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Review
. 2013 Feb 10;18(5):522-55.
doi: 10.1089/ars.2011.4391. Epub 2012 Aug 3.

The cystine/glutamate antiporter system x(c)(-) in health and disease: from molecular mechanisms to novel therapeutic opportunities

Jan Lewerenz  1 , Sandra J Hewett, Ying Huang, Maria Lambros, Peter W Gout, Peter W Kalivas, Ann Massie, Ilse Smolders, Axel Methner, Mathias Pergande, Sylvia B Smith, Vadivel Ganapathy, Pamela Maher
Affiliations
Review

The cystine/glutamate antiporter system x(c)(-) in health and disease: from molecular mechanisms to novel therapeutic opportunities

Jan Lewerenz et al. Antioxid Redox Signal. .

Abstract

The antiporter system x(c)(-) imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system x(c)(-) is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system x(c)(-), including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system x(c)(-). Moreover, the roles of system x(c)(-) in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system x(c)(-) inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System x(c)(-) is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system x(c)(-) in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS.

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Figures

FIG. 1.
FIG. 1.
Glutathione (GSH) metabolism. Cystine (CySS−) is taken up by system xc− (xc−). Intracellularly, CySS is reduced to cysteine (Cys) by thioredoxin reductase 1 (TRR1) or GSH. Glutamate cysteine ligase (GCL) catalyzes the synthesis of γ-glutamyl cysteine (γ-GC) from glutamate (Glu) and Cys, and glutathione synthase (GS) generates GSH by adding glycine (Gly). GSH reduces radicals (R•) nonenzymatically and organic hydroperoxides catalyzed by GSH peroxidase (GPx) and is thereby converted to GSH disulfide (GSSG). GSSG is recycled to GSH by GSH reductase (GR), a reaction that uses reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a co-factor. GSH S-transferase (GST) forms GSH adducts (GS-R) from organic molecules (R) and GSH, which along with GSH and GSSG are exported from the cell by multi-drug resistance proteins (MRP). The ecto-enzyme γ-glutamyl transferase (GGT) transfers the γ-glutamyl moiety of GSH to an acceptor amino acid (AA), leading to cysteinyl glycine (CysGly), which is cleaved by a dipeptidase (DP) to Cys and Gly. Both GGT and DP are membrane-bound enzymes. Cys is either taken up by cysteine transporters, among them, system alanine-serine-cysteine (ASC), or extracellularly oxidized to CySS−, which is again taken up by system xc−.
FIG. 2.
FIG. 2.
System xc−. System xc− is composed of the 4F2 heavy chain (4F2hc) and the light chain, xCT, which are linked by a disulfide bond (-S-S-). System xc− imports cystine (CySS−) in exchange for glutamate (Glu).
FIG. 3.
FIG. 3.
Phylogenetic analysis. Phylogenetic analysis of xCT/SLC7A11 orthologs and related heterodimeric amino acid transporter (HAT) proteins. xCT proteins from vertebrates, cephalochordata, hemichordata, and echinodermata; five putative Drosophila melanogaster LAT1/2 or xCT homologs; the three nonvertebrate HAT light-chain proteins from Caenorhabditis elegans and Schistosoma japonensis with a characterized function; and HAT orthologs from four evolutionarily separated vertebrates were included in the analysis. The distantly related SLC7A14 transporter was used as an outlier. Proteins were identified by BLAST search and used to generate multiple sequence alignments. The output was then used to generate a phylogenetic analysis using Maximum Likelihood and 100 bootstrap steps. The number of bootstrap iterations resulting in the branching shown is given as a number in front of each branch. The scale bar shows 0.2 (or 20%) sequence divergence (for methods see Supplementary Data [available online at www.liebertpub.com/ars]).
FIG. 4.
FIG. 4.
Transcriptional regulation of xCT expression. A variety of stimuli (see section I.B), for example, electrophiles, heavy metals, and reactive oxygen species (ROS), lead to activation of the nuclear factor NF-E2-related factor 2 (Nrf2), which binds to the electrophile response element (EpRE) within the xCT promoter region and activates transcription. Amino acid (AA) starvation leads to phosphorylation of eIF2α (peIF2α), which leads to the translational up-regulation of the transcription factor activating transcription factor 4 (ATF4). ATF4 activates the transcription of xCT by binding to the amino acid response element (AARE) contained in the xCT promoter. Bacterial lipopolysaccharides (LPS), tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), fibroblast growth factor-2 (FGF2), and erythropoietin (EPO) also increase the transcription of xCT through unknown or partially known signaling pathways. IL-1β acts via the IL-1 receptor (IL-1R), while FGF2 activates the FGF receptor 1 (FGFR1) and increases xCT transcription via PI3K and MEK. MicroRNA-26b directly targets xCT mRNA.
FIG. 5.
FIG. 5.
Regulation of system xc− by substrate availability. Glutamate released by system xc− is taken up by excitatory amino acid transporters (EAAT). Intracellular glutamate, which also can be synthesized from aspartate (Asp) and glutamine (Gln), fuels import of the anionic form of cystine (CySS−) by system xc−. Extracellular protons (H+) lead to the formation of neutral cystine (CySS), which is not accepted as a substrate by system xc−.
FIG. 6.
FIG. 6.
System xc− regulates the extracellular cystine/cysteine redox couple. Cystine (CySS−) imported by system xc− is intracellularly reduced to cysteine (Cys) by thioredoxin reductase 1 and/or GSH (TRR1/GSH). Cys can be directly exported by system ASC; therefore, system xc− changes the ratio of the extracellular CySS/Cys redox couple in favor of Cys.
FIG. 7.
FIG. 7.
Cell density and disulfide exchange of cystine with extracellular GSH regulate sensitivity of HT22 cells to system xc− inhibition by glutamate. (A) HT22 cells were seeded at the indicated densities per well in 96-well plates, and glutamate was added after 24 h. (B) After 24 h in culture, HT22 cells were treated with the indicated concentrations of GSH in the absence of glutamate in a normal medium (Ctrl), along with 10 mM glutamate (10 mM Glu) or with a medium exchanged for a cystine-free medium (-Cystine). (A/B) Survival was measured by the MTT assay after 24 h and normalized to cells not treated with glutamate (A) or GSH (B). Graphs represent the means of three independent experiments.
FIG. 8.
FIG. 8.
Oxidative glutamate toxicity—neuronal cell death induced by system xc− inhibition in vitro. In hippocampal HT22 cells, glutamate-mediated inhibition of cystine uptake via system xc− causes a decrease of intracellular GSH. GSH levels below 20% lead to an increase of ROS. In the early phase, de novo synthesis of RNA and proteins is necessary. One of the proteins induced is GADD45α. 12-lipoxygenase (12-LOX) and soluble guanylate cyclase are activated and mediate an accumulation of ROS and cGMP, and, subsequently, Ca++ influx. Ca++ induces cell death mediated by truncated BH3-interacting domain death agonist (Bid) and nuclear translocation of apoptosis-inducing factor (nAIF) (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)
FIG. 9.
FIG. 9.
System xc− expressed in macrophages regulates T-cell proliferation and activity through cysteine release. Macrophages import cystine (CySS−) via system xc−. Intracellularly, cystine is reduced to cysteine (Cys) by GSH or thioredoxin reductase 1 (TRR1/GSH), which is subsequently released to the extracellular space and taken up by T lymphocytes for GSH synthesis.
FIG. 10.
FIG. 10.
GSH-mediated chemoresistance in tumor cells though system xc−. In cancer cells, GSH is synthesized from cystine (CySS−) taken up via system xc−. GSH protects cells from ROS induced by anticancer drugs (Drug) and/or is used as a co-substrate for GSTs, which form GSH-drug adducts (GS-Drug). GSH adducts are exported from the cell by MRP.
FIG. 11.
FIG. 11.
System xc− sensitizes tumor cells to selenite toxicity. Cystine taken up by tumor cells is intracellularly reduced to cysteine, which is subsequently exported from the cell. Extracellular cysteine reduces selenite, probably to selenide, which is subsequently imported into the cell by an unknown transport mechanism, where it induces cytotoxicity.
FIG. 12.
FIG. 12.
The anatomy of the eye.
FIG. 13.
FIG. 13.
The anatomy of the retina. (a) Hematoxylin-eosin stained section of the retina. The outermost layer is the retinal pigment epithelium (RPE). The microvillous processes of RPE cells interdigitate with the outer segments (OS) of adjacent photoreceptor cells. The cell bodies of the photoreceptor cells, known as rods and cones, constitute the outer nuclear layer (ONL). Photoreceptor cells synapse in the outer plexiform layer (OPL) with bipolar cells. Bipolar cells, horizontal cells, and amacrine cells have their cell bodies in the inner nuclear layer (INL). Axons of the bipolar cells synapse in the inner plexiform layer (IPL) with dendrites of the ganglion cells (gcl). (b) Immunohistochemical labeling with antibodies against vimentin of the radially oriented fibers of Müller cells that span the entire retina.
FIG. 14.
FIG. 14.
Distribution of xCT immunoreactivity in the corneal epithelium and the ciliary body. Hematoxylin-eosin stained sections of (a) the cornea and (c) the ciliary body (cb). (a) The corneal epithelium (EP) is comprised of a layer of columnar cells (c) attached to the Bowman's membrane (BM), wing cells (w), and superficial cells (s). The other layers of the cornea, from outside to inside, are the stroma (ST), the Descemet's membrane (DM), and the endothelial layer (EN). (B/D) The distribution of xCT immunoreactivity in (b) the corneal epithelium and (d) the ciliary body (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars.)
FIG. 15.
FIG. 15.
Hypothetical roles of system xc−in diseases of the central nervous system. (Left panel) In the healthy brain, glutamate release by system xc− (here only depicted in astrocytes, as conflicting data about neuronal expression have been published) is balanced by glutamate uptake by EAATs, which leads to negligible activation of ionotropic glutamate receptors (GluR). (Right panel) In many disease states of the brain, oxidative stress is present (ROS), which might lead to the up-regulation of system xc− and subsequent increased glutamate release. Activated microglia may also contribute to the increase in glutamate release via system xc− (not shown). Simultaneously, EAATs are down-regulated. The increased extracellular glutamate concentration activates ionotropic glutamate receptors and induces excitotoxicity.
FIG. 16.
FIG. 16.
Glutamate released by system xc− modulates synaptic activity. Glutamate released into the synaptic cleft is taken up by astrocytic glutamate transporters (EAAT) Extrasynaptically, glutamate released by system xc− activates presynaptic metabotropic glutamate receptors 2 and 3 (mGluR2/3) and thereby reduces the release probability of vesicular glutamate into the synaptic cleft. Postsynaptically, glutamate released by system xc− activates metabotropic glutamate receptor 5 (mGluR5). As a result, long-term depression (LTD) is permitted. The cyst(e)ine pro-drug N-acetyl cysteine can increase extracellular cystine, thereby activating glutamate release by system xc− and subsequently increasing signaling through pre- and postsynaptic mGluRs.
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