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 sphingomyeli n pathway modulates LPS-initiated responses. For this purpose we investigat ed the effects of N-acetylsphingosine (C-2-ceramide) on LPS-induced product ion of NO and PGE(2) in murine RAW 264.7 macrophages and explored the signa ling pathways involved. We found that within a range of 10-50 muM, C-2-cera mide inhibited LPS-elicited NO synthase and cyclooxygenase-2 induction acco mpanied 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-dihydr oceramide, did not affect the LPS response. The nuclear translocation and D NA binding study revealed that ceramide can inhibit LPS-induced NF-kappaB a nd AP-I activation. The immunocomplex kinase assay indicated that I kappaB kinase activity stimulated by LPS was inhibited by ceramide, which concomit antly reduced the I kappaB alpha degradation caused by LPS within 1-6 h. In concert with the decreased cytosolic p65 protein level, LPS treatment resu lted in rapid nuclear accumulation of NF-kappaB subunit p65 and its associa tion with the cAMP-responsive element binding protein. Ceramide coaddition inhibited all the LPS responses. In addition, LPS-induced PKC and p38 mitog en-activated protein kinase activation were overcome by ceramide. In conclu sion, we suggest that ceramide inhibition of LPS-mediated induction of indu cible NO synthase and cyclooxygenase-2 is due to reduction of the activatio n of NF-kappaB and AP-1, which might result from ceramide's inhibition of L PS-stimulated I kappaB kinase, p38 mitogen-activated protein kinase, and pr otein kinase C.