Surfactant, comprised mainly of the phospholipid, phosphatidylcholine (PC), and key hydrophobic proteins, is deficient in ARDS. Recent studies suggest that surfactant replacement therapy might be useful in ARDS. The cytokine, tumor necrosis factor (TNF), plays a key role in the pathogenesis of acute lung injury, and has been shown to decrease the levels of surfactant PC. The major question addressed in this proposal is how TNF decreased lung PC content. Previous studies have shown that TNF decreases PC by increasing PC degradation. This proposal will address a complementary paradigm that TNF decreases PC synthesis. The biosynthesis of PC is regulated tightly in cells by the rate regulatory enzyme cytidylyltransferase (CT). The regulation of CT is largely-post translational, since CT is activated by fatty acids and inhibited by sphingolipids and enzyme phosphorylation. One effect of TNF is the generation of a novel class of bioactive lipid second messengers derived from sphingomyelin (SM) hydrolysis. These sphingolipid products include ceramide and sphingosine. TNF also activates multiple mitogen-activated protein (MAP) kinase pathways, including the p42/44 MAP pathway. Both p42/44 MAP kinase and sphingosine inhibit CT activity. Sphingosine can also stimulate p42/44 MAP kinase activity. Thus, the generation of sphingosine, in response to TNF activation of the SM hydrolysis pathway, might represent a novel effector mechanism for the inhibitory effects of TNF on surfactant PC synthesis. These observations led to the overall hypothesis that TNF decreases surfactant lipids, in part, by decreasing PC synthesis. In this project, we will determine if the negative effects of tumor necrosis factor (TNF) on CT activity are due to induction of the lipid inhibitor, sphingosine and/or activation of the p42/44 MAP kinase (AIM 1). We will also determine if activation of CT by glucocorticoids is mediated by altering specific fatty acids or sphingolipids and if these effects of glucocorticoid pre-treatment oppose inhibition of CT by TNF (AIM 2). These basic studies are especially relevant in view of ongoing clinical studies; for example, these studies might impact the development of newer treatment strategies directed at increasing surfactant synthesis such as modulation of sphingolipid generation. In addition, results that emerge from these studies could lead to modification of existing surfactant preparations used to treat ARDS.
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