High-fluoride promoted phagocytosis-induced apoptosis in a matured ameloblast-like cell line

Abstract

Endocytosis and phagocytosis are important physiologic activities occurring during ameloblast differentiation. We have previously found that excess fluoride inhibited ameloblasts endocytotic functions. Here, we hypothesized that increasing amounts of fluoride affects ameloblast phagocytotic function during their differentiation. Using cell culture, we first induced maturation of the mouse ameloblast-like LS8 cells by treatment with exogenous retinoic acid (RA) and dexamethasone (DEX). We measured their phagocytotic activity by fluorescent microscopy using a live cell visualization station. We found that with increasing amounts of fluoride exposure phagocytosis by LS8 cells matured by RA/DEX treatment and with fluoride (NaF) and only fluoride treated with LS8 cells demonstrating upregulated amounts of the phagocytotic protein markers, LAMP1 and CD68. The connection between phagocytosis and apoptosis was confirmed by the increased number of phagocytotic vacuole-like structures and the heterochromatin margination phenomenon observed in the RA/DEX NaF treated group. The increase in albumin uptake by ameloblasts was confirmed using whole organ culture of incisor tooth germs. Here, in fluoride treated mature ameloblasts, we observed higher albumin uptake, which was accompanied by decreased amounts of the apoptosis marker, Bcl-2 and up-regulated expression of CD68. From these observations, we infer that high doses of fluoride may cause apoptosis by increasing the uptake of large protein particles in matured ameloblasts and loss of Bcl-2 signals might be involved in this process.

Nanostructured Ti surfaces and retinoic acid/dexamethasone present a spatial framework for the maturation and amelogenesis of LS-8 cells

“…However, in RA medium, the proliferation of LS-8 cells was significantly suppressed and fewer cells and pseudopodia were observed on all modified Ti surfaces. Moreover, LS-8 cells in six-well culture plates showed a decrease in proliferation, suggesting that the recession of proliferation may be associated with LS-8 cell maturation with exogenous RA and DEX, independent of different surface chemical compositions 44,45 and nanostructure.…”

Retinoic Acid Excess Impairs Amelogenesis Inducing Enamel Defects

“…These variations, also observed in acquired enamel defects, have been proposed to be due to environmental excess in fluoride (Yang et al, 2015), or to other nutritional, environmental, or behavioral changes (Li et al, 2013), along with genetic makeup of an individual. Retinoids are regulators of skeletal growth and patterning, known to lead to skeletal fragility when given in excess (Henning et al, 2015).…”

“…Indeed, ameloblasts are highly sensitive to their environment (Simmer et al, 2010). Body stressors including high fever (Ryynänen et al, 2014), excess fluoride (Yang et al, 2015), and endocrine disrupters such as bisphenol A disrupt ameloblast function (Jedeon et al, 2014). It is likely that a variety of factors contribute to clinical heterogeneity found in AI and to acquired enamel defects such as molar incisor hypomineralization (MIH) or hypomineralized second primary molars (HSPM; Alaluusua, 2010; Jeremias et al, 2013).…”

Fluoride Induces Apoptosis in Mammalian Cells: In Vitro and In Vivo Studies

“…Fluoride promoted cytochromec release, up-regulation of UCP2, attenuation of ATP synthesis, and H2AX phosphorylation (γH2AX) (18). High amounts of fluoride induced more apoptosis/dead cells and reduced the expression of Bcl-2 decreased expression along with up-regulated expression of CD68 (15,22). Furthermore, a decreased expression and increased phosphorylation of Foxo1 in NaF-treated ameloblast cells has been shown (24).…”

“…Ameloblasts also showed high sensitivity to fluoride with respect to cytotoxicity, especifically apoptosis. Flow cytometry showed that both 10 μM and 20 μM NaF significantly increased the apoptotic index of ameloblast-lineage cells (15). Morphologically, high fluoride incubation damaged the ameloblast ultrastructure manifesting a series of intracellular stress responding cell organelle destruction, and a marked increase in the expression of apoptotic genes (16)(17)(18).…”