No abstract

Recently, we demonstrated that ceramide kinase, and its product, ceramide 1-phosphate (Cer-1-P), were mediators of arachidonic acid released in cells in response to interleukin-1␤ and calcium ionophore (Pettus, B. J., Bielawska, A., Spiegel, S., Roddy, P., Hannun, Y. A., and Chalfant, C. E. (2003) J. Biol. Chem. 278, 38206 -38213). In this study, we demonstrate that down-regulation of cytosolic phospholipase A 2 (cPLA 2 ) using RNA interference technology abolished the ability of Cer-1-P to induce arachidonic acid release in A549 cells, demonstrating that cPLA 2 is the key phospholipase A 2 downstream of Cer-1-P. Treatment of A549 cells with Cer-1-P (2.5 M) induced the translocation of full-length cPLA 2 from the cytosol to the Golgi apparatus/perinuclear regions, which are known sites of translocation in response to agonists. Cer-1-P also induced the translocation of the CaLB/C2 domain of cPLA 2 in the same manner, suggesting that this domain is responsive to Cer-1-P either directly or indirectly. In vitro studies were then conducted to distinguish these two possibilities. In vitro binding studies disclosed that Cer-1-P interacts directly with full-length cPLA 2 and with the CaLB domain in a calcium-and lipid-specific manner with a K Ca of 1.54 M. Furthermore, Cer-1-P induced a calcium-dependent increase in cPLA 2 enzymatic activity as well as lowering the EC 50 of calcium for the enzyme from 191 to 31 nM. This study identifies Cer-1-P as an anionic lipid that translocates and directly activates cPLA 2 , demonstrating a role for this bioactive lipid in the mediation of inflammatory responses.

In this study we addressed the role of sphingolipid metabolism in the inflammatory response. In a L929 fibroblast model, tumor necrosis factor-alpha (TNF) induced prostaglandin E2 (PGE2) production by 4 h and cyclooxygenase-2 (COX-2) induction as early as 2 h. This TNF-induced PGE2 production was inhibited by NS398, a COX-2 selective inhibitor. GC-MS analysis revealed that only COX-2-generated prostanoids were produced in response to TNF, thus providing further evidence of COX-2 selectivity. As sphingolipids have been implicated in mediating several actions of TNF, their role in COX-2 induction and PGE2 production was evaluated. Sphingosine-1-phosphate (S1P) induced both COX-2 and PGE2 in a dose-responsive manner with an apparent ED50 of 100-300 nM. The related sphingolipid sphingosine also induced PGE2, though with much less efficacy. TNF induced a 3.5-fold increase in sphingosine-1-phosphate levels at 10 min that rapidly returned to baseline by 40 min. Small interfering RNAs (siRNAs) directed against mouse SK1 decreased (typically by 80%) SK1 protein and inhibited TNF-induced SK activity. Treatment of cells with RNAi to SK1 but not SK2 almost completely abolished the ability of TNF to induce COX-2 or generate PGE2. By contrast, cells treated with RNAi to S1P lyase or S1P phosphatase enhanced COX-2 induction leading to enhanced generation of PGE2. Treatment with SK1 RNAi also abolished the effects of exogenous sphingosine and ceramide on PGE2, revealing that the action of sphingosine and ceramide are due to intracellular metabolism into S1P. Collectively, these results provide novel evidence that SK1 and S1P are necessary for TNF to induce COX-2 and PGE2 production. Based on these findings, this study indicates that SK1 and S1P could be implicated in pathological inflammatory disorders and cancer.

Despite the importance of prostaglandins, little is known about the regulation of prostanoid synthesis proximal to the activation of cytosolic phospholipase A 2 , the initial rate-limiting step. In this study, ceramide-1-phosphate (C-1-P) was shown to be a specific and potent inducer of arachidonic acid (AA) and prostanoid synthesis in cells. This study also demonstrates that two well established activators of AA release and prostanoid synthesis, the cytokine, interleukin-1␤ (IL-1␤), and the calcium ionophore, A23187, induce an increase in C-1-P levels within the relevant time-frame of AA release. Furthermore, the enzyme responsible for the production of C-1-P in mammalian cells, ceramide kinase, was activated in response to IL-1␤ and A23187. RNA interference targeted to ceramide kinase specifically down-regulated ceramide kinase mRNA and activity with a concomitant decrease of AA release in response to IL-1␤ and A23187. Down-regulation of ceramide kinase had no effect on AA release induced by exogenous C-1-P. Collectively, these results indicate that ceramide kinase, via the formation of C-1-P, is an upstream modulator of phospholipase A 2 activation. This study identifies previously unknown roles for ceramide kinase and its product, C-1-P, in AA release and production of eicosanoids and provides clues for potential new targets to block inflammatory responses.

Treatment of A549 cells with C 6 -ceramide resulted in a significant increase in the endogenous long chain ceramide levels, which was inhibited by fumonisin B1 (FB1), and not by myriocin (MYR). The biochemical mechanisms of generation of endogenous ceramide were investigated using A549 cells treated with selectively labeled C 6 -ceramides, [sphingosine-3- The results demonstrated that 3 H label was incorporated into newly synthesized long chain ceramides, which was inhibited by FB1 and not by MYR. Interestingly, the 14 C label was not incorporated into long chain ceramides. Taken together, these results show that generation of endogenous ceramide in response to C 6 -ceramide is due to recycling of the sphingosine backbone of C 6 -ceramide via deacylation/ reacylation and not due to the elongation of its fatty acid moiety. Moreover, the generation of endogenous long chain ceramide in response to C 6 -ceramide was completely blocked by brefeldin A, which causes Golgi disassembly, suggesting a role for the Golgi in the metabolism of ceramide. In addition, the generation of endogenous ceramide in response to short chain exogenous ceramide was induced by D-erythro-but not L-erythro-C 6 -ceramide, demonstrating the stereospecificity of this process. Interestingly, several key downstream biological activities of ceramide, such as growth inhibition, cell cycle arrest, and modulation of telomerase activity were induced by D-erythro-C 6 -ceramide, and not L-erythro-C 6 -ceramide (and inhibited by FB1) in A549 cells, suggesting a role for endogenous long chain ceramide in the regulation of these responses.