ATP-dependent choline phosphate-induced mitogenesis in fibroblasts involves activation of pp70 S6 kinase and phosphatidylinositol 3'-kinase through an extracellular site. Synergistic mitogenic effects of choline phosphate and sphingosine 1-phosphate.
In 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 potentiating effects required 50-100 microM ATP. The co-mitogenic effects of ATP were mimicked by adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), ADP, and UTP, but not by AMP or adenosine, indicating the mediatory role of a purinergic P2 receptor. Externally added ChoP acted on DNA synthesis without its detectable uptake into fibroblasts, indicating that ChoP can be a mitogen only if it is released from cells. Extracellular ATP (10-100 microM) induced extensive release of ChoP from fibroblasts. ChoP had negligible effects, even in the presence of ATP or insulin, on the activity state of p42/p44 mitogen-activated 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 treatments with the PI 3'-kinase inhibitor wortmannin only partially (approximately 40-50%) reduced the combined effects of ChoP, ATP, and insulin on DNA synthesis; in contrast, the pp70 S6 kinase inhibitor rapamycin almost completely inhibited these effects. ATP and insulin also potentiated, while rapamycin strongly inhibited, the mitogenic effects of sphingosine 1-phosphate (S1P). Furthermore, even maximally effective concentrations of ChoP and S1P synergistically stimulated DNA synthesis. The results indicate that in the presence of extracellular ATP and/or S1P, ChoP induces mitogenesis through an extracellular site by mechanisms involving the activation of pp70 S6 kinase and, to a lesser extent, PI 3'-kinase.
Chung
T
T
Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA.
Crilly
K S
KS
Anderson
W H
WH
Mukherjee
J J
JJ
Kiss
Z
Z
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis; D004357:Drug Synergism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006251:Harvey murine sarcoma virus / Q000235:genetics; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008246:Lysophospholipids; D051379:Mice; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D010767:Phosphorylcholine / Q000378:metabolism / Q000494:pharmacology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011090:Polyenes / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases; D020123:Sirolimus; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D000077191:Wortmannin
D000255:Adenosine Triphosphate; D000730:Androstadienes; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004247:DNA; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D007328:Insulin; D008246:Lysophospholipids; D019869:Phosphatidylinositol 3-Kinases; D010741:Phospholipases A; D010767:Phosphorylcholine; D017853:Phosphotransferases (Alcohol Group Acceptor); D011090:Polyenes; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D019893:Ribosomal Protein S6 Kinases; D020123:Sirolimus; D013110:Sphingosine; D013936:Thymidine; D000077191:Wortmannin; C060506:sphingosine 1-phosphate