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Research focused on transforming growth factor β (TGFβ) signaling in osteoblast is gradually increasing, whereas literature is rare in terms of fluorosis. This work aimed to investigate how TGFβ signaling participated in regulation of the osteoblast by different doses of fluoride treatment. Bone marrow stem cells (BMSCs) were developed into osteoblastic cells and exposed to 1, 4, and 16 mg/L F− with and without 10 ng/mL of TGFβ. Cell viability and differentiation state of osteoblast under different settings were measured by means of cell counting kit and analysis of alkaline phosphatase (ALP) activity as well as formation of mineral nodules. Real-time PCR was utilized to test expression of ALP and Runt-related transcription factor 2 (Runx2) at gene level. The gene expression of TGFβ signaling effectors was also investigated, such as TGFβ receptors (TβRs), smad3, and mitogen-activated protein kinases (MAPK). Results demonstrated that fluoride treatment exhibited action on osteoblast viability and osteogenic differentiation and upregulated expression of TβR2, smad3, and MAPK in this process. Administration of TGFβ strengthened ALP activity but attenuated formation of mineral nodules. Co-treatment of TGFβ and low-dose fluoride increased ALP activity compared to same dose of single fluoride treatment, whereas it inhibited mineral nodule formation. Administration of TGFβ reversed the suppression of high-dose fluoride on osteogenic differentiation of BMSCs. Taken together, studies revealed that TβR2 acted as a target for fluoride and TGFβ treatment on BMSCs, and smad3 and MAPK were involved in the mechanism of fluoride regulating osteogenic differentiation. Together, our data indicated that TGFβ receptor-mediated signaling through smad3 and MAPK was required for modulation of fluoride on osteoblast viability and differentiation, and activating TβR2-smad3 signaling pathway reversed suppression of osteoblasts differentiation by high dose of fluoride treatment.

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.

Overexposure to fluoride from environmental sources can cause serious public health problems. Disrupted osteoblast function and impaired bone formation were found to be associated with excessive fluoride exposure. A massive analysis of microRNAs (miRNAs) was used to figure out the possible pathways in which fluoride affects osteoblast function. MC3T3-E1 cells were treated with 8 mg/L of fluorine for 7 days. Total RNA of cells was extracted, and their integrity and purity were tested. RNA samples were analyzed by using miRNA array, including miRNA labeling, hybridization, scanning, and expression data analysis to compare the profiling of miRNA expression between control and fluoride-treated group. Transcriptome analysis console and enrichment analysis calculated by miRSystem were used to predict target genes and collect miRNAs pathway maps. Forty-five upregulated and 31 downregulated miRNAs expression were found in the fluoride-treated group, and most of the verified miRNAs were mature. The KEGG pathway enrichment analysis searched out 36 pathways that scored more than 0.1. These pathways mainly included intracellular signaling, cytokines, metabolism, and cytoskeleton-related pathways. Among them, the Wnt, insulin, TGF-beta, hedgehog, VEGF, and notch pathways in osteoblasts were those mainly affected by fluoride treatment. These results have shown a number of higher level systemic pathways activated by overexposure of fluoride in osteoblastic cells and verified that fluoride affected the molecular crosstalk in the osteoblasts.

In this paper, 3-aminobenzeneboronic acid functionalized Mn 2+ -doped ZnTe/ZnSe quantum dots (APBA-dQDs) were prepared. The APBA functional groups had strong binding ability with F – , resulting in the quenchment of dQDs photoluminescence (PL). Under the optimal condition, the fluorescence intensity of APBA-dQDs was related linearly to the concentration of F – in the range of 0.25–1.5 µmol/L with a detection limit of 0.1 µmol/L. The selectivity of fluorescence quenching of APBA-dQDs for F – was enhanced. Moreover, the proposed methodology for the sensing of F – at EM 560 nm in MC3T3-E1 osteoblastic cells was demonstrated and got a satisfactory results. The results indicate that the APBA-dQDs are promising candidates for intracellular in MC3T3-E1 osteoblastic cells. To the best of our knowledge, it was the first report of F – sensing by using the quenched fluorescence of APBA-dQDs in non-cancerous cells.

Parathyroid hormone (PTH), PTH-related peptide (PTHrP), and calcium-sensing receptor (CaSR) play important roles in maintaining calcium homeostasis. Here, we study the effect of fluoride on expression of PTH, PTHrP, and CaSR both in vitro and in vivo. MC3T3-E1 cells and Sprague-Dawley rats were treated with different concentrations of fluoride. Then, the free calcium ion concentration in cell culture supernatant and serum were measured by biochemical analyzer. The expression of PTH, PTHrP, and CaSR was analyzed by qRT-PCR and Western blot. We found that the low dose of fluoride increased ionized calcium (i[Ca(2+)]) and the high dose of fluoride decreased i[Ca(2+)] in cell culture supernatant. The low dose of fluoride inhibited the PTH and PTHrP expression in MC3T3-E1 cells. The high dose of fluoride improved the PTHrP expression in MC3T3-E1 cells. Interestingly, we found that NaF decreased serum i[Ca(2+)] in rats. Fluoride increased CaSR expression at both messenger RNA (mRNA) and protein levels in MC3T3-E1 cells and rats. The expression of PTHrP protein was inhibited by fluoride in rats fed regular diet and was increased by fluoride in rats fed low-calcium diet. Fluoride also increased the expression of PTH, NF-kappaB ligand (RANKL), and osteoprotegerin (OPG) in rats. The ratio of RANKL/OPG in rats fed low-calcium food in presence or absence of fluoride was significantly increased. These results indicated that fluoride might be able to affect calcium homeostasis by regulating PTH, PTHrP, and CaSR.

Exposure to excessive fluoride poses a threat to human health, including increased susceptibility to developing the skeletal fluorosis. Despite its recognized importance as an endemic disease, little is known about how fluoride directly impacts on osteoblasts. We previously reported that fluoride-stimulating monolayer-cultured osteoblast proliferation or inhibiting cell viability depended on fluoride-exposure concentration and period, both accompanied with active oxidative stress. The purpose of this study was to provide extra insight into skeletal fluorosis by comparing their regulation of oxidative stress in rats and OS732 cells (a human osteoblast-like cell line) cultured in the three-dimensional approach. Our in vivo and in vitro studies proved that exposure to fluoride promoted varying extents of oxidative stress. Three-dimensional cultured OS732 cells revealed the action of fluoride on cell viability from excitatory to inhibitory trend according to fluoride-exposure concentration and time. The study provided insight into the mechanism of skeletal fluorosis. Also, this study distinguished itself by identifying oxidative stress as a potential modulator of osteogenesis in skeletal fluorosis.

Osteoblast L-type voltage-dependent calcium channels (VDCC) play important roles in maintaining intracellular homeostasis and influencing multiple cellular processes. In particular, they contribute to the activities and functions of osteoblasts (OBs). In order to study how L-type VDCC modulate calcium ion (Ca(2+)) homeostasis and the expression of osteogenic transcription factors in OBs exposed to fluoride, MC3T3-E1 cells were exposed to a gradient of concentrations of fluoride (0, 2.0, 5.0, 10.0 mg/L) in combination with 10 μM nifedipine, a specific inhibitor of VDCC, for 48 h. We examined messenger RNA (mRNA) and protein levels of Cav1.2, the main subunit of VDCC, and c-fos, c-jun, runt-related transcription factor 2 (Runx2), osterix (OSX), and intracellular free Ca(2+) ([Ca(2+)]i) concentrations in MC3T3-E1 cells. Our results showed that [Ca(2+)]i levels increased in a dose-dependent manner with increase in concentration of fluoride. Meantime, results indicated that lower concentrations of fluoride (less than 5 mg/L, especially 2 mg/L) can lead to high expression of Cav1.2 and enhance osteogenic function, while high concentration of fluoride (10 mg/L) can induce decreased Cav1.2 and osteogenic transcriptional factors in MC3T3E1 cells exposed to fluoride. However, the levels of [Ca(2+)]i, Cav1.2, c-fos, c-jun, Runx2, and OSX induced by fluoride were significantly altered and even reversed in the presence of nifedipine. These results demonstrate that L-type calcium channels play a crucial role in Ca(2+) homeostasis and they affect the expression of osteogenic transcription factors in fluoride-treated osteoblasts.

We investigated the relationship between oxidative stress and osteoblasts viability in osteoblasts exposed to various concentrations of fluoride in this study. Primary calvarial osteoblasts from neonatal Kunming mice were cultured and subcultured to the third generation. Osteoblasts were incubated with sodium fluoride (0, 0.5, 1, 2, 4, 8, 12, and 20 mgF(-)/L) for 24, 48, and 72 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis showed cell viability significantly increased after osteoblasts exposed to low concentrations of fluoride (0.5 to approximately 2 mgF(-)/L) for 24 to approximately 72 h. Oxidative stress analysis showed that low concentration of fluoride excited lipid peroxidation in osteoblasts and increased activity of antioxidant enzymes in varying degrees. We demonstrated that changes of osteoblasts viability of the low-dose fluoride groups are different from those of high-dose fluoride groups; however, both low and high doses of fluoride caused active state of oxidative stress in osteoblasts, which suggesting that oxidative stress may be excited by the active osteoblasts viability induced by a low dose of fluoride.