. We now show that Cer-1-P prevents cell death in bone-marrow-derived macrophages (BMDMs) after withdrawal of macrophage colony-stimulating factor (M-CSF). Removal of M-CSF is known to induce apoptosis in these cells. Cer-1-P blocked activation of the caspase-9/caspase-3 pathway and prevented DNA fragmentation, indicating that the enhancement of cell survival was due to inhibition of apoptosis. M-CSF deprivation resulted in activation of acid sphingomyelinase (A-SMase), increased ceramide levels, and a decrease in intracellular Cer-1-P. Exogenously added Cer-1-P inhibited A-SMase in intact BMDMs at concentrations that also prevented apoptosis. Cer-1-P also inhibited A-SMase in cell homogenates, suggesting a possible direct physical interaction of Cer-1-P with the enzyme. In conclusion, these data demonstrate that Cer-1-P blocks apoptosis in BMDMs through inhibition of A-SMase, thereby reducing ceramide generation. This adds a new dimension to the understanding of the metabolic interrelationship of ceramides and Cer-1-P, and shows how altering the balance of intracellular levels of these mediators can affect cell survival. The breakdown of sphingomyelin (SM) produces bioactive sphingolipid metabolites, some of which are believed to act as second messengers that control critical cellular functions. For example, N -deacylation of SM generates sphingosine phosphocholine, which is mitogenic for fibroblasts (1). Stimulation of SMase activity produces ceramides, which can inhibit cell proliferation and are potent inducers of apoptosis (2-4). Ceramides have been shown to regulate several protein kinases, including ceramide-activated protein kinase (5, 6) and protein kinase C (7), or protein phosphatases of the 2 A family (8). In addition, ceramides are potent inhibitors of phospholipase D, both in cultured cells (9, 10) and in cell-free systems (11). Ceramides can be degraded by ceramidases to sphingosine, and this, in turn, can be phosphorylated by sphingosine kinase to produce sphingosine-1-phosphate (Sph-1-P). Both sphingosine and Sph-1-P have been implicated in the regulation of cell proliferation and death (12-15).Another important ceramide metabolite that can be generated through the action of ceramide kinase is ceramide-1-phosphate (Cer-1-P) (16, 17). Boudker and Futerman (18) characterized a phosphatase that specifically hydrolyzes Cer-1-P in plasma membranes, suggesting that ceramide and Cer-1-P can be interconverted in cells. More recently, Riboni et al. (19) observed that Cer-1-P can also be produced from the recycling of sphingosine produced from ganglioside catabolism, and Rile et al. (20) reported that Cer-1-P can be formed intracellularly in neutrophils. Critical biological functions have been attributed to Cer-1-P. We first found that short-chain acetyl (C 2 )-and octanoyl (C 8 )-Cer-1-P, as well as natural long-chain Cer-1-P, stimulated the incorporation of [ 3 H]thymidine into DNA in fibroblasts, and that this action did not involve conversion of Cer-1-P to Sph-1-P (21, 22). More recently, it was fo...

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C 2 -and C 6 -ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) abolished the stimulation of DNA synthesis by sphingosine 1-phosphate in rat fibroblasts. This inhibition by ceramide was partially prevented by insulin. C 2 -ceramide did not alter the stimulation of DNA synthesis by insulin and decreased the sphingosine-induced stimulation by only 16%. The ceramides did not significantly modify the actions of sphingosine or sphingosine 1-phosphate in decreasing cAMP concentrations. C 2 -and C 6 -ceramides blocked the activation of phospholipase D by sphingosine 1-phosphate, and this inhibition was not affected by insulin. Okadaic acid decreased the activation of phospholipase D by sphingosine 1-phosphate and did not reverse the inhibitory effect of C 2 -ceramide on this activation. Therefore, this effect of C 2 -ceramide is unlikely to involve the stimulation of phosphoprotein phosphatase activity. Sphingosine did not activate phospholipase D activity significantly after 10 min. C 2 -ceramide stimulated the conversion of exogenous [ 3 H]sphingosine 1-phosphate to sphingosine and ceramide in fibroblasts. Ceramides can inhibit some effects of sphingosine 1-phosphate by stimulating its degradation via a phosphohydrolase that also hydrolyzes phosphatidate. Furthermore, C 2 -and C 6 -ceramides stimulated ceramide production from endogenous lipids, and this could propagate the intracellular signal. This work demonstrates that controlling the production of ceramide versus sphingosine and sphingosine 1-phosphate after sphingomyelinase activation could have profound effects on signal transduction.

Sphingolipids are essential components of cell membranes, and many of them regulate vital cell functions. In particular, ceramide plays crucial roles in cell signaling processes. Two major actions of ceramides are the promotion of cell cycle arrest and the induction of apoptosis. Phosphorylation of ceramide produces ceramide 1-phosphate (C1P), which has opposite effects to ceramide. C1P is mitogenic and has prosurvival properties. In addition, C1P is an important mediator of inflammatory responses, an action that takes place through stimulation of cytosolic phospholipase A2, and the subsequent release of arachidonic acid and prostaglandin formation. All of the former actions are thought to be mediated by intracellularly generated C1P. However, the recent observation that C1P stimulates macrophage chemotaxis implicates specific plasma membrane receptors that are coupled to Gi proteins. Hence, it can be concluded that C1P has dual actions in cells, as it can act as an intracellular second messenger to promote cell survival, or as an extracellular receptor agonist to stimulate cell migration.

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We found that natural (long-chain) ceramide 1-phosphate can be dispersed into aqueous solution when dissolved in an appropriate mixture of methanol/dodecane (49:1, v/v). This solvent mixture facilitates the interaction of this phosphosphingolipid with cells. Under these conditions, incubation of EGFR T17 fibroblasts with natural ceramide 1-phosphate caused a potent stimulation of DNA synthesis. This effect was accompanied by an increase in the levels of proliferating-cell nuclear antigen. Concentrations of natural ceramide 1-phosphate that stimulated the synthesis of DNA did not inhibit adenylate cyclase activity, nor did they stimulate phospholipase D. Natural ceramide 1-phosphate did not alter the cellular phosphorylation state of tyrosine residues or of mitogen-activated protein kinase. Furthermore, natural ceramide 1-phosphate failed to induce the expression of the proto-oncogenes c-myc and c-fos. Both the stimulation of DNA synthesis and the induction of proliferating-cell nuclear antigen by natural ceramide 1-phosphate were inhibited by natural ceramides. This work suggests that the use of methanol and dodecane to deliver natural ceramide 1-phosphate to cells may be useful for elucidation of the biological function(s) and mechanism(s) of action of ceramide 1-phosphate.

In this report, we show for the first time that ceramide-1-phosphate (C1P) stimulates the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB) pathway, which is a major mechanism whereby growth factors promote cell survival. Also, C1P induced IjB phosphorylation, and enhanced the DNA binding activity of the transcription factor NF-jB. Apoptotic macrophages showed a marked reduction of Bcl-X L levels, and this was prevented by C1P. These findings suggest that C1P blocks apoptosis, at least in part, by stimulating the PI3-K/ PKB/ NF-jB pathway and maintaining the production of antiapoptotic Bcl-X L . Based on these and our previous observations, we propose a working model for C1P in which inhibition of acid sphingomyelinase and the subsequent decrease in ceramide levels would allow cell signaling through stimulation of the PI3-K/PKB pathway to promote cell survival.