Group V Phospholipase A2-dependent Induction of Cyclooxygenase-2 in Macrophages
Journal of Biological Chemistry
When exposed for prolonged periods of time (up to 20 h) to bacterial lipopolysaccharide (LPS) murine P388D 1 macrophages exhibit a delayed prostaglandin biosynthetic response that is entirely mediated by cyclooxygenase-2 (COX-2). Both the constitutive Group IV cytosolic phospholipase A 2 (cPLA 2 ) and the inducible Group V secretory phospholipase A 2 (sPLA 2 ) are involved in the cyclooxygenase-2-dependent generation of prostaglandins in response to LPS. Using the selective sPLA 2 inhibitor
... -acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane sulfonic acid (LY311727) and an antisense oligonucleotide specific for Group V sPLA 2 , we found that induction of COX-2 expression is strikingly dependent on Group V sPLA 2 , which was further confirmed by experiments in which exogenous Group V sPLA 2 was added to the cells. Exogenous Group V sPLA 2 was able to induce significant arachidonate mobilization on its own and to induce expression of the COX-2. None of these effects was observed if inactive Group V sPLA 2 was utilized, implying that enzyme activity is crucial for these effects to take place. Therefore, not only delayed prostaglandin production but also COX-2 gene induction are dependent on a catalytically active Group V sPLA 2 . COX-2 expression was also found to be blunted by the Group IV cPLA 2 inhibitor methyl arachidonyl fluorophosphonate, which we have previously found to block Group V sPLA 2 induction as well. Collectively, the results support a model whereby Group IV cPLA 2 activation regulates the expression of Group V sPLA 2 , which in turn is responsible for delayed prostaglandin production by regulating COX-2 expression. Phospholipase A 2 (PLA 2 ) 1 plays a key role in numerous cellular processes by regulating the release of arachidonic acid (AA) from membrane phospholipids. In turn, free AA can be converted into the prostaglandin (PG) precursor PGH 2 by the action of cyclooxygenases (COX). These two reactions constitute the regulatory checkpoints of the pathway leading to PG biosynthesis in mammalian cells. A considerable body of evidence suggests that specific coupling between certain PLA 2 and COX forms accounts for the differential regulation of the immediate and delayed responses (1-11). We have shown that the immediate platelet-activating factor-receptor-mediated phase of PGE 2 production in LPSprimed P388D 1 macrophages involves Group V sPLA 2 coupling to COX-2 (3). More recently, we have discovered that the exact same coupling of Group V sPLA 2 to COX-2 also regulates the delayed PGE 2 response of P388D 1 macrophages to LPS alone (4). Under the latter conditions, expression of both Group V sPLA 2 and COX-2 was markedly induced and correlated with ongoing AA release and PG biosynthesis, respectively (4), suggesting that the AA produced by Group V sPLA 2 was used by COX-2 to produce PGE 2 . Importantly, sPLA 2 expression could be abolished by pretreating the cells with the cPLA 2 inhibitor methyl arachidonyl fluorophosphonate, implying that a functionally active cPLA 2 is required for the delayed PGE 2 response to occur (4). In the current study we extend our previous observations on the delayed phase of PGE 2 in P388D 1 macrophages to further investigate the regulatory relationships between the three effectors of the response (i.e. cPLA 2 , sPLA 2 , and COX-2). We now demonstrate tight coupling between sPLA 2 and COX-2 for the delayed phase of PGE 2 generation by showing that COX-2 induction by LPS is dependent upon a catalytically active Group V sPLA 2 .