CaSR-Mediated hBMSCs Activity Modulation: Additional Coupling Mechanism in Bone Remodeling Compartment

Abstract

Near the bone remodeling compartments (BRC), extracellular calcium concentration (Ca²⁺_o) is locally elevated and bone marrow stromal cells (BMSCs) close to the BRC can be exposed to high calcium concentration. The calcium-sensing receptor (CaSR) is known to play a key role in maintaining extracellular calcium homeostasis by sensing fluctuations in the levels of extracellular calcium (Ca²⁺_o). When human BMSCs (hBMSCs) were exposed to various calcium concentrations (1.8, 3, 5, 10, 30 mM), moderate-high extracellular calcium concentrations (3-5 mM) stimulated proliferation, while a high calcium concentration (30 mM) inhibited the proliferation. Exposure to various calcium concentrations did not induce significant differences in the apoptotic cell fraction. Evaluation of multi-lineage differentiation potential showed no significant difference among various calcium concentration groups, except for the high calcium concentration (30 mM) treated group, which resulted in increased calcification after in vitro osteogenic differentiation. Treatment of NPS2143, a CaSR inhibitor, abolished the stimulatory effect on hBMSCs proliferation and migration indicating that CaSR is involved. These results suggest that the calcium concentration gradient near the BRC may play an important role in bone remodeling by acting as an osteoblast-osteoclast coupling mechanism through CaSR.

Keywords: bone remodeling; calcium-sensing receptor; osteoblast-osteoclast coupling; proliferation.

Figure 1

Basal level expression of calcium-sensing receptor (CaSR) in human dermal fibroblasts (hDFs) and human bone marrow stromal cells (hBMSCs) examined by western blot analysis (a) and the relative density of CaSR band normalized to α-tubulin (b) (n = 2).

Figure 2

Moderate-high calcium concentration stimulate hBMSC proliferation through CaSR. (a) hDFs and hBMSCs were treated with various calcium concentrations for 48 h and relative cell numbers were analyzed by the MTT assay (** p = 0.0038, *** p = 0.0002). (b) hBMSCs were treated with NPS2143, a CaSR antagonist. Relative cell density was determined by the MTT assay (* p = 0.0176, ** p = 0.0034, ** p = 0.0004). (c) During hBMSC exposure to various calcium concentrations for 48 h, BrdU was treated for the last 24 h. Proportion of BrdU-positive cells was compared as fold difference (* p = 0.0416). (d) Comparison of cell survival in response to various calcium concentrations based on the propidium iodide-incorporated apoptotic assay. hDFs and hBMSCs were exposed to various calcium concentrations for 48 h and subjected to propidium iodide (PI) staining. The ratio of PI-negative survival fractions in each group of cells was analyzed by fluorescence-activated cell sorting (FACS) and compared.

Figure 3

The continuance of hBMSCs proliferation after exposure to moderate-high calcium concentrations. (a) The schematic drawing of experiment procedure. hBMSCs were treated with various calcium concentrations for 48 h and sub-cultured to in standard media with normal calcium level. (b) Cell growth was evaluated after three days of seeding and compared between passages (P3 + Ca: * 1.8 vs. 3 mM p = 0.0293, * 1.8 vs. 5 mM p = 0.04481, P4: ** 1.8 vs. 3 mM p = 0.0030, ** 1.8 vs. 5 mM p = 0.0071).

Figure 4

Effect of various calcium concentrations on multi-lineage differentiation potentials of hBMSCs. hBMSCs were treated with various calcium concentrations for 48 h and cultured in differentiation induction media for four weeks. (a) Calcium depositions in hBMSCs subjected to osteogenesis were visualized by Alizarin Red S staining. The intensity of Alizarin Red S staining was evaluated by ImageJ software (* p = 0.0113). (b) hBMSCs subjected to adipogenesis was stained by Oil Red O. The extent of adipogenesis was determined by comparing the proportion of Oil Red O-positive cells normalized by the number of total nuclei. (c) After in vitro chondrogenesis, the cartilaginous matrix formation was confirmed by Safranin O staining.

Figure 5

Effect of Ca²⁺_o on hBMSC migration behavior. (a) hBMSCs in normal calcium media were seeded into millicells across the calcium concentration gradient established by media containing 5–30 mM calcium. After four hours of incubation, the migrated cells were fixed and stained with methyl violet (* p = 0.0324, ** p = 0.0089). (b) To investigate the involvement of CaSR in calcium-induced migration, the migration assay was performed in the presence or absence of 10 μM NPS2143 (* p = 0.0317, ** p = 0.0085). (c) hBMSCs were placed inside the millicell and the media inside and outside of the millicell was controlled as indicated. Ca indicates the media containing 5 mM calcium (* p = 0.0242, ** p = 0.0015, *** p = 0.0003). (d,e) hBMSCs were subjected to the wound closure assay with various calcium concentrations (1.8 mM vs. 3.0 mM: ** p = 0.0094, 1.8 mM vs. 5.0 mM: ** p= 0.0013, 1.8 mM vs. 10.0 mM: *** p = 0.00007, 3.0 mM vs. 5.0mM: * p = 0.0374, 3.0 mM vs. 10.0 mM: ** p = 0.0082).

Figure 5

Effect of Ca²⁺_o on hBMSC migration behavior. (a) hBMSCs in normal calcium media were seeded into millicells across the calcium concentration gradient established by media containing 5–30 mM calcium. After four hours of incubation, the migrated cells were fixed and stained with methyl violet (* p = 0.0324, ** p = 0.0089). (b) To investigate the involvement of CaSR in calcium-induced migration, the migration assay was performed in the presence or absence of 10 μM NPS2143 (* p = 0.0317, ** p = 0.0085). (c) hBMSCs were placed inside the millicell and the media inside and outside of the millicell was controlled as indicated. Ca indicates the media containing 5 mM calcium (* p = 0.0242, ** p = 0.0015, *** p = 0.0003). (d,e) hBMSCs were subjected to the wound closure assay with various calcium concentrations (1.8 mM vs. 3.0 mM: ** p = 0.0094, 1.8 mM vs. 5.0 mM: ** p= 0.0013, 1.8 mM vs. 10.0 mM: *** p = 0.00007, 3.0 mM vs. 5.0mM: * p = 0.0374, 3.0 mM vs. 10.0 mM: ** p = 0.0082).

Figure 6

The schematic drawing of BRC (bone remodeling compartment). Compartmentalization of the BRC by canopy cells resulted in the establishment of concentration gradient of the chemoattractant released by the action of bone-resorbing osteoclasts.