No abstract

No abstract

No abstract

No abstract

No abstract

The fi rst report of a biological effect for ceramide-1-phosphate (C1P) was by , which demonstrated that short chain (not naturally found in cells) C1P induced DNA synthesis in Rat-1 fi broblasts. Later, treatment of T17 fi broblasts with long chain, natural C1P was also demonstrated to induce a potent increase in DNA synthesis ( 2 ) and the levels of proliferating nuclear antigen. Following this line of research, a recent report demonstrated that C1P prevented programmed cell death in bone marrow-derived macrophages after withdrawal of macrophage colony-stimulating factor ( 3 ). Treatment of these cells with C1P effectively blocked the activation of caspases and prevented DNA fragmentation upon serum removal.Abstract Previously, our laboratory demonstrated that ceramide-1-phosphate (C1P) specifi cally activated group IVA cytosolic phospholipase A 2 (cPLA 2 ␣ ) in vitro. In this study, we investigated the chain length specifi city of this interaction. C1P with an acyl-chain of ≥ 6 carbons effi ciently activated cPLA 2 ␣ in vitro, whereas C 2 -C1P, was unable to do so. Delivery of C1P to cells via the newly characterized ethanol/dodecane system demonstrated a lipid-specifi c activation of cPLA 2 ␣ , AA release, and PGE 2 synthesis (EC 50 = 400 nM) when compared to structurally similar lipids. C1P delivered as vesicles in water also induced a lipid-specifi c increase in AA release. Mass spectrometric analysis demonstrated that C1P delivered via ethanol/dodecane induced a 3-fold increase in endogenous C1P with little metabolism to ceramide. C1P was also more effi ciently delivered (>3-fold) to internal membranes by ethanol/dodecane as compared to vesiculated C1P. Using this now established delivery method for lipids, C 2 -C1P was shown to be ineffective in the induction of AA release as compared with C 6 -C1P, C 16 -C1P, and C 18:1 C1P. Here, we demonstrate that C1P requires ≥ 6 carbon acyl-chain to activate cPLA 2 ␣ . Thus, published reports on the biological activity of C 2 -C1P are not via eicosanoid synthesis. Furthermore, this study demonstrates that the alcohol/dodecane system can be used to effi ciently de-

No abstract

a b s t r a c tWe previously demonstrated that ceramide-1-phosphate (C1P) stimulates fibroblast and macrophage proliferation, but the mechanisms involved in this action have only been partially described.Here we demonstrate that C1P induces translocation of protein kinase C-alpha (PKC-a) from the soluble to the membrane fraction of bone marrow-derived macrophages. Translocation of this enzyme was accompanied by its phosphorylation on Ser 657 residue. Activation of PKC-a was independent of prior stimulation of phosphatidylinositol-dependent or phosphatidylcholine-dependent phospholipase C activities, but required activation of sphingomyelin synthesis. Inhibition of PKC-a activation also blocked C1P-stimulated macrophage proliferation indicating that this enzyme is essential for the mitogenic effect of C1P.