S P has an anti apoptotic and

S1P has an anti-apoptotic and proliferative effect in ES cells and these S1P functions are contingent on the activation of MAPKs (Wong et al., 2007). Previously, it has been reported that S1P stimulates ERK, p38, or JNK phosphorylation, which is involved in the regulation of pluripotency of human ES cells (Pitson and Pebay, 2009) and mouse ES cells (Rodgers et al., 2009). However, in the present study, S1P activated ERK and JNK, but not p38, which may be explained by differences in concentration of S1P as well as differences in signaling mechanisms depending on the specific cell type. In addition, VEGF has also been shown to induce phosphorylation of MAPKs through Flk-1 (Ho and Kuo, 2007). These results suggest that the S1P receptor and Flk-1 share common downstream molecules, and thus, it is possible that the S1P-induced VEGF-dependent and -independent Flk-1 activation subsequently activates MAPKs, which mediate S1P-induced mouse ES cell proliferation. Consistent with these reports, in the present study, both S1P and VEGF-A164 elicited the ERK and JNK activation, and combination treatment of S1P and VEGF-A164 synergistically increased the phosphorylation level of ERK and JNK. S1P-induced ERK and JNK activation was blocked by S1P1/3 or Flk-1 inhibition. However, inhibition of VEGF action through VEGF-A164 Ab and VEGF siRNA did not affect on S1P-induced ERK and JNK activation, thereby suggesting that S1P-indcued ERK and JNK via S1P1/3 requires Flk-1 phosphorylation in absence of VEGF. It has been reported that the serum deprivation of mouse ES cell, which is important to develop defined culture systems in animal product-free conditions, induced Paclitaxel synchronization in G1 phase (Zhang et al., 2005). In the present study, the S1P treatment in absence of serum stimulates the ES cell proliferation through cyclin D1/CDK4 (early G1 phase) and cyclin E/CDK2 (late G1 phase) expression, which are important to drive progression through G1 past a point known as the restriction point and S-phase entry, respectively (Sherr and Roberts, 1999). Moreover, inhibition of ERK and JNK with pharmacological inhibitors blocked S1P-induced DNA synthesis and expression of CDK4/cyclin D1 and CDK2/cyclin E. Taken together, our observation suggests the possible role of S1P as a physiological regulator of the G1–S phase transition of mouse ES cells, which will provide valuable tools for modulating ES cell function and cell fate as well as development of defined culture systems. This is the first report that shows the S1P-induced increase in mouse ES cell proliferation through Flk-1 activation and related signaling cascade. A schematic illustration of the interrelationship between S1P and Flk-1 is shown in Fig. 9. Although many aspects remain entirely speculative, understanding the mechanisms by which S1P induces Flk-1 activation and cell proliferation will shed light on future therapeutic applications of ES cells in the regenerative medicine. In conclusion, S1P-elicited transactivation of Flk-1 mediated by S1P1/3-dependent β-arrestin/c-Src pathways stimulated proliferation through activation of ERK and JNK in mouse ES cells. The following are the supplementary materials related to this article.
Acknowledgment This work was supported by the Basic Science Research Program (2013-003879) through the National Research Foundation (NRF) of Korea by the Ministry of Education, Science and Technology, Republic of Korea.
Introduction Stem cells have critical roles not only in the maintenance of organ homeostasis but also in the development of tumors (Jordan et al., 2006). Recent findings confirm the idea that cells with stem cell properties are truly responsible for tumorigenesis (Dean et al., 2005). These stem cell-like cancer cells (also termed cancer stem cells, CSCs) have been defined as a subset of tumor cells with the ability to self-renew and give rise to phenotypically diverse tumor cell populations to drive tumorigenesis. Not surprisingly, CSCs have been identified in a wide variety of cancers thus far (Clevers, 2011).