Department of Surgery, Weill Cornell Medicine, New York, New York.
Vascular Biology Program, Boston Children's Hospital, Boston, Massachusetts.
Stem Cells. 2020 May;38(5):613-623. doi: 10.1002/stem.3145. Epub 2020 Jan 29.
Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule regulating organogenesis, angiogenesis, cell proliferation, and apoptosis. S1P is generated by sphingosine kinases (SPHK1 and SPHK2) through the phosphorylation of ceramide-derived sphingosine. Phenotypes caused by manipulating S1P metabolic enzymes and receptors suggested several possible functions for S1P in embryonic stem cells (ESCs), yet the mechanisms by which S1P and related sphingolipids act in ESCs are controversial. We designed a rigorous test to evaluate the requirement of S1P in murine ESCs by knocking out both Sphk1 and Sphk2 to create cells incapable of generating S1P. To accomplish this, we created lines mutant for Sphk2 and conditionally mutant (floxed) for Sphk1, allowing evaluation of ESCs that transition to double-null state. The Sphk1/2-null ESCs lack S1P and accumulate the precursor sphingosine. The double-mutant cells fail to grow due to a marked cell cycle arrest at G2/M. Mutant cells activate expression of telomere elongation factor genes Zscan4, Tcstv1, and Tcstv3 and display longer telomeric repeats. Adding exogenous S1P to the medium had no impact, but the cell cycle arrest is partially alleviated by the expression of a ceramide synthase 2, which converts excess sphingosine into ceramide. The results indicate that sphingosine kinase activity is essential in mouse ESCs for limiting the accumulation of sphingosine that otherwise drives cell cycle arrest.
鞘氨醇-1-磷酸（S1P）是一种生物活性脂质分子，可调节器官发生、血管生成、细胞增殖和凋亡。S1P 通过鞘氨醇激酶（SPHK1 和 SPHK2）将来源于神经酰胺的鞘氨醇磷酸化生成。操纵 S1P 代谢酶和受体的表型表明 S1P 在胚胎干细胞（ESCs）中可能具有几种功能，但 S1P 和相关鞘脂在 ESCs 中发挥作用的机制仍存在争议。我们设计了一项严格的测试，通过敲除 Sphk1 和 Sphk2 来创建无法产生 S1P 的细胞，以评估 S1P 在小鼠 ESCs 中的需求。为了实现这一目标，我们创建了 Sphk2 突变和 Sphk1 条件性突变（floxed）的品系，允许评估过渡到双缺失状态的 ESCs。Sphk1/2- 缺失的 ESCs 缺乏 S1P 并积累前体鞘氨醇。由于 G2/M 期的明显细胞周期停滞，双突变细胞无法生长。突变细胞激活端粒伸长因子基因 Zscan4、Tcstv1 和 Tcstv3 的表达，并显示更长的端粒重复序列。向培养基中添加外源性 S1P 没有影响，但通过表达将过量的鞘氨醇转化为神经酰胺的神经酰胺合酶 2，部分缓解了细胞周期停滞。结果表明，鞘氨醇激酶活性对于限制鞘氨醇的积累至关重要，否则鞘氨醇会导致细胞周期停滞。
