Inhibition of inducible prostaglandin E2 synthase by 15-deoxy-Δ12,14-prostaglandin J2 and polyunsaturated fatty acids

doi:10.1016/S0006-2952(02)00844-4

Biochemical Pharmacology

Volume 63, Issue 6, 15 March 2002, Pages 1183-1189

https://doi.org/10.1016/S0006-2952(02)00844-4 Get rights and content

Abstract

Prostaglandin E₂ synthase (PGE synthase) is one of the membrane-associated proteins in the eicosanoid and glutathione metabolism (MAPEG) family of microsomal enzymes and constitutes a novel inducible enzyme involved in inflammation and pyretic responses. We report, using a reversed-phase HPLC assay for the production of tritiated prostaglandin E₂ (PGE₂) by membranes from cells overexpressing human microsomal PGE synthase, that PGE synthase activity is inhibited effectively by 15-deoxy-Δ^12,14-prostaglandin J₂ and arachidonic acid. The anti-inflammatory compound 15-deoxy-PGJ₂ was considerably more potent at inhibiting PGE synthase ( $ic_{50} =0.3$ μM) than the closely related PGJ₂ or Δ¹²-PGJ₂, or the reaction product PGE₂. Arachidonic acid, docosahexaenoic acid, and eicosapentaenoic acid inhibited PGE synthase with a similar potency ( $ic_{50} =0.3$ μM) and were more potent inhibitors than various fatty acid analogues. The present results on the inducible PGE synthase extend observations on the ability to bind arachidonic acid to another member of the MAPEG family, and also suggest a novel mechanism of action for the anti-inflammatory effects of DHA, EPA, and 15-deoxy-PGJ₂.

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-Δ^12,14-prostaglandin J₂ (15-deoxy-PGJ₂).

Section snippets

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 [ $^{3} H$ ]-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 PGF_2α 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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Inhibition of inducible prostaglandin E2 synthase by 15-deoxy-Δ12,14-prostaglandin J2 and polyunsaturated fatty acids

Abstract

Introduction

Section snippets

Materials

Inhibitory effects of PUFAs on PGE synthase activity

Discussion

Acknowledgements

J. Biol. Chem.

Neurosci. Lett.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

Prostaglandins Other Lipid Mediat.

Cell

Biochem. Biophys. Res. Commun.

Prostaglandins as potentiators of increased vascular permeability in inflammation

Nature

The eicosanoids and their biochemical mechanisms of action

Biochem. J.

Characterization of a monoclonal antibody that neutralizes the activity of prostaglandin E2

J. Immunol.

Selective neutralization of prostaglandin E2 blocks inflammation, hyperalgesia, and interleukin 6 production in vivo

J. Exp. Med.

Impaired febrile response in mice lacking the prostaglandin E receptor subtype EP3

Nature

Identification of mu-class glutathione transferases M2-2 and M3-3 as cytosolic prostaglandin E synthases in the human brain

Neurochem. Res.

Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target

Proc. Natl. Acad. Sci. U.S.A.

Coordinate up- and down-regulation of glutathione-dependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells

Eur. J. Biochem.

Inhibition of inducible prostaglandin E₂ synthase by 15-deoxy-Δ^12,14-prostaglandin J₂ and polyunsaturated fatty acids

Characterization of a monoclonal antibody that neutralizes the activity of prostaglandin E₂

Selective neutralization of prostaglandin E₂ blocks inflammation, hyperalgesia, and interleukin 6 production in vivo

Impaired febrile response in mice lacking the prostaglandin E receptor subtype EP₃