Glutamate inhibits chondral mineralization through apoptotic cell death mediated by retrograde operation of the cystine/glutamate antiporter

Although we have previously demonstrated the functional significance of excitatory amino acid transporters as well as glutamate (Glu) receptors (GluRs) expressed by chondrocytes, little attention has been paid to the possible expression of the cystine/Glu antiporter responsible for the bi-directional transmembrane transport of Glu in chondrocytes to date. In organotypic cultured mouse embryonic metatarsals isolated before vascularization, the chondral mineralization was significantly decreased in the presence of Glu at a high concentration. Apoptotic cells were detected within the late proliferating and prehypertrophic chondrocytic layers in metatarsals cultured in the presence of Glu. A group III metabotropic GluR (mGluR) antagonist partially, but significantly, prevented the inhibition of mineralization by Glu in metatarsals without affecting the number of apoptotic cells. Both decreased mineralization and apoptosis by Glu were significantly prevented by the addition of the cystine/Glu antiporter inhibitor homocysteic acid, as well as reduced glutathione (GSH) and cystine. Expression of mRNA for xCT and 4F2hc subunits, which are components of the cystine/Glu antiporter, was seen in both cultured mouse metatarsals and rat costal chondrocytes. In chondrocytes cultured with Glu, a significant decrease was seen in intracellular GSH levels, together with increases in the number of apoptotic cells and the level of intracellular reactive oxygen species. These results suggest that Glu could regulate chondrogenic differentiation toward mineralization through a mechanism associated with apoptosis mediated by the depletion of intracellular GSH after the retrograde operation of the cystine/Glu antiporter, in addition to the activation of group III mGluR, in chondrocytes.     

Glutamate is a determinant of cellular proliferation through modulation of nuclear factor E2 p45-related factor-2 expression in osteoblastic MC3T3-E1 cells

Activation of particular glutamate (Glu) receptors is shown to promote cellular differentiation toward maturation during osteoblastogenesis. In the present study, we have evaluated the possible modulation by Glu of cellular proliferation in osteoblastic cells endowed to proliferate for self-renewal and to differentiate toward matured osteoblasts. Exposure to Glu significantly suppressed the proliferation activity at a concentration over 500 microM without inducing cell death in osteoblastic MC3T3-E1 cells before differentiation. The suppression by Glu occurred in a manner sensitive to the prevention by either cystine or reduced glutathione. Expression of mRNA was for the first time shown with the cystine/Glu antiporter composed of xCT and 4F2hc subunits in these undifferentiated osteoblastic cells. A significant decrease was seen in intracellular total glutathione levels in undifferentiated MC3T3-E1 cells cultured with Glu, indeed, whereas the cellular proliferation activity was drastically decreased by the addition of the glutathione depleter cyclohexene-1-one and the glutathione biosynthesis inhibitor L-buthionine-[S,R]-sulfoximine, respectively. Exposure to Glu led to a significant increase in mRNA expression of nuclear factor E2 p45-related factor 2 (Nrf2) together with the generation of reactive oxygen species, while a significant decrease was seen in the proliferation activity in MC3T3-E1 cells with stable overexpression of Nrf2. These results suggest that Glu could suppress the cellular proliferation toward self-renewal through a mechanism associated with the upregulation of Nrf2 expression in association with the depletion of intracellular glutathione after promoting the retrograde operation of the cystine/Glu antiporter in undifferentiated MC3T3-E1 cells.     

Functional expression of beta2 adrenergic receptors responsible for protection against oxidative stress through promotion of glutathione synthesis after Nrf2 upregulation in undifferentiated mesenchymal C3H10T1/2 stem cells

Adrenaline is believed to play a dual role as a neurotransmitter in the central nervous system and an adrenomedullary hormone in the peripheral tissues. In contrast to accumulating evidence for the involvement in endochondral ossification, osteoblastogenesis, and osteoclastogenesis, little attention has been paid to the role of adrenergic signals in the mechanisms underlying proliferation and differentiation of mesenchymal stem cells with self-renewal capacity and multi-potentiality to differentiate into osteoblast, chondrocyte, adipocyte, and myocyte lineages. Expression of mRNA was seen for different adrenergic receptor (AdR) subtypes, including beta(2)AdR, in the mesenchymal stem cell line C3H10T1/2 cells and mouse bone marrow mesenchymal stem cells before differentiation. Exposure to adrenaline not only increased cAMP formation, phosphorylation of cAMP responsive element (CRE) binding protein (CREB) on serine133 and CRE reporter activity in a manner sensitive to propranolol, but also rendered C3H10T1/2 cells resistant to the cytotoxicity of hydrogen peroxide, but not of either 2,4-dinitirophenol or tunicamycin. Adrenaline induced a rapid but transient increase in mRNA expression of the antioxidative gene nuclear factor E2 p45-related factor-2 (Nrf2) along with an increase in the cystine/glutamate antiporter subunit xCT mRNA expression. Hydrogen peroxide was less cytotoxic in cells overexpressing Nrf2, moreover, while adrenaline significantly increased xCT promoter activity with an increase in endogenous glutathione levels. These results suggest that adrenaline may selectively protect mesenchymal C3H10T1/2 cells from oxidative stress through a mechanism related to the promoted biosynthesis of glutathione in association with transient Nrf2 expression after activation of beta(2)AdR.     

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.     

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.     

Distribution of the cystine/glutamate antiporter system xc- in the brain, kidney, and duodenum.

System x(c)(-), one of the main transporters responsible for central nervous system cystine transport, is comprised of two subunits, xCT and 4F2hc. The transport of cystine into cells is rate limiting for glutathione synthesis, the major antioxidant and redox cofactor in the brain. Alterations in glutathione status are prevalent in numerous neurodegenerative diseases, emphasizing the importance of proper cystine homeostasis. However, the distribution of xCT and 4F2hc within the brain and other areas has not been described. Using specific antibodies, both xCT and 4F2hc were localized predominantly to neurons in the mouse and human brain, but some glial cells were labeled as well. Border areas between the brain proper and periphery including the vascular endothelial cells, ependymal cells, choroid plexus, and leptomeninges were also highly positive for the system x(c)(-) components. xCT and 4F2hc are also present at the brush border membranes in the kidney and duodenum. These results indicate that system x(c)(-) is likely to play a role in cellular health throughout many areas of the brain as well as other organs by maintaining intracellular cystine levels, thereby resulting in low levels of oxidative stress.     

xCt cystine transporter expression in HEK293 cells: pharmacology and localization

xCT, the core subunit of the system x(c)(-) high affinity cystine transporter, belongs to a superfamily of glycoprotein-associated amino acid transporters. Although xCT was shown to promote cystine transport in Xenopus oocytes, little work has been done with mammalian cells (Sato, H., Tamba, M., Ishii, T., and Bannai, S. J. Biol. Chem. 274, 11455-11458, 1999). Therefore, we have constructed mammalian expression vectors for murine xCT and its accessory subunit 4F2hc and transfected them into HEK293 cells. We report that this transporter binds cystine with high affinity (81 microM) and displays a pharmacological profile expected for system x(c)(-). Surprisingly, xCT transport activity in HEK293 cells is not dependent on the co-expression of the exogenous 4F2hc. Expression of GFP-tagged xCT indicated a highly clustered plasma membrane and intracellular distribution suggesting the presence of subcellular domains associated with combating oxidative stress. Our results indicate that HEK293 cells transfected with the xCT subunit would be a useful vehicle for future structure-function and pharmacology experiments involving system x(c)(-).     

Loss of Nrf2 in bone marrow-derived macrophages impairs antigen-driven CD8+ T cell function by limiting GSH and Cys availability

NF-E2-related factor 2 (Nrf2), known to protect against reactive oxygen species, has recently been reported to resolve acute inflammatory responses in activated macrophages. Consequently, disruption of Nrf2 promotes a proinflammatory macrophage phenotype. In the current study, we addressed the impact of this macrophage phenotype on CD8⁺ T cell activation by using an antigen-driven coculture model consisting of Nrf2^−/− and Nrf2^+/+ bone marrow-derived macrophages (BMDMΦ) and transgenic OT-1 CD8⁺ T cells. OT-1 CD8⁺ T cells encode a T cell receptor that specifically recognizes MHC class I-presented ovalbumin OVA(257–264) peptide, thereby causing a downstream T cell activation. Interestingly, coculture of OVA(257–264)-pulsed Nrf2^−/− BMDMΦ with transgenic OT-1 CD8⁺ T cells attenuated CD8⁺ T cell activation, proliferation, and cytotoxic function. Since the provision of low-molecular-weight thiols such as glutathione (GSH) or cysteine (Cys) by macrophages limits antigen-driven CD8⁺ T cell activation, we quantified the amounts of intracellular and extracellular GSH and Cys in both cocultures. Indeed, GSH levels were strongly decreased in Nrf2^−/− cocultures compared to wild-type counterparts. Supplementation of thiols in Nrf2^−/− cocultures via addition of glutathione ester, N-acetylcysteine, β-mercaptoethanol, or cysteine itself restored T cell proliferation as well as cytotoxicity by increasing intracellular GSH. Mechanistically, we identified two potential Nrf2-regulated genes involved in thiol synthesis in BMDMΦ: the cystine transporter subunit xCT and the modulatory subunit of the GSH-synthesizing enzyme γ-GCS (GCLM). Pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of the cystine antiporter xCT in Nrf2^+/+ BMDMΦ mimicked the effect of Nrf2^−/− BMDMΦ on CD8⁺ T cell function. Our findings demonstrate that reduced levels of GCLM as well as xCT in Nrf2^−/− BMDMΦ limit GSH availability, thereby inhibiting antigen-induced CD8⁺ T cell function.     

Human cystine/glutamate transporter: cDNA cloning and upregulation by oxidative stress in glioma cells

A human cDNA for amino acid transport system x(C)(-) was isolated from diethyl maleate-treated human glioma U87 cells. U87 cells expressed two variants of system x(C)(-) transporters hxCTa and hxCTb with altered C-terminus regions probably generated by the alternative splicing at 3'-ends. Both hxCTa and hxCTb messages were also detected in spinal cord, brain and pancreas, although the level of hxCTb expression appears to be lower than that of hxCTa in these tissues. When expressed in Xenopus oocytes, hxCTb required the heavy chain of 4F2 cell surface antigen (4F2hc) and exhibited the Na(+)-independent transport of L-cystine and L-glutamate, consistent with the properties of system x(C)(-). In agreement with this, 137 kDa band was detected by either anti-xCT or anti-4F2hc antibodies in the non-reducing condition in western blots, whereas it shifted to 50 kDa or 90 kDa bands in the reducing condition, indicating the association of two proteins via disulfide bands. We found that the expression of xCT was rapidly induced in U87 cells upon oxidative stress by diethyl maleate treatment, which was accompanied by the increase in the L-cystine uptake by U87 cells. Because of this highly regulated nature, xCT in glial cells would fulfill the task to protect neurons against oxidative stress by providing suitable amount of cystine to produce glutathione.     

The cystine/glutamate antiporter xCT is a key regulator of EphA2 S897 phosphorylation under glucose-limited conditions

EphA2, which belongs to the Eph family of receptor tyrosine kinases, is overexpressed in a variety of human cancers. Serine 897 (S897) phosphorylation of EphA2 is known to promote cancer cell migration and proliferation in a ligand-independent manner. In this study, we show that glucose deprivation induces S897 phosphorylation of EphA2 in glioblastoma cells. The phosphorylation requires the activity of the cystine/glutamate antiporter xCT and reactive oxygen species (ROS)-dependent ERK and RSK activation. Furthermore, depletion of EphA2 in glioblastoma cells leads to decreased cell viability under glucose starvation. Our results suggest a role of EphA2 in glioblastoma cell viability under glucose-limited conditions.     

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.     

Effect of oxygen on induction of the cystine transporter by bacterial lipopolysaccharide in mouse peritoneal macrophages

Amino acid transport in mouse peritoneal macrophages is mediated by several membrane carriers with different substrate specificity and sensitivity to environmental stimuli. We reported previously that transport activities of cystine and arginine in the macrophages were induced markedly by low concentrations of bacterial lipopolysaccharide (LPS). It is known that a variety of macrophage functions are affected by ambient oxygen tension. In this study, we have investigated the effects of oxygen on the induction of amino acid transport activity by LPS and found that the induction of cystine, but not arginine, transport activity was dependent on the ambient oxygen tension. When the macrophages were cultured with 2% O(2) in the presence of 1 ng/ml LPS, induction of cystine transport activity was reduced by approximately 70% compared with cells cultured under normoxic conditions. In macrophages, transport of cystine is mediated by a Na(+)-independent anionic amino acid transporter named system x(c)(-). System x(c)(-) is composed of two protein components, xCT and 4F2hc, and the expression of xCT was closely correlated with system x(c)(-) activity. A putative NF-kappaB binding site was found in the 5'-flanking region of the xCT gene, but the enhanced expression of xCT by LPS and oxygen was not mediated by NF-kappaB binding. An increase in intracellular GSH in macrophages paralleled induction of xCT, but not gamma-glutamylcysteine synthetase. These results suggest the importance of system x(c)(-) in antioxidant defense in macrophages exposed to LPS and oxidative stress.     

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.     

Cooperative action of glutamate transporters and cystine/glutamate antiporter system Xc- protects from oxidative glutamate toxicity

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress. In this paradigm, an excess of extracellular glutamate blocks the glutamate/cystine-antiporter system Xc-, depleting the cell of cysteine, a building block of the antioxidant glutathione. Loss of glutathione leads to the accumulation of reactive oxygen species and eventually cell death. We selected cells resistant to oxidative stress, which exhibit reduced glutamate-induced glutathione depletion mediated by an increase in the antiporter subunit xCT and system Xc- activity. Cystine uptake was less sensitive to inhibition by glutamate and we hypothesized that glutamate import via excitatory amino acid transporters and immediate re-export via system Xc- underlies this phenomenon. Inhibition of glutamate transporters by l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and DL-threo-beta-benzyloxyaspartic acid (TBOA) exacerbated glutamate-induced cell death. PDC decreased intracellular glutamate accumulation and exacerbated glutathione depletion in the presence of glutamate. Transient overexpression of xCT and the glutamate transporter EAAT3 cooperatively protected against glutamate. We conclude that EAATs support system Xc- to prevent glutathione depletion caused by high extracellular glutamate. This knowledge could be of use for the development of novel therapeutics aimed at diseases associated with depletion of glutathione like Parkinson's disease.     

Yokukansan,a Kampo Medicine,Protects PC12 Cells from Glutamate-Induced Death by Augmenting Gene Expression of Cystine/Glutamate Antiporter System Xc?

Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc⁻, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells). Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc⁻ subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook.