Surfactant, a surface-active mixture comprised of phosphatidylcholine (PC) and key hydrophobic proteins, is deficient in acute lung injury. The cytokine tumor necrosis factor alpha (TNFalpha),-plays a key role in the pathogenesis of sepsis-induced lung injury and decreases levels of surfactant PC. The major question addressed in this proposal is how TNFalpha decreases PC content. Previous studies have shown that TNFalpha decreases PC by increasing PC degradation. This proposal will address a complementary paradigm that TNFalpha decreases PC synthesis. The synthesis of PC is tightly regulated in cells by the rate-regulatory enzyme cytidylyltransferase (CT). CT is activated by fatty acids, but inhibited by other lipids such as ceramide and sphingosine. CT activity is also inhibited by enzyme phosphorylation induced by mitogen-activated protein (MAP) kinases. One effect of TNFalpha is the generation of ceramide derived from sphingomyelin (SM) hydrolysis. TNFalpha also activates multiple MAP kinase pathways, including the p42/44 MAP pathway. Ceramide rapidly deacylates to sphingosine, and sphingosine can trigger p42/44 MAP kinase activation. Further, ceramide activates cell proteases, which might degrade the CT enzyme. These observations led to the overall hypothesis that TNFalpha inhibits surfactant PC synthesis, in part, by decreasing CT activity via generation of the inhibitory lipid, ceramide. In this proposal, we will determine if the negative effects of TNFalpha on CT activity are due to induction of ceramide, and/or activation of the p42/44 MAP kinase (AIM 1), or ceramide-induced alteration of CT protein stability (AIM 2). We will also determine if fatty acids can counteract TNFalpha inhibitory effects as they stimulate CT activity and surfactant production in vitro. However, because exogenous fatty acids have mixed success and are potentially toxic in vivo, we will use a new strategy to counteract TNFalpha effects by administering very low-density lipoproteins (VLDL) with lipoprotein lipase (LPL). VLDL triglycerides are hydrolyzed to fatty acids by LPL. We will determine if activation of CT by VLDL and LPL is mediated by altering specific fatty acids or sphingolipids, and if these effects of lipoprotein pretreatment oppose inhibition of CT by TNFalpha (AIM 3). Our hypothesis will be tested by in vivo administration of TNFalpha and VLDL with analysis conducted in primary adult type II alveolar epithelial cells. These in vivo studies will be supplemented with a TNFalpha and lipoprotein-responsive type II (MLE-12) cell line. The unique contributions of this proposal impacting the field of surfactant metabolism include 1) delineation of a novel effector pathway linking TNFalpha-signaling with surfactant synthesis (AIM 1) 2) studies investigating CT protein stability which represent a new regulatory mechanism for this key surfactant enzyme (AIM 2) and 3) studies with potential clinical application by which lipoproteins modulate surfactant synthesis in the setting of cytokine-induced acute lung injury (AIM 3).
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