Introduction

Fluoride is a trace element that plays an important role in both bone formation and homeostasis of bone mineral metabolism [1]. Low level of fluoride intake can prevent dental caries. However, prolonged and excessive ingestion of fluoride can cause fluorosis, which affects both teeth and bones. The dental fluorosis is characterized by tooth discoloration and enamel damage. The skeletal fluorosis can cause the most serious consequence, such as crippling, osteoporosis, and osteosclerosis. The previous studies have suggested that fluoride can cause cytotoxicity in a concentration-dependent manner [2]. Meanwhile, the long-term exposure to this element affects the amount and catalytic properties of many enzymes. Low concentration of fluoride stimulates the osteoblast proliferation [3, 4]. At high dose, the toxic effect of fluoride includes inflammatory reactions [5], oxidative stress [6, 7], and DNA damage [8]. Fluoride induces apoptosis by alteration in expression of both Bax and Bcl-2 in osteoblast [3, 9].

The interaction between fluoride and calcium has been reported for years [10]. The calcium ion is an essential intracellular messenger and modulates a diverse range of cellular physiological functions, including muscle contraction[11], gene transcription [12], and cell proliferation[13]. Recent studies indicated that fluoride induces the intracellular calcium ion release and results in the elevation of reactive oxygen reactions that relates to the cytotoxicity [14]. The increased intracellular calcium ion is associated with the fluoride-induced apoptosis [15]. Chronic fluoride ingestion affects calcium ion homeostasis by reduction of the amount of calcium pump proteins [16]. In contrast, fluoride leads to increased cytosolic calcium concentrations in proximal tubules [17], fibroblasts [18], and osteoblasts [19]. However, the mechanism of effect of fluoride on calcium homeostasis has not been sufficiently established.

The extracellular fluid calcium concentration is tightly regulated by parathyroid hormone (PTH), a hormone that can stimulate bone resorption and release calcium from bone, stimulate calcium reabsorption from the renal tubules, and increase production of 1,25(OH)2D3 in kidney to enhance intestinal absorption of calcium [20]. PTH-related peptide (PTHrP), a peptide produced by tumors with close homology in the N-terminal sequence to PTH itself [21], was originally isolated as a causal factor for hypercalcemia of malignancy (HM). PTHrP has been shown to play a critical role in bone formation by promoting survival and differentiation of osteoblasts [22, 23]. The interaction between fluoride and PTH and/or PTHrP has not been sufficiently investigated.

We previous demonstrated that L-type calcium channels are important in fluoride-related calcium ion homeostasis in osteoblasts [24]. In the present study, we investigate the role of fluoride in the expression of PTH, PTHrP, and calcium-sensing receptor (CaSR).

Materials and Methods

Measurement of Free Calcium Ion Concentration

The osteoblastic cell line MC3T3-E1 was obtained from Cell Bank (Shanghai, China). MC3T3-E1 cells were cultured in alpha minimum essential medium (Gibco Laboratories, Grand Island, NY) supplemented with 10 % heat-inactivated fetal bovine serum and 100 U/mL of penicillin and 100 μg/mL of streptomycin (Sigma Chemicals, St. Louis, MO). The cells were maintained at 37 °C in a humidified incubator in an atmosphere of 5 % CO2-95 % air and passaged at a 1:5 ratio with trypsin every 3–5 days. Cells (1 × 104) were plated into 96-well plates and treated with different doses of NaF for 48 h. Then, the supernatant was collected and the concentration of free calcium ion was measured with biochemical analyzer (CS-400B, Dirui, China).

Animals and Treatment

Forty Sprague-Dawley male rats were purchased from Beijing Laboratory Animal Center in China. All procedures involving animals were carried out in strict accordance with the international standards of animal care guidelines and were approved by the local Care of Experimental Animals Committee. Rats were randomly divided into four groups. Each experimental group contained ten rats. Control group was feed regular food and water, and others were raised by either regular food and 100 mg/L NaF water, or low-calcium food and regular water, or low-calcium food and 100 mg/L NaF water for 2 months, respectively. The food and water were renewed every day during the experiment. At the end of the experiment, long bones (femoral and tibiofibula) were collected and the concentration of free calcium ion in serum was measured with biochemical analyzer as above (CS-400B, Dirui, China).

RNA Isolation and qRT-PCR

MC3T3-E1 cells were cultured in 5 % FCS for 24 h and then treated with α-MEM (5 % calf serum) containing fluoride at a dose of 0, 2, 5, and 10 mg/L for 48 h. The bone samples from animal experiments were immersed in liquid nitrogen and pulverized. Total RNA of both cells and bones was extracted using TRIzol reagent (Invitrogen Inc., USA) according to the manufacturer’s instructions. First-strand complementary DNA (cDNA) was synthesized from l μg of total RNA in the present of oligo (dT)18 primer and reverse transcriptase (Takara, Japan). Quantitative PCR was performed on ABI 7500 real-time PCR system (Applied Biosystems, CA). cDNA template, primers, and SYBR Premix Ex Tag (Takara, Japan) in a total of 20 μL were applied to the following PCR programs: 95 °C preheating for 1 min, followed by 40 cycles of 95 °C for 15 s (denaturation), 60 °C for 20 s (annealing), and 72 °C for 32 s (elongation). The real-time PCR primer sequences are listed in Table 1. Gene expression was analyzed using the comparative threshold cycle (ΔΔCt) method [25], and relative expression levels were quantified by normalizing to β-actin expression.

Table 1 The real-time PCR primer sequences
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Protein Isolation and Western Blot

MC3T3-E1 cells and pulverized bones were lysed with RIPA buffer (Beyotime, China). The protein concentration of the samples was measured with a bicinchoninic acid protein assay kit (BCA, Beyotime, China). For Western blot, an equivalent amount of protein from cells or bones was mixed with 5× loading buffer (4:1) and boiled at 100 °C for 10 min. Then, the samples were separated by SDS-PAGE and transferred to PVDF membranes (PALL, USA). Membranes were incubated overnight at 4 °C with primary antibodies (β-actin, 1:5000, Proteintech; PTH, 1:200, Santa Cruz; PTHrP, 1:200, Santa Cruz; CaSR, 1:100, Santa Cruz; osteoprotegerin (OPG), 1:100, Santa Cruz; RANKL, 1:600, Epitomics). After three washes with TBS/0.1 % Tween 20 for 5 min, the membranes were incubated with secondary antibodies for 1 h at room temperature [26]. The immune blot signals were visualized using the EasySee Western Blot Kit (TransGen, China). The protein expression levels were determined by densitometric scanning (Tanon-1600 gel image system, Shanghai, China).

Statistical Analysis

Statistical analyses of the above results were performed by a one-way analysis of variance (ANOVA) by using the SPSS program (version 13.0) for windows (SPSS, Chicago, IL, USA). When ANOVA indicated a significant difference, the further analysis was performed by using the Fisher protected least significant difference (PLSD). Differences are considered statistically significant if p < 0.05.

Results

Fluoride Affected the Concentration of Free Calcium Ion in MC3T3-E1 Cell Culture Supernatant and Serum in Rats

To determine the function of fluoride on free calcium ion, we either treated the MC3T3-E1 cells with different concentration of fluoride or fed the rats with or without fluoride. The concentration of ionized calcium (i[Ca2+]) in cell culture supernatant and serum was measured with biochemical analyzer. As shown in Fig. 1a, the concentration of i[Ca2+] in cell culture supernatant was increased at 2 mg/L dose of fluoride and was decreased at high dose of fluoride (5 and 10 mg/L), as compared with control group. Interestingly, we found that the concentration of i[Ca2+] in serum was decreased in rats fed 100 mg/L NaF in presence or absence of calcium supplement in food (Fig. 1b).

Fig. 1
figure 1

The concentration of free calcium in cell culture supernatant and serum. a The MC3T3-E1 cells were treated with different doses of NaF for 48 h. Then, the supernatant was collected and biochemical analyzer was used to measure the concentration of free calcium ion. b The concentration of free calcium ion in serum from rats fed fluoride

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Quantitation of CaSR, PTH, and PTHrP mRNA and Protein Expression

To examine the effect of fluoride on calcium receptors, we studied the expression of CaSR by both Western blot and qRT-PCR. As demonstrated in Table 2 and Fig. 2, fluoride increased CaSR expression at both messenger RNA (mRNA) and protein levels in MC3T3-E1 cells and rats. We next studied the expression of PTH in MC3T3-E1 cells and long bones. As shown in Table 3 and Fig. 3, the PTH expression was decreased at both mRNA and protein levels in MC3T3-E1 cells after fluoride treatment at 2 and 5 mg/L. High dose of fluoride decreased the expression of PTH both at mRNA and protein level in MC3T3-E1 cells. The Western blot in bone indicated that PTH was increased in rats fed fluoride (Fig. 3d). Table 4 and Fig. 4 show the function of fluoride on PTHrP expression. When compared with control group, the low dose of fluoride (2 mg/L) decreased the expression of PTHrP protein and the high dose of fluoride (10 mg/L) increased the expression of PTHrP protein in MC3T3-E1 cells. The fluoride with regular food inhibited the expression of PTHrP protein, while fluoride with low-calcium food increased the expression of PTHrP protein.

Table 2 The expression level of CaSR mRNA in MC3T3-E1 cells by RT-PCR
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Fig. 2
figure 2

Quantitation of CaSR mRNA and protein expression. a The expression level of CaSR mRNA in MC3T3-E1 cells by RT-PCR. b The expression level of CaSR protein in rats by Western blot analysis with β-actin as loading control. c The graph indicates the relative density of CaSR protein in rats

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Table 3 The expression level of PTH mRNA in MC3T3-E1 cells by RT-PCR
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Fig. 3
figure 3

Quantitation of PTH mRNA and protein expression. a The expression level of PTH mRNA in MC3T3-E1 cells by RT-PCR. b The expression level of PTH protein in MC3T3-E1 cells by Western blot analysis with β-actin as loading control. c The graph indicates the relative density of PTH protein in MC3T3-E1 cells. d The expression level of PTH protein in rats by Western blot analysis with β-actin as loading control. e The graph indicates the relative density of PTH protein in rats

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Table 4 The expression level of PTHrP mRNA in rats by RT-PCR
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Fig. 4
figure 4

Quantitation of PTHrP mRNA and protein expression. a The expression level of PTHrP protein in MC3T3-E1 cells by Western blot analysis with β-actin as loading control. b The graph indicates the relative density of PTHrP protein in MC3T3-E1 cells. c The expression level of PTHrP mRNA in rats by RT-PCR

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The Effect of Fluoride on RANKL and OPG

To determine the effect of fluoride on osteoclastogenesis, we examined the expression of OPG and RANKL in rats after treated with or without fluoride. The expression levels of OPG and RANKL were increased in rats with regular food and 100 mg/L NaF water, low-calcium food and regular water, and low-calcium food and 100 mg/L NaF water (Fig. 5). The ratio of RANKL/OPG in rats fed low-calcium food in presence or absence of fluoride was significantly increased, as compared with control group (Fig. 5).

Fig. 5
figure 5

Expression of RANKL and OPG in rats in presence of fluoride. a The expression level of RANKL and OPG protein by Western blot analysis with β-actin as loading control. b, c The graphs indicate the relative density of OPG and RANKL protein, respectively. d The ratio of RANKL and OPG

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Discussion

Previous studies have demonstrated that fluoride increases metabolic turnover of bones and affects the homeostasis of bone mineral metabolism by stimulating osteoblast proliferation, although the mechanism is not yet understood [1, 3, 27–29]. High fluoride ingestion can cause hypocalcemia in fluorotic patients by increased calcitonin, and then, PTH levels were raised in serum to maintain calcium levels.

In the present study, we investigated the role of fluoride in calcium homeostasis and osteogenic transcription factor expressions both in vitro and in vivo. It has been demonstrated that intracellular calcium concentration is 10,000 to 100,000 times less than extracellular calcium concentration [30]. In plasma, calcium exists in three different forms: ionized or the biologically active form, protein-bound calcium, and complexed to phosphate and citrate [31]. Our results showed that the concentration of free calcium ion was slightly increased at low dose of fluoride and decreased at high dose of fluoride in cell culture supernatant. Previous studies showed that low dose of fluoride does not alter viability in MC3T3-E1 cells [32, 33] and high dose of fluoride increases PTH secretion [39]. Our previous study indicated that PTH and PTHrP play a critical role in anabolic effect of fluoride on bone turnover in osteoblasts [40]. Fluoride affects PTH and PTHrP expression in our study. These results indicate that fluoride might affect calcium ion concentration by regulating the secretion of PTH and PTHrP. The extracellular CaSR is a G protein-coupled receptor that is activated by extracellular calcium ion and plays an important role in regulating calcium homeostasis [34, 35]. The CaSR is found in multiple tissues including the parathyroid gland, thyroid, kidney, and bone [35]. In the parathyroid cell, CaSR is coupled to a G protein belonging to the Gq/G11 subfamily [36]. The CaSR maintains a stable ionized calcium concentration in the blood by modulating PTH secretion. Additionally, the signaling through the CaSR suppresses parathyroid gene transcription and cell proliferation through posttranslational mechanism [37, 38]. Our study showed that fluoride led to increase CaSR expression at dose-dependent manner at both the mRNA and protein levels in MC3T3-E1 cells and rats. Therefore, fluoride plays critical roles in calcium homeostasis maintenance. Fluoride upregulates the CaSR expression by modulating the extracellular calcium, then leads to downregulate the PTH and PTHrP expression. But high expression level of PTH and PTHrP were showed in our results.. Further studies should be done to investigate if there are fluoride receptors in osteoblast and activate downstream transcription factors and stimulate the PTH and PTHrp expression.

The receptor activators of RANKL and OPG are key molecules in bone remodeling by regulating differentiation and activation of osteoclasts. Osteoblasts express RANKL which initiates osteoclastogenesis by activating a variety of downstream signaling pathways such as mitogen-activated protein kinases, NF-κB, AP-1, c-fos, and NFATc1 [41]. The ratio of OPGL/OPG determines the delicate balance between bone resorption and synthesis. The previous study indicated that the RANKL/OPG system seems to be affected directly or indirectly by fluoride [42]. In our study, fluoride increased RANKL and OPG expression. These results indicated that the calcium and high fluoride enhanced the activity of osteoclasts and bone remodeling.

In summary, we have presented data that indicate that fluoride has an effect on the regulation of CaSR expression by modulating the extracellular calcium and then regulates the secretion of PTH and PTHrP. CaSR is important in modulating the calcium ion concentration in the present of fluoride. But, the mechanism is not clear and needs further studies.