Shiro Bannai

The cystine/cysteine cycle: a redox cycle regulating susceptibility versus resistance to cell death.

The glutathione-dependent system is one of the key systems regulating cellular redox balance, and thus cell fate. Cysteine, typically present in its oxidized form cystine in the extracellular space, is regarded as the rate-limiting substrate for glutathione (GSH) synthesis. Cystine is transported into cells by the highly specific amino-acid antiporter system xc−. Since Burkitts Lymphoma (BL) cells display limited uptake capacity for cystine, and are thus prone to oxidative stress-induced cell death, we stably expressed the substrate-specific subunit of system xc−, xCT, in HH514 BL cells. xCT-overexpressing cells became highly resistant to oxidative stress, particularly upon GSH depletion. Contrary to previous predictions, the increase of intracellular cysteine did not affect the cellular GSH pool, but concomitantly boosted extracellular cysteine concentrations. Even though cells were depleted of bulk GSH, xCT overexpression maintained cellular integrity by protecting against lipid peroxidation, a very early event in cell death progression. Our results show that system xc− protects against oxidative stress not by elevating intracellular GSH levels, but rather creates a reducing extracellular environment by driving a highly efficient cystine/cysteine redox cycle. Our findings show that the cystine/cysteine redox cycle by itself must be viewed as a discrete major regulator of cell survival.

System xc− 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 γ-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.

Dopaminergic neurons of system xc−-deficient mice are highly protected against 6-hydroxydopamine-induced toxicity

Malfunctioning of system xc–, responsible for exchanging intracellular glutamate for extracellular cystine, can cause oxidative stress and excitotoxicity, both important phenomena in the pathogenesis of Parkinsons disease (PD). We used mice lacking xCT (xCT_/_ mice), the specific subunit of system xc˜, to investigate the involvement of this antiporter in PD. Although cystine that is imported via system xc˜ 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 xc˜ 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 xc˜.—Massie, A., Schallier, A., Kim, S. W., Fernando, R., Kobayashi, S., Beck, H., De Bundel, D., Vermoesen, K., Bannai, S., Smolders, I., Conrad, M., Plesnila, N., Sato, H., Michotte, Y. Dopaminergic neurons of system xc “‐deficient mice are highly protected against 6‐hydroxydopamine‐induced toxicity. FASEB J. 25, 1359–1369 (2011). www.fasebj.org

Cystathionine Is a Novel Substrate of Cystine/Glutamate Transporter: IMPLICATIONS FOR IMMUNE FUNCTION*

Background: System xc− is involved in various pathophysiological conditions, such as neurodegenerative disorders and cancer. Results: Extracellular cystathionine competitively inhibited cystine uptake and could be exchanged with intracellular glutamate via system xc−. Conclusion: Cystathionine is exclusively transported into immune tissues as the third physiological substrate of system xc−. Significance: Cystathionine can be exchanged with glutamate to reduce extracellular glutamate levels. 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 β-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−.

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 xc−. 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 xc− do not exacerbate the phenotypic consequences of SOD1 deficiency in postnatal mice under ordinary breeding conditions.

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.