Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts both extracellularly and intracellularly. The SPL gene encodes a mammalian S1P lyase that degrades S1P. Here, we have disrupted the SPL gene in mouse F9 embryonal carcinoma cells by gene targeting. This is the first report of gene disruption of mammalian S1P lyase. The SPL-null cells exhibited no S1P lyase activity, and intracellular S1P was increased approximately 2-fold, compared with wild-type cells. Treatment of F9 embryonal carcinoma cells with retinoic acid induces differentiation to primitive endoderm (PrE). An acceleration in this PrE differentiation was observed in the SPL-null cells. This effect was apparently caused by the accumulated S1P, since N,N-dimethylsphingosine, a S1P synthesis inhibitor, had an inhibitory effect on the PrE differentiation. Moreover, F9 cells stably expressing sphingosine kinase also exhibited an acceleration in the differentiation. Exogenous S1P had no effect on differentiation, indicating that intracellular but not extracellular S1P is involved. Moreover, we determined that expression of the SPL protein is up-regulated during the progression to PrE. We also showed that sphingosine kinase activity is increased in PrE-differentiated cells. These results suggest that intracellular S1P has a role in the PrE differentiation and that SPL may be involved in the regulation of intracellular S1P levels during this differentiation.

Sphingosine 1-phosphate (S1P) functions as a ligand for the S1P/EDG family receptors. For years, intracellular signaling roles for S1P have also been suggested, especially in cell proliferation. Now, we have generated several mouse F9 embryonic carcinoma cell lines varying in expression of the S1P-degrading enzyme, S1P lyase (SPL) and/or sphingosine kinase (SPHK1). All these cell lines accumulated S1P compared to the wild-type F9 cells, but the amounts varied. We investigated the ability of these cells to proliferate under low serum conditions, as measured by a thymidine uptake assay. Although F9 cells over-expressing SPHK1 did exhibit enhanced DNA synthesis, other S1P-accumulating cells ( SPL -null cells and SPL -null cells over-expressing SPHK1) did not. The overproduction of both SPL and SPHK1 resulted in the most striking mitogenic effect. Moreover, nM concentrations of sphingosine (or dihydrosphingosine) stimulated DNA synthesis in an SPL -dependent manner. These results indicate that products by the SPL pathway, not S1P itself, function in mitogenesis.

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Disulfide bonds of 2-isocyanatophenyl methyl disulfide and 2-endo-isocyanato-6-endo-(methyldisulfanyl)bicyclo[2.2.1]heptane showed neighboring group participation in formation of thiocarbamates. Natural Bond Orbital (NBO) analyses revealed that the unusual nucleophilicity requires a rigid through-space...