ATP stimulates mouse embryonic stem cell proliferation via protein kinase C, phosphatidylinositol 3-kinase/Akt, and mitogen-activated protein kinase signaling pathways

This study investigated the effect of ATP and its related signal cascades on the proliferation of mouse ESCs. ATP increased the level of [ ³H]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 P2X₃ agonist), ATP-γS (a P2Y agonist), and 2-methylthio-ATP (a P2X and P2Y agonist) stimulated [³H]thymidine incorporation. P2 purinoceptor antagonists (suramin, reactive blue 2) inhibited the ATP-induced increase in [³H]thymidine incorporation. Reverse transcription-polymerase chain reaction analysis revealed P2X₃, P2X₄, P2Y₁, and P2Y₂ 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 [³H]thymidine incorporation. ATP increased the level of intracellular cAMP and inositol phosphates. ATP translocated PKC α, δ, and ζ from the cytosol to the membrane compartment. ATP and its agonists increased [Ca ²⁺]_i. In addition, the ATP-induced increase in [ ³H]thymidine incorporation was completely inhibited by a combination of EGTA (extracellular Ca²⁺ 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 [³H]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.