J Pharmacol Sci 124, 307 – 312 (2014) Journal of Pharmacological Sciences © The Japanese Pharmacological Society Current Perspective The Role of in Arachidonic Acid Metabolism

Hiroyuki Nakamura1,* and Toshihiko Murayama1 1Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8675, Japan

Received November 22, 2013; Accepted January 17, 2014

Abstract. The arachidonic acid (AA) cascade is regulated mainly by the actions of two rate- limiting enzymes, phospholipase A2 (PLA2) and inducible cyclooxygenase-2 (COX-2). PLA2 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 PLA2 family, cyto- solic PLA2a (cPLA2a, 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 -1-phosphate and lactosylceramide activate cPLA2a directly, -1- phosphate induces COX-2 expression. Sphingomyelin has been shown to inhibit the activity of cPLA2a. In addition, several analogs including a therapeutic agent currently used clinically are also reported to be inhibitors of cPLA2a. This review explores the role of sphingolipids in the regulation of cPLA2a and COX-2.

Keywords: arachidonic acid cascade, cytosolic phospholipase A2a, cyclooxygenase, sphingolipid

1. Introduction bone of sphingoid base. Not only do sphingolipids play a structural role in the cellular membrane, but they have Arachidonic acid (AA) is a precursor of eicosanoids, also been implicated in various significant cell signaling including prostaglandins (PG) and thromboxanes, playing pathways and physiological processes (2). Recent studies important roles in many physiological and pathological have revealed that sphingolipids regulate cPLA2a and functions. The biosynthesis of these AA metabolites COX-2 as shown below. This review provides an over- occurs mainly by activation of phospholipase A2 (PLA2) view of our current understanding of the regulatory in response to a wide variety of stimuli. PLA2 catalyzes mechanisms of cPLA2a and COX-2 by sphingolipids. the hydrolysis of the sn-2 position of membrane glycero- phospholipids to liberate free AA and lysophospholipids 2. Sphingolipid metabolism (1). Mammalian cells have structurally diverse forms of 2+ PLA2 including secretory PLA2, Ca -independent PLA2, The first necessary step in the de novo pathway of and cytosolic PLA2 (cPLA2). Among these, group IVA ceramide generation involves palmitoyl-CoA and amino PLA2 (cPLA2a) is highly selective for glycerophos­ acid condensation, via the action of the enzyme pholipids containing AA. The released AA is converted serine palmitoyl transferase, to form dihydrosphingosine by the action of cyclooxygenase (COX)-1 and COX-2 (2). Following its synthesis, serine-derived dihydro- to PG. sphingosine is acylated via action of the ceramide syn- Sphingolipids are a class of lipids containing a back- thases to become dihydroceramide. Dihydroceramide is then desaturated to form ceramide. Ceramide may also *Corresponding author. [email protected] be generated by the breakdown of membrane sphingo- Published online in J-STAGE on March 4, 2014 doi: 10.1254/jphs.13R18CP myelin (SM) or via degradation of complex glycosphin- golipids by the action of sphingomyelinases and glucosyl Invited article , respectively. Degradation of ceramide is

307 308 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 membranes including the Golgi apparatus, endoplasmic removed from ceramide and the 18-carbon amino-alcohol reticulum, and nuclear envelope. The translocation of compound sphingosine is formed. Sphingosine then cPLA2a to the perinuclear membranes is important for its serves as the substrate for the sphingosine kinases, which functional coupling with COXs, which reside there. The are responsible for phosphorylating sphingosine at the activation of cPLA2a in cells requires sustained phos- primary hydroxyl group, resulting in the production of phorylation of Ser505 by extracellular signal-regulated sphingosine-1-phosphate (S1P). In lieu of being phos- kinase 1/2. The main role of the Ser505 phosphorylation phorylated by to S1P, sphingosine of cPLA2a is to promote membrane penetration of can be recycled back to ceramide via ceramide synthase– hydrophobic residues in the active-site rim by inducing a mediated reacylation. Ceramide is also phosphorylated conformational change in the enzyme; these enhanced by the action of ceramide kinase (CerK) to form hydrophobic interactions allow sustained membrane ceramide-1-phosphate (C1P) (Fig. 1). interaction of cPLA2a. Full activation of cPLA2a is achieved by additional phosphorylation at Ser515 and Ser727 2+ 3. Regulatory mechanisms of cPLA2a activity by Ca -calmodulin kinase II and mitogen-activated protein kinase-activated protein kinases, respectively. cPLA2a is widely expressed in mammalian cells and Phosphatidylinositol 4,5-bisphosphate binds with high mediates the production of functionally diverse lipids in affinity and specificity to cPLA2a, facilitating membrane response to extracellular stimuli. cPLA2a is highly selec- binding and activity. The binding site of phosphati- tive for glycerophospholipids containing AA (1). cPLA2a dylinositol 4,5-bisphosphate includes four lysine residues is regulated mainly by binding Ca2+, phosphorylation on (Lys488, Lys541, Lys543, and Lys544), which are located in serine residues, and interaction with lipids. The binding the highly basic region of the catalytic domain on the 2+ of Ca to the C2 domain of cPLA2a triggers transloca- side close to the membrane. In addition, sphingolipids tion of cPLA2a from the cytosol to the perinuclear regulate the activity of cPLA2a as shown below. Sphingolipids Regulate cPLA2a and COX-2 309

4. Regulatory mechanisms of cPLA2a activity by of cPLA2a (11). Exogenous ceramide is metabolized sphingolipids by various enzymes including CerK, ceramidases, SM synthases, and glucosylceramide synthases. The release 4.1. Role of C1P in the activity of cPLA2a of AA induced by C6-ceramide is inhibited by treatment The first report on the regulation of AA release and with CerK inhibitor in RBL-2H3 rat mast cells (12). the production of PG by C1P was by Chalfant and Thus, it seems that the release of AA induced by the co-workers (3). These authors demonstrated that C1P exogenous addition of ceramide is in part due to C1P potently and specifically stimulates AA release and PG generation. synthesis in A549 lung adenocarcinoma cells. In the same report, the authors showed that CerK via the pro- 4.3. Role of lactosylceramide (LacCer) in the activity duction of C1P is a mediator of AA release in response of cPLA2a to interleukin-1b (IL-1b) and calcium ionophore A23187. LacCer is a member of the glycosphingolipid family In a later report, the same group demonstrated that the and is known to be a bioactive lipid in various cell mechanism of C1P-stimulated AA release occurs through physio­logical processes. LacCer stimulates PECAM-1 direct activation of cPLA2a (4). In addition, C1P has (platelet endothelial cell adhesion molecule-1) expres- been shown to induce the translocation of cPLA2a and sion in the adhesion and diapedesis of monocytes/ its C2 domain to the Golgi apparatus. Our laboratory lymphocytes, which is inhibited by treatment with inhibi- 2+ has also reported that C1P activates cPLA2a directly tors of Ca -independent PLA2 or cPLA2a (13). Recently, and by a protein kinase C–dependent pathway (5). Sub- our laboratory has reported that down-regulation of sequently, Chalfant’s group showed that C1P increases cPLA2a using RNA interference abolishes the ability of the affinity of cPLA2a for membranes through interac- LacCer to induce AA release in cells, suggesting that tion with the C2 domain of cPLA2a (6). This enhanced cPLA2a is the key PLA2 downstream of LacCer (14). association of cPLA2a with membrane leads to an Treatment of cells with LacCer induces the translocation increase in the enzymatic activity of the enzyme. In of full-length cPLA2a from the cytosol to the Golgi addition, the C1P interaction site has been identified in apparatus. LacCer also induces the translocation of the a cationic b-groove of the C2 domain using the solved C2 domain of cPLA2a in the same manner. Interestingly, structure of cPLA2a in conjunction with site-directed LacCer interacts directly with full-length cPLA2a and mutagenesis (7). Specifically, the amino acids Arg57, with the C2 domain in a Ca2+-independent manner. 58 59 Arg , and Arg are required for the interaction of Furthermore, LacCer directly activates cPLA2a in vitro. cPLA2a with C1P. The mutation of these residues Thus, LacCer is a novel direct activator of cPLA2a. abolishes the ability of the enzyme to be activated and translocated in response to probably C1P formed by 4.4. Role of SM in the activity of cPLA2a A23187 or ATP (8). Recently, Arg59, Arg61, and His62 Our laboratory has reported that SM inhibits the activ- (an RxRH sequence) have also been shown to be essen- ity of cPLA2a and the release of AA (15). In that report, tial for the in vitro affinity of C1P and translocation in we showed that A23187-induced AA release was in- response to A23187 (9). These studies demonstrate creased in LY-A cells that are defective in the ceramide that C1P is required for the translocation of cPLA2a to transport protein CERT for SM synthesis, compared with intracellular membranes in response to stimuli and the that in control cells. On the other hand, increasing the subsequent production of AA. cellular SM level by treatment with an acid sphingo­ myelinase inhibitor decreased the release of AA in re- 4.2. Role of ceramide in the activity of cPLA2a sponse to stimuli including A23187 or platelet-activating Ceramide has been shown to interact directly with factor (PAF). In addition, in vitro study indicated that cPLA2a via the C2 domain, which causes improved SM disturbed the binding of cPLA2a to glycerophos­ membrane binding of cPLA2a, resulting in enhanced AA pholipids. These results suggest that SM at the bio­ release and PG synthesis (10). To determine the func- membrane plays important roles in regulating the tions of ceramide in cells, ceramide analogs consisting of cPLA2a-dependent release of AA by inhibiting the sphingosine linked by an amide bond to a short-chain binding of cPLA2a to glycerophospholipids. fatty acid, such as acetic acid (C2-ceramide), hexanoic acid (C6-ceramide), and octanoic acid (C8-ceramide), 5. Regulation of COX-2 expression by S1P are often used because they are cell-permeable. Both C2- and C8- were shown to enhance the S1P exerts a variety of biological responses through A23187-induced release of AA in Chinese hamster ovary extracellular specific receptors or intracellular mecha- cells, which was inhibited by treatment with an inhibitor nisms as a secondary messenger. The S1P family of 310 H Nakamura and T Murayama

G protein–coupled receptors, of which there are five (S1P1 – S1P5), couple to different alpha subunits of heterotrimeric G proteins such as Gai, Gaq, and Ga12/13. 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, phosphati- dylinositol-3 kinase, adenylyl cyclase, phospholipase C, Rho, and c-jun N-terminal kinase, resulting in various cellular responses (16). Fig. 2. Proposed mechanisms of AA metabolism by sphingolipids. 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 Our laboratory has reported recently that TNF-a- for at least 8 h. In contrast, COX-1 expression is not induced AA release is abolished by RNA interference affected by S1P. Subsequently, Ki et al. (18) have of cPLA2a or glucosylceramide synthase (14). TNF-a reported that Ga12 specifically regulates nuclear factor- induces the translocation of cPLA2a to the perinuclear kB–mediated COX-2 induction by S1P downstream of regions, which is inhibited by treatment with inhibitor of S1P1, S1P3, and S1P5, in a process mediated by the c-jun glucosylceramide synthase. TNF-a induces the activa- N-terminal kinase–dependent ubiquitination and degra- tion of b-1,4-galactosyl transferase and the formation of dation of IkBa. Although they showed that S1P-induced LacCer, which is generated via the galactosylation of COX-2 induction was not attenuated by RNA interfer- glucosylceramide produced from ceramide. In addition, ence of S1P2, another group has shown that S1P2 activa- TNF-a and IL-1b activate sphingomyelinase that hydro- tion induces COX-2 expression (19). In transforming lyzes SM to ceramide. Thus, it is supposed that, in growth factor b1–stimulated fibroblasts, S1P formed by response to pro-inflammatory cytokines, the level of SM the receptor stimulation induces COX-2 expression via as an inhibitor of cPLA2a is decreased, and C1P and S1P1 (20). Thus, subtypes of S1P receptor for COX-2 LacCer as activators are increased in the membrane, induction may depend on cell types or conditions. In thereby inducing the catalytic ability of cPLA2a to addition, S1P has been shown to activate nucleocyto­ release AA (Fig. 2). In addition, the induction of COX-2 plasmic shuttling of the mRNA-stabilizing protein in response to cytokine-stimulated S1P formation HuR, which in turn stabilizes COX-2 mRNA and elicits exhibits a significant synergistic response in the produc- COX-2 expression (21). Thus, S1P may increase COX-2 tion of PG. expression by inducing transcription of COX-2 mRNA and by stabilization of its mRNA. 7. Sphingolipid analogs as inhibitors of cPLA2a

6. The role of sphingolipid metabolism in the inflam- 7.1. FTY720 matory response FTY720 {2-amino-2-[2-(4-octylphenyl)ethyl]propane- 1,3-diol} is structurally similar to sphingosine and the lead Pro-inflammatory cytokines, including IL-1b and compound from which it was derived, ISP-1 (myriocin), tumor necrosis factor-a (TNF-a), regulate both cPLA2a was isolated from the culture broth of Isaria sinclairii and COX-2 via sphingolipid metabolism. The abilities of (25). Also known as , or by the trade name IL-1b or TNF-a to induce COX-2 expression are abro- GilenyaTM, FTY720 is currently used clinically to quell gated by RNA interference of sphingosine kinase 1, which the symptoms and slow the progression of multiple can be rescued by replenishment of S1P, indicating that sclerosis. FTY720 is phosphorylated by sphingosine the sphingosine kinase 1/S1P pathway is required up- kinase 2, and a large body of evidence suggests that stream of the induction of COX-2 (22, 23). IL-1b leads FTY720-phosphate, an analog of S1P, is the biologically to the formation of C1P via CerK, resulting in the activa- active form. FTY720-phosphate can bind to all known tion of cPLA2a. Postoperative ileus, a major cause of S1P receptors, except S1P2, and has been shown to morbidity after abdominal surgery, is characterized by regulate S1P1 actions that are crucial for lymphocyte intestinal dysmotility and inflammation. The levels of S1P migration and trafficking. and of C1P are increased in intestinal smooth muscle cells FTY720 has also been shown to inhibit antigen- from a rat model of intestinal surgical manipulation (24). induced release of AA and secretion of PGD2 (26). Sphingolipids Regulate cPLA2a and COX-2 311

Table 1. Properties of sphingolipids in AA metabolism Sphingolipids Functions Effective concentrations References

C1P promotion of cPLA2a translocation 2.5 – 30 mM 4, 5

Ceramide activation of cPLA2a in vitro 0.5 – 2.5 mM (10 – 50 mol%) 10

LacCer promotion of cPLA2a translocation 30 mM 14

SM inhibition of cPLA2a in vitro 2 mM (50 mol%) 15 S1P COX-2 gene expression 0.1 – 1 mM 18 stabilization of COX-2 mRNA 0.1 – 1 mM 21

FTY720 inhibition of cPLA2a in vitro 3 – 10 mM (1 – 3 mol%) 26

CERA-1 inhibition of cPLA2a in vitro 7.5 – 30 mM (2.5 – 10 mol%) 28

C5-DM-C1P inhibition of cPLA2a in vitro 5 mM (70 mol%) 29

C2-DE-C1P inhibition of cPLA2a in vitro 5 mM (70 mol%) 30

inhibition of cPLA2a translocation 10 mM 30 AA: arachidonic acid, C1P: ceramide-1-phosphate, LacCer: lactosylceramide, SM: sphingomyelin, S1P: sphingosine-1-

phosphate, cPLA2a: cytosolic phospholipase A2a.

Interestingly, these effects are independent of its phos- to glycerophospholipids in the lipid–protein overlay phorylation and S1P receptor functions. In addition, assay. Thus, C1P analogs may be useful for the develop- FTY720 but not FTY720-phosphate inhibits recombinant ment of therapeutics for various AA metabolism–related cPLA2a activity. Thus, the development of nonphos- diseases. phorylatable analogs of FTY720 that still target cPLA2a but would not affect lymphocyte trafficking and homing 8. Concluding remarks through S1P receptors might lead to novel potent and specific therapeutics. This review describes mechanisms by which various bioactive sphingolipids can differentially modulate the 7.2. C1P analogs AA metabolism. Table 1 lists regulators of cPLA2a A C1P analog, 1-methyl-2-(3-methoxyphenyl)-2- activity and COX-2 expression. C1P and LacCer mediate (octanoylamino)ethyl-disodium-phosphate, named phospho- cPLA2a activation by causing the translocation of ceramide analogue-1 (PCERA-1), was synthesized by cPLA2a to the Golgi apparatus, whereas SM inhibits Zor and co-workers (27). PCERA-1 has been shown cPLA2a activity. S1P increases COX-2 expression by to be an immune modulator with the ability to down- inducing transcription of COX-2 mRNA and by stabili- regulate the production of pro-inflammatory cytokines zation of its mRNA. Various sphingolipid analogs can such as TNF-a and IL-12p40 and to up-regulate the act as cPLA2a as inhibitors. Pro-inflammatory cytokines production of the anti-inflammatory cytokine IL-10, in increase cPLA2a activity and COX-2 expression via lipopolysaccharide-stimulated macrophages and in vivo. sphingolipid metabolism as shown in Fig. 2. However, In addition, PCERA-1 suppresses lipopolysaccharide- much remains to be understood and discovered with induced AA release and PGE2 production in RAW264.7 regard to the regulatory mechanisms of sphingolipid macrophages by inhibiting the enzymatic activity of metabolism enzymes. Hopefully, such approved under- cPLA2a (28). The inhibitory activity of PCERA-1 on standing will enable the development of agents aimed at the release of AA is attributed to its dephosphorylated decreasing the potential contribution of AA metabolism derivative, ceramide analogue-1 (CERA-1), which to human pathology. directly inhibits cPLA2a. Our laboratory has also synthesized novel di-methyl References (DM) or di-ethyl (DE) phosphate ester analogs of C1P with N-acyl chains of different lengths, and found 1 Murakami M, Taketomi Y, Miki Y, Sato H, Hirabayashi T, that short-N-acyl C1P analogs such as C2-DM-C1P, C5- Yamamoto K. Recent progress in phospholipase A2 research: DM-C1P, C6-DM-C1P, and C2-DE-C1P inhibited the From cells to animals to humans. Prog Lipid Res. 2011;50: 152–192. release of AA mediated by cPLA2a in cells (29, 30). a 2 Hannun YA, Obeid LM. Principles of bioactive lipid signaling: These analogs inhibit the enzymatic activity of cPLA2 lessons from sphingolipids. Nat Rev Mol Cell Biol. 2008;9: directly. Interestingly, C2-DE-C1P inhibits the transloca- 139–150. tion of cPLA2a in response to A23187 or PAF. In 3 Pettus BJ, Bielawska A, Spiegel S, Roddy P, Hannun YA, Chalfant addition, C2-DE-C1P disturbs the binding of the enzyme CE. Ceramide kinase meditates cytokine- and calcium ionophore- 312 H Nakamura and T Murayama

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