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EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES

ATP Stimulates Mouse Embryonic Stem Cell Proliferation via Protein Kinase C, Phosphatidylinositol 3-Kinase/Akt, and Mitogen-Activated Protein Kinase Signaling Pathways

Department of Veterinary Physiology, Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea

Key Words. ATP • Mitogen-activated protein kinases • Phosphatidylinositol 3-kinase/Akt • Protein kinase C • Embryonic stem cells

Correspondence: Ho Jae Han, D.V.M., Ph.D., Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea. Telephone: 82-62-530-2831; Fax: 82-62-530-2809; e-mail: hjhan{at}chonnam.ac.kr

Received November 24, 2006; accepted for publication August 3, 2006.
First published online in STEM CELLS EXPRESS   August 17, 2006.



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 P2X₁ and P2X₃ agonist), ATP-S (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 , , and from the cytosol to the membrane compartment. ATP and its agonists increased [Ca²⁺]_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 Ca²⁺ 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.

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