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Introduction: Recombinant DNA-produced amelogenin protein was compared with calcium hydroxide in a study of immature apex closure conducted in 24 young mongrel dogs., Methods: Root canals of maxillary and mandibular right premolars (n = 240) were instrumented and left open for 14 days. Canals were cleansed, irrigated, and split equally for treatment with recombinant mouse amelogenin (n = 120) or calcium hydroxide (n = 120)., Results: After 1, 3, and 6 months, the animals were sacrificed and the treated teeth recovered for histologic assessment and immunodetection of protein markers associated with odontogenic cells. After 1 month, amelogenin-treated canals revealed calcified tissue formed at the apical foramen and a pulp chamber containing soft connective tissue and hard tissue; amelogenin-treated canals assessed after 3- and 6-month intervals further included apical tissue functionally attached to bone by a periodontal ligament. In contrast, calcified apical tissue was poorly formed in the calcium hydroxide group, and soft connective tissue within the pulp chamber was not observed., Conclusions: The findings from this experimental strategy suggest recombinant amelogenin protein can signal cells to enhance apex formation in nonvital immature teeth and promote soft connective tissue regeneration., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Patients with Axenfeld-Rieger Syndrome (ARS) present various dental abnormalities, including hypodontia, and enamel hypoplasia. ARS is genetically associated with mutations in the PITX2 gene, which encodes one of the earliest transcription factors to initiate tooth development. Thus, Pitx2 has long been considered as an upstream regulator of the transcriptional hierarchy in early tooth development. However, because Pitx2 is also a major regulator of later stages of tooth development, especially during amelogenesis, it is unclear how mutant forms cause ARS dental anomalies. In this report, we outline the transcriptional mechanism that is defective in ARS. We demonstrate that during normal tooth development Pitx2 activates Amelogenin (Amel) expression, whose product is required for enamel formation, and that this regulation is perturbed by missense PITX2 mutations found in ARS patients. We further show that Pitx2-mediated Amel activation is controlled by chromatin-associated factor Hmgn2, and that Hmgn2 prevents Pitx2 from efficiently binding to and activating the Amel promoter. Consistent with a physiological significance to this interaction, we show that K14-Hmgn2 transgenic mice display a severe loss of Amel expression on the labial side of the lower incisors, as well as enamel hypoplasia-consistent with the human ARS phenotype. Collectively, these findings define transcriptional mechanisms involved in normal tooth development and shed light on the molecular underpinnings of the enamel defect observed in ARS patients who carry PITX2 mutations. Moreover, our findings validate the etiology of the enamel defect in a novel mouse model of ARS.

Cementum is the outer-, mineralized-tissue covering the tooth root and an essential part of the system of periodontal tissue that anchors the tooth to the bone. Periodontal disease results from the destructive behavior of the host elicited by an infectious biofilm adhering to the tooth root and left untreated, may lead to tooth loss. We describe a novel protocol for identifying peptide sequences from native proteins with the potential to repair damaged dental tissues by controlling hydroxyapatite biomineralization. Using amelogenin as a case study and a bioinformatics scoring matrix, we identified regions within amelogenin that are shared with a set of hydroxyapatite-binding peptides (HABPs) previously selected by phage display. One 22-amino acid long peptide regions referred to as amelogenin-derived peptide 5 (ADP5) was shown to facilitate cell-free formation of a cementum-like hydroxyapatite mineral layer on demineralized human root dentin that, in turn, supported attachment of periodontal ligament cells in vitro. Our findings have several implications in peptide-assisted mineral formation that mimic biomineralization. By further elaborating the mechanism for protein control over the biomineral formed, we afford new insights into the evolution of protein-mineral interactions. By exploiting small peptide domains of native proteins, our understanding of structure-function relationships of biomineralizing proteins can be extended and these peptides can be utilized to engineer mineral formation. Finally, the cementomimetic layer formed by ADP5 has the potential clinical application to repair diseased root surfaces so as to promote the regeneration of periodontal tissues and thereby reduce the morbidity associated with tooth loss.

Fully mature enamel is about 98% mineral by weight. While mineral crystals appear very early during its formative phase, the newly secreted enamel is a soft gel-like matrix containing several enamel matrix proteins of which the most abundant is amelogenin (Amelx). Histological analysis of mineralized dental enamel reveals markings called cross-striations associated with daily increments of enamel formation, as evidenced by injections of labeling dyes at known time intervals. The daily incremental growth of enamel has led to the hypothesis that the circadian clock might be involved in the regulation of enamel development. To identify daily rhythms of clock genes and Amelx, we subjected murine ameloblast cells to serum synchronization to analyze the expression of the circadian transcription factors Per2 and Bmal1 by real-time PCR. Results indicate that these key genetic regulators of the circadian clock are expressed in synchronized murine ameloblast cell cultures and that their expression profile follows a circadian pattern with acrophase and bathyphase for both gene transcripts in antiphase. Immunohistological analysis confirms the protein expression of Bmal and Cry in enamel cells. Amelx expression in 2-day postnatal mouse molars dissected every 4 hours for a duration of 48 hours oscillated with an approximately 24-hour period, with a significant approximately 2-fold decrease in expression during the dark period compared to the light period. The expression of genes involved in bicarbonate production (Car2) and transport (Slc4a4), as well as in enamel matrix endocytosis (Lamp1), was greater during the dark period, indicating that ameloblasts express these proteins when Amelx expression is at the nadir. The human and mouse Amelx genes each contain a single nonconserved E-box element within 10 kb upstream of their respective transcription start sites. We also found that within 2 kb of the transcription start site of the human NFYA gene, which encodes a positive regulator of amelogenin, there is an E-box element that is conserved in rodents and other mammals. Moreover, we found that Nfya expression in serum-synchronized murine ameloblasts oscillated with a strong 24-hour rhythm. Taken together, our data support the hypothesis that the circadian clock temporally regulates enamel development.

Periodontitis is a chronic-, infectious-disease of the human periodontium that is characterized by the loss of supporting tissues surrounding the tooth such as the periodontal ligament (PDL), cementum and alveolar bone. Regeneration of the periodontium is dependent on the participation of mesenchymal stem/stromal cells (MSC) resident in the PDL. Enamel matrix derivative (EMD), an extract from immature porcine enamel rich in amelogenin protein but that also contain bone morphogenetic protein (BMP), is used to treat periodontal defects. The effects of EMD on MSC cells of the PDL are not well characterized. In this in vitro study, we identify PDL progenitor cells from multiple individuals and demonstrate that EMD stimulates them. We show that the effect of EMD on cell proliferation and migration is mediated through the amelogenin it contains, while the differentiation of these progenitor cells to cell types of mineralized tissue is mainly due to BMP signaling., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Strepsirrhines are members of a primate suborder that has a distinctive set of features associated with the development of the dentition. Amelogenin (AMEL), the better known of the enamel matrix proteins, forms 90% of the secreted organic matrix during amelogenesis. Although AMEL has been sequenced in numerous mammalian lineages, the only reported strepsirrhine AMEL sequences are those of the ring-tailed lemur and galago, which contain a set of additional proline-rich tandem repeats absent in all other primates species analyzed to date, but present in some non-primate mammals. Here, we first determined that these repeats are present in AMEL from three additional lemur species and thus are likely to be widespread throughout this group. To evaluate the functional relevance of these repeats in strepsirrhines, we engineered a mutated murine amelogenin sequence containing a similar proline-rich sequence to that of Lemur catta. In the monomeric form, the MQP insertions had no influence on the secondary structure or refolding properties, whereas in the assembled form, the insertions increased the hydrodynamic radii. We speculate that increased AMEL nanosphere size may influence enamel formation in strepsirrhine primates.

Two point mutations (T21I and P40T) within amelogenin have been identified from human DNA sequences in 2 instances of amelogenesis imperfecta. We studied the folding and self-assembly of recombinant amelogenin (rM180) compared to the T21I and P40T mutants analogs. At pH 5.8 and 25°C, rM180 and the P41T mutant existed as monomers, whereas the T21I mutant formed small oligomers. At pH 8 and 25°C, all of the amelogenin samples formed nanospheres with hydrodynamic radii (R(H)) of around 15-16 nm. Upon heating to 37°C, particles of P41T increased in size (R(H) = 18 nm). During thermal denaturation at pH 5.8, both of the mutant proteins refolded more slowly than the wild-type (WT) rM180. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that the WT transformed to a partially folded conformation upon heating and remained stable. Thermal denaturation and refolding studies indicated that the mutants were less stable and exhibit a greater ability to prematurely aggregate compared to the WT. Our data suggest that in the case of P41T, alterations in the self-assembly of amelogenin are a consequence of destabilization of the secondary structure, while in the case of T21I they are a consequence of change in the overall hydrophobicity at the N-terminal region. We propose that alterations in the assembly (i.e. premature aggregation) of mutant amelogenins may have a profound effect on intra- and extracellular processes such as amelogenin secretion, proteolysis, and its interactions with nonamelogenins as well as with the forming mineral., (Copyright © 2011 S. Karger AG, Basel.)

Mutations in amelogenin sequence result in defective enamel, and the diverse group of genetically altered conditions is collectively known as amelogenesis imperfecta (AI). Despite numerous studies, the detailed molecular mechanism of defective enamel formation is still unknown. In this study, we have examined the biophysical properties of a recombinant murine amelogenin (rM180) and two point mutations identified from human DNA sequences in two cases of AI (T21I and P41T). At pH 5.8 and 25 °C, wild type (WT) rM180 and mutant P41T existed as monomers, and mutant T21I formed lower order oligomers. CD, dynamic light scattering, and fluorescence studies indicated that rM180 and P41T can be classified as a premolten globule-like subclass protein at 25 °C. Thermal denaturation and refolding monitored by CD ellipticity at 224 nm indicated the presence of a strong hysteresis in mutants compared with WT. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that WT transformed to a partially folded conformation upon heating and remained stable. The partially folded conformation formed by P41T, however, readily converted into a heterogeneous population of aggregates. T21I existed in an oligomeric state at room temperature and, upon heating, rapidly formed large aggregates over a very narrow temperature range. Thermal denaturation and refolding studies indicated that the mutants are less stable and exhibit poor refolding ability compared with WT rM180. Our results suggest that alterations in self-assembly of amelogenin are a consequence of destabilization of the intrinsic disorder. Therefore, we propose that, like a number of other human diseases, AI appears to be due to the destabilization of the secondary structure as a result of amelogenin mutations.

Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up >90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the (1)H-(15)N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the (1)H-(15)N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12 and Y12 near the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggests that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region. Starting from 2% acetic acid, where rp(H)LRAP was monomeric in solution, NaCl addition effected residue specific changes in molecular dynamics manifested by the reduction in intensity and disappearance of (1)H-(15)N HSQC cross peaks. As observed for the full-length protein, these perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different patterns of (1)H-(15)N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences contribute to the cell signaling properties attributable to LRAP but not to the full-length protein., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objectives: Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific amelogenin isoform, leucine-rich amelogenin peptide or LRAP. However, it is unclear if LAMP-1 is an amelogenin receptor for dental mesenchymal cells. The goal of this study was to determine if LAMP-1 serves as a cell surface binding site for full length amelogenin on tooth root/periodontium associated mesenchymal cells., Design: Murine dental follicle cells and cementoblasts (OCCM-30) were cultured for 2 days followed by addition of full length recombinant mouse amelogenin, rp(H)M180. Dose-response (0-100 microg/ml) and time course (0-120 min) assays were performed to determine the optimal conditions for live cell surface binding using immunofluorescent microscopy. A competitive binding assay was performed to determine binding specificity by adding Emdogain (1 mg/ml) to the media. An antibody against LAMP-1 was used to detect the location of LAMP-1 on the cell surface and the pattern was compared to cell surface bound amelogenin. Both amelogenin and cell surface LAMP-1 were immuno-co-localized to compare the amount and distribution pattern., Results: Maximum surface binding was achieved with 50 microg/ml of rp(H)M180 for 120 min. This binding was specific as demonstrated by competitive inhibition (79% lower) with the addition of Emdogain. The binding pattern for rp(H)M180 was similar to the distribution of surface LAMP-1 on dental follicle cells and cementoblasts. The high co-localization coefficient (0.92) for rp(H)M180 and LAMP-1 supports rp(H)M180 binding to cell surface LAMP-1., Conclusions: The data from this study suggest that LAMP-1 can serve as a cell surface binding site for amelogenin on dental follicle cells and cementoblasts., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology, where it is used as a local adjunct to periodontal surgery to stimulate regeneration of periodontal tissues lost to periodontal disease. The biological effect of EMD is through stimulation of local growth factor secretion and cytokine expression in the treated tissues, inducing a regenerative process that mimics odontogenesis. The major (>95%) component of EMD is Amelogenins (Amel). No other active components have so far been isolated from EMD, and several studies have shown that purified amelogenins can induce the same effect as the complete EMD. Amelogenins comprise a family of highly conserved extracellular matrix proteins derived from one gene. Amelogenin structure and function is evolutionary well conserved, suggesting a profound role in biomineralization and hard tissue formation. A special feature of amelogenins is that under physiological conditions the proteins self-assembles into nanospheres that constitute an extracellular matrix. In the body, this matrix is slowly digested by specific extracellular proteolytic enzymes (matrix metalloproteinase) in a controlled process, releasing bioactive peptides to the surrounding tissues for weeks after application. Based on clinical and experimental observations in periodontology indicating that amelogenins can have a significant positive influence on wound healing, bone formation and root resorption, several new applications for amelogenins have been suggested. New experiments now confirm that amelogenins have potential for being used also in the fields of endodontics, bone regeneration, implantology, traumatology, and wound care.

Using solution-state NMR spectroscopy, new insights into the early events governing amelogenin supramolecular self-assembly have been identified using sodium chloride and calcium chloride to trigger the association. Two-dimensional 1H-15N HSQC spectra were recorded for 15N- and 13C-labeled murine amelogenin as a function of increasing NaCl and CaCl2 concentration beginning with solution conditions of 2% acetic acid at pH 3.0, where amelogenin was monomeric. Residue specific changes in molecular dynamics, manifested by the reduction in intensity and disappearance of 1H-15N HSQC cross-peaks, were observed with the addition of either salt to the protein. With increasing NaCl concentrations, residues between T21 and R31 near the N-terminus were affected first, suggesting that these residues may initiate amelogenin dimerization, the first step in nanosphere assembly. At higher NaCl concentrations, more residues near the N-terminus (Y12-I51) were affected, and with further additions of NaCl, residues near the C-terminus (L141-T171) began to show a similar change in molecular dynamics. With increasing CaCl2 concentrations, a similar stepwise change in molecular dynamics involving essentially the same set of amelogenin residues was observed. As the concentration of either salt was increased, a concomitant increase in the estimated overall rotational correlation time (tau(c)) was observed, consistent with assembly. Self-assembly into a dimer or trimer was established with dynamic light scattering studies under similar conditions that showed an increase in diameter of the smallest species from 4.1 nm in the absence of salt to 10 nm in the presence of salt. These results suggest a possible stepwise interaction mechanism, starting with the N-terminus and followed by the C-terminus, leading to amelogenin nanosphere assembly.

Amelogenin is the predominant matrix protein in developing dental enamel. Making extensive use of residue specific 15N-labeled amino acids samples, the majority of the main and side chain resonances for murine amelogenin were assigned in 2% aqueous acetic acid at pH 3.0.

The enamel matrix protein amelogenin is secreted by ameloblasts into the extracellular space to guide the formation of highly ordered hydroxyapatite mineral crystallites, and, subsequently, is almost completely removed during mineral maturation. Amelogenin interacts with the transmembrane proteins CD63 and LAMP (lysosome-associated membrane protein) 1, which are involved in endocytosis. Exogenously added amelogenin has been observed to move rapidly into CD63/LAMP1-positive vesicles in cultured cells. In the present study, we demonstrate the protein region defined by amino acid residues 103-205 for CD63 interacts not only with amelogenin, but also with other enamel matrix proteins (ameloblastin and enamelin). A detailed characterization of binding regions in amelogenin, CD63 and LAMP1 reveals that the amelogenin region defined by residues PLSPILPELPLEAW is responsible for the interaction with CD63 through residues 165-205, with LAMP1 through residues 226-251, and with the related LAMP2 protein through residues 227-259. We predict that the amelogenin binding region is: (i) hydrophobic; (ii) largely disordered; and (iii) accessible to the external environment. In contrast, the binding region of CD63 is likely to be organized in a '7' shape within the mushroom-like structure of CD63 EC2 (extracellular domain 2). In vivo, the protein interactions between the secreted enamel matrix proteins with the membrane-bound proteins are likely to occur at the specialized secretory surfaces of ameloblast cells called Tomes' processes. Such protein-protein interactions may be required to establish short-term order of the forming matrix and/or to mediate feedback signals to the transcriptional machinery of ameloblasts and/or to remove matrix protein debris during enamel biomineralization.

C/EBPalpha is implicated to regulate mouse amelogenin gene expression during tooth enamel formation in vitro. Because enamel formation occurs during postnatal development and C/EBPalpha-deficient mice die at birth, we used the Cre/loxP recombination system to characterize amelogenin expression in C/EBPalpha conditional knock-out mice. Mice carrying the Cre transgene under the control of the human keratin-14 promoter show robust Cre expression in the ameloblast cell lineage. Mating between mice bearing the floxed C/EBPalpha allele with keratin-14-Cre mice generate C/EBPalpha conditional knock-out mice. Real-time PCR analysis shows that removal of one C/EBPalpha allele from the molar enamel epithelial organ of 3-day postnatal mice results in dramatic decrease in endogenous C/EBPalpha mRNA levels and coordinately altered amelogenin mRNA abundance. Conditional deletion of both C/EBPalpha alleles further diminishes C/EBPalpha mRNA levels; however, rather than ablating amelogenin expression, we observe wild-type amelogenin mRNA abundance levels. We examined C/EBPbeta and nuclear factor YA expression, two transcription factors that had previously been shown to modestly participate in amelogenin expression, in vitro but found no significant changes in either of their mRNA abundance levels comparing conditional knock-out mice with wild-type counterparts. Although the abundance of C/EBPdelta is also unchanged in C/EBPalpha conditional knock-out mice, in vitro we find that C/EBPdelta activates the mouse amelogenin promoter and synergistically cooperates with nuclear factor Y, suggesting that C/EBPdelta can functionally substitute for C/EBPalpha to produce an enamel matrix competent to direct biomineralization.

The amelogenin gene is tightly regulated at the temporal and spatial level in accord with the developmental requirement for tooth formation. Previous studies have shown that CCAAT/enhancer-binding protein alpha (C/EBPalpha) is a transactivator of the mouse X-chromosomal amelogenin gene. C/EBPalpha contains four highly conserved regions (CR) named CR1, CR2, CR3, and CR4. Transient transfection assays showed that CR2 in isolation had an exceptional capacity to enhance transcription from the 2.3 kb mouse amelogenin promoter. The remaining conserved regions of C/EBPalpha, either in isolation or in selected combinations, were less effective in amelogenin transactivation than the full length C/EBPalpha. Msx2 has previously been shown to antagonize C/EBPalpha through protein-protein interactions with C/EBPalpha, and the carboxyl-terminus of Msx2 is required for protein-protein interactions. Co-immunoprecipitation analyses identified that the carboxyl-terminal domain (residues 218-359) of C/EBPalpha is required for the C/EBPalpha-Msx2 protein-protein interactions.

Proteins of the developing enamel matrix include amelogenin, ameloblastin and enamelin. Of these three proteins amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes' processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which amelogenin, or degraded amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation.