J Vet Sci.  2014 Mar;15(1):133-140. 10.4142/jvs.2014.15.1.133.

Regulation of matrix metalloproteinase-9 protein expression by 1alpha,25-(OH)2D3 during osteoclast differentiation

• Gu JH
• Tong XS
• Chen GH
• Liu XZ
• Bian JC
• Yuan Y
• Liu ZP

• KMID: 1737619
• DOI: http://doi.org/10.4142/jvs.2014.15.1.133

Abstract

To investigate 1alpha,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1alpha,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1alpha,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1alpha,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.

Keyword

1alpha,25-(OH)2D3; bone lacunar resorption; MMP-9; osteoclast; TRAP

MeSH Terms

Acid Phosphatase/metabolism
Animals
Blotting, Western
Calcitriol/*pharmacology
Calcium Channel Agonists/pharmacology
*Cell Differentiation
Cell Line
Cell Proliferation
Gene Expression Regulation, Enzymologic/*drug effects
Isoenzymes/metabolism
Matrix Metalloproteinase 9/*genetics/metabolism
Mice
Osteoclasts/*cytology/*enzymology
Tetrazolium Salts
Thiazoles
Acid Phosphatase
Calcium Channel Agonists
Calcitriol
Isoenzymes
Tetrazolium Salts
Thiazoles
Matrix Metalloproteinase 9

Figure

• 

  Fig. 1 MTT analysis of RAW264.7 cell viability after treatment with 1α,25-(OH)2D3, receptor activator of nuclear factor κB ligand (RANKL), and macrophage colony-stimulating factor (M-CSF). The results are expressed as the mean ± standard error (SE). RANKL (30 µg/L) plus M-CSF (25 µg/L) promoted the proliferation of RAW264.7 cell (**p < 0.01 vs. group A). Additionally, 1α,25-(OH)2D3 inhibited the proliferation rate of RAW264.7 cells in a dose-dependent manner (†p < 0.05 vs. group B, ††p < 0.01 vs. group B).

• 

  Fig. 2 Morphology and number of TRAP-positive osteoclasts in RAW264.7 cells after 5 day of incubation with or without cytokines. No osteoclast formation was observed in group A (RAW264.7 cells cultured without any cytokines). In contrast, many osteoclasts (indicated by black arrows) were formed in groups B ~ E. (A) No cytokines. (B) 30 µg/L RANKL and 25 µg/L M-CSF. (C) 30 µg/L RANKL, 25 µg/L M-CSF, and 10-10 mol/L 1α, 25-(OH)2D3. (D) 30 µg/L RANKL, 25 µg/L M-CSF, and 10-9 mol/L 1α, 25-(OH)2D3. (E) 30 µg/L RANKL, 25 µg/L M-CSF, and 10-8 mol/L 1α, 25-(OH)2D3. Scale bars = 100 µm.

• 

  Fig. 3 The number of TRAP-positive multinucleated osteoclasts formed with or without M-CSF, RANKL, and 1α,25-(OH)2D3. No osteoclasts were formed in group A (RAW264.7 cells cultured without any cytokines). M-CSF and RANKL induced the formation of TRAP-positive multinucleated osteoclasts (groups B~E). In addition, 1α,25-(OH)2D3 increased the number of TRAP-positive multinucleated osteoclasts (groups C ~E). **p < 0.01 vs. group A, †p < 0.05 vs. group B, and ††p < 0.01 vs. group B.

• 

  Fig. 4 Resorption lacunae on bone slices observed with an XL30-ESEM environmental scanning electron microscope. No resorption lacunae were observed on bone slices incubated with cells from group A (RAW264.7 cells cultured without any cytokines). In contrast, many resorption lacunae (indicated with white arrows) were formed in bone slices incubated with cells from groups B ~ E. (A) No cytokines. (B) 30 µg/L RANKL and 25 µg/L M-CSF. (C) 30 µg/L RANKL, 25 µg/L M-CSF, and 10-10 mol/L 1α, 25-(OH)2D3. (D) 30 µg/L RANKL, 25 µg/L M-CSF, and 10-9 mol/L 1α, 25-(OH)2D3. (E) 30 µg/L RANKL, 25 µg/L M-CSF, and 10-8 mol/L 1α, 25-(OH)2D3. Scale bars = 50 µm.

• 

  Fig. 5 The area of lacunar resorption. No resorption lacunae were observed in bone slices incubated with cells from group A (RAW264.7 cells cultured without any cytokines). M-CSF and RANKL (group B) induced osteoclast bone resorption. Additionally, 1α,25-(OH)2D3 enhanced osteoclast bone resorption (groups C ~ E). **p < 0.01 vs. group A, †p < 0.05 vs. group B, and ††p < 0.01 vs. group B.

• 

  Fig. 6 Detection of MMP-9 protein expression in osteoclasts by Western blotting. The results are expressed as the mean ± SE. MMP-9 was expressed in each group of cells. Compared to group A (RAW264.7 cells cultured without any cytokines), RANKL and M-CSF enhanced the expression of MMP-9 (**p < 0.01 vs. control group). Furthermore, 1α,25-(OH)2D3 promoted the expression of MMP-9 in a dose-dependent manner (†p < 0.05 vs. group B and ††p < 0.01 vs. group B).

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