Cystine/glutamate transporter, system xc−, is involved in nitric oxide production in mouse peritoneal macrophages
Introduction
Macrophages produce large amounts of reactive oxygen species (ROS) and nitric oxide (NO) in response to inflammatory stimuli such as bacterial lipopolysaccharides (LPS) and interferon γ, and function to protect the host from bacterial infection [1,2]. In macrophages, NO synthase 2 (NOS2) is induced by various stimuli, including LPS and interferon γ, which cause the production of large amounts of NO. Recent studies have revealed that NO produced by macrophages functions, not only as an antimicrobial, tumoricidal, and tissue-damaging effector molecule operating in the innate immune system, but also as an effector of adaptive immune responses and cyto-protection [3]. Although NO itself plays multiple physiological roles, it also reacts with other gaseous molecules and is converted to reactive nitrogen oxide species (RNOS). Among the known RNOS, peroxynitrite (ONOO−), which is formed by the reaction of NO with superoxide, exerts oxidative damage to many biological molecules [4]. Acting in coordination, antioxidative and redox molecules protect cells against oxidative and nitrosative stress by eliminating ROS and RNOS.
Glutathione is the most abundant non-protein thiol that is produced in cells and plays pleiotropic roles, such as antioxidation and the detoxification of toxicants. The antioxidative functions of glutathione are effectively expressed by donating electrons to peroxides via glutathione peroxidase (GPX) [5]. Thus, an insufficiency of glutathione triggers the redox imbalance and makes cells more vulnerable to oxidative insults, leading to cell death.
Intracellular glutathione levels are maintained by de novo synthesis from constituent amino acids, glutamate, glycine, and cysteine that is catalyzed by the action of γ-glutamylcysteine synthetase (γGCS) and glutathione synthetase (GSS) as well as by recycling oxidized glutathione by glutathione reductase (GSR) in a NADPH-dependent manner [6]. A redox-sensitive transcriptional regulatory factor, Nrf2, regulates the expression of the enzymes that are involved in both the de novo synthesis and reductive recycling of the oxidized glutathione [7,8]. Among three substrate amino acids, cysteine is generally maintained at low levels in many types of cells and thus constitutes a rate-limiting factor for the synthesis of glutathione [9].
Several systems, including amino acid transporters and metabolic protein degradation, function to supply cysteine to cells [10]. In some types of cells, cysteine can be supplied via the transsulfuration pathway in association with methionine metabolism [11]. A reduced form of extracellular cysteine is transported into cells via neutral amino acid transporters, e.g., ASCTs and LAT2 [12,13]. The oxidized form of cysteine, i.e., cystine, is taken up by cells via system xc−, which is composed of two protein components; xCT (SLC7A11) and 4F2hc (SLC3A2) [14]. In many cultured cells, xCT is induced by various stimuli such as electrophilic agents [15], amino acid deprivation [16], bacterial lipopolysaccharide [17], and contributes to the maintenance of intracellular glutathione level. xCT is the main component of the system xc− and mediates the transport of cystine and cystathionine into cells in exchange with glutamate [14,18].
xCT is constitutively expressed in a limited number of organs, such as the thymus, spleen, and cerebral meninges [19]. Upon oxidative stress, xCT is induced in alveolar macrophages, the kidney, and liver, and hence a xCT deficiency aggravates the oxidative damage in these organs [[20], [21], [22], [23]]. xCT-deficient (KO) mice show healthy phenotypes except for a redox imbalance in the blood plasma [24]. Due to the oxidative conversion of cysteine to cystine in the culture medium, intracellular glutathione levels depend on the extent of cystine uptake via xCT in many types of cultured cells under routine culture conditions. Mouse embryonic fibroblasts (MEF) isolated from KO mice are unable to survive under routine culture conditions due to decreased glutathione levels [18,24].
We previously reported that the activities of system xc− and system y+, which mediates the transport of cationic amino acids, are strongly induced by a very small amount of LPS in macrophages [17,25]. Because arginine is a substrate of system y+ and a precursor of NO, it seems rational that system y+ and NOS2 are simultaneously induced by LPS. The question arises as to whether the simultaneous induction of system xc− and system y+ has any physiological and/or pathophysiological significance. In the present study, we compared NO production by peritoneal macrophages isolated from both KO and wild-type (WT) mice under various culture conditions.
Section snippets
Materials
All chemicals and agents were purchased from Sigma (St. Louis, MO, USA) or Wako Pure Chemicals Industries (Osaka, Japan), unless otherwise stated.
Mice
KO mice were generated, as described in a previous study using a gene-targeting technique [24]. The genotype was determined by PCR, as reported previously. After backcrossing the C57BL/6N mice more than 10 times, the resulting WT and KO mice were used in the experiments reported here. All of the mice had free access to standard rodent chow and water.
Production of nitric oxide in xCT-deficient macrophages
To investigate a role of xCT in NO production in macrophages, we measured the levels of nitrite, a stable metabolite of NO, in culture medium of LPS-stimulated macrophages isolated from xCT-deficient (KO) and wild-type (WT) mice. Nitrite levels were increased in the culture medium of both KO and WT macrophages in a time-dependent manner. However, nitrite levels in medium of KO macrophages were significantly lower than that of WT cells (Fig. 1A). In both macrophages, arginine uptake activity was
Discussion
The findings reported in this study show that NO production in xCT-deficient (KO) macrophages is significantly decreased compared to that in WT cells. xCT primarily functions as an amino acid transporter which mediates an exchange of cystine and glutamate on the plasma membrane. There are several possible explanations for how such a deficiency in a transporter could cause a decrease in NO production. One possibility is the inactivation of post-translational modification of the NOS2 protein.
Author contribution
SK performed most of the experiments, SH, TH, MS, RK assisted in some of the experiments, such as western blot of proteins and detection of oxidative stress of macrophages. SB, JF advised the design of some experiments and helped writing manuscript. SK, JF and HS designed the experiments and wrote the paper.
Conflicts of interest
The authors declared no conflicts of interest.
Acknowledgement
This work was supported by the JSPS KAKENHI Grant-in-Aid for Research Activity Start-up (16H06648) to SK.
References (39)
Nitric oxide synthase in innate and adaptive immunity: an update
Trends Immunol.
(2015)- et al.
Review: glutathione peroxidases
BBA - Gen. Subj
(2013) Glutathione biosynthesis and its inhibition
Methods Enzymol.
(1995)- et al.
Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages
J. Biol. Chem.
(2000) - et al.
Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress
Free Radic. Biol. Med.
(2009) - et al.
The SLC1 high-affinity glutamate and neutral amino acid transporter family
Mol. Aspect. Med.
(2013) - et al.
Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity
J. Biol. Chem.
(1999) - et al.
Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins
J. Biol. Chem.
(1999) - et al.
Electrophile response element-mediated induction of the cystine/glutamate exchange transporter gene expression
J. Biol. Chem.
(2002) - et al.
Transcriptional control of cystine/glutamate transporter gene by amino acid deprivation
Biochem. Biophys. Res. Commun.
(2004)
Cystathionine is a novel substrate of cystine/glutamate transporter: implications for immune function implications for immune function
J. Biol. Chem.
Aggravation of ischemia-reperfusion-triggered acute renal failure in xCT-deficient mice
Arch. Biochem. Biophys.
Enhanced expression of cystine/glutamate transporter in the lung caused by the oxidative-stress-inducing agent paraquat
Free Radic. Biol. Med.
Ascorbic acid prevents acetaminophen-induced hepatotoxicity in mice by ameliorating glutathione recovery and autophagy
Arch. Biochem. Biophys.
Redox imbalance in cystine/glutamate transporter-deficient mice
J. Biol. Chem.
Induction of cationic amino acid transport activity in mouse peritoneal macrophages by lipopolysaccharide
Biochim. Biophys. Acta Biomembr.
Methodologies for the application of monobromobimane to the simultaneous analysis of soluble and protein thiol components of biological systems
J. Biochem. Biophys. Meth.
Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides
J. Biol. Chem.
Nitric-oxide synthase forms N-NO-pterin and S-NO-Cys: implications for activity, allostery, and regulation
J. Biol. Chem.
Cited by (19)
Carnosine dipeptidase II (CNDP2) protects cells under cysteine insufficiency by hydrolyzing glutathione-related peptides
2021, Free Radical Biology and MedicineCitation Excerpt :Primary hepatocytes from xCT KO mice maintain normal levels of Cys and GSH and can survive under cultured conditions for certain periods, which is rationalized assuming that sufficient amounts of Cys are produced via the transsulfuration pathway [16]. On the other hand, peripheral macrophages isolated from xCT KO mice contain only low levels of Cys and GSH but are still viable, even though Cys is not fully compensated for by the transsulfuration pathway [17]. Thus, macrophages appear to be different from hepatocytes, but the issue of specifically how xCT KO macrophages survive remains unclear.
Ferroptosis in the tumor microenvironment: perspectives for immunotherapy
2021, Trends in Molecular MedicineNitric oxide produced by NOS2 copes with the cytotoxic effects of superoxide in macrophages
2021, Biochemistry and Biophysics ReportsCitation Excerpt :These observations were also consistent with the stimulated production of NO in SOD1 KO macrophages compared to WT macrophages. We previously reported that arginine uptake is stimulated by an LPS treatment in cultured mouse macrophages [18]. It has been also reported that the cationic amino acid transporter (CAT)-2, which is responsible for taking up arginine, is induced in stimulated macrophages [29].
Effect of chitosan oligosaccharide-conjugated selenium on improving immune function and blocking gastric cancer growth
2021, European Journal of PharmacologyCitation Excerpt :Macrophage senses conserved microbial structures and induces a proinflammatory response via the secretion of cytokines (Fang et al., 2019). Nitric oxide (NO) produced by macrophages functions, not only serves as an antimicrobial, tumoricidal and tissue-damaging effector molecules operating in the innate immune system, but also serves as an effector of adaptive immune responses and cyto-protection (Kobayashi et al., 2018). Interleukin (IL)-1β is a pleiotropic cytokine and a master mediator of inflammation, which plays a crucial role in the host defense against bacterial infection and tissue injury (Fang et al., 2019).