Fluorosed mouse ameloblasts have increased SATB1 retention and Gαq activity.

Yan Zhang,Sunita Ho,Orapin Horst,Ji-Yeon Kim,Pamela K Den Besten,Yukiko Nakano,Ralf J Radlanski,Li Zhu

doi:10.1371/journal.pone.0103994

Abstract

Dental fluorosis is characterized by subsurface hypomineralization and increased porosity of enamel, associated with a delay in the removal of enamel matrix proteins. To investigate the effects of fluoride on ameloblasts, A/J mice were given 50 ppm sodium fluoride in drinking water for four weeks, resulting serum fluoride levels of 4.5 µM, a four-fold increase over control mice with no fluoride added to drinking water. MicroCT analyses showed delayed and incomplete mineralization of fluorosed incisor enamel as compared to control enamel. A microarray analysis of secretory and maturation stage ameloblasts microdissected from control and fluorosed mouse incisors showed that genes clustered with Mmp20 appeared to be less downregulated in maturation stage ameloblasts of fluorosed incisors as compared to control maturation ameloblasts. One of these Mmp20 co-regulated genes was the global chromatin organizer, special AT-rich sequence-binding protein-1 (SATB1). Immunohistochemical analysis showed increased SATB1 protein present in fluorosed ameloblasts compared to controls. In vitro, exposure of human ameloblast-lineage cells to micromolar levels of both NaF and AlF3 led to a significantly increase in SATB1 protein content, but not levels of Satb1 mRNA, suggesting a fluoride-induced mechanism protecting SABT1 from degradation. Consistent with this possibility, we used immunohistochemistry and Western blot to show that fluoride exposed ameloblasts had increased phosphorylated PKCα both in vivo and in vitro. This kinase is known to phosphorylate SATB1, and phosphorylation is known to protect SATB1 from degradation by caspase-6. In addition, production of cellular diacylglycerol (DAG) was significantly increased in fluorosed ameloblasts, suggesting that the increased phosphorylation of SATB1 may be related to an effect of fluoride to enhance Gαq activity of secretory ameloblasts.

Highlights

Enamel is the highly mineralized protective outer covering of teeth, conferring strength, abrasion resistance, and protection of the teeth in the oral cavity [1]
A gene cluster containing Mmp20 was selected for further analysis, as we have previously showed that MMP20 expression was altered by fluoride in cultured human ameloblast-lineage cells [24]
Serum fluoride levels in mice exposed to 50 ppm sodium fluoride increased 4 fold compared with controls

Summary

Introduction

Enamel is the highly mineralized protective outer covering of teeth, conferring strength, abrasion resistance, and protection of the teeth in the oral cavity [1]. In vitro studies have shown diverse fluoride-induced effects on ameloblasts that include enhanced proliferation, apoptosis, upregulation of stress related proteins, and elevated F-actin [15,16,17,18], are related to treatment dose, duration and cell type. It is not yet clear whether fluoride at physiological levels can directly affect ameloblast function, or whether fluorosis results entirely from matrix mediated effects on mineralization [4,19]. To investigate whether fluoride influences activation of G protein in ameloblasts, we used the A/J mouse model, which is highly susceptible to dental fluorosis [21], to determine the potential effects of fluoride on ameloblasts, including Gaq activity

Materials and Methods

Results

Discussion