Here we report the molecular identification of membrane-bound glutathione (GSH)-dependent prostaglandin (PG) E 2 synthase (mPGES), a terminal enzyme of the cyclooxygenase (COX)-2-mediated PGE 2 biosynthetic pathway. The activity of mPGES was increased markedly in macrophages and osteoblasts following proinflammatory stimuli. cDNA for mouse and rat mPGESs encoded functional proteins that showed high homology with the human ortholog (microsomal glutathione S-transferase-like 1). mPGES expression was markedly induced by proinflammatory stimuli in various tissues and cells and was down-regulated by dexamethasone, accompanied by changes in COX-2 expression and delayed PGE 2 generation. Arg 110 , a residue well conserved in the microsomal GSH S-transferase family, was essential for catalytic function. mPGES was functionally coupled with COX-2 in marked preference to COX-1, particularly when the supply of arachidonic acid was limited. Increased supply of arachidonic acid by explosive activation of cytosolic phospholipase A 2 allowed mPGES to be coupled with COX-1. mPGES colocalized with both COX isozymes in the perinuclear envelope. Moreover, cells stably cotransfected with COX-2 and mPGES grew faster, were highly aggregated, and exhibited aberrant morphology. Thus, COX-2 and mPGES are essential components for delayed PGE 2 biosynthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer.

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Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E 2 synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE 2 biosynthetic pathway. GSHdependent 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 (Tyr 9 ), which is known to be critical for the activity of cytosolic GSH S-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 PGE 2 from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A 2 in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE 2 that plays a role in maintenance of tissue homeostasis. Biosynthesis of prostaglandin (PG)1 E 2 , the most common prostanoid with potent bioactivities, is regulated by three sequential steps of the cyclooxygenase (COX) pathway. Phospholipase A 2 (PLA 2 ) initiates this pathway by releasing arachidonic acid (AA) from membrane glycerophospholipids. Of more than 10 members of the PLA 2 family characterized to date, cytosolic PLA 2 (cPLA 2 ) and several secretory PLA 2 s are involved in supplying AA to either of the two COX isozymes, COX-1 and COX-2, depending upon the phases of cell activation (1-3). The constitutive COX-1 is mainly utilized in immediate PGE 2 biosynthesis, which occurs within several minutes after stimulation with Ca 2ϩ mobilizers, whereas the inducible COX-2 mediates the delayed PGE 2 biosynthesis, which lasts for several hours following proinflammatory stimuli. Although COX-1 and COX-2 have been reported to exhibit subtle differences in AA requirements in that COX-2 is favored over COX-1 at low AA concentrations (3-5) and subcellular localizations (6), their functional segregation in the PGE 2 biosynthetic response cannot be fully explained only by these aspects.The activity of PGES, which catalyzes conversion of COXderived PGH 2 to PGE 2 , has been detected in both cytosolic and microsomal fractions of various cells, and in most, if not all, cases it requires glutathione (GSH) for optimal activity (7-9). Although several groups have attempted to purify this critical enzyme to near homogeneity for the last 20 years (7-9), such trials have been unsuccessful. The PGES enzyme purified from human brain cytosol was identified as a GSH S-transferase (GST), which converts PGH 2 to PGE ...

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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 (PGs) and leukotrienes (LTs). 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 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, host defense, and atherosclerosis. The cytosolic PLA2 (cPLA2) family consists of 3 enzymes, among which cPLA2alpha plays an essential role in the initiation of AA metabolism. Intracellular activation of cPLA2alpha is tightly regulated by Ca2+ and phosphorylation. The Ca2+-independent PLA2 (iPLA2) family contains 2 enzymes and may play a major role in membrane phospholipid remodeling. The platelet-activating factor (PAF) acetylhydrolase (PAF-AH) family represents a unique group of PLA2 that contains 4 enzymes exhibiting unusual substrate specificity toward PAF and/or oxidized phospholipids. In this review, we will overview current understanding of the properties and functions of each enzyme belonging to the sPLA2, cPLA2, and iPLA2 families, which have been implicated in signal transduction.

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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 PGE 2 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 PGE 2 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 PGE 2 involved in both tissue homeostasis and disease.Biosynthesis of prostaglandin (PG) 1 E 2 , which is produced by a variety of cells and tissues and exhibits diverse bioactivities, is mediated by three enzymatic reactions involving phospholipase A 2 (PLA 2 ), cyclooxygenase (COX), and PGE synthase (PGES). In this biosynthetic pathway, arachidonic acid (AA) released from membrane phospholipids by cytosolic or secretory PLA 2 s is converted to PGH 2 by COX-1 or COX-2 and is then isomerized to PGE 2 by terminal PGES enzymes.The constitutive COX-1 mainly promotes immediate PG production elicited by agonists promptly mobilizing intracellular Ca 2ϩ , a situation in which a burst release of AA occurs (1-5).The inducible COX-2 is essential for delayed PG generation induced by proinflammatory stimuli, during which AA is gradually supplied over long periods, and also promotes immediate PG production if it already exists in cells primed by particular stimuli (1-5). Current studies employing isozyme-specific inhibitors and knockout mice have revealed that the two COXs play distinct roles in vivo (6 -10), and segregated utilization of these enzymes at the cellular level has been explained not only by their distinct expression profiles but also by subtle differences in their AA requirement, hydroperoxide sensitivity, and subcellular localization (4, 11-13). In addition, selective coupling with various terminal PG synthases has also been shown to influence crucially the utilization of the two COX isoforms during the different phases of cell activation (14 -16). PGES enzymes, which lie downstream of COXs, occur in multiple forms in mammalian cells (1). Among them, a perinuclear membrane-bound form of PGES belonging to the MAPEG (for membrane-associated proteins involved in eicosanoid and glutathione metabolism) family, which we herein cal...

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