Cloning, expression, and up-regulation of inducible rat prostaglandin e synthase during lipopolysaccharide-induced pyresis and adjuvant-induced arthritis.
We have cloned and expressed the inducible form of prostaglandin (PG) E synthase from rat and characterized its regulation of expression in several tissues after in vivo lipopoylsaccharide (LPS) challenge. The rat PGE synthase is 80% identical to the human enzyme at the amino acid level and catalyzes the conversion of PGH(2) to PGE(2) when overexpressed in Chinese hamster ovary K1 (CHO-K1) cells. PGE synthase activity was measured using [(3)H]PGH(2) as substrate and stannous chloride to terminate the reaction and convert all unreacted unstable PGH(2) to PGF(2alpha) before high pressure liquid chromatography analysis. We assessed the induction of PGE synthase in tissues from Harlan Sprague-Dawley rats after LPS-induced pyresis in vivo. Rat PGE synthase was up-regulated at the mRNA level in lung, colon, brain, heart, testis, spleen, and seminal vesicles. Cyclooxygenase (COX)-2 and interleukin 1beta were also up-regulated in these tissues, although to different extents than PGE synthase. PGE synthase and COX-2 were also up-regulated to the greatest extent in a rat model of adjuvant-induced arthritis. The RNA induction of PGE synthase in lung and the adjuvant-treated paw correlated with a 3.8- and 16-fold induction of protein seen in these tissues by immunoblot analysis. Because PGE synthase is a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family, of which leukotriene (LT) C(4) synthase and 5-lipoxygenase-activating protein are also members, we tested the effect of LTC(4) and the 5-lipoxygenase-activating protein inhibitor MK-886 on PGE synthase activity. LTC(4) and MK-886 were found to inhibit the activity with IC(50) values of 1.2 and 3.2 microm, respectively. The results demonstrate that PGE synthase is up-regulated in vivo after LPS or adjuvant administration and suggest that this is a key enzyme involved in the formation of PGE(2) in COX-2-mediated inflammatory and pyretic responses.
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Departments of Biochemistry and Molecular Biology and Pharmacology, Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec H9R 4P8, Canada. joseph_mancini@merck.com
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D000595:Amino Acid Sequence; D000818:Animals; D001169:Arthritis, Experimental / Q000201:enzymology*; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D004790:Enzyme Induction; D005334:Fever / Q000139:chemically induced* / Q000201:enzymology; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D015854:Up-Regulation
D018076:DNA, Complementary; D008070:Lipopolysaccharides; D011451:Prostaglandin-Endoperoxide Synthases