Sphingosine-1-phosphate-induced Flk-1 transactivation stimulates mouse embryonic stem cell proliferation through S1P(1)/S1P(3)-dependent beta-arrestin/c-Src pathways

Although recent findings showed that the bioactive lipid metabolites can regulate the ES cell functions, the physiological relevance of interaction between sphingosine-1-phosphate (S1P) and Flk-1 and its related signaling molecules are not yet clear in ES cell proliferation. In the present study, S1P(1-5) receptors were expressed in mouse ES cells and S1P increased S1P(1-3) receptor expression level. S1P treatment stimulated the cellular proliferation in S1P(1/3)-dependent manner, located in lipid rafts. In response to S1P, beta-arrestin was recruited to S1P1/3 receptor and c-Src was activated. S1P also increased the binding of S1P(1/3) receptor with Flk-1. Similar to responses for VEGF, S1P increased Flk-1 phosphorylation, which was blocked by beta-arrestin siRNA, and PP2, but not by VEGF-A(164) antibody or VEGF siRNA. In addition, S1P induced VEGF expression and VEGFR2 kinase inhibitor (SU1498) blocked the S1P-induced cellular proliferation. However, VEGF-A(164) antibody or VEGF siRNA partially blocked S1P-induced cellular proliferation, suggesting that both VEGF-dependent Flk-1 activation and VEGF-independent Flk-1 activation are involved in S1P-induced ES cell proliferation. S1P and VEGF-induced phosphorylation of ERK and JNK were blocked by pretreatment with SU1498. Moreover, inhibition of ERK and JNK blocked S1P-induced cellular proliferation. In conclusion, S1P-elicited transactivation of Flk-1 mediated by S1P1/3-dependent beta-arrestin/c-Src pathways stimulated mouse ES cell proliferation. (C) 2013 The Authors. Published by Elsevier B. V. All rights reserved.