Degradation of IkappaBalpha in activated RAW264.7 cells is blocked by the phosphatidylinositol 3-kinase inhibitor LY294002.
The mechanism by which lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) induces production of proinflammatory cytokines in murine macrophages, and the role of phosphatidylinositol 3-kinase (PI3-kinase) have not been well investigated. Activation of nuclear factor kappaB (NF-kappaB) is initiated by the phosphorylation of the inhibitory subunit, IkappaB, which targets IkappaB for degradation and leads to the release of active NF-kappaB. In this study we demonstrate that 2-(4-morpholinyl)-8-phenylchromone (LY294002), which inhibits PI3-kinase, specifically inhibited degradation of IkappaBalpha in RAW264.7 cells stimulated with interferon-gamma (IFN-gamma) plus LPS or IFN-gamma plus PMA. To elucidate the importance of this activity in RAW264.7 cells, we examined tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 production in the activated cells. Pretreatment of the cells with LY294002 resulted in the inhibition of TNF-alpha and IL-6 production in RAW264.7 cells stimulated with IFN-gamma plus LPS or IFN-gamma plus PMA. Furthermore, LY294002 inhibited the production of nitric oxide (NO) in RAW264.7 cells stimulated with IFN-gamma plus LPS or IFN-gamma plus PMA. LY294002 also inhibited inducible NO synthase (iNOS) mRNA expression in the activated RAW264.7 cells. In conclusion, the present results suggest that PI3-kinase is involved in the signal transduction pathway responsible for LPS- or PMA-mediated TNF-alpha and IL-6 production, and that LY294002 inhibits NO generation through blocking the degradation of IkappaBalpha in activated RAW264.7 cells.
Park
S J
SJ
Department of Oriental Pharmacy, Korea Institute of Oriental Pharmacy, College of Pharmacy, Wonkwang University, Chonbuk.
Lee
S C
SC
Hong
S H
SH
Kim
H M
HM
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006868:Hydrolysis; D020672:I-kappa B Proteins; D007371:Interferon-gamma / Q000494:pharmacology; D015850:Interleukin-6 / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D009025:Morpholines / Q000494:pharmacology*; D000072000:NF-KappaB Inhibitor alpha; D009569:Nitric Oxide / Q000096:biosynthesis; D000081082:Phosphoinositide-3 Kinase Inhibitors; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D002867:Chromones; D017931:DNA Primers; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D015850:Interleukin-6; D008070:Lipopolysaccharides; D009025:Morpholines; C000606205:Nfkbia protein, mouse; D000081082:Phosphoinositide-3 Kinase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D007371:Interferon-gamma; D013755:Tetradecanoylphorbol Acetate