Tooth enamel is the outer covering of tooth crowns, the hardest material in the mammalian body, yet fracture resistant. The extremely high content of 95 wt% calcium phosphate in healthy adult teeth is achieved through mineralization of a proteinaceous matrix that changes in abundance and composition. Enamel-specific proteins and proteases are known to be critical for proper enamel formation. Recent proteomics analyses revealed many other proteins with their roles in enamel formation yet to be unraveled. Although the exact protein composition of healthy tooth enamel is still unknown, it is apparent that compromised enamel deviates in amount and composition of its organic material. Why these differences affect both the mineralization process before tooth eruption and the properties of erupted teeth will become apparent as proteomics protocols are adjusted to the variability between species, tooth size, sample size and ephemeral organic content of forming teeth. This review summarizes the current knowledge and published proteomics data of healthy and diseased tooth enamel, including advancements in forensic applications and disease models in animals. A summary and discussion of the status quo highlights how recent proteomics findings advance our understating of the complexity and temporal changes of extracellular matrix composition during tooth enamel formation.

Tooth enamel forms in an ephemeral protein matrix where changes in protein abundance, composition and posttranslational modifications are critical to achieve healthy enamel properties. Amelogenin (AMELX) with its splice variants is the most abundant enamel matrix protein, with only one known phosphorylation site at serine 16 shown in vitro to be critical for regulating mineralization. The phosphorylated form of AMELX stabilizes amorphous calcium phosphate, while crystalline hydroxyapatite forms in the presence of the unphosphorylated protein. While AMELX regulates mineral transitions over space and time, it is unknown whether and when un-phosphorylated amelogenin occurs during enamel mineralization. This study aims to reveal the spatiotemporal distribution of the cleavage products of the most abundant AMLEX splice variants including the full length P173, the shorter leucine-rich amelogenin protein (LRAP), and the exon 4-containing P190 in forming enamel, all within the context of the changing enamel matrix proteome during mineralization. We microsampled permanent pig molars, capturing known stages of enamel formation from both crown surface and inner enamel. Nano-LC-MS/MS proteomic analyses after tryptic digestion rendered more than 500 unique protein identifications in enamel, dentin, and bone. We mapped collagens, keratins, and proteolytic enzymes (CTSL, MMP2, MMP10) and determined distributions of P173, LRAP, and P190 products, the enamel proteins enamelin (ENAM) and ameloblastin (AMBN), and matrix-metalloprotease-20 (MMP20) and kallikrein-4 (KLK4). All enamel proteins and KLK4 were near-exclusive to enamel and in excellent agreement with published abundance levels. Phosphorylated P173 and LRAP products decreased in abundance from recently deposited matrix toward older enamel, mirrored by increasing abundances of testicular acid phosphatase (ACPT). Our results showed that hierarchical clustering analysis of secretory enamel links closely matching distributions of unphosphorylated P173 and LRAP products with ACPT and non-traditional amelogenesis proteins, many associated with enamel defects. We report higher protein diversity than previously published and Gene Ontology (GO)-defined protein functions related to the regulation of mineral formation in secretory enamel (e.g., casein α-S1, CSN1S1), immune response in erupted enamel (e.g., peptidoglycan recognition protein, PGRP), and phosphorylation. This study presents a novel approach to characterize and study functional relationships through spatiotemporal mapping of the ephemeral extracellular matrix proteome.

The mechanisms that regulate post-natal growth of the craniofacial complex and that ultimately determine the size and shape of our faces are not well understood. Hippo signaling is a general mechanism to control tissue growth and organ size, and although it is known that Hippo signaling functions in neural crest specification and patterning during embryogenesis and before birth, its specific role in postnatal craniofacial growth remains elusive. We have identified the transcription factor FoxO6 as an activator of Hippo signaling regulating neonatal growth of the face. During late stages of mouse development, FoxO6 is expressed specifically in craniofacial tissues and FoxO6^-/- mice undergo expansion of the face, frontal cortex, olfactory component and skull. Enlargement of the mandible and maxilla and lengthening of the incisors in FoxO6^-/- mice are associated with increases in cell proliferation. In vitro and in vivo studies demonstrated that FoxO6 activates Lats1 expression, thereby increasing Yap phosphorylation and activation of Hippo signaling. FoxO6^-/- mice have significantly reduced Hippo Signaling caused by a decrease in Lats1 expression and decreases in Shh and Runx2 expression, suggesting that Shh and Runx2 are also linked to Hippo signaling. In vitro, FoxO6 activates Hippo reporter constructs and regulates cell proliferation. Furthermore PITX2, a regulator of Hippo signaling is associated with Axenfeld-Rieger Syndrome causing a flattened midface and we show that PITX2 activates FoxO6 expression. Craniofacial specific expression of FoxO6 postnatally regulates Hippo signaling and cell proliferation. Together, these results identify a FoxO6-Hippo regulatory pathway that controls skull growth, odontogenesis and face morphology. The basic question of how human faces develop, undergo morphogenesis and grow after birth to define our final characteristic shape has been studied from the earliest days of comparative vertebrate developmental research. While many studies have shown the factors and mechanisms that contribute to the cells and tissues of the face during embryology, fewer studies have determined mechanisms that promote face growth after birth and into childhood. In our quest to understand developmental mechanisms of facial growth we used murine gene expression and bioinformatics analyses combined with human 3D facial variations and genome-wide association studies to identify genes and variants controlling post-natal face growth. Bioinformatics analyses of mouse craniofacial gene expression identified FoxO6 as a transcription factor expressed at late stages of face development. Ablation of FoxO6 in the mouse resulted in specific anterior growth of the mouse face. The increased FOXO6 expression activated Hippo signaling to reduce face growth. These data indicate that changes in FOXO6 expression control face growth during early childhood.

An unresolved problem in tooth enamel studies has been to analyze simultaneously and with sufficient spatial resolution both mineral and organic phases in their three dimensional (3D) organization in a given specimen. This study aims to address this need using high-resolution imaging to analyze the 3D structural organization of the enamel matrix, especially amelogenin, in relation to forming enamel crystals. Chemically fixed hemi-mandibles from wild type mice were embedded in LR White acrylic resin, polished and briefly etched to expose the organic matrix in developing tooth enamel. Full-length amelogenin was labeled with specific antibodies and 10 nm immuno-gold. This allowed us to use and compare two different high-resolution imaging techniques for the analysis of uncoated samples. Helium ion microscopy (HIM) was applied to study the spatial organization of organic and mineral structures, while field emission scanning electron microscopy (FE-SEM) in various modes, including backscattered electron detection, allowed us to discern the gold-labeled proteins. Wild type enamel in late secretory to early maturation stage reveals adjacent to ameloblasts a lengthwise parallel alignment of the enamel matrix proteins, including full-length amelogenin proteins, which then transitions into a more heterogeneous appearance with increasing distance from the mineralization front. The matrix adjacent to crystal bundles forms a smooth and lacey sheath, whereas between enamel prisms it is organized into spherical components that are interspersed with rod-shaped protein. These findings highlight first, that the heterogeneous organization of the enamel matrix can be visualized in mineralized en bloc samples. Second, our results illustrate that the combination of these techniques is a powerful approach to elucidate the 3D structural organization of organic matrix molecules in mineralizing tissue in nanometer resolution.

Excess albumin in enamel is a characteristic of the prevalent developmental dental defect known as chalky teeth or molar hypomineralization (MH). This study uses proteomic analyses of pig teeth to discern between developmental origin and post-eruptive contamination and to assess the similarity to hypomineralized human enamel. Here, the objective is to address the urgent need for an animal model to uncover the etiology of MH and to improve treatment. Porcine enamel is chalky and soft at eruption; yet, it hardens quickly to form a hard surface and then resembles human teeth with demarcated enamel opacities. Proteomic analyses of enamel from erupted teeth, serum, and saliva from pigs aged 4 (n = 3) and 8 weeks (n = 2) and human (n = 4) molars with demarcated enamel opacities show alpha-fetoprotein (AFP). AFP expression is limited to pre- and perinatal development and its presence in enamel indicates pre- or perinatal inclusion. In contrast, albumin is expressed after birth, indicating postnatal inclusion into enamel. Peptides were extracted from enamel and analyzed by nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) after tryptic digestion. The mean total protein number was 337 in the enamel of all teeth with 13 different unique tryptic peptides of porcine AFP in all enamel samples but none in saliva samples. Similarities in the composition, micro-hardness, and microstructure underscore the usefulness of the porcine model to uncover the MH etiology, cellular mechanisms of albumin inclusion, and treatment for demarcated opacities.