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 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 F1alpha (PGF1alpha), prostaglandin F(2alpha) (PGF2alpha), 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 PGF1alpha and PGF2alpha>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 microm [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 PGF1alpha 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.
Trebino
Catherine E
CE
Inflammation, Pfizer Global Research and Development, Groton Laboratories, Pfizer Inc., Groton, Connecticut 06340, USA.
Eskra
James D
JD
Wachtmann
Timothy S
TS
Perez
Jose R
JR
Carty
Thomas J
TJ
Audoly
Laurent P
LP
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D015777:Eicosanoids / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D005838:Genotype; D007093:Imidazoles / Q000494:pharmacology; D007249:Inflammation; D019746:Intramolecular Oxidoreductases / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008861:Microsomes / Q000378:metabolism; D000071876:Prostaglandin-E Synthases; D011453:Prostaglandins / Q000378:metabolism; D013864:Thioglycolates / Q000494:pharmacology; D013929:Thromboxane B2 / Q000494:pharmacology; D013997:Time Factors
D015777:Eicosanoids; D005231:Fatty Acids, Unsaturated; D007093:Imidazoles; D008070:Lipopolysaccharides; D011453:Prostaglandins; D013864:Thioglycolates; C029166:dazoxiben; D016718:Arachidonic Acid; C008033:12-hydroxy-5,8,10-heptadecatrienoic acid; D013929:Thromboxane B2; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; C000605865:Ptges protein, mouse