Yu Haolan, Jiang Ningning, Yu XiuHua, Zhao Zhitao, Zhang Xiuyun, Xu Hui
Department of Regenerative Medical Science, School of Pharmaceutical Sciences, Jilin University, Changchun, 130021, People's Republic of China.
First Clinical Hospital, Jilin University, Changchun, 130021, People's Republic of China.
Toxicology. 2018 Jan 15;393:73-82. doi: 10.1016/j.tox.2017.11.009. Epub 2017 Nov 7.
Studies that have focused on the role TGFβ signaling plays in osteoclast activity are gradually increasing; however, literature is rare in terms of fluorosis. The aim of this study is to observe the role the TβR1/Smad3 pathway plays in fluoride regulating cellsosteoclast-like cells that are under the treatment of TGFβ receptor 1 kinase. The RANKL-mediated osteoclast-like cells from RAW264.7 cells were used as osteoclast precursor model. The profile of miRNA expression in fluoride-treated osteoclast-like cells exhibited 303 upregulated miRNAs, 61 downregulated miRNAs, and further drew 37 signaling pathway maps by KEGG and Biocarta pathway enrichment analysis. TGFβ and its downstream effectors were included among them. Osteoclast viability, formation and function were detected via MTT method, bone resorption pit and tartrate-resistant acid phosphatase (TRACP) staining, respectively. Results demonstrated that different doses of fluoride exhibited a biphasic effect on osteoclast cell viability, differentiation, formation and function. It indicated that a low dose of fluoride treatment stimulated them, but high dose inhibited them. SB431542 acted as TβR1 kinase inhibitor and blocked viability, formation and function of osteoclast-like cells regulated by fluoride. The expression of the osteoclast marker, RANK, and TβR1/Smad3 at gene and protein level was analyzed under fluoride with and without SB431542 treatment. Fluoride treatment indicated little effect on the RANK protein expression; however it significantly influenced TRACP expression in osteoclast-like cells. The stimulation of fluoride on the expression of Smad3 gene and phosphorylated Smad3 protein exhibited dose-dependent manner. SB431542 significantly impeded phosphorylation of Smad3 protein and TRACP expression in osteoclast-like cells that were exposed to fluoride. Our work demonstrated that TGFβ signaling played a key role in fluoride regulating osteoclast differentiation, formation and function. It elucidated that TβR1/Smad3 pathway participated in the mechanism of biphasic modulation of osteoclast mode regulated by fluoride.
关注转化生长因子β（TGFβ）信号传导在破骨细胞活性中作用的研究正在逐渐增加；然而，关于氟中毒的文献却很少。本研究的目的是观察TβR1/Smad3通路在氟调节经TGFβ受体1激酶处理的破骨样细胞中的作用。将RAW264.7细胞中RANKL介导的破骨样细胞用作破骨细胞前体模型。氟处理的破骨样细胞中miRNA表达谱显示有303个上调的miRNA、61个下调的miRNA，并通过KEGG和Biocarta通路富集分析进一步绘制了37个信号通路图。其中包括TGFβ及其下游效应分子。分别通过MTT法、骨吸收陷窝和抗酒石酸酸性磷酸酶（TRACP）染色检测破骨细胞活力、形成和功能。结果表明，不同剂量的氟对破骨细胞的活力、分化、形成和功能具有双相作用。这表明低剂量氟处理可刺激它们，而高剂量则抑制它们。SB431542作为TβR1激酶抑制剂，可阻断氟调节的破骨样细胞的活力、形成和功能。在有或无SB431542处理的氟环境下，分析破骨细胞标志物RANK以及基因和蛋白水平的TβR1/Smad3的表达。氟处理对RANK蛋白表达影响不大；然而，它显著影响破骨样细胞中TRACP的表达。氟对Smad3基因表达和磷酸化Smad3蛋白的刺激呈剂量依赖性。SB431542显著阻碍了暴露于氟的破骨样细胞中Smad3蛋白的磷酸化和TRACP的表达。我们的研究表明，TGFβ信号传导在氟调节破骨细胞分化、形成和功能中起关键作用。这阐明了TβR1/Smad3通路参与了氟对破骨细胞模式双相调节的机制。
