Redox outside the Box: Linking Extracellular Redox Remodeling with Intracellular Redox Metabolism*

Aerobic organisms generate reactive oxygen species as metabolic side products and must achieve a delicate balance between using them for signaling cellular functions and protecting against collateral damage. Small molecule (e.g. glutathione and cysteine)- and protein (e.g. thioredoxin)-based buffers regulate the ambient redox potentials in the various intracellular compartments, influence the status of redox-sensitive macromolecules, and protect against oxidative stress. Less well appreciated is the fact that the redox potential of the extracellular compartment is also carefully regulated and is dynamic. Changes in intracellular metabolism alter the redox poise in the extracellular compartment, and these are correlated with cellular processes such as proliferation, differentiation, and death. In this minireview, the mechanism of extracellular redox remodeling due to intracellular sulfur metabolism is discussed in the context of various cell-cell communication paradigms.

Aerobic organisms generate reactive oxygen species as metabolic side products and must achieve a delicate balance between using them for signaling cellular functions and protecting against collateral damage. Small molecule (e.g. glutathione and cysteine)-and protein (e.g. thioredoxin)-based buffers regulate the ambient redox potentials in the various intracellular compartments, influence the status of redox-sensitive macromolecules, and protect against oxidative stress. Less well appreciated is the fact that the redox potential of the extracellular compartment is also carefully regulated and is dynamic. Changes in intracellular metabolism alter the redox poise in the extracellular compartment, and these are correlated with cellular processes such as proliferation, differentiation, and death. In this minireview, the mechanism of extracellular redox remodeling due to intracellular sulfur metabolism is discussed in the context of various cell-cell communication paradigms.
Life on oxygen necessitated the evolution of antioxidant systems to protect against overoxidation and to combat reactive oxygen species (ROS) 2 generated as side products from a leaky electron transfer chain. ROS are also produced in a multitude of other oxygen-metabolizing reactions and, at low levels, serve in biological signaling pathways (1,2). At elevated levels, ROS are correlated with a plethora of complex diseases, including cardiovascular and neurodegenerative diseases and cancers (3). In addition to their widely recognized role in countering oxidative threats, antioxidant systems are vitally important for poising the intra-and extracellular redox milieus, both of which are maintained far from equilibrium. Small molecule-and proteinbased redox buffer systems, including GSH/GSSG, cysteine/ cystine, and oxidized/reduced thioredoxin, are used for thiolbased redox homeostasis essential for controlling cellular functions, e.g. gene expression, cell cycle progression, and apoptosis (4). In proteins, methionine and cysteine are the two common amino acids that can be reversibly oxidized, and their redox status can influence protein conformation and/or stabil-ity, enzymatic activity, and interactions with other protein and/or DNA partners. Oxidation of methionine leads to methionine sulfoxide, which can be reversed enzymatically, or to methionine sulfone, an overoxidized species that cannot be rescued (5). Oxidation of cysteine can lead to formation of sulfenic, sulfinic, or sulfonic acid, the latter of which represents an irreversible modification. In addition to small molecule repair systems, antioxidant enzymes are utilized for maintaining the integrity of functionally critical redox-active amino acids and include sulfiredoxin (6), methionine sulfoxide reductase (5), and thioredoxin (7), whereas enzymes such as superoxide dismutase, glutathione peroxidase, and catalase keep ROS levels at bay.
The cellular redox status governs the equilibrium between oxidized versus reduced states of cysteines and methionines. The more oxidizing environment of the endoplasmic reticulum (ER) and extracellular compartment permits a higher prevalence of oxidized cysteines. Indeed, an entire machinery exists in the ER for introducing disulfides for proper folding and functioning of secreted proteins. Similarly, the extent of steady-state thiol modifications (e.g. glutathionylation and cysteinylation) is modulated by the ambient redox potential of the cellular compartment. A shift in the steady-state redox potential driven, for example, by metabolic changes (e.g. sulfur amino acid paucity) or environmental insults (e.g. oxidative stress) can impact the distribution of reduced versus oxidized protein thiols. Indeed, based on the Nernst equation, for every 30-mV change in the redox potential, an ϳ10-fold shift in the equilibrium between protein dithiols and disulfides is expected. Proteins with regulatory thiol/disulfide switches are susceptible to shifts in redox state.
Although the importance of redox buffers for maintaining a reducing intracellular environment is well appreciated, the extracellular compartment is generally viewed as an oxidizing microenvironment that is redox-inert. Emerging studies are changing this view, however, by revealing that the extracellular redox environment is dynamic and is sculpted by intracellular metabolism. Because many signaling pathways involved in autocrine and paracrine regulation emanate from the cell surface, the dynamics of extracellular redox buffering are likely to have a profound effect on cell-cell communication and function. In this minireview, we focus on thiol-based redox buffering beyond the membrane and its regulation by a metabolic circuitry within the membrane.

Redox Nodes
The primary intracellular redox hubs that interface with redox-active thiol-containing macromolecular targets are oxidized/reduced thioredoxin, GSH/GSSG, and cysteine/cystine (Fig. 1). Estimates of the cytoplasmic redox potentials reveal that these systems are not in equilibrium with each other and suggest instead that they are likely to be under kinetic control (Fig. 1A). Thus, the steady-state redox potential for the oxidized/reduced thioredoxin couple in proliferating cells is between Ϫ270 and Ϫ280 mV and is largely unperturbed in response to cues such as differentiation and apoptosis (8). The GSH/GSSG redox potential ranges between Ϫ260 mV (in proliferating cells) and Ϫ220 mV (in differentiated cells) and is shifted further in the oxidative direction during apoptosis or in response to sulfur amino acid starvation (8,9). The cysteine/ cystine couple is the most oxidized at Ϫ160 mV in proliferating cells and Ϫ125 mV in differentiated cells but is unresponsive to cysteine deficiency (10). Because the GSH/GSSG redox couple is the most dynamic of the three redox nodes, it has been suggested that it functions as a switchable node that can be either oxidizing or reducing depending on the status of cells during the aging process, during the cell cycle, or in response to environmental triggers (11). In contrast, the relative stabilities of the reduced/oxidized thioredoxin and cysteine/cystine couples lend themselves better to their predominant roles as reducing and oxidizing nodes, respectively.
In the extracellular space, the cysteine/cystine redox couple is quantitatively the most significant. In normal human plasma, the concentrations of cysteine (8 -10 M) and cystine (40 -50 M) (12) are considerably higher the those of GSH (2.8 M) and GSSG (0.14 M) (13). These correspond to redox potentials of Ϫ80 mV for the cysteine/cystine couple and Ϫ140 mV for the GSH/GSSG couple. A linear age-dependent increase in the plasma cysteine/cystine redox potential (at a rate of 0.14 mV/year) is observed for individuals between 19 and 85 years of age, whereas the GSH/GSSG redox potential is stable for the first 45 years and thereafter becomes more oxidized at a linear rate of 0.7 mV/year (12). Mammalian cells in culture hold the extracellular cysteine/cystine redox potential at approximately Ϫ80 mV, which shifts in the reductive or oxidative direction depending on whether the cells are ready for proliferation or death ( Fig. 1A) (14,15).

Intracellular Sulfur Metabolism Modulates Extracellular Redox Potential
Cysteine is derived from methionine via the transsulfuration pathway or can be obtained via import of cysteine or cystine (followed by reduction) (Fig. 1B). The transsulfuration pathway comprises two pyridoxal 5Ј-phosphate-dependent enzymes, cystathionine ␤-synthase and ␥-cystathionase, which successively convert homocysteine, a byproduct of the methionine cycle, to cystathionine and cysteine, respectively. In liver, where this pathway is best characterized, it serves as an overflow route for disposal of excess sulfur and is activated by S-adenosylmethionine, an indicator of sulfur sufficiency (16). Flux through the transsulfuration pathway is also activated under oxidative stress conditions (17,18). Cystathionine ␤-synthase represents a regulatory focal point and is subjected to sumoylation (19) and allosteric (20,21), intrasteric (22), and hormonal (23,24) regulation. The second major homocysteine-utilizing enzyme, methionine synthase, conserves methionine via a transmethylation reaction. It utilizes 5-methyltetrahydrofolate as a substrate and is dependent on a vitamin B 12 cofactor for catalysis. Because both homocysteine-utilizing enzymes are dependent on B vitamins (B 12 , B 6 , and folic acid), sufficiency or deficiency of these nutrients can modulate flux through the sulfur pathway.
In contrast to the 1-10 mM steady-state concentrations of GSH found inside cells, cysteine concentrations are approximately an order of magnitude lower and limit GSH synthesis. The first of two steps in the GSH synthesis pathway is catalyzed by ␥-glutamylcysteine ligase, the rate-limiting enzyme. The second enzyme, GSH synthetase, catalyzes the ATP-dependent addition of glycine to ␥-glutamylcysteine to generate GSH. In addition to supporting antioxidant and xenobiotic clearance (via the action of GSTs) functions, GSH also serves as a repository of cysteine. GSH is rapidly turned over in many cell types, with half-lives varying from 2 to 6 h, which suggests a high export rate (25).
Members of the ABCC (ATP-binding cassette C) family of multidrug resistance-associated proteins (primarily MRP1 and MRP2 and, possibly, CFTR (cystic fibrosis transmembrane conductance regulator)) handle extrusion of GSH from cells (26). Under oxidative stress conditions, GSSG can be excreted via FIGURE 1. Changes in redox potentials and cellular function. A, the steadystate cytoplasmic redox potentials for the three major thiol-based buffers in differentiated cells are shown inside the box, whereas the extracellular redox potentials for the GSH/GSSG and cysteine/cystine redox couple are shown outside. Shifts in the reductive and oxidative directions are associated with proliferation and apoptosis, respectively. Trx red /Trx ox , reduced/oxidized thioredoxin. B, the GSH/cysteine cycle connects intra-and extracellular GSH and cysteine pools. Cysteine and cystine can be transported via Na ϩ -dependent and Na ϩ -independent transporters. Although the mechanisms of cystine reduction are not fully understood, it is reduced in the intracellular milieu, e.g. by GSH or thioredoxin. Alternatively, cysteine can be produced from homocysteine, which is a product of the methionine cycle, via the transsulfuration pathway catalyzed by cystathionine ␤-synthase (CBS) and ␥-cystathionase (CSE). Cysteine is incorporated into GSH via the actions of ␥-glutamylcysteine ligase (GCL) and glutathione synthetase (GS). The transmembrane GSH/cysteine cycle, shown in red, results in circulation of intracellular GSH to the outside, where it is cleaved by the actions of ␥-GT and a dipeptidase (DP). X and X-CH 3 represent a methyl group acceptor and a methylated acceptor, respectively. these transporters. Despite the large quantities of GSH that are secreted into plasma, the concentration of GSH in circulation is low. The ectoenzyme ␥-glutamyl transpeptidase (␥-GT), which is widely distributed in animal tissues (27), is important for maintaining low extracellular GSH. ␥-GT catalyzes the cleavage of GSH into glutamate and cysteinylglycine, and the latter is converted by a dipeptidase to its component amino acids. The amino acids released via this mechanism can remain in circulation or can be taken up locally or at a distance. The integration of intra-and extracellular sulfur metabolism via the transcellular GSH/cysteine cycle (Fig. 1B) permits modulation of the extracellular redox poise by intracellular metabolic changes. Potential mechanisms for influencing the extracellular redox potential include changing the rate of cystine consumption, glutathione extrusion, and glutathione cleavage. Systemically, the release of large quantities of GSH from the liver serves as a source of cysteine for extrahepatic tissues.
A comparison of postprandial plasma cysteine/cystine redox potential showed that a meal with sulfur amino acids causes a 10-mV reductive shift compared with one missing these amino acids (28). This study reveals both the dynamic nature of the systemic extracellular cysteine/cystine redox potential and its modulation by diet.

Cysteine/Cystine Transporters
Multiple transporters with tissue-specific distribution exist for the import of cystine, the predominant form of the amino acid in circulation, and for cysteine, which is present at substantially lower concentrations (29,30). Broadly, the transporters can be categorized into Na ϩ -dependent and Na ϩ -independent types (Fig. 1B). The X AG Ϫ or excitatory amino acid transporters are typically known for their high-affinity Na ϩ -dependent glutamate uptake activity and have been extensively characterized in the CNS. There are five excitatory amino acid transporter subtypes designated 1-5, with different cellular distributions. In contrast to the high affinity for glutamate (K m ϭ 9 M), the affinity for cystine (K m ϭ 470 M (31)) and cysteine (96 M (32)) is modest. Na ϩ -dependent uptake of cysteine occurs via system ASC (K m ϭ 17 M (32)), which also serves as a transporter for alanine, serine, and threonine.
System b 0ϩ catalyzes the Na ϩ -independent import of cystine (K m ϭ 30 M (33)) and is important in the renal absorption of cystine and neutral and dibasic amino acids. Mutations in system b 0ϩ lead to cystinuria. The Na ϩ -independent x C Ϫ antiporter uses the transmembrane glutamate gradient to drive import of cystine (K m ϭ 11 M (32)). The two amino acids are exchanged in a 1:1 stoichiometry. Inefficient import of cystine both by naïve T cells (34) and by neurons makes them metabolically dependent on dendritic cells (35) and astrocytes (36), respectively, for meeting their cysteine needs. In both instances of intercellular dependences, the transmembrane GSH/cysteine cycle furnishes extracellular cysteine for uptake.
Transporters that can release intracellular cysteine are poorly characterized. The system L-like neutral amino acid antiporter LAT2, which has limited tissue distribution, exchanges extracellular neutral amino acids (with K m values in the micromolar range) for intracellular cysteine and other amino acids (with millimolar K m ) (29, 37).

Redox Modulation in Extracellular Compartment
The cytoplasmic and extracellular redox potentials of the primary thiol buffers are vastly different (Fig. 1A) and influence the structure, stability, and function of macromolecules that reside within each compartment. The redox status of exofacial membrane proteins, receptors, and transporters should be sensitive to the cysteine/cystine redox couple and influence, in turn, their stability and/or function.
A number of studies have revealed systemic changes in the plasma or extracellular GSH/GSSG and cysteine/cystine redox couples. More oxidized potentials are correlated with aging, smoking, cardiovascular disease, and diabetes (11). Emerging evidence in several heterocellular systems that are metabolically coupled is revealing that the basis for extracellular redox climate dynamics is a change in intracellular redox metabolism. In the following section, a few examples of extracellular redox remodeling that occurs during intercellular communication that have been described by the author's laboratory are discussed.
An example of the interplay between redox metabolism and intercellular signaling involves cells of the adaptive immune system, which are mobilized into action when an infection overwhelms innate defense mechanisms. An early step in this process is engagement of naïve T cells with antigen-presenting cells in immune synapses, which elicits a flurry of signaling and leads to activation and subsequent clonal expansion of T cells. Very low expression of the x C Ϫ antiporter makes T cells inefficient in importing cystine and metabolically dependent on antigen-presenting cells for cysteine needed for activation (34,38). In contrast to naïve T cells, antigen-presenting cells, e.g. dendritic cells, express the x C Ϫ antiporter, are proficient in cystine uptake, and further up-regulate expression of x C Ϫ upon co-cul-FIGURE 2. Intercellular redox signaling and metabolite changes. A, redox remodeling during T cell activation. Induction of the x C Ϫ transporter in dendritic cells leads to increased consumption of cystine, which, via the ␥-glutamyl cycle, is converted to extracellular cysteine, which is imported by T cells and used in GSH biosynthesis. The reductive shift in the extracellular redox potential during T cell activation has been documented to increase the proportion of thiols on exofacial membrane protein domains in dendritic cells and T cells. B, activated T cells secrete large quantities of glutamate that are efficiently cleared via the X AG Ϫ transporter on astrocytes. In turn, this activates the x C Ϫ transporter, stimulating cystine consumption and GSH synthesis, secretion, and cleavage. Cysteine derived from extracellular GSH is taken up by neurons and converted to GSH and serves a neuroprotective function. Although the status of membrane protein thiols under these co-culture conditions has not been characterized, the reductive shift in the extracellular compartment is expected to increase the proportion of thiols versus disulfides and may be important in initiating or curtailing signaling pathways. The upward and downward red arrows indicate increases and decreases, respectively. ture with T cells (Fig. 2A) (14). The net outcome of these changes is a shift in the extracellular microenvironment to a more reducing potential. Metabolic labeling and pharmacological inhibition studies have revealed that the mechanism for conversion of extracellular cystine imported by dendritic cells to cysteine involves the transmembrane GSH/cysteine cycle. The increased cystine consumption and cysteine release by dendritic cells result in an approximately Ϫ30-mV decrease in the extracellular cysteine/cystine redox potential to Ϫ110 mV (14). In contrast, when dendritic cells and naïve T cells are cultured separately, the extracellular redox potentials are approximately Ϫ80 and Ϫ50 mV, respectively, consistent with dendritic cells being differentiated and T cells being fated for apoptosis in the absence of activation. The reductive remodeling of the extracellular environment is conducive for T cell proliferation and also causes a significant shift in the membrane thiol status as visualized by Alexa maleimide labeling of cell surface thiols (14). Hence, within 1 h of co-culture, a substantial increase in labeling is seen on the surfaces of dendritic cells and T cells, increasing by 4-and 8-fold, respectively in 48 h. The identities of the redox-sensitive proteins that are affected by the shift in the extracellular redox potential and the impact of this redox change on membrane protein functions and signaling pathways need to be elucidated.
Regulatory T cells thwart activation of effector T cells as part of a natural surveillance mechanism for keeping autoimmunity in check (39). One strategy for immunosuppression deployed by regulatory T cells is to interfere with extracellular redox remodeling during T cell activation (14,40). Remarkably, addition of cysteine to the culture medium at concentrations that build up during co-culture of naïve T cells and dendritic cells alleviates immunosuppression by regulatory T cells (14). Hence, a change in intracellular redox metabolism in dendritic cells that leads to a shift in the extracellular redox potential is a critical determinant of the outcome of T cell activation and hence immunity.
In the CNS, a different set of cellular players engage with infiltrating T cells when the blood-brain barrier is breached during injury. The ensuing neuroinflammatory response involves interactions between T cells and glial cells and, if controlled, is neuroprotective. Engagement of astrocytes and T cells results in significant increases in extracellular cysteine and GSH levels (Fig. 2B) (41). The mechanism of the resulting extracellular reductive shift is novel and involves cystine uptake that is driven by high concentrations of glutamate released by activated T cells. Intracellular accumulation of glutamate due to the activity of the X AG Ϫ transporter stimulates the x C Ϫ antiporter, which is driven by the transmembrane glutamate gradient, and leads to increased intracellular cysteine that stimulates GSH biosynthesis. In addition to the reductive shift in the extracellular redox poise, the enhanced availability of cysteine is an important arm of the neuroprotective response shaped by T cells because neurons are inefficient transporters of cystine. Cysteine exported by astrocytes is then imported by neurons and used for GSH synthesis, increasing antioxidant capacity.
As the above example illustrates, astrocytes play a critical role in neuronal redox homeostasis. In Alzheimer disease, extracellular deposition of amyloid ␤ (A␤), derived from the amyloid precursor protein, is seen in senile plaques. Repeated exposure of astrocytes to A␤ leads to enhanced uptake of cystine via the x C Ϫ transporter, decreased expression of cystathionine ␤-synthase, decreased intracellular GSH, and increased extracellular cysteine, which is derived via the transmembrane GSH/cysteine cycle (42). The net result of these metabolic changes is a 10-mV oxidative shift in the intracellular GSH/ GSSG redox potential and a 30-mV reductive shift in the extracellular cysteine/cystine redox potential. It has been speculated that chronic A␤-induced remodeling of the extracellular redox environment might stimulate microglial proliferation, enhance inflammation, and exacerbate neuronal degeneration (42). Unregulated accumulation of astrocyte-derived cysteine could lead to NMDA receptor excitotoxicity (43) and crossing of the threshold between a neuroprotective versus neurotoxic concentration of the amino acid.
Although the above examples illustrate a common theme of extracellular redox remodeling during cell-cell communication, they also reveal significant mechanistic differences in how this is achieved in different systems. Indeed, yet another strategy for controlling the extracellular redox potential appears to operate in cultured human colonic epithelial cells and involves cystine transporters but not intracellular GSH metabolism (44). A more complete picture of the identities of membrane proteins affected by redox changes is needed to understand how the extracellular redox environment influences cellular function.

Extracellular Redox Enzymes
Significant gaps exist in our understanding of how changes in the extracellular thiol/disulfide status of membrane proteins are catalyzed and how this process is regulated in mammals. H 2 O 2 reacts slowly with thiols to form a sulfenic acid intermediate, which can subsequently be converted in an enzyme-catalyzed or uncatalyzed reaction to a disulfide (Scheme 1) (45,46). Thiol peroxidases and peroxiredoxins can accelerate the same reaction (45). Alternatively, vitamin C or dehydoascorbate can react with thiols, leading to disulfide generation (Scheme 2) (47).
Some candidate proteinaceous catalysts that might be important in determining the dithiol/disulfide equilibrium on exofacial membrane protein domains include sulfhydryl oxi-dases and secreted thioredoxin/thioredoxin reductase. Proteindisulfide isomerases, which have been reported on the cell surface, do not catalyze the net generation of disulfide bonds. Instead, they shuffle bonds, exchanging one disulfide for another.
Members of the quiescin-sulfhydryl oxidase (QSOX) family are flavin-dependent enzymes involved in oxidative protein folding in the ER (48). QSOX is also found on the outer face of the plasma membrane in secreted fluids and in cell culture medium. QSOX catalyzes the reduction of molecular oxygen to H 2 O 2 using the two electrons released during conversion of a thiol pair to a disulfide in client proteins. A role for QSOXderived H 2 O 2 in signaling has been suggested. Secreted/exofacial QSOX might also participate in extracellular matrix formation and redox cycles pertinent to cell proliferation, development, and adhesion (48). It is not known if the levels and/or activity of QSOX is modulated under conditions in which extracellular redox remodeling occurs and, if so, how this process might be regulated.
Thioredoxin is a small disulfide oxidoreductase and reduces disulfide bonds on target proteins, including ribonucleotide reductase, peroxiredoxins, methionine sulfoxide reductase, and the transcription factor AP-1. Extracellular thioredoxin, which is secreted by a leaderless pathway, has anti-inflammatory effects (49). A truncated form of thioredoxin missing ϳ20 amino acids at the C terminus is also a potent cytokine and is mitogenic. However, its mitogenic activity is independent of an active site CXXC motif (50). This is in contrast to the extracellular effects of full-length thioredoxin, which require an intact active site motif. Thioredoxin is released in response to oxidative stress in a process that also requires an intact active site CXXC motif (51). Secreted thioredoxin interacts with the TNF receptor superfamily member CD30 on lymphocytes in a CXXC-dependent manner (52). Thioredoxin released by dendritic cells upon engagement with T cells (38) does not play a role in the ensuing reductive shift in the extracellular cysteine/ cystine redox potential (14).
For thioredoxin or related family members to function as disulfide reductases to regulate the dithiol/disulfide equilibrium of extracellular membrane protein domains, additional components, e.g. NADPH and thioredoxin reductase, are needed. Although normal and cancerous cells are reported to secrete thioredoxin reductase (53), the extracellular source of reductant necessary for completing this redox cycle is unknown.

Future Perspectives
Oxidative stress is widely invoked in conditions ranging from inflammation and aging to complex diseases. However, the specificity of the oxidative stress response in a given disease is generally very poorly understood. More problematic still are the limited insights into the redox changes that occur in the extracellular compartment with the potential to influence a host of paracrine and autocrine signaling pathways and transporter and receptor functions. These limitations explain, in part, the remarkable lack of success with the wide therapeutic use of antioxidants. However, the information gap represents an opportunity for elucidating the mechanisms underlying redox changes within and outside the cell, identifying targets that are modulated by these changes, and applying this information toward developing rational therapeutic strategies.