Lipid metabolites including prostaglandins, leukotrienes, other eicosanoids, and platelet activating factor are produced in a wide variety of cells. Their production is a key factor in the inflammatory response.
The aim of this project is to determine the basic mechanisms of prostaglandin regulation in inflammation by studying the prostaglandin pathways in the munne P388D1 macrophage-like cell line. During the course of previous grants, we have characterized the pathways for prostaglandin production and their control mechanisms in the P388D1 macrophages. We have identified five separate mechanisms that modulate prostaglandin production. The point of control appears to be focused on the first two steps in the prostaglandin pathway, i.e. the liberation of arachidonic acid (AA) by phospholipase A2s (PLA2) and oxidation of this AA by cyclooxygenases. We have identified the two PLA2s that participate in this pathway in P388D1 cells, i.e. the Group IV Ca2+-dependent cytosolic PLA2 and the Group V Ca2+-dependent secreted PLA2, and have shown that modulating their activity is the key to controlling prostaglandin production. We have expanded these studies in the current proposal to explore in depth how the activities of these two PLA2s are controlled. We will use confocal microscopy to follow changes in the subcellular location of the GIV PLA2 in response to changes in the levels of Ca2+, PIP2, and vimentin and ascertain how this affects prostaglandin production. We have also shown that phosphorylation processes play an important role in regulating the production of prostaglandins. We have now proposed a set of experiments that will determine how phosphorylation controls these processes. We will also explore how the extracellular GV PLA2 is controlled, i.e. via up-regulation, subcellular translocation, or activation. We will determine the factors that control the expression of GV PLA2. We will use the confocal procedures to follow the subcellular migration of this enzyme through out the course of the cellular activation.
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