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. 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. Transport of amino acids across plasma membrane is mediated by several transport systems in mammalian cells (1Christensen H.N. Physiol. Rev. 1990; 70: 43-77Crossref PubMed Scopus (963) Google Scholar). We have described a Na+-independent, cystine/glutamate exchange transport system, designated as system x–c, in various cultured cells like human fibroblasts and mouse peritoneal macrophages (2Bannai S. Kitamura E. J. Biol. Chem. 1980; 255: 2372-2376Abstract Full Text PDF PubMed Google Scholar, 3Watanabe H. Bannai S. J. Exp. Med. 1987; 165: 628-640Crossref PubMed Scopus (136) Google Scholar). Cells expressing system x–c 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 contributes to driving the cystine/cysteine cycle and to maintaining the redox balance between cystine and cysteine in the culture medium (6Sato H. Tamba M. Ishii T. Bannai S. J. Biol. Chem. 1999; 274: 11455-11458Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar). In cultured cells, the activity of system x–c is also demonstrated to be essential for maintaining the intracellular GSH levels (5Bannai S. Tateishi N. J. Membrane Biol. 1986; 89: 1-8Crossref PubMed Scopus (305) Google Scholar). Because GSH plays a central role in alleviating oxidative stress, system x–c 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 (6Sato H. Tamba M. Ishii T. Bannai S. J. Biol. Chem. 1999; 274: 11455-11458Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar), and the transport activity is thought to be mediated by xCT. The activity of system x–c is induced by various stimuli, including electrophilic agents like diethyl maleate (7Bannai S. J. Biol. Chem. 1984; 259: 2435-2440Abstract Full Text PDF PubMed Google Scholar), oxygen (4Bannai S. Sato H. Ishii T. Sugita Y. J. Biol. Chem. 1989; 264: 18480-18484Abstract Full Text PDF PubMed Google Scholar), hydrogen peroxide (8Bannai S. Sato H. Ishii T. Taketani S. Biochim. Biophys. Acta. 1991; 1092: 175-179Crossref PubMed Scopus (84) Google Scholar), bacterial lipopolysaccharide (LPS) 2The abbreviations used are: LPSlipopolysaccharideHPLChigh performance liquid chromatographyCySSTtotal non-protein-bound disulfide forms of cysteineCySSXthe mixed disulfide of cysteine and the other thiolsGSSTtotal non-protein-bound disulfide forms of GSHGSSYthe mixed disulfide of GSH and the other thiols2ME2-mercaptoethanolNACN-acetyl cysteineGFPgreen fluorescent proteinDIGdigoxigenin (9Sato H. Fujiwara K. Sagara J. Bannai S. Biochem. J. 1995; 310: 547-551Crossref PubMed Scopus (101) Google Scholar), and amino acid deprivation (10Sato H. Nomura S. Maebara K. Sato K. Tamba M. Bannai S. Biochem. Biophys. Res. Commun. 2004; 325: 109-116Crossref PubMed Scopus (127) Google Scholar). We have demonstrated that the induction of xCT by diethyl maleate is mediated by the electrophile response element located in the 5′-flanking region of the xCT gene and that the transcription factor Nrf2 binds to this element to activate the transcription of the xCT gene (11Sasaki H. Sato H. Kuriyama-Matsumura K. Sato K. Maebara K. Wang H. Tamba M. Itoh K. Yamamoto M. Bannai S. J. Biol. Chem. 2002; 277: 44765-44771Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). We have recently demonstrated that the induction of xCT by amino acid deprivation is mediated by two amino acid response elements located in the 5′-flanking region of the xCT gene and suggested that the transcription factor ATF4 is involved in the inducible transcription of the xCT gene (10Sato H. Nomura S. Maebara K. Sato K. Tamba M. Bannai S. Biochem. Biophys. Res. Commun. 2004; 325: 109-116Crossref PubMed Scopus (127) Google Scholar). lipopolysaccharide high performance liquid chromatography total non-protein-bound disulfide forms of cysteine the mixed disulfide of cysteine and the other thiols total non-protein-bound disulfide forms of GSH the mixed disulfide of GSH and the other thiols 2-mercaptoethanol N-acetyl cysteine green fluorescent protein digoxigenin Although it is obvious that system x–c plays a pivotal role in maintaining intracellular GSH level and modulating cystine/cysteine redox balance out of the cell in vitro, it is unknown whether system x–c functions similarly in vivo. To clarify the physiological role of system x–c in vivo, we generated a mouse model deficient in xCT. In the present study, we describe the generation and initial characterization of the mice unable to express xCT. Materials—l-[14C]Cystine was obtained from PerkinElmer Life Sciences. Monobromobimane was purchased from Molecular Probes, Inc. (Eugene, OR). All other chemicals and agents were purchased from Sigma or Wako Pure Chemical Industries, Ltd (Tokyo, Japan). Generation of xCT-null Mice—Genomic clones containing the mouse xCT gene were isolated from a 129/Sv genomic phage library (Stratagene). A 2.3-kb genomic fragment containing the translation initiation site and its 5′-flanking region was cut out with HindIII and NcoI, blunted with S1 nuclease, and inserted into modified pSVβ containing the GFP coding sequence. The fragment accompanying the GFP sequence was cut out and inserted into pLOXNATA, which contains neomycin resistance (neor) and thymidine kinase for selection of homologous recombination. Another 3.9-kb genomic fragment containing parts of exon 1 and intron 1 was cut out with NcoI and BamHI, blunted with Klenow fragment, and subcloned into pLOXNATA (Fig. 1). The targeting vector was linearized and transfected into E14 ES cells by the electroporation. Cells were cultured on growth-arrested neor embryonic fibroblasts under the G418 selection. Resistant colonies were picked on days 8 – 10, dissociated with trypsin, and divided into two aliquots. One aliquot was plated on a 96-well plate, and genomic DNA was isolated from another aliquot and screened by PCR. Through PCR analysis of ∼1,000 ES cell clones, we identified four clones that carried the homologous recombinant allele. These positive clones were expanded and genotyped by Southern blot analysis. The positive ES clone was injected into C57BL/6 blastcysts and chimeric mice were generated. Male chimeric mice were bred with C57BL/6 female mice. Germ line transmission of the targeted allele was determined by the presence of agouti mice in the offspring. Mice were genotyped by isolating DNA from tail biopsies and analyzed by Southern blotting using the probes shown in Fig. 1. To remove the neor cassette in the allele of the mice, we bred these mice with Cre recombinase-expressing mice (C57BL/6). Offspring heterozygous for the neor cassette deleted allele were interbred, and mice homozygous for the mutation were identified by PCR and Southern blot analysis. All mice were 129/Svj-C57BL/6 mixed background littermates from F1 heterozygote crosses. All experiments were performed in 8 – 12-week-old homozygous (xCT–/–), heterozygous (xCT+/–), and wild-type (xCT+/+) littermates. This study was approved by the Animal Care and Use Committee at the University of Tsukuba. Cell Culture and Cystine Uptake—Peritoneal macrophages elicited by 4% thioglycollate medium were collected and cultured as described previously (12Sato H. Kuriyama-Matsumura K. Hashimoto T. Sasaki H. Wang H. Ishii T. Mann G. Bannai S. J. Biol. Chem. 2001; 276: 10407-10412Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Embryonic fibroblasts were prepared from day 14 embryos and cultured in Iscove's Modified Dulbecco's Medium supplemented with Insulin-Transferrin-Selenium-G supplement (Invitrogen, Life Technologies, Inc.) and 10% fetal bovine serum at 37 °C in 5% CO2 and 95% air in the presence or absence of 50 μm 2-mercaptoethanol (2ME). For subsequent experiments, cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum with and without 2ME. Cells were plated at 2 × 105 cells/35-mm diameter dish, cultured for 24 h, and the activity of cystine transport was measured as described previously (2Bannai S. Kitamura E. J. Biol. Chem. 1980; 255: 2372-2376Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were rinsed three times in warmed (10 mm phosphate-buffered saline (137 mm NaCl, 3 mm KCl), pH 7.4, containing 0.01% CaCl2, 0.01% MgCl2·6H2O and 0.1% glucose), and then incubated in 0.5 ml of the warmed uptake medium at 37 °C for specified time periods. The uptake medium was PBSG containing l-[14C]cystine (0.05 mm and 0.1 μCi/0.5 ml). Uptake was terminated by rapidly rinsing cells three times with ice-cold PBS, and radioactivity in cells was determined. Cystine uptake was determined under conditions approaching initial rates of uptake, i.e. measuring uptake for cystine at 2 min. The uptake of cystine increased linearly during this incubation. Measurement of Intracellular Cysteine and GSH—The cysteine and GSH contents in cells were determined by the method of Cotgreave and Moldéus (13Cotgreave I.A. Moldéus P. J. Biochem. Biophys. Methods. 1986; 13: 231-249Crossref PubMed Scopus (229) Google Scholar) with a slight modification (14Sagara J. Miura K. Bannai S. J. Neurochem. 1993; 61: 1667-1671Crossref PubMed Scopus (136) Google Scholar). Cells were rapidly rinsed three times with ice-cold 20 mm HEPES-saline (137 mm NaCl, 3 mm KCl), pH 7.4, containing 0.01% CaCl2, 0.01% MgCl2·6H2O and 0.1% glucose, and incubated in the dark at room temperature for 10 min with 100 μl of 8 mm monobromobimane in 50 mm N-ethylmorpholine (pH 8) and 100 μl of 50 mm HEPES-saline, containing 0.01% CaCl2, 0.01% MgCl2·6H2O, and 0.1% glucose. Then 10 μl of 100% trichloroacetic acid were added. The protein precipitate was removed by centrifugation at 15,000 × g for 5 min, and bimane adducts of cysteine and GSH in the supernatant were analyzed by high performance liquid chromatography (HPLC). The HPLC separation was achieved on a steel column (4.6 × 100 mm) packed with 3-μm octadodecylsilica reversed phase material. The fluorescence at 480 nm was monitored with the at The was performed with in pH for 8 min. The was 1 the GSH in was measured by the method described previously Biochem. PubMed Scopus Google Scholar), which is on the of GSH in the of by the GSH The GSH from the was and the of the oxidized was for mouse xCT was digoxigenin by transcription from the linearized using and (Stratagene). was on a in the presence of membrane and with the probes in for at The were for 5 min at room temperature with 1 × 0.1% and then for min at °C with 0.1 × 0.1% The were using Measurement of in were with and was collected from the was and 100 plasma was into the containing 10 μl of acid and 10 of as min in the was and the The was and at × g for 20 min. The supernatant was its pH was to with 1 and 50 μl of the was by the amino acid Japan). In this cysteine was with and be determined. Measurement of and the of Cysteine and of GSH in cysteine and GSH 100 μl of the plasma was mixed with 100 μl of 8 mm monobromobimane in 50 mm pH and incubated in the dark at room temperature for 10 min. Then 10 μl of 100% trichloroacetic acid was added. The protein precipitate was removed by centrifugation at 15,000 × g for 5 min, and 20 supernatant was measured by HPLC as described of the total non-protein-bound disulfide forms of cysteine and of GSH were determined as described previously Biochem. PubMed Scopus Google Scholar). Briefly, plasma was with to and then by protein was removed by centrifugation and was to the supernatant to reduce disulfide The is by The thiols were with monobromobimane and the bimane adducts of cysteine and GSH were analyzed by is to cystine is the mixed disulfide of cysteine and the other and is to is the mixed disulfide of GSH and the other and be as a in the mixed However, the bimane adducts of and were in in HPLC and of and was to be Generation of xCT-null heterozygous for targeted of the xCT gene were obtained by the chimeric with wild-type female mice and agouti were screened for the presence of the xCT gene by PCR. Offspring heterozygous for the mutation were interbred, and mice homozygous for the allele were identified by Southern blot analysis (Fig. In the macrophages derived from these mice, for xCT was and the significant activity of system x–c was although the induction of the activity of system x–c by was significantly (Fig. and and of experiments demonstrated that the to be in the neor which was inserted in the between the xCT genomic We bred these mice with Cre recombinase-expressing mice to remove the neor cassette (Fig. Offspring heterozygous for the neor allele were interbred, and mice homozygous for the mutation were identified by PCR and Southern blot analysis (Fig. In these homozygous mice in Fig. the of for xCT was in and xCT is in the wild-type mice (Fig. In the peritoneal macrophages derived from these mice, no xCT was (Fig. of cystine transport was but the activity was by glutamate and was induced by that no activity of cystine transport via system x–c is in these cells (Fig. These that the homozygous mice xCT-null of xCT-null xCT–/– mice and and were fertile. were healthy in appearance at the of The of homozygous mixed background were The of the xCT–/– from with mixed background was that rates in the were no were in of the including and of and at the of 8 In no were in these mice of plasma amino acids from littermate and xCT–/– mice that the xCT–/– the concentration of cystine in plasma to the wild-type mice (Fig. Plasma amino acid other than cystine no significant Cysteine concentration was in the plasma of and xCT–/– mice, the xCT–/– the concentration of GSH of the wild-type mice (Fig. of the total non-protein-bound disulfide forms of cysteine i.e. cystine in plasma was higher in xCT–/– mice than in mice. the cystine concentration shown in Fig. the part of is thought to be was higher in xCT–/– mice but the of and GSH was in and xCT–/– mice. The results that the plasma of xCT–/– mice is in oxidized than that of wild-type mice. We have measured the GSH contents in the and of and xCT–/– mice at the of 8 – and significant between the two of and in the plasma of the and xCT–/– mice. Plasma of littermate and xCT–/– mice at the of 8 were and of and were of and as cysteine and GSH the to to of Embryonic from xCT-null to culture embryonic fibroblasts from the day 14 embryos of the littermate and xCT–/– mice. Cells derived from xCT–/– mice and under the routine culture cells derived from and mice However, xCT–/– cells were cultured in the presence of with 50 μm cells (Fig. In these embryonic the activity of cystine transport of the xCT–/– cells was compared with that of the and cells (Fig. and the activity and was by glutamate that the slight transport of cystine in the xCT–/– cells was mediated by system In these cells, the intracellular cysteine and GSH were measured in the presence or absence of 2ME. intracellular cysteine and GSH of xCT–/– cells were within 8 in the absence of (Fig. and activity of cystine uptake and intracellular cysteine and GSH in embryonic fibroblasts derived from the and xCT–/– mice. and cells were cultured in the absence of 50 μm and xCT–/– cells were cultured in the presence of 50 μm 2ME. of uptake of was the and and cells were cultured in the absence of 50 μm and xCT–/– cells were cultured in the presence of 50 μm 2ME. cells were cultured in or without 50 μm and the cell was prepared at the time The of intracellular cysteine and GSH were the Cell was in the presence of N-acetyl cysteine or E. shown in Fig. the of the xCT–/– cells cultured in the routine culture without 2ME. However, the concentration of for maintaining the cell was than 1 which was higher than that of 2ME. the other xCT–/– cells from the induced by the of although cells In the present study, we describe the generation of a mutation of the xCT gene by homologous in the The mutation results in the redox in plasma of the mice, i.e. a significant oxidative of the plasma cystine/cysteine redox balance and the in the plasma concentration of compared with of the wild-type mice. P. Biol. Med. 2002; PubMed Scopus Google Scholar) have the redox for the cystine/cysteine 2 is using the shown in is the for the redox is the is the is the of and is for the cystine/cysteine (pH is J. P. Biol. Med. PubMed Scopus Google Scholar). to by S. P. 2002; PubMed Scopus Google Scholar). shown in and the plasma of cystine and cysteine μm and μm on in the mice. The in these mice is the other in the xCT–/– mice, the of cystine and cysteine μm and μm on and is The between the and xCT–/– mice is P. Biol. Med. 2002; PubMed Scopus Google Scholar), that the for cystine/cysteine redox balance in the human at the of 20 is and a of cystine/cysteine redox balance with at a of the P. Biol. Med. 2002; PubMed Scopus Google Scholar). The oxidative of in the xCT–/– mice that the is in these mice. is the in plasma cystine in with a in plasma cysteine P. Biol. Med. 2002; PubMed Scopus Google Scholar). is that the results between the and xCT–/– mice at the of 8 i.e. significantly higher plasma cystine with in the plasma cysteine in the xCT–/– mice, compared with the mice. is thought that the plasma of cystine and cysteine determined by several including the of cysteine from or protein with oxidized of and with the intracellular of and the membrane transport and rates of cystine and cysteine K. E. 2002; PubMed Scopus Google Scholar). the activity of the system of cystine in the of the is important factor for the plasma cystine and cysteine S. Biochim. Biophys. Acta. 1984; PubMed Scopus Google Scholar). In the plasma of with significant of cystine is 1980; PubMed Scopus Google Scholar, Acta. 2001; PubMed Scopus Google Scholar). Thus, the system of cystine in the which is mediated by amino acid transport system plays a pivotal role for maintaining the plasma cystine/cysteine redox However, the results that xCT also functions as a system for the of cystine in xCT is in of the H. Tamba M. S. Sato K. K. M. Bannai S. J. 2002; PubMed Google Scholar), in (Fig. and in K. M. and S. These take part in the of cystine in Because xCT–/– mice system at least in part the of system in xCT–/– mice. In GSH contents in there was no difference between and xCT–/– mice. it is to plasma cysteine concentration. the other the plasma GSH level is significantly in xCT–/– mice. be that cystine at the high concentration in plasma of these mice with GSH to mixed disulfide in the This is by the shown in Fig. 5 the of GSH in xCT–/– mice was by the of significant difference in the of the plasma of GSH and a that xCT–/– mice have a in GSH The fibroblasts from the of the xCT–/– mice survive under the routine culture but in the presence of (Fig. described previously T. Bannai S. Sugita Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar), with cystine in the medium and a mixed disulfide of and which is up by cells neutral amino acid The mixed disulfide in cells is rapidly to cysteine and and the is released out of cells and with cystine In this cysteine is and cells survive and Because functions 50 μm is to the other high concentration of is required for maintaining the of cells derived from xCT–/– mice (Fig. is thought to be into the cell and is to with cells cultured with or cells cultured with although (Fig. GSH in these cells was In this cysteine is from the medium, and cells to of the of protein The results that cells from xCT–/– mice under the routine culture conditions die of stress, which is by antioxidant as GSH or E. xCT is a inducible i.e. it is induced by electrophilic agents (7Bannai S. J. Biol. Chem. 1984; 259: 2435-2440Abstract Full Text PDF PubMed Google Scholar), oxygen (4Bannai S. Sato H. Ishii T. Sugita Y. J. Biol. Chem. 1989; 264: 18480-18484Abstract Full Text PDF PubMed Google Scholar), and like and (9Sato H. Fujiwara K. Sagara J. Bannai S. Biochem. J. 1995; 310: 547-551Crossref PubMed Scopus (101) Google Scholar). The xCT–/– mice be to oxidative is the response of these mice to these in vivo. The oxidative of the plasma cystine/cysteine redox balance was in with increased P. Biol. Med. 2002; PubMed Scopus Google Scholar, H. P. PubMed Google Scholar) but also in with and in with or H. P. PubMed Google Scholar, P. E. J. PubMed Scopus Google Scholar, M. S. P. Biol. Med. PubMed Scopus Google Scholar). have in response to in the redox Physiol. Rev. 2001; Scopus Google Scholar). The plasma cystine/cysteine redox balance is significantly oxidized in the xCT–/– mice at the of is that in these mice. The xCT–/– mice be for and to oxidative We M. K. S. of for and G. and M. for We K. for the Cre recombinase-expressing mice