title: Overexpression of human prostaglandin G/H synthase-1 and -2 by recombinant vaccinia virus: inhibition by nonsteroidal anti-inflammatory drugs and biosynthesis of 15-hydroxyeicosatetraenoic acid.
abstract: Human prostaglandin G/H synthase (hPGHS)-1 and hPGHS-2, key enzymes in the formation of prostanoids from arachidonic acid, were expressed at high levels in COS-7 cells using a T7 RNA polymerase/vaccinia virus expression system. The open reading frame of hPGHS-2 cloned into vaccinia virus without its natural 5' and 3' untranslated regions directed only low levels of hPGHS-2 enzyme activity in COS-7 cells. High-level hPGHS-2 expression was achieved by appending the 3' untranslated region of hPGHS-1 to the hPGHS-2 open reading frame, with subsequent expression of the hybrid mRNA using vaccinia virus. Enzymatically active recombinant hPGHS-1 and hPGHS-2 were present as glycosylated proteins in the microsomal fraction prepared from infected cells, whereas recombinant hPGHS-1 and hPGHS-2 prepared from the microsomal fraction of cells treated with tunicamycin, an inhibitor of N-linked glycosylation, were enzymatically inactive. The major prostanoid products formed by microsomes from COS-7 cells containing either recombinant hPGHS-1 or hPGHS-2 after incubation with arachidonic acid were prostaglandin D2 and E2, with lower levels of prostaglandin F2 alpha and 6-keto-prostaglandin F1 alpha. A range of potencies were observed for various nonsteroidal anti-inflammatory drugs as inhibitors of prostaglandin E2 synthesis by hPGHS-1 and hPGHS-2. Recombinant hPGHS-1 and hPGHS-2 both produced 15- and 11-hydroxyeicosatetraenoic acid (HETE) from arachidonic acid, with 15-HETE production by hPGHS-2 being stimulated 5-fold by preincubation with aspirin. Chiral phase high performance liquid chromatography analysis showed that aspirin-treated hPGHS-2 produced 15(R)-HETE, with no detectable 15(S)-HETE.
authors.affiliation: Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
authors.forename: G P
authors.initials: GP
authors.lastname: O'Neill
authors.forename: J A
authors.initials: JA
authors.lastname: Mancini
authors.forename: S
authors.initials: S
authors.lastname: Kargman
authors.forename: J
authors.initials: J
authors.lastname: Yergey
authors.forename: M Y
authors.initials: MY
authors.lastname: Kwan
authors.forename: J P
authors.initials: JP
authors.lastname: Falgueyret
authors.forename: M
authors.initials: M
authors.lastname: Abramovitz
authors.forename: B P
authors.initials: BP
authors.lastname: Kennedy
authors.forename: M
authors.initials: M
authors.lastname: Ouellet
authors.forename: W
authors.initials: W
authors.lastname: Cromlish
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D016718:Arachidonic Acid / Q000494:pharmacology; D001241:Aspirin / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis*; D015151:Immunoblotting; D008861:Microsomes / Q000187:drug effects; D008969:Molecular Sequence Data; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D014616:Vaccinia virus / Q000276:immunology / Q000378:metabolism*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D006893:Hydroxyeicosatetraenoic Acids; D011994:Recombinant Proteins; D016718:Arachidonic Acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D011451:Prostaglandin-Endoperoxide Synthases; D001241:Aspirin