Ethanol potentiates the mitogenic effects of sphingosine 1-phosphate by a zinc- and calcium-dependent mechanism in fibroblasts

In mouse embryo NIH 3T3 fibroblasts, ethanol (60-80 mM) was found to enhanc e the stimulatory effects of sphingosine 1-phosphate (S1P) on both DNA synt hesis and cell proliferation. Well-detectable potentiating effects of ethan ol on SIP-induced mitogenesis required the presence of calcium (>1 mM) and zinc (20-40 mu M) in the incubation medium. The amphibian tetrapeptide bomb esin, which is known to mobilize intracellular calcium in fibroblasts, had no effect alone, but it approximately doubled the combined stimulatory effe cts of ethanol and S1P on DNA synthesis. The synergistic mitogenic effects of ethanol and S1P were also slightly enhanced, rather than inhibited, by t he alcohol dehydrogenase inhibitor 8-methylpyrazole (5 mM). Of the various growth regulatory enzymes examined, ethanol detectably enhanced the stimula tory effects of S1P on the phosphosphorylation (activation) of p42/p44 mito gen-activated protein (MAP) kinases, but not of p38 MAP kinase. Cotreatment of fibroblasts with ethanol for 10 min also enhanced the stimulatory effec ts of S1P on the activities of c-Raf-l kinase and p70 S6 kinase, but neithe r S1P nor ethanol had effects on phosphatidylinositol 3'-kinase and Akt/PKB kinase activities. Ethanol-plus-S1P-induced DNA synthesis was partially in hibited by both PD 98059 (50 mu M) and rapamycin (10 nM), inhibitors of p42 /p44 MAP kinase kinase and mTOR/p70 S6 kinases, respectively. The results i ndicate that in NIH 3T3 fibroblasts, ethanol can enhance the mitogenic effe cts of S1P by a zinc- and calcium-dependent mechanism involving both the ra pamycin-sensitive p70 S6 kinase-dependent and the c-Raf-1/MAP kinase-depend ent growth regulatory pathways. (C) 1999 Academic Press.