Regulation of cytosolic Phospholipase A2 activity plays a central role in cell responses

Abstract

Phospholipases A2 are enzymes that hydrolyse fatty acids from the sn-2 position of phospholipids, resulting in the release of free fatty acids and lysophospholipids. The sn-2 position of phospholipids in mammalian cells is enriched with arachidonic acid, which is a substrate for cyclooxygenases, lipoxygenases and cytochrome p450s, giving PLA2s an important role in the control of the synthesis of prostaglandins, leukotrienes and other eicosanoids. Arachidonic acid and its metabolites, the eicosanoids, have been implicated in a number of physiological and pathophysiological processes and is preferentially released by cytosolic phospholipase A2 (cPLA2), implicating that cPLA2 activity has to be tightly regulated. The aim of this thesis was to gain more insight in the regulation of cPLA2 in mitogen- and oxidative stress-induced cells, as well as in continuously cycling cells. Furthermore, the possible role of cPLA2 and the downstream arachidonic acid metabolising enzymes, cyclooxygenases and lipoxygenases, in cell cycle progression was investigated. The studies described in chapters 2, and 3 show that cPLA2 is activated through different signal transduction pathways depending on the stimulus in the extracellular environment. In response to epidermal growth factor (EGF), cPLA2 is predominantly activated through PKC-MEK-p42/44MAPK, while serum-induced cPLA2 activity is mainly mediated via the Raf-MEK-p42/44MAPK pathway. In contrast, direct activation of PKC by phorbol ester (PdBu) did not result in increased cPLA2 activity, while p42/44MAPK was activated via Raf-MEK and through MEK. Activation of cPLA2 by the oxidant hydrogen peroxide (H2O2) is partly mediated via Raf-MEK-p42/44MAPK and partly through a phosphorylation-independent mechanism involving peroxidised phospholipids. These results suggest that activation of cPLA2 is not only governed by post-translational modifications but, more importantly, by localisation of the signal transduction components at a certain time that determines whether cPLA2, at which place and what time cPLA2 will be activated. The cellular localisation of signal transduction components determines whether cPLA2 will be activated. However, understanding the function of cPLA2 in cells requires also knowledge of the activation of cPLA2 in a temporal manner. Therefore, the activity and function of cPLA2 was investigated during the ongoing cell cycle as described in chapters 4 and 5. cPLA2 activity was high in mitosis, decreasing rapidly in early G1. A small increase was observed in mid/late G1, followed by a strong increase at the G1/S transition. These changes in cPLA2 activity were not due to differences in cPLA2 protein expression, but due to p42/44MAPK mediated phosphorylation of the enzyme. Inhibition of cPLA2 activity in early G1 phase using ATK, an inhibitor for cPLA2, resulted in a marked reduction in DNA synthesis. Furthermore, inhibition of cyclooxygenases at different time points after mitosis did not have any effect on cell cycle progression from G1 to S phase, whereas inhibition of lipoxygenases resulted in G1 phase arrest. Moreover, lipoxygenases are pivotal for S phase progression, since no DNA synthesis occurred when lipoxygenases were inhibited. Thus it is important to understand the mechanism and function of cPLA2 regulation for the development and therapeutic use of drugs.