Abstract
MicroRNAs (miRNAs) are important regulators of embryonic stem cell (ESC) biology, and their study has identified key regulatory mechanisms. To find novel pathways regulated by miRNAs in ESCs, we undertook a bioinformatics analysis of gene pathways differently expressed in the absence of miRNAs due to the deletion of Dicer, which encodes an RNase that is essential for the synthesis of miRNAs. One pathway that stood out was Ca(2+) signaling. Interestingly, we found that Dicer(-/-) ESCs had no difference in basal cytoplasmic Ca(2+) levels but were hyperresponsive when Ca(2+) import into the endoplasmic reticulum (ER) was blocked by thapsigargin. Remarkably, the increased Ca(2+) response to thapsigargin in ESCs resulted in almost no increase in apoptosis and no differences in stress response pathways, despite the importance of miRNAs in the stress response of other cell types. The increased Ca(2+) response in Dicer(-)(/-) ESCs was also observed during purinergic receptor activation, demonstrating a physiological role for the miRNA regulation of Ca(2+) signaling pathways. In examining the mechanism of increased Ca(2+) responsiveness to thapsigargin, neither store-operated Ca(2+) entry nor Ca(2+) clearance mechanisms from the cytoplasm appeared to be involved. Rather, it appeared to involve an increase in the expression of one isoform of the IP(3) receptors (Itpr2). miRNA regulation of Itpr2 expression primarily appeared to be indirect, with transcriptional regulation playing a major role. Therefore, the miRNA regulation of Itpr2 expression offers a unique mechanism to regulate Ca(2+) signaling pathways in the physiology of pluripotent stem cells.