Cigarette smoking has a main role in respiratory diseases and deregulated ceramide signaling is critical for uncontrolled cell death (apoptosis), a hallmark of pathologic conditions that cause lung injury in these diseases. We propose that excessive ceramide generation, by a sphingomyelinase (SMase) that hydrolyzes sphingomyelin, is a primary event in the pathogenesis of lung injury in pulmonary diseases such as asthma, bronchiectasis, and chronic obstructive pulmonary disorders (COPD). Therefore, in a search for the specific SMase that is up-regulated by cigarette smoke (CS), we have recently cloned a novel nSMase, nSMase2. We showed that this nSMase2 is modulated by CS to elevate ceramide generation and apoptosis induction. Therefore, it links CS to ceramide generation and cell apoptosis at the molecular level. Based on our initial studies, we hypothesize that nSMase2 is a crucial regulator of CS-mediated ceramide signaling for apoptosis elevation in human airway epithelial cells and thus in lung injury.
We propose to test the specific hypothesis that the novel nSMase2 is a regulated enzyme, i.e., a specific target that is modulated by CS to generate ceramide, which mediates exacerbated-apoptosis in the lung. This proposal will focus on characterizing the cellular, molecular and biochemical mechanisms of activation of nSMase2 and its role in vivo in lung injury of mice exposed to CS. To test this hypothesis; first, we will use genetic knockdown, over-expression and dominant-negative mutant expression to identify key CS-induced biological responses of primary airway epithelial cells that are critically dependent on nSMase2. We will test the idea that nSMase2 is a direct positive regulator of ceramide generation and apoptosis first in primary airway epithelial cells and then in vivo in the lungs of mice exposed to CS. We will then elucidate nSMase2 interactions with other components of the apoptotic machinery, such as members of the Bcl2 and caspase families. Second, we will test the hypothesis that nSMase2 subcellular localization and thus, its ability to generate ceramide, is dictated by exposure to CS. Third, we will undertake structure/function analyses of nSMase2 to clarify the role of nSMase2 primary/secondary structure in its capacity to respond to CS; site-directed mutagenesis of nSMase2 will be used to determine which domain of the protein is necessary for its activation by CS, for its trafficking, for its interactions with other proteins, and for targeting to the plasma membrane for ceramide generation and augmentation of apoptosis. Through this comprehensive approach, we will elucidate the biological significance and apoptosis-related roles of this novel nSMase2 regulatory pathway and its molecular mechanism in lung epithelium destruction. Success of these studies will open new therapeutic avenues that target nSMase2-driven apoptotic processes in lung injury.
Therefore, the proposed project directly relates to the TRDRP research priorities. Understanding the mechanism of CS-induced pathological apoptosis in airway epithelial cells and in vivo, in lung injury of mice exposed to CS, is crucial to the development of therapeutic and pharmacological strategies to block the destructive properties of CS, thereby reducing damage to the lung of cigarette smokers. |