Redirection of eicosanoid metabolism in mPGES-1-deficient macrophages

Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H-2 (PGH(2)) 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 E-2 (PGE(2)) > thromboxane B-2 (TxB(2)) > 6-keto prostaglandin F-1 alpha (PGF1 alpha), prostaglandin F-2 alpha (PGF2 alpha), and prostaglandin D-2 (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 PGF1 alpha and PGF2 alpha > 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 pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 mu M [H-3] arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [H-3] PGE2 formation from mPGES-1 -/- macrophages compared with controls resulted in TxB2 and 6-keto PGF1 alpha becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[H-3] 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.