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

Abstract

AimsThe 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.MethodsCytosolic 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.ResultsOur 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.ConclusionsElevating [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.

Involvement of the calcium-sensing receptor in mineral trioxide aggregate-induced osteogenic gene expression in murine MC3T3-E1 cells

“…The increase in the concentration of calcium ions released from MTA over 3 days was ~0.7 mM and the average of increment of each day during experimental periods was ~0.3 mM in this culture system. Recently, osteoblast proliferation through CaSR and activation of phospholipase C in the condition of 2 mM extracellular calcium ions has been shown in primary rat calvarial cells 28) . Therefore, it was supposed that the calcium ions released from MTA might affect gene expressions of Runx2, type I collagen via CaSR.…”

Comparison of Phosphoinositide-Specific Phospholipase C Expression Panels of Human Osteoblasts Versus MG-63 and Saos Osteoblast-Like Cells

“…Functional studies have demonstrated that increased calcium levels activate PLC, finally resulting in the sustained elevation of calcium concentration (4). Activated PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2), a phosphorylated derivative of phosphatidylinositol that is mainly located in the inner half of the plasma membrane lipid bilayer (5-9), into inositol trisphosphate (IP3) and diacylglycerol (DAG).…”

Effect of Combined Action of Extracellular ATP and Elevated Calcium on Osteogenic Differentiation of Primary Cultures From Rat Calvaria

“…In addition, it has been shown that Ca 2+ , dose‐dependently, enhances proliferation of MC3T3 and human periosteal derived stem cells [Chai et al, ]. It also was reported that elevation of extracellular Ca 2+ stimulates the proliferation of rat primary osteoblasts involving store‐operated calcium entry and phosphorylation of ERK1/2 induced by activation of calcium‐sensing receptors [Ma et al, ; Hu et al, ]. ERK1/2 phosphorylation also occurred when MC3T3‐E1 cells were treated with PO 4 3− in the presence of Ca 2+ , but not in its absence [Khoshniat et al, ].…”