Extracellular signals play an essential role in dictating eukaryotic cell growth, proliferation and metabolism. TCR and costimulator-mediated signals drive T cell growth and clonal expansion, and coordinately reprogram metabolic cascades to fulfill the ensuing bioenergetic and biosynthetic demands (Fox et al., 2005
; Krauss et al., 2001
). While ectopic overexpression of Myc in transformed cell lines coordinately drives cell proliferation and proliferation-associated metabolic activities, including the catabolism of glucose and glutamine (Dang and Semenza, 1999
; Gordan et al., 2007
), whether the induction of endogenous Myc upon physiological stimulation plays a similar role has not been explored. We show that the activation of T cells rapidly switches metabolic programs from fatty acid β-oxidation and pyruvate oxidation via the TCA cycle to aerobic glycolysis, PPP and glutaminolysis. This metabolic reprogramming is associated with a global change in the metabolic transcriptome, which follows the induction of endogenous Myc and HIF1α upon activation. Based on the effects of acute genetic ablation of these transcription factors, we identified Myc as a critical metabolic reprogramming factor in T cells. Our studies shed light on the complex utilization of metabolic resources, pathways and intermediates that rationalize the strict dependence of T cell growth and proliferation on glutaminolysis, and indicate a novel glutaminolysis- and Myc-dependent de novo
polyamine biosynthetic pathway essential for T cell proliferation. While previous studies clearly demonstrate that Myc directly regulates T cell proliferation mainly through transcriptional control of cell cycle regulators (Dose et al., 2006
; Iritani et al., 2002
), our studies implicate Myc as an essential coordinator of T cell activation-induced cell growth, proliferation and several aspects of metabolic reprogramming.
T cell activation-induced glycolysis and glutaminolysis is reminiscent of the metabolic changes in tumor cells, where both aerobic glycolysis (the Warburg effect) and glutaminolysis are driven by aberrant oncogenic signals (Dang and Semenza, 1999
; Vander Heiden et al., 2009
). Notably, ectopic overexpression of Myc not only alters the transcription of several glycolytic genes but also preferentially switches the expression of PKM1 to PKM2 by modulating the alternative splicing of the PKM transcript (Dang and Semenza, 1999
; David et al., 2010
). While the transcription of metabolic enzymes and transporters involved in almost every step of glycolysis including PKM2 are induced in a Myc-dependent manner upon T cell activation, whether active T cells preferentially express PKM2 through alternative splicing remains to be determined. In contrast, the metabolic program associated with glutaminolysis in tumor cell lines and activated T cells is slightly different. In tumor cells, Myc controls glutamine uptake through transcription of SLC3a2, SLC5A1 and SLC7A1 and regulates the conversion of glutamine to glutamate through both transcriptional and posttranslational regulation of GLS1 (Gao et al., 2009
; Wise et al., 2008
). Corresponding steps in active T cells, however, are controlled by Myc-mediated transcription of SLC32a1, SLC32a2 and GLS2. Although recent studies indicate that the tumor suppressor p53 is required for the induction of GLS2 in a human tumor cell line (Hu et al., 2010
; Suzuki et al., 2010
), we have found that the up-regulation of GLS2 in murine T cells is p53-independent. Although the regulatory mechanisms involved are slightly different, the resemblance of tumor-associated and T cell activation-associated metabolic programs indicate that a general metabolic switch is required to support cell growth and proliferation in both pathological and physiological situations.
Glutamine has long been known to be an indispensible nutrient for proliferating cells (Kovacevic and McGivan, 1983
). Enhanced glutamine utilization in rat lymphocytes upon polyclonal mitogen stimulation is responsible for providing precursors for nucleotide biosynthesis (Newsholme et al., 1985a
). Metabolic flux analysis in a glioblastoma cell line further revealed that a major portion of glutamine-derived nitrogen and carbon are incorporated into amino acids and lactate, a fraction of which are secreted as metabolic by-products (DeBerardinis et al., 2007
). These findings indicate that glutamine is an important nitrogen- and carbon-donor for a variety of biosynthetic precursors. Supporting this notion, we found that activation of T cells triggers a Myc-dependent induction of glutaminolysis, which not only leads to the production of α-KG in the TCA cycle but is also coupled to nucleotide biosynthesis. Furthermore, the depletion of intracellular ornithine and polyamines in Myc-deficient activated T cells implicates a noncanonical, Myc-dependent polyamine biosynthetic pathway upon activation.
Early studies showed that overexpression of Myc in transformed cell lines induces the expression of ODC (Bello-Fernandez, et al., 1993
). While the protein level of ODC was not elevated, the mRNAs of all three metabolic enzymes downstream of ornithine were induced in a Myc-dependent manner upon T cell activation, leading to a massive increase in polyamine synthesis. The Myc-dependent induction of Aldh18a1, Prodh and OAT further indicates a novel Myc-dependent metabolic route upstream of ornithine that links glutamine and possibly proline to ornithine. Consistent with this, the Myc-dependent incorporation of 13
C-labeled glutamine carbon into ornithine after T cell activation indicates that activated T cells may acquire an alternative Myc-dependent pathway coupling glutaminolysis to polyamine biosynthesis to meet the markedly increased polyamine demands required for proliferation. Our results suggest that Myc-dependent glutamine catabolism not only replenishes the intermediate metabolites of the TCA cycle by producing the anaplerotic substrate α-KG, but also coordinates with Myc-dependent glucose catabolism to support amino acid, nucleotide and lipid biosynthesis. In addition, glutamine catabolism is tightly coupled to the biosynthesis of polyamines that are essential for T cell proliferation.