Excessive NaF Inhibits Vitamin D Receptor Expression in MC3T3-E1 Cells

Objective: The prolonged ingestion of excessively high amounts of fluoride leads to hypomineralization in bone and teeth. In this study, we examined effect of NaF at an excessive dose on the expression of mRNA for phenotypes related to osteoblastic function including mineralization in MC3T3-E1. Because vitamin D (VD) deficiency was intimately involved in hypomineralization such as osteomalacia, the effect on expression of mRNA for VD receptor (VDR) was examined. Methods: MC3T3-E1 cells were cultured for 6-72 hours in the presence of NaF (0.1-10 mM), and the expression of mRNA for VDR and osteoblastic functional markers, such as type 1 procollagen (Col), osteopontin (OPN), alkaline phosphatase (ALP), core binding factor alpha 1 (Cbfa1), parathyroid hormone receptor (PTHR), transforming growth factor beta (TGF) and basic fibroblast growth factor (bFGF) were analyzed by the reverse transcription-polymerase chain reaction procedure. The influences of phorbol 12-myristate 13-acetate (PMA, 1nM), a protein kinase C (PKC) activator, and calphostine C (0.1 and 10 nM), a PKC inhibitor on the expression of VDR mRNA were also examined. Calcium content in the cells layer was determined to estimate mineralization. Results: NaF (2-10 mM) dose-dependently inhibited mineralization, cell proliferation activity and expressions of mRNA for VDR, Col, OPN and ALP in the MC3T3-E1 cells. Time course study showed that inhibition of mRNA expression for VDR preceded inhibition of Col, OPN and ALP. On the contrary, expressions of mRNA for Cbfa1, PTHR, TGF and bFGF were unchanged by the treatment with NaF. NaF-inhibited expression of VDR mRNA was suppressed by the addition of PMA. Calphostine C inhibited VDR expression in MC3T3-E1 cells. These results suggested that NaF inhibited VDR expression through the PKC pathway. Conclusion: NaF at an excessive dose inhibits VDR expression in osteoblastic cells, which may be involved in hypomineralization in bone.