Magnesium Is a Key Regulator of the Balance between Osteoclast and Osteoblast Differentiation in the Presence of Vitamin D₃

Abstract

Magnesium (Mg) is crucial for bone health. Low concentrations of Mg inhibit the activity of osteoblasts while promoting that of osteoclasts, with the final result of inducing osteopenia. Conversely, little is known about the effects of high concentrations of extracellular Mg on osteoclasts and osteoblasts. Since the differentiation and activation of these cells is coordinated by vitamin D₃ (VD3), we investigated the effects of high extracellular Mg, as well as its impact on VD3 activity, in these cells. U937 cells were induced to osteoclastic differentiation by VD3 in the presence of supra-physiological concentrations (>1 mM) of extracellular Mg. The effect of high Mg concentrations was also studied in human bone-marrow-derived mesenchymal stem cells (bMSCs) induced to differentiate into osteoblasts by VD3. We demonstrate that high extra-cellular Mg levels potentiate VD3-induced osteoclastic differentiation, while decreasing osteoblastogenesis. We hypothesize that Mg might reprogram VD3 activity on bone remodeling, causing an unbalanced activation of osteoclasts and osteoblasts.

Keywords: biodegradable magnesium alloys; hematopoietic U937 cells; human bone-marrow mesenchymal stem cells; magnesium; osteoclasts; vitamin D3.

Figure 1

Effects of supra-physiological Mg concentrations on U937 cell-derived osteoclasts. U937 cells were differentiated to osteoclasts upon treatment with phorbol 12-myristate 13-acetate (PMA) and vitamin D₃ (VD3) for five days and simultaneously exposed to scalar concentrations of Mg ranging from 1 to 10 mM. The messenger RNA (mRNA) expression of transcription factors (A) and differentiation markers (B), both related to osteoclast differentiation, was then assessed using QRT-PCR. The results obtained are represented as histograms indicating analyzed genes on the x-axis and relative quantity of mRNA variations on the y-axis. Data are reported as means ± standard error of the mean (SEM) values deriving from a triplicate experiment. Asterisks indicate statistically significant results. p < 0.05.

Figure 2

Changes in morphology and proliferation rate in U937 cell-derived osteoclasts after exposure to supra-physiological Mg concentrations. U937 cells, under the experimental conditions described in Figure 1, were subjected to morphological analysis, performed by microscopic examination of May–Grünwald Giemsa-stained cytospins, and cell-cycle assessment, carried out by flow cytometry analysis of propidium iodide (PI)-stained cell suspensions. Panel A shows a couple of representative microscopic fields obtained with 1 and 10 mM Mg. The histogram presented in Panel B indicate the percentages of macrophages and osteoclasts detected in the same cell samples and relative statistical analysis. Histograms presented in Panel C indicate cell-cycle distribution and relative statistical analysis elicited by exposure to scalar concentrations of Mg ranging from 1 to 10 mM. Data are reported as means ± SEM values of a triplicate experiment. Asterisks indicate statistically significant results.

Figure 3

Effects of supra-physiological Mg concentrations determined on U937 cell-derived monocytes. U937 cells were differentiated to monocytes by stimulation with VD3 for five days, and contextually exposed to 1 and 10 mM Mg concentrations. Cell samples were then subjected to flow cytometry and QRT-PCR analysis of typical markers related to the monocyte–macrophage differentiation lineage. Panel A shows the results of flow cytometry represented as a histogram, indicating the analyzed surface antigen on the x-axis and the percentage of positive cells on the y-axis. Panel B shows the results of QRT-PCR, indicating analyzed genes on the x-axis and the relative quantity of mRNA variations on the y-axis. Data are represented as means ± SEM values of a triplicate experiment. Asterisks indicate statistically significant results. p < 0.05.

Figure 4

Effects of treatment with supra-physiological Mg concentrations on U937 cell-derived macrophages. U937 cells were differentiated to macrophages by exposure to PMA for two days, and simultaneously exposed to 1 and 10 mM Mg. Cell samples were then analyzed and the results presented as detailed in the legend of Figure 3. (A) Results from cell samples subjected to flow cytometry. (B) Results from cell samples subjected to QRT-PCR analysis.

Figure 5

Effects of supra-physiological Mg concentrations on VD3-induced osteoblastic differentiation. Alizarin Red staining was performed on bone-marrow-derived mesenchymal stem cells (bMSCs) cultured in 1, 3, 6, and 10 mM Mg added (OM) or not (CM) with the osteogenic cocktail for 14 days. Whole-well image (A) and photographs taken at 10× magnification (C) are shown. Absorbance was measured at 550 nm after acid extraction (B). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Effects of supra-physiological concentrations of Mg on the expression of RUNX2 and COL1A1 in bMSCs exposed to VD3. QRT-PCR was performed on RNA extracted from bMSCs cultured for four days in 1, 3, 6, and 10 mM Mg, added (OM) or not (CM) with the osteogenic cocktail. Primers designed on RUNX2 and COL1A1 sequence were used. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

(A) The bMSCs were treated for 30 min with H₂O₂ (50 μM) and then cultured for four days in culture medium (CM). RUNX2 expression was analyzed using QRT-PCR. The p-value was calculated vs. untreated cells; *** p < 0.001. (B) Reactive oxygen species (ROS) accumulation was quantified using 2′-7′-dichlorofluorescein diacetate (DCFH, Sigma-Aldrich). Cells were seeded into black-bottomed 96-well plates (Greiner Bio-One) and cultured in 1, 3, 6, and 10 mM Mg, added or not with the osteogenic cocktail for 24 h. Then, cells were washed with phosphate-buffered saline (PBS) and exposed to DCFH (20 µM). The rate of intracellular oxidative stress was evaluated by monitoring the emission at 529 nm of the DCFH dye using a GloMax^®-Multi Detection System (Promega, Madison, WI, USA). Three independent experiments were performed. Data are shown as means ± standard deviation.