ClC-3 chloride channel mediates the role of parathyroid hormone [1-34] on osteogenic differentiation of osteoblasts.
ClC-3 chloride channel mediates the role of parathyroid hormone [1-34] on osteogenic differentiation of osteoblasts.
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DOI：
10.1371/journal.pone.0176196
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Wang H
中科院分区：
文献类型：
--
作者：
Lu X;Ding Y;Niu Q;Xuan S;Yang Y;Jin Y;Wang H
不同浓度的甲状旁腺激素[1-34]（PTH[1-34]）对成骨细胞具有完全相反的作用。 PTH 间歇性刺激可在体外显着增加骨密度，主要通过蛋白激酶 A (PKA) 信号通路磷酸化 runt 相关转录因子 2 (Runx2)。根据最近的研究，ClC-3氯离子通道是一种重要的阴离子通道，也可以通过Runx2途径促进成骨。因此，我们研究的目的是研究 ClC-3 氯离子通道是否对 MC3T3-E1 细胞的 PTH 骨分化有影响。采用细胞计数试剂盒（CCK-8）和实时荧光定量PCR分别研究不同PTH刺激模式对MC3T3-E1细胞增殖和成骨相关基因表达的影响。我们发现PTH的最低抑制浓度为10−9 M，并且用10−9 M PTH处理时碱性磷酸酶（Alpl）和Runx2的表达处于最高水平。接下来，我们利用实时PCR和免疫荧光技术检测PTH处理下MC3T3-E1细胞中ClC-3的变化。结果显示，在 10−9 M 间歇性 PTH 给药时，ClC-3 氯离子通道的表达高于其他组。最后，我们利用ClC-3 siRNA技术检测ClC-3氯通道在PTH对成骨细胞成骨作用的影响中的作用，发现骨唾液蛋白（Ibsp）、骨钙素（Bglap）、osterix（Sp7）、Alpl和Runx2的表达明显减少，并形成矿化结节。根据以上数据，我们得出结论，成骨细胞中 ClC-3 氯离子通道的表达有助于它们对 PTH 刺激做出反应，从而介导成骨分化。
Different concentrations of parathyroid hormone [1–34] (PTH [1–34]) can have totally opposite effects on osteoblasts. Intermittent stimulation with PTH can significantly increase bone mineral density in vitro, mainly through the protein kinase A (PKA) signaling pathway, which phosphorylates runt-related transcription factor 2 (Runx2). The ClC-3 chloride channel, an important anion channel, can also promote osteogenesis via the Runx2 pathway based on recent studies. The purpose of our study, therefore, is to research whether the ClC-3 chloride channel has an effect on PTH osteodifferentiation in MC3T3-E1 cells. A cell counting kit (CCK-8) and real-time PCR were used to investigate the impact of different PTH stimulation modes on MC3T3-E1 cell proliferation and osteogenesis-related gene expression, respectively. We found that the minimum inhibitory concentration of PTH was 10−9 M, and the expression of alkaline phosphatase (Alpl) and Runx2 were at the highest levels when treated with 10−9 M PTH. Next, we used real-time PCR and immunofluorescence technique to detect changes in ClC-3 in MC3T3-E1 cells under PTH treatment. The results showed higher expression of the ClC-3 chloride channel at 10−9 M intermittent PTH administration than in the other groups. Finally, we used the ClC-3 siRNA technique to examine the role of the ClC-3 chloride channel in the effect of PTH on the osteogenesis of osteoblasts, and we found an obvious decrease in the expression of bone sialoprotein (Ibsp), osteocalcin (Bglap), osterix (Sp7), Alpl and Runx2, the formation of mineralization nodules as well. From the above data, we conclude that the expression of ClC-3 chloride channels in osteoblasts helps them respond to PTH stimulation, which mediates osteogenic differentiation.