Yoshikawa Yoshihiro, Yoshizawa Tatsuya, Domae Eisuke, Hieda Yohki, Takeyama Akira, Hirota Shuitsu, Kawamoto Akiyo, Goda Seiji, Tamura Isao, Kamada Aiko, Komasa Yutaka, Morita Shosuke, Yamagata Kazuya, Ikeo Takashi
Department of Biochemistry, Osaka Dental University, Osaka 573-1121, Japan.
Department of Medical Biochemistry, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
Arch Oral Biol. 2015 Sep;60(9):1319-26. doi: 10.1016/j.archoralbio.2015.06.001. Epub 2015 Jun 10.
BMP-2 induces osteoblast differentiation and activates osteoclast formation. Here, we investigated the role of Smad1, a molecule that signals downstream of BMP-2, in mediating the effects of BMP-2 on osteoclast differentiation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in osteoblasts.
The effects of 1,25(OH)2D3 and BMP-2 in osteoclasts were examined using polymerase chain reaction and Western blotting to measure changes in target gene and protein expression. Immunostaining was carried out to investigate the localization of the vitamin D receptor (VDR) in the nucleus in response to BMP-2.
Stimulation with both 1,25(OH)2D3 and BMP-2 resulted in significantly greater osteoclast formation and receptor activator of nuclear factor κB ligand (RANKL) mRNA expression compared to stimulation with 1,25(OH)2D3 alone. In addition, expression of the VDR protein was increased, enhancing the activity of 1,25(OH)2D3. Interestingly, knockdown of Smad1 resulted in reduced osteoclast formation, RANKL mRNA expression, and VDR protein expression compared with control cells. Costimulation with 1,25(OH)2D3 and BMP-2 enhanced VDR localization in the nucleus.
We found that BMP-2 induced Smad1 activation, thereby influencing the localization of VDR in the nucleus in the presence of 1,25(OH)2D3 and resulting in increased RANKL mRNA expression. These effects ultimately resulted in enhanced osteoclast differentiation.
骨形态发生蛋白-2（BMP-2）可诱导成骨细胞分化并激活破骨细胞形成。在此，我们研究了Smad1（一种在BMP-2下游发出信号的分子）在介导BMP-2对成骨细胞中1,25-二羟基维生素D3（1,25(OH)2D3）诱导的破骨细胞分化作用中的角色。
使用聚合酶链反应和蛋白质印迹法检测1,25(OH)2D3和BMP-2对破骨细胞的影响，以测量靶基因和蛋白质表达的变化。进行免疫染色以研究维生素D受体（VDR）在细胞核中对BMP-2的反应定位。
与单独用1,25(OH)2D3刺激相比，用1,25(OH)2D3和BMP-2共同刺激导致破骨细胞形成和核因子κB受体激活剂配体（RANKL）mRNA表达显著增加。此外，VDR蛋白表达增加，增强了1,25(OH)2D3的活性。有趣的是，与对照细胞相比，敲低Smad1导致破骨细胞形成、RANKL mRNA表达和VDR蛋白表达减少。1,25(OH)2D3和BMP-2共同刺激增强了VDR在细胞核中的定位。
我们发现BMP-2诱导Smad1激活，从而在存在1,25(OH)2D3的情况下影响VDR在细胞核中的定位，并导致RANKL mRNA表达增加。这些作用最终导致破骨细胞分化增强。
