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 (S1P) on phospholipase D (PLD)-mediated hydrolysis of phosphatidylethanolamine (PtdEtn), and evaluated the effects of the water-soluble product ethanolamine on S1P-induced DNA synthesis in NIH 3T3 cells. In [14C]ethanolamine-labelled cells, S1P (0.5-5 microM) 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 pretreatments (10 min) with the cell-permeant calcium chelator 1,2-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 phosphocholine (PCho), insulin, ATP or PMA. Ethanolamine (5-100 microM) did not modify the mitogenic effect of S1P alone, whereas at 50-100 microM concentrations it actually enhanced the mitogenic effect of PCho via a mitogen-activated protein (MAP) kinase-independent mechanism. In contrast, 5-20 microM concentrations of ethanolamine, which correspond to normal blood ethanolamine levels in humans, strongly inhibited DNA synthesis induced by S1P plus PCho via a MAP kinase-dependent mechanism; importantly, less or no inhibition was observed with 50-100 microM concentrations of ethanolamine. At 5-50 microM concentrations, ethanolamine also inhibited the synergistic mitogenic effects of both S1P plus insulin (22-27% inhibition) and PCho plus ATP (45-73% inhibition) but not those of S1P plus PMA or S1P plus ATP. The results indicate that S1P stimulates PLD-mediated hydrolysis of PtdEtn by a mechanism that may involve a regulatory protein kinase C 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 insulin, and particularly PCho.
Kiss
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The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA.
Crilly
K S
KS
Anderson
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WH
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D004247:DNA / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D019856:Ethanolamine / Q000378:metabolism / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006868:Hydrolysis; D007328:Insulin / Q000494:pharmacology; D008246:Lysophospholipids; D051379:Mice; D008934:Mitogens / Q000494:pharmacology; D008938:Mitosis / Q000187:drug effects*; D010713:Phosphatidylcholines / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010739:Phospholipase D; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005419:Flavonoids; D007328:Insulin; D008246:Lysophospholipids; D008934:Mitogens; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C060506:sphingosine 1-phosphate; D004533:Egtazic Acid; D019856:Ethanolamine; D000255:Adenosine Triphosphate; D004247:DNA; D010739:Phospholipase D; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one