Bone Morphogenetic Proteins(BMPs) are members of the TGF-β protein family,BMP/Smad Signaling pathways play important roles in viability and differentiation of osteoblast.Although the function of fluoride for osteoblast differentiation and viability has previously been characterized,the changes of BMP/Smad signaling pathway in osteoblasts by fluoride remains elusive.In this study, we investigated the role of BMP/Smad signaling pathway in fluoride-induced osteoblast-like Saos-2 cells differentiation.To determine whether the observed enhancement of Saos-2 cell differentiation by fluoride was mediated by increased proliferation or survival,we incubated Saos-2 cells to different concentrations of fluoride for 24h、48h、72h at 0mg/L、2 mg/L、4 mg/L、8 mg/L、16 mg/L、32 mg/L, the cell number was measured using an WST-based assay. To determine the effect of fluoride on osteoblast differentiation, Saos-2 cells were treated with 4mg/L fluoride for 72h and used real-time reverse transcription polymerase chain reaction (qRT-PCR) to measure expression of genes involved in osteoblast differentiation, including bone gla(BGP) and alkaline phosphatase(ALP).We found that fluoride enhanced the proliferation of Saos-2 cells in a dose-dependent manner and the higher expression of osteoblast marker genes.To further study whether Smads signaling acts directly on fluoride-induced differentiation of Saos-2 cells,we performed immunofluorescence against Smad1,Smad4,Smad5 and phosphorylated Smad1/5(p-Smad1/5) on Saos-2 cells during 4mg/L fluoride for 72h. Results from immunofluorescence showed that promotion effects of fluoride on Saos-2 cells differentiation were associated with the activation of BMP/Smad signaling pathway. Expression of p-Smad1/5, as an indicator of BMP activity,was higher after stimulation of Saos-2 cells differentiation. Smad4 as the common-mediator Smads,which plays key roles in the BMP/Smad Signaling pathways.Subsequently,we construct the plasmid expression vectors encoding the short hairpin RNA(shRNA) targeting Smad4 gene to block the BMP/Smad Signaling pathway,the expression of BGP and ALP detected by qRT-PCR were significantly down transfected by Smad4-shRNA. Even after transfection,the expression of BGP and BALP were also significantly down stimulated by 4mg/L fluoride for 72h.The present results reveal that activities of the TGF-β family were changed during the period of osteogenesis in the BMP/Smad pathway-specific manner,and that the activities of p-Smad1/5 were required for Saos-2 cells differentiation stimulated by fluoride.

• Legal notice:
  

  Copyright (c) Pielaszek Research, all rights reserved.
  The above materials, including auxiliary resources, are subject to Publisher's copyright and the Author(s) intellectual rights. Without limiting Author(s) rights under respective Copyright Transfer Agreement, no part of the above documents may be reproduced without the express written permission of Pielaszek Research, the Publisher. Express permission from the Author(s) is required to use the above materials for academic purposes, such as lectures or scientific presentations.
  In every case, proper references including Author(s) name(s) and URL of this webpage: https://science24.com/paper/28132 must be provided.

1. Analysis on the monitoring results of endemic drinking water¬-born fluorosis in China (2009-2011)