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.

Coupling of recombinant 5-lipoxygenase and leukotriene A4 hydrolase activities and transcellular metabolism of leukotriene A4 in Sf9 insect cells.

High-level expression of human leukotriene (LT) A4 hydrolase has been established in Sf9 insect cells using the recombinant baculovirus system. LTA4 hydrolase activity in this system is at least 50-fold higher than previously achieved in a bacterial cell system. Recombinant viral human LTA4 hydrolase (rvHLTA4h) was used for coinfection studies with recombinant viral 5-lipoxygenase (rvH5LO). When Sf9 cells expressing 5-lipoxygenase are incubated in the presence of A23187 and arachidonic acid, (5S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid 5-H(P)ETE and LTA4 are synthesized in a ratio of 5:1 for 5-H(P)ETE/LT. Coexpression of 5-lipoxygenase and LTA4 hydrolase in these insect cells results in the synthesis of 5-H(P)ETE, LTA4 and in addition LTB4, and the ratio shifts to 2:1 for 5-H(P)ETE/LT. The production of enzymically formed LTB4 after addition of arachidonic acid to the Sf9 cells coinfected with LTA4 hydrolase and 5-lipoxygenase is the first demonstration of channeling of arachidonic acid to LTB4 in an engineered intact cell system. This delineates a novel biological system to synthesize significant amounts of the potent chemotactic agent, LTB4. Studies in which Sf9 cells infected with rvH5LO were incubated with Sf9 cells infected with rvHLTA4h resulted in export of LTA4 from the rvH5LO cells and transcellular metabolism of LTA4 to LTB4 in the rvHLTA4h Sf9cells.