The death receptor Fas plays a critical role in the regulation of apoptosis. Activation of the Fas pathway causes apoptosis of lung epithelial cells, and Fas-induced apoptosis in these cells is sufficient to trigger the subsequent development of fibrosis. During the current funding period of this grant, we made the exciting novel observation that the Fas death pathway is redox regulated via the glutaredoxin/S-glutathionylation redox couple. Upon ligation of Fas, the thiol repair enzyme, glutaredoxin-1 (Grx1) is rapidly degraded via caspase 8/3, which causes increases in S-glutathionylation of Fas at Cysteine 294. S-glutathionylation of Fas promotes its recruitment into lipid rafts, and enhances binding of FasL, caspase activation and apoptosis, providing a feed forward amplification loop to enhance the strength of the apoptotic signal. Primary lung epithelial cells or fibroblasts that lack Grx1 display more S-glutathionylation of Fas and have enhanced caspase activity and FasL-induced apoptosis. Conversely, overexpression of Grx1 decreases S-glutathionylation of Fas and dampens FasL-induced caspase activation and apoptosis. These exciting new observations raise the intriguing possibility that the glutaredoxin/S-glutathionylation redox system also impact the extent of collagen deposition in the lung by controlling the extent of apoptosis in lung epithelial cells. The hypothesis of the current proposal is that increases in S-glutathionylation that occur as a result of caspase-dependent degradation of Grx1 play a causal role in apoptosis of epithelial cells and the subsequent development of pulmonary fibrosis, and that S-glutathionylation of Fas is a critical target in this process. We also speculate that augmentation of Grx1 in lung epithelium by reversing the increases in S-glutathionylation attenuates epithelial apoptosis, and dampens subsequent fibrogenesis.
In Specific Aim 1, we will elucidate the molecular basis of S-glutathionylation of Fas, and the mechanism whereby it strengthens the pro-apoptotic function of Fas.
In Specific Aim 2, we will determine whether patterns of S-glutathionylation and S-glutathionylation of Fas are increased in mouse models of fibrosis and patients with fibrotic lung disease.
In Specific Aim 3, we will determine whether mice that lack Grx1 systemically or in airway epithelium have an enhanced susceptibility to the development of pulmonary fibrosis, in association with increases in S-glutathionylation of Fas and caspase activation. We will address the requirement of Fas in the enhanced susceptibility to fibrogenesis.
In Specific Aim 4, we will address whether transgenic expression of Grx1 in lung epithelial cells confers protection against the development of fibrosis. Completion of the specific aims of this renewal application will provide important new information about the role of a recently discovered redox couple, glutaredoxin/S-glutathionylation in the development of pulmonary fibrosis.

Public Health Relevance

Epithelial cells that line the airways (breathing tubes) play an important role in the defense against inhaled materials, and are important in maintaining normal lung function. However, our laboratories have identified that epithelial cells play a negative role in promoting stiffening of the lung tissue, thereby decreasing lung function. We have identified some critical oxidative signals that promote this possibly negative event, by causing death of the epithelial cells, and prevent the normal repair process. This grant proposal has four specific aims to test the importance of these biochemical signals in lung stiffening, and will use both primary cell cultures from mouse airways, cell lines, and the construction of genetically altered mice. Completion of this project will provide new insights into the process of lung stiffening, and may lead to the development of new therapeutics to limit lung stiffening, and also potentially reverse this process.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL079331-08
Application #
8307763
Study Section
Lung Injury, Repair, and Remodeling Study Section (LIRR)
Program Officer
Eu, Jerry Pc
Project Start
2004-12-15
Project End
2014-02-28
Budget Start
2012-08-01
Budget End
2014-02-28
Support Year
8
Fiscal Year
2012
Total Cost
$372,488
Indirect Cost
$124,988
Name
University of Vermont & St Agric College
Department
Pathology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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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