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

[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 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

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

SOCE still occurs but is blocked by 2-APB in PMA-activated human neutrophils.

<p>(A) Spherical resting neutrophils visualised by DIC. (B) When PMA was added (2 µM for 5 min at 37°C), neutrophils were activated and spread on glass surface thereby flattening their morphology as observed by DIC. (C) After calcium store depletion by a calcium pump blocker TG (2 µM), external Ca²⁺ (1<i> </i>mM) was transiently removed 4 min later; then, further addition of 1 mM external Ca²⁺ resulted in obvious calcium entry, revealing the putative response for SOCE. (D) 2 µM TG was added in the absence of Ca²⁺, followed by readdition of 1 mM external Ca²⁺. (E) After calcium stores depletion by 2 µM TG, PMA-activated neutrophils were treated with 75 µM 2-APB. (F) 2 µM TG was added together with 75 µM 2-APB. (G, H) Calcium stores were triggered with G protein-coupled agonist FMLP (2 µM) in the presence/absence of calcium followed by removal/readdition of 1 mM external Ca²⁺.</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

U73122 completely blocks NEM or SNP-induced SOCE and inhibits PMA-elicited spreading of neutrophils.

<p>(A) representative tracings of [Ca²⁺]_c elevation induced by NEM (100 µM) in the presence or absence of U73122 (10 µM) in Ca²⁺-containing buffer. (B) Statistic results are means ± S.E.M (n = 30 from three independent experiments).^*<i>P</i><0.01, compared with NEM.^#<i>P</i><0.01, compared with SNP. (C) PMA-activated and adherent neutrophils image was obtained by DIC. (D) The effect of U73122 (10 µM) on morphology of PMA-activated neutrophils.</p

NEM or SNP results in SOCE via sulfhydryl modification in PMA-activated neutrophils.

<p>(A) Representative tracing of [Ca²⁺]_c elevation induced by 100 µM NEM in the presence of external Ca²⁺. (B) Representative tracings of 500 µM NEM inhibitory effect on [Ca²⁺]_c change caused by SNP (500 µM) within Ca²⁺-containing medium. (C) Typical tracings of [Ca²⁺]_c responses for PMA-activated neutrophils to 100 µM NEM or 500 µM SNP stimulation in Ca²⁺-free buffer. (D, E), [Ca²⁺]_c of PMA-pretreated neutrophils was triggered with NEM (100 µM) within Ca²⁺-free HBSS followed by readdition of 1 mM external Ca²⁺. (F) 1 mM external calcium was added to Ca²⁺-free PMA-activated neutrophils before application of stimulation, followed by addition of 100 µM NEM.</p

2-APB completely blocks SOCE in response to sulfhydryl modification in PMA-activated human neutrophils.

<p>All experiments were performed with cells in Ca²⁺-containing medium. (A) Typical tracings of [Ca²⁺]_c changes of PMA-activated neutrophils induced by 100 µM NEM in the presence of 2-APB at 0 µM (-□- curve), 75 µM (-○- curve), 150 µM (-△- curve), respectively. (B) Statistic data from three independent experiments (means±SEM, n = 30 cells). ^*<i>P</i><0.01, compared with 2-APB absence. (C, D) [Ca²⁺]_c increase was induced by NEM (100 µM) or SNP (500 µM) in PMA-activated neutrophils, followed by addition of 75 µM 2-APB at t = 197 or t = 314 s, resulting in return to baseline [Ca²⁺]_c.</p

NEM and SNP have no effect on [Ca²⁺]_c of resting human neutrophils.

<p>(A) Representative tracings of [Ca²⁺]_c responses to 100 µM NEM (black line) and 500 µM SNP (gray line). (B) Typical tracing of [Ca²⁺]_c change induced by 1 µM FMLP. For all experiments, resting human neutrophils were performed in suspension (1×10⁶/mL) within Ca²⁺-containing buffer.</p