Retinoic Acid Excess Impairs Amelogenesis Inducing Enamel Defects

Supawich Morkmued,Brigitte Schuhbaur,Pascal Dollé,Eric Mathieu,Virginie Laugel-Haushalter,Joseph Hemmerlé,Karen Niederreither,Agnès Bloch-Zupan

doi:10.3389/fphys.2016.00673

Abstract

Abnormalities of enamel matrix proteins deposition, mineralization, or degradation during tooth development are responsible for a spectrum of either genetic diseases termed Amelogenesis imperfecta or acquired enamel defects. To assess if environmental/nutritional factors can exacerbate enamel defects, we investigated the role of the active form of vitamin A, retinoic acid (RA). Robust expression of RA-degrading enzymes Cyp26b1 and Cyp26c1 in developing murine teeth suggested RA excess would reduce tooth hard tissue mineralization, adversely affecting enamel. We employed a protocol where RA was supplied to pregnant mice as a food supplement, at a concentration estimated to result in moderate elevations in serum RA levels. This supplementation led to severe enamel defects in adult mice born from pregnant dams, with most severe alterations observed for treatments from embryonic day (E)12.5 to E16.5. We identified the enamel matrix proteins enamelin (Enam), ameloblastin (Ambn), and odontogenic ameloblast-associated protein (Odam) as target genes affected by excess RA, exhibiting mRNA reductions of over 20-fold in lower incisors at E16.5. RA treatments also affected bone formation, reducing mineralization. Accordingly, craniofacial ossification was drastically reduced after 2 days of treatment (E14.5). Massive RNA-sequencing (RNA-seq) was performed on E14.5 and E16.5 lower incisors. Reductions in Runx2 (a key transcriptional regulator of bone and enamel differentiation) and its targets were observed at E14.5 in RA-exposed embryos. RNA-seq analysis further indicated that bone growth factors, extracellular matrix, and calcium homeostasis were perturbed. Genes mutated in human AI (ENAM, AMBN, AMELX, AMTN, KLK4) were reduced in expression at E16.5. Our observations support a model in which elevated RA signaling at fetal stages affects dental cell lineages. Thereafter enamel protein production is impaired, leading to permanent enamel alterations.

Highlights

Enamel formation is a unique biomineralization process involving a highly organized matrix protein scaffold deposition and degradation, leading to hydroxyapatite crystal nucleation generating a dense and tightly aligned network of hydroxyapatite crystals
To further characterize nutritional and environmental factors regulating enamel formation, we have focused on the role of retinoic acid (RA), the main active form of vitamin A that plays key roles during vertebrate development (Niederreither and Dollé, 2008)
To analyze RA-dependent tooth alterations, we employed a protocol where RA added to the food supply was administered to pregnant CD1 mice (Niederreither et al, 2002), at a concentration of 0.4 mg/g food beginning at E12.5 or later

Summary

Introduction

Enamel formation is a unique biomineralization process involving a highly organized matrix protein scaffold deposition and degradation, leading to hydroxyapatite crystal nucleation generating a dense and tightly aligned network of hydroxyapatite crystals. Ameloblasts are cells of ectodermal origin responsible for enamel development. These cells secrete enamel proteins, which are required for correct mineralization and structural maturation. Enamel formation is characterized by inductive, secretory, and maturation stages. At the secretory stage, polarized differentiated ameloblasts release enamel proteins, contributing to the enamel matrix (Bei, 2009). At the maturation stage, ameloblasts absorb water and organic matrix. Mature enamel is extremely strong, because of the density and fine organization of its crystal layers (Bei, 2009; Simmer et al, 2010)

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