Redirection of eicosanoid metabolism in mPGES-1-deficient macrophages

Abstract

Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E syntheses involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1-/- animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2) > thromboxane B2 (TxB2) > 6-keto prostaglandin F1α (PGF1α), prostaglandin F2α (PGF2α), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1-/- macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2 > 6-keto PGF1α and PGF2α > PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pro-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 μM [ 3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1-/-macrophages compared with controls resulted in TxB2 and 6-keto PGF1α becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1-/- samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1-/-cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.