Sphingosine Kinases Protect Murine ESCs from Sphingosine-induced Cell Cycle Arrest.

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 is 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 (CERS2), which converts excess sphingosine to ceramide. The results indicate that sphingosine kinase activity is essential in mESCs for limiting the accumulation of sphingosine that otherwise drives cell cycle arrest. © AlphaMed Press 2020 SIGNIFICANCE STATEMENT: The function of the S1P signaling pathway in embryonic stem cells (ESCs) has been unclear. We used a genetic approach to eliminate S1P from murine ESCs by deleting both sphingosine kinase orthologs. We found that loss of both kinases is incompatible with ESC proliferation, as the cells arrest at the G2/M checkpoint. However, the defect is not caused by lack of S1P but rather from accumulation of sphingosine, since the phenotype can be reverted by expression of ceramide synthase. The data are consistent with previous results from early zebrafish embryos, suggesting a key conserved role for limiting sphingosine levels in stem and progenitor cells.