Chain length specificity for activation of cPLA2α by C1P: use of the dodecane delivery system to determine lipid-specific effects

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-

Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels

“…The lipids were extracted, separated by TLC, and subjected to autoradiographic detection as described in the Materials and Methods section. The amounts of 32 P labeled d 18 20 ), the fact that the CERK-derived pool is playing such an important role can be attributed to the fact that synthesis of C1P by CERK is occurring in a specifi c location in the cell. More specifi cally, CERK is generating C1P in the trans--Golgi where cPLA 2 ␣ has been demonstrated to localize to upon stimulation.…”

Distinct receptor-mediated activities in macrophages for natural ceramide-1-phosphate (C1P) and for phospho-ceramide analogue-1 (PCERA-1)

“…C1P was shown to directly activate PLA 2 , implying that it has both receptordependent and receptor-independent activities (Pettus et al, 2004;Wijesinghe et al, 2008). PCERA-1 on the other hand, blocks LPS-induced PLA 2 activation and inhibits subsequent PGE 2 production (Goldsmith and Zor, unpublished).…”

The molecular basis of ceramide-1-phosphate recognition by C2 domains

“…Additionally, the R eq values for the deoxy-C1P surface were comparable to R eq values for the C2 domain to POPC vesicles alone, suggesting that the backbone hydroxyl group is an important determinant of C2 domain C1P binding. A549 cells were treated with exogenous lipid through a dodecane-ethanol vehicle to test the effects of each lipid on cPLA 2 ␣ translocation ( 38 ). Although membrane translocation was lower than in cells treated with the Ca 2+ ionophore A 23187 , the translocation was observed in ‫ف‬ 17% of cells when C1P was added to A549 cells but was not detectable in cells treated with vehicle alone or deoxy-C1P ( Fig.…”

“…Lipids were prepared as previously described ( 38 ). Briefl y, lipid mixtures were prepared by drying the allotted volume of chloroform-methanol (2:1) solubilized lipids under N 2 gas to make a 2.5 mM stock solution.…”