Department of Physiological Sciences, School of Medicine, University of Barcelona, Feixa Llarga s/n, 08907 L'Hospitalet del Llobregat, Barcelona, Spain.
Laboratory of Medicinal Chemistry (Associated Unit to CSIC), Faculty of Pharmacy and Food Sciences, and Institute of Biomedicine (IBUB), University of Barcelona, 08028 Barcelona, Spain.
Cells. 2019 Dec 19;9(1):18. doi: 10.3390/cells9010018.
Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca/ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology.
磷酸烯醇丙酮酸 (PEP) 浓度的变化会继发于葡萄糖供应的变化，从而调节 T 细胞中的钙信号，因为这种代谢物强烈抑制肌浆网 Ca/ATP 酶泵 (SERCA)。这种调节对于在肿瘤中确立免疫激活至关重要，因为 T 细胞和癌细胞争夺可用的营养物质。我们在这里检查了细胞溶质钙和下游效应途径的激活是否受葡萄糖和/或通过磷酸烯醇丙酮酸羧激酶 (PEPCK) 途径的分解代谢的影响，因为两者都被假设为 PEP 池提供燃料。我们的数据表明，在两种人结肠癌细胞系 HCT-116 和 SW480 中，细胞 PEP 与细胞外葡萄糖平行。PEP 与细胞溶质钙和 NFAT 活性相关，同时经典靶标 PTGS2 和 IL6 的转录上调被 CsA 预处理完全阻止。同样，直接将代谢物加载到细胞中会增加细胞溶质钙和 NFAT 活性。PEP 搅拌的细胞溶质钙也负责钙调蛋白 (CaM) 依赖性 c-Myc 丝氨酸 62 的磷酸化，导致活性增加，可能是通过增强蛋白质的稳定性。几种 c-Myc 靶蛋白的蛋白表达也与 PEP 水平相关。最后，作为 TCA 循环中间产物从分解代谢中直接贡献 PEP 的因子，PEPCK 参与该轴的情况受到了质疑，特别是在葡萄糖饥饿条件下。PEPCK 活性的抑制显示了 PEP 和钙水平的预期调节以及 NFAT 和 c-Myc 活性的后续调节。总之，这些结果表明，葡萄糖和 PEPCK 可以通过其对 PEP/Ca 轴的影响来调节 NFAT 和 c-Myc 活性，推进 PEP 作为一种第二信使来传递代谢、钙细胞信号和肿瘤生物学的作用。
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