Inositol 1,4,5-Trisphosphate Receptors Mediating Spontaneous Ca²⁺ Oscillation Favors Proliferation in Human Mesenchymal Stem Cells from Bone Marrow.

Although human mesenchymal stem cells (hMSCs) constitute a very small population of cells in bone marrow, they play an important role in the regulation of hematopoietic microenvironment. The self-renew and/or proliferation of hMSCs is believed to be particularly important in maintaining bone marrow niche; however, the regulation of this process is not fully understood. The present study was designed to investigate whether spontaneous Ca²⁺ oscillation mediated by inositol 1,4,5-trisphosphate (IP3) receptors participates in the proliferation regulation in cultured hMSCs from bone marrow using RT-PCR, cell proliferation assay, Western-blotting analysis. It was found that no gene expression for ryanodine-sensitive receptors was detected in undifferentiated hMSCs, while three subtypes of IP3 receptor genes (i.e. IP3R1, IP3R2, and IP3R3), and genes for sarco/endoplasmic reticulum Ca²⁺ ATPases (SERCA) (ATP2A1, ATP2A2, and ATP2A3) were expressed in these cells. The proliferation of hMSCs was reduced by inhibiting Ca²⁺ oscillation with the IP3 receptor antagonist 2-aminoethyl diphenylborinate (2-APB) or the SERCA inhibitor cyclopiazonic acid (CPA). In addition, inhibition of Mek/Erk and PI-3K/Akt signaling also decreased hMSCs proliferation. The relation of Ca²⁺ oscillation to the activity of these kinases was revealed by Western blotting analysis. Erk1/2 (Thr185/Tyr187) phosphorylation level was found to be reduced by directly inhibiting Ca²⁺ oscillation with 2-APB or CPA either in the presence or absence of serum. However, Akt (Thr308) phosphorylation was decreased only in the presence of serum, and serum-free starvation (2 h) eliminated Akt (Thr308) phosphorylation. Finally the calmodulin inhibitors W-7 and SKF-7171A were employed to further investigate whether this ubiquitous Ca²⁺ sensor is involved in the Ca²⁺ signal-mediated effect. Similarly, the phosphorylation level of Erk1/2 (Thr185/Tyr187) and Akt (Thr308) reduced upon the inhibition of calmodulin. In conclusion, our results demonstrate that IP3 receptors-mediated spontaneous Ca²⁺ oscillation and/or Ca²⁺/calmodulin signaling play(s) a crucial role in the regulation of cell proliferation mediated by multiple pro-proliferation signaling pathways (e.g. Mek/Erk and PI-3K/Akt). Importantly, Erk1/2 phosphorylation is sensitive to spontaneous Ca²⁺ oscillation, and therefore is responsible for the proliferation induced by Ca²⁺ oscillation in hMSCs.