Inhibition of inducible prostaglandin E2 synthase by 15-deoxy-Δ12,14-prostaglandin J2 and polyunsaturated fatty acids
Introduction
PGE2 has been shown to be a potent mediator of pain and inflammation [1], [2], [3], [4] and has been implicated in the development of pyresis [5]. The synthesis of PGE2 involves either cyclooxygenase-1 or the inducible cyclooxygenase-2, which convert arachidonic acid into PGH2, followed by the conversion of PGH2 to PGE2 by cytosolic [6], [7] or membrane-associated [8], [9], [10], [11], [12] PGE synthase. A membrane-associated, glutathione-dependent PGE synthase [8], [9], [10], [11], [12] has been shown recently to be up-regulated in cell lines and in macrophages following proinflammatory stimuli [9], [10], [11]. Induction of PGE synthase RNA was also reported in astrocytes treated with β-amyloid [8]. The inducible PGE synthase was found to couple better with cyclooxygenase-2 than cyclooxygenase-1 for the production of PGE2 when co-transfected into mammalian cells [10]. In addition, the mRNA for the inducible PGE synthase was found to be increased in several rat tissues following the administration of lipopolysaccharide in vivo[10], [12]. A marked increase in the production of the inducible PGE synthase at both the RNA and protein levels has also been observed in the rat adjuvant arthritis model [12], providing further support for a role of this enzyme in inflammatory responses. Therefore, PGE synthase appears to represent a major enzyme involved in cyclooxygenase-2-mediated PGE2 production and constitutes a potential target for therapeutic intervention. Inducible PGE synthase has been identified to be a member of the recently identified MAPEG superfamily, which includes 5-lipoxygenase activating protein (FLAP) and LTC4 synthase [13]. The homology between the members of the MAPEG family is reinforced by evidence of pharmacological cross-reactivity between these proteins [14], [15], including the inhibition of LTC4 synthase [16] and of PGE synthase [12] by the FLAP inhibitor MK-886. In the present report, we show that the activity of the inducible PGE synthase is also sensitive to inhibition by arachidonic acid and 15-deoxy-Δ12,14-prostaglandin J2 (15-deoxy-PGJ2).
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Materials
Chinese hamster ovary (CHO) cells were obtained from the American Type Culture Collection. Cell culture medium, serum, antibiotics, and lipofectamine were purchased from Life Technologies. Restriction enzymes, ligase, and complete protease cocktail were obtained from Boehringer Mannheim. Tritiated []-PGH2 (20 μCi/100 μL), a partially purified PGE synthase antibody, arachidonic acid, 5,6-dihydro-arachidonic acid, eicosapentaenoic acid (EPA), 11,14-EDA, 15(S)-HETE, U-51605, U-44069, 5-trans
Inhibitory effects of PUFAs on PGE synthase activity
PGE synthase activity was quantitated using an assay based on the conversion of tritiated PGH2 to PGE2 and using stannous chloride to terminate the reaction so as to reduce unreacted PGH2 to PGF2α prior to HPLC analysis. Typical chromatograms of the resolution of the reaction products are shown in Fig. 1 for membrane preparations from mock-transfected cells (Fig. 1A) or from cells expressing the inducible PGE synthase (Fig. 1B). Fig. 1C shows that the addition of 10 μM arachidonic acid strongly
Discussion
The novel findings of this study are that PUFAs, including arachidonic acid, EPA, and docosahexaenoic acid (DHA), as well as the hydrophobic PG 15-deoxy-PGJ2, are potent inhibitors of the inducible microsomal PGE synthase with ic50 values in the submicromolar range. LTC4 synthase, FLAP, and PGE synthase are members of the MAPEG superfamily [13]. It has been established previously that FLAP lacks enzymatic activity yet binds to arachidonic acid, suggesting that FLAP functions as a lipid transfer
Acknowledgements
The authors would like to thank Kevin Clark for his assistance in preparing the figures for this manuscript. The authors also thank M. David Percival, Zheng Huang, and David Claveau for many helpful discussions. This work was supported, in part, by an industrial post-doctoral fellowship to Omar Quraishi by the Natural Sciences and Engineering Research Council of Canada (NSERC).
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