Original article
Age-dependent oxidation of extracellular cysteine/cystine redox state (Eh(Cys/CySS)) in mouse lung fibroblasts is mediated by a decline in Slc7a11 expression

https://doi.org/10.1016/j.freeradbiomed.2018.02.026Get rights and content

Highlights

  • Aging is associated with decreased expression and activity of Slc7a11.

  • Decreased Slc7a11 is responsible for oxidation of extracellular Eh(Cys/CySS).

  • Slc7a11 restoration in old cells is sufficient to reduce extracellular Eh(Cys/CySS).

  • Regulation of extracellular Eh(Cys/CySS) by Slc7a11 is not dependent on glutathione.

Abstract

Aging is associated with progressive oxidation of the extracellular environment. The redox state of human plasma, defined by the concentrations of cysteine (Cys) and cystine (CySS), becomes more oxidized as we age. Recently, we showed that fibroblasts isolated from the lungs of young and old mice retain this differential phenotype; old cells produce and maintain 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. The purpose of the present study was to investigate the mechanistic link between Slc7a11 expression and extracellular Eh(Cys/CySS). Sulforaphane treatment or overexpression of Slc7a11 was used to increase Slc7a11 in lung fibroblasts from old mice, and sulfasalazine treatment or siRNA-mediated knock down was used to decrease Slc7a11 in young fibroblasts. Slc7a11 mRNA levels were measured by real-time PCR, Slc7a11 activity was determined by measuring the rate of glutamate release, Cys, CySS, glutathione (GSH) and its disulfide (GSSG) were measured by HPLC, and Eh(Cys/CySS) was calculated from the Nernst equation. The results showed that both Eh(Cys/CySS) and Eh(GSH/GSSG) were more oxidized in the conditioned media of old cells than in young cells. Up-regulation of Slc7a11 via overexpression or sulforaphane treatment restored extracellular Eh(Cys/CySS) in cultures of old cells, whereas down-regulation reproduced the oxidizing Eh(Cys/CySS) in young cells. Only sulforaphane treatment was able to increase total GSH and restore Eh(GSH/GSSG), whereas overexpression, knock down and sulfasalazine had no effect on these parameters. In addition, inhibition of GSH synthesis with buthionine sulfoximine had no effect on the ability of cells to restore their extracellular redox potential in response to an oxidative challenge. In conclusion, our study reveals Slc7a11 is the key regulator of age-dependent changes in extracellular Eh(Cys/CySS) in primary mouse lung fibroblasts, and its effects are not dependent on GSH synthesis.

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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