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Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2
Guadalupe Martel-Gallegos1, Griselda Casas-Pruneda, Filiberta Ortega-Ortega
1Department of Physiology, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico.
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.
Insights
Activation of P2X7 receptors (P2X7R) triggers calcium influx and a signaling cascade involving Pyk2 and ERK1/2 to produce reactive oxygen species (ROS) for pathogen defense.
Area of Science:
- Immunology
- Cell Signaling
- Biochemistry
Background:
- ATP-gated P2X7 receptors (P2X7R) on macrophages initiate reactive oxygen species (ROS) production through incompletely understood mechanisms.
- This study investigates the specific signaling pathway linking P2X7R stimulation to ROS generation in macrophages.
Purpose of the Study:
- To elucidate the signaling cascade responsible for ROS production following P2X7R activation.
- To identify key kinases and signaling molecules involved in this process.
Main Methods:
- J774 macrophage and HEK293 cells expressing P2X7R were stimulated with Bz-ATP.
- The role of extracellular calcium, various kinase inhibitors (MEK1/2, c-Src, PI3K, p38 MAPK, PKC), and specific signaling proteins (Pyk2, ERK1/2, calmodulin) were assessed.
- ROS production was measured, and kinase activation was confirmed using phospho-antibodies.
Main Results:
- ROS generation was strictly dependent on calcium influx through P2X7R.
- P2X7R stimulation activated Pyk2, but not calmodulin.
- Inhibition of MEK1/2 and c-Src abolished both ERK1/2 activation and ROS production.
- PI3K and p38 MAPK inhibitors had no significant effect on ROS generation.
- PKC inhibition reduced ERK1/2 activation but only slightly affected ROS production.
Conclusions:
- Purinergic stimulation via P2X7R leads to calcium entry and activates a PKC/c-Src/Pyk2/ERK1/2 pathway for ROS production.
- This pathway is independent of PI3K, p38 MAPK, and calmodulin.
- Understanding this ROS production mechanism is crucial for comprehending macrophage defense against microbial infections.
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