Redox status in mammalian cells and stem cells during culture in vitro: critical roles of Nrf2 and cystine transporter activity in the maintenance of redox balance

Abstract

Culturing cells and tissues in vitro has provided valuable insights into the molecular mechanisms regulating redox signaling in cells with implications for medicine. However, standard culture techniques maintain mammalian cells in vitro under an artificial physicochemical environment such as ambient air and 5% CO2. Oxidative stress is caused by the rapid oxidation of cysteine to cystine in culture media catalyzed by transition metals, leading to diminished intracellular cysteine and glutathione (GSH) pools. Some cells, such as fibroblasts and macrophages, express cystine transport activity, designated as system [Formula: see text], which enables cells to maintain these pools to counteract oxidative stress. Additionally, many cells have the ability to activate the redox sensitive transcription factor Nrf2, a master regulator of cellular defenses against oxidative stress, and to upregulate xCT, the subunit of the [Formula: see text] transport system leading to increases in cellular GSH. In contrast, some cells, including lymphoid cells, embryonic stem cells and iPS cells, express relatively low levels of xCT and cannot maintain cellular cysteine and GSH pools. Thus, fibroblasts have been used as feeder cells for the latter cell types based on their ability to supply cysteine. Other key Nrf2 regulated gene products include heme oxygenase 1, peroxiredoxin 1 and sequestosome1. In macrophages, oxidized LDL activates Nrf2 and upregulates the scavenger receptor CD36 forming a positive feedback loop to facilitate removal of the oxidant from the vascular microenvironment. This review describes cell type specific responses to oxygen derived stress, and the key roles that activation of Nrf2 and membrane transport of cystine and cysteine play in the maintenance and proliferation of mammalian cells in culture.

Keywords: 2-Mercaptoethanol; 4HNE, 4-hydroxynonenal; BCS, bathocuproine sulfonate; CD36; Cystine transporter; ES cells, embryonic stem cells; Embryonic stem cells; Feeder cells; Glutathione; HO-1, heme oxygenase 1; Keap1, Kelch-like ECH-associated protein 1; Lymphocytes; MRPs, multidrug resistance-associated proteins; Nrf2; Nrf2, nuclear factor erythroid 2-related factor 2; Oxygen; Prx1, peroxiredoxin 1; SQSTM1, sequestosome1; iPS cells; iPS cells, induced pluripotent stem cells; oxLDL, oxidized low density lipoprotein; xCT.

Graphical abstract

Fig. 1

Role of cystine transport in the maintenance of intracellular cysteine and GSH levels. Cysteine (CySH) is rapidly air-oxidized to cystine (CyS–SCy) in culture media catalyzed by reactions with transition metals. The specific cystine/glutamate Na⁺-independent transport system $x_{c^{-}}$ transports one cystine into cells in exchange for export of one glutamate molecule [14,23]. The cystine transporter is composed of a heterodimer, 4F2hc and xCT [28]. Induction of xCT expression by oxidative stress is regulated by Nrf2 while 4Fhc is constitutively expressed. Cystine is reduced to cysteine in cells, but effluxes from cells via neutral amino acid transporters including system L. A high concentration of glutamine in culture media contributes to the maintenance of high concentrations of intracellular glutamate, which facilitate efficient import of cystine via system $x_{c^{-}}$. GSH has a short half-life, since it is rapidly exported through transporters such as multidrug resistance-associated proteins (MRPs) [29]. GSH is cleaved into cysteinylglycine and amino acids by plasma membrane-bound enzymes [29].

Fig. 2

Fibroblasts serve as feeder cells by providing cysteine to co-cultured cells deficient in cystine transport activity. When human diploid fibroblasts are cultured in fresh medium containing 10% (v/v) fetal bovine serum, which has been oxidized during storage, they rapidly produce sulfhydryls in the medium [16]. These cells take up cystine via system $x_{c^{-}}$ and reduce it to cysteine intracellularly. Cellular cysteine in turn can efflux from cells and accumulate in the medium. Cysteine gradually unmasks cysteine residues in albumin, producing protein sulfhydryls via an SH/S-S exchange reaction. As fibroblasts release cysteine into the culture medium, these cells can be used as feeder cells to provide cysteine for other cells lacking cystine transporter activity.

Fig. 3

Cells deficient in cystine transport activity depend on cysteine for their survival and proliferation. Some cells such as murine lymphocytes and lymphoma L1210 cells do not express cystine transporters and largely depend on supply of extracellular cysteine for their survival. Supplemented cysteine is rapidly auto-oxidized in culture media in cell free conditions, but the presence of the copper chelator bathocuproine sulfonate (BCS) significantly prolongs the half-life of cysteine from about 30 min to 8 h. Addition of 50 µM cysteine to culture media every 24 h in the presence of 10 µM BCS supports continuous growth of L1210 murine lymphoma cells in RPMI1640 medium containing 10% (v/v) fetal calf serum [13]. Another method is to supplement a small thiol compound 2-mercaptoethanol (2ME) to the culture medium, which catalytically facilitates cysteine transport [19]. The mixed disulfide of cysteine and 2ME formed in the medium is stable and can be taken up by the cells via neutral amino acid transporters. It is reduced within the cells to cysteine and 2ME, with 2ME released back into medium. Due to its catalytic action, 2ME is routinely used at 20–50 µM concentrations for prolonged support of cell survival and growth of murine lymphocytes.

Fig. 4

Electrophilic agents and amino acid starvation up regulate xCT, resulting in increases in intracellular GSH. Diethylmaleate (DEM) is an electrophilic agent reactive with GSH and depletes GSH in human fibroblasts when added at 1 mM to culture media. However, when supplemented at 0.1 mM, it increases cellular GSH more than 2 fold within 15 h, preceded by an initial transient decrease over the first 3 h. DEM activates Nrf2 and upregulates expression of xCT, resulting in an increase in cystine uptake and cellular GSH [20,28,38]. Amino acid starvation activates transcription factor ATF4 leading upregulation of xCT expression [41].

Fig. 5

Activation of transcription factor Nrf2 by oxidized LDL and 4-hydroxynonenal in peritoneal macrophages. Both oxLDL and membrane permeable lipid metabolite 4HNE function as alarm signals for oxidative stress and activate Nrf2 leading to the upregulation of different endogenous antioxidants and CD36 expression in macrophages [21]. CD36 is a scavenger receptor which mediates roughly half of oxLDL uptake by macrophages, thereby contributing to the removal of toxic oxLDL from the micro-environment [21]. As oxLDL upregulates its receptor CD36 mainly via Nrf2 activation, the reaction provides a positive feedback loop. Exposure of murine peritoneal macrophages to elevated oxLDL concentrations leads to an upregulation of CD36 and accumulation of oil droplets in the cytoplasm [21]. In this context, Nrf2 facilitates foam cell formation under enhanced oxLDL loading [55,56]. Nrf2 activation in macrophages also upregulates antioxidant enzymes such as heme oxygenase-1 (HO-1) and peroxiredoxin 1 (Prx1) and the subunit of the $x_{c^{-}}$ transport system xCT to protect the cells from oxidative damage. Sequestosome1/p62 is known to modulate receptor mediated signaling and suppresses inflammation, however its antioxidant function remains to be defined [45,54].