Elevation of Extracellular Ca2+ Induces Store-Operated Calcium Entry via Calcium-Sensing Receptors: A Pathway Contributes to the Proliferation of Osteoblasts

Aims: The local concentration of extracellular Ca2+ ([Ca2+](o)) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca2+](o) induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca2+](o) to osteoblastic proliferation. Methods: Cytosolic Ca2+ concentration ([Ca2+](c)) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail. Results: Our data showed that elevating [Ca2+](o) evoked a sustained increase of [Ca2+](c) in a dose-dependent manner. This [Ca2+](c) increase was blocked by TMB-8 (Ca2+ release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or U73122 (a PLC inhibitor) strongly reduced the [Ca2+](o)-induced [Ca2+](c) increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca2+](o) resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca2+](o) significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, but not affected by Cav channels antagonists. Conclusions: Elevating [Ca2+](o) induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca2+ concentration.