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

<div><p>Aims</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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.</p></div

TG induced SOCE in rat calvarial osteoblasts.

<p>(A) After calcium store depletion by a calcium pump blocker TG (1 µM) in Ca²⁺-free buffer, addition of 2 mM external Ca²⁺ resulted in obvious calcium entry; then, further removal of external Ca²⁺ caused [Ca²⁺]_c decrease to baseline, suggesting the putative response for SOCE. (C, E) [Ca²⁺]_c increase was caused by TG (1 µM) in Ca²⁺-free HBSS, followed by application of 25 µM 2-APB or 20 µM BTP-2 during the high [Ca²⁺]_c plateau induced by re-addition of 2 mM external Ca²⁺, resulting in return to baseline [Ca²⁺]_c. Statistic data of ratio of F340/F380 before and after the application of Ca²⁺ free HBSS (B), 2-APB (D) and BTP-2 (F). * showed <i>P</i><0.05. (G) 1 µM TG was added after pretreatment with 25 µM 2-APB or 20 µM BTP-2 for 15 min, then, further addition of 2 mM external Ca²⁺ had no effect on [Ca²⁺]_c change. (H) Summary of the ratio of F340/F380 at 400 s from experiments shown in (G), * showed <i>P</i><0.05.</p

[Ca²⁺]_o-induced [Ca²⁺]_c increase was dependent on the activation of CaSR/PLC signaling in rat calvarial osteoblasts.

<p>(A) Representative tracings of [Ca²⁺]_c changes induced by elevating [Ca²⁺]_o (10 mM) alone (control) and in the presence of NPS2143 (10 µM), U73122 (5 µM) or U73343 (5 µM). Such reagents were added 15 min before application of the elevation of [Ca²⁺]_o. (B) Summary of the changes in F340/F380 at 250 s after the elevation of [Ca²⁺]_o from experiments shown in (A), * showed <i>P</i><0.05, compared with control in each group. (C) Typical tracings of [Ca²⁺]_c responses induced by induced by 2 mM spermine in the presence (black) and absence (red) of external Ca²⁺. Cells were pretreated with 25 µM 2-APB (blue) or 20 µM BTP-2 (purple) for 15 min prior to spermine (2 mM) in Ca²⁺-containing HBSS. (D) Representative tracings of [Ca²⁺]_c changes in response to 2 mM spermine in the presence of NPS2143 (10 µM), U73122 (5 µM) or U73343 (5 µM) in Ca²⁺-containging HBSS. Such reagents were added 15 min before adding spermine. (E) Summary of the changes in F340/F380 at 400 s after the stimulation with spermine in the presence Ca²⁺ free HBSS, 2-APB, BTP-2, NPS2143, U73122 or U73343 from experiments shown in C and D, * showed <i>P</i><0.05 comparing with control (spermine alone) in each group.</p

[Ca²⁺]_o-induced SOCE was involved in the high [Ca²⁺]_o-induced proliferation in rat calvarial osteoblasts.

<p>(A) Osteoblasts were cultured in medium containing different levels of [Ca²⁺]_o or in a medium with 2 µM BAPTA-AM+10 mM [Ca²⁺]_o. Typical cell morphological images were captured at 0 h, 24 h and 48 h and 72 h using a 10× objective. (B) Statistic data of cell numbers from experiments shown in (A). Each group of cells were grown in triplicate dishes and counted at different time points by measuring at least five regions (1 mm×1 mm grids). *<i>P</i><0.05, compared with control group (normal DMEM medium); # <i>P</i><0.05, compared with 10 mM [Ca²⁺]_o group. (C) Summary of absorbance (A₄₉₀) in each group. Absorbance (A₄₉₀) assessed by MTS assay is proportional to the number of living cells. Osteoblasts were incubated for 72 h in culturing medium with different levels of [Ca²⁺]_o or in a medium with 2 µM BAPTA-AM+10 mM [Ca²⁺]_o (n = 7 for each case), * showed <i>P</i><0.05, compared with [Ca²⁺]_o  = 1.8 mM group; # showed <i>P</i><0.05, compared with [Ca²⁺]_o  = 10 mM group. (D) Statistic data of cell numbers in each group at different time points. Osteoblasts were cultured in medium containing 10 mM [Ca²⁺]_o alone or together with 2-APB (25 µM), BTP-2 (20 µM), TMB-8 (50 µM), NPS2143 (10 µM), U73122 (5 µM), U73343 (5 µM), nifedipine (10 µM) and verapamil (10 µM), respectively. * showed <i>P</i><0.05 in comparison with [Ca²⁺]_o  = 10 mM group. (E) Summary of absorbance (A₄₉₀) measured after culturing for 72 h in [Ca²⁺]_o = 10 mM medium alone or 2-APB (25 µM), BTP-2 (20 µM), TMB-8 (50 µM), NPS2143 (10 µM), U73122 (5 µM), U73343 (5 µM), nifedipine (10 µM) and verapamil (10 µM) (n = 7 for each case), respectively. * showed <i>P</i><0.05 compared with [Ca²⁺]_o = 10 mM group.</p

[Ca²⁺]_o-induced [Ca²⁺]_c increase was blocked by 2-APB, BTP-2 and TMB-8 in rat calvarial osteoblasts, respectively.

<p>(A) Typical tracings of [Ca²⁺]_c responses resulted from elevating [Ca²⁺]_o (10 mM) in the absence (control) and in the presence of 2-APB (25 µM), BTP-2 (20 µM), or TMB-8 (50 µM). Such reagents were added for 15 min before the elevation of [Ca²⁺]_o. (B) Summary of the changes in F340/F380 at 250 s after the elevation of [Ca²⁺]_o from experiments shown in (A), * showed <i>P</i><0.05 comparing with control. (C, E, G) Representative tracings showing the effects of application of Ca²⁺ free HBSS, 25 µM 2-APB or 20 µM BTP-2 on the high [Ca²⁺]_c plateau induced by elevating [Ca²⁺]_o. Statistic data of the ratio of F340/F380 before and after the application of Ca²⁺ free HBSS (D), 2-APB (F) and BTP-2 (H), * showed <i>P</i><0.05.</p

Nifedipine or verapamil had no effect on [Ca²⁺]_o-induced [Ca²⁺]_c increase in rat calvarial osteoblasts.

<p>(A) Representative tracings of [Ca²⁺]_c changes caused by elevating [Ca²⁺]_o (10 mM) alone (control) and in the presence of nifedipine (10 µM) or verapamil (10 µM). Nifedipine or verapamil was added for 15 min before elevating [Ca²⁺]_o. (B) Summary of the changes in F340/F380 at 250 s after the elevation of [Ca²⁺]_o from experiments shown in (A). (C) Typical tracings of [Ca²⁺]_c changes caused by elevating [K⁺]_o (100 mM) alone (control) and in the presence of Ca²⁺ free HBSS, nifedipine (10 µM) or verapamil (10 µM). Nifedipine or verapamil was added for 15 min before elevating [K⁺]_o. (D) Summary of the peak values of increase in F340/F380 after the elevation of [K⁺]_o from experiments shown in (C).</p

Spatiotemporal Characteristics of Intercellular Calcium Wave Communication in Micropatterned Assemblies of Single Cells

Micropatterned substrates offer a unique possibility to define and control spatial organization of biological cells at the microscale, which greatly facilitates investigations of the cell-to-cell communication in vitro. Here, we developed a simple micropatterning strategy to resolve various spatiotemporal characteristics of intercellular calcium wave (ICW) communication among isolated BV-2 microglial cells. By using a single-ring assembly, we found that the direction of the initial transmitter secretion was strongly correlated with the site of the cell at which the mechanical stimulus triggering the ICWs was imposed. By using multiring assemblies, we observed that the response ratio of the same outmost cells 160 μm away from the center increased from 0% in the single-ring assembly to 9.6% in the four-ring assembly. This revealed that cells located in the interring acted as regenerative amplifiers for the ICWs generated by the central cell. By using a special oval-type micropattern, we found that calcium mobilization in lamellipodia of a fusiform BV-2 microglia cell occurred 2.9 times faster than that in the middle part of the cell, demonstrating a higher region-specific sensitivity of lamellipodia to the transmitter. Taken together, our micropatterning strategy opened up new experimental prospects to study ICWs and revealed novel spatiotemporal characteristics of ICW communication including stimulation site-dependent secretion, regenerative propagation, and region-specific cell sensitivity

Spatiotemporal Characteristics of Intercellular Calcium Wave Communication in Micropatterned Assemblies of Single Cells

Micropatterned substrates offer a unique possibility to define and control spatial organization of biological cells at the microscale, which greatly facilitates investigations of the cell-to-cell communication in vitro. Here, we developed a simple micropatterning strategy to resolve various spatiotemporal characteristics of intercellular calcium wave (ICW) communication among isolated BV-2 microglial cells. By using a single-ring assembly, we found that the direction of the initial transmitter secretion was strongly correlated with the site of the cell at which the mechanical stimulus triggering the ICWs was imposed. By using multiring assemblies, we observed that the response ratio of the same outmost cells 160 μm away from the center increased from 0% in the single-ring assembly to 9.6% in the four-ring assembly. This revealed that cells located in the interring acted as regenerative amplifiers for the ICWs generated by the central cell. By using a special oval-type micropattern, we found that calcium mobilization in lamellipodia of a fusiform BV-2 microglia cell occurred 2.9 times faster than that in the middle part of the cell, demonstrating a higher region-specific sensitivity of lamellipodia to the transmitter. Taken together, our micropatterning strategy opened up new experimental prospects to study ICWs and revealed novel spatiotemporal characteristics of ICW communication including stimulation site-dependent secretion, regenerative propagation, and region-specific cell sensitivity

Spatiotemporal Characteristics of Intercellular Calcium Wave Communication in Micropatterned Assemblies of Single Cells

Micropatterned substrates offer a unique possibility to define and control spatial organization of biological cells at the microscale, which greatly facilitates investigations of the cell-to-cell communication in vitro. Here, we developed a simple micropatterning strategy to resolve various spatiotemporal characteristics of intercellular calcium wave (ICW) communication among isolated BV-2 microglial cells. By using a single-ring assembly, we found that the direction of the initial transmitter secretion was strongly correlated with the site of the cell at which the mechanical stimulus triggering the ICWs was imposed. By using multiring assemblies, we observed that the response ratio of the same outmost cells 160 μm away from the center increased from 0% in the single-ring assembly to 9.6% in the four-ring assembly. This revealed that cells located in the interring acted as regenerative amplifiers for the ICWs generated by the central cell. By using a special oval-type micropattern, we found that calcium mobilization in lamellipodia of a fusiform BV-2 microglia cell occurred 2.9 times faster than that in the middle part of the cell, demonstrating a higher region-specific sensitivity of lamellipodia to the transmitter. Taken together, our micropatterning strategy opened up new experimental prospects to study ICWs and revealed novel spatiotemporal characteristics of ICW communication including stimulation site-dependent secretion, regenerative propagation, and region-specific cell sensitivity

Mechanism underlying mechanical stimulation-elicited ICWs among BV-2 microglia.

<p><b>①</b> A range of nucleotides, including ATP, ADP and UDP etc., are released into extracellular space in response to mechanical stimulus. <b>②</b> P2Y_12/13 receptors localized on the membrane of neighboring cells can sense ATP/ADP released from the stimulated cell, then activate PLC/IP₃/Ca²⁺ signaling. <b>③</b> Upon sensing and responding to released ATP/ADP, Ca²⁺ mobilization sequentially occur in neighboring cells, thus perform as intercellular calcium waves. <b>④</b> ATP cannot activate P2Y_12/13 receptors directly. Ecto-NTPase located on the plasma membrane could catalyze hydrolysis of ATP and generates ADP that predominantly activates P2Y_12/13 receptors.</p