Supriya Jayadev

(1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor of ceramidase

In this study, we have examined the cellular and biochemical activities of the ceramide analog (1S,2R)-Derythro-2-(N-myristoylamino)-1-phenyl-1-propanol (Derythro-MAPP). Addition of 5 μMD-e-MAPP to HL-60 human promyelocytic leukemia cells resulted in a concentration- and time-dependent growth suppression accompanied by an arrest in the G/G phase of the cell cycle; thus mimicking the action of exogenous ceramides. Its enantiomer L-e-MAPP was without effect. Two lines of evidence suggested that D-e-MAPP may not function as a direct analog of ceramide. First, D-e-MAPP possesses a stereochemical configuration opposite to that of D-erythro-ceramide. Second, D-e-MAPP failed to activate ceramide-activated protein phosphatase in vitro. Therefore, we examined if D-e-MAPP functioned indirectly by modulating endogenous ceramide levels. The addition of D-e-MAPP to cells, but not L-e-MAPP, caused a time- and concentration-dependent elevation in endogenous ceramide levels reaching greater than 3-fold over baseline following 24 h of treatment. Both D-e-MAPP and L-e-MAPP underwent similar uptake by HL-60 cells. D-e-MAPP was poorly metabolized, and remained intact in cells, whereas L-e-MAPP underwent a time- and concentration-dependent metabolism; primarily through N-deacylation. In vitro, L-e-MAPP was metabolized by alkaline ceramidase to an extent similar to that seen with C-ceramide. D-e-MAPP was not metabolized. Instead, D-e-MAPP inhibited alkaline ceramidase activity in vitro with an IC of 1-5 μM. D-e-MAPP did not modulate the activity of other ceramide metabolizing enzymes in vitro or in cells, and it was a poor inhibitor of acid ceramidase (IC > 500 μM). Finally, D-e-MAPP inhibited the metabolism of L-e-MAPP in cells. These studies demonstrate that D-e-MAPP functions as an inhibitor of alkaline ceramidase in vitro and in cells resulting in elevation in endogenous levels of ceramide with the consequent biologic effects of growth suppression and cell cycle arrest. These studies point to an important role for ceramidases in the regulation of endogenous levels of ceramide.

Phospholipase A2 is necessary for tumor necrosis factor alpha-induced ceramide generation in L929 cells.

The role of cytosolic phospholipase A2 (cPLA2) in the regulation of ceramide formation was examined in a cell line (L929) responsive to the cytotoxic action of tumor necrosis factor α (TNFα). In L929 cells, the addition of TNFα resulted in the release of arachidonate, which was followed by a prolonged accumulation of ceramide occurring over 5–12 h and reaching 250% over base line. The formation of ceramide was accompanied by the hydrolysis of sphingomyelin and the activation of three distinct sphingomyelinases (neutral Mg2+-dependent, neutral Mg2+-independent, and acidic enzymes). The variant cell line C12, which lacks cPLA2, is resistant to the cytotoxic action of TNFα. TNFα was able to activate nuclear factor κB in both the wild-type L929 cells and the C12 cells. However, TNFα was unable to cause the release of arachidonate or the accumulation of ceramide in C12 cells. C6-ceramide overcame the resistance to TNFα and caused cell death in C12 cells to a level similar to that in L929 cells. The introduction of the cPLA2 gene into C12 cells resulted in partial restoration of TNFα-induced arachidonate release, ceramide accumulation, and cytotoxicity. This study suggests that cPLA2 is a necessary component in the pathways leading to ceramide accumulation and cell death.

The sphingomyelin cycle: The flip side of the lipid signaling paradigm

Publisher Summary Several investigations on the sphingomyelin (SM) cycle offer an initial and important insight into regulated metabolism of sphingolipids and on possible second messenger/cell regulatory roles for sphingolipid derived molecules as exemplified by ceramide. These investigations have two major implications. First, they illustrate the potential role of regulated sphingolipid metabolism in the diverse processes of signal transduction and cell regulation. Accordingly, the SM cycle may serve as a prototype of sphingolipid signal transduction. Investigations conducted over the past several years have already begun implicating sphingosine, sphingosine phosphate, and sphingosine phosphorylcholine as specific regulators of diverse functions (such as calcium mobilization, thymidine uptake, and cell motility). Therefore, an investigation of sphingolipids in a signaling modality may promise the discovery of novel second messengers and the elucidation of important signal transduction pathways. A second implication of these investigations relates to the specific insight that an investigation of the SM/ceramide pathway is beginning to shed on cellular mechanisms involved in growth suppression and apoptosis. Unlike the study of mitogenesis where intensive investigation has identified multiple oncogenes, oncogene products, signal transduction pathways, and transcription factors involved in the mitogenic response, much less insight has been generated in the research of growth inhibitory mechanisms.