Background: Skeletal fluorosis has become a public health issue in recent years as its serious impact on patients’ life expectancy. Bone morphogenetic protein 2 (BMP2) plays a key role in promoting osteogenesis. However, the mechanism of BMP2-Wnt/B-catenin axis in skeletal fluorosis needs further exploration.
Methods: The RT-qPCR and western blot assay were carried out to examine the mRNA and protein levels. Cell viability was measured by MTT assay. A commercial ALP assay kit was used to detect ALP activities. Alizarin Red staining was performed to measure the formation of mineralized nodules. Methylation-specific PCR (MSP) was performed to measure the methylation level of BMP2.
Results: Fluoride promoted the expression of osteogenic marker genes (OPN, OCN, OSX and RUNX2) and induced the proliferation and differentiation of MC3T3-E1 cells. Fluoride induced hypomethylation and high expression of BMP2. Furthermore, knockdown of BMP2 reversed the promoting effect of fluoride on osteogenic differentiation of MC3T3-E1. The expression of B-catenin, glycogen synthase kinase 3B (GSK3B), wingless/integrated 3a (Wnt3a), low-density lipoprotein receptor-related protein 5 (LRP5) and dishevelled 1 (Dv1) were increased in osteoblasts treated with fluoride, however, knockdown of BMP2 reversed this phenomenon. Simultaneous knockdown of BMP2 and B-catenin significantly inhibited the differentiation of osteoblasts induced by fluoride.
Conclusion: Fluoride contributed to proliferation and differentiation of osteoblasts through BMP2-Wnt/B-catenin axis, providing a feasible theoretical basis for the treatment of skeletal fluorosis.
Keywords: BMP2; Fluoride; Osteoblasts; Skeletal fluorosis; Wnt/?-catenin.
*Original abstract online at https://www.sciencedirect.com/science/article/abs/pii/S0009279722000758?via%3Dihub
Fluorine is a necessary trace element for the normal development of human organs, but long-term exposure of fluorine will result a fluorosis [1,2]. Appropriate amount of fluoride has positive effects on teeth and bones, but long-term excessive intake can cause severe damage , leading to dental fluorosis and skeletal fluorosis [, , , ]. Patients with skeletal fluorosis display multiple bone lesions, such as osteoporosis, osteosclerosis, calcification of ligaments and osteopenia , which bring a lot of inconvenience to patients’ life and even affect their life expectancy. Fluorosis is particularly prevalent in underdeveloped nations, and also become a widely epidemic and a public health problem in some parts of China . Thus, it is of vital importance to investigate bone-related regulatory gene expressions in chronic fluorosis and the pathogenesis of skeletal fluorosis.
DNA methylation is one of the main modes of epigenetic inheritance, which regulates gene expression by affecting chromatin in diverse skeletal diseases [, , ]. Excessive fluoride has been shown to induce abnormal DNA methylation in mice or cells, suggesting that DNA methylation may be a new mechanism in the process of fluorosis [1,13]. Bone morphogenetic protein 2 (BMP2) is a member of the BMPs family and plays a central role in early embryogenesis, bone development, and osteoblastic differentiation [14,15]. It was reported that abnormal methylation in BMP2 promoter region might downregulate the expression of osteoblast markers involved in bone formation . In addition, Ma et al. indicated that fluoride could decrease the methylation level of specific CpGs in the DNA promoter region and up-regulate the expression level of BMP2 . We hypothesized that genetic or epigenetic alterations in BMP2 may be a risk factor for skeletal fluorosis based on the foregoing evidence, but more investigation was needed.
The Wnt/B-catenin signaling pathway plays key roles in controlling the differentiation of bone marrow stem cells (BMSCs) . When the Wnt/B-catenin pathway is activated, the key molecule B-catenin is released from a complex composed of adenomatous polyposis coli (APC), glycogen synthase kinase 3B (GSK3B), and axin, thereby regulating target gene expression . In addition, BMP signaling can also stimulate differentiation of BMSCs, such as BMP2 . A previous study suggested that B-catenin signaling was activated via BMP2 to induce osteoblast differentiation . BMP2 promoted bone regeneration of BMSCs in diabetic by promoting the Wnt signaling pathway through regulating B-catenin and GSK3B expression . All of these findings revealed that BMP2 stimulated the Wnt/B-catenin signaling pathway, which is important in osteoblastic differentiation and bone formation. However, whether the BMP2-Wnt/B-catenin axis is involved in the pathogenesis of skeletal fluorosis remains to be verified.
In this study, we not only indicated that fluoride induced the hypopethylation of BMP2, but also found that knockdown of BMP2 decreased B-catenin expression in osteoblasts treated with fluoride. Based on these findings and rationales, we aimed to investigate the effects of the BMP2-Wnt/B-catenin axis on the growth of treated cells, providing feasible theoretical support for better prevention and treatment of skeletal fluorosis.
Cell culture and treatments
Murine osteoblast MC3T3-E1 cells were obtained from American Type Culture Collection (ATCC, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, CA, USA) containing 10% fetal bovine serum (FBS, Invitrogen, CA, USA) with 1% penicillin-streptomycin. Cells were maintained at 37?°C under humidified condition with 5% CO2. After reaching to a steady state of exponential growth, the cells were treated with sodium fluoride (NaF) (purity: 99.0%, Wako Pure Chemical Industries Ltd.,
Fluoride promoted proliferation and differentiation of MC3T3-E1 cells
To investigate the role of fluoride on the proliferation and differentiation of osteoblasts, MC3T3-E1 cells were treated with NaF at different concentrations (0, 125, 250, 500, 1000 uM) for different time (24, 48, 72, 96 h). Results from MTT assays showed that fluoride treatment induced similar uptrends on cell viability at 48 and 72 h (Fig. 1A). Treatment with NaF at the concentration of 500 and 1000 uM significantly increased the ALP activities of cells (Fig. 1B), indicating that Over 500 uM
Skeletal fluorosis patients have been more broadly dispersed in recent years, and the disease has become a serious public health problem worldwide . Although previous studies suggested that methionine combined with vitamin E , choline , taurine  and pomegranate juice  might have application value in the treatment of skeletal fluorosis, it was difficult to find effective target therapies due to the unclear pathophysiology of skeletal fluorosis . In this study, we
In conclusion, our findings suggested that hypomethylation of BMP2 was linked to the pathogenesis of skeletal fluorosis by promoting the proliferation and differentiation of osteoblasts, which was mediated via activating the Wnt/B-catenin pathway in vitro. These observations provided a feasible theoretical basis for the treatment of skeletal fluorosis.
This work was supported by National Natural Science Foundation of China (No. 81660521, No. 81660324) and Autonomous Region’s 13th Five-Year Key Discipline-Basic Medicine.