J Vet Sci.  2013 Dec;14(4):405-412. 10.4142/jvs.2013.14.4.405.

Inhibitory effects of osteoprotegerin on osteoclast formation and function under serum-free conditions

• Fu YX
• Gu JH
• Zhang YR
• Tong XS
• Zhao HY
• Yuan Y
• Liu XZ
• Bian JC
• Liu ZP

• KMID: 1712306
• DOI: http://doi.org/10.4142/jvs.2013.14.4.405

Abstract

The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor kappaB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor kappaB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions.

Keyword

activation; differentiation; osteoclast; osteoprotegerin; serum-free

MeSH Terms

Acid Phosphatase/genetics/metabolism
Animals
Avian Proteins/*pharmacology
Bone Marrow Cells/drug effects/*metabolism
Cells, Cultured
Ducks
Embryo, Nonmammalian/drug effects/metabolism
Isoenzymes/genetics/metabolism
Macrophage Colony-Stimulating Factor/metabolism
Osteoclasts/cytology/*drug effects/*metabolism
Osteoprotegerin/*pharmacology
RANK Ligand/metabolism
Real-Time Polymerase Chain Reaction
Receptor Activator of Nuclear Factor-kappa B/genetics/metabolism
Acid Phosphatase
Avian Proteins
Isoenzymes
Macrophage Colony-Stimulating Factor
Osteoprotegerin
RANK Ligand
Receptor Activator of Nuclear Factor-kappa B

Figure

• 

  Fig. 1 Inhibitory effects of OPG on TRAP-positive multinucleated osteoclast differentiation in M-CSF- and RANKL-stimulated duck embryo bone marrow and RAW 264.7 cells. (A~A4) Duck embryo bone marrow cells were suspended in α-MEM with FBS (v/v 10%) and then seeded in 96-well culture plates. After a 24-h cultivation period, the α-MEM was replaced with serum-free medium containing M-CSF (25 ng/mL) and RANKL (30 ng/mL). The cells were cultured up to 5 days. Next, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells in the presence of M-CSF and RANKL, and incubated for another 3 days. (B~B4) RAW 264.7 cells were suspended in α-MEM with FBS (v/v 10%) and then seeded in 96-well culture plates. After a 24-h culturing period, the α-MEM was replaced with serum-free medium with M-CSF (25 ng/mL) and RANKL (30 ng/mL). The cells were then incubated for 48 h. Next, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells in the presence of M-CSF and RANKL, and the cells were incubated for another 3 days. At the end of the incubation period, the two types of cells were fixed and stained for TRAP. (A and B) control, (A1 and B1) 10 ng/mL OPG, (A2 and B2) 20 ng/mL OPG, (A3 and B3) 50 ng/mL OPG, (A4 and B4) 100 ng/mL OPG. ×50 (A~A4), ×100 (B~B4). Scale bars = 500 µm (A~A4), 200 µm (B~B4). (C and D) TRAP-positive multinucleated cells (TRAP+) were observed under a light microscope and counted. The results are expressed as the mean ± SEM. **p < 0.01 and *p < 0.05 vs. the control groups. OPG: osteoprotegerin, TRAP: tartrate-resistant acid phosphatase, M-CSF: macrophage colony stimulatory factor, RANKL: receptor activator for nuclear factor κB ligand, α-MEM: minimum essential medium alpha, FBS: fetal bovine serum.

• 

  Fig. 2 Suppressive effects of OPG on F-actin ring formation in duck embryo osteoclasts. Duck embryo bone marrow cells were suspended in α-MEM with FBS (v/v 10%) and then seeded in 12-well culture plates. After incubating for 24 h, the α-MEM was replaced with serum-free medium containing M-CSF (25 ng/mL) and RANKL (30 ng/mL). The cells were cultured for up to 5 days. Next, 0, 10, 20, 50, and 100 ng/mL OPG were added to the different groups of cells in the presence of M-CSF and RANKL. The cells were incubated for another 3 days. At the end of the cultivation period, the cells were fixed and stained for TRITC-phalloidin. The F-actin rings were observed using an inverted phase contrast fluorescence microscope with appropriate filters. (A) control, (B) 10 ng/mL OPG, (C) 20 ng/mL OPG, (D) 50 ng/mL OPG, (E) 100 ng/mL OPG. ×400. Scale bars = 100 µm (A~E).

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  Fig. 3 Inhibition of osteoclasts pit resorption by OPG. (A~A5) Duck embryo bone marrow cells were suspended in α-MEM with FBS (v/v 10%) and then seeded in 48-well culture plates with bovine cortical bone slices. After 24 h, the α-MEM was replaced with serum-free medium supplemented with M-CSF (25 ng/mL) and RANKL (30 ng/mL). The cells were cultured for up to 5 days. Next, 0, 10, 20, 50, 100 ng/mL OPG were added to the different groups of cells in the presence of M-CSF and RANKL, and incubated for another 3 days. At the end of the incubation period, resorbed areas were examined using an environmental scanning electron microscope. The groups were as follows: (A) control, (A1) 10 ng/mL OPG, (A2) 20 ng/mL OPG, (A3) 50 ng/mL OPG, (A4) 100 ng/mL OPG, and (A5) bone slices without cells. ×1,000. Scale bars = 50 µm. (A6) The histogram represents the volume of pit resorption. The results are expressed as the mean ± SEM. **p < 0.01 and *p < 0.05 vs. the control groups.

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  Fig. 4 Effects of OPG on the expression of TRAP and RANK. RAW264.7 cells were suspended in α-MEM with FBS (v/v 10%) and then seeded in 6-well culture plates. After 24 h, the α-MEM was replaced with serum-free medium containing M-CSF (25 ng/mL) and RANKL (30 ng/mL). The cells were then incubated for 48 h. (A and B) For the concentration gradient study, 0, 10, 20, 50, and 100 ng/mL OPG were added in the presence of M-CSF and RANKL to various groups of cells. OPG treatment was sustained for 30 min. The control cells (#) were treated with M-CSF and RANKL for another 30 min (C and D). For the time gradient study, 100 ng/mL OPG were added in the presence of M-CSF (25 ng/mL) and RANKL (30 ng/mL) to the different groups of cells. OPG treatment was sustained for 15, 30, 60, and 120 min. The control cells (#) were treated with M-CSF (25 ng/mL) and RANKL (30 ng/mL) without OPG for another 15 min. At each indicated period, TRAP and RANK mRNA expression levels were measured with real-time PCR. Results are expressed as the mean ± SEM. **p < 0.01 and *p < 0.05 vs. the control groups (#).

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