Redirection of Eicosanoid Metabolism in mPGES-1-deficient Macrophages

Abstract

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 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 pre-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. 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 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 pre-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. Modulation of prostaglandin metabolism is at the center of current anti-inflammatory therapies. Non-steroidal anti-inflammatory drugs and aspirin-related drugs block the activity of cyclooxygenases and their ability to convert arachidonic acid (AA) 1The abbreviations used are: AA, arachidonic acid; mPGES, microsomal prostaglandin E synthase; PGE2, prostaglandin E2; PGH2, prostaglandin H2; PGF1α, prostaglandin F1α; PGF2α, prostaglandin F2α; PGD2, prostaglandin D2; PGI2, prostaglandin I2; TxB2, thromboxane B2; TxA2, thromboxane A2; LPS, lipopolysaccharide; HPLC, high pressure liquid chromatography; Cox, cyclooxygenase; TxAS, thromboxane synthase; EIA, enzyme immunoassay; PGIS, prostaglandin I synthase; PBS, phosphate-buffered saline; HHT, 12-hydroxyheptadecatrienoic acid. into PGH2. PGH2 can be subsequently metabolized by terminal prostaglandin synthases to the corresponding biologically active PGs, namely, PGI2, thromboxane A2, PGD2, PGF2α, and PGE2. 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We that the absence of mPGES-1 expression in macrophages be by to prostaglandins PGE2. murine macrophages isolated from wild type and mPGES-1-deficient animals were to diverse inhibitors pro-inflammatory stimuli. expression of and mPGES-1–/– cells in of prostaglandin metabolic was of mPGES-1 in the absence of mPGES-1 results suggest that PGH2 metabolism can be to biologically active prostaglandins PGE2, that the mPGES-1 enzyme is a release of PGH2 of and mice were as (3Trebino C.E. Stock J.L. Gibbons C.P. Naiman B.N. Wachtmann T.S. Umland J.P. Pandher K. Lapointe J-M. Saha S. Roach M.L. Carter D. Thomas N.A. Durtschi B.A. McNeish J.D. Hambor J.E. Jakobsson P.-J. Carty T.J. Perez J.R. Audoly L.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9044-9049Crossref PubMed Scopus (510) Google Scholar). animals used in were an were by the and and with of were then and as J.M. Wicks J.R. Audoly L.P. J. Clin. Investig. 2002; 110: 651-658Crossref PubMed Scopus (346) Google Scholar). 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The following were used for of prostaglandin antibody antibody PGIS, antibody and TxAS, antibody Eicosanoid were for PGE2, 6-keto PGF1α of TxB2 of PGF2α, and by to the The of by was Eicosanoid macrophages were to at a of in of for was and the cells were for were with and of 10 μm arachidonic acid and of [3H]arachidonic acid was subsequently for a of at The were and to of the phase of the was then a of eicosanoid of 6-keto PGF1α, TxB2, PGF2α, PGE2, and of and and of was to of in a at for the was to a with of acid in the was then a with of followed by of and of The and were then with of and with of followed by of resulting in a in was then using a The of phase was using a to and the with of acid and using a into the with at a of A to at a of was used to the The was with at and The of the of and of the were from to of of was in of and the was using a were from are as the were by of with a of PGE2 in of wild type macrophages with a increase in the release of PGE2 into the cellular the of and a at the In contrast, mPGES-1–/– cells significant increase in PGE2 formation at of in mPGES-1–/– the of PGE2 we to further profile prostaglandin formation were isolated and for PGE2, 6-keto PGF1α of TxB2 of PGF2α, and No significant was detected between and mPGES-1–/– from cells with In contrast, incubation with a significant increase in in the isolated from wild type with PGE2 were by in isolated from mPGES-1–/– TxB2 the most prostaglandin in mPGES-1–/– samples. The order of prostaglandin abundance in mPGES-1–/– was as TxB2 > 6-keto PGF1α > PGF2α > > PGE2. In contrast, in was as PGE2 > TxB2 > 6-keto PGF1α > PGF2α > We detected an increase in the of TxB2, 6-keto PGF1α, PGF2α, and in mPGES-1–/– to characterize the for altered pattern of we the expression of the terminal prostaglandin synthases and cyclooxygenases isoforms for the formation of the most abundant from and macrophages with in a of to the that have to of prostaglandin we profiled the induction of PGIS, and by were detected at of the mPGES-1 resulted in a significant of mPGES-1 expression Importantly, the absence of mPGES-1 expression in significant of PGIS, or as by data for and of macrophages with or have a significant or in or mPGES-1–/– samples. In contrast, expression of mPGES-1 and Cox-2 in was several at multiple following incubation The pattern of Cox-2 expression between and mPGES-1–/– with the absence of at the expression both mPGES-1 and Cox-2 are in response to an inflammatory their of expression expression of Cox-2 at and to by In contrast, mPGES-1 expression a an at and a at The the pattern of expression of these two at mPGES-1 is to the of and Cox-2 have to of during an suggest that murine macrophages their prostaglandin production to and to a and in the absence of mPGES-1 further the role of mPGES-1 in inflammatory cells were to 10 μm from to The 10 of were with the of production with PGE2 production in mPGES-1–/– was compared with wild type mPGES-1–/– and significant of TxB2 and 6-keto PGF1α and the 10 of of both of these prostaglandins were detected in mPGES-1–/– both 10 further profile the of mPGES-1 eicosanoid were using a incubation by treatment with The profile the reduction in PGE2 production by in 6-keto PGF1α, TxB2, and PGF2α A and were altered in mPGES-1–/– samples. Our results suggest that a that with was in mPGES-1–/– by to wild type samples. results that the absence of mPGES-1 can be by metabolic prostaglandins PGE2, and HHT, at the cellular of of a the of following of the prostaglandin we the of a prostaglandin type and mPGES-1–/– macrophages with of the thromboxane synthase were to 10 μm arachidonic acid following incubation with The were for cells of the TxB2 were in mPGES-1–/– compared with a corresponding increase in PGE2 The for the TxB2 and PGE2 were and results suggest that or can of prostaglandin metabolism in of murine macrophages with or a combination of and a in prostaglandins released into the with PGE2 the most abundant The induction of Cox-2 and mPGES-1 expression with the release of PGE2. also the specific increase in microsomal PGE synthase activity (3Trebino C.E. Stock J.L. Gibbons C.P. Naiman B.N. Wachtmann T.S. Umland J.P. Pandher K. Lapointe J-M. Saha S. Roach M.L. Carter D. Thomas N.A. Durtschi B.A. McNeish J.D. 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In that the absence or of a PGH2 metabolic enzyme results in an altered metabolic profile in murine macrophages. of key metabolic enzymes support the that the of mPGES-1-deficient macrophages is in to in the expression profile of prostaglandin metabolic data that PGH2 metabolism a major metabolic for PGH2, terminal prostaglandin synthases have the to the PGH2 by Importantly, formation of PGE2 is a significant The combination of and approaches in these further the importance of mPGES-1 in PGE2 synthesis and provides insights into the contribution of mPGES-1 to PGH2 metabolism.