Date on Master's Thesis/Doctoral Dissertation

5-2020

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Watson, Walter H.

Committee Co-Chair (if applicable)

Roman, Jesse

Committee Member

Roman, Jesse

Committee Member

States, J. Christopher

Committee Member

Cai, Lu

Committee Member

Chen, Shao-yu

Committee Member

Hoyle, Gary W.

Author's Keywords

Slc7a11; redox potential; redox state; fibroblast; idiopathic pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by extracellular matrix deposition by fibroblasts. Aging and oxidative stress increase the susceptibility to IPF. Redox couples, cysteine/cystine (Cys/CySS) and glutathione/glutathione disulfide (GSH/GSSG), and their redox potentials (Eh) quantify oxidative stress. Fibroblasts from old mice maintain more oxidized extracellular Eh(Cys/CySS) than young mice. Microarray shows down-regulation of Slc7a11 potentially mediates this age-related oxidation. Slc7a11 is the key component of system Xc-, an antiporter that imports CySS and exports glutamate. The first aim of this dissertation is to investigate the mechanistic link between Slc7a11 expression and extracellular Eh(Cys/CySS). The second aim is to evaluate the effects of aging on the redox states of intracellular proteins and whether Slc7a11 contributes to the age-dependent effects. The last aim is to compare SLC7A11 expression, extracellular Eh(Cys/CySS) and intracellular Eh(GSH/GSSG) between human lung fibroblasts from IPF and non-IPF donors and to explore their association with pro-fibrotic gene expression. Slc7a11 expression was manipulated by pharmacological and genetic methods. Reduced and oxidized forms of Cys residues were labelled by Iodoacetyl Tandem Mass Tags. The ratio of oxidized/reduced forms (i.e., redox state) of a Cys residue was determined by multiplexed tandem mass spectrometry. Eh(Cys/CySS) and Eh(GSH/GSSG) were more oxidized in conditioned media of old fibroblasts. Up-regulation of Slc7a11 reduced extracellular Eh(Cys/CySS) for old fibroblasts. Inhibition of GSH synthesis had no effect on the ability of cells to restore their extracellular Eh(Cys/CySS). Redox states of 151 proteins changed with aging. Slc7a11 over-expression restored redox states of 104 proteins. Ingenuity Pathway Analysis showed these 104 proteins were involved in pathways of protein translation initiation, ubiquitin-proteasome-mediated degradation and integrin-cytoskeleton-associated signaling. Slc7a11 expression was lower in IPF fibroblasts. Extracellular Eh(Cys/CySS) was more oxidized and expression of pro-fibrotic genes was higher in IPF fibroblasts. In conclusion, Slc7a11 is the key regulator of extracellular Eh(Cys/CySS). Its effects are independent of GSH synthesis. Aging results in changes of redox states of proteins involved in protein turnover and cytoskeleton dynamics. Up-regulating Slc7a11 restores changes of protein redox states due to aging. Decreased SLC7A11 might represent a susceptibility factor for developing tissue disrepair and fibrosis in IPF.

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