Importance of melastatin-like transient receptor potential 7 and cations (magnesium, calcium) in human osteoblast-like cell proliferation

Abstract

Bone tissue in the adult is continuously being remodelled, and overall bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation. Adequate osteoblastic proliferation is essential for both appropriate formation and for regulation of resorption, and thereby the maintenance of bone remodelling equilibrium.

Objectives: Here, we have investigated the roles of melastatin-like transient receptor potential 6 and 7 (TRPM6, TRPM7), which are calcium (Ca2+) and magnesium (Mg2+) conducting channels, during proliferation of human osteoblasts.

Results: Genetic expression of TRPM6 and TRPM7 was shown in human osteoblast-like MG-63, SaOS and U2-OS cells, and reduction of extracellular Mg2+ or Ca2+ led to a decrease of cell proliferation. Concomitant reduction of both ions further accentuated reduction of cell proliferation. Expression of TRPM7 channels was increased under conditions of reduced extracellular Mg2+ and Ca2+ levels whereas expression of TRPM6 was not modified, suggesting compensatory mechanisms afforded by TRPM7 in order to maintain intracellular ion homeostasis. Pre-incubation of cells in reduced extracellular Mg2+ conditions led to activation of Ca2+ and Mg2+ influx. Reduction of TRPM7 expression by specific siRNA prevented latter influx and inhibited cell proliferation.

Conclusions: Our results indicate that extracellular Mg2+ and Ca2+ deficiency reduces the proliferation of human osteoblastic cells. Expression and activity of TRPM7 is modulated by extracellular Mg2+ and Ca2+ availability, indicating that TRPM7 channels are involved in intracellular ion homeostasis and proliferation of osteoblasts.

Figure 1

Analysis of genetic expression of TRPM6 and TRPM7 channels in human osteoblast‐like cells. Total RNA from MG‐63, SaOS and U2‐OS cells was isolated and subjected to RT‐PCR using specific primers for TRPM6 (NM_017662) and TRPM7 (NM_017672) channels. Results are representative data obtained from three independent isolations of RNA. Ladder of 100 bp.

Figure 2

Effect of extracellular Mg²⁺ on MG‐63 proliferation. Cells were incubated for 48 h in culture media containing different concentrations of Ca²⁺ and increasing concentrations of Mg²⁺, and cell proliferation was determined by MTT assays. Results are expressed as (a) ratio of absorbance compared to initial MTT result or (b) as the relative effect of Mg²⁺ compared to conditions without Mg²⁺. Values for normal DMEM/F12 were 2.12 ± 0.21 relative to the initial MTT. Data are means ± SEM of at least three individual experiments.

Figure 3

Effect of extracellular Ca²⁺ on MG‐63 proliferation. Cells were incubated for 48 h in culture media containing different concentrations of Mg²⁺ and increasing concentrations of Ca²⁺, and cell proliferation was determined by MTT assays. Results are expressed as (a) ratio of the absorbance compared to initial MTT value or (b) as relative effect of Ca²⁺ compared to conditions with 0.1 mm Ca²⁺. Values for normal DMEM/F12 were 2.12 ± 0.21. Data are means ± SEM of at least three individual experiments.

Figure 4

Effect of extracellular Mg²⁺ and Ca²⁺ on DNA synthesis. Cells were incubated for 24 or 48 h in DME/F12 containing different concentrations of Mg²⁺ and Ca²⁺. The evaluation of DNA synthesis was performed by BrdU incorporation as described in the Materials and Methods section. As positive control for the stimulation of DNA synthesis, cells were incubated with 25 ng/mL PDGF‐BB in DMEM/F12. Values are the mean ± SEM of percentages compared to DMEM/F12 medium from three to four individual experiments. *P < 0.05, †P < 0.02, ‡P < 0.01, §P < 0.001 compared to DMEM/F12.

Figure 5

Expression of TRPM6 and TRPM7 under Mg²⁺‐ and/or Ca²⁺‐ reduced culture conditions. MG‐63 cells were incubated for 48 h in DME/F12 containing 1 mm Ca²⁺ without Mg²⁺, in DME/F12 containing 0.8 mm Mg²⁺ and 0.1 mm Ca²⁺, in DME/F12 containing 0.1 mm Ca²⁺ without Mg²⁺ or in DMEM/F12 (1 mm Ca²⁺ and 0.8 mm Mg²⁺). Total RNA was isolated from cells cultured under previous conditions, and levels of TRPM6 and TRPM7 transcripts were determined by semiquantitative RT‐PCR (a) or real‐time PCR (b) as described in the Materials and Methods section. Relative levels of TRPM7 expression compared to those under control conditions (DMEM/F12) are expressed as the mean ± SEM of three independent experiments. *P < 0.008, Student's t‐test.

Figure 6

Activation of TRPM7 under low extracellular Mg²⁺ levels. (a) Cells were loaded for a 2‐h incubation period with Fluo‐3 or Magnesium Green in HBSS with 1 mm Ca²⁺ without Mg²⁺ or HBSS without Ca²⁺ with 0.8 mm Mg²⁺ (right panel: inset). Thereafter, cells were transferred to Ca²⁺‐free and Mg²⁺‐free HBSS and Ca²⁺ (left panel) or Mg²⁺ (right panel) were added to the incubation medium (final concentration of 3 mm and 5 mm respectively). Measurements of intracellular Ca²⁺ or Mg²⁺ were performed as described in the Materials and Methods section. (b) MG‐63 cells were transfected with specific siRNA against TRPM7 for 48 h. RT‐PCR and RQ‐PCR were performed to evaluate expression levels of TRPM6, TRPM7 and β‐2‐microglobulin. *P < 0.001. (c) Cells were transfected with siTRPM7(2) for 48 h and were loaded for a 2‐h incubation period with Fluo‐3 in HBSS with 1 mm Ca²⁺ without Mg²⁺. Thereafter, cells were transferred to Ca²⁺‐ and Mg²⁺‐free HBSS and Ca²⁺ was added to the incubation medium (final concentration of 3 mm). Right panel: non‐transfected cells loaded with Fluo‐3 in HBSS with 1 mm Ca²⁺ without Mg²⁺ were first incubated with 30 µm SKF‐96365 and Ca²⁺ was added to the incubation medium. Data are means ± SEM of three to four individual experiments with cumulating analysis of between 10 and 20 cells per field.

Figure 7

Effect of reducing TRPM7 expression on cell proliferation. Cells were transfected with specific siRNAs against TRPM7 [siTRPM7(1) and siTRPM7(2)] or control negative siRNA [siRNA(–)] and cell proliferation was determined after 72 h for MG‐63 cells (a) or U2‐OS cells (b) and compared to MTT activity following 24 h of incubation with siRNA (MTT initial). Data are means ± SEM of three individual experiments. *P < 0.05 and †P < 0.001 compared to cell proliferation with siRNA(–).