Hyperosmotic Stimulus Down-regulates $1{\\alpha}$, 25-dihydroxyvitamin $D_3$-induced Osteoclastogenesis by Suppressing the RANKL Expression in a Co-culture System원문보기
The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects $1{\alpha}$, 25-dihydroxyvitamin $D_3$ ($1{\alpha},25(OH)_2D_3$)-induced osteoclastogenesis is not clear. In the present study, the ef...
The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects $1{\alpha}$, 25-dihydroxyvitamin $D_3$ ($1{\alpha},25(OH)_2D_3$)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on $1{\alpha},25(OH)_2D_3$-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 $1{\alpha},25(OH)_2D_3$-induced tartrate-resistant acid phosphatase-positive multinucleated cells and $1{\alpha},25(OH)_2D_3$-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor ${\kappa}B$ ligand (RANKL) and Runx2 expressions were down-regulated in response to $1{\alpha},25(OH)_2D_3$. Knockdown of Runx2 inhibited $1{\alpha},25(OH)_2D_3$-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 $1{\alpha},25(OH)_2D_3$-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.
The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects $1{\alpha}$, 25-dihydroxyvitamin $D_3$ ($1{\alpha},25(OH)_2D_3$)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on $1{\alpha},25(OH)_2D_3$-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 $1{\alpha},25(OH)_2D_3$-induced tartrate-resistant acid phosphatase-positive multinucleated cells and $1{\alpha},25(OH)_2D_3$-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor ${\kappa}B$ ligand (RANKL) and Runx2 expressions were down-regulated in response to $1{\alpha},25(OH)_2D_3$. Knockdown of Runx2 inhibited $1{\alpha},25(OH)_2D_3$-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 $1{\alpha},25(OH)_2D_3$-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.
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제안 방법
After culturing, the attached cells were re moved from the plate by the addition of a 4% sodium hypo chlorite solution. Images of the pit were captured using a digital camera attached to a microscope at X100 magnifica tion, and the total area of the resorption pits was analyzed using the Image Meta Morph Program (Universal Imaging, Downingtown, PA, USA). The data are presented as means± SEM of four samples.
4A, 4B). To clarify how TonEBP works in the process of 1a, 25(OH)2D3- induced osteoclastogenesis, TonEBP overexpression and si lence of TonEBP expression into cal.,arial osteoblastic cells were checked, and also applied in BMMs in the presence of 1a, 25(OH)2D3. As shown in Fig.
대상 데이터
Parallel PCR analysis was run for the housekeeping gene B- actin to normalize data for differences in mRNA quantity and integrity. All primers were obtained from BIONEER (Kyung-gi - Do, Republic of Korea). PCR products were separated on 1.
The 1a, 25(OH)2D3, sucrose, tartrate-re si stant acid phosphatase (TRAP) stain ing kit, collagenase, and most of the chemicals were pur chased from Sigma-Aldrich (St Louis, MO). ICR mice were purchased from Samtacho (O-san, Kyung-gi-Do, Republic of Korea). Sodium dodecyl sulfate (SDS) was obtained from Bio-Rad Laboratories (Hercules, CA, USA).
ICR mice were purchased from Samtacho (O-san, Kyung-gi-Do, Republic of Korea). Sodium dodecyl sulfate (SDS) was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Anti-Runx2 and anti-RANKL were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
이론/모형
These down- reg ulations of RANKL and Runx2 protein suggest that hyper osmotic exposure to osteoblastic cells inhibits RANKL ex pression via Runx2, eventually interupting osteoclastogenesis in the co-culture system. RANKL expression was examined after disrupting the Runx2 production using the siRNA method. As expected, an upregulated level of 1a, 25 (OH)2D3-induced RANKL expression in mouse cal.
The results are expressed as mean±SE. The statistical significance between comparable groups was determined using the Kruskal-Wallis and Bon- ferroni's test. In statistical tests, the p-value<0.
성능/효과
4D). From these results, it could be speculated that the TonEBP gene may be capable of down-regulation the RANKL expression by suppressing the Runx2 at the upstream level, which in turn leads to in hibition of 1a, 25(OH)2D3-induced osteoclast differentiation.
Of these possible contributors to the hyperosmotic effect of the osteoblastic cells, we employed TonEBP, which is a transcriptional factor for the cellular accumulation of compatible osmolytes under the circumstances of hyper osmotic stimulus, because it is reasonable to assume that TonEBP is involved in processes affecting tissue hydration and osmotic environment, as well as a variety of physiologic functions. Our results demonstrate that a hyperosmotic stimulus of 50 mM sucrose enhances the expression of TonEBP, following the suppression of the Runx2, which, in turn, down-regulates 1a, 25(OH)2D3-induced RANKL ex pression as expected (Fig. 4A, 4B). Additionally, we con firmed that the 1a, 25(OH)2D3-induced RANKL protein lev el was suppressed when TonEBP was overexpressed in the cal.
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