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.
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.
O'Neill
G P
GP
Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
Mancini
J A
JA
Kargman
S
S
Yergey
J
J
Kwan
M Y
MY
Falgueyret
J P
JP
Abramovitz
M
M
Kennedy
B P
BP
Ouellet
M
M
Cromlish
W
W
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*
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