EXTRACELLULAR SPHINGOSINE 1-PHOSPHATE STIMULATES FORMATION OF ETHANOLAMINE FROM PHOSPHATIDYLETHANOLAMINE - MODULATION OF SPHINGOSINE 1-PHOSPHATE-INDUCED MITOGENESIS BY ETHANOLAMINE

In this work, we determined the effects of sphingosine 1-phosphate (S1 P) on phospholipase D (PLD)-mediated hydrolysis of phosphatidylethanol amine (PtdEtn), and evaluated the effects of the water-soluble product ethanolamine on S1P-induced DNA synthesis in NIH 3T3 cells. In [C-14] ethanolamine-labelled cells, S1P (0.5-5 mu M) stimulated PLD-mediated hydrolysis of PtdEtn 1.5-2.1-fold. Down-regulation of protein kinase C by chronic (24 h) treatment of cells with 300 nM PMA, or pretreatment s (10 min) with the cell-permeant calcium chelator bis-(O-aminophenoxy )-ethane-N,N,N',N'-tetra-acetic acid tetra-acetoxymethyl ester led to the inhibition of S1P-induced PtdEtn hydrolysis. S1P alone was a weak inducer of DNA synthesis, but its effects were enhanced by phosphochol ine (PCho), insulin, ATP or PMA. Ethanolamine (5-100 mu M) did not mod ify the mitogenic effect of S1P alone, whereas at 50-100 mu M concentr ations it actually enhanced the mitogenic effect of PCho via a mitogen -activated protein (MAP) kinase-independent mechanism. In contrast, 5- 20 mu M concentrations of ethanolamine, which correspond to normal blo od ethanolamine levels in humans, strongly inhibited DNA synthesis ind uced by S1P plus PCho via a MAP kinase-dependent mechanism; importantl y, less or no inhibition was observed with 50-100 mu M concentrations of ethanolamine. At 5-50 mu M concentrations, ethanolamine also inhibi ted the synergistic mitogenic effects of both S1P plus insulin (22-27% inhibition) and PCho plus ATP (45-73% inhibition) but not those of S1 P plus PMA or S1P plus ATP. The results indicate that S1P stimulates P LD-mediated hydrolysis of PtdEtn by a mechanism that may involve a reg ulatory protein kinase isoform. Increased formation of ethanolamine by PLD-mediated. PtdEtn hydrolysis or by other means may be required for maximal stimulation of DNA synthesis by S1P in the presence of insuli n, and particularly PCho.