Activation of p38 mitogen‐activated protein kinase and partial reactivation of the cell cycle by cis‐4‐methylsphingosine direct postmitotic neurons towards apoptosis

Abstract

As shown before in three different cell types, cis -4-methylsphingosine is a synthetic, membrane permeable, pro-drug, that is taken up by cells and phosphorylated to a metabolically stable cis -4-methylsphingosine-phosphate. The synthetic compound mimicked the mitogenic effect of sphingosine-1-phosphate (S1P) in Swiss 3T3 fibroblasts, but induced apoptosis in B104 neuroblastoma cells. We now investigated its effect in differentiated primary cultured neurons. In contrast to S1P, which had no effect on growth of these postmitotic cells, cis -4-methylsphingosine-phosphate induced apoptosis. Interestingly, both compounds stimulated extracellular regulated kinase (ERK) and also p38 mitogen-activated protein kinase (MAPK). Additionally, both compounds induced an increased expression of cyclin D1 but not of cyclin E. Our results document that the different physiological effects, apoptosis in the case of the accumulating metabolically stable synthetic compound vs. no apoptosis in the case of the short-living S1P, rely only on nuances of impact. In other words both sphingoid phosphates affect similar pathways albeit in a sustained and more pronounced manner in case of the metabolically stable synthetic compound. Experiments with several pharmacological inhibitors indicate that cis -4-methylsphingosine-phosphate-induced neuronal apoptosis is mediated on the one hand by a caspase dependent and p38 MAPK forwarded pathway and on the other hand by an abortive reactivation of the cell cycle, a caspase independent process.

Subcellular Origin of Sphingosine 1-Phosphate Is Essential for Its Toxic Effect in Lyase-deficient Neurons

“…When added to the culture medium, this analog is taken up and mainly phosphorylated to the respective cis-4-methylsphingosine phosphate, which accumulates intracellularly, because it is poorly cleaved (if at all) by S1P lyase (10). Intriguingly, this compound promoted proliferation of quiescent Swiss 3T3 fibroblasts (11), as does S1P (12), but induced apoptosis in postmitotic terminally differentiated primary cultured neurons (13).…”

“…Both sphingoid phosphates affected similar pathways. However, the effect of the synthetic accumulated compound was more pronounced and persistent when compared with the more transient and less pronounced effect of the short living physiological counterpart (13). We therefore assumed that conditions that allow sufficient accumulation of S1P in primary cultured neurons should end up in neuronal apoptosis.…”

“…The effect of S1P on the viability of lyase-deficient neurons was compared with the previously described neuronal apoptosis induced by cis-4-methylsphingosine (via cis-4-methylsphingosine phosphate) (13). As depicted in Fig.…”

“…This prediction was based on experimental results indicating that the different physiological effects, apoptosis in the case of the accumulating metabolically stable synthetic compound versus no apoptosis in the case of the short living S1P, rely only on nuances of impact (13). Both sphingoid phosphates affected similar pathways.…”

“…6 (D-F) clearly indicate that the partial simultaneous reduction of the expression of these three S1P receptors did not have any significant impact on apoptosis induced by S1P in lyase-deficient neurons. S1P Partially Reactivates the Cell Cycle in Lyase-deficient Neurons-We have shown previously that cis-4-methylsphingosine phosphate-induced neuronal apoptosis involves an inappropriate reactivation of the cell cycle (13). To determine whether S1P-induced apoptosis in lyase-deficient neurons involves a similar mechanism, we analyzed the expression of cyclin D1 and cyclin E. Cyclin D1 and its associated kinase, cyclin-dependent kinase 4, are major regulators of early steps in cell cycle progression, e.g.…”