Gallium ions promote osteoinduction of human and mouse osteoblasts via the TRPM7/Akt signaling pathway

Abstract

Gallium (Ga) ions have been widely utilized for biomedical applications; however, their role in osteoblast regulation is not completely understood. The aim of the present study was to investigate the potential effect of Ga ions on osteoinduction in two osteoblast cell lines and to explore the underlying mechanisms. Human hFOB1.19 and mouse MC3T3‑E1 osteoblasts were treated with Ga nitride (GaN) at different concentrations, and the degree of osteoinduction was assessed. Ga ion treatment was found to increase alkaline phosphatase activity and accelerate calcium nodule formation, as assessed using ALP activity assay and Alizarin red S staining. Moreover, upregulated expression levels of osteogenic proteins in osteoblasts were identified using western blotting and reverse transcription‑quantitative PCR. Western blotting was also performed to demonstrate that the biological action of Ga ions was closely associated with the transient receptor potential melastatin 7/Akt signaling pathway. Furthermore, it was found that Ga ions did not cause osteoblast apoptosis, as indicated via flow cytometry, but promoted osteoclast proliferation, migration or invasion. The present study investigated the potential role of Ga ions in regulating osteoinduction of osteoblasts, thereby providing a promising strategy for the treatment of osteoporosis.

Keywords: gallium; osteoinduction; osteoblasts; transient receptor potential melastatin 7; osteoporosis.

Figure 1.

Effect of different Ga ion concentrations on MC3T3-E1 and hFOB1.19 cell ALP activity. ALP activity of (A) MC3T3-E1 and (B) hFOB1.19 cells following treatment with different concentrations of GaN for 7 days. *P<0.05 and **P<0.01 vs. control; ^##P<0.01 vs. osteoinduction. Ga, gallium; ALP, alkaline phosphatase; GaN, gallium nitride.

Figure 2.

Effect of Ga ions on the Akt signaling pathway in MC3T3-E1 and hFOB1.19 cells. (A) Akt and p-Akt expression levels in MC3T3-E1 cells following treatment with GaN (10⁻⁴ M) for 0, 7 or 14 days. (B) Akt and p-Akt expression levels in hFOB1.19 cells following treatment with GaN (10⁻⁴ M) for 0, 3 or 7 days. Ga, gallium; p, phosphorylated; GaN, gallium nitride.

Figure 3.

Effect of TRPM7 siRNAs on TRPM7 protein expression in MC3T3-E1 and hFOB1.19 cells. (A) Total and (B) membrane TRPM7 protein expression levels in MC3T3 cells following transfection with TRPM7 siRNAs. (C) Total and (D) membrane TRPM7 protein expression levels in hFOB1.19 cells following transfection with TRPM7 siRNAs. TRPM7, transient receptor potential melastatin 7; siRNA, small interfering RNA.

Figure 4.

Effect of Ga ions on ALP activity and calcium nodule formation in MC3T3-E1 and hFOB1.19 cells. Cells were treated with GaN (10⁻⁴ M), with or without pre-treatment with TRPM7 siRNA (transfection for 96 h) or LY294002 (10 µM). ALP activity of (A) MC3T3-E1 and (B) hFOB1.19 cells. (C) Representative images of Alizarin Red S staining of MC3T3-E1 cells (scale bar, 100 µm). **P<0.01. Ga, gallium; ALP, alkaline phosphatase; siRNA, small interfering RNA; GaN, gallium nitride; TRPM7, transient receptor potential melastatin 7.

Figure 5.

Effect of Ga ions on osteogenic-related protein expression in MC3T3-E1 and hFOB1.19 cells. Cells were treated with GaN (10⁻⁴ M), with or without pre-treatment with TRPM7 siRNA (transfection for 96 h) or LY294002 (10 µM). Protein expression levels of (A) Runx2, Osterix, Akt, p-Akt after 7 days, (B) OCN, OPN, Akt and p-Akt after 14 days in MC3T3-E1 cells. Protein expression levels of (C) MMP2, MMP9, Akt, p-Akt after 3 days, (D) Runx2, Akt and p-Akt after 7 days in hFOB1.19 cells. (E) Expression level of VEGF protein in hFOB1.19 cells. Akt and p-Akt were measured throughout the treatment process. (F) Relative mRNA expression levels of Runx2, Osterix, OPN and OCN in MC3T3-E1 cells. (G) Relative mRNA expression levels of MMP2, MMP9, Runx2 and VEGF in hFOB1.19 cells. **P<0.01. Ga, gallium; Runx2, Runt-related transcription factor 2; p, phosphorylated; OCN, osteocalcin; OPN, osteopontin; GaN, gallium nitride; siRNA, small interfering RNA; MMP, matrix metallopeptidase; VEGF, vascular endothelial growth factor; TRPM7, transient receptor potential melastatin 7.

Figure 6.

Effect of Ga ions on MC3T3-E1 and hFOB1.19 cell proliferation. MC3T3-E1 cells were treated with GaN (10⁻⁴ M), with or without pre-treatment with TRPM7 siRNA (transfection for 96 h) or LY294002 (10 µM) for 0, 7 or 14 days. hFOB1.19 cells were treated with GaN (10⁻⁴ M), with or without pre-treatment with siRNA (siRNA transfection for 96 h) or LY294002 (10 µM) for 0, 3 or 7 days. (A) MC3T3-E1 and (B) hFOB1.19 cell proliferation. (C) MC3T3-E1 and (D) hFOB1.19 cell apoptosis. **P<0.01. Ga, gallium; GaN, gallium nitride; siRNA, small interfering RNA; OD, optical density; TRPM7, transient receptor potential melastatin 7.

Figure 7.

Effect of Ga ions on hFOB1.19 cell migration. hFOB1.19 cells were treated with GaN (10⁻⁴ M) for 24 h, with or without pre-treatment with TRPM7 siRNA (transfection for 96 h) or LY294002 (10 µM). (A) Representative images of the Transwell migration assay (scale bar, 200 µm). (B) Quantification of cell migration. **P<0.01. Ga, gallium; GaN, gallium nitride; siRNA, small interfering RNA; TRPM7, transient receptor potential melastatin 7.

Figure 8.

Effect of Ga ions on hFOB1.19 cell invasion. hFOB1.19 cells were treated with GaN (10⁻⁴ M) for 24 h, with or without pre-treatment with TRPM7 siRNA (transfection for 96 h) or LY294002 (10 µM). (A) Representative images of the Transwell invasion assay (scale bar, 200 µm). (B) Quantification of cell invasion. **P<0.01. Ga, gallium; GaN, gallium nitride; siRNA, small interfering RNA; TRPM7, transient receptor potential melastatin 7.