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A6350 - Alterations in the Expression of Slc7a11 in Murine Lung Fibroblasts Is Responsible for Aging-Dependent Oxidation of Cysteine/Cystine Redox State (Eh Cys/CySS)
Author Block: J. Ritzenthaler, Y. Zhang, T. J. Burke, J. Otero, J. Roman, W. H. Watson; Medicine, University of Louisville, Louisville, KY, United States.
Rationale: The redox state of human plasma, defined by the concentrations of cysteine (Cys) and cystine (CySS), becomes more oxidized with age, and this may contribute to increased susceptibility to environmental insults. We previously showed that fibroblasts isolated from the lungs of young and old mice retained this differential phenotype; old cells produced and maintained a more oxidizing extracellular redox potential (Eh Cys/CySS) than young cells. Microarray analysis identified down-regulation of Slc7a11, the light subunit of the CySS/glutamate transporter, as a potential mediator of age-related oxidation in these cells. Thus, we set out to investigate the mechanistic link between Slc7a11 expression and extracellular Eh(Cys/CySS). Methods: Sulforaphane treatment or transient transfection with a plasmid encoding Slc7a11 were used to increase expression levels of Slc7a11 in lung fibroblasts isolated from old (18-24 mo. old) C57BL/6 mice, wherease sulfasalazine treatment or siRNA-mediated knock down were used to decrease activity and expression of Slc7a11 in lung fibroblasts isolated from young (3-4 mo. old) mice. L-buthionine-sulfoximine (BSO) was used to inhibit cellular glutathione (GSH) synthesis. Slc7a11 mRNA levels were measured by qPCR, concentrations of Cys, CySS, GSH, glutathione disulfide (GSSG) and CySSG were measured by HPLC, and their redox potentials (Eh) were calculated from the Nernst equation. Results: Both extracellular Eh Cys/CySS and Eh GSH/GSSG were more oxidized in old cells than in young cells. Up-regulation of Slc7a11 via pharmacological or genetic means resulted in a more reducing extracellular Eh Cys/CySS, whereas its down-regulation produced a more oxidizing Eh(Cys/CySS). Up-regulation via sulforaphane increased total GSH and restored Eh GSH/GSSG, while up-regulation via overexpression plasmid had no effect on these parameters. Neither siRNA-mediated knock down of Slc7a11 nor sulfasalazine inhibition of Slc7a11 activity influenced total GSH. Inhibition of GSH synthesis with BSO had no effect on the ability of cells to restore their extracellular redox potential in response to an oxidative challenge. Alterations in the expression of Slc7a11 not only resulted in oxidation of Eh Cys/CySS, but also in upregulation of fibronectin, a matrix glycoprotein implicated in tissue disrepair. Conclusions: These observations reveal that Slc7a11 is the key regulator of extracellular Eh Cys/CySS in lung fibroblasts, and its effects are not dependent on intracellular GSH synthesis. Down regulation of Slc7a11 may be responsible for oxidative stress and upregulation of fibronectin in aging lungs, thereby increasing susceptibility to tissue disrepair after injury.