Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2 |
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Authors: | Guadalupe Martel-Gallegos,Griselda Casas-Pruneda,Filiberta Ortega-Ortega,Sergio Sá nchez-Armass,Jesú s Alberto Olivares-Reyes,Becky Diebold,Patricia Pé rez-Cornejo,Jorge Arreola |
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Affiliation: | 1. Department of Physiology, School of Medicine, Universidad Autónoma de San Luis Potosí, San Luis Potosí, SLP 78210, Mexico;2. Faculty of Chemical Sciences, Universidad Autónoma de San Luis Potosí, San Luis Potosí, SLP 78290, Mexico;3. Department of Biochemistry, CINVESTAV-IPN, México, DF 07360, Mexico;4. Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA;5. Physics Institute, Universidad Autónoma de San Luis Potosí, San Luis Potosí, SLP 78290, Mexico |
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Abstract: | BackgroundActivation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.MethodsJ774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.ResultsROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.ConclusionsPurinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.General significanceROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections. |
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Keywords: | P2X7R, P2X7 receptor channels LPS, lipopolysaccharide IL-1β, interleukin-1β TNFα, tumor necrosis factor α ROS, reactive oxygen species JNK, c-Jun N-terminal kinase ERK1/2, extracellular regulated kinases 1 and 2 MAPKs, mitogen-activated protein kinases PI3K, phosphatidylinositol 3 kinase Bz-ATP, 2&prime ,3&prime -O-(4-benzoyl-benzoyl) ATP PMA, phorbol myristate acetate DPI, diphenyleneiodonium SB203580, 4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine SB239063, trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol Gö 6983, 2-(1-(3 dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1h-indol-3-yl)maleimide PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one PP2, 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl) pyrazolo[3,4-d]pyrimidine PF431396, N-Methyl-N-[2-[[[2-[(2,3-dihydro-2- oxo-1H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]methanesulfonamide DCFH2DA, 2&prime ,7&prime -dichlorodihydrofluorescein diacetate DCF, 2&prime ,7&prime -dichlorofluorescein Fura-2 AM, Fura-2-acetoxymethylester L-012, 8-amino-5-chloro-7-phenylpyrido [3,4-d]pyridazine-1,4(2H,3H) dione DMSO, dimethlysulfoxide HBSS, Hanks's balanced salt solution SS, saline solution WB, Western blot EGFR, Epidermal Growth Factor Receptor PLD, phospholipase D |
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