β-catenin signaling pathway

Toxicology Letters 225 (2014) 34–42

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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

Fluoride promotes osteoblastic differentiation through canonical Wnt/␤-catenin signaling pathway Leilei Pan a,1 , Xiaoguang Shi b,1 , Shuang Liu a , Xiaoying Guo a,∗ , Ming Zhao a , Ruoxin Cai a , Guifan Sun a a Department of Occupational and Environmental Health, Liaoning Provincial Key Laboratory of Arsenic Biological Effect and Poisoning, School of Public Health, China Medical University, Shenyang, People’s Republic of China b Department of Endocrinology and Metabolism, First Affiliated Hospital, China Medical University, Shenyang, People’s Republic of China

h i g h l i g h t s • Fluoride promoted osteoblastic proliferation and differentiation in primary cultured rat osteoblasts. • Fluoride promoted Akt and GSK3␤ phosphorylation and activated the canonical Wnt/␤-catenin signaling pathway in osteoblast. • Wnt/␤-catenin signaling pathway was involved in fluoride-induced osteoblastic differentiation.

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Article history: Received 22 September 2013 Received in revised form 21 November 2013 Accepted 22 November 2013 Available online 1 December 2013 Keywords: Fluoride Osteoblastic differentiation Canonical Wnt/␤-catenin signaling

a b s t r a c t Although fluoride is known to stimulate bone formation, the underlying mechanisms are not fully understood. Recent studies have implicated the Wnt/␤-catenin pathway as a major signaling cascade in bone biology. Our earlier studies highlighted a probable role of canonical Wnt pathway in bone formation of chronic fluoride-exposed rats, but the mechanism remains unclear. The current study determined the involvement of Wnt/␤-catenin signaling in fluoride-induced osteoblastic differentiation. Using primary rat osteoblasts, we demonstrated that fluoride significantly promoted osteoblasts proliferation and alkaline phosphate (ALP) expression as well as the mRNA expression levels of bone differentiation markers, including type I collagen (COL1A1), ALP and osteonectin. We further found fluoride induced phosphorylations at serine 473 of Akt and serine 9 of glycogen synthase kinase-3␤ (GSK3␤), which resulted in GSK-3␤ inhibition and subsequently the nuclear accumulation of the ␤-catenin, as shown by Western blot and immunofluorescence analysis. Moreover, fluoride also induced the expression of Wnt-targeted gene runt-related transcription factor 2 (Runx2). Importantly, the positive effect of fluoride on ALP activity and mRNA expressions of COL1A1, ALP, osteonection and Runx2 was abolished by DKK-1, a blocker of the Wnt/␤-catenin receptor. Taken together, these findings suggest that fluoride promotes osteoblastic differentiation through Akt- and GSK-3␤-dependent activation of Wnt/␤-catenin signaling pathway in primary rat osteoblasts. Our findings provide novel insights into the mechanisms of action of fluoride in osteoblastogenesis. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction It has been reported that fluoride has anabolic effect on bone metabolism and stimulates osteoblastic bone formation in vitro and in vivo (Grynpas et al., 2000). Bone histomorphometric studies have indicated that the effect of fluoride to increase bone mass was due entirely to an increase in bone formation, and that the

∗ Corresponding author at: Department of Occupational and Environmental Health, School of Public Health, China Medical University, 92 North 2nd Road, Heping District, Shenyang 110001, People’s Republic of China. Tel.: +86 24 23256666x5405. E-mail address: [email protected] (X. Guo). 1 These authors contributed equally to this work. 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.11.029

stimulation of bone formation was mediated through an increase in the osteoblast number (Briancon and Meunier, 1981; Harrison et al., 1981). In several in vitro models, fluoride was shown to enhance both the growth and mineralization of osteoblast (Farley et al., 1983; Wergedal et al., 1988; Qu et al., 2008). There is evidence that the anabolic action of fluoride is exerted partly through MAPK mitogenic signal pathway in osteoblasts (Lau and Baylink, 2003). However, the precise molecular mechanisms underlying the observed anabolic effects of fluoride on bone cells are still under investigation. Recently, the role of Wnt signaling pathway in bone formation has been elucidated, and it appears as an important signaling cascade for osteoblast differentiation, bone formation and homeostasis (Baron and Kneissel, 2013). In the canonical Wnt signaling

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pathway, binding of Wnt ligand to its transmembrane Frizzled (Fzd) receptor and the co-receptors low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) results in the destruction of a complex that consist of glycogen synthase kinase-3␤ (GSK-3␤), axin and adenomatous polyposis coli (APC) (Mao et al., 2001). The destruction of this complex leads to the stabilization and nuclear translocation of active dephosphorylated ␤-catenin, which, in turn, activates the lymphoid enhancer factor-1 (LEF)/T cell factors (TCFs) to induce the transcription of key osteoblastic genes that are Wnt targets (Bennett et al., 2005; Gaur et al., 2005). The importance of Wnt signaling in osteogenesis has been widely reported in several in vitro and in vivo studies. Gain-of-function mutations of the LRP5 gene in mice exhibited an enhanced osteoblastic activity, a reduction in osteoblast apoptosis, and high bone mass phenotype (Boyden et al., 2002; Little et al., 2002; Van Wesenbeeck et al., 2003). Conversely, a family carrying a loss-of-function mutations in this gene was associated with low bone mass, which is secondary to decreased osteoblast proliferation and function, and cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG) (Gong et al., 2001). These findings indicate that Wnt/␤-catenin signaling pathways are important in bone formation. Recently, we have demonstrated that fluoride induced ␤catenin mRNA and protein expression in the bones of rats, which suggested that the Wnt/␤-catenin signaling may be involved in the fluoride-induced bone formation (Guo et al., 2011). However, the precise mechanisms between fluoride and the Wnt/␤-catenin signaling pathway, which is associated bone cell proliferation and differentiation, have been unclear at least in the case of osteogenic process. Thus in the present study, to settle this issue, we examine whether fluoride activates Wnt signaling in primary rat osteoblasts and if Wnt/␤-catenin pathway is required during fluoride-induced osteoblastic differentiation. 2. Materials and methods 2.1. Chemicals and reagents Sodium fluoride was purchased from Wako Pure Chemical Industries (Osaka, Japan). ␣-Modified minimal essential medium (␣-MEM, Gibco, Life Technologies, USA), penicillin-streptomycin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tritron X-100, SDS, methanol, and dimethylsulfoxide (DMSO) were purchased from Sigma–Aldrich. Fetal bovine serum (FBS) was purchased from HyClone (Thermo Scientific, Logan, UT, USA). Recombinant human Dickkopf-related protein 1 (DKK-1) was purchased from R&D systems (Minneapolis, MN). ALP staining kit and the bicinchoninic acid (BCA) protein assay kit was obtained from Beyotime Institute of Biotechnology (Shanghai, China). RNAiso, PrimeScript® RT reagent kit and SYBR® Premix Ex TaqTM II were obtained from the Takara Biotechnology Company (Dalian, China). The enhanced chemiluminescence (ECL) kit was purchased from Thermo scientific (Billerica, MA, USA). Antibodies were obtained as follows: GSK-3␤ and phospho-GSK-3␤ (Ser9) monoclonal antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-active-␤-catenin antibody (clone 8E7) was purchased from Millipore (Billerica, MA). Anti-Akt, antiphospho-Akt (Ser473 ), Runx2, ␤-actin antibodies and secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, TX, USA). All other chemicals used were of analytical grade. 2.2. Cell culture Primary rat osteoblasts were isolated from the calvarias of 1day-old Sprague-Dawley rats by sequential enzymatic digestion,

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as described previously (Hefley et al., 1981). In brief, the calvarias, consisting of frontal and parietal bones, were dissected aseptically, free of sutures, and subjected to consecutive digestions at 37 ◦ C in Hefley’s buffer containing 2 mg/ml collagenase and 0.25% trypsin. Cells and debris released during the first two, 20-min digestions, were discarded and cells obtained from the third, 60-min digestions were plated in ␣-MEM supplemented with 10% FBS, 2 mM l-glutamine, 1% penicillin/streptomycin in a humidified 5% CO2 incubator at 37 ◦ C. Cells were grown for several days until reaching ∼90% confluence, and were then trypsinized and plated in the same medium for subsequent experimental procedures. For all experiments primary osteoblasts used were between 2th and 5th passage. 2.3. Cell proliferation Effect of fluoride on cell proliferation was measured by MTT assay. Briefly, osteoblasts were seeded in 96-well plates at a density of 1 × 103 cells per well. After 24 h incubation, the cells were treated with fluoride at various concentrations for 72 h. Then 50 ␮l MTT (5 mg/mL) was added to each wells and the mixture was incubated for 4 h at 37 ◦ C. The mixture was replaced with 150 ␮l dimethyl sulfoxide (DMSO) to dissolve formazan crystals. After shaking for 10 min, absorbance was measured on SpectraMax Plus 384 microplate reader (Molecular Devices Co., Sunnyvale, CA, USA) at a test wavelength of 570 nm and a reference wavelength of 630 nm. Cell viability was calculated as a percentage of viable cells in fluoride-treated group vs. untreated control by following equation. Cell viability (%) = [OD (fluoride) − OD (blank)]/[OD (control) − OD (blank)] × 100. 2.4. Alkaline phosphatase (ALP) staining assay For the ALP staining assay, osteoblasts were seeded in 24-well plates and treated with various concentrations of fluoride for 7 days. After treatments, cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and washed three times with deionized water. Cells were incubated in a mixture of nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate for 1 h, washed with deionized water, and observed under a light microscope. The resulting blue, insoluble, granular dye deposit indicated sites of ALP activity. Stained cells were photographed with Zeiss AxioCam digital camera. 2.5. RNA extraction and real-time quantitative RT-PCR Total RNAs were isolated from osteoblasts using RNAisoTM Plus (TakaRa, Dalian, China). Five microgram of total RNA from each sample was reverse-transcribed into single-stranded cDNA using PrimeScript® RT reagent kit (TakaRa, Dalian, China). Quantitative real-time PCRs of ALP, COL1A1, osteonectin, ␤-catenin, Runx2 and GAPDH were performed on an equal amount of cDNA using SYBR® Premix Ex TaqTM II according to the instructions of the manufacturer (TakaRa, Dalian, China). The primers used were as follows: ALP (Forward: 5 -TGA TCA CTC CCA CGT TTT CA-3 and Reverse 5 -GCT GTG AAG GGC TTC TTG TC-3 ; NM 013059), COL1A1 (Forward: 5 -TCC TGC CGA TGT CGC TAT C-3 and Reverse 5 -CAA GTT CCG GTG TGA CTC GT-3 ; NM 053304), osteonectin (Forward: 5 GAA GAG ATG GTG GCG GAG-3 and Reverse 5 -ACA GGC AGG GGG CAA TGT ATT TG-3 ; NM 012656), ␤-catenin (Forward: 5 -GCC AGT GGA TTC CGT ACT GT-3 and Reverse 5 -GAG CTT GCT TTC CTG ATT GC-3 ; NM 053357), Runx2 (Forward: 5 -TAA CGG TCT TCA CAA ATC CTC-3 and Reverse 5 -GGC GGT CAG AGA ACA AAC TA-3 ; NM 001146038.1) and GAPDH (Forward: 5 -ATG GCC TTC CGT GTT CCT AC-3 and Reverse 5 -CAC CTT CTT GAT GTC ATC ATA CTT G-3 ; NM 017008). Reactions were performed in triplicate on ABI PRISM

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7500 Real-Time PCR System (Applied Biosystems) using the following thermal conditions: 95 ◦ C for 30 s, followed by 40 cycles of 95 ◦ C for 5 s, 60 ◦ C for 30 s, and 72 ◦ C for 30 s, and a dissociation program of 95 ◦ C for 15 s, 60 ◦ C for 30 s, and 95 ◦ C for 15 s. GAPDH was amplified as an internal control. Melting curve analysis was included to assure that only one PCR product was formed. The relative amount of RNA was calculated by the 2−Ct method. 2.6. Western blot analysis After the treatment, the osteoblasts were rinsed with ice-cold PBS and lysed in 100 ␮l lysis buffer (150 mM NaCl, 50 mM Tris–HCl, 1% NP-40, 0.5% SDS, pH 7.4). The lysates were cleared by centrifugation at 20,000 × g for 5 min at 4 ◦ C. The supernatants were collected and the protein concentration was measured. Protein was subjected to electrophoresis in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane using a semi-dry transfer system (Trans-Blot SD Semi-Dry Transfer Cell, Bio-Rad Laboratories, Hercules, CA, USA). Then the membranes were blocked in 5% BSA in PBST buffer for 2 h at room temperature and incubated with appropriate primary antibodies in 5% BSA overnight at 4 ◦ C. The primary antibodies used include anti-phospho-GSK3␤ (Ser-9) (1:1000), anti-GSK3␤ (1:1000), anti-phospho-Akt (Ser-473) (1:1000), anti-Akt (1:1000), anti-active-␤-catenin (1:500), antiRunx2 (1:200) and ␤-actin (1:4000) antibody. After three times washing with PBST for 3 min, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) in 1% BSA for 90 min at room temperature. After same washing steps, the signal was visualized by the enhanced chemiluminescence (ECL) and exposed to X-ray film. Optical densitometric scans of the membranes were performed using ImageJ software (NIH, Bethesda, MD, USA). Relative levels are expressed as a ratio of treated over control, after correction to the housekeeping protein. The results depicted in each figure are representative of at least three separate cell preparations. Each experiment was repeated three times. 2.7. Immunofluorescence staining for ˇ-catenin nuclear translocation Primary rat osteoblasts were grown on glass coverslips and incubated with fluoride for 72 h. The cells were washed with cold PBS twice, fixed in ice-cold methanol and permeabilized with 0.5% Triton X-100 for 15 min. Cells were then blocked with 0.5% bovine serum albumin for 1 hr at room temperature. Samples were then incubated with mouse monoclonal anti-dephosphorylated ␤-catenin antibody (1:500) overnight at 4 ◦ C followed by incubation with TRITC-conjugated secondary antibody and DAPI. The signal was visualized by confocal fluorescence microscopy (Olympus, Japan). 2.8. Statistical analysis Results were expressed as means ± standard error (S.E.). Data were analyzed by one-way analysis of variance (ANOVA), using SPSS software ver. 10.0. Significant changes were classified as *p < 0.05 and **p < 0.01. Each experiment was performed at least three times, and the representative graphs are shown. 3. Results 3.1. Fluoride promoted osteoblasts proliferation and differentiation To examine the effects of fluoride on cell proliferation, primary osteoblasts were treated with fluoride (10−8 –10−3 M) for 72 h. MTT

assay showed that fluoride (10−6 –10−5 M) significantly stimulate the proliferation of primary osteoblasts (p < 0.05) (Fig. 1A). Then the effect of fluoride on alkaline phosphatase (ALP) expression, an early marker of osteoblast differentiation, was observed. As shown in Fig. 1B, 10−7 –10−4 M fluoride significantly induced the expression of ALP in osteoblasts on day 7 compared with untreated control. The fluoride-induced osteoblast differentiation was further confirmed by its activation on the mRNA levels of bone differentiation marker genes, including ALP, type I collagen (COL1A1) and osteonectin as determined by quantitative real-time RT-PCR (qRT-PCR). The cultures treated with fluoride demonstrated higher gene expression compared to the control cultures (Fig. 1C). These results suggested that fluoride promotes osteoblasts proliferation and differentiation in vitro.

3.2. Fluoride promoted Akt and GSK3ˇ phosphorylation and activated the canonical Wnt/ˇ-catenin signaling pathway in osteoblast Activation of the canonical Wnt signaling pathway is known to dephosphorylation and nuclear translocation of ␤-catenin. To evaluate whether fluoride activates the Wnt signaling pathway, we initially analyzed the effect of fluoride on the levels of ␤-catenin mRNA and protein in osteoblasts by qRT-PCR and Western blot. Fluoride (10−7 –10−5 M) induced a significant increase in the ␤catenin mRNA level, which peaked at 10−6 M (Fig. 2A). As shown in Fig. 2, fluoride increased ␤-catenin protein level after treatment for 72 h (Fig. 2B) and at concentration from 10−7 to 10−6 M, with a maximum increase with the 10−7 M dose, as compared with control (Fig. 2C). The effect of fluoride on the nuclear translocation of ␤-catenin was further confirmed by immunofluorescence assay in primary osteoblasts. Immunofluorescence analysis showed that fluoride significantly increased ␤-catenin translocation into nucleus in osteoblasts (Fig. 2D). ␤-Catenin mainly existed in the cytoplasm in control cells, but after treatment with 10−7 M fluoride for 72 h, the staining was more intense and localized in the nuclear compared with control, representing activated Wnt/␤-catenin signaling. These findings demonstrate that fluoride activates Wnt/␤-catenin signaling by promoting nuclear localization of ␤-catenin protein through suppression of degradation of the protein as well as by induction of RNA synthesis. Because GSK3␤ phosphorylates ␤-catenin and prevent LEF/TCFactivated transcription activity, we therefore next investigated whether the activation of Wnt/␤-catenin signaling mediated by fluoride involves GSK3␤ phosphorylation in osteoblasts. Osteoblasts were treated with 10−7 M fluoride for 24–72 h, lysed for immunoblotting using anti-phospho-GSK3␤ (Ser-9) antibodies. As shown in Fig. 3A, fluoride enhanced the phosphorylation levels of GSK3␤ Ser9 in a time-dependent manner, and the maximum increase was at 72 h. These data suggest that the activation of Wnt/␤-catenin signaling induced by fluoride is dependent on GSK3␤ activity. To test whether fluoride-mediated GSK3␤dependent activation of Wnt/␤-catenin signaling proceeds through the activation of the PI3K/Akt pathway, osteoblasts were treated with 10−7 M fluoride for 24–72 h, lysed for immunoblotting using anti-phospho-Akt (Ser-473) antibodies. As shown in Fig. 3B, fluoride enhanced the phosphorylation levels of Akt ser473 in a time-dependent manner, and the maximum increase was also at 72 h. These findings demonstrate an association between fluorideinduced Akt ser473 phosphorylation and GSK3␤ activity, which may be an important component of canonical Wnt signaling activation in osteoblasts. To test whether Dickkopf-1 (DKK-1), a potent Wnt antagonist, has any effect on fluoride-induced GSK3␤ phosphorylation and ␤catenin, the osteoblasts were pretreated with 0.5 ␮g/ml DKK-1 for

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Fig. 1. Fluoride enhanced cell proliferation and differentiation in rat primary osteoblasts. (A) Effect of fluoride on the cell proliferation of primary rat osteoblasts. Osteoblasts were seeded onto 96-well plate at a density of 1 × 103 cells/well and incubated with various concentrations of fluoride (10−8 –10−3 M) for 72 h. The proliferation of osteoblasts was determined by MTT assay as described in Section 2. Each value is the mean ± S.E. of three independent experiments. (B) Effect of fluoride on ALP expression of primary rat osteoblasts. Confluent osteoblasts were cultured with various concentrations of fluoride (10−7 –10−4 M) for 7 d. Osteoblast was stained with Naphthol AS-MX phosphate to examine the expression of ALP (magnification 10×). Scale bar, 400 ␮m. (C) Effects of different concentrations of fluoride on the mRNA expression of ALP, COL1A1 and OCN in primary rat osteoblasts. Osteoblasts were treated with fluoride (10−7 –10−4 M) or without fluoride for 3 days. Total RNAs were extracted from cultures to perform quantitative RT-PCR for bone differentiation markers: ALP, COL1A1 and osteonectin. GAPDH served as an internal control. Values are expressed as the fold of increase to control and are means ± S.E. of three independent experiments, each with triplicate samples. *p < 0.05 or **p < 0.01 vs. control value.

1 h followed by exposure to 10−7 M fluoride for a further 72 h. The phospho-GSK3␤ and ␤-catenin, induced by fluoride, was markedly inhibited by DKK1 as shown in Fig. 4, indicating that fluoride could act on the Wnt receptor.

fluoride activated Wnt/␤-catenin signaling, and stimulated downstream gene expression of ␤-catenin such as Runx2 in primary rat osteoblasts. Collectively, our results strongly suggest that fluoride increases osteoblastic differentiation through the activation of the Wnt/␤-catenin signaling pathway.

3.3. Fluoride induced osteoblastic differentiation via canonical Wnt/ˇ-catenin signaling and the expression of Wnt target genes

4. Discussion

Having shown that fluoride activates Wnt/␤-catenin signaling pathway in osteoblasts, we then investigated whether this effect may have significant impact on osteoblast differentiation. The cultured osteoblasts were pretreated with DKK-1 for 1 h before treatment with 10−7 M of fluoride. Indeed, pretreatment of cells with DKK-1 effectively decreased the fluoride-stimulated ALP activity (Fig. 5A) and the mRNA levels of osteogenic transcription factors to the control levels (Fig. 5B). This specific blockage further confirmed the fluoride-activated Wnt/␤-catenin signaling was via the receptor. These results indicate that activation of Wnt/␤catenin signaling is involved in osteoblastic differentiation induced by fluoride. We next examined the effect of fluoride on Runx2 expression, a known target gene of ␤-catenin, in osteoblasts. Osteoblasts were treated with 10−7 –10−4 M fluoride for 72 h, and qRT-PCR was performed to determine Runx2 mRNA expression. As shown in Fig. 6A, fluoride (10−7 and 10−6 M) significantly up-regulated Runx2 mRNA expression levels. In addition, as shown in Fig. 6B, 10−6 M fluoride increased the protein expression level of Runx2 (Fig. 6B). As expected, this induction was markedly blocked by the pretreatment of DKK-1 (Fig. 6C). These results indicate that

Consistent with previous studies (Farley et al., 1983; Wergedal et al., 1988; Qu et al., 2008), our results showed that fluoride at concentration ranging from 0.1 to 100 ␮M significantly enhanced both the proliferation and differentiation of primary rat osteoblasts. We found fluoride promoted osteoblast differentiation by increasing the activity of ALP and the expressions of osteoblastic differentiation makers such as ALP, COL1A1, osteonectin and Runx2 in rat primary osteoblasts. By stimulating osteoblast growth and differentiation, fluoride promotes bone formation and increase trabecular bone mineral density (BMD) (Riggs et al., 1990). However, the precise molecular mechanisms by which fluoride exerts its anabolic action on bone are incompletely understood. Wnt/␤-catenin signaling pathway has currently been recognized as an important regulator of bone mass and osteoblast differentiation, which provides a rationale for its investigation in action of fluoride. Our previous study has shown increased expressions of Wnt signaling pathway-related genes such as ␤-catenin in chronic fluoride-treated rats bones (Guo et al., 2010). Wang and Liu et al. have recently reported that decreased Sclerostin and Dickkopf-1 levels, the potent antagonists of Wnt/␤-catenin signaling pathway, were found in serum of fluorosis patients and

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Fig. 2. Fluoride activated Wnt/␤-catenin signaling pathway in primary rat osteoblast. (A) Effect of fluoride on ␤-catenin mRNA expression in osteoblasts. Osteoblasts were incubated with and without fluoride for 72 h. Total RNAs were extracted from cultured osteoblasts to perform real-time RT-PCR to determine the expression of ␤-catenin mRNA. The results shown are the mean ± S.E. from three separate experiments. (B) Representative blots showing fluoride increases ␤-catenin protein expression in osteoblasts. Osteoblasts were treated with fluoride (10−7 M) for the indicated times (24–72 h) and harvested for immunoblotting using anti-␤-catenin and anti-␤-actin antibodies. The lower panel denotes the mean ± S.E. of three experiments for each condition determined from densitometry relative to ␤-actin. **p < 0.01 vs. control value. (C) Representative blots showing effects of different concentration of fluoride on ␤-catenin protein expression. Osteoblasts were treated with various concentrations of fluoride (10−7 –10−4 M) for 72 h and harvested for immunoblotting using anti-␤-catenin and anti-␤-actin antibodies. Data are represented as mean ± S.E. (n = 3). *p < 0.05 and **p < 0.01 vs. control values. (D) Fluoride induces ␤-catenin nuclear translocation in osteoblasts. Osteoblasts were treated with 10−7 M fluoride for 72 h, followed by the immunofluorescence staining for ␤-catenin (red) using anti-dephosphorylated ␤-catenin. The nuclei were stained with DAPI (blue). A representative result from the three independent experiments is shown. ␤-Catenin immunostaining was predominantly localized in the nucleus after treatments with fluoride. Arrows indicate nuclear localization of ␤-catenin (violet) (Magnification 40×). Scale bar, 50 ␮m.

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Fig. 3. Activation of Wnt/␤-catenin signaling by fluoride is GSK-3␤ and Akt-dependent in osteoblasts. (A) Fluoride increased the phosphorylation of GSK3␤ at Ser9 in a time-dependent manner. Fluoride (10−7 M) was applied onto cultured osteoblasts for different times as indicated. Total GSK3␤ and its phosphorylated form (p-GSK3␤) were revealed by specific antibodies in a Western blot analysis (upper panel). The intensities of the protein bands were quantified and calculated as percentages of the control (lower panel). (B) Fluoride increased the phosphorylation of Akt at Ser473 in a time-dependent manner. Fluoride (10−7 M) was applied onto cultured osteoblasts for different times as indicated. Total Akt and its phosphorylated form (p-Akt) were revealed by specific antibodies in a Western blot analysis (upper panel). The quantification from the blots was shown by a densitometer (lower panel). Values are expressed as the fold of increase to control culture. *p < 0.05 and **p < 0.01 vs. control cells. Data shown are representative of three separate experiments.

fibroblasts exposed to fluoride (Wang et al., 2013; Liu et al., 2012). These findings, therefore, provided evidence that Wnt signaling was involved in the bone anabolic effect of fluoride. However, the relationship between fluoride and Wnt/␤-catenin activation has not been broadly identified. This leads us to investigate the mechanism underlying this regulation using cultured osteoblasts derived from newborn rat calvaria. Here we demonstrated that, indeed, fluoride activated the Wnt/␤-catenin signaling pathway in osteoblasts. By Western blot and immunofluorescence analysis, we found fluoride increased

the expression of ␤-catenin protein, which was predominantly localized in the nucleus. A key negative regulator of ␤-catenin in the canonical Wnt signaling pathway is the serine/threonine protein kinase GSK3␤, which phosphorylates and promotes the degradation of ␤-catenin in quiescent cells (Wu and Pan, 2010). It has been shown that phosphorylation of GSK-3␤ leads to inhibition of GSK-3␤ thus resulting in the stimulation of the Wnt pathway (Mao et al., 2001). Furthermore, GSK3␤ is a downstream component of the PI3K/Akt signaling pathway. The phosphorylation of Akt at serine 473 residues results in its activation and, therefore,

Fig. 4. Inhibitory effect of DKK-1 on fluoride-induced phosphorylation of GSK3␤ and ␤-catenin in primary cultured osteoblasts. Osteoblasts were pretreated with 0.5 ␮g/ml DKK-1 for 1 h and incubated with or without fluoride (10−7 M) in the presence or absence of the DKK1. GSK3␤ or its phosphorylated form (p-GSK3␤), ␤-catenin and ␤-actin were revealed by specific antibodies by Western blot (left panel). The quantification from the blots was shown by a densitometer (right panel). **p < 0.01 vs. control cells. ## p < 0.01 vs. fluoride-treated cells.

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Fig. 5. DKK-1 blocks fluoride-induced osteoblastic differentiation. Osteoblasts were pretreated with 0.5 ␮g/ml DKK-1 for 1 h and incubated with or without fluoride (10−7 M) in the presence or absence of the DKK1. (A) Inhibition by DKK-1 of fluoride-mediated increase of ALP staining in osteoblasts (magnification 10×). ALP staining was determined after the application of fluoride (10−7 M), onto cultured osteoblasts for 7 days, with or without the pretreatment of DKK-1 (0.5 ␮g/ml), as indicated. (B) mRNA levels of bone differentiation markers: ALP, COL1A1 and osteonectin, were also measured by real-time RT-PCR. Values are expressed as the fold of increase to control and are means ± S.E., where n = 3. **p < 0.01 vs. control cells; ## p < 0.01 vs. fluoride-treated cells.

promotes GSK3␤ phosphorylation at serine 9 thereby inhibiting GSK3␤ activity and activating the canonical Wnt signaling (Fukumoto et al., 2001). In this study, we found fluoride slightly increased the phosphorylation level of GSK-3␤ on serine 9 after treatment with fluoride for 48 h. However, marked stimulation of GSK-3␤ phosphorylation was observed at 72 h. In addition, we also found the time course of Akt phosphorylation at serine 473 coincided with phosphorylation of GSK-3␤, suggesting that PI3K/Akt signaling is also involved. These observations indicate that fluoride induced the phosphorylation of Akt at serine 473 and subsequently activated the phosphorylation of GSK-3␤ at serine 9, leading to inhibition of GSK-3␤ activity and activation of Wnt/␤-catenin signaling. Further experiments are required to completely understand the precise mechanisms through which activation of Wnt/␤-catenin is mediated by fluoride. Having shown that fluoride activates Wnt/␤-catenin signaling in osteoblasts, we investigated the functional implication of Wnt/␤-catenin signaling induced by fluoride in osteoblasts. Several molecules, including the DKK family negatively regulate canonical Wnt signaling (Morvan et al., 2006) and DKK-1 is a powerful antagonist of canonical Wnt/␤-catenin signaling. DKK-1 functions via direct binding to LPR5/6, and as such it prevents the binding of Wnt ligands to its receptors for signal induction (Brott and Sokol, 2002; Morvan et al., 2006). The present finding that the increased osteoblastic differentiation induced by fluoride was blunted by DKK1 indicates that this effect is mediated by activation of the canonical Wnt signaling pathway. This mechanism is consistent with the recent finding that canonical Wnt/␤-catenin signaling contributes to the promotion of osteoblastic differentiation in vivo (Tamura et al., 2010). Previous studies have shown that fluoride

acts on osteoprogenitor cells and osteoblasts to favor osteoblastogenesis in part via activation of MAPK or BMP/Smads multiple signaling pathways (Lau and Baylink, 2003; Huo et al., 2013). The present data provide another mechanism by which fluoride promotes osteoblastic differentiation via Wnt/␤-catenin signaling. In addition to this effect, our data reveal that fluoride increased Runx2 and other osteoblast phenotypic genes, including ALP, type I collagen (COL1A1) and osteonectin, which control osteoblast differentiation via activation of Wnt/␤-catenin signaling. This indicates that activation of Wnt/␤-catenin signaling is implicated in the increased osteoblast gene expression induced by fluoride. These results therefore identify Wnt/␤-catenin signaling as an important mechanism that is involved in fluoride-induced osteoblastic differentiation in vitro. Recently, several molecules have been shown to exert a bone anabolic effect in part via canonical Wnt signaling (Cheng et al., 2008; Wan et al., 2008). The present data reveal that fluoride is another agent that may exert bone anabolism in part by enhancing Wnt/␤-catenin signaling pathway. Runx2 is the main transcription factor that plays an essential role in osteoblast differentiation and bone formation. The molecular mechanism of Runx2 has been demonstrated to directly stimulate the transcription of osteoblast-related genes such as osteocalcin (OCN), osteopontin and type I collagen by binding to specific enhancer regions (Komori, 2010; Prince et al., 2001). Several significant observations show that Runx2 gene is a direct target of the canonical Wnt signaling pathway for the stimulation of bone formation (Gaur et al., 2005; Day et al., 2005). Previous study reported that the anabolic effects of fluoride on bone formation were attributed to the increased expression of Runx2 (Zhou et al.,

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Fig. 6. Fluoride induces the expression of Runx2 in cultured osteoblasts. (A) Fluoride at different concentrations (10−7 –10−4 M) was applied onto cultured osteoblasts for 72 h, total RNAs were extracted from the cultures to perform quantitative RT-PCR for the expression of Runx2 mRNAs. The mRNA levels were calculated as percentages of the control level. Data are expressed as mean ± S.E. of three independent experiments. *p < 0.05; **p < 0.01 vs. control cells. (B) Effect of fluoride on Runx2 protein levels. Cultured osteoblasts were treated with fluoride (10−7 M and 10−6 M) for 48 h. The expressions of Runx2 were revealed by specific antibodies in a Western blot analysis (upper panel). The quantification from the blots was shown by a densitometer (lower panel). (C) DKK-1 blocks fluoride-induced Runx2 expression. Cultured osteoblasts were pretreated with 0.5 ␮g/ml DKK-1 for 1 h and incubated with or without fluoride (10−7 M) for 48 h, in the presence or absence of the DKK1, as indicated. The expressions of Runx2 were revealed by specific antibodies in a Western blot analysis (left panel). The quantification from the blots was shown by a densitometer (right panel).

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