Biological Trace Element Research

, Volume 148, Issue 1, pp 117–121

The Influence of Fluoride on the Expression of Inhibitors of Wnt/β-Catenin Signaling Pathway in Rat Skin Fibroblast Cells

Authors

  • Xiao-Li Liu
    • Department of Occupational and Environmental Health, School of Public Health, Tongji Medical CollegeHuazhong University of Science and Technology
  • Chang-Cheng Li
    • Department of Occupational and Environmental Health, School of Public Health, Tongji Medical CollegeHuazhong University of Science and Technology
    • Department of Occupational and Environmental Health, School of Public Health, Tongji Medical CollegeHuazhong University of Science and Technology
  • Cai-Yan Cui
    • Department of Occupational and Environmental Health, School of Public Health, Tongji Medical CollegeHuazhong University of Science and Technology
  • Yu-Zeng Zhang
    • Department of Occupational and Environmental Health, School of Public Health, Tongji Medical CollegeHuazhong University of Science and Technology
  • Yun Liu
    • Department of Occupational and Environmental Health, School of Public Health, Tongji Medical CollegeHuazhong University of Science and Technology
Article

DOI: 10.1007/s12011-012-9333-9

Cite this article as:
Liu, X., Li, C., Liu, K. et al. Biol Trace Elem Res (2012) 148: 117. doi:10.1007/s12011-012-9333-9

Abstract

The effective therapy of fluoride-induced bone diseases requires an understanding of the mechanism of the disorders. Changes in the inhibitors of the Wnt/β-catenin pathway, Dickkopf-1 (Dkk-1) and Sclerostin (SOST), were studied in supernatants harvested from rat skin fibroblasts cultured with varied doses of fluoride. The contents of SOST and Dkk-1 in fibroblast supernatants were assessed at four exposure time-points and investigated by using the method of ELISA. Compared to the relevant controls (0 mg F/L), a significant decrease of the concentrations of SOST and Dkk-1 was observed as the fluoride concentration increased. Compared to the relevant time controls (24 h), a significant decrease of the concentrations of SOST and Dkk-1 was observed with the extension of time. Our results suggest that the Wnt/β-catenin pathway inhibitors Dkk-1 and SOST play an important role in skeletal fluorosis. They can be used as important indications for diagnosing bone metabolism changes caused by fluoride exposure and therapeutic targets in diseases resulting from fluoride exposure.

Keywords

FluorideFibroblastSclerostinDickkopf-1

Introduction

Long-time fluoride intake in high concentration can result in fluoride-induced bone injuries. Skeletal fluorosis often leads to osteosclerosis of the skeleton with significant long-term difficulties, including impaired neck and lumbar mobility, aching of the axial skeleton, kyphosis, and painful lower extremities, ultimately causing crippling and incapacitation [1]. Extraperiosteal calcification and ossification play an important role in the development of skeletal fluorosis. Fibroblast, acting as the main-type cell of extraperiosteal soft tissue which includes the tendon and the point linking up the ligament, plays an important role in extraperiosteal ossification. Under the non-physiological condition, fibroblast can perform osteogenesis function, so it belongs to the category of inducible osteogenic precursor cell. Under fluoride exposure, fibroblast can perform its potential osteogenesis function.

Studies have also established that Wnt/β-catenin activity is essential for normal osteogenesis [2, 3]. The Wnt/β-catenin pathway is mainly composed of Wnt ligands, Wnt receptors, β-catenin, and nuclear transcription factors (the T-cell factors/lymphoid enhancer factor (TCFs/LEF)). Wnts initiate signaling by binding to a member of the Frizzled family and low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6, which are Wnt/β-catenin pathway receptors), leading to the downregulation of glycogen synthase kinase-3 (GSK-3) activity [4]. Inactivation of GSK-3 increases β-catenin levels in the cytosol. The cytosolic β-catenin is transferred to the nucleus and forms complexes with members of the TCFs/LEF class of DNA-binding proteins [4]. These complexes modulate the transcriptional activity of target promoters [4].

Enhancement of the Wnt/β-catenin pathway caused by the overexpression or deficiency of Wnt/β-catenin pathway inhibitors is associated with increased bone formation in mice and humans [5, 6]. The gene regulated by the Wnt/β-catenin pathway is involved in metabolism, proliferation, cell cycle, and apoptosis. Initiation of the Wnt/β-catenin pathway is modulated by soluble Wnt/β-catenin pathway inhibitors, including Dickkopf (Dkk-1) and Sclerostin (SOST), which regulate the Wnt/β-catenin pathway at the level of the Wnt/Frizzled/LRP interaction. Sclerostin, encoded by the Sost gene, is a secreted cysteine knot protein among the DNA family, and it emerged as a potent inhibitor of bone growth [79]. Dkk-1, as the endogenous secreted protein, is the first identified member of the Dickkopf family of secreted proteins that includes Dkk-1, Dkk-2, Dkk-3, Dkk-4, and a related protein, soggy.

The inhibitors, Dkk-1 and SOST, compete with the Wnt/β-catenin pathway for binding to LRP5/6, thus inhibiting the activation of the Wnt/β-catenin pathway in vitro [1012]. However, the mechanism of Dkk-1 and SOST's function remains to be elucidated in vitro.

In view of this characteristic, we have studied the changes of SOST and Dkk-1, acting as the inhibitors of the Wnt/β-catenin pathway, in fibroblasts exposed to different doses of fluoride for different lengths of time. We aim to determine the reference indications of fluoride exposure and want to provide a theoretical basis for the prevention and treatment of skeletal fluorosis.

Materials and Methods

Cell Cultures

Normal skin was isolated from a 1- to 3-day-old rat, and the skin was dissected free from adherent soft tissue. The skin was washed in phosphate-buffered saline and was cut into pieces (about 1 mm3). The pieces were placed in six-well plates which were put upside down in a humidified incubator containing of 5% CO2 at 37°C for 2 h. After 2 h, the Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin were added to the plates. Cells were harvested with trypsinase when they got 80% confluence and seeded into 25-cm2 culture flasks. Cells were used in the experiment at passages three–six. Then, the cells were harvested with trypsinase when they got 100% confluence and were seeded into 24-well plates at a density of 4 × 104 cells/well. After incubation for 24 h, the culture medium was replaced with fresh media containing the same medium supplemented with 0.0001, 0.001, 0.01, 0.1, 1, 10, and 20 mg F/L plus 6% FBS. After 24, 48, 72, and 96 h incubation, the culture supernatants were harvested.

ELISA

The concentrations of SOST and Dkk-1 in cell culture supernatants were measured using the ELISA kits.

Statistical Analysis

All data were presented as mean values with standard deviation. Data were analyzed by one-way ANOVA and are shown in Tables 1 and 2. Student Newman–Kuels multiple range and least significant difference procedures were employed to compare the means between different groups. Differences were considered to be statistically significant at P < 0.05.
Table 1

Concentration of SOST in fibroblasts of different fluoride exposures over time

Fluoride concentration (mg/L)

Concentration of SOST (μg/L)

24 h

48 h

72 h

96 h

0

25.5 ± 1.73

25.8 ± 1.64

26.8 ± 0.82

22.6 ± 0.99

0.0001

22.2 ± 2.49

20.6 ± 1.20a

24.5 ± 2.64

21.4 ± 0.80

0.001

22.8 ± 3.62

20.7 ± 1.71a

24.3 ± 2.68

18.1 ± 1.73a, h

0.01

24.9 ± 2.36

19.2 ± 1.18a, g

24.2 ± 2.88

20.1 ± 0.33d, g

0.1

23.9 ± 3.80

24.6 ± 2.02

24.9 ± 4.93

20.4 ± 0.72e, i

1

23.8 ± 0.47

24.6 ± 2.16

23.3 ± 3.10b

19.8 ± 1.50f, j

10

25.4 ± 1.99

25.1 ± 1.09

23.8 ± 1.93c

22.6 ± 2.51k

20

25.5 ± 2.52

27.2 ± 0.45

25.6 ± 1.71

24.2 ± 1.19

Data are mean ± SD; n = 6

aCompared to 0 mg/L, P = 0.000

bCompared to 0 mg/L, P = 0.033,

cCompared to 0 mg/L, P = 0.049

dCompared to 0 mg/L, P = 0.003

eCompared to 0 mg/L, P = 0.009

fCompared to 0 mg/L, P = 0.001

gCompared to 24 h: P = 0.000

hCompared to 24 h: P = 0.006

iCompared to 24 h: P = 0.050

jCompared to 24 h: P = 0.003

kCompared to 24 h: P = 0.023

Table 2

Concentration of Dkk-1 in fibroblasts of different fluoride exposures over time

Fluoride concentration (mg/L)

Concentration of Dkk-1 (μg/L)

24 h

48 h

72 h

96 h

0

24.1 ± 3.00

20.4 ± 1.27

25.1 ± 1.14

26.4 ± 1.79

0.0001

24.3 ± 1.14

19.7 ± 2.49e

24.1 ± 1.60

25.8 ± 1.21

0.001

26.4 ± 2.07

16.3 ± 3.71a, e

23.3 ± 2.49f

25.0 ± 1.05

0.01

24.5 ± 2.49

17.3 ± 1.18e

21.1 ± 0.68b, g

22.1 ± 0.94b, j

0.1

24.2 ± 1.36

22.3 ± 0.81

22.9 ± 0.94c

19.6 ± 1.31b, e

1

25.1 ± 1.87

23.3 ± 2.28

21.8 ± 0.43d, h

20.5 ± 0.79b, e

10

23.1 ± 1.81

23.6 ± 1.55

21.0 ± 1.77b, i

23.5 ± 0.74b

20

26.0 ± 1.94

23.5 ± 1.69

18.3 ± 1.80b, e

21.3 ± 1.12b, e

Data are mean ± SD; n = 6

aCompared to 0 mg/L, P = 0.029

bCompared to 0 mg/L, P = 0.000

cCompared to 0 mg/L, P = 0.026

dCompared to 0 mg/L, P = 0.001

eCompared to 24 h, P = 0.000

fCompared to 24 h, P = 0.044

gCompared to 24 h, P = 0.003

hCompared to 24 h, P = 0.002

iCompared to 24 h, P = 0.029

jCompared to 24 h, P = 0.039

Results

As seen in Table 1, the concentration of SOST in fibroblasts of the 0.0001, 0.001, and 0.01 mg F/L groups at 48 h was significantly lower than that of 0 mg F/L group. The concentration of SOST in fibroblasts of the 1 and 10 mg F/L groups at 72 h and that of the 0.001, 0.01, 0.1, and 1 mg F/L groups at 96 h were also significantly lower than that of 0 mg F/L group. The concentration of SOST in fibroblasts of the 0.001, 0.01, 0.1, 1, and 10 mg F/L groups at 96 h and that of the 0.01 mg F/L groups at 48 h were significantly lower than that of the groups at 24 h, respectively.

As seen in Table 2, the concentration of Dkk-1 in fibroblasts of the 0.001 mg F/L group at 48 h was significantly lower than that of 0 mg F/L group. The concentration of Dkk-1 in fibroblasts of the 0.01, 0.1, 1, 10, and 20 mg F/L groups at 72 h and that of the 0.01, 0.1, 1, 10, and 20 mg F/L groups at 96 h were also significantly lower than that of 0 mg F/L group. The concentration of Dkk-1 in fibroblasts of the 0.0001, 0.001, and 0.01 mg F/L groups at 48 h, that of the 0.001, 0.01, 1, 10, and 20 mg F/L groups at 72 h, and that of the 0.01, 0.1, 1, and 20 mg F/L groups at 96 h were significantly lower than that of the groups at 24 h, respectively.

In the present study, we evaluated the concentrations of SOST and Dkk-1 in fibroblasts of different fluoride exposures over time. Compared to the relevant controls (0 mg F/L), a significant decrease of the concentrations of SOST and Dkk-1 was observed as the fluoride concentration increased. Compared to the relevant controls (24 h), a significant decrease of the concentrations of SOST and Dkk-1 was observed with the extension of time.

Discussion

Fluorine is an essential trace element and is mainly distributed in the hard tissues, such as the bones and teeth. The accumulation of fluorine in the bone influences normal bone metabolism, causing abnormal bone metabolism and clinical pathological changes and ultimately leading to fluoride-induced bone injury [13].

At the cell surface, two groups of secreted protein that are capable of regulating the Wnt/β-catenin pathway with distinct modes of action are present [14]. One consists of protein that modulates the Wnt/β-catenin pathway by directly binding to the Wnt/β-catenin pathway ligands. This group includes members of the secreted frizzled-related protein [1517]. The second group of Wnt/β-catenin pathway inhibitors is composed of the structurally unrelated Dickkopf (Dkk) and SOST/sclerostin families [10, 18]. These proteins inhibit the Wnt/β-catenin pathway by directly competing with Wnt/β-catenin for binding to the Wnt receptor LRP5/6 [11, 19].

Both Dkk-1 and SOST have been shown to be directly involved in bone growth regulation [14]. The loss-of-function mutation of Sost gene can result in complete or partial loss of sclerostin function, which is responsible for sclerosteosis or Van Buchem disease, two similar types of autosomal recessive craniotubular hyperostosis [20]. Some studies about knockout and transgenic mouse support the role of Dkk-1 as the endogenous regulator of the Wnt/β-catenin pathway in bones. The loss-of-function mutation of Dkk-1 has increased the bone mass in mice, whereas mice that overexpress Dkk-1 in osteoblasts have reduced bone mass [21].

Although the identical target of Dkk-1 and SOST is LRP5/6, there might not be some interaction effect because they are structurally unrelated proteins. Some studies have indicated that Dkk-1 and SOST are mutually independent when they act as the regulatory factors of LRP5/6 [21]. Dkk-1 inhibits the Wnt/β-catenin pathway by binding to the third β-propeller domain of LRP5/6, which interferes with the formation of the Wnt/LRP/Fz trimolecular complex. Sclerostin binds to the first and/or second β-propeller domain of LRP5 and, being similar to Dkk-1, prevents the formation of a Wnt/LRP/Fz trimolecular complex [21]. Although we know both Dkk-1 and SOST can bind to LRP5/6, we need to further determine whether they act synergistically or competitively.

Fluoride is a trace element widely distributed in the environment in a wide range of concentrations. It can be found in water, soil, atmosphere, food, and so on [22]. There are many high-fluoride areas distributed in the world, such as China and India. The serum fluoride, urinary fluoride, and other tissue fluoride levels of the population in high fluoride areas are closely related to their intake of fluoride. According to a spot study, the content of serum fluoride is 0.46 ± 0.22 mg/L, the content of urinary fluoride is 2.72 ± 0.16 mg/L, and the content of air fluoride is 2.08 ± 1.01 mg/m3 in a fluoride-exposed population in an aluminum factory in China [13]. Other studies also indicated that the fluoride levels in the urine of children from high fluoride areas in China are 2.04 ± 0.89 mg/L while the fluoride concentration in drinking water is 1.8 mg/L [23]. According to Zhou Zhen-rong, the content of serum fluoride of the patients with skeletal fluorosis is 0.22–0.53 mg/L [24]. However, the content of serum fluoride can fluctuate in a wide range in patients with skeletal fluorosis with the difference of three to eight times. The permissible limit of fluoride in drinking water is 1.0 mg/L according to WHO (1997). In India, 30% of the habitations of the country were found to have more than 1.5 mg/L fluoride concentration in drinking water, and 11% were found to have the concentration at toxic level (about 3.0 mg/L) [25]. In Southwest China, the fluoride content of hot spring water in some areas is 1.02–6.907 mg/L, which is not suitable for drinking [26]. Then, in our experiment, the fluoride exposure condition of 0.0001–20 mg/L is close to the fluoride in vivo population in high fluoride areas, and the effect of fluoride exposure on fibroblasts can be possible in a naturally fluoride-polluted environment in China and India.

There is little research about the changes of SOST and Dkk-1 in fibroblasts following the treatment with fluoride. In our study, the fibroblast cell lines were cultured, and the protein content of SOST and Dkk-1 in different time and fluoride-treated concentrations were observed using the ELISA method. Seven groups of different fluoride concentrations (0.0001, 0.001, 0.01, 0.1, 1, 10, 20 mg F/L) and four time groups (24, 48, 72, 96 h) were set. Our studies have shown that the concentrations of both Dkk-1 and SOST in the high-F and low-F exposed groups were significantly lower than those in the control group and the concentrations of both Dkk-1 and SOST decreased significantly with the extension of time.

The results of our study also indicate that long time exposure of high fluoride can cause a concurrent decrease in Dkk-1 and SOST acting as Wnt/β-catenin pathway inhibitors, which might contribute to weaken the inhibitory action of the Wnt/β-catenin pathway. The Wnt/β-catenin pathway is activated, and the activity is enhanced, leading to the the inactivity of GSK-3. Thus, the degradation of β-catenin which acts as an important intracellular molecule in the Wnt/β-catenin pathway is suppressed. The activated Wnt/β-catenin pathway can result in the acceleration of bone transformation and osteogenesis which might be responsible for skeletal fluorosis.

Osteosclerosis is a serious medical problem during skeletal fluorosis. The studies about current and potential treatments for osteosclerosis that interact with the Wnt/β-catenin pathway are indispensable. The Wnt/β-catenin pathway inhibitors Dkk-1 and SOST, because of their roles in suppressing new bone formation, are considered highly promising therapeutic targets. Ongoing work will undoubtedly identify their potential druggable targets within the Wnt/β-catenin pathway.

According to our results, Dkk-1 and SOST might be early sensitive biomarkers of fluoride-induced bone injuries. But some fundamental regulatory mechanisms—how these inhibitors recognize LRP5/6 and what changes may occur in LRP after their function, etc.—are still not entirely clear and need further in-depth study.

Acknowledgment

This research was sponsored by the National Natural Science Foundation of China (NSFC) (no. 81072255).

Conflicts of interest

The authors declare that they have no conflict of interest.

Copyright information

© Springer Science+Business Media, LLC 2012