The role of Slc7a11 in controlling extracellular and intracellular redox environments of lung fibroblasts - potential targets for intervention in aging and idiopathic pulmonary fibrosis.

Author

Yuxuan Zheng, University of LouisvilleFollow

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 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 (E_h) quantify oxidative stress. Fibroblasts from old mice maintain more oxidized extracellular E_h(Cys/CySS) than young mice. Microarray shows down-regulation of Slc7a11 potentially mediates this age-related oxidation. Slc7a11 is the key component of system X_c^-, 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 E_h(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 E_h(Cys/CySS) and intracellular E_h(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. E_h(Cys/CySS) and E_h(GSH/GSSG) were more oxidized in conditioned media of old fibroblasts. Up-regulation of Slc7a11 reduced extracellular E_h(Cys/CySS) for old fibroblasts. Inhibition of GSH synthesis had no effect on the ability of cells to restore their extracellular E_h(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 E_h(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 E_h(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.

Recommended Citation

Zheng, Yuxuan, "The role of Slc7a11 in controlling extracellular and intracellular redox environments of lung fibroblasts - potential targets for intervention in aging and idiopathic pulmonary fibrosis." (2020). Electronic Theses and Dissertations. Paper 3428.
https://doi.org/10.18297/etd/3428