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 [3], leading to dental fluorosis and skeletal fluorosis [[4], [5], [6], [7]]. Patients with skeletal fluorosis display multiple bone lesions, such as osteoporosis, osteosclerosis, calcification of ligaments and osteopenia [8], 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 [9]. 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 [[10], [11], [12]]. 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 [16]. 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 [17]. 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) [18]. 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 [19]. In addition, BMP signaling can also stimulate differentiation of BMSCs, such as BMP2 [20]. A previous study suggested that B-catenin signaling was activated via BMP2 to induce osteoblast differentiation [21]. BMP2 promoted bone regeneration of BMSCs in diabetic by promoting the Wnt signaling pathway through regulating B-catenin and GSK3B expression [22]. 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.