y11 receptor (P2Y11R) is an ATP-sensitive GPCR playing an immunomodulatory role in human dendritic cells (DC). We hypothesized that P2Y11R stimulation modulated the pro-inflammatory responses of human cardiac fibroblasts (HCF) to Hypoxia/Reoxygenation (H/R) mainly by acting on their secretome. P2Y11R stimulation in HCF at the onset of reoxygenation significantly limited H/R-induced proliferation (-19%) and pro-inflammatory cytokines and ATP secretion (-44% and -83% respectively). Exposure of DC to HCF secretome increased their expression of CD83, CD25 and CD86, suggesting a switch from immature to mature phenotype. Under LPS stimulation, DC had a pro-inflammatory profile (high IL-12/IL-10 ratio) that was decreased by HCF secretome (-3,8-fold), indicating induction of a tolerogenic profile. Moreover, P2Y11R inhibition in HCF led to high IL-12 secretion in DC, suggesting that the immunomodulatory effect of HCF secretome is P2Y11R-dependant. HCF secretome reduced H/R-induced cardiomyocytes death (-23%) through RISK pathway activation. P2Y11R inhibition in HCF induced a complete loss of HCF secretome protective effect, highlighting the cardioprotective role of P2Y11R. Our data demonstrated paracrine interactions between HCF, cardiomyocytes and DC following H/R, suggesting a key role of HCF in the cellular responses to reperfusion. These results also demonstrated a beneficial role of P2Y11R in HCF during H/R and strongly support the hypothesis that P2Y11R is a modulator of I/R injury.


Assuntos
Traumatismo por Reperfusão Miocárdica/genética , Miocárdio/metabolismo , Receptores Purinérgicos P2/genética , Traumatismo por Reperfusão/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Hipóxia/genética , Hipóxia/patologia , Fatores Imunológicos/metabolismo , Interleucina-12/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Comunicação Parácrina/genética , Receptores Purinérgicos P2/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
17.
Int J Mol Sci ; 19(7)2018 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-30029501

RESUMO

Uridine 5'-diphosphate (UDP)-activated purinergic receptor P2Y6 is a member of a G-protein-coupled purinergic receptor family that plays an important role in mammalian innate immunity. However, the role of the P2Y6 receptor (P2Y6R) in fish immunity has not been investigated. In this report, we characterized a P2Y6R gene from Japanese flounder (Paralichthys olivaceus) and examined its role in fish innate immunity. Sequence analysis reveals that the Japanese flounder P2Y6R protein is conserved and possesses four potential glycosylation sites. Quantitative real-time RT-PCR analysis shows that P2Y6R is broadly distributed in all examined Japanese flounder tissues with dominant expression in the liver. In addition, P2Y6R gene expression was up-regulated in head kidney macrophages (HKMs) upon lipopolysaccharides (LPS) and poly(I:C) stimulations but down-regulated by LPS challenge in peripheral blood leukocytes (PBLs). Furthermore, pharmacological inhibition of the endogenous P2Y6 receptor activity by the potently selective P2Y6R antagonist, MRS 2578, greatly up-regulated pro-inflammatory cytokine interleukin (IL)-1ß, IL-6 and TNF-α gene expression in PBL cells treated with UDP. Moreover, LPS- and poly(I:C)-induced gene expression of IL-1ß and TNF-α in Japanese flounder PBL cells was attenuated significantly by inhibition of P2Y6R activity with antagonist MRS 2578. Collectively, we, for the first time, showed the involvement of functional purinergic P2Y6R in fish innate immunity.


Assuntos
Linguado/imunologia , Linguado/metabolismo , Imunidade Inata , Receptores Purinérgicos P2/metabolismo , Difosfato de Uridina/farmacologia , Sequência de Aminoácidos , Animais , Citocinas/genética , Citocinas/metabolismo , Linguado/sangue , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Inflamação/imunologia , Inflamação/patologia , Isotiocianatos/farmacologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Filogenia , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Análise de Sequência de Proteína , Tioureia/análogos & derivados , Tioureia/farmacologia
18.
J Clin Neurosci ; 56: 156-162, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30045810

RESUMO

Purinergic signaling in spinal cord microglia plays an important role in the pathogenesis of neuropathic pain. Among all P2 receptors, P2Y6 receptor is expressed in rat dorsal spinal cord. However, it's not clear that the role of P2Y6 receptor in the chronic constriction injury (CCI) model of neuropathic pain rats. We evaluated the effect of repeated intrathecal administration of MRS2578 (selective P2Y6 receptor antagonist) on CCI-induced nociceptive behaviors in rats. After CCI, MRS2578 (10-11-10-4 M) was administration. The thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were assessed. The expression of P2Y6 receptor and Iba-1 at rat dorsal spinal cord was observed by using RT-PCR. We found that intrathecal injection of MRS2578 suppressed CCI-induced mechanical allodynia and thermal hyperalgesia with a dose-dependent manner. The CCI rats presented increased expression of P2Y6 receptor and Iba-1 at the mRNA level in the ipsilateral dorsal spinal cord than that in sham group. Treatment with either minocycline or SB203580 effectively inhibited P2Y6 receptor expression compared to CCI rats. Intrathecal injection of UDP enhanced mechanical and thermal allodynia than that in CCI group. To the further study, intrathecal injection of UDP causes mechanical allodynia and thermal hyperalgesia in naive rats. The increased expression of P2Y6 receptor and Iba-1 were observed in UDP-treated rats. Intrathecal injection of MRS2578 alleviates pain response in UDP-treated rats. These observations suggested that P2Y6 receptor in dorsal spinal cord contribute to mechanical allodynia and thermal hyperalgesia in CCI-induced neuropathic pain.


Assuntos
Neuralgia/metabolismo , Receptores Purinérgicos P2/metabolismo , Nervo Isquiático/lesões , Animais , Isotiocianatos/farmacologia , Isotiocianatos/uso terapêutico , Masculino , Neuralgia/tratamento farmacológico , Antagonistas Purinérgicos/farmacologia , Antagonistas Purinérgicos/uso terapêutico , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/genética , Nervo Isquiático/metabolismo , Tioureia/análogos & derivados , Tioureia/farmacologia , Tioureia/uso terapêutico
19.
EBioMedicine ; 32: 72-83, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29887330

RESUMO

Although psychotropic drugs act on neurons and glial cells, how glia respond, and whether glial responses are involved in therapeutic effects are poorly understood. Here, we show that fluoxetine (FLX), an anti-depressant, mediates its anti-depressive effect by increasing the gliotransmission of ATP. FLX increased ATP exocytosis via vesicular nucleotide transporter (VNUT). FLX-induced anti-depressive behavior was decreased in astrocyte-selective VNUT-knockout mice or when VNUT was deleted in mice, but it was increased when astrocyte-selective VNUT was overexpressed in mice. This suggests that VNUT-dependent astrocytic ATP exocytosis has a critical role in the therapeutic effect of FLX. Released ATP and its metabolite adenosine act on P2Y11 and adenosine A2b receptors expressed by astrocytes, causing an increase in brain-derived neurotrophic factor in astrocytes. These findings suggest that in addition to neurons, FLX acts on astrocytes and mediates its therapeutic effects by increasing ATP gliotransmission.


Assuntos
Depressão/tratamento farmacológico , Fluoxetina/administração & dosagem , Proteínas de Transporte de Nucleotídeos/genética , Receptor A2B de Adenosina/genética , Receptores Purinérgicos P2/genética , Trifosfato de Adenosina/metabolismo , Animais , Antidepressivos/administração & dosagem , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Depressão/genética , Depressão/metabolismo , Depressão/patologia , Exocitose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo
20.
Front Immunol ; 9: 1159, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29937766

RESUMO

Adenosine triphosphate (ATP) is known to induce cell death in T lymphocytes at high extracellular concentrations. CD4+ and CD8+ T lymphocytes have a differential response to ATP, which in mice is due to differences in the P2X7 receptor expression levels. By contrast, we observed that the difference in human CD4+ and CD8+ T lymphocyte response toward the synthetic ATP-analog BzATP is not explained by a difference in human P2X7 receptor expression. Rather, the BzATP-induced human P2X7 receptor response in naïve and immune-activated lymphocyte subtypes correlated with the expression of another ATP-binding receptor: the human P2Y11 receptor. In a recombinant expression system, the coexpression of the human P2Y11 receptor counteracted BzATP-induced human P2X7 receptor-driven lactate dehydrogenase release (a marker of cell death) and pore formation independent of calcium signaling. A mutated non-signaling human P2Y11 receptor had a similar human P2X7 receptor-inhibitory effect on pore formation, thus demonstrating that the human P2X7 receptor interference was not caused by human P2Y11 receptor signaling. In conclusion, we demonstrate an important species difference in the ATP-mediated cell death between mice and human cells and show that in human T lymphocytes, the expression of the human P2Y11 receptor correlates with human P2X7 receptor-driven cell death following BzATP stimulation.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2/metabolismo , Linfócitos T/fisiologia , Animais , Sinalização do Cálcio , Morte Celular , Células Cultivadas , Difosfonatos/farmacologia , Humanos , Camundongos , Naftalenossulfonatos/farmacologia , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptor Cross-Talk , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7/genética , Transgenes/genética
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