Calcium sensing receptor expression in ovine amniotic fluid mesenchymal stem cells and the potential role of R-568 during osteogenic differentiation

Abstract

Amniotic fluid-derived stem (AFS) cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR), a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs) and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT), cell number, Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM), potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining) and augmented intracellular calcium and Inositol trisphosphate (IP3) levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps prompting the use of calcimimetics in bone regenerative medicine.

Figure 1. CaSR identification in oAFMSCs.

(A) Western blot analysis shows expression of CaSR in oAFMSCs comparable to sheep fetal calvarial osteoblast (OBs) used as a positive control. β-actin was used as an internal control. Figure (B) shows the shift of mean fluorescence intensity of CaSR in oAFMSCs (filled histogram) as compared to the mean fluorescence intensity of secondary antibody alone (open histogram). (C) A representative cell in a bright field and the diffuse expression of CaSR obtained by IDEAS software (Amnis). Magnification 40x.

Figure 2. Evaluation of the optimum concentration of calcimimetics on oAFMSCs treated for 14 days.

(A) MTT assay performed at day 14 on oAFMSCs shows a reduction in cell viability upon increasing the concentrations of R-568 or S-568 (0.1–10 µM) (*p<0.01 vs Basal). (B) Decreasing cell number trend on day 14 at enhanced concentrations of calcimimetics R-568 and S-568 (0.1–10 µM) (*p<0.001 vs Basal). The number of cells is normalized per cm². All histograms represent the mean ± SD of three independent experiments. (C) Bright field images of oAFMSCs at increased concentrations of calcimimetics showing the cell morphology and the accumulations of vacuoles at high concentrations (enlargement in the right corner). Magnification 10x.

Figure 3. Estimation of osteogenic differentiation by Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) staining.

(A) ALP activity performed on day 7 of oAFMSC culture at increased concentrations of R-568 and S-568 (0.1–10 µM) (*p<0.05 vs Basal; **p<0.01 vs Basal). ALP activities were calculated with p-nitrophenol standard and normalized per mg of proteins. (B) ARS staining performed on day 14 of oAFMSC culture at increased concentrations of R-568 and S-568 (0.1–10 µM) (*p<0.01 vs Basal; **p<0.01 vs Basal). All histograms represent the mean ± SD of three independent experiments. The results are normalized per total cell number/well. (C) Representative images of ARS assay after 14 days of treatment with calcimimetics. Note the presence of mineral deposits after treatment with R-568 (1 µM). High concentrations (5 and 10 µM) of calcimimetics lead to a change in cell morphology with the presence of vacuoles (enlargement in the right corner). Magnification 10x.

Figure 4. Effect of calcimimetics on ALP activity.

ALP activity, detected at day 7, on oAFMSCs treated with calcimimetics pre-incubated (30 minutes) or not with Calhex-231 (1 µM, *p<0.0001 2.5 mM CaCl₂ vs Basal 1.8 mM CaCl₂ and R-568 vs Basal 1.8 mM CaCl₂; ^§p<0.01 R-568+2.5 mM CaCl₂ vs S-568+2.5 mM CaCl₂; **p<0.001 R-568+2.5 mM CaCl₂ vs 2.5 mM CaCl₂ and R-568+Calhex+2.5 mM CaCl₂ vs R-568+2.5 mM CaCl₂; ^#p<0.002 R-568+Calhex vs R-568+1.8 mM CaCl₂). ALP activities were calculated with p-nitrophenol standard and normalized per mg of proteins.

Figure 5. Effect of calcimimetics on ARS assay.

(A) Ovine AFMSCs were treated with calcimimetics (1 µM) for 14 days and the mineralization was quantified at the end of treatment by reading the absorbance. The cells showed higher mineralization in response to CaSR activator R−568 (1 µM, *p<0.01), while S−568 did not lead to any mineralization. The mineral deposits produced by the culture with R−568 were significantly reduced in the presence of Calhex−231 (**p<0.05) and R-568 significantly increased ARS compared to S−568 (^#p<0.05 R-568 vs S-568). The results are normalized per total cell number/well. (B) Representative full plate and microscope images of ARS assay after 14 days of treatment. Magnification 10x.

Figure 6. CaSR protein expression in oAFMSCs treated with calcimimetics.

(A) Western blot analysis of oAFMSCs cultured with R-568 or S-568. Whole cell lysates were separated by SDS-PAGE, immunoblotted, and revealed with anti-CaR antibody. Equivalent amounts (40 µg) of total protein were loaded in each lane. β-actin was used as an internal control. *p<0.001 OBs vs Basal; **p<0.001 R-568 vs Basal; ^#p<0.01 R−568 vs S−568. (B) Representative graph derived from Imaging flow cytometry (Amnis) shows the shift of mean fluorescence intensity (MFI) of CaSR in oAFMSCs treated with R−568 (red curve) versus basal condition (green filled curve), while treatment with S-568 did not cause any shift (blue curve). The MFI of secondary antibody alone (Alexa-488, CTRL-) is indicated by the empty green curve.

Figure 7. Imaging flow cytometry analysis of CaSR expression in oAFMSCs evaluated intracellularly and at membrane level.

Shown here in a permeabilizing condition (A–B) is the CaSR expression after treatment for 14 days with R-568 or S-568 in the presence or absence of Calhex-231 (*p<0.05 R-568 vs Basal; **p<0.02 R-568+Calhex vs R-568). The histograms in B (left), related to the MFI bars in (A), show the CaSR distribution in 5×10³-acquired-oAFMSCs/sample. Representative single cell images in B (right). The same experiments were performed in non-permeabilizing conditions to detect CaSR membrane localization (*p<0.01 R-568 vs Basal; **p<0.05 R-568+Calhex vs R-568) (C–D). Dead cells were excluded by 7-Aminoactinomycin D (7-AAD) staining. Data analyses were performed by IDEAS software (Amnis). The results shown by the bars (A–C) are expressed as a fold increase and are calculated by subtracting the MFI value of negative events (MFI of secondary antibody alone, CTRL-) from that of positive events. Data represent three separate experiments.

Figure 8. Intracellular Ca²⁺ and IP3 variations in oAFMSCs stimulated with R-568.

Temporal changes in intracellular Ca²⁺ levels, expressed as f/f₀ ratio (see Material and Methods), were measured in oAFMSCs. In panel (A) the representative traces showed calcium variations in cells treated with 1 µM R-568. In panel (B) the traces showed the cell response in presence of 1 µM Calhex-231. In panel (C) the IP3 levels were expressed as ng/ml/5×10⁵ cells. CaCl₂ was used at 2.5 mM; Basal indicates 1.8 mM CaCl₂. *p<0.02 CaCl₂ vs Basal and CaCl₂+Calhex-231 vs CaCl₂; **p<0.05 R-568 vs Basal and R-568+Calhex-231 vs R-568.