Excessive epithelial apoptosis and lack of proper epithelial restitution are believed to be critical to fibrogenesis. The death receptor Fas plays a cardinal role in epithelial apoptosis, and the subsequent development of pulmonary fibrosis. Similarly, changes in the oxidative environment, as well as endoplasmic reticulum (ER) stress are believed to be important in the pathogenesis of idiopathic pulmonary fibrosis (IPF), suggesting a causal link between redox and ER perturbations, Fas, epithelial apoptosis, and fibrogenesis. During the current funding cycle of this grant we discovered that the potency of Fas as a death receptor is enhanced following its S-glutathionylation, a form of protein oxidation. S-glutathionylation represents the conjugation of the antioxidant molecule glutathione to reactive protein cysteines (PSSG). We recently unraveled that in lung epithelial cells not all Fas is expressed on the surface but that a latent pool of Fas which is not fully processed into the ligand binding form, is localized in the ER. In response to stimulation of surface Fas with FasL, the protein disulfide isomerase, ERp57, induces rapid oxidative processing of Fas within the ER. We speculate that hydrogen peroxide (H2O2) produced during this process is responsible for S-glutathionylation, in a reaction that is catalyzed by glutathione S-transferase P (GSTP). These discoveries illuminate a new dimension of Fas- induced apoptosis "ligand-triggered oxidative processing of latent Fas in the ER" as a regulatory mechanism to regulate the strength of cell death. The exact oxidative events triggered within the ER remain unknown, and the functional role of H2O2 generated within the ER for epithelial apoptosis, and subsequent pathogenesis of fibrosis are unclear. The central hypothesis to be addressed herein is that ERp57-catalyzed processing of Fas leads to increases of H2O2 content in the ER. Increases in H2O2 are in turn required to permit GSTP-catalyzed S-glutathionylation of Fas, augmenting epithelial apoptosis, thereby leading to pulmonary fibrosis.
In Specific Aim #1 we will determine the functional requirement of ERp57 in oxidative processing, and subsequent S- glutathionylation of Fas in lung epithelium, and the resultant development of pulmonary fibrosis.
Specific Aim #2 seeks to explore the functional requirement of GSTP in S-glutathionylation of Fas, and the subsequent development of pulmonary fibrosis.
In Specific Aim #3 we will assess the functional importance of H2O2 generated in the ER in Fas-dependent epithelial apoptosis and subsequent fibrogenesis. We will use complementary cell culture and mouse transgenic approaches, coupled to detailed analysis of these processes in lung tissues from patients with IPF as well as NSIP, using innovative and clinically relevant strategies. Completion of proposed experiments is likely to exert a substantial impact given that Fas, ER stress, and glutathione redox perturbations have been independently linked to the pathogenesis of fibrosis, and methodologies aimed at assessing or targeting S-glutathionylation may prove to be clinically relevant as diagnostic tools and potential therapeutics.

Public Health Relevance

This project seeks to identify the mechanisms whereby lung epithelial cells die, and the relevance this plays for the development of a scar in the lung. Specifically we will examine the importance of oxidants generated in a specific organelle, known as the endoplasmic reticulum (ER) in activating the cell death receptor, Fas in lung epithelial cells. The importance of the oxidant, hydrogen peroxide in this process will be determined by removing the enzyme that leads to its formation within the ER of epithelial cells. We also will use approaches to quench hydrogen peroxide from the ER and determine the impact for development of a scar. We will also determine whether these approaches stop the further progression of existing scars. We also will take advantage of a drug (TLK199) which is already in clinical trials to see if this drug can be used in setting of lung scarring, and will evaluate lng tissues for patients with idiopathic pulmonary fibrosis and non-specific interstitial pneumonia. This project is important in that it may demonstrate that therapeutics to prevent scars should be directed to normalizing oxidative processes in epithelial cells, and may therefore provide new opportunities to stop the process of scarring in the future.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CVRS-G (02))
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Eu, Jerry Pc
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University of Vermont & St Agric College
Schools of Medicine
United States
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Anathy, Vikas; Aesif, Scott W; Hoffman, Sidra M et al. (2014) Glutaredoxin-1 attenuates S-glutathionylation of the death receptor fas and decreases resolution of Pseudomonas aeruginosa pneumonia. Am J Respir Crit Care Med 189:463-74
Janssen-Heininger, Yvonne M W; Nolin, James D; Hoffman, Sidra M et al. (2013) Emerging mechanisms of glutathione-dependent chemistry in biology and disease. J Cell Biochem 114:1962-8
Roberson, Elle C; Tully, Jane E; Guala, Amy S et al. (2012) Influenza induces endoplasmic reticulum stress, caspase-12-dependent apoptosis, and c-Jun N-terminal kinase-mediated transforming growth factor-? release in lung epithelial cells. Am J Respir Cell Mol Biol 46:573-81
Anathy, Vikas; Roberson, Elle C; Guala, Amy S et al. (2012) Redox-based regulation of apoptosis: S-glutathionylation as a regulatory mechanism to control cell death. Antioxid Redox Signal 16:496-505
Aesif, Scott W; Anathy, Vikas; Kuipers, Ine et al. (2011) Ablation of glutaredoxin-1 attenuates lipopolysaccharide-induced lung inflammation and alveolar macrophage activation. Am J Respir Cell Mol Biol 44:491-9
Janssen-Heininger, Yvonne M W; Aesif, Scott W; van der Velden, Jos et al. (2010) Regulation of apoptosis through cysteine oxidation: implications for fibrotic lung disease. Ann N Y Acad Sci 1203:23-8
Alcorn, John F; Ckless, Karina; Brown, Amy L et al. (2010) Strain-dependent activation of NF-kappaB in the airway epithelium and its role in allergic airway inflammation. Am J Physiol Lung Cell Mol Physiol 298:L57-66
Anathy, Vikas; Aesif, Scott W; Guala, Amy S et al. (2009) Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas. J Cell Biol 184:241-52
Alcorn, John F; van der Velden, Jos; Brown, Amy L et al. (2009) c-Jun N-terminal kinase 1 is required for the development of pulmonary fibrosis. Am J Respir Cell Mol Biol 40:422-32
Janssen-Heininger, Yvonne M W; Poynter, Matthew E; Aesif, Scott W et al. (2009) Nuclear factor kappaB, airway epithelium, and asthma: avenues for redox control. Proc Am Thorac Soc 6:249-55

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