J Bone Metab.  2012 Nov;19(2):111-120. 10.11005/jbm.2012.19.2.111.

alpha-Tocopheryl Succinate Inhibits Osteoclast Formation by Suppressing Receptor Activator of Nuclear Factor-kappaB Ligand (RANKL) Expression and Bone Resorption

• Kim HN
• Lee JH
• Jin WJ
• Lee ZH

• KMID: 1975984
• DOI: http://doi.org/10.11005/jbm.2012.19.2.111

Abstract

OBJECTIVE
Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte/macrophage lineage during normal and pathological bone turnover. Recently, several studies revealed that alpha-tocopheryl succinate (alphaTP-suc) have demonstrated potent anti-cancer activities in vitro and in vivo. However, the effects of alphaTP-suc on osteoclast formation and bone resorption remain unknown. Thus, in this study, we examined the effects of alphaTP-suc on osteoclast differentiation and bone resorbing activity in inflammatory bone loss model.
METHODS
Osteoclast differentiation assay was performed by cocultures of mouse bone marrow cells and calvarial osteoblasts in culture media including interleukin-1 (IL-1). Osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP). The level of receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). ICR mice were administered an intraperitoneal injections of alphaTP-suc or dimethyl sulfoxide (DMSO) 1 day before the implantation of a freeze-dried collagen sponge loaded with phosphate-buffered saline (PBS) or IL-1 over the calvariae and every other day for 7 days. The whole calvariae were obtained and analyzed by micro-computed tomography (CT) scanning, and stained for TRAP.
RESULTS
alphaTP-suc inhibits osteoclast formation in cocultures stimulated by IL-1 and decreased the level of expression of RANKL mRNA in osteoblasts. In addition, administered intraperitoneal injections of alphaTP-suc prevented IL-1-mediated osteoclast formation and bone loss in vivo.
CONCLUSION
Our findings suggest that alphaTP-suc may have therapeutic value for treating and preventing bone-resorptive diseases, such as osteoporosis.

Keyword

Alpha-tocopheryl succinate; Interleukin-1; Osteoclast; Osteoporosis; RANKL

MeSH Terms

Acid Phosphatase
Animals
Bone Marrow Cells
Bone Resorption
Coculture Techniques
Collagen
Culture Media
Cytokines
Dimethyl Sulfoxide
Injections, Intraperitoneal
Interleukin-1
Isoenzymes
Mice
Mice, Inbred ICR
Osteoblasts
Osteoclasts
Osteoporosis
Porifera
RANK Ligand
Receptor Activator of Nuclear Factor-kappa B
RNA, Messenger
Safrole
Skull
Succinic Acid
Acid Phosphatase
Collagen
Culture Media
Cytokines
Dimethyl Sulfoxide
Interleukin-1
Isoenzymes
RANK Ligand
RNA, Messenger
Receptor Activator of Nuclear Factor-kappa B
Safrole
Succinic Acid

Figure

• 

  Fig. 1 Effects of alpha-tocopheryl succinate (αTP-suc) on osteoclast differentiation in coculture. (A) Tartrate-resistant acid phosphatase (TRAP) staining of cocultures in the presence of interleukin-1 (IL-1; 15 ng/mL) for 7 days with or without αTP-suc (10 µM or 20 µM). (B) Bone marrow cells and calvarial osteoblasts were cultured with IL-1 (15 ng/mL) in the presence or absence of αTP-suc, αTP acetate (ace) or αTP. After 7 days, cells were fixed and stained for TRAP. TRAP-positive multinucleated cells containing three or more nuclei were counted as osteoclasts. *P < 0.05 versus dimethyl sulfoxide-treated control.

• 

  Fig. 2 Effects of alpha-tocopheryl succinate (αTP-suc) on receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclast formation in bone marrow-derived macrophages. (A) Tartrate-resistant acid phosphatase (TRAP) staining of osteoclast precursor cultures in the presence of 30 ng/mL macrophage colony-stimulating factor (M-CSF) and 100 ng/mL RANKL for 4 days with or without αTP-suc (20 µM) or αTP (20 µM). (B) Bone marrow-derived macrophages were cultured with the indicated doses of αTP-suc, αTP-ace or αTP in the presence of M-CSF and RANKL. After 4 days, TRAP-positive multinucleated cells containing three or more nuclei were counted as osteoclasts.

• 

  Fig. 3 Effects of alpha-tocopheryl succinate (αTP-suc) on interleukin-1 (IL-1)-induced receptor activator of nuclear factor-kappaB ligand (RANKL) expression in osteoblasts. (A and B) Mouse calvarial osteoblasts were pretreated with αTP-suc (20 µM), αTP (20 µM) or dimethyl sulfoxide (DMSO) for 12 h and then cultured with IL-1 (15 ng/mL) or 1α,25(OH)2D3 (VD; 10 nM) for 24 h. (A) The mRNA levels of the indicated genes were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). (B) The indicated protein amounts were determined by using enzyme Immunoassay (ELISA) kits in cell lysates (RANKL) and in cell culture media (osteoprotegerin and prostaglandin E2). *P < 0.05.

• 

  Fig. 4 Effects of alpha-tocopheryl succinate (αTP-suc) on IL-1-induced bone destruction in vivo. (A-C) A collagen sponge treated with PBS or IL-1 (2 µg) was implanted over the calvarial bone of 5-week-old mice. αTP-suc (80 mg/kg body weight) or vehicle (DMSO) was administered intraperitoneally for 7 days. (A) Whole calvariae were fixed and stained for TRAP to identify eroded surfaces (top), and presented as 3D micro-CT images (bottom). (B) Calvarial bone mineral content was measured by micro-CT analysis. (C) Osteoclast number was analyzed by histomorphometric analysis. *P < 0.05 versus control group; #P < 0.05 versus group treated with IL-1 only.

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