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6. a cysteine tank, is an important component of redox signaling pathways and plays an essential role in T cell function. An increase in intracellular GSH levels is needed for the proliferative response of T cells to mitogens and antigens (24). Perturbation of intracellular GSH levels and the GSH/GSSG redox status dramatically affects DNA synthesis, T cell proliferation, and the cytotoxic T cell response (8,17). Low cysteine levels allow activation of NF-Bdependent transcription in the early G1phase of the cell cycle, whereas in the late G1and S phases, IL-2dependent cell proliferation is correlated with higher cysteine/GSH levels (3). GSH depletion restricts cell-cycle progression from the G1to the S phase (13). Cysteine, the limiting amino acid for GSH synthesis, can be obtained from metabolism through the transsulfuration pathway, or via transport. The ASC system imports cysteine, whereas the xC-antiporter uses the transmembrane glutamate gradient to drive import of cystine into the cell, where it is subsequently reduced to cysteine (Fig. 1). The xC-cystine transporter is composed of two subunits, the transmembrane xCT light chain, which houses the transporter activity, and the regulatory extracellular 4F2 heavy chain (22). In the extracellular compartment, cysteine exists predominantly in its oxidized form, and the concentration of plasma cysteine (10 to 25 M) is significantly lower than of cystine (100 to 200 M) (28). == FIG. 1. == Glutathione homeostasis in T cells by the xC-transporter and the transsulfuraton pathway.PPG, SAS, and azaserine are inhibitors of -cystathionase, the xC-transporter, and the neutral amino acid transporter ASC, respectively. CBS, CSE, and GCL denote cystathionine -synthase, -cystathionase, and -glutamylcysteine ligase, respectively. Nave T cells reportedly lack the xC-transporter and are thus metabolically dependent on antigen-presenting cells (APCs) for meeting their cysteine needs during activation and proliferation (1,16,29). Endowed with the xC-transporter, APCs take up cystine, and using a convoluted metabolic route involving conversion to GSH followed by its secretion and cleavage, furnish extracellular cysteine for uptake by T cells (29). GSH levels in APCs influence T cell response patterns, with low Choline bitartrate GSH favoring a Th1- over a Th2-associated cytokine-secretion pattern (19). The contribution, if any, of the transsulfuration pathway, which converts methionine to cysteine, to redox metabolism in nave T cells is not known. Methionine is an essential amino acid that is converted via the methionine cycle to homocysteine. Two enzymes in the transsulfuration pathway, cystathionine -synthase (CBS) and -cystathionase, convert homocysteine to cysteine and play a quantitatively significant role in supplying cysteine needed for GSH synthesis in several cell types (5,15). In the present study, we evaluated changes in the transsulfuration pathway and the xC-system during transformation of nave T cells to the activated state. We demonstrate, by using metabolic labeling and pharmacologic inhibition studies, the presence of an intact transsulfuration pathway in both nave and activated T cells and in Jurkat cells. We found that the expression of xCT is induced on T cell activation, weaning T cells off their metabolic dependence on APCs. Under oxidative-stress conditions, the transsulfuration pathway is upregulated in nave but not transformed T cells, whereas Rabbit Polyclonal to DDX50 its inhibition enhances cellular susceptibility to death in both nave and transformed cells. == Materials and Methods == == Mice and cell lines == Male BALB/c mice (7 to 10 weeks) were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained in pathogen-free animal facilities at the University of Michigan. The University’s Committee on Use and Care of Animals approved the protocol for animal handling used in this study. Jurkat cells were obtained from ATCC (Manassas, VA). == Isolation Choline bitartrate and preparation of murine primary cells == CD3+T lymphocytes were prepared from lymph nodes and spleen Choline bitartrate that were harvested and mashed. T cells were enriched by negative selection on Choline bitartrate T cell columns (R&D Systems, Minneapolis, MN) as per the vendor’s protocol (6). Forin vitroactivation, as isolated nave T cells were suspended in RPMI medium containing 2.5% heat-inactivated fetal bovine serum (FBS),.