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1.
Oral Oncol ; 109: 104808, 2020 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-32540611

RESUMO

OBJECTIVES: To assess functional expression of the P2Y2 nucleotide receptor (P2Y2R) in head and neck squamous cell carcinoma (HNSCC) cell lines and define its role in nucleotide-induced epidermal growth factor receptor (EGFR) transactivation. The use of anti-EGFR therapeutics to treat HNSCC is hindered by intrinsic and acquired drug resistance. Defining novel pathways that modulate EGFR signaling could identify additional targets to treat HNSCC. MATERIALS AND METHODS: In human HNSCC cell lines CAL27 and FaDu and the mouse oral cancer cell line MOC2, P2Y2R contributions to extracellular nucleotide-induced changes in intracellular free Ca2+ concentration and EGFR and extracellular signal-regulated kinase (ERK1/2) phosphorylation were determined using the ratiometric Ca2+ indicator fura-2 and immunoblot analysis, respectively. Genetic knockout of P2Y2Rs using CRISPR technology or pharmacological inhibition with P2Y2R-selective antagonist AR-C118925 defined P2Y2R contributions to in vivo tumor growth. RESULTS: P2Y2R agonists UTP and ATP increased intracellular Ca2+ levels and ERK1/2 and EGFR phosphorylation in CAL27 and FaDu cells, responses that were inhibited by AR-C118925 or P2Y2R knockout. P2Y2R-mediated EGFR phosphorylation was also attenuated by inhibition of the adamalysin family of metalloproteases or Src family kinases. P2Y2R knockout reduced UTP-induced CAL27 cell proliferation in vitro and significantly reduced CAL27 and FaDu tumor xenograft volume in vivo. In a syngeneic mouse model of oral cancer, AR-C118925 administration reduced MOC2 tumor volume. CONCLUSION: P2Y2Rs mediate HNSCC cell responses to extracellular nucleotides and genetic or pharmacological blockade of P2Y2R signaling attenuates tumor cell proliferation and tumorigenesis, suggesting that the P2Y2R represents a novel therapeutic target in HNSCC.

2.
Brain Res Bull ; 151: 25-37, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30472151

RESUMO

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by three major histopathological markers: amyloid-ß (Aß) plaques, neurofibrillary tangles and gliosis in the central nervous system (CNS). It is now accepted that neuroinflammatory events in the CNS play a crucial role in the development of AD. This review focuses on neuroinflammatory signaling mediated by purinergic receptors (P1 adenosine receptors, P2X ATP-gated ion channels and G protein-coupled P2Y nucleotide receptors) and how therapeutic modulation of purinergic signaling influences disease progression in AD patients and animal models of AD.


Assuntos
Doença de Alzheimer/metabolismo , Receptores Purinérgicos/metabolismo , Receptores Purinérgicos/fisiologia , Trifosfato de Adenosina , Doença de Alzheimer/patologia , Animais , Sistema Nervoso Central/fisiologia , Modelos Animais de Doenças , Humanos , Placa Amiloide/metabolismo , Purinas/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Transdução de Sinais/fisiologia
3.
Mol Biol Cell ; 28(21): 2887-2903, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28835374

RESUMO

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, ß3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation ß5/ß3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y2R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αIIbß3 integrins and (RGD)P2Y2R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y2R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y2R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αIIbß3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα13 protein from binding to the cytoplasmic domain of the ß3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y2R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation.


Assuntos
Infecções por Hantavirus/metabolismo , Integrina beta3/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Animais , Células CHO , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Cricetulus , Células Endoteliais , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Orthohantavírus/isolamento & purificação , Humanos , Cadeias beta de Integrinas/metabolismo , Integrina beta3/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores Purinérgicos P2Y2/genética , Semaforinas/metabolismo , Transdução de Sinais
4.
J Biol Chem ; 292(40): 16626-16637, 2017 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-28798231

RESUMO

Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1ß and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1ß, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R-/-). P2X7R-mediated IL-1ß release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1ß release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28-/-, IFNγ-/-, NOD.H-2h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.


Assuntos
Células Epiteliais/metabolismo , Interleucina-1beta/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Piridinas/farmacologia , Receptores Purinérgicos P2X7/metabolismo , Síndrome de Sjogren/metabolismo , Glândula Submandibular/metabolismo , Tetrazóis/farmacologia , Animais , Antígenos CD28/genética , Antígenos CD28/metabolismo , Modelos Animais de Doenças , Células Epiteliais/patologia , Inflamassomos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/genética , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Potássio/metabolismo , Receptores Purinérgicos P2X7/genética , Síndrome de Sjogren/genética , Síndrome de Sjogren/patologia , Sódio/metabolismo , Glândula Submandibular/patologia
5.
Cell Mol Life Sci ; 74(4): 731-746, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27652381

RESUMO

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y2 receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y2 receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y2 Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y2 Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y2 receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y2 RGE mutant receptors. Consistent with the role of P2Y2 receptors in vasodilation, receptor knockdown and overexpression of P2Y2 RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y2 receptor in shear stress-induced cytoskeletal alterations in HUVECs.


Assuntos
Actinas/metabolismo , Células Endoteliais/citologia , Receptores Purinérgicos P2Y2/metabolismo , Fibras de Estresse/metabolismo , Actinas/ultraestrutura , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrinas/metabolismo , Mutação , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores Purinérgicos P2Y2/genética , Fibras de Estresse/ultraestrutura , Estresse Mecânico , Cicatrização
6.
Neuropharmacology ; 104: 169-79, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26519903

RESUMO

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of memory and cognitive ability and is a serious cause of mortality. Many of the pathological characteristics associated with AD are revealed post-mortem, including amyloid-ß plaque deposition, neurofibrillary tangles containing hyperphosphorylated tau proteins and neuronal loss in the hippocampus and cortex. Although several genetic mutations and risk factors have been associated with the disease, the causes remain poorly understood. Study of disease-initiating mechanisms and AD progression in humans is inherently difficult as most available tissue specimens are from late-stages of disease. Therefore, AD researchers rely on in vitro studies and the use of AD animal models where neuroinflammation has been shown to be a major characteristic of AD. Purinergic receptors are a diverse family of proteins consisting of P1 adenosine receptors and P2 nucleotide receptors for ATP, UTP and their metabolites. This family of receptors has been shown to regulate a wide range of physiological and pathophysiological processes, including neuroinflammation, and may contribute to the pathogenesis of neurodegenerative diseases like Parkinson's disease, multiple sclerosis and AD. Experimental evidence from human AD tissue has suggested that purinergic receptors may play a role in AD progression and studies using selective purinergic receptor agonists and antagonists in vitro and in AD animal models have demonstrated that purinergic receptors represent novel therapeutic targets for the treatment of AD. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Encefalite/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Humanos , Microglia/metabolismo , Placa Amiloide/metabolismo
8.
PLoS One ; 10(5): e0123641, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25955532

RESUMO

Transforming growth factor-ß (TGF-ß) is a multi-functional cytokine with a well-described role in the regulation of tissue fibrosis and regeneration in the liver, kidney and lung. Submandibular gland (SMG) duct ligation and subsequent deligation in rodents is a classical model for studying salivary gland damage and regeneration. While previous studies suggest that TGF-ß may contribute to salivary gland fibrosis, the expression of TGF-ß signaling components has not been investigated in relation to mouse SMG duct ligation-induced fibrosis and regeneration following ductal deligation. Following a 7 day SMG duct ligation, TGF-ß1 and TGF-ß3 were significantly upregulated in the SMG, as were TGF-ß receptor 1 and downstream Smad family transcription factors in salivary acinar cells, but not in ductal cells. In acinar cells, duct ligation also led to upregulation of snail, a Smad-activated E-cadherin repressor and regulator of epithelial-mesenchymal transition, whereas in ductal cells upregulation of E-cadherin was observed while snail expression was unchanged. Upregulation of these TGF-ß signaling components correlated with upregulation of fibrosis markers collagen 1 and fibronectin, responses that were inhibited by administration of the TGF-ß receptor 1 inhibitors SB431542 or GW788388. After SMG regeneration following a 28 day duct deligation, TGF-ß signaling components and epithelial-mesenchymal transition markers returned to levels similar to non-ligated controls. The results from this study indicate that increased TGF-ß signaling contributes to duct ligation-induced changes in salivary epithelium that correlate with glandular fibrosis. Furthermore, the reversibility of enhanced TGF-ß signaling in acinar cells of duct-ligated mouse SMG after deligation indicates that this is an ideal model for studying TGF-ß signaling mechanisms in salivary epithelium as well as mechanisms of fibrosis initiation and their resolution.


Assuntos
Transdução de Sinais/genética , Glândula Submandibular/metabolismo , Glândula Submandibular/patologia , Fator de Crescimento Transformador beta/genética , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Benzamidas/farmacologia , Biomarcadores/metabolismo , Caderinas/genética , Caderinas/metabolismo , Modelos Animais de Doenças , Fibrose , Ligadura , Masculino , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fatores de Transcrição da Família Snail , Glândula Submandibular/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima/genética
9.
Biol Cell ; 107(1): 1-21, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25179475

RESUMO

Alzheimer's disease (AD) is the most common cause of dementia, affecting more than 10% of people over the age of 65. Age is the greatest risk factor for AD, although a combination of genetic, lifestyle and environmental factors also contribute to disease development. Common features of AD are the formation of plaques composed of beta-amyloid peptides (Aß) and neuronal death in brain regions involved in learning and memory. Although Aß is neurotoxic, the primary mechanisms by which Aß affects AD development remain uncertain and controversial. Mouse models overexpressing amyloid precursor protein and Aß have revealed that Aß has potent effects on neuroinflammation and cerebral blood flow that contribute to AD progression. Therefore, it is important to consider how endogenous signalling in the brain responds to Aß and contributes to AD pathology. In recent years, Aß has been shown to affect ATP release from brain and blood cells and alter the expression of G protein-coupled P2Y receptors that respond to ATP and other nucleotides. Accumulating evidence reveals a prominent role for P2Y receptors in AD pathology, including Aß production and elimination, neuroinflammation, neuronal function and cerebral blood flow.


Assuntos
Doença de Alzheimer/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Família Multigênica , Receptores Purinérgicos P2Y/genética
10.
Am J Physiol Cell Physiol ; 307(1): C83-96, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24760984

RESUMO

Hyposalivation resulting from salivary gland dysfunction leads to poor oral health and greatly reduces the quality of life of patients. Current treatments for hyposalivation are limited. However, regenerative medicine to replace dysfunctional salivary glands represents a revolutionary approach. The ability of dispersed salivary epithelial cells or salivary gland-derived progenitor cells to self-organize into acinar-like spheres or branching structures that mimic the native tissue holds promise for cell-based reconstitution of a functional salivary gland. However, the mechanisms involved in salivary epithelial cell aggregation and tissue reconstitution are not fully understood. This study investigated the role of the P2Y2 nucleotide receptor (P2Y2R), a G protein-coupled receptor that is upregulated following salivary gland damage and disease, in salivary gland reconstitution. In vitro results with the rat parotid acinar Par-C10 cell line indicate that P2Y2R activation with the selective agonist UTP enhances the self-organization of dispersed salivary epithelial cells into acinar-like spheres. Other results indicate that the P2Y2R-mediated response is dependent on epidermal growth factor receptor activation via the metalloproteases ADAM10/ADAM17 or the α5ß1 integrin/Cdc42 signaling pathway, which leads to activation of the MAPKs JNK and ERK1/2. Ex vivo data using primary submandibular gland cells from wild-type and P2Y2R(-/-) mice confirmed that UTP-induced migratory responses required for acinar cell self-organization are mediated by the P2Y2R. Overall, this study suggests that the P2Y2R is a promising target for salivary gland reconstitution and identifies the involvement of two novel components of the P2Y2R signaling cascade in salivary epithelial cells, the α5ß1 integrin and the Rho GTPase Cdc42.


Assuntos
Agregação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Glândula Parótida/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y2/efeitos dos fármacos , Glândula Submandibular/efeitos dos fármacos , Uridina Trifosfato/farmacologia , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glândula Parótida/citologia , Glândula Parótida/metabolismo , Fosforilação , Inibidores de Proteases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , Receptores Purinérgicos P2Y2/deficiência , Receptores Purinérgicos P2Y2/genética , Glândula Submandibular/citologia , Glândula Submandibular/metabolismo , Transfecção , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/metabolismo
11.
Mol Neurobiol ; 49(2): 1031-42, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24193664

RESUMO

Neuroinflammation is a prominent feature in Alzheimer's disease (AD) and activation of the brain's innate immune system, particularly microglia, has been postulated to both retard and accelerate AD progression. Recent studies indicate that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is an important regulator of innate immunity by assisting in the recruitment of monocytes to injured tissue, neutrophils to bacterial infections and eosinophils to allergen-infected lungs. In this study, we investigated the role of the P2Y2R in progression of an AD-like phenotype in the TgCRND8 mouse model that expresses Swedish and Indiana mutations in amyloid precursor protein (APP). Our results indicate that P2Y 2 R expression is upregulated in TgCRND8 mouse brain within 10 weeks of age and then decreases after 25 weeks of age, as compared to littermate controls expressing low levels of the P2Y 2 R. TgCRND8 mice with homozygous P2Y 2 R deletion survive less than 5 weeks, whereas mice with heterozygous P2Y 2 R deletion survive for 12 weeks, a time point when TgCRND8 mice are fully viable. Heterozygous P2Y 2 R deletion in TgCRND8 mice increased ß-amyloid (Aß) plaque load and soluble Aß1-42 levels in the cerebral cortex and hippocampus, decreased the expression of the microglial marker CD11b in these brain regions and caused neurological deficits within 10 weeks of age, as compared to age-matched TgCRND8 mice. These findings suggest that the P2Y2R is important for the recruitment and activation of microglial cells in the TgCRND8 mouse brain and that the P2Y2R may regulate neuroprotective mechanisms through microglia-mediated clearance of Aß that when lost can accelerate the onset of an AD-like phenotype in the TgCRND8 mouse.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Modelos Animais de Doenças , Receptores Purinérgicos P2Y2/deficiência , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/patologia , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Receptores Purinérgicos P2Y2/genética
12.
J Biomed Sci Eng ; 7(14): 1105-1121, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25657827

RESUMO

Vascular endothelial cadherin (VE-cadherin) mediates homophylic adhesion between endothelial cells and is an important regulator of angiogenesis, blood vessel permeability and leukocyte trafficking. Rac1, a member of the Rho family of GTPases, controls VE-cadherin adhesion by acting downstream of several growth factors, including angiopoietin-1 and vascular endothelial growth factor (VEGF). Here we show that UTP-induced activation of the Gq protein-coupled P2Y2 nucleotide receptor (P2Y2R) in human coronary artery endothelial cells (HCAECs) activated Rac1 and caused a transient complex to form between P2Y2R, VE-cadherin and VEGF receptor-2 (VEGFR-2). Knockdown of VE-cadherin expression with siRNA did not affect UTP-induced activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but led to a loss of UTP-induced Rac1 activation and tyrosine phosphorylation of p120 catenin, a cytoplasmic protein known to interact with VE-cadherin. Activation of the P2Y2R by UTP also caused a prolonged interaction between p120 catenin and vav2 (a guanine nucleotide exchange factor for Rac) that correlated with the kinetics of UTP-induced tyrosine phosphorylation of p120 catenin and VE-cadherin. Inhibitors of VEGFR-2 (SU1498) or Src (PP2) significantly diminished UTP-induced Rac1 activation, tyrosine phosphorylation of p120 catenin and VE-cadherin, and association of the P2Y2R with VE-cadherin and p120 catenin with vav2. These findings suggest that the P2Y2R uses Src and VEGFR-2 to mediate association of the P2Y2R with VE-cadherin complexes in endothelial adherens junctions to activate Rac1.

13.
J Neurochem ; 125(6): 885-96, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23550835

RESUMO

The pro-inflammatory cytokine interleukin-1ß (IL-1ß), whose levels are elevated in the brain in Alzheimer's and other neurodegenerative diseases, has been shown to have both detrimental and beneficial effects on disease progression. In this article, we demonstrate that incubation of mouse primary cortical neurons (mPCNs) with IL-1ß increases the expression of the P2Y2 nucleotide receptor (P2Y2R) and that activation of the up-regulated receptor with UTP, a relatively selective agonist of the P2Y2R, increases neurite outgrowth. Consistent with the accepted role of cofilin in the regulation of neurite extension, results indicate that incubation of IL-1ß-treated mPCNs with UTP increases the phosphorylation of cofilin, a response absent in PCNs isolated from P2Y2R(-/-) mice. Other findings indicate that function-blocking anti-αv ß3/5 integrin antibodies prevent UTP-induced cofilin activation in IL-1ß-treated mPCNs, suggesting that established P2Y2R/αv ß3/5 interactions that promote G12 -dependent Rho activation lead to cofilin phosphorylation involved in neurite extension. Cofilin phosphorylation induced by UTP in IL-1ß-treated mPCNs is also decreased by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), suggesting a role for P2Y2R-mediated and Gq-dependent calcium mobilization in neurite outgrowth. Taken together, these studies indicate that up-regulation of P2Y2Rs in mPCNs under pro-inflammatory conditions can promote cofilin-dependent neurite outgrowth, a neuroprotective response that may be a novel pharmacological target in the treatment of neurodegenerative diseases.


Assuntos
Córtex Cerebral/citologia , Interleucina-1beta/farmacologia , Neurônios/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Integrina alfaVbeta3/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neuritos/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Fosforilação , Cultura Primária de Células , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y2/genética , Receptores de Vitronectina/metabolismo , Regulação para Cima , Uridina Trifosfato/farmacologia
14.
CNS Neurol Disord Drug Targets ; 11(6): 722-38, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22963441

RESUMO

P2Y receptors for extracellular nucleotides are coupled to activation of a variety of G proteins and stimulate diverse intracellular signaling pathways that regulate functions of cell types that comprise the central nervous system (CNS). There are 8 different subtypes of P2Y receptor expressed in cells of the CNS that are activated by a select group of nucleotide agonists. Here, the agonist selectivity of these 8 P2Y receptor subtypes is reviewed with an emphasis on synthetic agonists with high potency and resistance to degradation by extracellular nucleotidases that have potential applications as therapeutic agents. In addition, the recent identification of a wide variety of subtype-selective antagonists is discussed, since these compounds are critical for discerning cellular responses mediated by activation of individual P2Y receptor subtypes. The functional expression of P2Y receptor subtypes in cells that comprise the CNS is also reviewed and the role of each subtype in the regulation of physiological and pathophysiological responses is considered. Other topics include the role of P2Y receptors in the regulation of blood-brain barrier integrity and potential interactions between different P2Y receptor subtypes that likely impact tissue responses to extracellular nucleotides in the CNS. Overall, current research suggests that P2Y receptors in the CNS regulate repair mechanisms that are triggered by tissue damage, inflammation and disease and thus P2Y receptors represent promising targets for the treatment of neurodegenerative diseases.


Assuntos
Barreira Hematoencefálica/metabolismo , Sistema Nervoso Central/metabolismo , Inflamação/metabolismo , Doenças Neurodegenerativas/metabolismo , Agonistas do Receptor Purinérgico P2Y/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y/fisiologia , Animais , Sistema Nervoso Central/efeitos dos fármacos , Humanos , Receptores Purinérgicos P2Y/efeitos dos fármacos , Receptores Purinérgicos P2Y/metabolismo
15.
Am J Physiol Cell Physiol ; 303(7): C790-801, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22875784

RESUMO

Inflammation of the salivary gland is a well-documented aspect of salivary gland dysfunction that occurs in Sjogren's syndrome (SS), an autoimmune disease, and in γ-radiation-induced injury during treatment of head and neck cancers. Extracellular nucleotides have gained recognition as key modulators of inflammation through activation of cell surface ionotropic and metabotropic receptors, although the contribution of extracellular nucleotides to salivary gland inflammation is not well understood. In vitro studies using submandibular gland (SMG) cell aggregates isolated from wild-type C57BL/6 mice indicate that treatment with ATP or the high affinity P2X7R agonist 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) induces membrane blebbing and enhances caspase activity, responses that were absent in SMG cell aggregates isolated from mice lacking the P2X7R (P2X7R(-/-)). Additional studies with SMG cell aggregates indicate that activation of the P2X7R with ATP or BzATP stimulates the cleavage and release of α-fodrin, a cytoskeletal protein thought to act as an autoantigen in the development of SS. In vivo administration of BzATP to ligated SMG excretory ducts enhances immune cell infiltration into the gland and initiates apoptosis of salivary epithelial cells in wild-type, but not P2X7R(-/-), mice. These findings indicate that activation of the P2X7R contributes to salivary gland inflammation in vivo, suggesting that the P2X7R may represent a novel target for the treatment of salivary gland dysfunction.


Assuntos
Mediadores da Inflamação/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Glândula Submandibular/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/fisiologia , Animais , Epitélio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glândulas Salivares/metabolismo
16.
Mol Neurobiol ; 46(1): 96-113, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22467178

RESUMO

Extracellular nucleotides induce cellular responses in the central nervous system (CNS) through the activation of ionotropic P2X and metabotropic P2Y nucleotide receptors. Activation of these receptors regulates a wide range of physiological and pathological processes. In this review, we present an overview of the current literature regarding P2X and P2Y receptors in the CNS with a focus on the contribution of P2X7 and P2Y(2) receptor-mediated responses to neuroinflammatory and neuroprotective mechanisms.


Assuntos
Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Inflamação/patologia , Nucleotídeos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Animais , Humanos , Fármacos Neuroprotetores/metabolismo
17.
Purinergic Signal ; 8(3): 559-78, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22528682

RESUMO

Purinergic signaling plays a unique role in the brain by integrating neuronal and glial cellular circuits. The metabotropic P1 adenosine receptors and P2Y nucleotide receptors and ionotropic P2X receptors control numerous physiological functions of neuronal and glial cells and have been implicated in a wide variety of neuropathologies. Emerging research suggests that purinergic receptor interactions between cells of the central nervous system (CNS) have relevance in the prevention and attenuation of neurodegenerative diseases resulting from chronic inflammation. CNS responses to chronic inflammation are largely dependent on interactions between different cell types (i.e., neurons and glia) and activation of signaling molecules including P2X and P2Y receptors. Whereas numerous P2 receptors contribute to functions of the CNS, the P2Y(2) receptor is believed to play an important role in neuroprotection under inflammatory conditions. While acute inflammation is necessary for tissue repair due to injury, chronic inflammation contributes to neurodegeneration in Alzheimer's disease and occurs when glial cells undergo prolonged activation resulting in extended release of proinflammatory cytokines and nucleotides. This review describes cell-specific and tissue-integrated functions of P2 receptors in the CNS with an emphasis on P2Y(2) receptor signaling pathways in neurons, glia, and endothelium and their role in neuroprotection.


Assuntos
Fármacos Neuroprotetores , Receptores Purinérgicos P2Y2/fisiologia , Animais , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiologia , Endotélio/fisiologia , Humanos , Inflamação/patologia , Neuroglia/fisiologia , Neurônios/fisiologia , Receptores Purinérgicos P2X/fisiologia , Transdução de Sinais/fisiologia
18.
J Neurochem ; 121(2): 228-38, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22353164

RESUMO

Amyloid ß-protein (Aß) deposits in brains of Alzheimer's disease patients generate proinflammatory cytokines and chemokines that recruit microglial cells to phagocytose Aß. Nucleotides released from apoptotic cells activate P2Y(2) receptors (P2Y(2) Rs) in macrophages to promote clearance of dead cells. In this study, we investigated the role of P2Y(2) Rs in the phagocytosis and clearance of Aß. Treatment of mouse primary microglial cells with fibrillar (fAß(1-42) ) and oligomeric (oAß(1-42) ) Aß(1-42) aggregation solutions caused a rapid release of ATP (maximum after 10 min). Furthermore, fAß(1-42) and oAß(1-42) treatment for 24 h caused an increase in P2Y(2) R gene expression. Treatment with fAß(1-42) and oAß(1-42) aggregation solutions increased the motility of neighboring microglial cells, a response inhibited by pre-treatment with apyrase, an enzyme that hydrolyzes nucleotides. The P2Y(2) R agonists ATP and UTP caused significant uptake of Aß(1-42) by microglial cells within 30 min, which reached a maximum within 1 h, but did not increase Aß(1-42) uptake by primary microglial cells isolated from P2Y(2) R(-/-) mice. Inhibitors of α(v) integrins, Src and Rac decreased UTP-induced Aß(1-42) uptake, suggesting that these previously identified components of the P2Y(2) R signaling pathway play a role in Aß phagocytosis by microglial cells. Finally, we found that UTP treatment enhances Aß(1-42) degradation by microglial cells, but not in cells isolated from P2Y(2) R(-/-) mice. Taken together, our findings suggest that P2Y(2) Rs can activate microglial cells to enhance Aß clearance and highlight the P2Y(2) R as a therapeutic target in Alzheimer's disease.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Movimento Celular/efeitos dos fármacos , Microglia/metabolismo , Nucleotídeos/metabolismo , Nucleotídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Agonistas do Receptor Purinérgico P2Y , Receptores Purinérgicos P2Y2/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Separação Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Integrina alfa5/farmacologia , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Neurofibrilas/metabolismo , Fagocitose/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Uridina Trifosfato/farmacologia , Proteínas rac de Ligação ao GTP/fisiologia , Quinases da Família src/fisiologia
19.
Artigo em Inglês | MEDLINE | ID: mdl-25774333

RESUMO

P2Y receptors are G protein-coupled receptors (GPCRs) that are activated by adenine and uridine nucleotides and nucleotide sugars. There are eight subtypes of P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14), which activate intracellular signaling cascades to regulate a variety of cellular processes, including proliferation, differentiation, phagocytosis, secretion, nociception, cell adhesion, and cell migration. These signaling cascades operate mainly by the sequential activation or deactivation of heterotrimeric and monomeric G proteins, phospholipases, adenylyl and guanylyl cyclases, protein kinases, and phosphodiesterases. In addition, there are numerous ion channels, cell adhesion molecules, and receptor tyrosine kinases that are modulated by P2Y receptors and operate to transmit an extracellular signal to an intracellular response.

20.
Mol Neurobiol ; 41(2-3): 356-66, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20387013

RESUMO

Acute inflammation is important for tissue repair; however, chronic inflammation contributes to neurodegeneration in Alzheimer's disease (AD) and occurs when glial cells undergo prolonged activation. In the brain, stress or damage causes the release of nucleotides and activation of the G(q) protein-coupled P2Y(2) nucleotide receptor subtype (P2Y(2)R) leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y(2)R activation induces the phosphorylation of the epidermal growth factor receptor (EGFR), a response dependent upon the presence of a SH3 binding domain in the intracellular C terminus of the P2Y(2)R that promotes Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes. Other studies indicate that P2Y(2)R activation increases astrocyte migration. P2Y(2)R activation by UTP increases the expression in astrocytes of alpha(V)beta(3/5) integrins that bind directly to the P2Y(2)R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y(2)R, an interaction required for G(o) and G(12) protein-dependent astrocyte migration. In rat primary cortical neurons (rPCNs) P2Y(2)R expression is increased by stimulation with interleukin-1beta (IL-1beta), a pro-inflammatory cytokine whose levels are elevated in AD, in part due to nucleotide-stimulated release from glial cells. Other results indicate that oligomeric beta-amyloid peptide (Abeta(1-42)), a contributor to AD, increases nucleotide release from astrocytes, which would serve to activate upregulated P2Y(2)Rs in neurons. Data with rPCNs suggest that P2Y(2)R upregulation by IL-1beta and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1beta-upregulated P2Y(2)Rs in rPCNs increases the phosphorylation of cofilin, a cytoskeletal protein that stabilizes neurite outgrowths. Thus, activation of pro-inflammatory P2Y(2)Rs in glial cells can promote neuroprotective responses, suggesting that P2Y(2)Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases.


Assuntos
Encéfalo/citologia , Neuroglia/metabolismo , Neurônios/metabolismo , Receptores Purinérgicos P2/metabolismo , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Citoesqueleto/metabolismo , Inflamação/metabolismo , Microvasos/metabolismo , Dados de Sequência Molecular , Neuroglia/citologia , Neurônios/citologia , Estrutura Secundária de Proteína , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y2 , Transdução de Sinais/fisiologia
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