ATP stimulates mouse embryonic stem cell proliferation via PKC, PI3K/Akt, and MAPKs signaling pathways

¹ Department of Veterinary Physiology, Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea

^* To whom correspondence should be addressed. E-mail: hjhan{at}chonnam.ac.kr.

	Abstract

This study investigated the effect of adenosine 5' triphosphate (ATP) and its related signal cascades on the proliferation of mouse embryonic stem (ES) cells. ATP increased the level of [³H] thymidine/BrdU incorporation and the number of cells in both a timeand dose-dependent manner. AMP-CPP (a P2X₁ and P2X₃ agonist), ATP-S (a P2Y agonist), and 2-MesATP (a P2X and P2Y agonist) stimulated [³H] thymidine incorporation. P2 purinoceptor antagonists (suramin, reactive blue 2) inhibited the ATPinduced increase in [³H] thymidine incorporation. RT-PCR analysis revealed P2X₃, P2X₄, P2Y₁, and P2Y₂ expression in mouse ES cells. Adenylate cyclase inhibitor (SQ 22536), PLC inhibitors (neomycin or U 73122), and 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 BAPTA-AM (intracellular Ca²⁺ chelator). ATP phosphorylated Akt and p44/42 MAPKs in a time-dependent manner, and either suramin or RB2 blocked the ATP-induced phosphorylation of Akt. Suramin, RB2, the 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 MEK inhibitor (PD 98059). Suramin, RB2, PD 98059, and wortmannin blocked the ATP-induced increase in the cyclin D1, cyclin E, CDK2, and CDK4 levels. In conclusion, ATP stimulates mouse ES cell proliferation through PKC, PI3K/Akt, and MAPKs via the P2 purinoceptors.

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