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Chia, Shi Biao

Cellular, Molecular, and Biomedical Sciences Graduate Program

2020

Ph. D.

Asthma is a complex lung disorder that affects more than 200 million people across the globe. About 10% of asthmatics have severe disease accompanied by structural airway remodeling, including subepithelial fibrosis, airway smooth muscle cell hyperplasia, goblet metaplasia, and increased vascularization. Oxidative stress has been well-linked to asthma pathogenesis; however, the precise redox mechanisms governing the pathological states are slowly being teased apart in the recent years. Protein S-glutathionylation (PSSG) is a posttranslational modification where a three amino acid-peptide, glutathione, forms a disulfide bond with reactive cysteines of a protein thereby potentially changing the protein's biological functions. Glutaredoxins are members of the thioredoxin family and glutaredoxin-1 (GLRX) is the main deglutathionylating enzyme in the cells. We have previously demonstrated that GLRX status regulates the outcome of bleomycin- or AdTGFB-induced pulmonary fibrosis. However, whether GLRX status similarly regulates house dust mite (HDM)-induced fibrotic airway remodeling remains unexplored. Furthermore, mechanisms whereby glutathionylated proteins contribute to airway fibrosis also remain to be unraveled. In this dissertation, I demonstrated exacerbated fibrotic airway remodeling of Glrx-/- mice after HDM exposure. Glrx-/- airway epithelial cells have elevated epithelial-to-mesenchymal transition-like phenomenon, enhanced TGFB1 signaling, NOTCH pathway activation and altered differentiation potential when cultured under air-liquid interface conditions, with Glrx-/- mouse tracheal basal cells differentiating more readily to acetylated tubulin+ cells at the expense of Scgb1a1+ secretory cells. Enrichment of glutathionylated proteins from HDM-exposed mouse lungs revealed various glutathionylated extracellular matrix proteins, including glutathionylated collagen 1A1 (COL1A1-SSG). Importantly, COL1A1-SSG conferred resistance to collagenase-mediated degradation, and increased matrix stiffness. Exposure of epithelial cells to TGFB1 induced COL1A1-SSG, and fibroblasts grown on a glutathionylated collagen matrix display an activated, proliferative phenotype and increased expression of extracellular matrix crosslinking enzyme, lysyl oxidase like-2 (Loxl2). Fibroblasts grown on a glutathionylated collagen matrix also produced extracellular oxidants and released glutathione. Overall these findings point to a putative feedforward loop whereby glutathionylation of collagen, fibroblasts activation and resultant release of hydrogen peroxide and glutathione sustain an increased oxidative environment that drives airway fibrosis.