The phosphorylated derivative of sphingosine, sphingosine-1-phosphate, is a short-living metabolite of ultimate ceramide degradation and was shown to act as an intracellular signaling molecule, stimulating cell proliferation in quiescent Swiss 3T3 fibroblasts and inducing the release of calcium from intracellular stores (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell. Biol. 114, 155-167). In the present study, 24-h treatment of Swiss 3T3 fibroblasts with the synthetic sphingosine analogue cis-4-methylsphingosine resulted in proliferation of quiescent Swiss 3T3 fibroblasts that was 3-fold stronger than that of equimolar sphingosine-1-phosphate. The phosphorylated derivative of cis-4-methylsphingosine accumulated drastically in the cells. Simultaneous treatment with the sphingosine kinase inhibitor L-threo-sphinganine reduced both the amount of phosphorylated cis-4-methylsphingosine and cell proliferation induced by this compound by about 50%, indicating that the phosphorylated derivative mediated the proliferative stimulus. The mitogenic effect of cis-4-methylsphingosine was associated with a mobilization of intracellular calcium in Swiss 3T3 fibroblasts that was similar to that induced by sphingosine-1-phosphate.The results demonstrate that the phosphorylated derivative of cis-4-methylsphingosine mimics the previously reported mitogenic action of sphingosine-1-phosphate in Swiss 3T3 cells, and the stronger effect most likely corresponds to the unusual accumulation of this compound.
Sphingolipids (SL),1 i.e. glycosphingolipids and sphingomyelin, are structural molecules of the plasma membrane of eukaryotic organisms. Glycosphingolipids are involved in intercellular and cell-matrix interactions and membrane binding of antibodies, bacteria, and viruses (1). Sphingolipid catabolism converges on ceramide, which is consecutively degraded to sphingosine and sphingosine-1-phosphate (SPP). Evidence emerged that these three compounds function as intracellular second messengers with wide spectrum activity in signal transduction and cell growth regulation (2-4). Although sphingosine is widely known to be growth inhibitory and cytotoxic, it was shown to stimulate growth in Swiss 3T3 fibroblasts (5). However, several lines of evidence suggest that the mitogenic effect of sphingosine is mediated, at least to a substantial part, by SPP. SPP was shown to stimulate proliferation in quiescent Swiss 3T3 fibroblasts by an activation of the mitogen-activated protein kinase (Raf-1/MKK/MAP kinase) pathway (6). In these cells, exogenous SPP triggers mobilization of calcium from intracellular stores as well as an activation of phospholipase D, and both events are thought to be involved in mediating the proliferative response of Swiss 3T3 cells (7-9). Furthermore, activation of sphingosine kinase in response to growth factors resulted in a rapid and transient increase of endogenous SPP in Swiss 3T3 fibroblasts (10, 11), suggesting that SPP production mediates the proliferative stimulus. The int...
