Korean J Physiol Pharmacol.  2010 Jun;14(3):169-176. 10.4196/kjpp.2010.14.3.169.

Hyperosmotic Stimulus Down-regulates 1alpha, 25-dihydroxyvitamin D3-induced Osteoclastogenesis by Suppressing the RANKL Expression in a Co-culture System

• Tian YS
• Jeong HJ
• Lee SD
• Kong SH
• Ohk SH
• Yoo YJ
• Seo JT
• Shin DM
• Sohn BW
• Lee SI

• KMID: 2285399
• DOI: http://doi.org/10.4196/kjpp.2010.14.3.169

Abstract

The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on 1alpha,25(OH)2D3-induced osteoclastogenesis was investigated in an osteoblast-preosteoclast co-culture system. Serial doses of sucrose were applied as a mechanical force. These hyperosmotic stimuli significantly evoked a reduced number of 1alpha,25(OH)2D3-induced tartrate-resistant acid phosphatase-positive multinucleated cells and 1alpha,25(OH)2D3-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor kappaB ligand (RANKL) and Runx2 expressions were down-regulated in response to 1alpha,25(OH)2D3. Knockdown of Runx2 inhibited 1alpha,25(OH)2D3-induced RANKL expression in osteoblastic cells. Finally, the hyperosmotic stimulus induced the overexpression of TonEBP in osteoblastic cells. These results suggest that hyperosmolarity leads to the down-regulation of 1alpha,25(OH)2D3-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.

Keyword

Hyperosmotic stimulus; TonEBP; Osteoblast; RANKL; Runx2

MeSH Terms

Bone Remodeling
Coculture Techniques
Down-Regulation
Osteoblasts
RANK Ligand
Sucrose
RANK Ligand
Sucrose

Figure

• 

  Fig. 1. Effect of hyperosmotic stimulus of sucrose on 1α,25(OH)2D3-induced osteoclast differentiation in a co-culture system. Mouse BMMs were co-cultured with calvarial osteoblastic cells in the presence of 1α,25(OH)2D3 and the indicated concentration of sucrose. (A) Preosteoclast cells underwent differentiation into TRAP-positive multinuclear cells in the presence of 1α,25(OH)2D3, and different concentrations of sucrose. (B) The number of TRAP-positive multinuclear cells was counted. (C) Cell viability was determined by a MTT assay. Results are presented as the mean values±SEM (n=4; ∗p<0.05, ∗∗p<0.001 versus 1α,25(OH)2D3 only). (D) Osteoclasts had bone resorbing activity on calcium phosphate apatite-coated plates. (E) Total areas of formation of pit resorption. Osteoclast differentiation was significantly inhibited as the increase in sucrose concentration. Bar indicates mean±SEM (n=5; ∗p<0.05, ∗∗p < 0.001 versus 1α,25(OH)2D3 only).

• 

  Fig. 2. Effect of hyperosmotic stimulus on the genes of osteoblast. Osteoblastic cells were cultured with 1α,25(OH)2D3 and the indicated sucrose concentration. (A) Total RNA was extracted, and then mRNA expression was analyzed by RT-PCR. (B) Bar graph shows the quantitative analysis of RANKL/β-actin mRNA. (C, D) For quantitative analysis, sRANKL and OPG productions were determined by ELISA. The bar graph shows the concentration level of RANKL and OPG protein. (E) Osteoblast marker genes were amplified by RT-PCR using RNA the primary osteoblastic cells that were exposed to the hyperosmotic stimulus. Bar represents the mean±SEM (n=4; ∗p<0.05 versus 1α,25(OH)2D3 only).

• 

  Fig. 3. Effects of hyperosmotic stimulus in 1α,25(OH)2D3-induced Runx2 expression. (A) Western blot analysis of Runx2 expression in osteoblastic cells stimulated with 1α, 25(OH)2D3 in the presence of indicated sucrose concentration. (B) Densitometric analysis of Runx2 and β-actin expression was used to normalize Runx2 data. (C) Western blot analysis of the levels of 1α,25(OH)2D3-induced RANKL and Runx2 expression in osteoblastic cells transfected with Runx2 siRNA and selected with G418-neomycine. Bar represents the mean±SEM (n=6; ∗p<0.05, ∗∗p < 0.001 versus 1α,25(OH)2D3 only).

• 

  Fig. 4. Changes in the expression of TonEBP and Runx2 by hyperosmotic stimulus. Expression of TonEBP by hyperosmotic stress inhibits 1α,25(OH)2D3-induced RANKL expression in osteoblastic cells. (A) Western blot analysis of TonEBP expression in osteoblastic cells treated with 1α,25(OH)2D3 and 25 and 50 mM sucrose addition to the culture medium for 2 days. (B) TonEBP was stimulated by hyperosmotic stress caused by the addition of sucrose. (C) Western blot analysis of TonEBP expression levels in osteoblastic cells transfected with TonEBP (overexpression) or TonEBP siRNA. (D) The levels of RANKL expression were analyzed by RT-PCR using osteoblastic cells transfected with TonEBP siRNA and TonEBP in the presence or absence of 1α,25(OH)2D3. (E) The down-regulation in Runx2 expression by the stimulation of TonEBP (in response to the addition of 50 mM sucrose to the culture medium) in osteoblastic cells and TonEBP expression in osteoblastic cells transfected with Runx2 siRNA. Bar represents the mean±SEM (n=3; ∗p<0.05, ∗∗p<0.005 versus 1α,25(OH)2D3 only).

Reference

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