Kdo2‐Lipid A stimulated GIVA Phospholipase A2 catalyzed AA release is Phosphatidic Acid Phosphohydrolase‐1 Dependent

Abstract

Macrophages respond to toll‐like receptor 4 (TLR‐4) agonists, such as LPS and Kdo2‐Lipid A, by upregulating cyclooxygenase 2 (COX‐2) and releasing prostaglandins. Group IVA phospholipase A2 (GIVA PLA2) catalyzes the deacylation of membrane‐bound arachidonic acid (AA) from the sn‐2 position of glycerophospholipids, which can then be further metabolized by COX‐2 in the synthesis of prostaglandins. Phosphatidic acid phosphohydrolase‐1 (PAP‐1) is a Mg+2 dependent, membrane‐associated protein involved in the synthesis of triacylglycerol. We show herein that GIVA PLA2‐mediated AA release and COX‐2 expression through TLR‐4 signaling mechanism requires PAP‐1. PAP‐1 chemical inhibition decreased COX‐2 mRNA transcript production, COX‐2 protein expression and AA release from macrophages. Addition of diacylglycerol, the end‐product of PAP‐1 catalysis, enhanced AA release from uninhibited cells and restored the AA release in inhibited cells. TLR‐4 activation increased the levels of endogenous cellular DAG within two minutes of stimulation and this increase was sensitive to PAP‐1 inhibition. Furthermore, PA phosphohydrolase activity assays showed that PAP‐1 activity translocated from the cytosolic fraction to the membrane fraction within two minutes of stimulation. DAG add‐back experiments demonstrate that TLR‐4 induced COX‐2 expression is enhanced by the addition of exogenous DAG. Additionally, PAP‐1 chemical inhibition reduced the synergistic release of AA from cells stimulated with TLR‐4 agonist and ATP, while not affecting the magnitude of synergistic activation.