Original articleAge-dependent oxidation of extracellular cysteine/cystine redox state (Eh(Cys/CySS)) in mouse lung fibroblasts is mediated by a decline in Slc7a11 expression
Graphical abstract
Introduction
Reversible reduction and oxidation (redox) of the sulfur-containing amino acid cysteine (Cys) is exploited for a large number of biological processes [1]. Redox reactive Cys can be found as the free amino acid, as part of the thiol antioxidant glutathione (γ-glutamylcysteinylglycine; GSH), or as functional/regulatory sites within proteins [2]. Cys and its oxidized form, cystine (CySS), constitute a redox couple that can be expressed in terms of its redox potential, or Eh value. Likewise, GSH and its disulfide form, abbreviated GSSG, comprise another redox couple. These 2 couples are functionally connected but differentially regulated [3]. Cys and CySS are present in greater concentrations than GSH and GSSG outside of cells, whereas GSH and GSSG predominate within the intracellular compartment [4], [5]. In addition, each couple and each compartment are maintained at different redox potentials [6]. Therefore, it is important to specify which compartment is being considered when reporting redox potentials. Both intracellularly and extracellularly Cys/CySS and GSH/GSSG function as redox buffers to maintain redox homeostasis [2] and resist or facilitate oxidation of protein thiols to change protein functions and transduce signals [7], [8]. Thus, changes in redox potential can have a dramatic effect on cellular function. For example, oxidation of extracellular Eh(Cys/CySS) suppressed proliferation and inhibited signal transduction in Caco2 cells [9], [10], increased pro-inflammatory IL-1β in human monocytic U937 cells [11], and stimulated proliferation and pro-fibrotic gene expression in mouse lung fibroblasts [12].
Oxidation of the extracellular space is reflected in changes in plasma redox potentials. In vivo studies have shown that plasma Eh(Cys/CySS) was oxidized in mice with bleomycin-induced lung injury [13], and in rats with kainic acid and pilocarpine-induced epilepsy [14]. In humans, plasma Eh(Cys/CySS) was found to be more oxidized in adults chronically exposed to arsenic [15], adults acutely exposed to acetaminophen [16], and in children with autism [17]. Thus, oxidation of the extracellular environment, or redox stress, is associated with disease processes and environmental or pharmacological exposures.
Aging is a risk factor for development of a number of chronic diseases. One way in which aging may promote disease development or progression is by changing the set-point of the redox buffering systems. Aging is associated with a steady oxidation of plasma Eh(Cys/CySS) [18], but the mechanisms responsible are unclear. Cells in culture maintain an Eh(Cys/CySS) remarkably close to the redox potential of plasma [10], [19], [20], suggesting that cells are actively involved in controlling their immediate extracellular redox environment. Recently, we found that lung fibroblasts from old mice (24 months old) produced an extracellular Eh(Cys/CySS) that was more oxidized than that produce by their young counterparts (2 months old) [21].
Differential gene expression analysis revealed that Slc7a11 was down-regulated in old mouse lung fibroblasts [21]. Slc7a11 (also called xCT) is the light chain of system Xc- which transports CySS into cells and exports glutamate with 1:1 as the exchange ratio [22]. Previous studies have suggested that Slc7a11 expression is linked to control of the extracellular Cys/CySS redox state. Mice lacking Slc7a11 have a more-oxidizing extracellular Eh(Cys/CySS), as evidenced by an increase in their plasma CySS concentrations that is not balanced by a corresponding increase in plasma Cys [23]. Conversely, stimulation of B cell differentiation is accompanied by an upregulation of Slc7a11 and an increase in extracellular Cys concentration [24]. In the latter study there was also an increase in intracellular GSH, consistent with other studies showing that Slc7a11 activity supports intracellular GSH levels by supplying Cys, which is the rate-limiting amino acid for its synthesis [25]. The purpose of the present study was to determine whether down-regulation of Slc7a11 in fibroblasts from old mice was sufficient to explain the oxidation of the extracellular redox environment associated with aging, and to determine whether synthesis of intracellular GSH was a pre-requisite for this effect.
Section snippets
Reagents
Reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Corning (Manassas, VA) unless otherwise specified.
Primary lung fibroblasts culture
Lung fibroblasts were isolated from young (3 months) or old (24 months) female C57BL/6 mice as described previously [21]. Animal use was approved by the Institutional Animal Care and Use Committee of the University of Louisville. DMEM with 10% FBS and 1% antibiotic-antimycotic solution were used for regular cell culture [26]. Fibroblasts between passage numbers 8 and 15 were used in
Manipulation of Slc7a11 by pharmacological agents
Consistent with our previous studies [21], primary lung fibroblasts from old mice had lower expression of Slc7a11 (Fig. 1A) and more oxidized extracellular Eh(Cys/CySS) redox potential (Fig. 1B) relative to fibroblasts from young mice. To begin to assess whether expression level of Slc7a11 was responsible for the observed differences in the extracellular redox states of young and old fibroblasts, we treated old fibroblasts with sulforaphane, an Nrf2 inducer known to increase expression of
Discussion
The current studies confirm the finding that Slc7a11 expression was lower in primary lung fibroblasts from old mice than in those from young mice, and that this was associated with increased oxidation of extracellular Eh(Cys/CySS) redox potential [21]. We have now extended those findings by investigating the mechanistic link between these two observations. We found that up-regulation of Slc7a11 expression by either sulforaphane treatment or transient transfection was sufficient to restore the
Acknowledgments
Research reported in this publication was supported by Veterans Affairs Grant 5I01 BX000216-02 (Roman), National Institutes of Health grants R01 AA019953 (Roman) and U01 HL121807 (Roman), the National Institute on Alcohol Abuse and Alcoholism under Award no. P50AA024337-8305 (Roman), and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant no. P20GM113226-6176 (Watson).
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