Surfactant, a surface-active mixture comprised primarily of phosphatidylcholine (PC) and key proteins, isdeficient in acute lung injury. Tumor necrosis factor alpha (TNFa) pjays a key role in sepsis-inducedacute lung injury anal decreases surfactant PC synthesis. The major question addressed in thisproposal is how TNFa decreases PC synthesis. Prior studies in the Pi's laboratory have shown thatthe bioactive sphingolipid, ceramide, generated in response to TNFa activation of the sphingomyelin(SM) hydrolysis pathway, is one important mechanism whereby TNFa exerts its inhibitory effects onsurfactant PC synthesis. This proposal will expand on these observations by investigating the molecularbasis by which TNFa-ceramide signaling inhibits PC synthesis. The synthesis of PC in cells is tightlyregulated by the rate-regulatory enzyme cytidylyltransferase (CT). CT activity is inhibited by ceramideand by enzyme phosphorylation induced by mitogen-activated protein (MAP) Kinases; CT protein is alsodegraded by calpains and the ubiquitin-proteasome. The physiologic role of CT phosphorylation andregulation of CT protein stability, however, remain largely unknown. One effect of TNFa is the activation multiple kinase pathways, including p42/44 MAP kinase.TNFa also activates calpains and the ubiquitin-proteasome system. These systems are also activatedby ceramide. Thus, we will specifically test the hypothesis that TNFa inhibits surfactant PCsynthesis by decreasing CTactivjty via coordinate activation of specific kinases and proteinasesin response to generation of the inhibitory lipid, ceramide. In this proposal, we will determine if thenegative effects of TNFa on CT activity are due to ceramide activation of the p42/44 MAP kinase (AIM1). 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 toidentify the regions within the CT primary structure that are molecular targets for site-specificphosphorylation and proteolytic cleavage. We will then determine if enhanced CT phosphorylationaffects vulnerability of the enzyme to proteolysis. Finally, we will perform deletional and site-directedmutagenesis of CT to generate enzyme mutants, that when expressed in vivo, are less sensitive tophosphorylation and proteolytic modification in the setting of TNFa exposure. Our hypothesis will betested by in vivo administration of TNFa with analysis conducted in primary type II alveolar epithelialcells. These studies will be supplemented with a TNFa-respqnsive type II (MLE-12) cell line. The uniquecontributions of this proposal impacting the field of lung injury include 1) delineation of novel kinasepathways linking TNFa-ceramide signaling with surfactant synthesis, 2) studies investigating CT proteinstability 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 mutantsthat exhibit robust catalytic activity and are proteinase and kinase-resistant in the setting of cytokine-induced acute lung injury.
Showing the most recent 10 out of 43 publications
