Loss of the cystine/glutamate antiporter in melanoma abrogates tumor metastasis and markedly increases survival rates of mice.

The cystine/glutamate antiporter, system xc- , is essential for the efficient uptake of cystine into cells. Interest in the mechanisms of system xc- function soared with the recognition that system xc- presents the most upstream node of ferroptosis, a recently described form of regulated necrosis relevant for degenerative diseases and cancer. Since targeting system xc- hold the great potential to efficiently combat tumor growth and metastasis of certain tumors, we disrupted the substrate-specific subunit of system xc- , xCT (SLC7A11) in the highly metastatic mouse B16F10 melanoma cell line and assessed the impact on tumor growth and metastasis. Subcutaneous injection of tumor cells into the syngeneic B16F10 mouse melanoma model uncovered a marked decrease in the tumor-forming ability and growth of KO cells compared to control cell lines. Strikingly, the metastatic potential of KO cells was markedly reduced as shown in several in vivo models of experimental and spontaneous metastasis. Accordingly, survival rates of KO tumor-bearing mice were significantly prolonged in contrast to those transplanted with control cells. Analyzing the in vitro ability of KO and control B16F10 cells in terms of endothelial cell adhesion and spheroid formation revealed that xCT expression indeed plays an important role during metastasis. Hence, system xc- emerges to be essential for tumor metastasis in mice, thus qualifying as a highly attractive anticancer drug target, particularly in light of its dispensable role for normal life in mice.

Cystine/glutamate transporter, system xc-, is involved in nitric oxide production in mouse peritoneal macrophages.

The amino acid transport system xc- is important for maintaining intracellular glutathione levels and extracellular redox balance. The main component of system xc-, xCT, is strongly induced by various stimuli, including oxidative stress and bacterial lipopolysaccharides (LPS) in macrophages. In the present study, we investigated the production of nitric oxide by LPS-stimulated mouse peritoneal macrophages isolated from both xCT-deficient and wild-type mice. After culturing macrophages in the presence of LPS for 24-48 h, nitrite levels in the medium of xCT-deficient macrophages were significantly decreased compared to that of wild-type cells. However, the transport activity of arginine, a precursor of nitric oxide, and the expression of nitric oxide synthase 2 in xCT-deficient macrophages were similar to those of wild-type cells. When wild-type macrophages were cultured in the medium that contained no cystine, nitric oxide production was decreased to the level similar to that of the xCT-deficient macrophages. When xCT-deficient macrophages were cultured with 2-mercaptoethanol, intracellular cysteine levels were increased and nitrite accumulation in the medium was significantly increased. On the other hand, when these cells were cultured with buthionine sulfoximine, an inhibitor of glutathione synthesis, nitrite accumulation in the medium was essentially unchanged, although intracellular glutathione levels were very low. Reactive oxygen species levels in xCT-deficient macrophages were higher than those of wild-type cells, and treatment with LPS caused an increase in oxidative stress in both cells. These results suggest that intracellular cysteine supplied by xCT contributes to nitric oxide production and the reduction of oxidative stress in macrophages.

The ferroptosis inducer erastin irreversibly inhibits system xc- and synergizes with cisplatin to increase cisplatin's cytotoxicity in cancer cells.

System xc- was recently described as the most upstream node in a novel form of regulated necrotic cell death, called ferroptosis. In this context, the small molecule erastin was reported to target and inhibit system xc-, leading to cysteine starvation, glutathione depletion and consequently ferroptotic cell death. Although the inhibitory effect of erastin towards system xc- is well-documented, nothing is known about its mechanism of action. Therefore, we sought to interrogate in more detail the underlying mechanism of erastin's pro-ferroptotic effects. When comparing with some well-known inhibitors of system xc-, erastin was the most efficient inhibitor acting at low micromolar concentrations. Notably, only a very short exposure of cells with low erastin concentrations was sufficient to cause a strong and persistent inhibition of system xc-, causing glutathione depletion. These inhibitory effects towards system xc- did not involve cysteine modifications of the transporter. More importantly, short exposure of tumor cells with erastin strongly potentiated the cytotoxic effects of cisplatin to efficiently eradicate tumor cells. Hence, our data suggests that only a very short pre-treatment of erastin suffices to synergize with cisplatin to efficiently induce cancer cell death, findings that might guide us in the design of novel cancer treatment paradigms.

Cystathionine is a novel substrate of cystine/glutamate transporter: implications for immune function.

The cystine/glutamate transporter, designated as system xc(-), is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system xc(-), xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues, such as the thymus and spleen. Although cystine and glutamate are the well established substrates of system xc(-) and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analyzed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged as being absent specifically in the thymus and spleen of xCT-deficient mice. No expression of either cystathionine ß-synthase or cystathionine γ-lyase was observed in the thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knock-out cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation. We thus conclude that cystathionine is a novel physiological substrate of system xc(-) and that the accumulation of cystathionine in immune tissues is exclusively mediated by system xc(-).

Enhanced expression of cystine/glutamate transporter in the lung caused by the oxidative-stress-inducing agent paraquat.

In mammalian cultured cells, the activity of a cystine/glutamate transporter, designated System xc(-), has been shown to be essential for maintaining intracellular glutathione levels and the extracellular cystine/cysteine redox balance. The substrate-specific subunit of this transporter, xCT, is strongly induced by various stimuli, including oxidative stress, which suggests that xCT is one of the adaptive cellular defense systems against these types of stress. Embryonic fibroblasts from xCT-deficient mice fail to survive unless a cysteine precursor, N-acetylcysteine, is present. However, it is unclear whether xCT has similar functions in vivo because xCT-deficient mice are apparently normal. In this study, we investigated the phenotype of the xCT-deficient mice under paraquat-induced oxidative stress. At a paraquat dose of 45mg/kg, the survival rate of the xCT-deficient mice was significantly lower than that of the wild-type mice. Under this condition, total glutathione (the reduced form of glutathione (GSH)+the oxidized form of GSH) levels in the lungs of the xCT-deficient mice were lower than those in the lungs of the wild-type mice. Histopathological examinations showed that paraquat administration worsened the alveolar structure of the xCT-deficient mice compared with the wild-type mice. After paraquat treatment, obvious 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal reactivity was detected in the lungs of the xCT-deficient mice. Although xCT expression was slightly detectable in the lungs of the normal wild-type mice, paraquat administration induced xCT mRNA expression in the lung. Constitutive expression of xCT mRNA was detected in alveolar macrophages isolated from the pulmonary lavage fluid of the wild-type mice, and paraquat administration strongly enhanced xCT mRNA expression in these cells. GSH levels in bronchoalveolar lavage fluid were significantly higher in the paraquat-treated wild-type mice than in the paraquat-treated xCT-deficient mice. These results suggest that xCT contributes to the maintenance of glutathione levels in lungs and the glutathione redox state as a protective system against paraquat toxicity in vivo.

Loss of system x(c)- does not induce oxidative stress but decreases extracellular glutamate in hippocampus and influences spatial working memory and limbic seizure susceptibility.

System x(c)- exchanges intracellular glutamate for extracellular cystine, giving it a potential role in intracellular glutathione synthesis and nonvesicular glutamate release. We report that mice lacking the specific xCT subunit of system x(c)- (xCT(-/-)) do not have a lower hippocampal glutathione content, increased oxidative stress or brain atrophy, nor exacerbated spatial reference memory deficits with aging. Together these results indicate that loss of system x(c)- does not induce oxidative stress in vivo. Young xCT(-/-) mice did however display a spatial working memory deficit. Interestingly, we observed significantly lower extracellular hippocampal glutamate concentrations in xCT(-/-) mice compared to wild-type littermates. Moreover, intrahippocampal perfusion with system x(c)- inhibitors lowered extracellular glutamate, whereas the system x(c)- activator N-acetylcysteine elevated extracellular glutamate in the rat hippocampus. This indicates that system x(c)- may be an interesting target for pathologies associated with excessive extracellular glutamate release in the hippocampus. Correspondingly, xCT deletion in mice elevated the threshold for limbic seizures and abolished the proconvulsive effects of N-acetylcysteine. These novel findings sustain that system x(c)-) is an important source of extracellular glutamate in the hippocampus. System x(c)(-) is required for optimal spatial working memory, but its inactivation is clearly beneficial to decrease susceptibility for limbic epileptic seizures.

The dynamics of cysteine, glutathione and their disulphides in astrocyte culture medium.

Glutathione (GSH) plays an important neuroprotective role, and its synthesis depends on the amount of available cysteine (CSH) in the cells. Various kinds of evidence suggest that astrocytes can provide CSH or GSH to neurons, but the delivery mechanism of the thiol-compounds has not been elucidated. In this study, the dynamics of CSH, GSH and their disulphides in astrocyte culture medium were investigated by following the time-course of concentration changes and by computer simulation and curve fitting to experimental data using a mathematical model. The model consists of seven reactions and three transports, which are grouped into four categories: autoxidation of thiols into disulphides, thiol-disulphide exchange and reactions of thiols with medium components, as well as the cellular influx and efflux of thiols and disulphides. The obtained results are interpreted that cystine (CSSC) after entering astrocyte is reduced to CSH, most of which is released to medium and autoxidized to CSSC. The efflux of GSH was estimated to be considerably slower than that of CSH, and most of the excreted GSH is converted to cysteine-glutathione disulphide principally through the thiol-disulphide exchange. The results seem to indicate that astrocytes provide neurons mainly with CSH, rather than GSH, as the antioxidant material for neuroprotection.

Dopaminergic neurons of system x(c)⁻-deficient mice are highly protected against 6-hydroxydopamine-induced toxicity.

Malfunctioning of system x(c)(-), responsible for exchanging intracellular glutamate for extracellular cystine, can cause oxidative stress and excitotoxicity, both important phenomena in the pathogenesis of Parkinson's disease (PD). We used mice lacking xCT (xCT(-/-) mice), the specific subunit of system x(c)(-), to investigate the involvement of this antiporter in PD. Although cystine that is imported via system x(c)(-) is reduced to cysteine, the rate-limiting substrate in the synthesis of glutathione, deletion of xCT did not result in decreased glutathione levels in striatum. Accordingly, no signs of increased oxidative stress could be observed in striatum or substantia nigra of xCT(-/-) mice. In sharp contrast to expectations, xCT(-/-) mice were less susceptible to 6-hydroxydopamine (6-OHDA)-induced neurodegeneration in the substantia nigra pars compacta compared to their age-matched wild-type littermates. This reduced sensitivity to a PD-inducing toxin might be related to the decrease of 70% in striatal extracellular glutamate levels that was observed in mice lacking xCT. The current data point toward system x(c)(-) as a possible target for the development of new pharmacotherapies for the treatment of PD and emphasize the need to continue the search for specific ligands for system x(c)(-).

Conflicting effects of N-acetylcysteine on purified neurons derived from rat cortical culture.

We examined the protective effects of N-acetylcysteine (NAC) on the death of glia-free neurons in culture. Under normoxic conditions, the protection by NAC was observed only in cystine-free but not complete medium. When the cells were cultured under hypoxic conditions, NAC much elongated their survival even in the presence of cystine. H2O2 was found to be generated to considerable concentration in the presence of both NAC and cystine, and the administration of catalase prevented the cell death. These results suggest that the harmful effect of NAC is because of H2O2 generated by autoxidation of cysteine, which derives from the reaction between NAC and cystine. The present results raise the possibility that NAC can act as either antioxidant or prooxidant depending on the milieu.

System x(c)- and thioredoxin reductase 1 cooperatively rescue glutathione deficiency.

GSH is the major antioxidant and detoxifier of xenobiotics in mammalian cells. A strong decrease of intracellular GSH has been frequently linked to pathological conditions like ischemia/reperfusion injury and degenerative diseases including diabetes, atherosclerosis, and neurodegeneration. Although GSH is essential for survival, the deleterious effects of GSH deficiency can often be compensated by thiol-containing antioxidants. Using three genetically defined cellular systems, we show here that forced expression of xCT, the substrate-specific subunit of the cystine/glutamate antiporter, in gamma-glutamylcysteine synthetase knock-out cells rescues GSH deficiency by increasing cellular cystine uptake, leading to augmented intracellular and surprisingly high extracellular cysteine levels. Moreover, we provide evidence that under GSH deprivation, the cytosolic thioredoxin/thioredoxin reductase system plays an essential role for the cells to deal with the excess amount of intracellular cystine. Our studies provide first evidence that GSH deficiency can be rescued by an intrinsic genetic mechanism to be considered when designing therapeutic rationales targeting specific redox enzymes to combat diseases linked to GSH deprivation.

Aggravation of ischemia-reperfusion-triggered acute renal failure in xCT-deficient mice.

This study examined the question of whether deficiency of xCT, a cystine-transporter gene, exacerbates ischemia-reperfusion-induced acute renal failure (ARF). Two weeks after the right nephrectomy of male mice at 16-18weeks of age, the left renal vessels were clamped for 45min to induce renal ischemia. After (24h) induction of ischemia, xCT(-/-) mice had elevated concentrations of blood urea nitrogen and creatinine indicative of ARF, while in xCT(+/-) and xCT(+/+) mice, these parameters did not differ from the sham-operated mice. Immunohistochemical analyses of kidneys using antibodies against the oxidative stress markers revealed stronger staining in xCT(-/-) mice compared with xCT(+/+) mice. Induction of xCT mRNA in the kidneys of xCT(+/+) mice was demonstrated using reverse transcriptase (RT)-PCR analysis and was further confirmed using quantitative RT-PCR. These data provide the first in vivo evidence that xCT is induced by oxidative stress and helps prevent ischemia-reperfusion injury to kidneys.

Time-dependent changes in striatal xCT protein expression in hemi-Parkinson rats.

Altered glutamate signaling is associated with Parkinson's disease. To study the involvement of the cystine/glutamate antiporter in the pathogenesis of Parkinson's disease, we developed new polyclonal antibodies recognizing xCT, the specific subunit of this antiporter. The striatal xCT protein expression level was investigated in a hemi-Parkinson rat model, using semiquantitative western blotting. We observed time-dependent changes after a unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway with increased expression levels in the deafferented striatum after 3 weeks. Twelve weeks postlesion, expression levels returned to normal. These data suggest, for the first time, an involvement of the cystine/glutamate antiporter in determining the aberrant glutamate neurotransmission in the striatum of a parkinsonian brain.

Deficiency of the cystine-transporter gene, xCT, does not exacerbate the deleterious phenotypic consequences of SOD1 knockout in mice.

Because glutathione scavenges reactive oxygen species (ROS) and also donates electrons to antioxidative systems, it may compensate for the oxidative stress caused by SOD1 deficiency. The cystine/glutamate transporter, which consists of two proteins, xCT and 4F2hc, has been designated system x (c) (-) . This transporter system plays a role in the maintenance of glutathione levels in mammalian cells. In the present study, we created SOD1 (-/-); xCT (-/-) double-knockout mice by intercrossing xCT-knockout and SOD1-knockout animals. We determined if the double-knockout mice express the phenotypic characteristics unique to SOD1 (-/-) mice-increased oxidative stress and the production of autoantibodies against erythrocytes. We also compared the phenotype of the double-knockout mice with those of the single-knockout and wild-type mice. Although two major antioxidative systems were found to be defective in the SOD1 (-/-); xCT (-/-) mice, relative to the SOD1 (-/-) mice, no functional deficits were observed. Based on these results, it appears that defects in system x (c) (-) do not exacerbate the phenotypic consequences of SOD1 deficiency in postnatal mice under ordinary breeding conditions.

Induction of cystine/glutamate transporter in bacterial lipopolysaccharide induced endotoxemia in mice.

BACKGROUND: Cystine/glutamate transporter, system xc-, contributes to the maintenance of intracellular glutathione levels and the redox balance in the extracellular space. The main component of the transporter, xCT, is known to be strongly induced by various stimuli like oxidative stress in mammalian cultured cells. We examined the expression of xCT mRNA in vivo in the experimental endotoxemia. METHODS: Northern blot analysis and in situ hybridization were used to investigate the expression of xCT mRNA in the tissues of the mice exposed to bacterial lipopolysaccharide (LPS). RESULTS: Northern blot analysis revealed that xCT mRNA was constitutively expressed in the brain, thymus, and spleen, and that the expression of xCT mRNA was strongly up-regulated in thymus and spleen by the administration of a sublethal dose of LPS. In addition to brain, thymus, and spleen, xCT mRNA was detected also in the bronchiolar epithelium of the lung by the administration of the lethal dose of LPS. CONCLUSION: xCT is induced in some specific tissues by the administration of LPS. The results suggest that cystine/glutamate transporter plays an important role under the inflammatory conditions.

Expression and function of cystine/glutamate transporter in neutrophils.

Reactive oxygen species (ROS) produced by neutrophils are essential in the host defense against infections but may be harmful to neutrophils themselves. Glutathione (GSH) plays a pivotal role in protecting cells against ROS-mediated oxidant injury. Cystine/glutamate transporter, designated as system xc- and consisting of two proteins, xCT and 4F2hc, is important to maintain GSH levels in mammalian-cultured cells. In the present paper, we have investigated system xc- in neutrophils. In human peripheral blood neutrophils, neither the activity of system xc- nor xCT mRNA was detected. The activity was induced, and xCT mRNA was expressed when they were cultured in vitro. The mRNA expression was much enhanced in the presence of opsonized zymosan or PMA. In contrast, mouse peritoneal exudate neutrophils, immediately after preparation, exhibited system xc- activity and expressed xCT mRNA. The activity and the expression were heightened further when they were cultured. Peritoneal exudate cells (mostly neutrophils) from xCT-deficient (xCT-/-) mice had lower cysteine content than those from the wild-type mice. GSH levels in the xCT-/-cells decreased rapidly when they were cultured, whereas those in the wild-type cells were maintained during the culture. Apoptosis induced in culture was enhanced in the xCT-/-cells compared with the wild-type cells. These results suggest that system xc- plays an important role in neutrophils when they are activated, and their GSH consumption is accelerated.

Beneficial effect of antioxidants in purified neurons derived from rat cortical culture.

Brain cell suspensions obtained from cerebrum of fetal rats were cultured and after 5 days neurons were separated from the residual cells. These purified neurons, which were replated on the dish, started to die within 24 h in culture. Glutathione content of these neurons decreased rapidly to less than one-tenth of the initial level after 24 h. In the presence of alpha-tocopherol, a well-known antioxidant, the neurons survived for at least 3 days, though glutathione content remained very low. Butylated hydroxyanisol had similar effect, but ascorbic acid and uric acid had no or very little effect. Serotonin, which is assumed to have an antioxidant activity, kept the neurons alive for 3 days. These results suggest that neurons separated from the other types of cells cannot survive due to the oxidative stress, which may otherwise be neutralized by a mechanism involving glutathione, and that antioxidants including serotonin has a beneficial effect on these purified neurons.

Redox imbalance in cystine/glutamate transporter-deficient mice.

Cystine/glutamate transporter, designated as system x(-)(c), mediates cystine entry in exchange for intracellular glutamate in mammalian cells. This transporter consists of two protein components, xCT and 4F2 heavy chain, and the former is predicted to mediate the transport activity. This transporter plays a pivotal role for maintaining the intracellular GSH levels and extracellular cystine/cysteine redox balance in cultured cells. To clarify the physiological roles of this transporter in vivo, we generated and characterized mice lacking xCT. The xCT(-/-) mice were healthy in appearance and fertile. However, cystine concentration in plasma was significantly higher in these mice, compared with that in the littermate xCT(-/-) mice, while there was no significant difference in plasma cysteine concentration. Plasma GSH level in xCT(-/-) mice was lower than that in the xCT(-/-) mice. The embryonic fibroblasts derived from xCT(-/-) mice failed to survive in routine culture medium, and 2-mercaptoethanol was required for survival and growth. When 2-mercaptoethanol was removed from the culture medium, cysteine and GSH in these cells dramatically decreased, and cells started to die within 24 h. N-Acetyl cysteine also rescued xCT(-/-)-derived cells and permitted growth. These results demonstrate that system x(-)(c) contributes to maintaining the plasma redox balance in vivo but is dispensable in mammalian development, although it is vitally important to cells in vitro.

Transcriptional control of cystine/glutamate transporter gene by amino acid deprivation.

Recent studies have demonstrated that depletion of amino acids results in the induction of several genes and that a genomic cis-element termed amino acid response element (AARE) is required for the induction. System x(c)(-) is an anionic amino acid transport system highly specific for cystine and glutamate, and its activity is known to be induced by cystine deprivation. This transporter is composed of two protein components, xCT and 4F2 heavy chain, and xCT is thought to mediate the transport activity. In the present study, the molecular mechanism for the induction of xCT by amino acid deprivation has been investigated. In mouse NIH3T3 cells, the activity of system x(c)(-) and xCT mRNA is induced not only by deprivation of cystine but also by deprivation of other amino acids. Two AAREs, each located in the opposite direction with an intervening sequence, were found in the 5'-flanking region of the mouse xCT gene. Promoter analysis revealed that both AAREs were necessary for the maximal induction of xCT mRNA in response to the amino acid deprivation. Glucose deprivation had no effect on the induction of the activity of system x(c)(-). Electrophoretic mobility shift assay showed that ATF4, but not ATF2, is involved in the amino acid control of xCT expression. These results demonstrate that xCT is a new member of the proteins whose transcriptional control by the amino acid deprivation is mediated by AARE.

Expression of the activity of cystine/glutamate exchange transporter, system x(c)(-), by xCT and rBAT.

The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells.

Vitamin C inhibits diethylmaleate-induced L-cystine transport in human vascular smooth muscle cells.

Adaptive increases in intracellular glutathione (GSH) in response to oxidative stress are mediated by induction of L-cystine uptake via the anionic amino acid transport system x(c)(-). The recently cloned transporter xCT forms a heteromultimeric complex with the heavy chain of 4F2 cell surface antigen (4F2hc/CD98). Depletion of GSH by the electrophile diethylmaleate (DEM) induces the activity and expression of xCT in peritoneal macrophages. We here examine the effects of vitamin C on induction of xCT by DEM in human umbilical artery smooth muscle cells. DEM caused time- (3-24 h) and concentration- (25-100 microM) dependent increases in L-cystine transport, with GSH depleted by 50% after 6 h and restored to basal values after 24 h. xCT mRNA levels increased after 4 h DEM treatment with negligible changes detected for 4F2hc mRNA. DEM caused a rapid (5-30 min) phosphorylation of p38(MAPK). Inhibition of p38(MAPK) by SB203580 (10 microM) enhanced DEM-induced increases in L-cystine transport and GSH, whereas inhibition of p42/p44(MAPK) (PD98059, 10 microM) had no effect. Pretreatment of cells with vitamin C (100 microM, 24 h) attenuated DEM-induced adaptive increases in L-cystine transport and GSH levels. Inhibition of p38(MAPK), but not p42/p44(MAPK), reduced the cytoprotective action of vitamin C. Our findings suggest that DEM induces activation of xCT via intracellular signaling pathways involving p38(MAPK), and that vitamin C, in addition to its antioxidant properties, may modulate this signaling pathway to protect smooth muscle cells from injury.