mPGES-1 : a key regulator of fever and neonatal respiratory depression

Abstract: Prostaglandins are potent lipid mediators, synthesized de novo from arachidonic acid (AA) upon cell activation. AA is oxidized by the cyclooxygenase isoenzymes (COX-1 and COX-2) to form PGH2, the common substrate for downstream enzymes involved in prostaglandin biosynthesis. COX-1 is constitutively expressed in most cells and regarded as housekeeping protein. In contrast, expression of COX-2 is markedly increased by pro-inflammatory cytokines at sites of inflammation. PGH2 is converted into biologically active prostanoids like PGD2, PGE2, PGF2alpha, TXA2 and PGI2 by specific enzymes in a cell-specific manner. The isomerization of PGH2 to PGE2, a potent mediator of pain and inflammation, is specifically catalyzed by human microsomal prostaglandin E synthase-1 (mPGES-1), a member of the MAPEG (membrane associated proteins in eicosanoid and glutathione metabolism) superfamily. High levels of PGE2 have been found in numerous disease states. Human mPGES-1 was expressed as an N-terminal-histidine-tagged protein in E. coli. The membrane bound enzyme was solubilized using Triton X-100 and purified to apparent homogeneity using a combination of hydroxyapatite and immobilized metal affinity chromatography. Purified mPGES-1 exhibited high glutathione (GSH)-dependent catalytic activity for the conversion of both PGH2 to PGE2 and, PGG2 to 15-hydroperoxy-PGE2. Moreover, mPGES-1 also exhibited GSH-dependent peroxidase activity towards cumene hydroperoxide and 5-HpETE as well as low but significant glutathione transferase activity, possibly reflecting a relationship to other members of the MAPEG family. A 10 Å projection map of mPGES-1 determined using electron crystallography as well as hydrodynamic studies of mPGES-1-Triton X-100 complex, independently demonstrated the trimeric organization of mPGES-1. The role of mPGES-1 in endotoxin-induced fever, as well as aseptic, cytokine-dependent, inflammation-induced fever was investigated. In response to intraperitoneal injection of lipopolysaccharide (LPS), wildtype DBA/1lacJ mice developed a robust fever with markedly increased PGE2 levels in the cerebrospinal fluid (CSF) and significant LPS-induced mPGES-1 activity in membrane fractions isolated from brain tissues. In contrast, the mPGES-1 knockout mice did not develop fever and, the PGE2 levels in the CSF did not differ significantly from the saline-treated wildtype mice, suggesting a critical role for mPGES-1 in the development of endotoxin-induced fever. In a cytokine-dependent fever model, subcutaneous injection of turpentine induced biphasic fever in wildtype mice, whereas mPGES-1 knockout mice displayed a core body temperature similar to the saline-treated wildtype mice, indicating that mPGES-1 activity was indispensable for the induction of cytokine-dependent fever. mPGES-1 did not, however, mediate hyperthermia induced by psychological stress. The role of mPGES-1 in neonatal respiratory depression was investigated using 9-day old DBA/1lacj mice. Wildtype mice treated with IL-1beta exhibited a reduced respiratory frequency during normoxia as well hyperoxia compared to saline treated mice. This effect of IL-1beta was attenuated in mPGES-1 knockout mice. Moreover, IL-1beta treatment induced apneas, irregular breathing pattern and reduced the anoxic survival of the wildtype mice, and these effects were attenuated in mice lacking mPGES-1. Both IL-1beta and hypoxia treatment synergistically induced a rapid 4-fold mPGES-1 activity in the brainstem of wildtype mice compared to the saline treatment. These results suggest a central role for mPGES-1 in the regulation of neonatal breathing. Taken together, these findings provide further support of mPGES-1 as an attractive target for the development of anti-inflammatory and anti-pyretic drugs. It is also possible that such drugs could be used in neonates at risk for respiratory suppression.