OVEREXPRESSION OF HUMAN PROSTAGLANDIN G H SYNTHASE-1 AND SYNTHASE-2 BY RECOMBINANT VACCINIA VIRUS - INHIBITION BY NONSTEROIDAL ANTIINFLAMMATORY DRUGS AND BIOSYNTHESIS OF 15-HYDROXYEICOSATETRAENOIC ACID/

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 e xpression system. The open reading frame of hPGHS-2 cloned into vaccin ia virus without its natural 5' and 3' untranslated regions directed o nly low levels of hPGHS-2 enzyme activity in COS-7 cells. High-level h PGHS-2 expression was achieved by appending the 3' untranslated region of hPGHS-1 to the hPGHS-2 open reading frame, with subsequent express ion of the hybrid mRNA using vaccinia virus. Enzymatically active reco mbinant hPGHS-1 and hPGHS-2 were present as glycosylated proteins in t he microsomal fraction prepared from infected cells, whereas recombina nt 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 micro somes from COS-7 cells containing either recombinant hPGHS-1 or hPGHS- 2 after incubation with arachidonic acid were prostaglandins D-2 and E (2), With lower levels of prostaglandin F-2 alpha, and 6-keto-prostagl andin F-1 alpha. A range of potencies were observed for various nonste roidal anti-inflammatory drugs as inhibitors of prostaglandin E(2) syn thesis by hPGHS-1 and hPGHS-2. Recombinant hPGHS-1 and hPGHS-2 both pr oduced 15- and 11-hydroxyeicosatetraenoic acid (HETE) from arachidonic acid, with 15-HETE production by hPGHS-2 being stimulated 5-fold by p reincubation with aspirin. Chiral phase high performance liquid chroma tography analysis showed that aspirin-treated hPGHS-2 produced 15(R)-H ETE, with no detectable 15(S)-HETE.