Fluoride induces hypomethylation of BMP2 and activates osteoblasts through the Wnt/β-catenin signaling pathway

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/beta-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 differen-tiation of MC3T3-E1. The expression of beta-catenin, glycogen synthase kinase 3 beta (GSK3 beta), wingless/integrated 3 alpha (Wnt3 alpha), 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 beta-catenin significantly inhibited the differentiation of osteoblasts induced by fluoride. Conclusion: Fluoride contributed to proliferation and differentiation of osteoblasts through BMP2-Wnt/beta-catenin axis, providing a feasible theoretical basis for the treatment of skeletal fluorosis.