Korean J Physiol Pharmacol.  2013 Feb;17(1):65-71. 10.4196/kjpp.2013.17.1.65.

TRPM7 Is Essential for RANKL-Induced Osteoclastogenesis

• Yang YM
• Jung HH
• Lee SJ
• Choi HJ
• Kim MS
• Shin DM

• KMID: 1432765
• DOI: http://doi.org/10.4196/kjpp.2013.17.1.65

Abstract

The transient receptor potential melastatin type 7 (TRPM7) channel is a widely expressed non-selective cation channel with fusion to the C-terminal alpha kinase domain and regarded as a key regulator of whole body Mg2+ homeostasis in mammals. However, the roles of TRPM7 during osteoclastogenesis in RAW264.7 cells and bone marrow-derived monocyte/macrophage precursor cells (BMMs) are not clear. In the present study, we investigate the roles of TRPM7 in osteoclastogenesis using methods of small interfering RNA (siRNA), RT-PCR, patch-clamp, and calcium imaging. RANKL (receptor activator of NF-kappaB ligand) stimulation did not affect the TRPM7 expression and TRPM7-mediated current was activated in HEK293, RAW264.7, and BMM cells by the regulation of Mg2+. Knock-down of TRPM7 by siTRPM7 reduced intracellular Ca2+ concentration ([Ca2+]i) increases by 0 mM [Mg2+]e in HEK293 cells and inhibited the generation of RANKL-induced Ca2+ oscillations in RAW264.7 cells. Finally, knock-down of TRPM7 suppressed RANKL-mediated osteoclastogenesis such as activation and translocation of NFATc1, formation of multinucleated cells, and the bone resorptive activity, sequentially. These results suggest that TRPM7 plays an essential role in the RANKL-induced [Ca2+]i oscillations that triggers the late stages of osteoclastogenesis.

Keyword

Calcium signaling; Osteoclastogenesis; RANKL; TRPM7

MeSH Terms

Calcium
Calcium Signaling
HEK293 Cells
Homeostasis
Mammals
NF-kappa B
Phosphotransferases
RNA, Small Interfering
Calcium
NF-kappa B
Phosphotransferases
RNA, Small Interfering

Figure

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  Fig. 1 Expression of and activation of endogenous TRPM7 channels in HEK293, RAW264.7, and BMMs. (A) Whole cells lysates were collected from cells stimulated with RANKL for indicated time. TRPM7 was blotted with its antibody. (B) mRNA expression of TRPM7 were decreased after transfection of siTRPM7 in HEK293 and RAW264.7 cells. (C) Activation of endogenous TRPM7-mediated currents by a voltage ramp (-100 mV to +100 mV in 50-ms intervals, Vh=0 mV), used to determine current-voltage relations in cells. TRPM7 currents were activated by 0 mM [Mg2+]e and these effects diminished in TRPM7 knock-down cells. (D) The amplitude of endogenous TRPM7-mediated currents at -80 and +80 mV in HEK293 and RAW264.7 cells. Data were expressed as the mean±SEM. **p<0.01, *p<0.05 compared with control.

• 

  Fig. 2 Effects of deletion of TRPM7 on [Ca2+]i increases mediated by RANKL stimulation and removal of extracellular Mg2+. (A) Cells were transfected with siTRPM7 using lipofectamine 2000. After 72 h of transfection, whole mRNA was collected using Trizol method. Beta-actin is used as loading control. (B) Control and siTRPM7 treated cells were treated with RANKL. After 48 h of RANKL stimulation, [Ca2+]i in single cell was measured using Fura-2 fluorescence dye. To confirm cell's viability, 1 mM ATP was used. (C) Application of 0 mM [Mg2+]e induced [Ca2+]i increases and it was repeated by the second application of 0 mM [Mg2+]e in HEK293 cells. [Ca2+]i increases also inhibited by 100 µM Gd3+ and transfected siTRPM7. (D) Application of 0 mM [Mg2+]e induced [Ca2+]i increases in the absence or presence of RANKL and it was inhibited by 100 µM Gd3+ and transfected siTRPM7 in RAW264.7 cells. (E) The degree of [Ca2+]i increases by 0 mM [Mg2+]e in HEK293 and RAW264.7 cells. Data were expressed as the mean±SEM. ***p<0.001 compared with 0 mM [Mg2+]e treated control. n.s., not significant.

• 

  Fig. 3 Effects of deletion of TRPM7 on NFATc1 and bone resorptive activity. (A and B) Cells were transfected with sicontrol and siTRPM7 in the presence or absence of RANKL for 48 h. To confirm the localization and expression of NFATc1, cells were fixed with 4% paraformaldehyde (PFA) and whole lysates were collected using RIPA lysis buffer respectively. Localization inside the cell and NFATc1 expression in whole lysates was confirmed with antibody for NFATc1. (C) To verify the formation of MNCs, each sample was incubated for 6 days in the presence of RANKL. TRAP staining was performed as described in methods. And then MNCs in each well was counted. (D) To determine the bone resorptive activity, cells were seeded on bone-slice covered plate in the presence of RANKL for 15 days. After incubation, whole image of each well was taken to calculate mineralized area as described in methods. Data were normalized to the expression level in RANKL treated control and expressed as the mean±SEM. **p<0.01, *p<0.05 compared with RANKL treated control.

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