Yang Chen, Wang Yan, Xu Hui
School of Pharmaceutical Sciences, Jilin University, Changchun, P. R. China.
PLoS One. 2017 Jan 26;12(1):e0170674. doi: 10.1371/journal.pone.0170674. eCollection 2017.
This study aimed to preliminary investigate the role of activin receptor-like kinase (ALK) 5 as one of TGF-βR1 subtypes in bone turnover and osteoblastic differentiation induced by fluoride. We analyzed bone mineral density and the expression of genes related with transforming growth factor-β1(TGF-β1) signaling and bone turnover in rats treated by different concentrations of fluoride with or without SB431542 in vivo. Moreover, MTT assay, alkaline phosphatase staining, RT-PCR, immunocytochemical analysis and western blot analysis were used to detect the influence on bone marrow stem cells (BMSC) after stimulating by varying concentration of fluoride with or without SB431542 in vitro. The in vivo study showed SB431542 treatment affected bone density and gene expression of rats, which indicated TGF-β1 and ALK5 might take part in fluoride-induced bone turnover and bone formation. The in vitro study showed low concentration of fluoride improved BMSC cells viability, alkaline phosphatase activity, and osteocalcin protein expression which were inhibited by high concentration of fluoride. The gene expression of Runx2 and ALK5 in cells increased after low concentration fluoride treatment which was also inhibited by high concentration of fluoride. Fluoride treatment inhibited gene and protein expression of Samd3 (except 1 mgF-/L). Compared with fluoride treatment alone, cells differentiation was inhibited with SB431542 treatment. Moreover, the expression of Runx2, ALK5 and Smad3 were influenced by SB431542 treatment. In conclusion, this preliminary study indicated that fluoride regulated osteoblastic TGFβ1 signaling in bone turnover and cells differentiation via ALK5.
本研究旨在初步探讨激活素受体样激酶（ALK）5作为转化生长因子-β受体1（TGF-βR1）亚型之一在氟诱导的骨转换和成骨细胞分化中的作用。我们分析了体内给予不同浓度氟（加或不加SB431542）处理的大鼠的骨密度以及与转化生长因子-β1（TGF-β1）信号传导和骨转换相关基因的表达。此外，采用MTT法、碱性磷酸酶染色、逆转录-聚合酶链反应（RT-PCR）、免疫细胞化学分析和蛋白质印迹分析，检测体外给予不同浓度氟（加或不加SB431542）刺激后对骨髓干细胞（BMSC）的影响。体内研究表明，SB431542处理影响大鼠的骨密度和基因表达，这表明TGF-β1和ALK5可能参与氟诱导的骨转换和骨形成。体外研究表明，低浓度氟可提高BMSC细胞活力、碱性磷酸酶活性和骨钙素蛋白表达，而高浓度氟则抑制这些指标。低浓度氟处理后细胞中Runx2和ALK5的基因表达增加，高浓度氟也抑制这一现象。氟处理抑制了Samd3的基因和蛋白表达（1mgF⁻/L除外）。与单独氟处理相比，SB431542处理抑制细胞分化。此外，SB431542处理影响Runx2、ALK5和Smad3的表达。总之，这项初步研究表明，氟通过ALK5调节成骨细胞TGFβ1信号传导，参与骨转换和细胞分化。
