Adhesion of cells to an extracellular matrix (ECM) is involved in a number of biological phenomena including wound healing, embryogenesis, blood coagulation, and metastasis. The long term goal of this project is to elucidate the molecular events that commence with the cell attachment stage of cell-substrate adhesion and regulate the ensuing cell spreading and cell migration stages. We have demonstrated that during attachment of cells to collagen of fibronectin the cognate receptors become clustered and activate cytosolic phospholipase A2 by phosphorylation and translocation to the membrane where arachidonic acid (AA) is release from phospholipids. AA can be oxidized by lipoxygenase into metabolites that turn on the production of diacylglycerol followed by the translocation of protein kinase C to the membrane to activate actin polymerization and the cell spreading stage of adhesion. This information and preliminary work leads to the following hypothesis: Cell spreading and cell migration are regulated respectively by two lipid second messenger pathways which are branches of AA metabolism; lipoxygenase (LOX) and cyclooxygenase (COX) metabolites separately activate sequences of kinases and small GTP-binding proteins leading respectively to the polymerization of actin essential for spreading and bundling of actin filaments essential for migration. Pharmacological, biochemical and molecular biological strategies will be used to evaluate the hypothesis by exploring the following specific aims: 1. Determine which COX metabolites upregulate cAMP to increase PKA activity for actin bundling and migration. 2. Determine whether the amount of COX that is ectopically expressed or expressed in response to a calcium signal determines the level of cAMP and the rate of migration. 3. Establish if and where the GTP-binding proteins, Cdc42, Rac and Rho are involved in the LOX and COX pathways. 4. Ascertain how the metabolites of the three lipoxygenases might regulate the production of diacylglycerol which is essential for PKC activation in the cell spreading stage of adhesion.
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