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

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 H(2)O(2)-induced proliferation of mouse ES cells and its related signaling molecules. AA release was maximally increased in response to 10(-4) M H(2)O(2) for 1 h. In addition, H(2)O(2) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and the phosphorylation of protein kinase C (PKC), p44/42, p38 mitogen-activated protein kinase (MAPK), and JNK/SAPK. Moreover, H(2)O(2) 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 H(2)O(2)-induced cytosolic phospholipase A(2) (cPLA(2)) activation and AA release. H(2)O(2) increased NF-kappaB phosphorylation to induce an increase in the levels of cyclooxygenase (COX)-2 proteins. Subsequently, H(2)O(2) stimulated PGE(2) synthesis, which was reduced by the inhibition of NF-kappaB activation. Moreover, each H(2)O(2) or PGE(2) increased DNA synthesis and the number of cells. However, H(2)O(2)-induced increase in DNA synthesis was inhibited by the suppression of cPLA(2) pathway. In conclusion, H(2)O(2) increased AA release and PGE(2) production by the upregulation of cPLA(2) and COX-2 via Ca(2+)/PKC/MAPKs and EGFR transactivation, subsequently proliferation of mouse ES cells.