Arachidonic acid release by H2O2 mediated proliferation of mouse embryonic stem cells: Involvement of Ca2+/PKC and MAPKs‐induced EGFR transactivation

Abstract

Reactive oxygen species (ROS) generated by a variety of endogenous factors and roles in embryonic stem (ES) cells has yet to be identified. Thus, we examined role of arachidonic acid (AA) in H₂O₂-indued proliferation of mouse ES cells and its related signaling molecules. AA release was maximally increased in response to 10⁻⁴ M H₂O₂ for 1 h. In addition, H₂O₂ increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the phosphorylation of protein kinase C (PKC), p44/42, p38 mitogen-activated protein kinase (MAPK), and JNK/SAPK. Moreover, H₂O₂ induced an increase in the phosphorylation of epidermal growth factor receptor (EGFR), which was blocked by the inhibition of p44/42 or p38 MAPKs. The inhibition of each signal molecule with specific inhibitors blocked H₂O₂-induced cytosolic phospholipase A₂ (cPLA₂) activation and AA release. H₂O₂ increased NF-κB phosphorylation to induce an increase in the levels of cyclooxygenase (COX)-2 proteins. Subsequently, H₂O₂ stimulated PGE₂ synthesis, which was reduced by the inhibition of NF-κB activation. Moreover, each H₂O₂ or PGE₂ increased DNA synthesis and the number of cells. However, H₂O₂-induced increase in DNA synthesis was inhibited by the suppression of cPLA₂ pathway. In conclusion, H₂O₂ increased AA release and PGE₂ production by the upregulation of cPLA₂ and COX-2 via Ca²⁺/PKC/MAPKs and EGFR transactivation, subsequently proliferation of mouse ES cells. J. Cell. Biochem. 106: 787–797, 2009.