title: ATP stimulates mouse embryonic stem cell proliferation via protein kinase C, phosphatidylinositol 3-kinase/Akt, and mitogen-activated protein kinase signaling pathways.
abstract: This study investigated the effect of ATP and its related signal cascades on the proliferation of mouse ESCs. ATP increased the level of [(3)H]thymidine/5-bromo-2'-deoxyuridine incorporation and the number of cells in both a time- and dose-dependent manner. AMP-CPP (a P2X(1) and P2X(3) agonist), ATP-gammaS (a P2Y agonist), and 2-methylthio-ATP (a P2X and P2Y agonist) stimulated [(3)H]thymidine incorporation. P2 purinoceptor antagonists (suramin, reactive blue 2) inhibited the ATP-induced increase in [(3)H]thymidine incorporation. Reverse transcription-polymerase chain reaction analysis revealed P2X(3), P2X(4), P2Y(1), and P2Y(2) expression in mouse ESCs. Adenylate cyclase inhibitor (SQ 22536), phospholipase C inhibitors (neomycin or U 73122), and protein kinase C (PKC) inhibitors (bisindolylmaleimide I or staurosporine) inhibited the ATP-induced increase in [(3)H]thymidine incorporation. ATP increased the level of intracellular cAMP and inositol phosphates. ATP translocated PKC alpha, delta, and zeta from the cytosol to the membrane compartment. ATP and its agonists increased [Ca(2+)](i). In addition, the ATP-induced increase in [(3)H]thymidine incorporation was completely inhibited by a combination of EGTA (extracellular Ca(2+) chelator) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (intracellular Ca(2+) chelator). ATP phosphorylated Akt and p44/42 mitogen-activated protein kinases (MAPKs) in a time-dependent manner, and either suramin or reactive blue 2 (RB2) blocked the ATP-induced phosphorylation of Akt. Suramin, RB2, the phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), or the Akt inhibitor inhibited the phosphorylation of p44/42 MAPKs. The ATP-induced increase in [(3)H]thymidine incorporation was inhibited by wortmannin, the Akt inhibitor, and the MAPK kinase inhibitor (PD 98059). Suramin, RB2, PD 98059, and wortmannin blocked the ATP-induced increase in the cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 levels. In conclusion, ATP stimulates mouse ESC proliferation through PKC, PI3K/Akt, and MAPKs via the P2 purinoceptors.
authors.affiliation: Department of Veterinary Physiology, Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea.
authors.forename: Jung Sun
authors.initials: JS
authors.lastname: Heo
authors.forename: Ho Jae
authors.initials: HJ
authors.lastname: Han
mesh_terms: D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000819:agonists / Q000494:pharmacology*; D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D058919:Purinergic P2 Receptor Antagonists; D015398:Signal Transduction / Q000187:drug effects*; D013936:Thymidine / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
chemical_list: D004791:Enzyme Inhibitors; D058919:Purinergic P2 Receptor Antagonists; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D048052:Mitogen-Activated Protein Kinase 3; D010738:Type C Phospholipases; D001973:Bromodeoxyuridine; D013936:Thymidine