Influence of Calcium Supplementation against Fluoride-Mediated Osteoblast Impairment in Vitro: Involvement of the Canonical Wnt/β-Catenin Signaling Pathway

Fluoride (F) is capable of promoting abnormal proliferation and differentiation in primary cultured mouse osteoblasts (OB cells), although the underlying mechanism responsible remains rare. This study aimed to explore the roles of wingless and INT-1 (Wnt) signaling pathways and screen appropriate doses of calcium (CA²⁺) to alleviate the sodium fluoride (NaF)-induced OB cell toxicity. For this, we evaluated the effect of dickkopf-related protein 1 (DKK1) and CA²⁺ on mRNA levels of wingless/integrated 3a (Wnt3a), low-density lipoprotein receptor-related protein 5 (LRP5), dishevelled 1 (Dv1), glycogen synthase kinase 3β (GSK3β), β-catenin, lymphoid enhancer binding factor 1 (LEF1), and cellular myelocytomatosis oncogene (cMYC), as well as Ccnd1 (Cyclin D1) in OB cells challenged with 10^-6 mol/L NaF for 24 h. The demonstrated data showed that F significantly increased the OB cell proliferation rate. Ectogenic 0.5 mg/L DKK1 significantly inhibited the proliferation of OB cells induced by F. The mRNA expression levels of Wnt3a, LRP5, Dv1, LEF1, β-catenin, cMYC, and Ccnd1 were significantly increased in the F group, while significantly decreased in the 10^-6 mol/L NaF + 0.5 mg/L DKK1 (FY) group. The mRNA expression levels of Wnt3a, LRP5, β-catenin, and cMYC were significantly decreased in the 10^-6 mol/L NaF + 2 mmol/L CaCl₂ (F+CaII) group. The protein expression levels of Wnt3a, Cyclin D1, cMYC, and β-catenin were significantly increased in the F group, whereas they were decreased in the F+CaII group. However, the mRNA and protein expression levels of GSK3β were significantly decreased in the F group while significantly increased in the F+CaII group. In summary, F activated the canonical Wnt/β-catenin pathway and changed the related gene expression and β-catenin protein location in OB cells, promoting cell proliferation. CA²⁺ supplementation (2 mmol/L) reversed the expression levels of genes and proteins related to the canonical Wnt/β-catenin pathway.