The Role of Sphingolipids in Arachidonic Acid Metabolism

Transcription

1 J Pharmacol Sci 124, (2014) Journal of Pharmacological Sciences The Japanese Pharmacological Society Current Perspective The Role of Sphingolipids in Arachidonic Acid Metabolism Hiroyuki Nakamura 1, * and Toshihiko Murayama 1 1 Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba , Japan Received November 22, 2013; Accepted January 17, 2014 Abstract. The arachidonic acid (AA) cascade is regulated mainly by the actions of two ratelimiting enzymes, phospholipase A 2 (PLA 2 ) and inducible cyclooxygenase-2 (COX-2). PLA 2 acts to generate AA, which serves as the precursor substrate for COX-2 in the metabolic pathway leading to prostaglandin production. Amongst more than 30 members of the PLA 2 family, cytosolic PLA 2 a (cpla 2 a, group IVA) plays a major role in releasing AA from cellular membranes. Sphingolipids are a novel class of bioactive lipids that play key roles in the regulation of several cellular processes including growth, differentiation, inflammatory responses, and apoptosis. Recent studies implicated a regulatory function of sphingolipids in prostaglandin production. Whereas ceramide-1-phosphate and lactosylceramide activate cpla 2 a directly, sphingosine-1- phosphate induces COX-2 expression. Sphingomyelin has been shown to inhibit the activity of cpla 2 a. In addition, several sphingolipid analogs including a therapeutic agent currently used clinically are also reported to be inhibitors of cpla 2 a. This review explores the role of sphingolipids in the regulation of cpla 2 a and COX-2. Keywords: arachidonic acid cascade, cytosolic phospholipase A 2 a, cyclooxygenase, sphingolipid 1. Introduction *Corresponding author. nakahiro@faculty.chiba-u.jp Published online in J-STAGE on March 4, 2014 doi: /jphs.13R18CP Invited article Arachidonic acid (AA) is a precursor of eicosanoids, including prostaglandins (PG) and thromboxanes, playing important roles in many physiological and pathological functions. The biosynthesis of these AA metabolites occurs mainly by activation of phospholipase A 2 (PLA 2 ) in response to a wide variety of stimuli. PLA 2 catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate free AA and lysophospholipids (1). Mammalian cells have structurally diverse forms of PLA 2 including secretory PLA 2, Ca 2+ -independent PLA 2, and cytosolic PLA 2 (cpla 2 ). Among these, group IVA PLA 2 (cpla 2 a) is highly selective for glycerophospholipids containing AA. The released AA is converted by the action of cyclooxygenase (COX)-1 and COX-2 to PG. Sphingolipids are a class of lipids containing a backbone of sphingoid base. Not only do sphingolipids play a structural role in the cellular membrane, but they have also been implicated in various significant cell signaling pathways and physiological processes (2). Recent studies have revealed that sphingolipids regulate cpla 2 a and COX-2 as shown below. This review provides an overview of our current understanding of the regulatory mechanisms of cpla 2 a and COX-2 by sphingolipids. 2. Sphingolipid metabolism The first necessary step in the de novo pathway of ceramide generation involves palmitoyl-coa and amino acid serine condensation, via the action of the enzyme serine palmitoyl transferase, to form dihydrosphingosine (2). Following its synthesis, serine-derived dihydrosphingosine is acylated via action of the ceramide synthases to become dihydroceramide. Dihydroceramide is then desaturated to form ceramide. Ceramide may also be generated by the breakdown of membrane sphingomyelin (SM) or via degradation of complex glycosphingolipids by the action of sphingomyelinases and glucosyl ceramidases, respectively. Degradation of ceramide is 307

2 308 H Nakamura and T Murayama Fig. 1. Shingolipid metabolic pathway and chemical structures of sphingolipids. A: Sphingolipid metabolic pathway. B: Chemical structures of sphingolipids. carried out by the ceramidases, whereby the acyl chain is removed from ceramide and the 18-carbon amino-alcohol compound sphingosine is formed. Sphingosine then serves as the substrate for the sphingosine kinases, which are responsible for phosphorylating sphingosine at the primary hydroxyl group, resulting in the production of sphingosine-1-phosphate (S1P). In lieu of being phosphorylated by sphingosine kinase to S1P, sphingosine can be recycled back to ceramide via ceramide synthase mediated reacylation. Ceramide is also phosphorylated by the action of ceramide kinase (CerK) to form ceramide-1-phosphate (C1P) (Fig. 1). 3. Regulatory mechanisms of cpla 2 a activity cpla 2 a is widely expressed in mammalian cells and mediates the production of functionally diverse lipids in response to extracellular stimuli. cpla 2 a is highly selective for glycerophospholipids containing AA (1). cpla 2 a is regulated mainly by binding Ca 2+, phosphorylation on serine residues, and interaction with lipids. The binding of Ca 2+ to the C2 domain of cpla 2 a triggers translocation of cpla 2 a from the cytosol to the perinuclear membranes including the Golgi apparatus, endoplasmic reticulum, and nuclear envelope. The translocation of cpla 2 a to the perinuclear membranes is important for its functional coupling with COXs, which reside there. The activation of cpla 2 a in cells requires sustained phosphorylation of Ser 505 by extracellular signal-regulated kinase 1/2. The main role of the Ser 505 phosphorylation of cpla 2 a is to promote membrane penetration of hydrophobic residues in the active-site rim by inducing a conformational change in the enzyme; these enhanced hydrophobic interactions allow sustained membrane interaction of cpla 2 a. Full activation of cpla 2 a is achieved by additional phosphorylation at Ser 515 and Ser 727 by Ca 2+ -calmodulin kinase II and mitogen-activated protein kinase-activated protein kinases, respectively. Phosphatidylinositol 4,5-bisphosphate binds with high affinity and specificity to cpla 2 a, facilitating membrane binding and activity. The binding site of phosphatidylinositol 4,5-bisphosphate includes four lysine residues (Lys 488, Lys 541, Lys 543, and Lys 544 ), which are located in the highly basic region of the catalytic domain on the side close to the membrane. In addition, sphingolipids regulate the activity of cpla 2 a as shown below.

3 Sphingolipids Regulate cpla 2a and COX Regulatory mechanisms of cpla 2 a activity by sphingolipids 4.1. Role of C1P in the activity of cpla 2 a The first report on the regulation of AA release and the production of PG by C1P was by Chalfant and co-workers (3). These authors demonstrated that C1P potently and specifically stimulates AA release and PG synthesis in A549 lung adenocarcinoma cells. In the same report, the authors showed that CerK via the production of C1P is a mediator of AA release in response to interleukin-1b (IL-1b) and calcium ionophore A In a later report, the same group demonstrated that the mechanism of C1P-stimulated AA release occurs through direct activation of cpla 2 a (4). In addition, C1P has been shown to induce the translocation of cpla 2 a and its C2 domain to the Golgi apparatus. Our laboratory has also reported that C1P activates cpla 2 a directly and by a protein kinase C dependent pathway (5). Subsequently, Chalfant s group showed that C1P increases the affinity of cpla 2 a for membranes through interaction with the C2 domain of cpla 2 a (6). This enhanced association of cpla 2 a with membrane leads to an increase in the enzymatic activity of the enzyme. In addition, the C1P interaction site has been identified in a cationic b-groove of the C2 domain using the solved structure of cpla 2 a in conjunction with site-directed mutagenesis (7). Specifically, the amino acids Arg 57, Arg 58, and Arg 59 are required for the interaction of cpla 2 a with C1P. The mutation of these residues abolishes the ability of the enzyme to be activated and translocated in response to probably C1P formed by A23187 or ATP (8). Recently, Arg 59, Arg 61, and His 62 (an RxRH sequence) have also been shown to be essential for the in vitro affinity of C1P and translocation in response to A23187 (9). These studies demonstrate that C1P is required for the translocation of cpla 2 a to intracellular membranes in response to stimuli and the subsequent production of AA Role of ceramide in the activity of cpla 2 a Ceramide has been shown to interact directly with cpla 2 a via the C2 domain, which causes improved membrane binding of cpla 2 a, resulting in enhanced AA release and PG synthesis (10). To determine the functions of ceramide in cells, ceramide analogs consisting of sphingosine linked by an amide bond to a short-chain fatty acid, such as acetic acid (C2-ceramide), hexanoic acid (C6-ceramide), and octanoic acid (C8-ceramide), are often used because they are cell-permeable. Both C2- and C8-ceramides were shown to enhance the A23187-induced release of AA in Chinese hamster ovary cells, which was inhibited by treatment with an inhibitor of cpla 2 a (11). Exogenous ceramide is metabolized by various enzymes including CerK, ceramidases, SM synthases, and glucosylceramide synthases. The release of AA induced by C6-ceramide is inhibited by treatment with CerK inhibitor in RBL-2H3 rat mast cells (12). Thus, it seems that the release of AA induced by the exogenous addition of ceramide is in part due to C1P generation Role of lactosylceramide (LacCer) in the activity of cpla 2 a LacCer is a member of the glycosphingolipid family and is known to be a bioactive lipid in various cell physio logical processes. LacCer stimulates PECAM-1 (platelet endothelial cell adhesion molecule-1) expression in the adhesion and diapedesis of monocytes/ lymphocytes, which is inhibited by treatment with inhibitors of Ca 2+ -independent PLA 2 or cpla 2 a (13). Recently, our laboratory has reported that down-regulation of cpla 2 a using RNA interference abolishes the ability of LacCer to induce AA release in cells, suggesting that cpla 2 a is the key PLA 2 downstream of LacCer (14). Treatment of cells with LacCer induces the translocation of full-length cpla 2 a from the cytosol to the Golgi apparatus. LacCer also induces the translocation of the C2 domain of cpla 2 a in the same manner. Interestingly, LacCer interacts directly with full-length cpla 2 a and with the C2 domain in a Ca 2+ -independent manner. Furthermore, LacCer directly activates cpla 2 a in vitro. Thus, LacCer is a novel direct activator of cpla 2 a Role of SM in the activity of cpla 2 a Our laboratory has reported that SM inhibits the activity of cpla 2 a and the release of AA (15). In that report, we showed that A23187-induced AA release was increased in LY-A cells that are defective in the ceramide transport protein CERT for SM synthesis, compared with that in control cells. On the other hand, increasing the cellular SM level by treatment with an acid sphingomyelinase inhibitor decreased the release of AA in response to stimuli including A23187 or platelet-activating factor (PAF). In addition, in vitro study indicated that SM disturbed the binding of cpla 2 a to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cpla 2 a-dependent release of AA by inhibiting the binding of cpla 2 a to glycerophospholipids. 5. Regulation of COX-2 expression by S1P S1P exerts a variety of biological responses through extracellular specific receptors or intracellular mechanisms as a secondary messenger. The S1P family of

4 310 H Nakamura and T Murayama G protein coupled receptors, of which there are five (S1P 1 S1P 5 ), couple to different alpha subunits of heterotrimeric G proteins such as Ga i, Ga q, and Ga 12/13. S1P receptor expression patterns, along with the Ga subunits to which each receptor couples, dictate the activation of different downstream targets that occur upon receptor activation, including the activation of Rac, extracellular signal-regulated kinase, phosphatidylinositol-3 kinase, adenylyl cyclase, phospholipase C, Rho, and c-jun N-terminal kinase, resulting in various cellular responses (16). Various investigators have demonstrated that S1P regulates COX-2 expression. Davaille et al. (17) have reported that S1P treatment causes strong induction of COX-2, which peaks after 3 h and remains elevated for at least 8 h. In contrast, COX-1 expression is not affected by S1P. Subsequently, Ki et al. (18) have reported that Ga 12 specifically regulates nuclear factorkb mediated COX-2 induction by S1P downstream of S1P 1, S1P 3, and S1P 5, in a process mediated by the c-jun N-terminal kinase dependent ubiquitination and degradation of IkBa. Although they showed that S1P-induced COX-2 induction was not attenuated by RNA interference of S1P 2, another group has shown that S1P 2 activation induces COX-2 expression (19). In transforming growth factor b1 stimulated fibroblasts, S1P formed by the receptor stimulation induces COX-2 expression via S1P 1 (20). Thus, subtypes of S1P receptor for COX-2 induction may depend on cell types or conditions. In addition, S1P has been shown to activate nucleocytoplasmic shuttling of the mrna-stabilizing protein HuR, which in turn stabilizes COX-2 mrna and elicits COX-2 expression (21). Thus, S1P may increase COX-2 expression by inducing transcription of COX-2 mrna and by stabilization of its mrna. 6. The role of sphingolipid metabolism in the inflammatory response Pro-inflammatory cytokines, including IL-1b and tumor necrosis factor-a (TNF-a), regulate both cpla 2 a and COX-2 via sphingolipid metabolism. The abilities of IL-1b or TNF-a to induce COX-2 expression are abrogated by RNA interference of sphingosine kinase 1, which can be rescued by replenishment of S1P, indicating that the sphingosine kinase 1/S1P pathway is required upstream of the induction of COX-2 (22, 23). IL-1b leads to the formation of C1P via CerK, resulting in the activation of cpla 2 a. Postoperative ileus, a major cause of morbidity after abdominal surgery, is characterized by intestinal dysmotility and inflammation. The levels of S1P and of C1P are increased in intestinal smooth muscle cells from a rat model of intestinal surgical manipulation (24). Fig. 2. Proposed mechanisms of AA metabolism by sphingolipids. Our laboratory has reported recently that TNF-ainduced AA release is abolished by RNA interference of cpla 2 a or glucosylceramide synthase (14). TNF-a induces the translocation of cpla 2 a to the perinuclear regions, which is inhibited by treatment with inhibitor of glucosylceramide synthase. TNF-a induces the activation of b-1,4-galactosyl transferase and the formation of LacCer, which is generated via the galactosylation of glucosylceramide produced from ceramide. In addition, TNF-a and IL-1b activate sphingomyelinase that hydrolyzes SM to ceramide. Thus, it is supposed that, in response to pro-inflammatory cytokines, the level of SM as an inhibitor of cpla 2 a is decreased, and C1P and LacCer as activators are increased in the membrane, thereby inducing the catalytic ability of cpla 2 a to release AA (Fig. 2). In addition, the induction of COX-2 in response to cytokine-stimulated S1P formation exhibits a significant synergistic response in the production of PG. 7. Sphingolipid analogs as inhibitors of cpla 2 a 7.1. FTY720 FTY720 {2-amino-2-[2-(4-octylphenyl)ethyl]propane- 1,3-diol} is structurally similar to sphingosine and the lead compound from which it was derived, ISP-1 (myriocin), was isolated from the culture broth of Isaria sinclairii (25). Also known as Fingolimod, or by the trade name Gilenya TM, FTY720 is currently used clinically to quell the symptoms and slow the progression of multiple sclerosis. FTY720 is phosphorylated by sphingosine kinase 2, and a large body of evidence suggests that FTY720-phosphate, an analog of S1P, is the biologically active form. FTY720-phosphate can bind to all known S1P receptors, except S1P 2, and has been shown to regulate S1P 1 actions that are crucial for lymphocyte migration and trafficking. FTY720 has also been shown to inhibit antigeninduced release of AA and secretion of PGD 2 (26).

5 Sphingolipids Regulate cpla 2a and COX Table 1. Properties of sphingolipids in AA metabolism Sphingolipids Functions Effective concentrations References C1P promotion of cpla 2a translocation mm 4, 5 Ceramide activation of cpla 2a in vitro mm (10 50 mol%) 10 LacCer promotion of cpla 2a translocation 30 mm 14 SM inhibition of cpla 2a in vitro 2 mm (50 mol%) 15 S1P COX-2 gene expression mm 18 stabilization of COX-2 mrna mm 21 FTY720 inhibition of cpla 2a in vitro 3 10 mm (1 3 mol%) 26 CERA-1 inhibition of cpla 2a in vitro mm ( mol%) 28 C5-DM-C1P inhibition of cpla 2a in vitro 5 mm (70 mol%) 29 C2-DE-C1P inhibition of cpla 2a in vitro 5 mm (70 mol%) 30 inhibition of cpla 2a translocation 10 mm 30 AA: arachidonic acid, C1P: ceramide-1-phosphate, LacCer: lactosylceramide, SM: sphingomyelin, S1P: sphingosine-1- phosphate, cpla 2a: cytosolic phospholipase A 2a. Interestingly, these effects are independent of its phosphorylation and S1P receptor functions. In addition, FTY720 but not FTY720-phosphate inhibits recombinant cpla 2 a activity. Thus, the development of nonphosphorylatable analogs of FTY720 that still target cpla 2 a but would not affect lymphocyte trafficking and homing through S1P receptors might lead to novel potent and specific therapeutics C1P analogs A C1P analog, 1-methyl-2-(3-methoxyphenyl)-2- (octanoylamino)ethyl-disodium-phosphate, named phosphoceramide analogue-1 (PCERA-1), was synthesized by Zor and co-workers (27). PCERA-1 has been shown to be an immune modulator with the ability to downregulate the production of pro-inflammatory cytokines such as TNF-a and IL-12p40 and to up-regulate the production of the anti-inflammatory cytokine IL-10, in lipopolysaccharide-stimulated macrophages and in vivo. In addition, PCERA-1 suppresses lipopolysaccharideinduced AA release and PGE 2 production in RAW264.7 macrophages by inhibiting the enzymatic activity of cpla 2 a (28). The inhibitory activity of PCERA-1 on the release of AA is attributed to its dephosphorylated derivative, ceramide analogue-1 (CERA-1), which directly inhibits cpla 2 a. Our laboratory has also synthesized novel di-methyl (DM) or di-ethyl (DE) phosphate ester analogs of C1P with N-acyl chains of different lengths, and found that short-n-acyl C1P analogs such as C2-DM-C1P, C5- DM-C1P, C6-DM-C1P, and C2-DE-C1P inhibited the release of AA mediated by cpla 2 a in cells (29, 30). These analogs inhibit the enzymatic activity of cpla 2 a directly. Interestingly, C2-DE-C1P inhibits the translocation of cpla 2 a in response to A23187 or PAF. In addition, C2-DE-C1P disturbs the binding of the enzyme to glycerophospholipids in the lipid protein overlay assay. Thus, C1P analogs may be useful for the development of therapeutics for various AA metabolism related diseases. 8. Concluding remarks This review describes mechanisms by which various bioactive sphingolipids can differentially modulate the AA metabolism. Table 1 lists regulators of cpla 2 a activity and COX-2 expression. C1P and LacCer mediate cpla 2 a activation by causing the translocation of cpla 2 a to the Golgi apparatus, whereas SM inhibits cpla 2 a activity. S1P increases COX-2 expression by inducing transcription of COX-2 mrna and by stabilization of its mrna. Various sphingolipid analogs can act as cpla 2 a as inhibitors. Pro-inflammatory cytokines increase cpla 2 a activity and COX-2 expression via sphingolipid metabolism as shown in Fig. 2. However, much remains to be understood and discovered with regard to the regulatory mechanisms of sphingolipid metabolism enzymes. Hopefully, such approved understanding will enable the development of agents aimed at decreasing the potential contribution of AA metabolism to human pathology. References 1 Murakami M, Taketomi Y, Miki Y, Sato H, Hirabayashi T, Yamamoto K. Recent progress in phospholipase A 2 research: From cells to animals to humans. Prog Lipid Res. 2011;50: Hannun YA, Obeid LM. Principles of bioactive lipid signaling: lessons from sphingolipids. Nat Rev Mol Cell Biol. 2008;9: Pettus BJ, Bielawska A, Spiegel S, Roddy P, Hannun YA, Chalfant CE. Ceramide kinase meditates cytokine- and calcium ionophore-

6 312 H Nakamura and T Murayama induced arachidonic acid release. J Biol Chem. 2003;278: Pettus BJ, Bielawska A, Subramanian P, Wijesinghe DS, Maceyka M, Leslie CC, et al. Ceramide-1-phosphate is a direct activator of cytosolic phospholipase A 2. J Biol Chem. 2004; 279: Nakamura H, Hirabayashi T, Shimizu M, Murayama T. Ceramide-1-phosphate activates cytosolic phospholipase A 2a directly and by PKC pathway. Biochem Pharmacol. 2006;71: Sabramanian P, Stahelin RV, Szulc Z, Bielawska A, Cho W, Chalfant CE. Ceramide 1-phosphate acts as a positive allosteric activator of group IVA cytosolic phospholipase A 2a and enhances the interaction of the enzyme with phosphatidylcholine. J Biol Chem. 2005;280: Stahelin RV, Subramanian P, Vora M, Cho W, Chalfant CE. Ceramide-1-phosphate binds group IVA cytosolic phospholipase A 2 via a novel site in the C2 domain. J Biol Chem. 2007; 282: Lamour NF, Subramanian P, Wijesinghe DS, Stehelin RV, Bonventre JV, Chalfant CE. Ceramide 1-phosphate is required for the translocation of group IVA cytosolic phospholipase A 2 and prostaglandin synthesis. J Biol Chem. 2009;284: Ward KE, Bhardwaj N, Vora M, Chalfant CE, Lu H, Stahelin RV. The molecular basis of ceramide-1-posphate recognition by C2 domain. J Lipid Res. 2013;54: Huwiler A, Johansen B, Skarstad A, Pfeilschifter J. Ceramide binds to the CaLB domain of cytosolic phospholipase A 2 and facilitates its membrane docking and arachidonic acid release. FASEB J. 2001;15: Shimizu M, Tada E, Makiyama T, Yasufuku K, Moriyama Y, Fujino H, et al. Effects of ceramide, ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A 2a; additional role of ceramide-1-phosphate in phosphorylation and Ca 2+ signaling. Cell Signal. 2009;21: Ji JE, Kim SK, Ahn KH, Choi JM, Jung SY, Jung KM, et al. Ceramide induces serotonin release from RBL-2H3 mast cell through calcium mediated activation of phospholipase A 2. Prostaglandins Other Lipid Mediat. 2011;94: Gong N, Wei H, Chowdhury SH, Chatterjee S. Lactosylceramide recruits PKCa/e and phospholipase A 2 to stimulate PECAM-1 expression in human monocytes and adhesion to endothelial cells. Proc Natl Acad Sci U S A. 2004;101: Nakamura H, Moriyama Y, Makiyama T, Emori S, Yamashita H, Murayama T. Lactosylceramide interacts with and activates cytosolic phospholipase A 2a. J Biol Chem. 2013;288: Nakamura H, Wakita S, Suganami A, Tamura Y, Hanada K, Murayama T. Modulation of the activity of cytosolic phospholipase A 2a (cpla 2a) by cellular sphingolipids and inhibition of cpla 2a by sphingomyelin. J Lipid Res. 2010;51: Orr Gandy KA, Obeid LM. Targeting the sphingosine kinase/ sphingosine 1-phosphate pathway in disease; Review of sphingosine kinase inhibitors. Biochim Biophys Acta. 2013;1831: Davaille J, Gallois C, Habib A, Li L, Mallat A, Tao J, et al. Antiproliferative properties of sphingosine 1-phosphate in human hepatic myofibroblasts. J Biol Chem. 2000;275: Ki SH, Choi MJ, Lee CH, Kim SG. Ga 12 specifically regulates COX-2 induction by sphingosine 1-phosphate. J Biol Chem. 2007;282: Skoura A, Sanchez T, Claffey K, Mandala SM, Proia RL, Hla T. Essential role of sphingosine 1-phosphate receptor 2 in pathological angiogenesis of the mouse retina. J Clin Invest. 2007; 117: Kawashima T, Yamazaki R, Matsuzawa Y, Yamaura E, Takabatake M, Otake S, et al. Contrary effects of sphingosine- 1-phosphate on expression of a-smooth muscle actin in transforming growth factor b1-stimulated lung fibroblasts. Eur J Pharmacol. 2012;696: Johann AM, Weigert A, Eberhardt W, Kuhn AM, Barra V, von Knethen A, et al. Apoptic cell-derived sphingosine-1-phosphate promotes HuR-dependent cyclooxygenase-2 mrna stabilization and protein expression. J Immunol. 2008;180: Pettus BJ, Bielawski J, Porcelli AM, Reames DL, Johnson KR, Morrow J, et al. The sphingosine kinase 1/sphingosine- 1-phosphate pathway mediates COX-2 induction and PGE 2 production in response to TNF-a. FASEB J. 2003;17: Billich A, Bornancin F, Mechtcheriakova D, Natt F, Huesken D, Baumruker T. Basal and induced sphiingosine kinase 1 activity in A549 carcinoa cells: function in cell survival and IL-1beta and TNF-alpha induced production of inflammatory mediators. Cell Signal. 2005;17: Dragusin M, Wehner S, Kelly S, Wang E, Merrill AH Jr, Kalff JC, et al. Effect of sphingosine-1-phosphate and ceramid-1- phosphate on rat intestinal smooth muscle cells: implications for postoperative ileus. FASEB J. 2006;20: Adachi K, Chiba K. FTY720 story. Its discovery and the following accelerated development of sphingosine 1-phosphate receptor agonists as immunomodulators based on reverse pharmacology. Perspect Medicin Chem. 2007;1: Payne SG, Oskeritzian CA, Griffiths R, Subramanian P, Barbour SE, Chalfant CE, et al. The immunosuppressant drug FTY720 inhibits cytosolic phospholipase A 2 independently of sphingosine-1-phosphate receptors. Blood. 2007;109: Avni D, Philosoph A, Meijler MM, Zor T. The ceramide-1- phosphate analogue PCERA-1 modulates tumor necrosis factoralpha and interleukin-10 production in macrophages via the camp-pka-creb pathway in a GTP-dependent manner. Immunology. 2010;129: Goldsmith M, Daka A, Lamour NF, Mashiach R, Glucksam Y, Meijler MM, et al. A ceramide analog inhibits cpla 2 activity and consequent PGE 2 formation in LPS-stimulated macrophages. Immunol Lett. 2011;135: Makiyama T, Nagasaka N, Houjyo Y, Yamaura E, Nakamura H, Koide Y, et al. Newly synthetic ceramide-1-phosphate analogs; their uptake, intracellular localization, and roles as an inhibitor of cytosolic phospholipase A 2a and inducer of cell toxicity. Biochem Pharmacol. 2010;80: Makiyama T, Nakamura H, Nishida A, Murayama T. C2-deethyl-ceramide-1-phosphate as an inhibitor of group IVA cytosolic phospholipase A 2. Eur J Pharmacol. 2012;697: