Yu Shun Tian; Hyun Joo Jeong; Sang-Do Lee; Seok Heui Kong; Seung-Ho Ohk; Yun-Jung Yoo; Jeong-Taeg Seo; Dong Min Shin; Byung-Wha Sohn; Syng-Ill Lee

doi:10.4196/kjpp.2010.14.3.169

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Tian, Jeong, Lee, Kong, Ohk, Yoo, Seo, Shin, Sohn, and Lee: Hyperosmotic Stimulus Down-regulates 1α, 25-dihydroxyvitamin D3-induced Osteoclastogenesis by Suppressing the RANKL Expression in a Co-culture System

Original Article

Korean J Physiol Pharmacol 2010;14(3):169-176.

Published online: 18 February 2010

DOI: https://doi.org/10.4196/kjpp.2010.14.3.169

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

Yu Shun Tian^1,^3,⁵, Hyun Joo Jeong^1,^3,⁵, Sang-Do Lee⁷, Seok Heui Kong^2,⁵, Seung-Ho Ohk⁶, Yun-Jung Yoo^1,^3,^4,⁵, Jeong-Taeg Seo^1,^3,^4,⁵, Dong Min Shin^1,^3,^4,⁵, Byung-Wha Sohn^2,⁵, Syng-Ill Lee^1,^3,^4,^5,

¹Department of Oral Biology, Yonsei University, Seoul 120-752, Korea

²Department of Orthodontics, Yonsei University, Seoul 120-752, Korea

³Oral Science Research Center, Yonsei University, Seoul 120-752, Korea

⁴Brain Korea 21 Project of Dental Science, Yonsei University, Seoul 120-752, Korea

⁵College of Dentistry, Yonsei University, Seoul 120-752, Korea

⁶Department of Oral Microbiology, College of Dentistry, Chonnam National University, Gwangju 500-757, Korea

⁷Department of Physiology, College of Medicine, Chungnam National University, Daejeon 305-764, Korea

Corresponding to: Syng-Ill Lee, Department of Oral Biology, Oral Science Research Center, Brain Korea 21 Project of Dental Science, College of Dentistry, Yonsei University, 134, Shinchon-dong, Seodaemoon-gu, Seoul 120-752, Korea. (Tel) 82-2-2228-3052, (Fax) 82-2-364-1085, (E-mail) lsi@yuhs.ac

Received 12 May 2010 Revised 25 May 2010 Accepted 7 June 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects 1α, 25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on 1α,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 1α,25(OH)₂D₃-induced tartrate-resistant acid phosphatase-positive multinucleated cells and 1α,25(OH)₂D₃-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor κB ligand (RANKL) and Runx2 expressions were down-regulated in response to 1α,25(OH)₂D₃. Knockdown of Runx2 inhibited 1α,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 1α,25(OH)₂D₃-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP over-expression in osteoblastic cells.

Keywords: Hyperosmotic stimulus, TonEBP, Osteoblast, RANKL, Runx2

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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).

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