Fluoride Action Network


Excess consumption of fluoride during the development of tooth enamel will cause dental fluorosis, but the exact molecular mechanisms remain to be elucidated. Circadian rhythm is implicated in many physiological processes and various diseases. There is increasing evidence indicates that ameloblast differentiation is under the control of clock genes. However, it has not been reported whether fluoride regulates ameloblast differentiation through clock genes and the downstream PPAR?. To explore the effect of fluoride on ameloblast differentiation and the underlying regulatory mechanism, we used both rat dental fluorosis model and an ameloblast cell line LS8 to conduct a series of experiments. Our results showed that fluoride significantly reduced the expression of PCNA, RUNX2 and MMP9 in rat ameloblasts and LS8 cells (P < 0.05). Fluoride increased nuclear translocation of ?-catenin in vivo and in vitro, and 0.1 ?g/ml Dkk1 pretreatment ameliorated the decreased expression of CXXC5, RUNX2 and MMP9 induced by fluoride. Furthermore, we found fluoride significantly inhibited the expression of Clock, Bmal1, Per2 and PPAR? in rat mandibular ameloblasts and LS8 cells by immunostaining, qPCR and Western blot (P < 0.05). Flow cytometry analysis showed that fluoride promoted ROS generation. Remarkably, 50 ?M resveratrol significantly ameliorated the inhibitory effect of fluoride on ameloblast differentiation markers, clock genes and PPAR?, and inhibited the Wnt/?-catenin signaling (P < 0.05). Taken together, these findings suggested that excessive fluoride promoted ROS generation, leading to the inhibition of clock genes, which resulted in reduced PPAR? and activated Wnt/?-catenin signaling pathway, thus inhibiting ameloblast differentiation and matrix degradation. This study provides a better understanding of the molecular mechanism of enamel defects in dental fluorosis.



Excess consumption of fluoride during the development of tooth enamel will cause dental fluorosis. The typical manifestations of dental fluorosis are striations, mottling, and even discoloration or pitting on the enamel surface in severe cases [1]. Dental fluorosis is endemic in several Asian countries, such as China, India and Pakistan, and it’s a serious public health issue around the world [2]. A large number of studies have been conducted to explore the pathogenesis of enamel defects in dental fluorosis, but the exact molecular mechanisms remain to be elucidated.

Ameloblasts, important cells responsible for enamel production, undergo three sequential stages called the presecretory stage, the secretory stage and the maturation stage during differentiation. The nuclei of ameloblasts in the presecretory stage separate distally from the dental papilla and the cytoplasm are filled with organelles necessary for the synthesis and secretion of enamel proteins. During the secretory stage, tall columnar ameloblasts secrete mainly enamel proteins for enamel development, whereas ameloblasts become short and cubic in shape at the maturation stage, reabsorbing secreted proteins and promoting enamel mineralization [3,4]. There are a multitude of stage-specific genes acting to regulate ameloblasts patterning and differentiation, including matrix metallopeptidase 20 (MMP20), an early protease whose role is to cleave accumulated enamel proteins in the secretory stage, and kallikrein 4 (KLK4), a late protease which degrades the retained organic matrix in maturation stages [5]. Furthermore, RUNX2 has been confirmed as an essential transcription factor for the maturation stage of ameloblasts. Runx2-knockout mice exhibit incomplete enamel structure and mineralization, as well as decrease in enamel protein expression [6]. Similarly, matrix metalloprotein 9 (MMP9), also known as gelatinase B, is involved in the hydrolysis of enamel proteins during tooth development. Studies have shown that MMP9 deficient mice have abnormal tooth morphology, immature ameloblasts differentiation and a lack of ameloblasts polarization. In addition, MMP9 has been identified to be a novel Runx2 target gene, and knockdown Runx2 results in reduced expression of MMP9 in MC3T3 cells [7]. A number of studies have shown that fluoride can affect enamel mineralization by inhibiting the expressions of maturation stage specific genes in ameloblasts, however, the precise regulatory mechanisms are unclear [[8], [9], [10]].

Canonical Wnt/?-catenin cascade has been shown to play an important role in ameloblasts differentiation and function during amelogenesis. Studies have demonstrated that constitutively activated ?-catenin in ameloblasts delays mineralization and causes hypomineralization in the incisor enamel [11]. Importantly, there is an opposed interplay between ?-catenin expression and peroxisome proliferator-activated receptors gamma (PPAR?) activity. The previous researches have shown that activation of the canonical Wnt/?-catenin pathway inhibits PPAR? expression, and vice versa [12]. Interestingly, a link between PPAR? and circadian rhythms has also been established [13].

Circadian rhythm, a self-sustaining molecular oscillator, is implicated in many cellular and physiological processes and various diseases such as cancer, obesity, arthritis and so on [14]. At the molecular level, several genes named “clock genes” constitute auto-regulatory transcription translation feedback loops which drive the rhythmic expression of clock-regulated genes in central and peripheral tissues. The core transcription factors of this loop are brain and muscle aryl hydrocarbon receptor nuclear translocation-like 1 (Bmal1) and circadian locomotor output cycles kaput (Clock). After the heterodimerization of Bmal1 and Clock complex in the cytoplasm translocates into the nucleus, it enhances the expression of period (Per) and cryptochrome (Cry), the negative components of the loop. Following the Per/Cry dimer translocates back into the nucleus, it conversely inhibits the transcriptional activity of Bmal1/Clock complex. There is increasing evidence indicates that clock genes are expressed in enamel forming ameloblasts and ameloblast differentiation is under the control of clock genes [15]. Furthermore, Zheng et al. reported that KLK4 and RUNX2 are downstream targets of clock genes [16].

Previously, Shusterman et al. reported that fluoride upregulated the Wnt/?-catenin signaling in murine ameloblast-lineage cells [17]. However, it is still not known whether fluoride affects ameloblast differentiation through PPAR? and clock genes. In this study, we used both rat dental fluorosis model and an ameloblast cell line LS8 to explore the effect of fluoride on ameloblast differentiation and the underlying regulatory mechanism. We found that fluoride suppressed ameloblasts differentiation through clock genes/PPAR? and the Wnt/?-catenin signaling pathway. Furthermore, resveratrol (RES) could ameliorate fluoride-induced ameloblast differentiation inhibition via clock genes/PPAR? and the Wnt/?-catenin signaling pathway.

Section snippets

Animals and treatment

Eighteen six-week-old male Sprague-Dawley rats were purchased from Beijing Huafukang Bioscience Co., LTD. The rats were randomly divided into three groups of 6 animals each after acclimatization. The rats in the control group were given distilled water to drink, and the other two groups were given drinking water containing 50 mg/L and 100 mg/L F? for a period of 6 weeks. The doses of fluoride were determined according to the previously reported studies of dental fluorosis in rats [18]. During

Fluoride contents and dental fluorosis in rats

Fluoride contents in serum and urine are considered as indicators of fluoride levels in the body. We firstly detected ionic fluoride levels in serum and urine in the three groups and found that the mean fluoride contents in serum and urine were 0.06 ?g/mL and 5.58 ?g/mL, respectively, in the control group. Markedly higher fluoride concentration in serum and urine was found in the fluoride groups than that of control group (P < 0.01) (Fig. 1. A&B).

The rat incisor enamel in the control group


Fluoride is widely existed in the environment and can enter the body through drinking water, polluted air or foodstuffs. Long-term excessive intake of fluoride causes fluorosis and the main symptoms are dental fluorosis and skeletal fluorosis. The pathological change of dental fluorosis is enamel defects with reduced mineral content and greater protein levels in enamel [21]. Although numerous studies have been conducted to explore the underlying pathogenesis, the precise molecular mechanisms

Author statement

Tingling Zou, conceptualization, investigation, data curation, writing original draft. Lan Ma, formal analysis, validation. Lili Gu, resources, validation. Shuhua Xi, resources, validation. Kaiqiang Zhang, conceptualization, supervision. Xiaoying Guo, conceptualization, supervision, funding acquision.

Compliance with ethical standards

All experiments were performed according to the norms of the local ethics committee at China Medical University in accordance with the national guidelines for animal use and care


This work was supported by a grant from the National Natural Science Foundation of China (NSFC) (No. 82073497).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


We thank Professor Malcolm L. Snead (Department of Biomedical Sciences, University of Southern California, USA) for donation of LS8 cells.

References (38)

*Original full-text article online at: https://www.sciencedirect.com/science/article/abs/pii/S0009279722003507?via%253Dihub