Gallium modulates osteoclastic bone resorption in vitro without affecting osteoblasts

Abstract

Background and purpose: Gallium (Ga) has been shown to be effective in the treatment of disorders associated with accelerated bone loss, including cancer-related hypercalcemia and Paget's disease. These clinical applications suggest that Ga could reduce bone resorption. However, few studies have studied the effects of Ga on osteoclastic resorption. Here, we have explored the effects of Ga on bone cells in vitro.

Experimental approach: In different osteoclastic models [osteoclasts isolated from long bones of neonatal rabbits (RBC), murine RAW 264.7 cells and human CD14-positive cells], we have performed resorption activity tests, staining for tartrate resistant acid phosphatase (TRAP), real-time polymerase chain reaction analysis, viability and apoptotic assays. We also evaluated the effect of Ga on osteoblasts in terms of proliferation, viability and activity by using an osteoblastic cell line (MC3T3-E1) and primary mouse osteoblasts.

Key results: Gallium dose-dependently (0-100 microM) inhibited the in vitro resorption activity of RBC and induced a significant decrease in the expression level of transcripts coding for osteoclastic markers in RAW 264.7 cells. Ga also dramatically reduced the formation of TRAP-positive multinucleated cells. Ga down-regulated in a dose-dependant manner the expression of the transcription factor NFATc1. However, Ga did not affect the viability or activity of primary and MC3T3-E1 osteoblasts.

Conclusions and implications: Gallium exhibits a dose-dependent anti-osteoclastic effect by reducing in vitro osteoclastic resorption, differentiation and formation without negatively affecting osteoblasts. We provide evidence that this inhibitory mechanism involves down-regulation of NFATc1 expression, a master regulator of RANK-induced osteoclastic differentiation.

Figure 1

Effect of Ga on unfractioned rabbit bone cells (RBC). Unfractioned RBC were prepared and cultured for 4 days either on dentin slices (A, B, D) or glass coverslips (C) in the presence of Ga at the indicated concentrations. (A) The resorption activity of osteoclasts in the presence or absence of Ga was seen by the formation of typical resorption lacunae observed by scanning electron microscopy with backscattered electrons (bar = 100 µm). (B) Cells were treated with graded concentrations of Ga and quantitative analysis of osteoclastic resorption was performed with a semi automatic image analyser. Osteoclastic resorption is expressed in relative activity compared with untreated cells. *P < 0.05 as compared with untreated cells; a: P < 0.05 as compared with 2.5 µM-treated cells; b: P < 0.05 as compared with 10 µM-treated cells; c: P < 0.05 as compared with 25 µM-treated cells. (C) Cell viability in the presence or absence of Ga was visualized by fluorescent microscopy after CTG staining (bar = 50 µm). (D) Cells were treated with graded concentrations of Ga and MTS activity was evaluated. As a positive control of cell death, cells were treated with actinomycin-D (Act-D; 5 µg·mL⁻¹) or its vehicle (DMSO). Results are expressed as relative MTS activity as compared with untreated cells. *P < 0.05 significantly different from vehicle treated cells. Ga, gallium; MTS, methyl tetrazolium salt.

Figure 4

Effect of Ga on the total number of TRAP-positive cells. Unfractioned RBC (A, B, C), RAW 264.7 (D, E, F) and human CD14-positive cells (G, H, I) were established on glass coverslips and treated or not with Ga at the indicated concentrations for 4 days (RBC) and 6 days (RAW 264.7 and CD14+). Cells were stained for TRAP activity and observed under a light microscope (bar = 30 µm). The total numbers of TRAP-positive cells were manually scored (C, F, I). Results were expressed as relative number of TRAP-positive cells compared with untreated cells (*P < 0.05 significantly different from untreated cells; a: P < 0.05 significantly different from 2.5 µM-treated cells; b: P < 0.05 significantly different from 10 µM-treated cells; c: P < 0.05 significantly different from 25 µM-treated cells; d: P < 0.05 significantly different from 50 µM-treated cells). Ga, gallium; TRAP, tartrate resistant acid phosphatase; RBC, rabbit bone cells.

Figure 3

Effect of Ga on RAW 264.7 viability. (A) RAW 264.7 cultured with (RANK-L) or without RANK-L (CTRL) were treated with 100 µM Ga (+) or its vehicle (−) for 6 days. As a positive control for cell death, cells were treated for 48 h with 5 µg·mL⁻¹ Act-D (+) or its vehicle (−). MTS activity was determined and results are expressed as relative MTS activity compared with untreated cells (*P < 0.05 significantly different from corresponding untreated cells). (B) Cells cultured with RANK-L were treated with 100 µM Ga or its vehicle for 6 days. Staurosporin (1 µM; 12 h) was used as a positive control for apoptotic cell death. Chromatin condensation was demonstrated by Hoechst 33342 staining as indicated in the Materials and Methods. Samples were observed with a fluorescent microscope (bar = 30 µm). Ga, gallium; RANK-L, receptor activator of nuclear factor kappa B-Ligand; MTS, methyl tetrazolium salt.

Figure 2

Effects of Ga on RAW 264.7 differentiation. (A) RAW 264.7 cells were established in the presence (RANK-L) or absence (CTRL) of 50 ng·mL⁻¹ of RANK-L for 6 days. Cells were stained for TRAP activity and observed under a light microscope (bar = 50 µm). Quantitative analysis of transcripts was performed by real-time polymerase chain reaction (RT-PCR) using primers specific for TRAP, CTR, CTK, RANK and OC-STAMP. Results are reported as fold change in gene expression relative to untreated cells after normalization against GAPDH. *P < 0.05 significantly different from corresponding untreated cells. (B) RAW 264.7 cells cultured concomitantly with RANK-L (50 ng·mL⁻¹) and different doses of Ga (0–50 µM–100 µM) for 6 days. Quantitative analysis of transcripts was performed by RT-PCR using primers specific for TRAP, CTR, RANK, CTK and OC-STAMP. Results are reported as fold change in gene expression relative to untreated cells after normalization against GAPDH. *P < 0.05 significantly different from corresponding untreated cells; a: P < 0.05 significantly different from 50 µM-treated cells. Ga, gallium; RANK, receptor activator of nuclear factor kappa B; RANK-L, receptor activator of nuclear factor kappa B-Ligand; CTK, cathepsin K; CTR, calcitonin receptor; OC-STAMP, osteoclastic stimulatory transmembrane protein; GAPDH, glyceraldehyde 3 phosphate deshydrogenase; RT-PCR, real-time polymerase chain reaction.

Figure 5

Effect of Ga on the expression of NFATc1. RAW 264.7 cells were cultured concomitantly with RANK-L (50 ng·mL⁻¹) and different doses of Ga (0, 50 µM, 100 µM) for 12 and 24 h. Quantitative analysis of transcripts was performed by RT-PCR using primers specific for NFATc1 as described in the Materials and Methods. Results are reported as fold change in gene expression relative to time zero after normalization against the housekeeping gene GAPDH. *P < 0.05 significantly different from equivalent untreated cells; a: P < 0.05 significantly different from 50 µM-treated cells. Ga, gallium; NFATc1, nuclear factor of activated T cell c1; GAPDH, glyceraldehyde 3 phosphate deshydrogenase.

Figure 6

Effect of Ga on osteoblastic viability and proliferation. Murine MC3T3-E1 osteoblastic cells (A, B) and murine primary calvaria-derived osteoblasts (C, D) were cultured as described in the Materials and Methods. After overnight incubation, cells were treated with 100 µM Ga or 5 µg·mL⁻¹ Act-D (+) or their respective vehicles (−) for 48 h. Osteoblastic viability was evaluated by MTS activity. Results are expressed in relative MTS activity compared with untreated cells (*P < 0.05 significantly different from untreated cells) (A, C). Osteoblastic proliferation was quantified by scoring cells manually after Trypan blue staining. Results are expressed as relative cell number as compared with untreated cells (B, D). *P < 0.05 significantly different from corresponding untreated cells. Ga, gallium; MTS, methyl tetrazolium salt.

Figure 7

Effect of Ga on alkaline phosphatase (ALP) activity. Murine MC3T3-E1 osteoblastic cells (A, B) and murine primary calvaria-derived osteoblasts (C, D) were cultured for 12 days and 4 days respectively. Then, cells were treated with 100 µM Ga or 100 ng·mL⁻¹ BMP-2 (+) or their respective vehicles (−) for 48 h. Total protein content was determined with a Coomassie assay. Results are expressed as relative protein content as compared with untreated cells (A, C). ALP activity was spectrophotometrically measured. Results are expressed as relative ALP activity as compared with untreated cells (B, D). *P < 0.05 significantly different from corresponding untreated cells. Ga, gallium; BMP-2, bone morphogenetic protein-2.

Figure 8

Effect of Ga on the expression of osteoblastic markers. Cultures of murine MC3T3-E1 osteoblastic cells were treated for 8 days with 100 µM Ga. Several markers of osteoblasts (Alp1, alkaline phosphatase, Ocn, osteocalcin; Osx, osterix; Opn, osteopontin; Runx2, Run related transcription factor 2) were measured by RT-PCR. Results are reported as fold change in gene expression relative to untreated cells after normalization against GAPDH. *P < 0.05, significantly different from the equivalent untreated cells. Ga, gallium; RT-PCR, real-time polymerase chain reaction; GAPDH, glyceraldehyde 3 phosphate deshydrogenase.