Both Wnt/β-catenin and Ihh signaling pathways control the proliferation and differentiation of osteoblasts and chondrocytes at multiple stages
[12][13][14][15][44][45][46]. One study tested the genetic relationship between Wnt and Hh signaling by generating double mutant mice with results revealing that Wnt/β-catenin signaling acts downstream of Hh signaling in enhancing bone formation
[67], in agreement with another study
[68]. Subsequently, the expression of genes associated with different stages of osteoblast differentiation was also examined, showing that β-catenin is required downstream of Ihh in promoting osteoblast maturation
[67]. It has been demonstrated that Wif1 (Wnt inhibitory factor 1) exerts biological effects by mediating and regulating Shh/Wnt/β-catenin signaling
[69], as well as Wif1 can effectively block the activation of the canonical Wnt signaling pathway in chondrocytes by binding to Wnt ligands (Wnt3a, etc.)
[69][70][71], speculating Hh signaling may exert inhibitory effects on downstream Wnt/β-catenin signaling through Wif1 in the pathogenesis of skeletal fluorosis. At the same time, the Wnt/β-catenin signaling is also interactively regulated with the PI3K/Akt signaling. It was shown that PI3K/Akt negatively regulates Gsk-3β activity, thereby inhibiting the phosphorylation of β-catenin
[72], which suggests a role for Wnt/β-catenin signaling acting downstream of PI3K/Akt. To determine this role, the researchers further analyzed the expression of Wnt-regulated genes, such as Dkk1 and Sfrp1 (secreted frizzled-related protein 1), showing that PI3K signaling activates these genes by peptide-mediated α5β1 integrin priming in mesenchymal skeletal cells
[73]. Moreover, PI3K/Akt signaling is also regulated by insulin-related signaling. IGF-1 is a principal growth-promoting signal for vertebrate skeletal development
[74] and as a specific ligand can activate the PI3K/Akt signaling pathway by binding and phosphorylating the membrane IGF-1 receptor (IGF-1R), leading to osteoblast differentiation and proliferation
[75].
At present, studies on the relevant signaling pathways involved in skeletal fluorosis are still predominantly single lineage studies. Nevertheless, active osteogenesis and accelerated bone turnover as crucial processes in the progression of skeletal fluorosis are regulated by sophisticated networks of multiple signaling pathways. The profound exploration of the interactive regulatory mechanisms among signaling pathways in skeletal fluorosis will facilitate the development of specific targeted therapeutic measures.
3. Mechanism of Stress Pathways in Skeletal Fluorosis
3.1. Effect of Endoplasmic Reticulum Stress on Skeletal Fluorosis
The endoplasmic reticulum (ER) is essential for protein synthesis and secretion in eukaryotic cells
[76]. When cells are subjected to certain external stimuli, the ER generates a series of regulatory mechanisms, generating endoplasmic reticulum stress (ERS)
[77]. At the same time, ERS can protect cells from damage by stimulating the unfolded protein response (UPR)
[78] or initiating the apoptotic program to ensure the survival of the organism
[79][80]. Proteomic studies have revealed that the expression of immunoglobulin heavy chain binding protein (BiP), also known as glucose-regulated protein 78 (GRP78), protein disulfide isomerase (PDI), proteasome 26S ATPase, and thioredoxin (Trx) is up-regulated in fluoride-stained osteoblasts, and these proteins play key roles in protein folding of ER
[81]. The results provide clues that ERS and UPR may be involved in the pathogenesis of skeletal fluorosis. URP is mediated by Bip and three response sensing proteins, including protein kinase-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6)
[82].
PERK is a serine/threonine-protein kinase that can be activated by accumulating misfolded or unfolded proteins on the ER
[83]. It can reduce the newly synthesized proteins of the ER by activating eukaryotic initiation factor 2a (eIF2a) and activating transcription factor 4 (ATF4) pathways, thereby reducing ER load, and even inducing C/EBP homologous protein (CHOP) protein synthesis to stimulate the apoptotic program
[84][85][86][87]. In addition, PERK can also directly activate nuclear factor erythroid 2-related factor 2 (Nrf2) to antagonize apoptosis to protect cell survival
[88]. A study has shown that fluoride exposure activates the PERK signaling pathway, leading to activation of ATF4 and Nrf2 and up-regulating the expression of genes related to bone turnover in osteoblasts
[89]. Meanwhile, the study also showed that ATF6 and IRE1 signaling factors mediated a less pronounced effect of ERS, suggesting that fluoride exposure mediates osteoblast damage mainly through the PERK signaling pathway
[89].
To further clarify the role of the PERK signaling pathway, changes in osteogenic and osteolytic gene expression were studied in osteoblastic cell lines before and after PERK gene interference, and the results showed that fluoride stimulated the protein expression of PERK, Nrf2, osteoprotegerin (OPG), and Runx2 in PERK siRNA-transfected cells to a certain extent
[90]. The above findings suggest the vital role of the PERK/Nrf2 pathway in the activation mechanism of fluorine-induced osteoblast and osteoclast. Nevertheless, studies on the IRE1 pathway and ATF6 pathway concerning skeletal fluorosis are still scarce and need to be further explored to provide new ideas and theoretical basis for preventing and treating skeletal fluorosis.
3.2. Effect of Oxidative Stress on Skeletal Fluorosis
Oxidative stress is considered to be an essential mechanism in the pathogenesis of fluorosis. Intake of large amounts of fluoride can cause an imbalance between antioxidant defense mechanisms and free radical levels, leaving cells in a state of oxidative stress
[91]. In fluorosis, oxidative stress increases reactive oxygen species (ROS) and is accompanied by reduced antioxidant enzyme activity and increased lipid peroxides
[92][93]. Nevertheless, excessive ROS can disrupt the dynamic balance between bone formation of osteoblasts and bone resorption of osteoclasts, leading to the development of skeletal fluorosis
[94]. A study has shown that osteoblasts treated with low fluorine concentrations are in a low-level oxidative stress state and maintain redox homeostasis by activating the nuclear factor E2-related factor 2-antioxidant responsive element (Nrf2-ARE) signaling pathway, which prevents cells from apoptosis or death
[95]. In contrast, when treated with high concentrations (4.00 mmol/L) of fluoride, the intracellular antioxidant system and signaling pathways were disrupted or disintegrated, leaving the cells in a decompensated state, and undergoing severe oxidative damage, even inducing apoptosis in osteoblasts
[95]. Another study noted that intracellular ROS levels increased significantly after sodium fluoride treatment, and osteoblasts presented apoptotic morphological changes such as chromatin condensation and DNA fragmentation
[96].
In addition, fluoride can likewise participate in the regulation of osteoclast proliferation through the oxidative stress pathway. Calcineurin (CaN) is a serine/threonine phosphatase dependent on calcium (Ca) and calmodulin (CaM), which is involved in the regulation of osteoclast differentiation and proliferation
[97]. Animal experiments have demonstrated that excessive fluoride exposure led to increased CaN mRNA and protein expression levels and serum CaN activity in rat bone tissue, but its upstream regulators Ca and CaM had no significant differences between the control and fluoride-infected groups
[98][99]. However, malondialdehyde (MDA) levels of the fluoride-infected group were significantly higher than the control group, and oxidative stress levels were also positively correlated with CaN activity
[99][100], suggesting excess fluoride may stimulate elevated CaN activity in the organism through the oxidative stress pathway, further leading to increased osteoclast production in rat bone tissue.
The above studies have illustrated that fluoride can induce osteoblast apoptosis and osteoclast proliferation through the oxidative stress pathway, providing evidence for the pathogenesis of skeletal fluorosis. However, the molecular mechanisms of fluoride effects on osteoclasts are still relatively poorly investigated, and how fluoride affects osteoclast proliferation and differentiation through the oxidative stress pathway has not been comprehensively clarified, and further in-depth studies are necessary. Besides, since both endoplasmic reticulum stress and oxidative stress are associated with the pathogenesis of skeletal fluorosis, further studies are needed to explore the interrelationship between them to provide new insights into the pathogenesis of skeletal fluorosis.
4. Mechanism of Epigenetics in Skeletal Fluorosis
4.1. Effect of DNA Methylation on Skeletal Fluorosis
DNA methylation is one of the earliest and most common epigenetic modifications, and it plays an important role in the pathogenesis of skeletal fluorosis. P16 protein is a key factor of cell cycle regulation in the G1/S phase, competitively inhibits the cell progression from G1 phase to S phase and blocks abnormal activation of osteoblasts
[101][102]. In fluorosis model experiments, promoter hypermethylation of the p16 gene inhibited its mRNA transcription and protein expression, while decreased p16 protein expression reduced its G1/S phase blocking effect, potentially providing an important molecular mechanism for the altered proliferative capacity and cell cycle distribution of osteoblasts caused by fluorine
[103]. In addition, research has revealed that fluoride causes promoter DNA hypermethylation of the BMP1, METAP2, MMP11, and BACH1 genes, then RNA expression levels are down-regulated, thereby promoting skeletal fluorosis
[104].
An animal experiment has shown that estrogen receptor α (ERα) mRNA expression is enhanced when the promoter methylation level of the ERα gene is inhibited in osteoblasts, thereby promoting osteoblast proliferation and differentiation
[105]. In addition, the promoter methylation level of the ERα gene is negatively correlated with urinary fluoride concentration, suggesting that the pathological bone changes induced by fluoride exposure in men were related to the promoter hypomethylation of the ERα gene
[106]. In conclusion, due to the diversity of fluoride regulation of bone damage and cellular DNA methylation, further works on the epigenetics of cellular regulation-related genes in fluorosis are needed.
4.2. Effect of Non-Coding RNAs on Skeletal Fluorosis
Following extensive research on the molecular mechanisms of abnormal bone metabolism in skeletal fluorosis at the molecular biology level, the role of microRNAs (miRNAs) is gradually being discovered. miRNAs as non-coding RNAs are post-transcription regulators of gene expression. miRNAs negatively regulate the expression of their target mRNAs and can inhibit the expression of mRNAs
[107]. Fluoride exposure in human osteosarcoma cells can affect the expression of genes related to bone metabolism through the miRNA pathway
[108]. Cyclin D1 is a protein that regulates the cell cycle by facilitating the cell transition from G1 phase to S phase and accelerating cell proliferation
[109]. Down-regulation of mir-486-3p can affect the expression of cyclin D1 and further regulate osteoblast proliferation and activation
[110]. Studies have shown that fluoride exposure induces the down-regulation of miR-4755-5p and Let-7c-5p, promoting fluoride-induced osteoblast proliferation and activation by regulating cyclin D1 expression
[111][112].
It was pointed out that miR-29a promotes the differentiation process of osteoblasts by targeting and inhibiting the expression of Dkk-1
[113]. The specific mechanism is that the canonical Wnt signaling pathway can induce transcription of miR-29a, which can enhance the Wnt signaling pathway by downregulating Dkk-1 (an antagonist of Wnt signaling), thereby regulating the osteoblast differentiation process
[113][114]. Another study also showed that the expression level of miR-27 was positively correlated with that of β-catenin, and it could activate Wnt signaling through the accumulation of β-catenin protein, thereby promoting osteoblast differentiation
[115]. This suggests that miR-27 could be a possible target for the development of drugs against skeletal fluorosis. In conclusion, the regulatory networks of non-coding RNAs in fluorosis are complex, and further investigations are needed to elucidate their regulatory mechanisms on specific target mRNAs. Meanwhile, exploring the interactions between miRNAs and other signaling pathways may provide new insights into the therapeutic strategies for skeletal fluorosis.
5. Conclusions
In summary, skeletal fluorosis is a chronic and progressive endemic disease with severe risks to human health. Exposure to a specific duration or dose of fluoride can alter osteoblasts, osteoclasts, and chondrocytes’ function, differentiation, and proliferation and lead to skeletal fluorosis by disrupting the balance between bone formation and resorption. Signaling pathways, stress pathways, epigenetics, and interactive regulatory mechanisms play crucial roles in the pathogenesis above. With the development of multi-level and multi-faceted studies in recent years, considerable progress has been made at the molecular biological level in understanding the pathogenesis of skeletal fluorosis. Unfortunately, although a great deal of research has been carried out, there are no clear and effective treatments for patients with skeletal fluorosis.
The treatment of skeletal fluorosis in traditional Chinese medicine (TCM) focuses on reducing fluoride concentrations in the body and improving autogenous regulatory mechanisms, while western medicine aims to reduce the toxicity of fluoride to the body and repair the damage through specific interventions
[116]. Studies have suggested that chemical elements such as calcium and magnesium can antagonize fluoride toxicity by binding fluoride in the intestine to form low-solubility complexes for excretion, thereby reducing fluoride absorption
[117]. Antioxidants have also been used to treat skeletal fluorosis, and concomitant intake of calcium, vitamin C, and vitamin D can antagonize fluoride toxicity
[118]. Additionally, some components of Chinese herbal medicine have antioxidant effects and can also effectively antagonize the oxidative stress caused by fluorosis, thereby reducing the degree of fluorosis
[94][119]. Considering the combination of TCM and Western medical treatment ideas for skeletal fluorosis may yield better effects. At the same time, it is more important to continue in-depth research on the complex pathogenesis of skeletal fluorosis and explore new targeted therapies at the molecular level to treat skeletal fluorosis.
Since there is no specific effective treatment to cure skeletal fluorosis completely, prevention and control of skeletal fluorosis by using safe drinking water and reducing the use of high fluoride coal burning is currently considered the ideal approach
[2]. Particularly, clarifying the pathogenesis of skeletal fluorosis and exploring effective targets against fluorine toxicity are of great importance to scientifically prevent and control the occurrence, development, and prevalence of skeletal fluorosis and reduce the risk to human health.