The function, activation and inhibition of phosphatidic acid phosphohydrolase-1 and Group IVA phospholipase A2 in toll-like receptor-4 activated macrophages

Macrophages are large, single nucleated leukocytes that participate in the innate and adaptive immune systems through the release of bioactive agents, phagocytosis of unwanted particles and antigen presentation. The macrophage immune response to invading microorganisms begins with the binding of the bacterial endotoxin lipopolysacchride (LPS) to the toll-like receptor-4 (TLR-4), which initiates intracellular signal transduction cascades that subsequently activate a variety of inflammatory proteins, including phosphatidic acid phosphohydrolase-1 (PAP-1) and the group IVA phospholipase A2 (GIVA PLA2). PAP-1 is a Mg+2-dependent membrane-associated enzyme that catalyzes the hydrolysis of phosphatidic acid (PA) into diacylglycerol (DAG), while GIVA PLA2 catalyzes the release of arachidonate from the sn-2 position of membrane phospholipids yielding lysophospholipid and free arachidonic acid (AA). The purpose of my research has been to better understand the molecular mechanism of the cellular regulation and function of PAP-1 and GIVA PLA2 through the use of a macrophage model. We began by characterizing the expression and activation of key inflammatory proteins that participate in this pathway, including cyclooxygenase-2 (COX-2), PAP-1 and GIVA PLA2. We observed that the chemical inhibition of cellular PAP-1 results in the loss of COX-2 upregulation in TLR-4 activated macrophages. We observed through HPLC-MS analysis of TLR-4 activated macrophage supernatants that PAP-1 was also necessary for the activation of cellular GIVA PLA2. Supplementation of macrophages with exogenous DAG, the product of PAP-1 hydrolysis, restored COX-2 expression and GIVA PLA2 activity. To ensure this was not the result of inhibitor promiscuity, we confirmed that PAP-1 inhibition does not result direct GIVA PLA2 activation through cellular stimulation with Ca+2 agonists, such as ionomycin and ATP. Surprisingly, PAP-1 inhibition reduced the absolute amount of AA released from cells synergistically stimulated with ATP and Kdo 2-Lipid A, a TLR-4 specific agonist, while not affecting the magnitude of synergy enhancement. Furthermore, our data suggests that two bioactive lipids, phosphatidylinositol 4,5-bisphosphate and ceramide 1-phosphate, play regulatory roles in the activation of GIVA PLA2 in TLR-4 activated macrophages. Since GIVA PLA2 has become a target in the generation of future anti-inflammatory drugs, our laboratory has been developing a novel class of GIVA PLA2 substrate-analogue chemical inhibitors. Using HPLC-MS to measure metabolite release for the purpose of evaluating inhibitor efficacy, we concluded that inhibitor structures that contain a 2-oxoamide moiety spaced at the delta or gamma position relative to that of the carboxylic acid function group demonstrate maximal cellular potency and specificity.