Surfactant, a surface-active mixture comprised primarily of phosphatidylcholine (PC) and key proteins, is deficient in acute lung injury. Tumor necrosis factor alpha (TNF() plays a key role in sepsis-induced acute lung injury and decreases surfactant PC synthesis. The major question addressed in this proposal is how TNF( decreases PC synthesis. Prior studies in the PI's laboratory have shown that the bioactive sphingolipid, ceramide, generated in response to TNF( activation of the sphingomyelin (SM) hydrolysis pathway, is one important mechanism whereby TNF( exerts its inhibitory effects on surfactant PC synthesis. This proposal will expand on these observations by investigating the molecular basis by which TNF(-ceramide signaling inhibits PC synthesis. The synthesis of PC in cells is tightly regulated by the rate-regulatory enzyme cytidylyltransferase (CT). CT activity is inhibited by ceramide and by enzyme phosphorylation induced by mitogen-activated protein (MAP) Kinases; CT protein is also degraded by calpains and the ubiquitin-proteasome. The physiologic role of CT phosphorylation and regulation of CT protein stability, however, remain largely unknown. One effect of TNF( is the activation of multiple kinase pathways, including p42/44 MAP kinase. TNF( also activates calpains and the ubiquitin-proteasome system. These systems are also activated by ceramide. Thus, we will specifically test the hypothesis that TNF( inhibits surfactant PC synthesis by decreasing CT activity via coordinate activation of specific kinases and proteinases in response to generation of the inhibitory lipid, ceramide. In this proposal, we will determine if the negative effects of TNF( on CT activity are due to ceramide activation of the p42/44 MAP kinase (Aim 1). We will also determine if ceramide destabilizes CT protein by activation of calpains and the ubiquitin- proteasome (Aim 2). Our hypothesis will be tested using molecular and biochemical approaches to identify the regions within the CT primary structure that are molecular targets for site-specific phosphorylation and proteolytic cleavage. We will then determine if enhanced CT phosphorylation affects vulnerability of the enzyme to proteolysis. Finally, we will perform deletional and site-directed mutagenesis of CT to generate enzyme mutants, that when expressed in vivo, are less sensitive to phosphorylation and proteolytic modification in the setting of TNF( exposure. Our hypothesis will be tested by in vivo administration of TNF( with analysis conducted in primary type II alveolar epithelial cells. These studies will be supplemented with a TNF(-responsive type II (MLE-12) cell line. The unique contributions of this proposal impacting the field of lung injury include 1) delineation of novel kinase pathways linking TNF(-ceramide signaling with surfactant synthesis, 2) studies investigating CT protein stability which represent a relatively new regulatory mechanism for this key surfactant enzyme, and 3) for the first time, studies directed at stimulating surfactant synthesis by expression of novel CT mutants that exhibit robust catalytic activity and are proteinase and kinase-resistant in the setting of cytokine- induced acute lung injury. ? ? ?

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Lung Injury, Repair, and Remodeling Study Section (LIRR)
Program Officer
Golden, AL
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University of Iowa
Internal Medicine/Medicine
Schools of Medicine
Iowa City
United States
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