Ceramide inhibits lipopolysaccharide-mediated nitric oxide synthase and cyclooxygenase-2 induction in macrophages: effects on protein kinases and transcription factors.
The goal of this study was to elucidate whether triggering the sphingomyelin pathway modulates LPS-initiated responses. For this purpose we investigated the effects of N-acetylsphingosine (C(2)-ceramide) on LPS-induced production of NO and PGE(2) in murine RAW 264.7 macrophages and explored the signaling pathways involved. We found that within a range of 10-50 microM, C(2)-ceramide inhibited LPS-elicited NO synthase and cyclooxygenase-2 induction accompanied by a reduction in NO and PGE(2) formation. By contrast, a structural analog of C(2)-ceramide that does not elicit functional activity, C(2)-dihydroceramide, did not affect the LPS response. The nuclear translocation and DNA binding study revealed that ceramide can inhibit LPS-induced NF-kappaB and AP-1 activation. The immunocomplex kinase assay indicated that IkappaB kinase activity stimulated by LPS was inhibited by ceramide, which concomitantly reduced the IkappaBalpha degradation caused by LPS within 1-6 h. In concert with the decreased cytosolic p65 protein level, LPS treatment resulted in rapid nuclear accumulation of NF-kappaB subunit p65 and its association with the cAMP-responsive element binding protein. Ceramide coaddition inhibited all the LPS responses. In addition, LPS-induced PKC and p38 mitogen-activated protein kinase activation were overcome by ceramide. In conclusion, we suggest that ceramide inhibition of LPS-mediated induction of inducible NO synthase and cyclooxygenase-2 is due to reduction of the activation of NF-kappaB and AP-1, which might result from ceramide's inhibition of LPS-stimulated IkappaB kinase, p38 mitogen-activated protein kinase, and protein kinase C.
Hsu
Y W
YW
Department of Pharmacology, National Taiwan University College of Medicine, Taipei, Taiwan.
Chi
K H
KH
Huang
W C
WC
Lin
W W
WW
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000276:immunology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000276:immunology; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D051546:Cyclooxygenase 2; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000276:immunology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D007166:Immunosuppressive Agents; D007527:Isoenzymes; D008070:Lipopolysaccharides; C064769:N-acetylsphingosine; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D018808:Transcription Factor AP-1; D051996:Transcription Factor RelA; D014157:Transcription Factors; D000072000:NF-KappaB Inhibitor alpha; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D017346:Protein Serine-Threonine Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D013110:Sphingosine