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Fluoride promotes viability and differentiation of osteoblast-like Saos-2 cells via BMP/Smads signaling pathway.

ABSTRACT: The BMP/Smad signaling pathway plays an important role in the viability and differentiation of osteoblast; however, it is not clear whether this pathway is involved in the fluoride-induced osteoblast differentiation. In this study, we investigated the role of BMP/Smad signaling pathway in fluoride-induced osteoblast-like Saos-2 cells differentiation. Cells were exposed to fluoride of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mM), and cell proliferation was determined using WST assays. The expression of osteoblast marker genes such as osteocalcin (BGP) and bone alkaline phosphatase (BALP) were detected by qRT-PCR. We found that fluoride enhanced the proliferation of Saos-2 cells in a dose-dependent manner and 0.2 mM of fluoride resulted in a higher expression of osteoblast marker genes. In addition, immunofluorescence analysis showed that the promotion effects of 0.2 mM of fluoride on Saos-2 cells differentiation were associated with the activation of the BMP/Smad pathway. Expression of phosphorylated Smad1/5(p-Smad1/5) was higher in cells exposed to 0.2 mM of fluoride. Plasmid expression vectors encoding the short hairpin RNA (shRNA) targeting Smad4 gene were used to block the BMP/Smad pathway, which resulted in a significantly reduced expression of BGP and BALP as well as their corresponding mRNA. The mRNA levels after transfection remained low even in the presence of fluoride. The present results reveal that BMP/Smad signaling pathway was altered during the period of osteogenesis, and that the activities of p-Smad1/5 were required for Saos-2 cells viability and differentiation induced by fluoride.

SUBMITTER: Huo L

PROVIDER: S-EPMC3763164 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Fluoride promotes viability and differentiation of osteoblast-like Saos-2 cells via BMP/Smads signaling pathway.

Huo Liangliang L Liu Kangkang K Pei Junrui J Yang Yanmei Y Ye Yan Y Liu Yang Y Sun Jing J Han Hepeng H Xu Weimin W Gao Yanhui Y

Biological trace element research 20130807 1

The BMP/Smad signaling pathway plays an important role in the viability and differentiation of osteoblast; however, it is not clear whether this pathway is involved in the fluoride-induced osteoblast differentiation. In this study, we investigated the role of BMP/Smad signaling pathway in fluoride-induced osteoblast-like Saos-2 cells differentiation. Cells were exposed to fluoride of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mM), and cell proliferation was determined using WST ass ...[more]

PMID: 23918166

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Project description:BackgroundBone morphogenetic proteins (BMPs) are involved in a plethora of cellular processes in embryonic development and adult tissue homeostasis. Signaling specificity is achieved by dynamic processes involving BMP receptor oligomerization and endocytosis. This allows for spatiotemporal control of Smad dependent and non-Smad pathways. In this study, we investigate the spatiotemporal regulation within the BMP-induced Smad transcriptional pathway.Methodology/principal findingsHere we discriminate between Smad signaling events that are dynamin-dependent (i.e., require an intact endocytic pathway) and dynamin-independent. Inhibition of dynamin-dependent endocytosis in fluorescence microscopy and fractionation studies revealed a delay in Smad1/5/8 phosphorylation and nuclear translocation after BMP-2 stimulation of C2C12 cells. Using whole genome microarray and qPCR analysis, we identified two classes of BMP-2 induced genes that are differentially affected by inhibition of endocytosis. Thus, BMP-2 induced gene expression of Id1, Id3, Dlx2 and Hey1 is endocytosis-dependent, whereas BMP-2 induced expression of Id2, Dlx3, Zbtb2 and Krt16 is endocytosis-independent. Furthermore, we demonstrate that short term inhibition of endocytosis interferes with osteoblast differentiation as measured by alkaline phosphatase (ALP) production and qPCR analysis of osteoblast marker gene expression.Conclusions/significanceOur study demonstrates that dynamin-dependent endocytosis is crucial for the concise spatial activation of the BMP-2 induced signaling cascade. Inhibition of endocytic processes during BMP-2 stimulation leads to altered Smad1/5/8 signaling kinetics and results in differential target gene expression. We show that interfering with the BMP-2 induced transcriptional network by endocytosis inhibition results in an attenuation of osteoblast differentiation. This implies that selective sensitivity of gene expression to endocytosis provides an additional mechanism for the cell to respond to BMP in a context specific manner. Moreover, we suggest a novel Smad dependent signal cascade induced by BMP-2, which does not require endocytosis.

Project description:Bone regeneration has always been a hot topic for orthopedic surgeons. The role of polydopamine coating in promoting bone regeneration has attracted much attention. Static magnetic field (SMF) is considered an effective and noninvasive treatment for enhancing bone regeneration. However, the effect of polydopamine combined with SMF on bone regeneration on scaffolds is not clear. The aim of this study was to investigate the effects and potential mechanism of polydopamine coating combined with SMF on bone regeneration in three-dimensional printed scaffolds. The polydopamine coating (pTi group) was applied onto porous Ti6Al4V scaffolds (Ti group). Surface characterization was performed by scanning electron microscopy. The 100 mT SMF environment (pTi-SMF group) was established to enhance osteogenic differentiation of human bone-derived mesenchymal stem cells (hBMSCs) on polydopamine coating scaffolds. The cell viability and proliferation were significantly enhanced in the SMF environment (pTi-SMF vs. Ti: P=0.005). Improved morphology (pTi-SMF vs. pTi: P=0.024, pTi-SMF vs. Ti: P=0.001) and adhesion (Ti: x̅±s=1.585±0.324; pTi: x̅±s=2.164±0.314; pTi-SMF: x̅±s=4.634±0.247, P<0.001) of hBMSCs were observed in the pTi-SMF group. The high expression of osteogenesis-related RNA and protein (ALP: Ti, x̅±s=1.249±0.218; pTi, x̅±s=2.503±0.209; pTi-SMF, x̅±s=2.810±0.246. OCN: Ti, x̅±s=1.483±0.304; pTi, x̅±s=3.636±0.322; pTi-SMF, x̅±s=4.641±0.278. Runx2: Ti, x̅±s=1.372±0.227; pTi, x̅±s=3.054±0.229; pTi-SMF, x̅±s=3.914±0.253) was found in the pTi-SMF group (pTi-SMF vs. Ti: P<0.001). Proteomics was applied to explore the osteogenic mechanism of polydopamine coating combined with SMF. A total of 147 different proteins were identified between the pTi-SMF and Ti group. The osteogenic effect might be associated with the BMP-Smads signaling pathway (pTi-SMF vs. Ti: BMPR1A, P=0.001; BMPR2, P<0.001; Smad4, P=0.001; Smad1/5/8, P=0.008). In conclusion, the osteogenic differentiation of hBMSCs on polydopamine coating scaffolds could be enhanced by SMF stimulation by upregulation of the BMP-Smads signaling pathway.

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Fluoride promotes viability and differentiation of osteoblast-like Saos-2 cells via BMP/Smads signaling pathway.

Publications

Fluoride promotes viability and differentiation of osteoblast-like Saos-2 cells via BMP/Smads signaling pathway.

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