Unregulated ceramide signaling causes uncontrolled cellular apoptosis, a hallmark in the pathogenesis of lung injury in pulmonary diseases such as asthma, bronchiectasis, and chronic obstructive pulmonary disorders. Our studies have demonstrated that reactive oxygen species (ROS) are key participants in ceramide generation and apoptosis. Yet, currently there is little insight regarding the cellular and molecular mechanisms linking oxidant exposure with ceramide and apoptosis, and ultimately with development of lung injury. We have shown that human airway epithelial cells exposed to ROS generate excessive ceramide, which functions as a potent inducer of cell death in these cells. We therefore proposed under the previous funding period that increased ceramide is a primary cause of the cellular injury observed with lung diseases. Our search for the link between ROS and ceramide production yielded a novel neutral sphingomyelinase, nSMase2, which was isolated from monkey lung tissue and human airway epithelial cells. Our preliminary studies suggested that nSMase2 is the main target sphingomyelinase (out of a large family of sphingomyelinases (SMases)) that is activated by oxidant exposure, and induces upregulated ceramide production, leading to upregulated cell death and lung injury. Having linked nSMase2 to oxidative stress and ceramide-induced lung injury, we now hypothesize that nSMase2 is in fact a crucial switch that initiates and regulates the ceramide-induced cellular death in lung epithelial/alveolar destruction. To prove this hypothesis, our current proposal seeks to explore and define the cellular, molecular, and biochemical mechanisms of nSMase2 activation and also to demonstrate its role in vivo using a lung injury model of cigarette smoke (CS) exposure in mice or rats.
In Specific Aim 1, we will investigate the role of nSMase2 in ceramide generation and induction of cell death using primary human and monkey airway epithelial cells as well as mice [or rats] exposed to CS-induced oxidative stress.
In Specific Aim 2, we will determine which protein-protein interactions are involved in the function, cellular localization and activation of nSMase2 and its modulation under oxidative stress. Finally, in Specific Aim 3, we will define the structure-function of nSMase2, characterizing how oxidative stress leads to changes in its hyper-reactive Cys residues, specific phosphorylation sites, and ubiquitinylation, and how these changes may be linked to nSMase2 interactions with proteins in the ceramide signaling pathway. Through these comprehensive studies, we will elucidate the biological significance and molecular mechanisms of the novel nSMase2 regulatory pathway, specifically its role in cell death and epithelial/alveolar destruction. Success of these studies will open new therapeutic avenues that target nSMase2-driven processes in lung injury and pulmonary diseases.
Our present studies focus on the characterization of the cellular, molecular and biochemical mechanisms involved in the activation of the novel nSMAse2 and its roles in lung injury in vivo using mice as a model organism. Success of these studies will open new therapeutic avenues that target nSMase2-driven processes in lung injury and pulmonary diseases.
|Goldkorn, Tzipora; Filosto, Simone; Chung, Samuel (2014) Lung injury and lung cancer caused by cigarette smoke-induced oxidative stress: Molecular mechanisms and therapeutic opportunities involving the ceramide-generating machinery and epidermal growth factor receptor. Antioxid Redox Signal 21:2149-74|
|Filosto, Simone; Baston, David S; Chung, Samuel et al. (2013) Src mediates cigarette smoke-induced resistance to tyrosine kinase inhibitors in NSCLC cells. Mol Cancer Ther 12:1579-90|
|Filosto, Simone; Becker, Cathleen R; Goldkorn, Tzipora (2012) Cigarette smoke induces aberrant EGF receptor activation that mediates lung cancer development and resistance to tyrosine kinase inhibitors. Mol Cancer Ther 11:795-804|
|Filosto, Simone; Ashfaq, Majid; Chung, Samuel et al. (2012) Neutral sphingomyelinase 2 activity and protein stability are modulated by phosphorylation of five conserved serines. J Biol Chem 287:514-22|
|Zeki, Amir A; Kenyon, Nicholas J; Goldkorn, Tzipora (2011) Statin drugs, metabolic pathways, and asthma: a therapeutic opportunity needing further research. Drug Metab Lett 5:40-4|
|Filosto, Simone; Khan, Elaine M; Tognon, Emiliana et al. (2011) EGF receptor exposed to oxidative stress acquires abnormal phosphorylation and aberrant activated conformation that impairs canonical dimerization. PLoS One 6:e23240|
|Filosto, Simone; Castillo, Sianna; Danielson, Aaron et al. (2011) Neutral sphingomyelinase 2: a novel target in cigarette smoke-induced apoptosis and lung injury. Am J Respir Cell Mol Biol 44:350-60|
|Filosto, Simone; Fry, William; Knowlton, Anne A et al. (2010) Neutral sphingomyelinase 2 (nSMase2) is a phosphoprotein regulated by calcineurin (PP2B). J Biol Chem 285:10213-22|
|Goldkorn, Tzipora; Filosto, Simone (2010) Lung injury and cancer: Mechanistic insights into ceramide and EGFR signaling under cigarette smoke. Am J Respir Cell Mol Biol 43:259-68|
|Levy, Michal; Khan, Elaine; Careaga, Milo et al. (2009) Neutral sphingomyelinase 2 is activated by cigarette smoke to augment ceramide-induced apoptosis in lung cell death. Am J Physiol Lung Cell Mol Physiol 297:L125-33|
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