Insulin stimulates Ca2+ uptake via PKC, cAMP, and p38 MAPK in mouse embryonic stem cells

Embryonic stem (ES) cells are provided as a powerful tool for developmental biology and have been shown to respond to insulin. However, little is known about the effect of insulin on [Ca2+]i regulation in the ES cells, although many cellular functions are tightly regulated by [Ca2+]i. Therefore, we examined the effect of insulin on Ca2+ uptake and its related signal pathways in the mouse ES cells. Mouse ES cells expressed alkaline phosphatase (AP), transcription factor Oct-4, and stage-specific embryonic antigen-1 (SSEA-1). Insulin increased the Ca2+ uptake in a time- and dose-dependent manner and the effect was blocked by L-type Ca2+ channel blockers, nifedifine and methoxyverapamil. Genistein or herbimycin A (tyrosine kinase inhibitors), wortmannin (PI-3K inhibitor), and staurosporine or bisindolylmaleimide I (PKC inhibitors) completely prevented insulin-induced increase of Ca2+ uptake. Wortmannin blocked insulin-induced PKC activation, but SQ 22536 (adenylate cyclase inhibitor) did not. Insulin also rapidly increased formation of inositol phosphates (IPs). We examined the involvement of MAPKs in mediating the effect of insulin on Ca2+ uptake. SB 203580 (p38 MAPK inhibitor) but not PD 98059 (p44/42 MAPKs inhibitor) blocked insulin-induced increase of Ca2+ uptake. Insulin significantly increased the phosphorylation of p38 MAPK but not p44/42 MAPKs. In addition, genistein, PKI, and bisindolylmaleimide I blocked the phosphorylation of p38 MAPK by insulin, suggesting a causal relationship. In conclusion, insulin partially stimulated Ca2+ uptake via PKC, cAMP, and p38 MAPK signaling pathways in mouse ES cells.