The objective of this proposal is to study the regulation and function of three Group IV cytosolic phospholipases A2 (cPLA2s), cPLA2a, cPLA2 and cPLA2? in lung cells. These enzymes contain a calcium- phospholipid binding domain and a characteristic Ser/Asp catalytic dyad. The founding member, cPLA2a, releases arachidonic acid for the production of prostaglandins and leukotrienes. These potent lipid mediators have diverse roles in regulating both normal and pathological processes. Pro-inflammatory lipid mediators (leukotrienes, thromboxane A2) produced as a result of cPLA2a activation regulate the development of pulmonary fibrosis, acute lung injury, allergic reactions and arthritis. In contrast prostaglandins E2 and I2 protect against fibrosis, hypertension and diabetes. Little is known about cPLA2 and cPLA2?, which are expressed together with cPLA2a in lung epithelial cells and fibroblasts. Eicosanoid production is not completely ablated in cPLA2a knockout mice or in cPLA2a-deficient lung fibroblasts. cPLA2? was identified as the PLA2 responsible for calcium-induced fatty acid release and prostaglandin E2 production in lung fibroblasts isolated from the cPLA2a knockout mouse. cPLA2a, cPLA2 and cPLA2? exhibit distinct enzymatic properties and subcellular localization suggesting differences in their regulation and function. In response to increases in intracellular calcium, cPLA2a translocates from the cytosol to Golgi, and cPLA2? translocates to ruffles and endosomes. In contrast, cPLA2 constitutively localizes to mitochondria and early endosomes. cPLA2 and cPLA2? are the first PLA2s identified on endosomes, where we hypothesize they regulate endosome trafficking. The specific aims of this proposal involve using purified cPLA2a, cPLA2 and cPLA2? to identify domains and residues that regulate membrane binding and hydrolytic activity. The role of calcium and polyphosphoinositides in regulating these processes in vitro and in cells will be investigated. The regulation of endosome trafficking by cPLA2 and cPLA2? in BEAS-2B cells and lung fibroblasts will be investigated using shRNA knockdown and small molecule PLA2 inhibitors. The lipid mediators produced as a result of cPLA2a and cPLA2? activation in primary lung fibroblasts and epithelial cells will be comprehensively analyzed. Since cPLA2?, unlike cPLA2a, releases linoleic acid, we hypothesize that it initiates the formation of 13(S)-hydroxy-octadecadienoic acid through 15- lipoxygenase. Using a conditional knockout mouse model, cPLA2? will be inducibly deleted from lung epithelial cells, which prominently express cPLA2?. The mouse model will be used to determine the role of cPLA2? in mediating lipid mediator production, and in regulating allergen-induced inflammation and airway hyper-responsiveness. Results from this proposal will provide detailed information about the regulation and function of Group IV cPLA2s in lung cells. PUBLIC HEALTH RELEVANCE. Members of the Group IV cytosolic phospholipase A2 family initiate the production of potent biologically active lipid metabolites that have diverse functional effects. Lipid mediators regulate physiological processes but also contribute to the pathological consequences of inflammatory diseases. Elucidating the properties of these enzymes will provide a better understanding of the regulation of lipid mediator production, and for development of mechanisms for controlling disease.

R01HL061378

1999-01-01

2013-07-31