Type 3 inositol 1,4,5-trisphosphate receptor negatively regulates apoptosis during mouse embryonic stem cell differentiation.

Ca(2+) signals generated by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are crucial for cellular processes such as apoptosis and differentiation. However, the exact roles of IP(3)Rs and their contributions to Ca(2+) signals in pluripotent embryonic stem (ES) cell behaviors remain largely unknown. In this study, we showed that the expression of type 3 IP(3)R (IP(3)R3) was transiently downregulated with a concomitant increase in apoptosis at the early differentiation stage of murine ES cells. Knockdown of IP(3)R3 by small interfering RNA increased apoptosis in differentiating cells but not in undifferentiated ES cells. Moreover, IP(3)R3 overexpression had the opposite effect. Consistently, IP(3)R3 knockdown altered Ca(2+) oscillations in differentiating cells but not in undifferentiated ES cells. The apoptosis in differentiating IP(3)R3-knockdown cells was decreased by chelating intracellular Ca(2+) with BAPTA-AM and increased in control ones. Furthermore, IP(3)R3 knockdown led to a suppression of the expression of mesodermal and mesoendodermal but not ectodermal markers. The differentiation suppressions were further confirmed by the impaired differentiation of mesodermal and some of the endodermal but not ectodermal derivatives. Such defects were partially because of the increased apoptosis in Flk-1(+) cells. These findings provide the first demonstration of the important role of IP(3)R3 in the regulation of apoptosis in early differentiating ES cells and subsequent lineage commitments through modulation of Ca(2+) signals.