Ceramide 1-Phosphate Is a Direct Activator of Cytosolic Phospholipase A2

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.

Role of sphingomyelin and ceramide in the regulation of the activity and fatty acid specificity of group V secretory phospholipase A2

“…Nevertheless, the effect of ceramide on the fatty acid specificity of sPLA 2 V is consistently observed in defined PC liposomes. It is of interest to note that ceramide phosphate, which has been reported to directly interact with, and activate cytosolic PLA 2 [35], is actually inhibitory to sPLA 2 V (Fig. 7) as well as to LCAT [16].…”

Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy

“…Another observation is the presence of 2 different organelles in the cytoplasm of the A549 cell, not clearly visible on the image obtained with the confocal microscope (Fig.1B) but now visible with the x100 immersion objective. This observation is not surprising as the cytoplasm of the cells has a complex organisation and different structures can be found such as the actin or microtubules 38,39 , endoplasmic reticulum 39,40 , the mitochondria 41 or the Golgi apparatus 42,43 .…”

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

“…A crucial discovery was the direct implication of C1P in the inflammatory response. Chalfant and co-workers were the first to demonstrate that the mechanism whereby C1P stimulates AA release occurs through direct activation of cPLA 2 (Pettus et al, 2004), and that C1P is a positive allosteric activator of group IV cPLA 2 . C1P was also shown to act in coordination with S1P to ensure maximal production of prostaglandins.…”

“…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).…”