Apoptotic Effect of Sphingosine 1-Phosphate and Increased Sphingosine 1-Phosphate Hydrolysis on Mesangial Cells Cultured at Low Cell Density∗

The lipid mediator sphingosine 1-phosphate (S1P) may alter the proliferation of mesangial cells during pathophysiological processes. Here, S1P stimulated proliferation of rat mesangial cells and phosphorylation of MAPKs at subconfluent cell density. Both effects were inhibited by pertussis toxin treatment. Mesangial cells expressed several S1P receptors of theendothelial differentiation gene family: EDG-1, -3, -5, and -8. Conversely, S1P induced apoptosis at low cell density (2 × 104cells/cm2), which was demonstrated by flow cytometry and Hoechst staining. Apoptosis was observed also in quiescent or growing cells and was not reverted by lysophosphatidic acid or platelet-derived growth factor. S1P enhanced phosphorylation of SAPKs. Incubation with [33P]S1P, [3H]S1P, and [3H]sphingosine demonstrated increased S1P hydrolysis, resulting in enhanced intracellular sphingosine levels and decreased S1P levels. A rise in total ceramide levels was also observed; however, ceramide did not originate from [3H]sphingosine, and S1P-induced apoptosis was not inhibited by fumonisin B, precluding involvement of de novoceramide synthesis in apoptosis. Therefore, we suggest that sphingosine accumulation and decreased S1P are primarily responsible for S1P-induced apoptosis. In conclusion, incubation of low-density mesangial cells with S1P results in apoptosis, presumably due to increased S1P hydrolysis.