J Dent Anesth Pain Med.  2018 Dec;18(6):349-359. 10.17245/jdapm.2018.18.6.349.

Propofol promotes osteoclastic bone resorption by increasing DC-STAMP expression

• Kim EJ
• Kim HJ
• Baik SW
• Kim KH
• Ryu SJ
• Kim CH
• Shin SW

• KMID: 2430212
• DOI: http://doi.org/10.17245/jdapm.2018.18.6.349

Abstract

BACKGROUND
Propofol is an intravenous anesthetic which has antioxidant effects due to its similarity in molecular structure to α-tocopherol. It has been reported that α-tocopherol increases osteoclast fusion and bone resorption. Here, we investigated the effects of propofol on signaling pathways of osteoclastogenic gene expression, as well as osteoclastogenesis and bone resorption using bone marrow-derived macrophages (BMMs).
METHODS
BMMs were cultured with macrophage colony-stimulating factor (M-CSF) alone or M-CSF plus receptor activator of nuclear factor kappa B ligand (RANKL) in the presence of propofol (0-50 µM) for 4 days. Mature osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP) and the numbers of TRAP-positive multinucleated osteoclasts were counted. To examine the resorption activities of osteoclasts, a bone resorption assay was performed. To identify the mechanism of action of propofol on the formation of multinucleated osteoclasts, we focused on dendritic cell-specific transmembrane protein (DC-STAMP), a protein essential for pre-osteoclastic cell fusion.
RESULTS
Propofol increased the formation of TRAP-positive multinucleated osteoclasts. In addition, the bone resorption assay revealed that propofol increased the bone resorption area on dentin discs. The mRNA expression of DC-STAMP was upregulated most strongly in the presence of both RANKL and propofol. However, SB203580, a p38 inhibitor, significantly suppressed the propofol/RANKL-induced increase in mRNA expression of DC-STAMP.
CONCLUSION
We have demonstrated that propofol enhances osteoclast differentiation and maturation, and subsequently increases bone resorption. Additionally, we identified the regulatory pathway underlying osteoclast cell-cell fusion, which was enhanced by propofol through p38-mediated DC-STAMP expression.

Keyword

Bone Resorption; Cell Fusion; Osteoclasts; Propofol; p38 MAP Kinase

MeSH Terms

Acid Phosphatase
Antioxidants
Bone Resorption*
Cell Fusion
Dentin
Gene Expression
Macrophage Colony-Stimulating Factor
Macrophages
Molecular Structure
Osteoclasts*
p38 Mitogen-Activated Protein Kinases
Propofol*
RANK Ligand
RNA, Messenger
Acid Phosphatase
Antioxidants
Macrophage Colony-Stimulating Factor
Propofol
RANK Ligand
RNA, Messenger
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 The effect of propofol on BMM cell viability and proliferation as determined by MTT assay. (A) Propofol did not have a cytotoxic effect on BMMs. (B) Cell proliferation was similar at all concentration of propofol (0–100 µM).

• 

  Fig. 2 Propofol markedly augmented the size of osteoclast and subsequently increased bone resorption. (A–C) BMMs were cultured with M-CSF alone or M-CSF plus RANKL with propofol (0–50 µM) for 4 days, and TRAP staining was performed on the resulting osteoclasts. (A) Example images are shown. (B) Quantification of TRAP-positive multinucleated osteoclasts (cells containing more than four nuclei). (C) Quantification of the size of osteoclasts. OC, osteoclast; M, M-CSF; M+R, M-CSF+RANKL. (D–E) To examine the resorption activities of osteoclasts, a bone resorption assay was performed using dentin discs. The resorption pits were visualized and quantified by laser scanning of cell-removed dentin discs. (*, P < 0.05).

• 

  Fig. 3 Propofol has little effect on the expression of c-Fos and NFATc1. (A) BMMs were cultured in the presence or absence of RANKL (100 ng/ml) or propofol (50 mM) for 4 days. Expression of c-Fos and NFATc1 were evaluated by western blotting at day 0, 2 and 4. (B) The mRNA levels of c-Fos and NFATc1 were evaluated by RT-PCR. The mRNA expression of TRAP served as an osteoclastic differentiation marker. (C) The protein expression of c-Fos and NFATc1 at various concentrations of propofol (0–100 µM) was determined by western blotting.

• 

  Fig. 4 Propofol increases osteoclast size through the activation of p38 and DC-STAMP expression. (A) Following incubation of BMM cells with propofol for the indicated times, phosphorylation of p38 was evaluated by western blotting. (B) The mRNA expression of DC-STAMP was analyzed by RT-PCR after treatment with the indicated combinations of RANKL (100 ng/ml), propofol (50 µM) and a p38 inhibitor, SB203580 (20 µM) for 2 days. (C) Quantitative real-time PCR analysis of DC-STAMP. (D) The representative TRAP-stained images are shown (top) and the sizes of mature osteoclasts were measured (bottom). Data are mean ± SD (*, P < 0.05). OC, osteoclast.

• 

  Fig. 5 A schematic diagram for propofol-induced large osteoclast formation. The engagement of RANKL to RANK leads to pivotal osteoclastogenic gene expression via the c-Fos/NFATc1 transcription factor axis. Although propofol does not alter these major differentiation pathways, it functions as a positive regulator of osteoclastogenesis by upregulating phosphorylation of p38 and subsequently increasing DC-STAMP expression.

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