Involvement of Phospholipid Hydroperoxide Glutathione Peroxidase in the Modulation of Prostaglandin D2 Synthesis

Abstract

Antigenic cross-linking of the high affinity IgE receptors on mast cells induced the synthesis of prostaglandin D2 (PGD2). The production of PGD2 in L9 cells, which overexpressed non-mitochondrial phospholipid glutathione peroxidase (PHGPx), was only one-third that in the control line of cells (S1 cells). The reduction in the formation of PGD2 in L9 cells was reversed upon inhibition of PHGPx activity by buthionine sulfoximine. Experiments with inhibitors demonstrated that prostaglandin H synthase-2 (PGHS-2) was the isozyme responsible for the production of PGD2 upon cross-linking of IgE receptors. The conversion of radiolabeled arachidonic acid to prostaglandin H2 (PGH2) was strongly inhibited in L9 cells, whereas the rate of conversion of PGH2 to PGD2 was the same in L9 cells and S1 cells, indicating that PGHS was inactivated in L9 cells. The PGHS activity in L9 cells was about half that in S1 cells. However, PGHS activity in L9 cells increased to the level in S1 cells upon the addition of the hydroperoxide 15-hydroperoxyeicosatetraenoic acid or of 3-chloroperoxybenzoic acid. These results suggest that non-mitochondrial PHGPx might be involved in the inactivation of PGHS-2 in nucleus and endoplasmic reticulum via reductions in levels of the hydroperoxides that are required for full activation of PGHS. Therefore, it appears that PHGPx might function as a modulator of the production of prostanoids, in addition to its role as an antioxidant enzyme. Antigenic cross-linking of the high affinity IgE receptors on mast cells induced the synthesis of prostaglandin D2 (PGD2). The production of PGD2 in L9 cells, which overexpressed non-mitochondrial phospholipid glutathione peroxidase (PHGPx), was only one-third that in the control line of cells (S1 cells). The reduction in the formation of PGD2 in L9 cells was reversed upon inhibition of PHGPx activity by buthionine sulfoximine. Experiments with inhibitors demonstrated that prostaglandin H synthase-2 (PGHS-2) was the isozyme responsible for the production of PGD2 upon cross-linking of IgE receptors. The conversion of radiolabeled arachidonic acid to prostaglandin H2 (PGH2) was strongly inhibited in L9 cells, whereas the rate of conversion of PGH2 to PGD2 was the same in L9 cells and S1 cells, indicating that PGHS was inactivated in L9 cells. The PGHS activity in L9 cells was about half that in S1 cells. However, PGHS activity in L9 cells increased to the level in S1 cells upon the addition of the hydroperoxide 15-hydroperoxyeicosatetraenoic acid or of 3-chloroperoxybenzoic acid. These results suggest that non-mitochondrial PHGPx might be involved in the inactivation of PGHS-2 in nucleus and endoplasmic reticulum via reductions in levels of the hydroperoxides that are required for full activation of PGHS. Therefore, it appears that PHGPx might function as a modulator of the production of prostanoids, in addition to its role as an antioxidant enzyme. reactive oxygen species phospholipid hydroperoxide glutathione peroxidase classical glutathione peroxidase prostaglandin prostaglandin H synthase phospholipase A2 rat basophile leukemia cells immunoglobulin leukotriene dinitrophenyl human serum albumin phosphate-buffered saline Dulbecco's modified Eagle's medium fetal calf serum polyvinylidene difluoride Tris-buffered saline plus Tween 20 thin-layer chromatography buthionine sulfoximine hydroperoxyeicosatetraenoic acid 3-chloroperoxybenzoic acid 5,6-carboxy-2′,7′-dichlorofluorescein diacetate phosphatidylcholine hydroperoxide polyacrylamide gel electrophoresis Reactive oxygen species (ROS),1 such as the superoxide anion, hydrogen peroxide, and hydroxy radicals, are constantly formed in aerobic cells as a result of mitochondrial respiration and reactions catalyzed by enzymes such as NADH/NADPH oxidase, xanthine oxidase, monooxidases, and cyclooxygenase (1Cerutti P.A. 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Prostaglandins are important mediators both in host defense mechanisms and in inflammation. Their production is stimulated by low levels of ROS via the activation of prostaglandin H synthase (PGHS) and/or phospholipase A2 in intact cells (6Harlan J.M. Callahan K.S. J. Clin. Invest. 1984; 74: 442-448Crossref PubMed Scopus (102) Google Scholar, 7Marshall P.J. Lands W.E. J. Lab. Clin. Med. 1986; 108: 525-534PubMed Google Scholar, 8Sporn P.H. Peters G.M. Simon R.H. Am. Rev. Respir. Dis. 1988; 137: 49-56Crossref PubMed Scopus (36) Google Scholar, 9Taylor L. Menconi M.J. Polgar P. J. Biol. Chem. 1983; 258: 6855-6857Abstract Full Text PDF PubMed Google Scholar). Prostaglandin D2 (PGD2) is one of the major products formed in mast cells from arachidonic acid by PGHS in response to pharmacological or physiological stimulation, such as treatment with a calcium ionophore, oxidative stress, or cross-linking of IgE receptors (10Lewis R.A. Soter N.A. Diamond P.T. Austen K.F. Oates J.A. Roberts L. J. 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These sequential reactions are catalyzed by PGHS, the enzyme that is the primary enzymatic regulator of the biosynthesis of prostanoids (15Vane J. Nature. 1994; 367: 215-216Crossref PubMed Scopus (696) Google Scholar). The cyclooxygenase activity of PGHS requires reaction of PGHS peroxidase with hydroperoxide to generate a tyrosyl radical species, which is the active form of PGHS (16Wei C. Kulmacz R.J. Tsai A.L. 1995; PubMed Scopus Google Scholar, A.L. B. J.A. P.J. Kulmacz R.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Tsai A.L. P.J. Kulmacz R.J. PubMed Scopus Google Scholar). Thus, it that the cellular synthesis of prostaglandin is in by intracellular levels of hydroperoxide I. PubMed Scopus Google Scholar). levels of hydroperoxides be by enzymes such as glutathione of and are in and cells. is in the and is in H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the reduction of acid hydroperoxides and hydrogen glutathione as a PHGPx is in and mitochondrial H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, 1994; Scholar, H. H. Nakagawa Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and is the only enzyme that C. 1985; PubMed Scopus Google J. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and J. Biol. Chem. Full Text PDF PubMed Google in and for the of in reduction of the which is required for activation of the cyclooxygenase activity of PGHS, is it that of glutathione levels the production of prostaglandin in Am. J. Google Scholar). the which intracellular PGHS activity in I. PubMed Scopus Google Scholar). These results suggest that might the activity of PGHS in the cellular Therefore, or PHGPx might be responsible for the of the formation of are in the role of PHGPx in the of synthesis PHGPx with in which the synthesis of of rat basophile leukemia cells, in which a or a form of PHGPx was overexpressed H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. H. Nakagawa Y. Biochem. Res. PubMed Scopus Google Scholar). The of PHGPx in L9 cells, which overexpressed the form of was in the and as with that in cells (S1 the of PHGPx in mitochondrial from L9 and S1 cells The of PHGPx in the mitochondrial of cells, which overexpressed the form of was that in the same from L9 cells. These of cells a with which to to the of PHGPx to the synthesis of that overexpressed non-mitochondrial PHGPx to to oxidative damage H. H. Nakagawa Y. Biochem. Res. PubMed Scopus Google Scholar). production of catalyzed by was inhibited as a result of levels of intracellular hydroperoxides H. H. Nakagawa Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in cells that overexpressed mitochondrial the of intracellular hydroperoxides induced by mitochondrial damage was and the cells cells that overexpressed non-mitochondrial PHGPx to by damage to to oxidative H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google and to a of that induced the of H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the cells that overexpressed non-mitochondrial or mitochondrial PHGPx to the of of PHGPx in the formation of cells PGD2 in response to by cross-linking of the high affinity immunoglobulin receptors or to calcium L. Am. J. 1995; Google Scholar, PubMed Scopus (21) Google Scholar). that non-mitochondrial PHGPx the production of PGD2 via of in levels of hydroperoxides in the nucleus and endoplasmic in from rat a from 5,6-carboxy-2′,7′-dichlorofluorescein diacetate and from acid was from serum albumin and from IgE dinitrophenyl was from PGH2 was from the control line of cells (S1 L9 and cells, which overexpressed non-mitochondrial PHGPx H. H. Nakagawa Y. Biochem. Res. PubMed Scopus Google and cells, which overexpressed mitochondrial PHGPx H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). These of rat basophile leukemia cells in a of and in Dulbecco's modified Eagle's medium with fetal calf serum and cells in with and for of to medium cells cells a of and for with phosphate-buffered saline cells with a of cells in of that for with cross-linking was by with for The medium and for to of cells. for of the with as H. Kitahara J. Nakagawa Y. J. Biochem. (Tokyo ). 1999; 125: 90-95Crossref PubMed Scopus (8) Google Scholar, H. H. Nakagawa Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells in that and for and with for for for enzyme cells by cross-linking IgE as by sequential treatment with and for with and with for The cells with and in with a from cells by on a polyacrylamide as by Nature. 227: PubMed Scopus Google and to a polyvinylidene difluoride membrane for in and in a protein as H. Kitahara J. Nakagawa Y. J. Biochem. (Tokyo ). 1999; 125: 90-95Crossref PubMed Scopus (8) Google Scholar). The membrane with the was for by with in that and Tween 20 The membrane was for with or PGHS-2 that with to an the membrane with it was with or The of to on the membrane was with an cells by cross-linking IgE receptors by sequential treatment with and with and for The in of and that and and in a and by to the H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). from for cells with acid for cells with that serum The cells with a of and receptors by the addition of cells on and the medium was by of with of to the medium for of was to and the was in a of a of and and to a The was with of a of and as by and B. J. Res. Full Text PDF PubMed Google Scholar). The rate of production of was from results of with a cells with for and with for of the cells with and in that and 20 acid for The was and with of The of arachidonic acid in the The was to and the was in a of a of and and to a The was as by and B. J. Res. Full Text PDF PubMed Google Scholar). The rate of production of was from results of with the The rate of production of PGD2 from prostaglandin as cells, which stimulated by cross-linking of IgE in that and prostaglandin H2 for The medium was and the level of PGD2 was by enzyme cells by cross-linking IgE receptors by sequential treatment with and cells with and with a The cells in and that and and in a The enzymatic reaction was in of that 20 and of protein in a for addition of acid The reaction was by addition of a of and acid with for The was to a The was with a of and acid The products by with from results of with the in of and The was and the was for PHGPx activity was by phosphatidylcholine hydroperoxide as the to H. H. Nakagawa Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). an 5,6-carboxy-2′,7′-dichlorofluorescein diacetate to levels of intracellular sequential treatment with and cells with and with in for was to the the cells, and was oxidized to the by The of in cells was with a of with with serum albumin as the The production of was in cells (S1 and in cells that overexpressed non-mitochondrial PHGPx by with arachidonic acid S1 cells prostaglandin D2 (PGD2) upon activation with whereas production of of prostaglandin was of PGD2 by enzyme treatment of cells with cells PGD2 to a level of cells. The level of PGD2 in S1 cells by was that in cells. The production of PGD2 was inhibited by the of non-mitochondrial PHGPx L9 and and of S1 cells that the level of a and the level of PGD2 synthesis of PGD2 in L9 cells was for with These results that non-mitochondrial PHGPx might be involved in of the formation of PGD2 by cells in response to of the of prostaglandin D2 by a control line of cells and by cells that overexpressed non-mitochondrial cells and L9 cells by cross-linking IgE receptors and by sequential treatment with and S1 cells and L9 cells for with and with for the and PGD2 was to the of buthionine sulfoximine on the production of PGD2 in to or of the production of PGD2 was to the of PHGPx the activity of by the level of glutathione in cells. the level of glutathione to about of that in cells H. Kitahara J. Nakagawa Y. J. Biochem. (Tokyo ). 1999; 125: 90-95Crossref PubMed Scopus (8) Google Scholar). The production of PGD2 in S1 cells was by treatment with However, treatment with a in the production of PGD2 in L9 cells in response to activation by The of formation of PGD2 in L9 cells was upon inhibition of PHGPx and the level of PGD2 to that in S1 cells. These results that the reduction in the rate of production of PGD2 was to the of PHGPx activity in of buthionine sulfoximine on the production of prostaglandin D2 by a control line of cells and by cells that overexpressed non-mitochondrial cells and L9 cells for with or buthionine sulfoximine with for and with for of PGD2 the to for in a cells and L9 cells for with or buthionine sulfoximine with for and with for of PGD2 the to for S1 cells in for cells). The activity of PHGPx in cells was increased by the with of PHGPx in of S1 and cells and The production of PGD2 in cells with was cells, one-third of that in S1 cells. to the PGHS isozyme responsible for production of PGD2 in cells inhibitors of PGHS The production of PGD2 in response to was by treatment with a of production of PGD2 was treatment with which is a of both and in the release of arachidonic acid was cells and cells with the inhibitors of and PGHS-2 was by with and of PGHS in L9 cells the same levels as in S1 cells. of was both in S1 and L9 cells by the of PGHS-2 was in both S1 and L9 cells by the with was in the of the in the level of S1 and L9 cells. of PGHS-2 in response to activation by was to of intracellular glutathione by treatment with These results that PGHS-2 was responsible for the production of PGD2 in cells that by cross-linking of IgE receptors. the of and and PGHS-2 in both and PGHS-2 was in as with whereas of in was to that in The production of PGD2 was addition of arachidonic acid or prostaglandin H2 (PGH2) to the medium in an to the in the synthesis of PGD2 in L9 cells inhibition The rate of formation of PGD2 from acid was of PGD2 from arachidonic acid was in S1 cells the with of the production of PGD2 was in L9 cells the same treatment with L9 cells to as PGD2 as S1 cells. conversion of PGH2 to catalyzed by PGD2 was in S1 or in L9 cells with and in of the of the conversion was S1 and L9 cells These results that the activity of PGD2 synthase was in L9 cells and S1 cells. PGHS-2 activity was to the of the activation of PGHS-2 in the of production of PGD2 in L9 cells. the of of PHGPx on PGHS the activity of PGHS in a from cells and from cells with arachidonic and the of was of in of PGHS activity S1 cells and L9 cells. by an in PGHS activity in the from S1 cells, such activation of PGHS was in the from L9 cells. the of hydroperoxides on the activity of PGHS in an to inhibition of the activity of PGHS might to levels of hydroperoxides in cells PGHS activity in from cells, in the of of the acid hydroperoxide or of 3-chloroperoxybenzoic acid both of which as Tsai A.L. Kulmacz R.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Kulmacz R.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of form of in from S1 and L9 cells and However, was to the of PGHS which the form of and was These results that the level of in to the activity of PGHS by the The with or in the on hydroperoxides L9 cells and S1 cells. the of S1 cells, the PGHS activity increased with in the of and it in the of L9 cells, PGHS was as the level of was with activity These results that L9 cells required S1 cells for activation of PGHS. with the PGHS activity increased with in the of indicating that the PGHS. The activity and in S1 and L9 cells, activity in a control line of cells and in cells that overexpressed non-mitochondrial from control cells and L9 cells that with for and with for The for PGHS activity as in the are as to the for S1 cells and are as of results from in a of on PGHS activity in cells. cells and L9 cells with for and with for with with a and The PGHS activity in the was in the of of and from control cells and L9 cells that with for and with for The for PGHS activity as in the are as to the for S1 cells and are as of results from The levels of in S1 and L9 cells by the diacetate with in S1 cells was that in stimulated L9 cells. These results that the levels of in L9 cells in S1 cells with The of with of L9 cells that with was the same as that in S1 cells with These results that the of L9 cells to increased the level of intracellular PHGPx is overexpressed in the of cells as a result of with a that PHGPx H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the production of PGD2 in cells in to the of mitochondrial PHGPx on the production of PGD2 to that in L9 cells, the formation of PGD2 was only in cells that with and strongly induced the production of PGD2 in S1 cells whereas production of PGD2 was in L9 cells in cells. The of non-mitochondrial PHGPx inhibited the production of PGD2 in response strongly that of mitochondrial These results that PHGPx in in of the synthesis of cells activity and release of leukotriene upon activation by the production of by L9 cells a with and that the production of was in L9 cells. However, the production of by cells was to that by S1 cells These results that mitochondrial PHGPx was involved in the of prostaglandin synthesis in the of leukotriene the the of of non-mitochondrial PHGPx in cells, which PGD2 with of products such as and in response to or PubMed Scopus (21) Google Scholar). The of non-mitochondrial PHGPx in cells the of the production of PGD2 in response to by IgE of PGHS are is in cells. from cells, is in of upon treatment with a of and H.R. PubMed Scopus Google Scholar). The cells in the and PGHS-2 in of the levels of of and induced PGHS-2 the control line of cells (S1 and cells that overexpressed non-mitochondrial L9 cells is that both and PGHS-2 by of in human and in human PGHS-2 C. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, H. B.R. J. P. 1995; PubMed Scopus Google Scholar). that the production of PGD2 was both by and by an of which with the active of PGHS-2 with that of I. 1994; PubMed Scopus Google that PGHS-2 was responsible for the production of PGD2 in cells upon by cells, PGH2 is to the form of PGD2 which requires glutathione as a for its PGH2 was to PGD2 the same rate in both S1 and L9 cells the production of PGD2 from arachidonic acid was in L9 cells the activity of PGHS was in L9 cells with These results that inhibition of the synthesis of PGD2 was to inhibition of the of PGH2 to PGD2 was to inhibition of the production of PGH2 from arachidonic acid by in phospholipase A2 activity was involved in the of PGD2 the of release of arachidonic acid by S1 cells and L9 cells the same is a that the cyclooxygenase activity of PGHS requires reaction of the peroxidase with hydroperoxide (16Wei C. Kulmacz R.J. Tsai A.L. 1995; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). activity was to about in the of PGHS-2 and in the of R.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). hydroperoxides by to generate the and a which is with the from the of oxidized species, a tyrosyl radical from the is as the active species that hydrogen from arachidonic acid to the cyclooxygenase reaction A.L. B. J.A. P.J. Kulmacz R.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Tsai A.L. P.J. Kulmacz R.J. PubMed Scopus Google Scholar). Therefore, the of hydroperoxides on the production of in an to inhibition of the activity of PGHS was to levels of hydroperoxides in cells. The low level activity of PGHS in L9 cells was increased upon treatment of cells with the and The activity of PGHS was and in S1 cells and L9 cells, and the activity was the same in the PGHS, and enzymatic activity a and in S1 cells and L9 cells, L9 cells or S1 cells for PGHS indicating that the intracellular level of hydroperoxides was in cells. low level of hydroperoxides in cells was demonstrated in L9 cells, as with S1 cells, intracellular hydroperoxides by H. H. Nakagawa Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with to in levels of intracellular with The in the levels of intracellular induced by was by of non-mitochondrial by PHGPx was by the addition of results that non-mitochondrial PHGPx was responsible for the reduction of lipid hydroperoxides and of the activity of PGHS in cells. that levels of intracellular hydroperoxides are by of intracellular of the and levels of PHGPx and in S1 and L9 cells H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. H. Nakagawa Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The levels of in S1 and L9 cells the of to PHGPx was in S1 cells and only in L9 cells H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). and PGHS-2 in the and endoplasmic reticulum I. M.K. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google and are in the and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PGHS-2 in the the result that the synthesis of PGH2 upon by the to which phospholipase A2 and from the J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, R. R. J. Biol. Chem. Full Text PDF PubMed Google PGHS in endoplasmic reticulum PGH2 in stimulated cells. are a major physiological of which be mitochondrial The ROS in of membrane lipids the of such as acid hydroperoxides and hydrogen PHGPx is in and mitochondrial of rat C. C. Biochem. Med. PubMed Scopus Google Scholar). a demonstrated that of mitochondrial of non-mitochondrial that via the mitochondrial cells to the of by of or by treatment with or H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). However, of PHGPx in the mitochondrial induced of the production of PGD2 in cells that stimulated by or The of PHGPx in the from L9 cells was and that from S1 and cells, the of PHGPx in the mitochondrial from L9 and from S1 cells However, the of PHGPx in the mitochondrial from cells was that from S1 and L9 cells, and the of PHGPx in the from L9 and cells the same level H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). PHGPx that a be Therefore, it is that PHGPx in and of cells be and the to and to full activity of These results that inactivation of PGHS-2 was to high level of PHGPx in the nucleus and endoplasmic of non-mitochondrial PHGPx in and of L9 cells was same as that in S1 cells H. Nakagawa Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The activity of PHGPx in cells that in medium was that in S1 cells, and production of PGD2 with in cells was one-third of that in S1 cells. These suggest that the PHGPx the production of the formation of from arachidonic acid the R. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Peters G.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). conversion requires of protein which is to the and arachidonic acid to R.A. P.J. Nature. PubMed Scopus Google Scholar). The of non-mitochondrial PHGPx the production of cells that stimulated with H. H. Nakagawa Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of mitochondrial PHGPx on the rate of production of Thus, PHGPx in the in might be for of the level of hydroperoxides required for the activation of results suggest that PHGPx in the nucleus might an important role in of the of arachidonic the synthesis of and the synthesis of prostanoids via the reduction of lipid hydroperoxides that from mitochondrial activity and J.A. J. Biochem. Res. 1999; PubMed Scopus Google Scholar). Thus, of arachidonic acid by PGHS to in addition to as The that non-mitochondrial PHGPx might be involved in the of cellular and via of the production of and for