Thromboxane A synthase-independent production of 12-hydroxyheptadecatrienoic acid, a BLT2 ligand

12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) has long been considered a by-product of thromboxane A2 (TxA2) biosynthesis with no biological activity. Recently, we reported 12-HHT to be an endogenous ligand for BLT2, a low-affinity leukotriene B4 receptor. To delineate the biosynthetic pathway of 12-HHT, we established a method that enables us to quantify various eicosanoids and 12-HHT using LC-MS/MS analysis. During blood coagulation, 12-HHT levels increased in a time-dependent manner and were relatively higher than those of TxB2, a stable metabolite of TxA2. TxB2 production was almost completely inhibited by treatment with ozagrel, an inhibitor of TxA synthase (TxAS), while 12-HHT production was inhibited by 80–90%. Ozagrel-treated blood also exhibited accumulation of PGD2 and PGE2, possibly resulting from the shunting of PGH2 into synthetic pathways for these prostaglandins. In TxAS-deficient mice, TxB2 production during blood coagulation was completely lost, but 12-HHT production was reduced by 80–85%. HEK293 cells transiently expressing TxAS together with cyclooxygenase (COX)-1 or COX-2 produced both TxB2 and 12-HHT from arachidonic acid, while HEK293 cells expressing only COX-1 or COX-2 produced significant amounts of 12-HHT but no TxB2. These results clearly demonstrate that 12-HHT is produced by both TxAS-dependent and TxAS-independent pathways in vitro and in vivo. 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) has long been considered a by-product of thromboxane A2 (TxA2) biosynthesis with no biological activity. Recently, we reported 12-HHT to be an endogenous ligand for BLT2, a low-affinity leukotriene B4 receptor. To delineate the biosynthetic pathway of 12-HHT, we established a method that enables us to quantify various eicosanoids and 12-HHT using LC-MS/MS analysis. During blood coagulation, 12-HHT levels increased in a time-dependent manner and were relatively higher than those of TxB2, a stable metabolite of TxA2. TxB2 production was almost completely inhibited by treatment with ozagrel, an inhibitor of TxA synthase (TxAS), while 12-HHT production was inhibited by 80–90%. Ozagrel-treated blood also exhibited accumulation of PGD2 and PGE2, possibly resulting from the shunting of PGH2 into synthetic pathways for these prostaglandins. In TxAS-deficient mice, TxB2 production during blood coagulation was completely lost, but 12-HHT production was reduced by 80–85%. HEK293 cells transiently expressing TxAS together with cyclooxygenase (COX)-1 or COX-2 produced both TxB2 and 12-HHT from arachidonic acid, while HEK293 cells expressing only COX-1 or COX-2 produced significant amounts of 12-HHT but no TxB2. These results clearly demonstrate that 12-HHT is produced by both TxAS-dependent and TxAS-independent pathways in vitro and in vivo. 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) was identified as a product of prostaglandin H2 (PGH2), which is biosynthesized from arachidonic acid (AA). Until recently, 12-HHT has been considered merely a by-product of thromboxane A2 (TxA2) biosynthesis (1John H. Cammann K. Schlegel W. Development and review of radioimmunoassay of 12-S-hydroxyheptadecatrienoic acid.Prostaglandins Other Lipid Mediat. 1998; 56: 53-76Crossref PubMed Scopus (12) Google Scholar). The cleavage of PGH2 into 12-HHT and malondialdehyde (MDA) is catalyzed by thromboxane A synthase (TxAS), the enzyme responsible for TxA2 biosynthesis (2Smith W.L. Urade Y. Jakobsson P.J. Enzymes of the cyclooxygenase pathways of prostanoid biosynthesis.Chem. Rev. 2011; 111: 5821-5865Crossref PubMed Scopus (350) Google Scholar, 3Simmons D.L. Botting R.M. Hla T. Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition.Pharmacol. Rev. 2004; 56: 387-437Crossref PubMed Scopus (1336) Google Scholar). During aggregation of human and mouse platelets, 12-HHT and TxA2 are produced in a 1:1 ratio in larger amounts than other prostaglandins (PG) (4Vincent J.E. Zijlstra F.J. van Vliet H. Determination of the formation of thromboxane B2 (TxB2), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT) and 12L-hydroxy-5,8,10,14 eicosatrienoic acid (HETE) from arachidonic acid and of the TxB2:HHT, TxB2:HETE and (TxB2 +HHT):HETE ratio in human platelets. Possible use in diagnostic purposes.Prostaglandins Med. 1980; 5: 79-84Abstract Full Text PDF PubMed Scopus (14) Google Scholar, 5Frohberg P. Drutkowski G. Wobst I. Monitoring eicosanoid biosynthesis via lipoxygenase and cyclooxygenase pathways in human whole blood by single HPLC run.J. Pharm. Biomed. Anal. 2006; 41: 1317-1324Crossref PubMed Scopus (27) Google Scholar, 6Beving H. Eksborg S. Nordlander R. Olsson P. Olsson U. Effects on cyclo-oxygenase of low and high dose aspirin.Thromb. Res. 1990; 59: 227-235Abstract Full Text PDF PubMed Scopus (5) Google Scholar, 7Best L.C. Jones P.B. McGuire M.B. Russell R.G. Thromboxane B2 production and lipid peroxidation in human blood platelets.Adv. Prostaglandin Thromboxane Res. 1980; 6: 297-299PubMed Google Scholar, 8Greenwald J.E. Alexander M.S. Van Rollins M. Wong L.K. Bianchine J.R. Argentation thin layer chromatography of arachidonic acid metabolites isolated from human platelets.Prostaglandins. 1981; 21: 33-39Crossref PubMed Scopus (9) Google Scholar, 9Hall E.R. Tai H.H. Purification of thromboxane synthetase and evidence of two distinct mechanisms for the formation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid by porcine lung microsomes.Biochim. Biophys. Acta. 1981; 665: 498-503Crossref PubMed Scopus (22) Google Scholar, 10Stuart M.J. Allen J.B. Arachidonic acid metabolism in the neonatal platelet.Pediatrics. 1982; 69: 714-718Crossref PubMed Google Scholar, 11Sadowitz P.D. Setty B.N. Stuart M. The platelet cyclooxygenase metabolite 12-L-hydroxy-5, 8, 10-hepta-decatrienoic acid (HHT) may modulate primary hemostasis by stimulating prostacyclin production.Prostaglandins. 1987; 34: 749-763Crossref PubMed Scopus (15) Google Scholar, 12Sweeney F.J. Pereira M.J. Eskra J.D. Carty T.J. The use of 12-hydroxyheptadecatrienoic acid (HHT) as an HPLC/spectrophotometric marker for cyclooxygenase pathway activity in resident rat peritoneal cells.Prostaglandins Leukot. Med. 1987; 26: 171-177Abstract Full Text PDF PubMed Scopus (12) Google Scholar). Nonenzymatic conversion of PGH2 to 12-HHT in vitro has also been reported to occur via a process that involves glutathione and heme (13Shimizu T. Kondo K. Hayaishi O. Role of prostaglandin endoperoxides in the serum thiobarbituric acid reaction.Arch. Biochem. Biophys. 1981; 206: 271-276Crossref PubMed Scopus (104) Google Scholar). Although PGs and TxA2 mediate various physiological and pathophysiological roles in many tissues and cells through the activation of their specific G protein-coupled receptors (GPCR), the biological roles of 12-HHT have yet to be elucidated (1John H. Cammann K. Schlegel W. Development and review of radioimmunoassay of 12-S-hydroxyheptadecatrienoic acid.Prostaglandins Other Lipid Mediat. 1998; 56: 53-76Crossref PubMed Scopus (12) Google Scholar). In contrast, the biological roles of TxA2 have been studied in detail. TxA2 stimulates platelet aggregation and increases blood pressure by inducing the contraction of vascular smooth muscles through activation of the TxA2-specific GPCR, TP (14Sellers M.M. Stallone J.N. Sympathy for the devil: the role of thromboxane in the regulation of vascular tone and blood pressure.Am. J. Physiol. Heart Circ. Physiol. 2008; 294: H1978-H1986Crossref PubMed Scopus (68) Google Scholar). For these reasons, ozagrel, an inhibitor of TxAS, is used clinically as a therapeutic drug to improve brain blood circulation after brain hemorrhage (15Izumo T. Suzuki G. Chen Z. Fujii Y. Kamei C. Effects of certain cerebral circulation activating drugs on regional cerebral blood flow in rats.Methods Find. Exp. Clin. Pharmacol. 1999; 21: 279-283Crossref PubMed Scopus (4) Google Scholar). Recently, we discovered that 12-HHT functions as an endogenous ligand for BLT2, a low-affinity receptor for leukotriene B4 (16Okuno T. Iizuka Y. Okazaki H. Yokomizo T. Taguchi R. Shimizu T. 12(S)-Hydroxyheptadeca-5Z, 8E, 10E-trienoic acid is a natural ligand for leukotriene B4 receptor 2.J. Exp. Med. 2008; 205: 759-766Crossref PubMed Scopus (147) Google Scholar, 17Yokomizo T. Leukotriene B4 receptors: novel roles in immunological regulations.Adv. Enzyme Regul. 2011; 51: 59-64Crossref PubMed Scopus (64) Google Scholar). Although the physiological functions of the 12-HHT/BLT2 axis in vivo remain unclear, we reported that BLT2 plays a protective role in murine inflammatory colitis (18Iizuka Y. Okuno T. Saeki K. Uozaki H. Okada S. Misaka T. Sato T. Toh H. Fukayama M. Takeda N. et al.Protective role of the leukotriene B4 receptor BLT2 in murine inflammatory colitis.FASEB J. 2010; 24: 4678-4690Crossref PubMed Scopus (81) Google Scholar). To understand the physiological functions of 12-HHT and BLT2, the molecular mechanism of 12-HHT biosynthesis needs to be clarified. In this report, we show that 12-HHT is produced by TxAS-dependent and TxAS-independent pathways in vitro and in vivo using TxAS-deficient mice and various inhibitors of enzymes responsible for the biosynthesis 12-HHT. Arachidonic acid, 12-HHT, 12-HETE, LTB4, TxB2, PGD2, PGE2, PGH2, ozagrel, and rabbit anti-TxAS antibody (Ab) were purchased from Cayman Chemical (Ann Arbor, MI). Aspirin and thrombin were from Sigma. Goat anti-COX-1 (C-20) and goat anti-COX-2 (C-20) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-goat IgG-HRP was from Jackson ImmunoResearch Laboratories (West Grove, PA). TxAS-knockout (KO) mice were generated using a strategy similar to that described previously for the generation of PGI2 synthase-deficient mice (19Yokoyama C. Yabuki T. Shimonishi M. Wada M. Hatae T. Ohkawara S. Takeda J. Kinoshita T. Okabe M. Tanabe T. Prostacyclin-deficient mice develop ischemic renal disorders, including nephrosclerosis and renal infarction.Circulation. 2002; 106: 2397-2403Crossref PubMed Scopus (108) Google Scholar). A clone of the murine TxAS gene was isolated from the 129SVJ murine genomic library in the Lambda FIX II vector (Stratagene, La Jolla, CA) with murine TxAS cDNA used as a probe. The targeting vector (supplementary Fig. IV) was designed to replace the Xba I-Xho I fragment, including exons 12 and 13, with a gene conferring neomycin resistance. Kpn I-Xba I fragment containing exon 11 was inserted into Kpn I/Xba I site between hsv-thymidine kinase and the neomycin-resistant genes of pPTN plasmid. A 7 kb fragment downstream of exon 13 was inserted at the Xho I-Not I site, which is upstream of the neomycin resistance gene. The targeting vector was transfected into the R1 embryonic stem cells by electroporation, and successful homologous recombination was confirmed by Southern blot and PCR analyses (data not shown). Positive clones were injected into eight cell-stage embryos of C57BL/6J mice and implanted into pseudopregnant ICR mice. Chimeric mice were generated and backcrossed eight times with C57BL/6J (Clea Japan Inc., Japan). All animal studies and procedures were approved by the Ethics Committees for Animal Experiments of Kyushu University. Blood was collected from healthy volunteers who had been free of drugs for at least two weeks. Ethical approval was obtained from the Juntendo University Research Ethics Committee to use human blood. After incubation at 37°C for the indicated times, the blood was centrifuged at 5,000 g for 10 min to separate the serum A of serum was with of containing acid and a of which as To platelets, of human blood was collected into of was obtained by at g for 10 were isolated from by at g for 10 with 12 and in human in were for min at were by the of of thrombin in 12 at and by the of of containing acid and a of was used to from mouse whole blood as described by the was using II of cDNA was used to TxAS and using the for for and for min for a of or for for and for for a of The of the used are as TxAS, and and The were by on a blood was collected from the and at 37°C for and After at 5,000 g for 10 min at the was as 10 was with of containing acid and a of eicosanoids as The cDNA of human COX-1 and COX-2 were a from of The of COX-1 was by PCR using a and an with and and into the site of H. K. J. for with a novel PubMed Scopus Google Scholar). The of COX-2 was by PCR using a and an with and and into the site of The of TxAS C. Suzuki K. Y. T. S. R. Tanabe T. of human thromboxane synthase using a PubMed Scopus Google was into the site in of the of these enzymes were confirmed by embryonic cells were in containing serum and at 37°C in The cells were transfected with using to the HEK293 cells transfected with vector were with and 10 and centrifuged at g for 10 min at The were and were using a were in and were for min at in and to After with in the were with primary antibodies or TxAS in and with antibodies After with the were with a and an HEK293 cells were at a with of After the cells were with and with 10 arachidonic acid in of at The were by the of of containing acid and a of eicosanoids as HEK293 cells transfected TxAS were in of and for was at for After a min at PGH2 in of was to the to the The was at and by the of of containing A was by were with to a of and on The was with containing acid, containing acid, and containing The were with of containing was by LC-MS/MS as described previously Y. T. N. Shimizu T. A method for eicosanoids and using Biochem. PubMed Scopus Google Scholar). For LC-MS/MS a chromatography of a a a and a with an were of was injected into the an at a flow of after the was to the to the a Japan). of was by a using A and at of and The of A and were acid and acid The flow was the was at and the was into a All were in a 12-HHT and eicosanoids were by The were for 12-HHT, for TxB2, and for and For a of eicosanoids was used as the 12-HHT was not 12-HHT was using and were by the studies have that 12-HHT is produced during blood coagulation M. J. Prostaglandin A the of and of PubMed Scopus Google Scholar, M. J. T. and of two prostaglandin endoperoxides that platelet PubMed Scopus Google Scholar). To we collected human blood from healthy volunteers and 12-HHT and eicosanoid levels during blood coagulation I and is in Fig. The of 12-HHT in the serum increased in a time-dependent manner during blood coagulation and was higher than that of TxB2, a stable metabolite of TxA2 in two volunteers Fig. similar levels of production of 12-HHT and TxB2 (supplementary Fig. the of the on 12-HHT of human blood with almost completely inhibited 12-HHT production in volunteers and also the of and TxAS in 12-HHT production during blood of human whole blood with a inhibited 12-HHT and TxB2 production in a with a of 10 inducing almost of 12-HHT and TxB2 production of blood with ozagrel, an inhibitor of TxAS, also inhibited 12-HHT and TxB2 production in a Although 10 almost completely inhibited TxB2 12-HHT production was only inhibited treatment at 10 also increased the production of PGD2 and The of and were volunteers I and we production of 12-HHT and eicosanoids in human from two volunteers with similar results Fig. of COX-1 and TxAS in human was confirmed by (data not shown). treatment 12-HHT production in a time-dependent and the of 12-HHT was higher than that of TxB2 at after also in PGD2 and the amounts of PGD2 and were than those of 12-HHT and TxB2 of human with 10 almost completely inhibited TxB2 and 12-HHT production was inhibited by Fig. production of PGD2 and was increased by These results that two biosynthetic pathways of 12-HHT TxAS-dependent and are in human and that treatment increases PGD2 and production by shunting PGH2 to the PGD2 and synthetic the production of 12-HHT and eicosanoids during blood coagulation in TxAS-deficient mice. The of TxAS in mice was confirmed by of whole blood cells whole blood was collected from and TxAS-deficient mice, and 12-HHT and eicosanoid levels were in the In mouse the of 12-HHT and TxB2 increased in a time-dependent manner and of and after 10 min of incubation In the serum from mice, 12-HHT was after min of while no TxB2 was In and PGD2 levels in the serum of mice were higher than in the serum of mice. These results that 12-HHT is produced during blood coagulation in mice in manner similar to that in and These results also show that significant amounts of 12-HHT are biosynthesized in the blood in the of TxAS, and that PGH2 by TxAS is used for the production of PGD2 and To the of TxAS and to the production of 12-HHT, we 12-HHT and eicosanoid levels after arachidonic acid treatment of HEK293 cells transfected with the for COX-1 or COX-2 with or HEK293 cells not or of enzyme in transfected HEK293 cells was confirmed by 12-HHT production was in cells with or TxAS after the of 10 arachidonic acid while TxB2 production was only in cells expressing both COX-1 and TxAS production was in cells expressing COX-1 and of COX-1 and TxAS production A significant of production was which was not by of or TxAS Recently, et reported that HEK293 cells produced a significant of after incubation with 10 arachidonic acid Y. M. S. R. T. H. Taguchi R. K. K. et and of novel Full Text Full Text PDF PubMed Scopus Google that arachidonic acid is to in HEK293 possibly by endogenous that TxAS is for 12-HHT production but for TxB2 production in To TxAS-independent production of 12-HHT, HEK293 cells were with the of arachidonic In cells expressing both COX-1 and TxAS, with 10 only inhibited 12-HHT while TxB2 production was completely inhibited In 12-HHT production was not by with in cells expressing only COX-1 These results show that TxAS the production of 12-HHT from PGH2 produced by COX-1 and that 12-HHT is produced by TxAS-dependent and TxAS-independent similar using COX-2 and 12-HHT production was in cells with or TxAS after the of 10 arachidonic acid 12-HHT production in cells expressing COX-2 was higher than in cells expressing both COX-2 and TxAS TxB2 production was only in cells with COX-2 and TxAS and production was only in cells expressing COX-2 These results also that TxAS is not for 12-HHT production but is for TxB2 production in as is the for To TxAS-independent production of 12-HHT, HEK293 cells were with In cells expressing both COX-2 and TxAS, with 10 not 12-HHT production but completely inhibited TxB2 production with also increased that PGH2 was used for the production of Fig. production was not by the with ozagrel, that the pathway is of the These results that TxAS is for TxA2 production but not for 12-HHT To TxAS-independent 12-HHT we the conversion of PGH2 into TxB2 and 12-HHT using a of HEK293 cells of PGH2 with a of HEK293 cells that had been transfected with TxAS in the accumulation of this was completely inhibited by the In of cells not PGH2 into TxA2. These results show that TxAS is for the conversion of PGH2 to TxA2. contrast, conversion of PGH2 into 12-HHT was in the of this was not by treatment of PGH2 into 12-HHT was in the of cells than and this was to of PGH2 into and PGD2 was also in and this was not to treatment PGD2 and The conversion of PGH2 into and PGD2 was in cells and was increased by treatment to a similar to that in These results clearly show that TxAS is for the conversion of PGH2 into the conversion of PGH2 into 12-HHT, PGD2, and is possibly The reduced conversion of PGH2 into PGD2 and in cells be by the conversion of PGH2 into TxA2 by 12-HHT was identified by and as an product of arachidonic acid metabolism downstream of M. of by the of Full Text PDF PubMed Google Scholar). TxAS the conversion of PGH2 to 12-HHT, and in an TxA2 stimulates platelet aggregation and vascular through activating the TP a of the Jones S. of and of prostanoid of Rev. 2011; PubMed Scopus Google Scholar). TxA2 has also been to a role in of vascular smooth and regulation S. in roles by mice in their PubMed Scopus Google Scholar). the physiological roles and biosynthetic pathway of 12-HHT remain In this report, we show that 12-HHT is produced by both TxAS-dependent and TxAS-independent pathways in vitro and in vivo using Until the conversion of 12-HHT from PGH2 by heme or reduced glutathione had been reported only in an in vitro (13Shimizu T. Kondo K. Hayaishi O. Role of prostaglandin endoperoxides in the serum thiobarbituric acid reaction.Arch. Biochem. Biophys. 1981; 206: 271-276Crossref PubMed Scopus (104) Google Scholar, K. S. S. on prostaglandin with to production of acid Biophys. Res. 2008; PubMed Scopus Google Scholar). et reported the successful of TxAS-deficient mice with and et mice and resistance to 2004; PubMed Scopus Google and 12-HHT production by with arachidonic acid using chromatography Although reported that the of 12-HHT in was reduced but not completely lost, not this in detail. In this report, we used LC-MS/MS to quantify the levels of 12-HHT and various eicosanoids using TxAS-deficient mice. During blood coagulation, 12-HHT levels of and which are high to endogenous BLT2, a for 12-HHT (16Okuno T. Iizuka Y. Okazaki H. Yokomizo T. Taguchi R. Shimizu T. 12(S)-Hydroxyheptadeca-5Z, 8E, 10E-trienoic acid is a natural ligand for leukotriene B4 receptor 2.J. Exp. Med. 2008; 205: 759-766Crossref PubMed Scopus (147) Google Scholar). In the blood of and the levels of 12-HHT were higher than those of TxB2 Fig. and the of 12-HHT was to TxB2 in (supplementary Fig. These be to the levels of TxAS in platelets. In blood produced amounts of TxB2 and 12-HHT (supplementary Fig. than the blood of and Fig. and the of TxB2 and 12-HHT generation analysis. Although we not the of 12-HHT in is to that generated 12-HHT is on the of results are this is the to show TxAS-independent 12-HHT production using TxAS-deficient mice. Recently, et reported that is to and PGH2 to 12-HHT P. S. O. and 2011; PubMed Scopus Google Scholar). is to the other The other of this is the of and TxAS TxAS or treatment a in PGD2 and levels and and These results that in the or of TxAS, PGH2 produced from by is into and PGD2, possibly through is by the that PGH2 is to 12-HHT, PGD2, and through synthetic pathways in (data not and that the levels of other enzymes are low in and The of TxAS for 12-HHT generation to be in whole blood Fig. and than in and cells Fig. possibly TxAS is higher in than in transfected The other is of and TxAS in and In platelets, PGH2 generated by COX-1 is used for TxA2 and 12-HHT generation in a TxAS-dependent and TxAS in transiently transfected cells are PGH2 generated by be and to 12-HHT of The 12-HHT generation in cells expressing only is than in cells expressing both and TxAS Fig. possibly conversion of 12-HHT is than TxAS-dependent generation of 12-HHT. In to the of the of cells and cells a of 12-HHT possibly by generation of 12-HHT. cells PGD2 and than cells In PGH2 not be to and the PGH2 be to PGD2 and The use of TxAS inhibitors as an in that arachidonic metabolism PGI2 and other protective the therapeutic of these have been M. P. drugs in PubMed Scopus Google of TxAS also results in accumulation of PGH2, which TP receptors at higher et mice and resistance to 2004; PubMed Scopus Google Scholar). Although of TxAS and TP may be a treatment J. thromboxane A2 synthetase and thromboxane receptor in Clin. Res. Google the production of PGs in this with TxAS including and using LC-MS/MS be a to the in pathways by inhibitors of enzymes of the arachidonic In we that 12-HHT is produced by TxAS-independent pathway in to a TxAS-dependent pathway both in vitro and in vivo. from mice significant amounts of 12-HHT. and PGD2 production in mouse serum was higher than that in that PGH2 is into the synthetic pathways for and used LC-MS/MS to quantify 12-HHT and eicosanoid levels and the novel shunting of PGH2 from the TxA2 synthetic pathway to synthetic pathways the of The Y. T. and M. of for their in the LC-MS/MS the for and of Kyushu University for K. T. K. and Y. for and of for and with arachidonic acid leukotriene B4 receptor cyclooxygenase G protein-coupled receptor human embryonic 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid malondialdehyde prostaglandin thromboxane A2 thromboxane A synthase thromboxane B2