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 muM, C-2-ceramide inhibited LPS-elicited NO synthase and cyclooxygenase-2 induction accompanied by a reduction in NO and PGE, 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-I activation. The immunocomplex kinase assay indicated that I kappaB kinase activity stimulated by LPS was inhibited by ceramide, which concomitantly reduced the I kappaB alpha 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 I kappaB kinase, p38 mitogen-activated protein kinase, and protein kinase C.