The physiological importance of phospholipase D signalling

Hormones, neurotransmitters and growth factors regulate the body s functions by activating processes in cells that produce signals that control the way a cell grows, divides, moves or secretes. One of these signalling pathways involves activation of the enzyme phospholipase D which produces the messenger phosphatidic acid. Studies in cells in tissue culture have suggested that this pathway is activated in many responses and is involved in processes such as how inflammation is activated, how cells move and how they secrete factors. There is a need to understand how these pathways are regulated and the precise roles played by this signalling event, since distortion of the regulation is implicated in inflammatory disorders and other diseases including cancer. The only way to understand this physiological role is to generate and study a genetically modified mouse model. We will isolate cells from the mice and thereby characterise the role and regulation of the signalling. This work has the potential to identify targets for future therapeutic intervention.
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Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine generating phosphatidic acid (PtdOH) as its lipid product. This reaction is stimulated by ligation of receptors for a variety of hormones, neurotransmitter, cytokines and growth factors. PtdOH binds to and activates a number of intracellular targets including phosphatidylinositol-4-phosphate-5-kinase, mTOR, S6kinase, sphingosine kinase1 and the NADPH oxidase. Activation of PLD signalling has been implicated in the regulation of membrane trafficking events, e.g. exocytosis and cytoskeletal events such as adhesion and migration; other reports have suggested roles in regulating glucose transport, superoxide generation, apoptosis, proliferation and differentiation. The two mammalian PLD genes are expressed in all tissues, but their regulation and physiological functions appear to differ. The lack of isoform-specific inhibitors and the limitations of cell line studies have prevented a clear definition of the physiological roles of this ubiquitous signalling pathway. This has pointed to the need to generate genetic models to examine PLD regulation and function. This application builds upon MRC-funded preliminary work that generated a PLD2 knock out mouse that demonstrated effects upon neutrophil responsiveness and brain lipids, in addition defects in ras signaling were detected in derived MEFs . In this programme we will determine the physiological importance of PLD2 by studying the mouse, isolated cells and derived MEFS. The importance of PLD signalling in adhesion, migration and superoxide generation will be examined in isolated neutrophils, whilst mast cells will be used to examine the regulated secretion of inflammatory mediators. By making use of knockouts in other signalling enzymes, particularly PI-3-kinases and PIP-5-kinases to generate double knockouts, we will investigate the regulation and importance of signalling cross talk in physiological regulation. These studies will define the importance of PLD signalling pathways in physiological systems and may point to potential therapeutic targets in inflammatory disorders and other diseases.