Flouride Promotes Viability and Differentiation of Osteoblast-Like Saos-2 Cells Via BMP/Smads Signaling Pathway

Abstract

The BMP/Smad signaling pathway plays an important role in the viability and differentiation of osteoblast; however, it is not clear whether this pathway is involved in the fluoride-induced osteoblast differentiation. In this study, we investigated the role of BMP/Smad signaling pathway in fluoride-induced osteoblast-like Saos-2 cells differentiation. Cells were exposed to fluoride of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mM), and cell proliferation was determined using WST assays. The expression of osteoblast marker genes such as osteocalcin (BGP) and bone alkaline phosphatase (BALP) were detected by qRT-PCR. We found that fluoride enhanced the proliferation of Saos-2 cells in a dose-dependent manner and 0.2 mM of fluoride resulted in a higher expression of osteoblast marker genes. In addition, immunofluorescence analysis showed that the promotion effects of 0.2 mM of fluoride on Saos-2 cells differentiation were associated with the activation of the BMP/Smad pathway. Expression of phosphorylated Smad1/5(p-Smad1/5) was higher in cells exposed to 0.2 mM of fluoride. Plasmid expression vectors encoding the short hairpin RNA (shRNA) targeting Smad4 gene were used to block the BMP/Smad pathway, which resulted in a significantly reduced expression of BGP and BALP as well as their corresponding mRNA. The mRNA levels after transfection remained low even in the presence of fluoride. The present results reveal that BMP/Smad signaling pathway was altered during the period of osteogenesis, and that the activities of p-Smad1/5 were required for Saos-2 cells viability and differentiation induced by fluoride.

Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism

“…Given the fluorinated nature of triazoles, it is plausible that free fluoride contributed to these effects. This is indicated by the effect of fluoride on osteoblast proliferation that we observed, which is consistent with previous studies (31)(32)(33). However, the finding that fluconazole did not elicit a similar response in osteoblasts suggests that this effect is specific to voriconazole.…”

“…As these findings are similar to the clinical syndrome of skeletal fluorosis in humans, a pathogenic mechanism has been suggested whereby chronic daily consumption of trifluorinated voriconazole molecules leads to excess plasma fluoride, which then promotes the development of periostitis by enhancing the activity of osteoblasts (11,13,17). Although this is consistent with the known clinical and cellular effects of fluoride (19,20,31,32,40), this hypothesis has not been directly evaluated for voriconazole-associated periostitis. Therefore, the goal of this study was to evaluate this hypothesis by assessing the effects of voriconazole exposure on osteoblastic activity in vitro.…”

“…Fluoride has the ability to stimulate new bone formation and to augment bone mass significantly, primarily by enhancing osteoblastic activity (31)(32)(33). Since voriconazole is a trifluorinated molecule and elevated plasma fluoride concentrations have been observed in patients with voriconazole-associated periostitis, it has been proposed that circulating free fluoride and its effects on osteoblasts may be involved in the mechanisms of voriconazoleinduced periostitis.…”

“…In mammals, UPR signaling involves three ER-localized signal transducers, namely, inositol-requiring enzyme 1 (IRE1), double-stranded RNA-activated Prklike ER kinase (PERK), and activating transcription factor 6 (ATF6) (28,29); roles for IRE1 and PERK during osteoblast differentiation have been described (27,30). Fluoride, similar to other Wnt agonists, was documented previously to have anabolic effects on bone metabolism and to stimulate osteoblastic bone formation in vitro through activation of the canonical Wnt/␤-catenin signaling pathway (31)(32)(33) and the endoplasmic reticulum (ER) stress response and subsequent unfolded protein response (UPR) (34,35).…”

Differential Effects of Fluoride During Osteoblasts Mineralization in C57BL/6J and C3H/HeJ Inbred Strains of Mice

“…The most common fluoride effects related to bone cells are the following: induction of apoptosis, induction of oxidative damage/stress, and regulation of the mitogenactivated protein kinase (MAPK). In addition, fluoride can promote osteoblast differentiation and proliferation through activation of BMP/Smad pathway [42].…”