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 syn thase from rat and characterized its regulation of expression in several ti ssues after in vivo lipopoylsaccharide (LPS) challenge. The rat PGE synthas e is 80% identical to the human enzyme at the amino acid level and catalyze s the conversion of PGH(2) to PGE(2) when overexpressed in Chinese hamster ovary K1 (CHO-K1) cells. PGE synthase activity was measured using [H-3]PGH( 2) as substrate and stannous chloride to terminate the reaction and convert all unreacted unstable PGH(2) to PGF(2 alpha) before high pressure liquid chromatography analysis. We assessed the induction of PGE synthase in tissu es from Harlan Sprague-Dawley rats after LPS-induced pyresis in vivo. Rat P GE synthase was up-regulated at the mRNA level in lung, colon, brain, heart , testis, spleen, and seminal vesicles. Cyclooxygenase (COX)-2 and interleu kin 1 beta were also up-regulated in these tissues, although to different e xtents 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 i nduction of PGE synthase in lung and the adjuvant-treated paw correlated wi th a 3.8- and 16-fold induction of protein seen in these tissues by immunob lot analysis. Because PGE synthase is a member of the membrane-associated p roteins in eicosanoid and glutathione metabolism (MAPEG) family, of which l eukotriene (LT) C-4 synthase and 5-lipoxygenase-activating protein are also members, we tested the effect of LTC4 and the 5-lipoxygenase-activating pr otein inhibitor MK-886 on PGE synthase activity. LTC4 and MK-886 were found to inhibit the activity with IC50 values of 1.2 and 3.2 muM, respectively. The results demonstrate that PGE synthase is up-regulated in vivo after LP S 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 resp onses.