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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