Related Experiment Videos
Signaling by eNOS through a superoxide-dependent p42/44 mitogen-activated protein kinase pathway
W Wang1, S Wang, E V Nishanian
1Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
American Journal of Physiology. Cell Physiology
|July 10, 2001
Summary
Endothelial nitric oxide synthase (eNOS) expression boosts tumor necrosis factor-alpha (TNF-alpha) via superoxide (O(2)(-)) and p42/44 mitogen-activated protein kinase (MAPK) signaling. This pathway is crucial for eNOS-mediated TNF-alpha production.
Area of Science:
- Cellular Biology
- Molecular Biology
- Immunology
Background:
- Endothelial nitric oxide synthase (eNOS) plays a role in cellular signaling.
- Mitogen-activated protein kinases (MAPK) are involved in reactive oxygen species (ROS) signaling and cytokine regulation.
- The precise mechanism linking eNOS-derived ROS to TNF-alpha production remains to be fully elucidated.
Purpose of the Study:
- To investigate the role of MAPK signaling in eNOS-mediated superoxide production and subsequent tumor necrosis factor-alpha (TNF-alpha) upregulation.
- To determine which specific MAPK pathways are activated by eNOS-derived superoxide.
- To elucidate the signaling cascade connecting eNOS activity to TNF-alpha expression.
Main Methods:
- Transfection of U-937 cells with eNOS and its mutants.
- Stimulation with phorbol 12-myristate 13-acetate (PMA) and redox-cycling agents.
- Measurement of TNF-alpha production.
- Assessment of MAPK phosphorylation (p42/44 MAPK and p38 MAPK) via Western blotting.
- Inhibition of MAPK pathways using PD-98059.
Main Results:
- eNOS expression upregulated PMA-induced TNF-alpha production via a superoxide-dependent mechanism.
- Phenazine methosulfate increased both TNF-alpha and p42/44 MAPK phosphorylation.
- eNOS-induced TNF-alpha upregulation correlated with p42/44 MAPK activation, not p38 MAPK.
- A catalytically inactive eNOS mutant failed to activate p42/44 MAPK.
- Inhibition of p42/44 MAPK blocked eNOS-mediated TNF-alpha upregulation.
Conclusions:
- Superoxide produced by eNOS activates the p42/44 MAPK pathway.
- This p42/44 MAPK activation is a critical step in the signaling cascade leading to increased TNF-alpha production.
- eNOS-derived superoxide signaling to TNF-alpha production is mediated through the p42/44 MAPK pathway.