Background: Activation 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.
Methods: J774 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.
Results: ROS 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.
Conclusions: Purinergic 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 significance: ROS 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.
Keywords: 2-(1-(3 dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1h-indol-3-yl)maleimide; 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; 2′,3′-O-(4-benzoyl-benzoyl) ATP; 2′,7′-dichlorodihydrofluorescein diacetate; 2′,7′-dichlorofluorescein; 4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine; 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl) pyrazolo[3,4-d]pyrimidine; 8-amino-5-chloro-7-phenylpyrido [3,4-d]pyridazine-1,4(2H,3H) dione; Bz-ATP; Calcium; DCF; DCFH(2)DA; DMSO; DPI; EGFR; ERK1/2; Epidermal Growth Factor Receptor; Fura-2 AM; Fura-2-acetoxymethylester; Gö6983; HBSS; Hanks's balanced salt solution; IL-1β; JNK; L-012; LPS; MAPKs; Macrophage; Mitogen-activated protein kinase; N-Methyl-N-[2-[[[2-[(2,3-dihydro-2- oxo-1H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]methanesulfonamide; Oxidative stress; P2X7 receptor channels; P2X7R; PD98059; PF431396; PI3K; PLD; PMA; PP2; Purinergic receptor; ROS; SB203580; SB239063; SS; TNFα; WB; Western blot; c-Jun N-terminal kinase; dimethlysulfoxide; diphenyleneiodonium; extracellular regulated kinases 1 and 2; interleukin-1β; lipopolysaccharide; mitogen-activated protein kinases; phorbol myristate acetate; phosphatidylinositol 3 kinase; phospholipase D; reactive oxygen species; saline solution; trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol; tumor necrosis factor α.
Copyright © 2013 Elsevier B.V. All rights reserved.