ATP-DEPENDENT CHOLINE PHOSPHATE-INDUCED MITOGENESIS IN FIBROBLASTS INVOLVES ACTIVATION OF PP70 S6 KINASE AND PHOSPHATIDYLINOSITOL 3'-KINASETHROUGH AN EXTRACELLULAR SITE - SYNERGISTIC MITOGENIC EFFECTS OF CHOLINE-PHOSPHATE AND SPHINGOSINE 1-PHOSPHATE

serum-starved NIH 3T3 clone 7 fibroblasts, choline phosphate (ChoP) (0 .5-1 mM) and insulin synergistically stimulate DNA synthesis. Here we report that ATP also greatly enhanced the mitogenic effects of ChoP (0 .1-1 mM) both in the absence and presence of insulin; maximal potentia ting effects required 50-100 mu M ATP. The co-mitogenic effects of ATP were mimicked by adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-0- (2-thiodiphosphate), ADP, and UTP, but not by AMP or adenosine, indica ting the mediatory role of a purinergic P-2 receptor. Externally added ChoP acted on DNA synthesis without its detectable uptake into fibrob lasts, indicating that ChoP can be a mitogen only if it is released fr om cells. Extracellular ATP (10-100 mu m) induced extensive release of ChoP from fibroblasts. ChoP had negligible effects, even in the prese nce of ATP or insulin, on the activity state of p42/p44 mitogen-activa ted protein kinases, while in combination these agents stimulated the activity of phosphatidylinositol 3'-kinase (PI 3'-kinase). Expression of a dominant negative mutant of the p85 subunit of PI 3'-kinase or tr eatments with the PI 3'-kinase inhibitor wortmannin only partially (si milar to 40-60%) reduced the combined effects of ChoP, ATP, and insuli n on DNA synthesis; in contrast, the pp70 S6 kinase inhibitor rapamyci n almost completely inhibited these effects. ATP and insulin also pote ntiated, while rapamycin strongly inhibited, the mitogenic effects of sphingosine 1-phosphate (S1P). Furthermore, even maximally effective c oncentrations of ChoP and S1P synergistically stimulated DNA synthesis . The results indicate that in the presence of extracellular ATP and/o r S1P, ChoP induces mitogenesis through an extracellular site by mecha nisms involving the activation of pp70 S6 kinase and, to a lesser exte nt, PI 3'-kinase.