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 permittedgrowth. 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.Transport of amino acids across plasma membrane is mediated by several transport systems in mammalian cells (1). We have described a Na ϩ -independent, cystine/glutamate exchange transport system, designated as system x c Ϫ , in various cultured cells like human fibroblasts and mouse peritoneal macrophages (2, 3). Cells expressing system x c Ϫ take up cystine in the medium into the cell, and reduce it to cysteine (thiol form), which is in turn used for the synthesis of GSH and proteins. A part of cysteine is released back into the medium via neutral amino acid transport systems, and the cysteine is rapidly oxidized to cystine by oxygen in the medium. Thus, a series of these transports and redox reactions constitutes cystine/cysteine cycle across the plasma membrane. The activity of system x c Ϫ contributes to driving the cystine/ cysteine cycle and to maintaining the redox balance between cystine and cysteine in the culture medium (4). In cultured cells, the activity of system x c Ϫ is also demonstrated to be essential for maintaining the intracellular GSH levels (5). Because GSH plays a central role in alleviating oxidative stress, system x c Ϫ is regarded as a constituent of the antioxidant defense systems, at least in cultured cells. This transporter is composed of two protein components, xCT and the heavy chain of 4F2 antigen (6), and the transport activity is thought to be mediated by xCT. The activity of system x c Ϫ is induced by various stimuli, including electrophilic agents like diethyl maleate (7), oxygen (4), hydrogen peroxide (8), bacterial lipopolysacchar...

In mammalian cultured cells, the cystine/glutamate exchange transport mediated by system x c ؊ is important to maintain intracellular GSH levels. System x c ؊ consists of two protein components, xCT and the heavy chain of 4F2 antigen. The activity of system x c ؊ is induced by various stimuli, including electrophilic agents like diethyl maleate. In the present study, we have investigated the mechanism of the transcriptional regulation of xCT mRNA by diethyl maleate. The xCT gene consisted of twelve exons and sequence analysis identified four electrophile response element (EpRE)-like sequences between ؊230 and ؊1 in the 5-flanking region, designated EpRE-1 to EpRE-4. To identify sequences mediating the constitutive and induced expression of xCT, a series of 5-deletion mutants created from the 5-flanking region were cloned into a luciferase reproter vector and transfected into BHK21 cells. The 5-deletion analysis revealed that the sequence between ؊116 and ؊82 is essential for the basal expression and the sequence between ؊226 and ؊116 containing EpRE-1 is essential in response to diethyl maleate. Mutational analysis demonstrated that EpRE-1 is critically involved in the response to diethyl maleate. Other stress agents like arsenite, cadmium, and hydroquinone seemed to induce system x c ؊ activity via the same sequence. Furthermore, the experiments using the mouse embryonic fibroblasts derived from the Nrf2-deficient mice revealed that the induction of xCT gene by electrophilic agents is mediated by Nrf2. EpRE occurs in a broad spectrum of genes for the proteins that are involved in the defense against xenobiotics and regulates their expression. The present results have demonstrated that xCT is a novel member of this protein family.

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