Date of Award

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular, Molecular and Biomedical Sciences

First Advisor

Yvonne M. Janssen-Heininger

Abstract

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.

Language

en

Number of Pages

212 p.

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