Redox Imbalance in Cystine/Glutamate Transporter-deficient Mice

Abstract

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...

Dissection of mitogenic and neurodegenerative actions of cystine and glutamate in malignant gliomas

“…Although glioma cells appear to be highly dependent on system X c À -mediated cystine incorporation required for glutathione production and thus the scavenging of reactive oxygen species (Chung et al, 2005;Chung and Sontheimer, 2009;Pham et al, 2010), normal cells might be less vulnerable. This notion is supported by genetic deletion studies, that is, xCT-deficient mice are healthy in appearance and fertile (Sato et al, 2005;Shih et al, 2006;Liu et al, 2007). Moreover, resistance of tumor cells to a variety of anticancer drugs is often associated with increased glutathione levels (Huang et al, 2005).…”

Regulation of Neuronal Glutathione Synthesis

“…The former subunit correlates more directly with the system xc-activity expressed in regions facing the CSF, suggesting a role in redox buffering of the cysteine / cystine balance in the CSF (65,66). Mice lacking the xCT subunit were recently reported to show no change in brain GSH contents (67). Mature neurons mainly take up cysteine via system X AG -(43, 48, 54), whereas immature neurons take up cystine (68) via system xc-, for GSH synthesis.…”

Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

“…Mutations in the light as well as the heavy subunit of system b 0,ϩ lead to cystinuria (4,5), whereas mutations in the light subunit y ϩ LAT1 cause lysinuric protein intolerance (6,7). Another light subunit, xCT that mediates cysteine uptake and glutamate efflux (8,9), is involved in vivo in cocaine relapse (10) and maintenance of the plasma redox balance (11). LAT1, the light subunit of system L, is overexpressed in certain primary human tumors.…”