P2X(7)/P2Z purinoreceptor-mediated activation of transcription factor NFAT in microglial cells
ABSTRACT ATP is released from neurons and other cell types during several physiological and stress conditions under which it exerts various biological effects upon binding to purinoreceptors. A rather peculiar purinoreceptor called P2X7/P2Z is expressed on microglial and other myeloic cells. Although increasing evidence implicates an important role for P2Z in inflammatory processes, little information exists about underlying signaling pathways. Here, we report that in N9 microglial cells, extracellular ATP potently activates nuclear factor of activated T cells (NFAT), a central transcription factor involved in cytokine gene expression. ATP activated NFAT rapidly (within 1 min), whereas activation of nuclear factor kappaB was much delayed, with strikingly distinct kinetics. During ATP stimulation, both NFAT-1 and NFAT-2 were activated by a calcineurin-dependent pathway that required the influx of extracellular calcium ions. Based on the pharmacological profile, NFAT activation was specifically mediated by P2Z and not by other purinoreceptors. N9 cells that lacked P2Z but still expressed P2Y purinoreceptors failed to respond to NFAT activation. We conclude that P2Z-mediated NFAT activation may represent a novel mechanism by which extracellular ATP can modulate early inflammatory gene expression within the nervous and immune system.
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- "In addition, sustained activation of P2X 7 Rs may generate non-selective pores that are permeable to small molecules up to 900 Da in size (Virginio et al., 1999; Di Virgilio et al., 2001). Although expression of the P2X 7 R is primarily associated with immune and hematopoietic cells (Surprenant et al., 1996; Di Virgilio et al., 2001), its mRNA or protein has been identified in all brain cell types in the CNS (Ferrari et al., 1999; Choi et al., 2007; Yu et al., 2008). Importantly, the P2X 7 R is highly expressed on microglia and activation of these receptors is correlated with release of the proinflammatory cytokines IL-1β (Ferrari et al., 1997; Lister et al., 2007) and TNFα (Hide et al., 2000; Lister et al., 2007). "
ABSTRACT: Activation and proliferation of glial cells and their progenitors is a key process of neuroinflammation associated with many neurodegenerative disorders. Under neuropathological conditions where glial cell activation and proliferation is evident, controlling the population of glia might be of therapeutic importance. The proliferative action of the cytokine tumor necrosis factor alpha (TNFα) on microglia has been reported, but the molecular mechanism of TNFα regulation of glial cell proliferation is largely unknown. Using a model of organotypic hippocampal-entorhinal cortex (HEC) slice culture, we investigated the role of ATP-P2X(7) receptor signaling in glial proliferation by TNFα. Populations of proliferating cells in HEC culture were labeled with 5-bromo-2'-deoxyuridine (BrdU). Treatment with TNFα induced strong expression of P2X(7) receptor mRNA and immunoreactivity in BrdU+ cells while markedly increasing proliferation of BrdU+ cells. In addition, TNFα increased aquaporin 4 (AQP4) expression, an ion channel involved in glial proliferation. The proliferative action of TNFα was attenuated by blocking the P2X(7) receptors with the specific antagonists oxATP, BBG, and KN62, or by lowering extracellular ATP with ATP hydrolysis apyrase. Basal proliferation of BrdU+ cells was also sensitive to blockade of ATP-P2X(7) signaling. Furthermore, TNFα activation of P2X(7) receptors appear to regulate AQP4 expression through protein kinase C cascade and down regulation of AQP4 expression can reduce TNFα-stimulated BrdU+ cell proliferation. Taken together, these novel findings demonstrate the importance of ATP-P2X(7) signaling in controlling proliferation of glial progenitors under the pathological conditions associated with increased TNFα.Glia 04/2012; 60(4):661-73. DOI:10.1002/glia.22302 · 6.03 Impact Factor
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- "Astrocytic NFAT is involved in initiation and maintenance of injury, disease, or aging-mediated neuroinflammatory processes (Pérez-Ortiz et al., 2008; Sama et al., 2008; Abdul et al., 2009). However, relatively few studies have documented expression and function of NFAT in microglia, the resident immune cell of the brain (Ferrari et al., 1999; Kataoka et al., 2009). This study has identified NFAT isoform expression in primary murine microglia cultures and verified that it is involved in regulating proinflammatory gene expression similar to its role in other immune cells. "
ABSTRACT: The transcription factor family, nuclear factor of activated T cells (NFAT), regulates immune cell phenotype. Four different calcium/calmodulin-regulated isoforms have been identified in the periphery, but isoform expression in microglia, the resident immune cells of the CNS, has not been fully defined. In this study microglial NFAT isoform expression and involvement in regulating inflammatory responses in murine primary microglia culture was examined. Western blot analysis demonstrated robust detection of NFATc1 and c2 isoforms in microglia. Electrophoretic mobility shift assays demonstrated increased NFAT-DNA binding from nuclear extracts of lipopolysaccharide (LPS) stimulated microglia. Moreover, LPS-stimulated microglia behaved similarly to T cell receptor agonist antibody-stimulated Jurkat cells demonstrating a transient increase in NFAT-driven luciferase reporter gene expression. LPS-induced NFAT-luciferase activity in microglia was attenuated by pretreatment with tat-VIVIT, a cell-permeable NFAT inhibitory peptide. Furthermore, LPS-mediated secretion of microglial cytokines, TNF-alpha and MCP-1, was decreased by treatment with tat-VIVIT but not with tat-VEET, a negative control peptide. These results demonstrate that NFAT plays a role in regulating proinflammatory responses in cultured murine microglia.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 07/2010; 30(28):9641-6. DOI:10.1523/JNEUROSCI.0828-10.2010 · 6.75 Impact Factor
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- "Stimulation of P2X 7 receptors increases protein tyrosine phosphorylation   ultimately leading to MAPK pathway activation. Many events downstream of P2X 7 receptor activation are dependent upon extracellular calcium influx  , and activation of MAPK pathways by P2X 7 receptors may involve calcium signaling. In RAW 264.7 macrophages, the calcium-dependent kinase Pyk2, which facilitates Ras activation, is tyrosine phosphorylated in response to treatment with BzATP  , potentially linking calcium fluxes, Ras activation, and MAPK pathways with P2X 7 receptors. "
ABSTRACT: P2X receptors are ATP-gated cation channels that mediate fast excitatory transmission in diverse regions of the brain and spinal cord. Several P2X receptor subtypes, including P2X(7), have the unusual property of changing their ion selectivity during prolonged exposure to ATP, which results in a channel pore permeable to molecules as large as 900 daltons. The P2X(7) receptor was originally described in cells of hematopoietic origin, and mediates the influx of Ca(2+) and Na(+) and Ca(2+) and Na(+) ions as well as the release of proinflammatory cytokines. P2X(7) receptors may affect neuronal cell death through their ability to regulate the processing and release of interleukin-1beta, a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X(7), a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X(7) receptors provides an inflammatory stimulus, and P2X(7) receptor-deficient mice have substantially attenuated inflammatory responses, including models of neuropathic and chronic inflammatory pain. Moreover, P2X(7) receptor activity, by regulating the release of proinflammatory cytokines, may be involved in the pathophysiology of depression. Apoptotic cell death occurs in a number of vascular diseases, including atherosclerosis, restenosis, and hypertension, and may be linked to the release of ATP from endothelial cells, P2X(7) receptor activation, proinflammatory cytokine production, and endothelial cell apoptosis. In this context, the P2X(7) receptor may be viewed as a gateway of communication between the nervous, immune, and cardiovascular systems.Cardiovascular Psychiatry and Neurology 06/2009; 2009:861324. DOI:10.1155/2009/861324