The role of TGFβ receptor 1-smad3 signaling in regulating the osteoclastic mode affected by fluoride

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.

Keywords: Fluoride; Osteoclast; RAW264.7 cells; Smad3; Transforming growth factor beta; Transforming growth factor beta receptor1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzamides / pharmacology
  • Cell Differentiation / drug effects
  • Cell Survival / drug effects
  • Dioxoles / pharmacology
  • Extracellular Matrix Proteins* / antagonists & inhibitors
  • Extracellular Matrix Proteins* / genetics
  • Fluorides / toxicity*
  • Gene Expression / drug effects
  • Mice
  • MicroRNAs / genetics
  • Osteoclasts / cytology
  • Osteoclasts / drug effects*
  • Osteoclasts / metabolism
  • RAW 264.7 Cells
  • Signal Transduction / drug effects
  • Smad3 Protein / genetics
  • Smad3 Protein / metabolism*
  • Stem Cells
  • Transforming Growth Factor beta* / antagonists & inhibitors
  • Transforming Growth Factor beta* / genetics


  • 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide
  • Benzamides
  • Dioxoles
  • Extracellular Matrix Proteins
  • MicroRNAs
  • Smad3 Protein
  • Smad3 protein, mouse
  • Transforming Growth Factor beta
  • betaIG-H3 protein
  • Fluorides