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.


Introduction
Bone is constantly remodeling and maintaining homeostasis between formation and resorption. Reducing formation or increasing resorption may lead to bone loss, osteoporosis, eventually debilitating fractures [1][2][3]. Osteoblasts play a pivotal role in bone formation and mineralization by secreting bone matrix components and providing factors essential for osteoclast differentiation [4][5][6]. In the bone microenvironment, the resorptive action of osteoclasts results in a local increase of extracellular calcium concentration ([Ca 2+ ] o ) which can reach levels as high as 40 mM [7]. This high level of [Ca 2+ ] o has been suggested to regulate bone formation by stimulating osteoblastic proliferation, chemotaxis, differentiation and mineralization [8][9][10]. Indeed, in vitro studies showed that high [Ca 2+ ] o promoted proliferation in a number of osteoblast cell lines including rat calvarial osteoblasts [10].
In various cell types, the store operated calcium entry (SOCE) determines sustained cytosolic calcium concentration ([Ca 2+ ] c ) increase which is critical in regulating a variety of cellular functions including secretion, apoptosis, and more specifically proliferation [11][12][13][14]. SOCE is activated in response to a reduction of Ca 2+ concentration in the intracellular endoplasmic reticulum (ER) stores. Under physiological conditions, receptormediated activation of the phospholipase C (PLC) induces the generation of inositol 1,4,5-trisphosphate (IP 3 ) and subsequently triggers IP 3 receptor-related Ca 2+ release from ER, which may stimulate SOCE in turn [15]. The SOCE phenomenon was described in some osteoblast-like cells by previous studies [16][17][18]. Furthermore, it found that SOCE initiated by the stimulus of platelet-derived growth factor was involved in the proliferation of osteoblast-like MG-63 cells [18]. With respect to high [Ca 2+ ] oinduced osteoblastic proliferation, the underlying intracellular signaling is largely unclear. Especially, it remains unknown whether the elevation of [Ca 2+ ] o can induce SOCE, and whether high [Ca 2+ ] o -induced osteoblastic proliferation is conducted through SOCE in osteoblasts.

Ethics Statement
The animal protocol in this study conformed to the Guide for the Care and Use of Laboratory Animals (the Guide, NRC 2011), and it was also approved by the Institutional Animal Care and Use Committee at Nankai University (Approval ID 201009080081).

Osteoblasts isolation and culture
Rat calvarial osteoblasts were isolated and cultured as previously described [33,34]. Briefly, anesthetized new born Wistar rats (3-day-old) were sacrificed by decapitation. Then, bone skulls were isolated from the soft tissue, and digested with collagenase. Calvarial cells were released by repeated digestion with trypsin. The isolated osteoblasts were cultured in DMEM medium containing 10% FBS at 37uC with 5% CO 2 .
[Ca 2+ ] c was measured with calcium imaging system built on an inverted fluorescence microscope (Olympus IX51). The Ca 2+ indicator fura-2 was alternately excited at 340 nm and 380 nm with a Lambda 10-2 sutter. Fluorescence images (filtered at 515 nm625 nm) were captured by a CCD camera (CoolSNAP fx-M) and quantitated with MetaFluor. [Ca 2+ ] c was represented by the ratio of fluorescence intensity at 340 nm/fluorescence intensity at 380 nm (F340/F380). At least three independent experiments were done for each condition. One curve of calcium changes was plotted as the representation of other similar traces. Ca 2+ -free HBSS solution was made by substituting MgCl 2 for CaCl 2 at the same concentration.

Proliferation Assay
The proliferation of osteoblasts was assessed by morphological observations and direct cell counting. The number of viable cells in proliferation was further determined by MTS assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay kit) and ATP assay (CellTiter-Glo Luminescent Cell Viability Assay kit), respectively. For morphological observations, osteoblasts were plated in 35 mm culture dishes (,5610 4 cells/dish) with DMEM containing 5% FBS at 37uC. Then, the pretreated-cells in each dish were monitored by an inverted light microscope (Olympus IX51) at 0, 24, 48 and 72 h in turn. In the meantime, the cell numbers in each dish were measured from at least five regions (1 mm61 mm grids) at the indicated time. For MTS and ATP assays, osteoblasts were seeded into 96-well plate at ,1610 4 cells/ well at 37uC in DMEM with 5% FBS and incubated overnight before treating with or without test agents for 72 h. The MTS assay was performed by directly adding 20 ml of the AQueous One Solution Reagent to culture wells (100 ml/well), incubating for 4 h and then recording the absorbance at 490 nm (A 490 ) with an ELISA reader (Bio-Rad Imark Microplate Reader). The ATP assay was carried out by adding 100 ml of the CellTiter-Glo Reagent (Buffer plus Substrate) to each well, then mixing contents for 2 minutes on an orbital shaker to induce cell lysis. After that the plate was incubated for 10 minutes to stabilize luminescent signal. The luminescent signal was measured by a luminometer (GloMax Multi Jr Detection System, Promega, USA). The ATP concentration in each well was derived from the standard curve.

Statistical analysis
All data passed the normality test and were presented as mean 6 standard deviation. The statistical comparison between two groups was carried out using Student's t-test (Origin 8.0), and the analysis for multiple groups was using Dunnett's test (SPSS 18.0, one-way ANOVA). P,0.05 was considered to be statistically significant. The values of half maximal effective concentration (EC 50 ) were calculated according to the dose-response curve fitting with the logistic equation: Y~Y max {Y min 1z(x=EC50) n zY min , where Y is the response, Y max is the asymptotic maximum, Y min is the asymptotic minimum, x is the extracellular calcium concentration and n is the Hill coefficient.

Thapsigargin induced SOCE in rat calvarial osteoblasts
Firstly, we checked the ability of generating SOCE in rat calvarial osteoblasts with ER Ca 2+ -pump blocker thapsigargin (TG), a drug widely used to test SOCE. It was seen from Figure Figure 1G and H). Taken together, these data confirmed the existence of SOCE in rat calvarial osteoblasts and the efficient inhibition of 2-APB and BTP-2 on SOCE.

Voltage-gated calcium channels did not contribute to [Ca 2+ ] o -induced [Ca 2+ ] c increase
Because rat calvarial osteoblasts expressed voltage-gated calcium (Cav) channels [38,39], we tested whether Cav channels contributed to [Ca 2+ ] o -induced [Ca 2+ ] c increase. It found that pretreatment the cells with Cav blockers nifedipine (10 mM) [27,40] Figure 3B). To verify the effectiveness of these two Cav blockers, a high [K + ] o experiment was performed as positive control. Data showed that elevating [K + ] o from 0 mM to 100 mM triggered a rapid increase of [Ca 2+ ] c (black line, Figure 3C), which was known to be attributed to Ca 2+ entry through Cav channels (blue line, Figure 3C Figure 4A and B) when cells were pretreated with 50 mM TMB-8 (a calcium release inhibitor) [ Figure 4C Figure 6D and E, Figure S1 and S2). These data indicated that the CaSR activation-induced SOCE participated in the process of high [Ca 2+ ] o -promoted cell proliferation in rat calvarial osteoblasts.

Discussion
In the present study, we found that elevating [ (Figure 2A). This [Ca 2+ ] c increase played key roles in cell proliferation ( Figure 6B; experiment with BAPTA-AM). We further showed that extracellular Ca 2+ entry contributed mostly to the sustained [Ca 2+ ] c increase ( Figure 4C). In most cell types, SOCE mediated extracellular Ca 2+ entry for ER Ca 2+ store refilling [11], and more importantly, the SOCE phenomenon was found in several osteoblastic cell lines including rat calvarial osteoblasts [16][17][18]. On the other hand, some papers reported the functional expression of Cav channels in osteoblasts, which regulated the cell proliferation and differentiation dependent on the type and expression level of Cav channels [38,39]. Thus, Cav channels may also serve as candidate channels accounting for Ca 2+ capacitive entry. We addressed these two presumptions in this study. Our data showed that SOCE blockers 2-APB, BTP-2, or Ca 2+ release inhibitor TMB-8 almost abolished   (Figure 4), whereas Cav channels antagonists had little effects (Figure 3). These results suggested that [Ca 2+ ] o -induced [Ca 2+ ] c increase was most likely through the activation of SOCE route rather than Cav channels. With respect to the molecular components of SOCE conducting Ca 2+ entry, some transient receptor potential (TRP) channels especially TRPC subfamily were considered for candidate SOCE machinery in osteoblasts as well as other cell types. Many members of TRP channels had been identified to exist in osteoblasts and served as SOCE machinery [43][44][45]. For instance, reports showed that the TRPC1 channel played a key role in SOCE and cell proliferation induced by platelet-derived growth factor in osteoblast-like MG-63 cell line [18], while TRPC3 was related to vitamin D-induced SOCE in chick skeletal muscle [46] and ROS 17/2.8 rat osteoblastic cells [47]. However, which member of TRPC channels actually contributed to elevated [Ca 2+ ] o -triggered [Ca 2+ ] i increases needs further investigation.
In terms of how extracellular Ca 2+ activated SOCE, it was known that extracellular Ca 2+ could stimulate G protein-PLC pathway by activating CaSR in various cell types. Then the following production of IP 3 caused Ca 2+ release. Functional CaSR was expressed in different types of osteoblast-like cells including primary rat calvarial osteoblasts [22][23][24][25][26][27][28]. However, it is not clear whether CaSR activation can cause SOCE in osteoblasts. In the present study, we found that elevated [Ca 2+ ] o -induced [Ca 2+ ] c increase was strongly blocked by specific CaSR antagonist NPS2143 and PLC inhibitor U73122. Similar responses were observed when the cells were stimulated with spermine, a specific CaSR agonist ( Figure 5). Furthermore, the EC 50 [19]. These data together indicated that extracellular Ca 2+ -induced SOCE was dependent on the activation of CaSR-related PLC/IP 3 pathway.
Another interesting finding of the present study was that we demonstrated the contribution of SOCE and CaSR-related PLC/ IP 3 [9,10,[48][49][50][51]. But how activation of these signal pathways leads to osteoblastic proliferation is still unclear now. As reported, high [Ca 2+ ] o activated PLC, probably through a Gprotein-coupled receptor mechanism, which then caused accumulation of IP 3 and mobilization of intracellular calcium, subsequently activated calcium/calmodulin/CaMKII signaling [9]. Furthermore, in primary human osteoblasts and MG-63 cells, high [Ca 2+ ] o -stimulated proliferation was dependent on sustained activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2, but other mitogen-activated protein (MAP) kinase signal pathways, p38 MAP kinase and SAPK/JNK, were not activated by [Ca 2+ ] o in osteoblasts [10]. ERK pathway was also found to participate in mediating bone morphogenetic protein (BMP)-2 gene expression induced by elevated [Ca 2+ ] o in human dental pulp cells [48]. High [Ca 2+ ] o was required for phosphate-dependent ERK1/2 phosphorylation and regulation of mineralizationassociated genes in osteoblasts [49]. Besides, other signal pathways including calcineurin/NFAT and cAMP/PKA were involved in other cell functions such as gene expression induced by high extracellular calcium [50,51]. In addition to the above mechanisms, the route of SOCE also played an important role in proliferation by inducing sustained [Ca 2+ ] c increase in various cell types including embryonic stem cells and smooth muscle cells [12][13][14]. Therefore, it's necessary to concern about its role in the high [Ca 2+ ] o -induced proliferation of osteoblasts. In this study, our data showed that intracellular calcium chelator BAPTA-AM reversed the high [Ca 2+ ] o -induced proliferation. 2-APB, BTP-2, TMB-8, NPS2143 or U73122 also reduced the [Ca 2+ ] o -induced proliferation ( Figure 6), indicating the contribution of CaSR/PLC activation-evoked SOCE to high [Ca 2+ ] o -induced proliferation of osteoblasts. In contrast, nifedipine or verapamil had little influence on the proliferation, suggesting that Cav channels were not involved. These results added a new insight to the current understanding on the role of extracellular calcium in osteoblasts proliferation.
In summary, we demonstrated that the elevation of [Ca 2+ ] o could stimulate CaSR, activate PLC, then trigger SOCE and consequently result in a sustained increase of [Ca 2+ ] c . This process was involved in osteoblastic proliferation induced by high level of extracellular Ca 2+ concentration. These findings may lead to new insights in the mechanisms of osteoblastic proliferation, and could provide some cellular basis for physiological regulation of bone remodeling.