Abstract
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 [Ca 2+] i regulation in the ES cells, although many cellular functions are tightly regulated by [Ca 2+] i. Therefore, we examined the effect of insulin on Ca 2+ 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 Ca 2+ uptake in a time- and dose-dependent manner and the effect was blocked by L-type Ca 2+ 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 Ca 2+ 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 Ca 2+ uptake. SB 203580 (p38 MAPK inhibitor) but not PD 98059 (p44/42 MAPKs inhibitor) blocked insulin-induced increase of Ca 2+ 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 Ca 2+ uptake via PKC, cAMP, and p38 MAPK signaling pathways in mouse ES cells.
Published Version
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