J Vet Sci.  2015 Jun;16(2):151-156. 10.4142/jvs.2015.16.2.151.

Involvement of the Ca2+ signaling pathway in osteoprotegerin inhibition of osteoclast differentiation and maturation

• Fu Y
• Gu J
• Wang Y
• Yuan Y
• Liu X
• Bian J
• Liu ZP

• KMID: 2164523
• DOI: http://doi.org/10.4142/jvs.2015.16.2.151

Abstract

The purpose of this study was to determine whether the Ca2+ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-kappaB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca2+ concentration [Ca2+]i and phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca2+]i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca2+ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca2+ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.

Keyword

Ca2+ signaling pathway; osteoclast; osteoclastogenesis; osteoprotegerin

MeSH Terms

Animals
Calcium/*metabolism
*Calcium Signaling
*Cell Differentiation/drug effects
Cell Line
Cell Survival/drug effects
Gene Expression Regulation/drug effects
Macrophage Colony-Stimulating Factor/metabolism
Mice
Osteoclasts/*cytology/*drug effects/*metabolism
Osteoprotegerin/*pharmacology
RANK Ligand/metabolism
Calcium
Macrophage Colony-Stimulating Factor
Osteoprotegerin
RANK Ligand

Figure

• 

  Fig. 1 Effect of osteoprotegerin (OPG) on [Ca2+]i in macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL)-treated osteoclasts. [Ca2+]i in the osteoclasts following treatment with 0, 10, 20, 50, and 100 ng/mL OPG was analyzed by flow cytometry. Fluorescent intensities showed that OPG reduced [Ca2+]i in the osteoclasts. Results are expressed as the mean ± SEM for three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group (#M-CSF + RANKL, 0 ng/mL OPG).

• 

  Fig. 2 Effect of 2-APB on [Ca2+]i in osteoclasts. [Ca2+]i in osteoclasts derived from RAW264.7 cells was analyzed by flow cytometry for the different treatment groups as indicated. Fluorescent intensities represent [Ca2+]i. It was found that 2-APB (50 ng/mL) reduces [Ca2+]i in M-CSF + RANKL-induced osteoclasts. [Ca2+]i was further lowered following treatment with 100 ng/mL OPG. Results are expressed as the mean ± SEM of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group (#M-CSF + RANKL, 0 ng/mL OPG, and 0 ng/mL 2-APB).

• 

  Fig. 3 Effect of OPG on Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation in M-CSF + RANKL-induced osteoclasts. The phosphorylation levels of CaMKII in the osteoclasts following treatment with 0, 10, 20, 50, and 100 ng/mL OPG were determined by Western blotting. Band intensities represented the level of phosphorylated (p)-CaMKII relative to CaMKII. These finding showed that OPG reduces the phosphorylation of CaMKII in a dose-dependent manner. Results are expressed as the mean ± SEM for three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group (M-CSF + RANKL, 0 ng/mL OPG).

• 

  Fig. 4 Effect of KN93 on CaMKII phosphorylation in osteoclasts. The levels of p-CaMKII in osteoclasts differentiated from RAW264.7 cells were measured by Western blotting. Band intensities represent the levels of p-CaMKII relative to CaMKII in the different treatment groups as indicated. These findings showed that KN93 (10 ng/mL) reduces the levels of p-CaMKII in M-CSF + RANKL-induced osteoclasts. The levels of p-CaMKII were further decreased following treatment with OPG (100 ng/mL). Results are expressed as the mean ± SEM of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control groups (con: M-CSF + RANKL, 0 ng/mL OPG, and 0 µM KN93).

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