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.

MicroRNAs (miRNAs) have been suggested to play important roles in cell proliferation, apoptosis, and differentiation. In this study, we examined miRNA expression profiles during 3T3-L1 pre-adipocyte differentiation. We constructed miRNA libraries from pre-and post-differentiated 3T3-L1 cells, and identified the expression of 77 previously reported miRNAs and 3 new miRNAs. Next, we investigated the expression levels of 102 miRNAs, including those identified in the libraries, during adipogenesis by Northern blot analysis. Sixty-five miRNAs were detected and the expression of 21 miRNAs was up-or downregulated during adipogenesis. Intriguingly, changes in the miRNA expression pattern were observed at day 9, when lipid droplets were visible, but not at days 1, 2, or 5 after the induction of differentiation. Antisense inhibition of the up-regulated miRNAs did not affect 3T3-L1 pre-adipocyte differentiation. Although these miRNAs may be involved in modulating adipocyte function, mild down-modulations of the up-regulated miRNAs do not appear to affect 3T3-L1 pre-adipocyte differentiation.

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Objective. Membrane-associated prostaglandin E synthase (mPGES) is a recently identified terminal enzyme of the arachidonic acid cascade, which converts PGH 2 to PGE 2 in rheumatoid arthritis synovial fibroblasts (RASFs). This study was undertaken to investigate factors regulating the expression of mPGES.Methods. RASFs were treated with interleukin-1␤ (IL-1␤), indomethacin, NS-398, rofecoxib, or meloxicam. The effects of PGE 2 and selective agonists for PGE 2 receptor subtypes (EP1, EP2, EP3, and EP4) were also studied. Expression of mPGES messenger RNA (mRNA) and protein was measured by Northern and Western blot analysis, respectively. EP receptor mRNA expression in RASFs was determined by reverse transcriptase-polymerase chain reaction. Production of PGE 2 and cAMP was measured by enzyme-linked immunosorbent assay.Results. The enhanced expression of mPGES mRNA and protein in IL-1␤-stimulated RASFs was attenuated by the addition of indomethacin, NS-398, rofecoxib, or meloxicam. This reduction of expression was reversed by PGE 2 . IL-1␤-induced PGES activity, measured by conversion of PGH 2 to PGE 2 , was decreased by rofecoxib. EP2 and EP4 receptor mRNA was detected in RASFs. EP2 and EP4 agonists, as well as PGE 2 , restored the inhibitory effect of rofecoxib on mPGES expression. The effect of PGE 2 was mimicked by forskolin, a direct activator of adenylate cyclase. Intracellular cAMP was increased by IL-1␤ and was inhibited by rofecoxib. Conclusion. Enhancement of mPGES expression by PGE 2 via the EP2/EP4 receptors with an increase in cAMP may play an important role in articular inflammation in patients with RA. It also seems that cyclooxygenase 2 (COX-2) inhibitors decrease PGE 2 production not only by direct inhibition of COX-2, but also by reducing mPGES expression in activated RASFs.Prostaglandin E synthase (PGES) catalyzes the final step of PGE 2 production, converting PGH 2 to PGE 2 . PGES exists downstream of cyclooxygenase (COX) (1,2). At least 2 forms of PGES, membraneassociated PGES (mPGES) (3-5) and cytosolic PGES (cPGES) (6), have recently been cloned and characterized. In cotransfection experiments, functional coupling between inducible mPGES and COX-2, as well as between constitutive cPGES and COX-1, has been demonstrated (4,6).Originally called microsomal glutathione S-transferase 1-like 1 (MGST1-L1), mPGES is a glutathione-dependent enzyme that shows coordinated induction with COX-2 in various cells and tissues by inflammatory stimuli (3,4). We recently showed that mPGES and COX-2 were induced by interleukin-1␤ (IL-1␤) on the perinuclear membrane in rheumatoid arthritis synovial fibroblasts (RASFs), causing an increase in PGE 2 production at sites of inflammation (7).

Microsomal prostaglandin E synthase-1 (mPGES-1) catalyzes the conversion of cyclooxygenase-derived prostaglandin (PG) H 2 to PGE 2 . Increased amounts of mPGES-1 were detected in inflamed intestinal mucosa from patients with inflammatory bowel disease (IBD) .