Ichiro Kudo

Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis

Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E2 synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE2 biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge. Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex. Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol. A tyrosine residue near the N terminus (Tyr9), which is known to be critical for the activity of cytosolic GSHS-transferases, was essential for PGES activity. The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment. cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE2 from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A2 in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE2 that plays a role in maintenance of tissue homeostasis.

Phospholipase A2 enzymes

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate arachidonic acid (AA), a precursor of eicosanoids including prostaglandins and leukotrienes. The same reaction also produces lysophosholipids, which represent another class of lipid mediators. So far, at least 19 enzymes that possess PLA2 activity have been identified and cloned in mammals. The secretory PLA2 (sPLA2) family, in which 10 isozymes have been identified, consists of low-molecular weight, Ca2+-requiring secretory enzymes that have been implicated in a number of biological processes, such as modification of eicosanoid generation, inflammation, and host defense. The cytosolic PLA2 (cPLA2) family consists of three enzymes, among which cPLA2alpha has been paid much attention by researchers as an essential component of the initiation of AA metabolism. The activation of cPLA2alpha is tightly regulated by Ca2+ and phosphorylation. The Ca2+-independent PLA2 (iPLA2) family contains two enzymes and may play a major role in phospholipid remodeling. The platelet-activating factor (PAF) acetylhydrolase (PAF-AH) family contains four enzymes that exhibit unique substrate specificity toward PAF and/or oxidized phospholipids. Degradation of these bioactive phospholipids by PAF-AHs may lead to the termination of inflammatory reaction and atherosclerosis.

Functional Coupling Between Various Phospholipase A2s and Cyclooxygenases in Immediate and Delayed Prostanoid Biosynthetic Pathways

Several distinct phospholipase A2s (PLA2s) and two cyclooxygenases (COXs) were transfected, alone or in combination, into human embryonic kidney 293 cells, and their functional coupling during immediate and delayed prostaglandin (PG)-biosynthetic responses was reconstituted. Signaling PLA2s, i.e. cytosolic PLA2 (cPLA2) (type IV) and two secretory PLA2s (sPLA2), types IIA (sPLA2-IIA) and V (sPLA2-V), promoted arachidonic acid (AA) release from their respective transfectants after stimulation with calcium ionophore or, when bradykinin receptor was cotransfected, with bradykinin, which evoked the immediate response, and interleukin-1 plus serum, which induced the delayed response. Experiments on cells transfected with either COX alone revealed subtle differences between the PG-biosynthetic properties of the two isozymes in that COX-1 and COX-2 were favored over the other in the presence of high and low exogenous AA concentrations, respectively. Moreover, COX-2, but not COX-1, could turn on endogenous AA release, which was inhibited by a cPLA2 inhibitor. When PLA2 and COX were coexpressed, AA released by cPLA2, sPLA2-IIA and sPLA2-V was converted to PGE2 by both COX-1 and COX-2 during the immediate response and predominantly by COX-2 during the delayed response. Ca2+-independent PLA2 (iPLA2) (type VI), which plays a crucial role in phospholipid remodeling, failed to couple with COX-2 during the delayed response, whereas it was linked to ionophore-induced immediate PGE2 generation via COX-1 in marked preference to COX-2. Finally, coculture of PLA2 and COX transfectants revealed that extracellular sPLA2s-IIA and -V, but neither intracellular cPLA2 nor iPLA2, augmented PGE2 generation by neighboring COX-expressing cells, implying that the heparin-binding sPLA2s play a particular role as paracrine amplifiers of the PG-biosynthetic response signal from one cell to another.

Cellular Prostaglandin E2 Production by Membrane-bound Prostaglandin E Synthase-2 via Both Cyclooxygenases-1 and -2

Current evidence suggests that two forms of prostaglandin (PG) E synthase (PGES), cytosolic PGES and membrane-bound PGES (mPGES) -1, preferentially lie downstream of cyclooxygenase (COX) -1 and -2, respectively, in the PGE2 biosynthetic pathway. In this study, we examined the expression and functional aspects of the third PGES enzyme, mPGES-2, in mammalian cells and tissues. mPGES-2 was synthesized as a Golgi membrane-associated protein, and spontaneous cleavage of the N-terminal hydrophobic domain led to the formation of a truncated mature protein that was distributed in the cytosol with a trend to be enriched in the perinuclear region. In several cell lines, mPGES-2 promoted PGE2 production via both COX-1 and COX-2 in the immediate and delayed responses with modest COX-2 preference. In contrast to the marked inducibility of mPGES-1, mPGES-2 was constitutively expressed in various cells and tissues and was not increased appreciably during tissue inflammation or damage. Interestingly, a considerable elevation of mPGES-2 expression was observed in human colorectal cancer. Collectively, mPGES-2 is a unique PGES that can be coupled with both COXs and may play a role in the production of the PGE2 involved in both tissue homeostasis and disease.

Recent advances in molecular biology and physiology of the prostaglandin E2-biosynthetic pathway.

Prostanoids represent a group of lipid mediators that are produced from arachidonic acid via the cyclooxygenase pathway. Once formed, the prostanoids are released from the cells and act on their cognate receptors on cell surfaces to exert their biological actions. Of these, prostaglandin E(2) (PGE(2)) is the most common prostanoid, being produced by a wide variety of cells and tissues and has a broad range of bioactivity. Recent advance in this field has led to identification and characterization of a number of enzymes that play roles in the biosynthesis of PGE(2), namely phospholipase A(2), cyclooxygenase and terminal PGE synthase. Each of these three reactions can be rate-limiting and involves multiple enzymes/isozymes that can act in different phases of cell activation and exhibit distinct functional coupling. In this review, we will overview a recent understanding of the molecular biology, regulatory mechanisms, and physiological functions of these enzymes.

Prostaglandin E synthase

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin (PG)H2 to PGE2, occurs in multiple forms with distinct enzymatic properties, modes of expression, cellular and subcellular localizations and intracellular functions. Cytosolic PGES (cPGES) is a cytosolic protein that is constitutively expressed in a wide variety of cells and tissues and is associated with heat shock protein 90 (Hsp90). Membrane-associated PGES (mPGES), the expression of which is stimulus-inducible and is downregulated by anti-inflammatory glucocorticoids, is a perinuclear protein belonging to the microsomal glutathione S-transferase (GST) family. These two PGESs display distinct functional coupling with upstream COXs in cells; cPGES is predominantly coupled with the constitutive COX-1, whereas mPGES is preferentially linked with the inducible COX-2. Several cytosolic GSTs also have the capacity to convert PGH2 to PGE2 in vitro. Accumulating evidence has suggested that mPGES participates in various pathophysiological states in which COX-2 is involved, implying that mPGES represents a potential novel target for drug development.

Fas-induced Arachidonic Acid Release Is Mediated by Ca2+-independent Phospholipase A2 but Not Cytosolic Phospholipase A2, Which Undergoes Proteolytic Inactivation

Fas-mediated apoptosis of human leukemic U937 cells was accompanied by increased arachidonic acid (AA) and oleic acid release from membrane glycerophospholipids, indicating phospholipase A2 (PLA2) activation. During apoptosis, type IV cytosolic PLA2 (cPLA2), a PLA2 isozyme with an apparent molecular mass of 110 kDa critical for stimulus-coupled AA release, was converted to a 78-kDa fragment with concomitant loss of catalytic activity. Cleavage of cPLA2 correlated with increased caspase-3-like protease activity in apoptotic cells and was abrogated by a caspase-3 inhibitor. A mutant cPLA2 protein in which Asp522 was replaced by Asn, which aligns with the consensus sequence of the caspase-3 cleavage site (DXXD↓X), was resistant to apo-ptosis-associated proteolysis. Moreover, a COOH-terminal deletion mutant of cPLA2 truncated at Asp522 comigrated with the 78-kDa fragment and exhibited no enzymatic activity. Thus, caspase-3-mediated cPLA2 cleavage eventually leads to destruction of a catalytic triad essential for cPLA2 activity, thereby terminating its AA-releasing function. In contrast, the activity of type VI Ca2+-independent PLA2 (iPLA2), a PLA2 isozyme implicated in phospholipid remodeling, remained intact during apoptosis. Inhibitors of iPLA2, but neither cPLA2 nor secretory PLA2 inhibitors, suppressed AA release markedly and, importantly, delayed cell death induced by Fas. Therefore, we conclude that iPLA2-mediated fatty acid release is facilitated in Fas-stimulated cells and plays a modifying although not essential role in the apoptotic cell death process.

Cytosolic Phospholipase A2 Is Required for Cytokine-induced Expression of Type IIA Secretory Phospholipase A2 That Mediates Optimal Cyclooxygenase-2-dependent Delayed Prostaglandin E2 Generation in Rat 3Y1 Fibroblasts

Activation of rat fibroblastic 3Y1 cells with interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) induced delayed prostaglandin (PG) E2 generation over 6–48 h, which occurred in parallel with de novo induction of type IIA secretory phospholipase A2 (sPLA2) and cyclooxygenase (COX)-2, without accompanied by changes in the constitutive expression of type IV cytosolic PLA2(cPLA2) and COX-1. Types V and IIC sPLA2s were barely detectable in these cells. Studies using an anti-type IIA sPLA2 antibody, sPLA2 inhibitors, and a type IIA sPLA2-specific antisense oligonucleotide revealed that IL-1β/TNFα-induced delayed PGE2 generation by these cells was largely dependent on inducible type IIA sPLA2, which was functionally linked to inducible COX-2. Delayed PGE2 generation was also suppressed markedly by the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3), which attenuated induction of type IIA sPLA2, but not COX-2, expression. AACOCF3inhibited the initial phase of cytokine-stimulated arachidonic acid release, and supplementing AACOCF3-treated cells with exogenous arachidonic acid partially restored type IIA sPLA2 expression. These results suggest that certain metabolites produced by the cPLA2-dependent pathway are crucial for the subsequent induction of type IIA sPLA2 expression and attendant delayed PGE2generation. Some lipoxygenase-derived products might be involved in this event, since IL-1β/TNFα-induced type IIA sPLA2induction and PGE2 generation were reduced markedly by lipoxygenase, but not COX, inhibitors. In contrast, Ca2+ionophore-stimulated immediate PGE2 generation was regulated predominantly by the constitutive enzymes cPLA2and COX-1, even when type IIA sPLA2 and COX-2 were maximally induced after IL-1β/TNFα treatment, revealing functional segregation of the constitutive and inducible PG biosynthetic enzymes.

Biosynthesis of anandamide and N-palmitoylethanolamine by sequential actions of phospholipase A2 and lysophospholipase D

Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2a were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.

Functional Association of Type IIA Secretory Phospholipase A2 with the Glycosylphosphatidylinositol-anchored Heparan Sulfate Proteoglycan in the Cyclooxygenase-2-mediated Delayed Prostanoid-biosynthetic Pathway

An emerging body of evidence suggests that type IIA secretory phospholipase A2(sPLA2-IIA) participates in the amplification of the stimulus-induced cyclooxygenase (COX)-2-dependent delayed prostaglandin (PG)-biosynthetic response in several cell types. However, the biological importance of the ability of sPLA2-IIA to bind to heparan sulfate proteoglycan (HSPG) on cell surfaces has remained controversial. Here we show that glypican, a glycosylphosphatidylinositol (GPI)-anchored HSPG, acts as a physical and functional adaptor for sPLA2-IIA. sPLA2-IIA-dependent PGE2 generation by interleukin-1-stimulated cells was markedly attenuated by treatment of the cells with heparin, heparinase or GPI-specific phospholipase C, which solubilized the cell surface-associated sPLA2-IIA. Overexpression of glypican-1 increased the association of sPLA2-IIA with the cell membrane, and glypican-1 was coimmunoprecipitated by the antibody against sPLA2-IIA. Glypican-1 overexpression led to marked augmentation of sPLA2-IIA-mediated arachidonic acid release, PGE2 generation, and COX-2 induction in interleukin-1-stimulated cells, particularly when the sPLA2-IIA expression level was suboptimal. Immunofluorescent microscopic analyses of cytokine-stimulated cells revealed that sPLA2-IIA was present in the caveolae, a microdomain in which GPI-anchored proteins reside, and also appeared in the perinuclear area in proximity to COX-2. We therefore propose that a GPI-anchored HSPG glypican facilitates the trafficking of sPLA2-IIA into particular subcellular compartments, and arachidonic acid thus released from the compartments may link efficiently to the downstream COX-2-mediated PG biosynthesis.