Berdyshev Evgeny V, Gorshkova Irina A, Usatyuk Peter, Zhao Yutong, Saatian Bahman, Hubbard Walter, Natarajan Viswanathan
The University of Chicago, Biological Sciences Division, Department of Medicine, 929 E. 57th Street, Room W403M, Chicago, IL 60637, USA.
Cell Signal. 2006 Oct;18(10):1779-92. doi: 10.1016/j.cellsig.2006.01.018. Epub 2006 Mar 10.
Sphingosine kinase 1 (SK1) is one of the two known kinases, which generates sphingosine-1-phosphate (S1P), a potent endogenous lipid mediator involved in cell survival, proliferation, and cell-cell interactions. Activation of SK1 and intracellular generation of S1P were suggested to be part of the growth and survival factor-induced signaling, and overexpression of SK1 provoked cell tumorigenic transformation. Using a highly selective and sensitive LC-MS/MS approach, here we show that SK1 overexpression, but not SK2, in different primary cells and cultured cell lines results in predominant upregulation of the synthesis of dihydrosphingosine-1-phosphate (DHS1P) compared to S1P. Stable isotope pulse-labeling experiments in conjunction with LC-MS/MS quantitation of different sphingolipids demonstrated strong interference of overexpressed SK1 with the de novo sphingolipid biosynthesis by deviating metabolic flow of newly formed sphingoid bases from ceramide formation toward the synthesis of DHS1P. On the contrary, S1P biosynthesis was not directly linked to the de novo sphingoid bases transformations and was dependent on catabolic generation of sphingosine from complex sphingolipids. As a result of SK1 overexpression, migration and Ca2+-response of human pulmonary artery endothelial cells (HPAEC) to stimulation with external S1P, but not thrombin, was strongly impaired. In contrast, selective increase in intracellular content of DHS1P or S1P through the uptake and phosphorylation of corresponding sphingoid bases had no effect on S1P-induced signaling or facilitation of wound healing. Furthermore, infection of human bronchial epithelial cells (HBEpC) with RSV A-2 virus increased SK1-mediated synthesis of DHS1P and S1P, whereas TNF-alpha enhanced only S1P production in HPAEC. These findings uncover a new functional role for SK1, which can control survival/death (DHS1P-S1P/ceramides) balance by targeting sphingolipid de novo biosynthesis and selectively generating DHS1P at a metabolic step preceding ceramide formation.
鞘氨醇激酶1（SK1）是已知的两种激酶之一，它能生成1-磷酸鞘氨醇（S1P），这是一种强大的内源性脂质介质，参与细胞存活、增殖及细胞间相互作用。SK1的激活及S1P在细胞内的生成被认为是生长和存活因子诱导信号传导的一部分，而SK1的过表达会引发细胞的致瘤转化。通过一种高度选择性和灵敏的液相色谱-串联质谱（LC-MS/MS）方法，我们在此表明，在不同原代细胞和培养细胞系中，SK1而非SK2的过表达导致二氢鞘氨醇-1-磷酸（DHS1P）的合成相较于S1P出现显著上调。结合LC-MS/MS对不同鞘脂进行定量的稳定同位素脉冲标记实验表明，过表达的SK1通过使新形成的鞘氨醇碱的代谢流从神经酰胺形成转向DHS1P的合成，对鞘脂从头生物合成产生强烈干扰。相反，S1P生物合成与鞘氨醇碱的从头转化并无直接关联，而是依赖于复杂鞘脂分解代谢产生鞘氨醇。由于SK1过表达，人肺动脉内皮细胞（HPAEC）对外部S1P刺激而非凝血酶刺激的迁移和Ca2+反应受到强烈损害。相比之下，通过摄取和磷酸化相应鞘氨醇碱选择性增加细胞内DHS1P或S1P的含量，对S1P诱导的信号传导或伤口愈合促进并无影响。此外，呼吸道合胞病毒A-2感染人支气管上皮细胞（HBEpC）会增加SK1介导的DHS1P和S1P合成，而肿瘤坏死因子-α仅增强HPAEC中S1P的产生。这些发现揭示了SK1的一种新功能，即通过靶向鞘脂从头生物合成并在神经酰胺形成之前的代谢步骤选择性生成DHS1P，来控制存活/死亡（DHS1P-S1P/神经酰胺）平衡。
