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Negative regulation by phosphatidylinositol 3-kinase of inducible nitric oxide synthase expression in macrophages
M J Díaz-Guerra1, A Castrillo, P Martín-Sanz
1Instituto de Bioquímica, Facultad de Farmacia, 28040 Madrid, Spain.
Abstract:
Triggering of the macrophage cell line RAW 264.7 with LPS promotes a transient activation of phosphatidylinositol 3-kinase (PI3-kinase). Incubation of activated macrophages with wortmannin and LY294002, two inhibitors of PI3-kinase, increased the amount of inducible nitric oxide synthase (iNOS) and the synthesis of nitric oxide. Treatment with wortmannin promoted a prolonged activation of NF-kappaB in LPS-treated cells as well as an increase in the promoter activity of the iNOS gene as deduced from transfection experiments using a 1.7-kb fragment of the 5' flanking region of the iNOS gene. Cotransfection of cells with a catalytically active p110 subunit of PI3-kinase impaired the responsiveness of the iNOS promoter to LPS stimulation, whereas transfection with a kinase-deficient mutant of p110 maintained the up-regulation in response to wortmannin. These results indicate that PI3-kinase plays a negative role in the process of macrophage activation and suggest that this enzyme might participate in the mechanism of action of antiinflammatory cytokines.
Insights
Phosphatidylinositol 3-kinase (PI3-kinase) negatively regulates macrophage activation. Inhibiting PI3-kinase increases nitric oxide production and enhances NF-kappaB activation, suggesting a role in anti-inflammatory cytokine action.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Lipopolysaccharide (LPS) transiently activates phosphatidylinositol 3-kinase (PI3-kinase) in RAW 264.7 macrophages.
- Macrophage activation involves complex signaling pathways influencing inflammatory responses.
Purpose of the Study:
- To investigate the role of PI3-kinase in modulating macrophage activation by LPS.
- To determine PI3-kinase's influence on inducible nitric oxide synthase (iNOS) and NF-kappaB signaling.
Main Methods:
- RAW 264.7 macrophage cell line stimulation with LPS.
- Inhibition of PI3-kinase using wortmannin and LY294002.
- Measurement of iNOS and nitric oxide synthesis.
- NF-kappaB activation assays and iNOS promoter activity analysis via transfection.
Main Results:
- PI3-kinase inhibition by wortmannin and LY294002 increased iNOS expression and nitric oxide production.
- Wortmannin treatment prolonged NF-kappaB activation and enhanced iNOS gene promoter activity.
- Modulating PI3-kinase catalytic activity affected iNOS promoter responsiveness to LPS and wortmannin.
Conclusions:
- PI3-kinase acts as a negative regulator in LPS-induced macrophage activation.
- These findings suggest PI3-kinase is involved in the mechanism of anti-inflammatory cytokines.