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

Abstract

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 (Ca 2+ ) to alleviate the sodium fluoride (NaF)-induced OB cell toxicity. For this, we evaluated the effect of dickkopf-related protein 1 (DKK1) and Ca 2+ 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 CaCl 2 (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. Ca 2+ supplementation (2 mmol/L) reversed the expression levels of genes and proteins related to the canonical Wnt/β-catenin pathway.

7,8-Dihydroxyflavone modulates bone formation and resorption and ameliorates ovariectomy-induced osteoporosis

“…Thus, the content of the β-catenin protein in normal cells is generally kept very low. In the presence of Wnt ligands, on the other hand, GSK3β is phosphorylated to yield inactive p-GSK3β, and the degrading complex is depolymerized, which allows β-catenin to avoid the degradation by the proteasome and accumulate in the cytoplasm ( Chen et al, 2021 ; Wang et al, 2019 ). The accumulated β-catenin translocates into the nucleus, binds with T-cell-specific transcription factor/lymphoid enhancer binding factor, and activates a series of target gene, such as cyclin D1, Runx2, and Osterix.…”

Dkk1 inhibits malignant transformation induced by Bmi1 via the β‐catenin signaling axis in WB‐F344 oval cells

“…In our results, compared with their levels in the WB-F344 cell control group and the Bmi1-overexpressing group, the expression of AFP and ALB was decreased in the WB- downstream Ca2+-sensitive metabolic processes [37]. A certain amount of Ca2+ supplementation can reverse Dkk1-mediated Wnt/β-Catenin/canonical pathway-related genes and proteins in primary cultured mouse osteoblasts [38].…”

“…These oscillatory Ca2+ signals are thought to transmit the information encoded in the extracellular stimulus to downstream Ca2+‐sensitive metabolic processes [37]. A certain amount of Ca2+ supplementation can reverse Dkk1‐mediated Wnt/β‐catenin/canonical pathway‐related genes and proteins in primary cultured mouse osteoblasts [38].…”

Prostate cancer‐secreted CCN3 uses the GSK3β and β‐catenin pathways to enhance osteogenic factor levels in osteoblasts

“…The intracellular enzyme, GSK3β, is an important component in the promotion of osteoblast expansion and function through Wnt signaling 24,25 . GSK3β is therefore a potential therapeutic target in bone diseases 26 .…”