Effect of phosphate group on remineralization of early enamel caries regulated by amelogenin peptide.

OBJECTIVE: To show the effect of the phosphate group on the remineralization process of early enamel caries mediated by amelogenin peptide. METHODS: Freshly extracted, completed, and crack-free bovine teeth were used to create artificial early enamel caries, which were randomly divided into four groups: Group A: fluorination remineralized solution treatment group; Group B: pure remineralized solution treatment group. Group C: 100 g/ml recombinant Amelogenin peptide remineralized solution treatment group (with single phosphate group on N-terminus); Group D: 100 g/ml non-phosphorylated recombinant Amelogenin peptide remineralized solution treatment group (without single phosphate group on N-terminus). For 12 days, fresh remineralized solutions were replaced daily. Transverse microradiography (TMR) was used after remineralization to determine mineral loss and demineralization depth before and after each sample's remineralization. Each sample's depth of remineralization and mineral acquisition were then determined. RESULTS: The recombinant amelogenin peptide group significantly outperformed the non-phosphorylated amelogenin peptide group in terms of mineral acquisition and mineralization depth (P<0.05). CONCLUSIONS: The recombinant Amelogenin's solitary phosphate group at the N-terminus helps recombinant Amelogenin to encourage the remineralization process of early enamel caries.

Peptide analysis of tooth enamel - A sex estimation tool for archaeological, anthropological, or forensic research.

Proteomics has become an attractive method to study human and animal material, biological profile, and origin as an alternative to DNA analysis. It is limited by DNA amplification in ancient samples and its contamination, high cost, and limited preservation of nuclear DNA. Currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. Proteomics provides a new, seemingly simple, and relatively non-expensive way of sex estimation without the risk of contamination. Proteins can be preserved in hard teeth tissue (enamel) for tens of thousands of years. It uses two sexually distinct forms of the protein amelogenin in tooth enamel detectable by liquid chromatography-mass spectrometry; the protein amelogenin Y isoform is present in enamel dental tissue only in males, while amelogenin isoform X can be found in both sexes. From the point of view of archaeological, anthropological, and forensic research and applications, the reduced destruction of the methods used is essential, as well as the minimum requirements for sample size.

Mechanistic Insights into Bioengineered Antibiofilm Enamel Pellicles.

Dental caries remains the most widespread chronic disease worldwide. Basically, caries originates within biofilms accumulated on dental enamel. Despite the nonrenewable nature of the enamel tissue, targeted preventive strategies are still very limited. We previously introduced customized multifunctional proteinaceous pellicles (coatings) for controlling bacterial attachment and subsequent biofilm succession. Stemmed from our whole proteome/peptidome analysis of the in vivo acquired enamel pellicle, we designed these pellicles using hybrid mixtures of the most abundant and complementary-acting antimicrobial and antifouling proteins/peptides for synergetic suppression of early biofilms. In conjugating these domains synthetically, their bioinhibitory efficacy was remarkably boosted. Herein, we sought to explore the key structure-function relationship of these potent de novo hybridized conjugates in comparison with their individual domains, solely or in physical mixtures. Specifically, we interrelated the following facets: physicochemical and 3-dimensional folding characteristics via molecular dynamics simulations, adopted secondary structure by circular dichroism, immobilization capacity on enamel through high-spatial resolution multiphoton microscopy, and biofilm suppression potency. Our data showed consistent associations among the increased preference for protein folding structures, α-helix content, and enamel-immobilization capacity; all were inversely correlated with the attached bioburden. The expressed phenotypes could be explained by the adopted strongly amphipathic helical conformation upon conjugation, mediated by the highly anionic and acidic N-terminal pentapeptide shared region/motif for enhanced immobilization on enamel. In conclusion, conjugating bioactive proteins/peptides is a novel translational approach to engineer robust antibiofilm pellicles for caries prevention. The adopted α-helical conformation is key to enhance the antibiofilm efficacy and immobilization capacity on enamel that are promoted by certain physicochemical properties of the constituent domains. These data are valuable for bioengineering versatile therapeutics to prevent/arrest dental caries, a condition that otherwise requires invasive treatments with substantial health care expenditures.

Conditional knockout of transient receptor potential melastatin 7 in the enamel epithelium: Effects on enamel formation.

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.

Response of TGF-ß isoforms in epithelial-mesenchymal transition of enamel epithelial cells.

OBJECTIVE: During enamel formation, transforming growth factor-beta (TGF-ß) isoforms exhibit different activities for gene expression, apoptosis, and endocytosis. This study aimed to investigate the differential response of TGF-ß isoforms to epithelial-mesenchymal transition (EMT) in enamel epithelial cells. DESIGN: Using a mouse enamel epithelial cell line (mHAT9d) cultured in the presence of each TGF-ß isoform, (1) the morphological changes in EMT were explored, (2) EMT-related genes were analyzed by next-generation sequencing (NGS), (3) TGF-ß pathway for EMT was identified by inhibition experiments, and (4) the expression of the TGF-ß receptor gene in response to the binding affinity of the TGF-ß isoform were analyzed. RESULTS: EMT was observed in mHAT9d cultured in the presence of TGF-ß1 and ß3 but not TGF-ß2. The expression of both epithelial and mesenchymal marker genes was observed in mHAT9d exhibiting EMT. NGS analysis suggested extracellular signal-regulated kinase (ERK) and Rho pathways as TGF-ß signaling pathways associated with EMT. However, EMT in mHAT9d cultured in the presence of TGF-ß1 or ß3 occurred even in presence of an ERK1/2 inhibitor and was suppressed by Rho-kinase inhibitor. The expression of co-receptors for TGF-ß signaling in mHAT9d cells reduced following stimulation with each TGF-ß isoform. In contrast, endoglin levels increased following TGF-ß1 or ß3 stimulation, but no change was noted in response to TGF-ß2. CONCLUSIONS: We propose that in TGF-ß-stimulated enamel epithelial cells, EMT mainly occurred via the Rho signaling pathway, and the differences in response across TGF-ß isoforms were due to their endoglin-mediated binding affinity for the TGF-ß receptor.

Odontogenesis-Associated Phosphoprotein (ODAPH) Overexpression in Ameloblasts Disrupts Enamel Formation via Inducing Abnormal Mineralization of Enamel in Secretory Stage.

Odontogenesis-associated phosphoprotein (ODAPH) is a recently discovered enamel matrix protein. Our previous study demonstrated that knockouting out Odaph in mice resulted in enamel hypomineralization. To further investigate the effect of Odaph on enamel mineralization, we constructed an Odaph overexpression mouse model, controlled by an amelogenin promoter. Our histological analysis of OdaphTg mice revealed that the enamel layer was thinner than in WT mice. An uneven, thinner enamel layer was confirmed using micro-computed tomography (uCT). It was subsequently found that the Tomes' processes lost their normal morphology, resulting in the loss of the enamel prism structure. These results indicate that Odaph overexpression in ameloblasts led to enamel dysplasia. In conjunction with this, Odaph overexpression hindered Amelx secretion, and may result in endoplasmic reticulum stress. Interestingly, uCT revealed that enamel had higher mineral density at the secretory stage; due to this, we did the histological staining for the mineralization-related proteins Alkaline phosphatase (ALPL) and Runt-related transcription factor 2 (RUNX2). It was observed that these proteins were up-regulated in OdaphTg mice versus WT mice, indicating that Odaph overexpression led to abnormal enamel mineralization. To confirm this, we transfected ameloblast-like cell line (ALC) with Odaph overexpression lentivirus in vitro and identified that both Alpl and Runx2 were strikingly upregulated in OE-mus-Odaph versus OE-NC cells. We concluded that the ectopic overexpression of Odaph in ameloblasts led to abnormal enamel mineralization. In summary, Odaph profoundly influences amelogenesis by participating in enamel mineralization.

Validation of a cariogenic biofilm model by evaluating the effect of fluoride on enamel demineralization.

In vitro biofilm models have been extensively used, but only few of the models available to date had been validated in terms of the dose-response effect of anti-caries and/or antimicrobial substances. Additionally, none of the validated models allow the use of microliter volumes of the treatment solutions, needed mainly to test (screen) novel but expensive substances under development. This study aimed at modifying an in vitro cariogenic Streptococcus mutans biofilm model and validating it by assessing the dose-response effect of fluoride on enamel demineralization. S. mutans cariogenic biofilms were developed on saliva-coated enamel slabs previously bonded to acrylic holders fixed to a lid of a culture plate. Biofilms were incubated 8 h/day in culture medium supplemented with 1% sucrose and then overnight in culture medium with glucose 0.1 mM. Biofilms were also treated 2×/day with 2.0 mL of solutions containing 0, 125, 275 and 1250 µg F/mL (n = 10/group). The replaced culture medium was used to: determine the biofilm acidogenicity; estimate the demineralization of enamel; and monitor the fluoride concentration. At 144 h, biofilms were collected for fluoride concentration analyses, and the fluoride uptake by enamel was determined in each slab. The model showed a dose-response effect of fluoride (R2 = 0.96, p < 0.001) between enamel demineralization and the fluoride concentration of the treatments. Water-soluble and bound biofilm fluoride concentrations (p < 0.007), as well as the firmly-bound fluoride concentration found in enamel (p < 0.0001), increased in a dose-dependent manner. Our model constitutes a validated approach that would allow the assessment of the anticaries potential of novel biotechnological strategies, as in the case of expensive salivary peptides, because it would allow to test the treatment solutions using smaller volumes.

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel.

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed "shedding", while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel "dahlite" crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.

Mouse Dspp frameshift model of human dentinogenesis imperfecta.

Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5' mutations affecting an N-terminal targeting sequence and 3' mutations that shift translation into the - 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp-1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a DsppP19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. DsppP19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. DsppP19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp-1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp+/+ and DsppP19L dentin. The Dspp-1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5' and 3' Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.

Ezh2 knockout in mesenchymal cells causes enamel hyper-mineralization.

Enhancer of zeste homolog 2 (EZH2) is the catalytic core of polycomb repressive complex 2 (PRC2), which primarily methylates lysine 27 on histone H3 (H2K27me3), generating transcriptionally suppressed heterochromatin. Since EZH2 suppresses expression of genes involved in dentin formation, we examined the role of EZH2 in tooth development. Intriguingly, microCT analysis of teeth from mice with conditional Ezh2 knockout in uncommitted mesenchymal cells showed hyper-mineralization of enamel, which is produced by the epithelial-lineage cells, ameloblasts. Scanning electron microscopy analysis and nano-indentation of the incisor enamel from knockout mice revealed smaller inter-rod spaces and higher hardness compared to wild type enamel, respectively. Interestingly, expression of the calcium channel subunit gene, Orai2, was decreased compared to its competitor, Orai1, both in knockout mouse incisors and the ex vivo culture of ameloblasts with the surrounding tissues under EZH2 inhibition. Moreover, histological analysis of incisor from knockout mice showed decreased ameloblastin and expedited KLK4 expression in the ameloblasts. These observations suggest that EZH2 depletion in dental mesenchymal cells reduces enamel matrix formation and increases enamel protease activity from ameloblasts, resulting in enamel hyper-mineralization. This study demonstrates the significant role of the suppressive H3K27me3 mark for heterochromatin on enamel formation.

Enamel-like tissue regeneration by using biomimetic enamel matrix proteins.

Enamel regeneration currently -is limited by our inability to duplicate artificially its complicated and well-aligned hydroxyapatite structure. The initial formation of enamel occurs in enamel organs where the ameloblasts secret enamel extracellular matrix formed a unique gel-like microenvironment. The enamel extracellular matrix is mainly composed by amelogenin and non-amelogenin. In this study, an innovative strategy was proposed to regenerate enamel-like tissue by constructing a microenvironment using biomimetic enamel matrix proteins (biomimetic EMPs) composed of modified leucine-rich amelogenin peptide (mLRAP) and non-amelogenin analog (NAA). Impressively, the regenerated enamel in this biomimetic EMPs on etched enamel surface produced prismatic structures, and showed similar mechanical properties to natural enamel. The results of X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) showed that regenerated crystal was hydroxyapatite. Molecular dynamics simulation analysis showed the binding energy between mLRAP and NAA were electrostatic forces and Van der Walls. These results introduced a promising strategy to induce crystal growth of enamel-like hydroxyapatite for biomimetic reproduction of materials with complicated hierarchical microstructures.

Expression pattern of transcriptional enhanced associate domain family member 1 (Tead1) in developing mouse molar tooth.

The Hippo pathway is essential for determining organ size by regulating cell proliferation. Previous reports have shown that impairing this pathway causes abnormal tooth development. However, the precise expression profile of the members of the transcriptional enhanced associate domain family (Tead), which are key transcription factors mediating Yap function, during tooth development is unclear. In this study, among the four isoforms of Tead (Tead1 - 4), only the expression of Tead1 mRNA was observed using semiquantitative RT- PCR in murine developing tooth germ at E16.5. The expression level of Tead1 mRNA in the excised murine mandibular molar tooth germ was significantly higher at E16.5 than at other developmental stages, as determined using quantitative PCR. We found that the mRNA expression of connective tissue growth factor (Ctgf), which is one of the Yap target genes directly controlling cell growth, changed consistently with that of Tead1 in developing molars. Fluorescent immunostaining revealed that Tead1 protein was expressed in both epithelial cells and mesenchymal cells of the dental lamina and dental epithelium, including the primary enamel knot during the cap stage. During the early bell stage (E16.5), Tead1 was expressed intensely in the inner and outer enamel epithelium, including the secondary enamel knot and the neighboring mesenchymal cells. Tead1 then specifically localized to the inner and outer enamel epithelium, which is responsible for enamel formation during the bell stage. These expression patterns were consistent with those of Yap, Taz, and Ctgf protein in developing molars. These results suggest that Tead1 acts as a mediator of the biological functions of Yap, such as the morphogenesis of cusp formation, during tooth development.

Maturation stage enamel defects in Odontogenesis-associated phosphoprotein (Odaph) deficient mice.

BACKGROUND: Mutation in Odontogenesis-associated phosphoprotein (ODAPH) has been reported to cause recessive hypomineralized amelogenesis imperfecta (AI) in human. However, the exact role of ODAPH in amelogenesis is still unknown. RESULTS: ODAPH was identified as a novel constituent of the atypical basal lamina located at the interface between maturation ameloblasts and the enamel by dual immunofluorescence staining of ODAPH and LAMC2. Odaph knockout mice were generated to explore the function of ODAPH in amelogenesis. Odaph-/- mice teeth showed severely attrition and reduced enamel mineralization. Histological analysis showed from transition or early-maturation stage, ameloblasts were rapidly shortened, lost cell polarity, and exhibited cell pathology. Abundant enamel matrix marked by amelogenin was retained. Temporary cyst-like structures were formed between flattened epithelial cells and the enamel from maturation stage to eruption. The integrity of the atypical basal lamina was impaired indicated by the reduced diffuse expression of LAMC2 and AMTN. The expression of maturation stage related genes of Amtn, Klk4, Integrinß6 and Slc24a4 were significantly decreased. CONCLUSIONS: Our results suggested Odaph played vital roles during amelogenesis by maintaining the integrity of the atypical basal lamina in maturation stage, which may contribute to a better understanding of the pathophysiology of human AI.

Consequences of NaCT/SLC13A5/mINDY deficiency: good versus evil, separated only by the blood-brain barrier.

NaCT/SLC13A5 is a Na+-coupled transporter for citrate in hepatocytes, neurons, and testes. It is also called mINDY (mammalian ortholog of 'I'm Not Dead Yet' in Drosophila). Deletion of Slc13a5 in mice leads to an advantageous phenotype, protecting against diet-induced obesity, and diabetes. In contrast, loss-of-function mutations in SLC13A5 in humans cause a severe disease, EIEE25/DEE25 (early infantile epileptic encephalopathy-25/developmental epileptic encephalopathy-25). The difference between mice and humans in the consequences of the transporter deficiency is intriguing but probably explainable by the species-specific differences in the functional features of the transporter. Mouse Slc13a5 is a low-capacity transporter, whereas human SLC13A5 is a high-capacity transporter, thus leading to quantitative differences in citrate entry into cells via the transporter. These findings raise doubts as to the utility of mouse models to evaluate NaCT biology in humans. NaCT-mediated citrate entry in the liver impacts fatty acid and cholesterol synthesis, fatty acid oxidation, glycolysis, and gluconeogenesis; in neurons, this process is essential for the synthesis of the neurotransmitters glutamate, GABA, and acetylcholine. Thus, SLC13A5 deficiency protects against obesity and diabetes based on what the transporter does in hepatocytes, but leads to severe brain deficits based on what the transporter does in neurons. These beneficial versus detrimental effects of SLC13A5 deficiency are separable only by the blood-brain barrier. Can we harness the beneficial effects of SLC13A5 deficiency without the detrimental effects? In theory, this should be feasible with selective inhibitors of NaCT, which work only in the liver and do not get across the blood-brain barrier.

Epithelial loss of mitochondrial oxidative phosphorylation leads to disturbed enamel and impaired dentin matrix formation in postnatal developed mouse incisor.

The formation of dentin and enamel matrix depends on reciprocal interactions between epithelial-mesenchymal cells. To assess the role of mitochondrial function in amelogenesis and dentinogenesis, we studied postnatal incisor development in K320E-TwinkleEpi mice. In these mice, a loss of mitochondrial DNA (mtDNA), followed by a severe defect in the oxidative phosphorylation system is induced specifically in Keratin 14 (K14+) expressing epithelial cells. Histochemical staining showed severe reduction of cytochrome c oxidase activity only in K14+ epithelial cells. In mutant incisors, H&E staining showed severe defects in the ameloblasts, in the epithelial cells of the stratum intermedium and the papillary cell layer, but also a disturbed odontoblast layer. The lack of amelogenin in the enamel matrix of K320E-TwinkleEpi mice indicated that defective ameloblasts are not able to form extracellular enamel matrix proteins. In comparison to control incisors, von Kossa staining showed enamel biomineralization defects and dentin matrix impairment. In mutant incisor, TUNEL staining and ultrastructural analyses revealed differentiation defects, while in hair follicle cells apoptosis is prevalent. We concluded that mitochondrial oxidative phosphorylation in epithelial cells of the developed incisor is required for Ca2+ homeostasis to regulate the formation of enamel matrix and induce the differentiation of ectomesenchymal cells into odontoblasts.

Optimization of Method for Human Sex Determination Using Peptidome Analysis of Teeth Enamel from Teeth of Different Biological Generation, Archeological Age, and Degrees of Taphonomic Preservation.

Determination of biological sex to human remains is a fundamental requirement in anthropological, archeological, and forensic anthropological studies. Sex determination based on morphological criteria is significantly limited in the cases of juvenile remains and adult skeletons in a poor state of preservation. Regular attempts have been made to use alternative techniques to resolve this issue, including analysis of tooth enamel peptides by liquid chromatography/mass spectrometry. Optimization of this method involving acid etching of tooth enamel for 10 min followed by desalting of the products of etching on SDB-RPS StageTips microcolumns and analysis of desalted sample (1/3) by liquid chromatography/mass spectrometry allowed reliable sex determination to fossil remains within a wide range of archeological and biological ages without destructing analyzed teeth. Increasing the duration of enamel etching ensured a 2 to 3-fold increase in the total number of identified peptides and, more importantly, in the number of identified fragments of amelogenin Y isoform specific for male teeth, which facilitated reliable sex determination of fossil remains. The suggested technique was tested with 8 permanent and 15 deciduous teeth of different archaeological age and different degree of preservation. Two amelogenin Y-specific peptide sequences were identified. One of these peptides [SM(+15.99)IRPPYS)] was found in all male-derived samples without exception; the other peptide [IRPPYSS(+79.97)], which contained phosphorylated Ser66 residue, was found only in the enamel from deciduous teeth, which suggests that phosphorylation of Ser66 plays a role in the enamel formation in deciduous teeth.

Pitx2-Sox2-Lef1 interactions specify progenitor oral/dental epithelial cell signaling centers.

Epithelial signaling centers control epithelial invagination and organ development, but how these centers are specified remains unclear. We report that Pitx2 (the first transcriptional marker for tooth development) controls the embryonic formation and patterning of epithelial signaling centers during incisor development. We demonstrate using Krt14Cre /Pitx2flox/flox (Pitx2cKO ) and Rosa26CreERT/Pitx2flox/flox mice that loss of Pitx2 delays epithelial invagination, and decreases progenitor cell proliferation and dental epithelium cell differentiation. Developmentally, Pitx2 regulates formation of the Sox2+ labial cervical loop (LaCL) stem cell niche in concert with two signaling centers: the initiation knot and enamel knot. The loss of Pitx2 disrupted the patterning of these two signaling centers, resulting in tooth arrest at E14.5. Mechanistically, Pitx2 transcriptional activity and DNA binding is inhibited by Sox2, and this interaction controls gene expression in specific Sox2 and Pitx2 co-expression progenitor cell domains. We demonstrate new transcriptional mechanisms regulating signaling centers by Pitx2, Sox2, Lef1 and Irx1.

ADAM10 is Expressed by Ameloblasts, Cleaves the RELT TNF Receptor Extracellular Domain and Facilitates Enamel Development.

MMP20 cleaves cadherins and may facilitate cell movement, however MMP20 is not known to cleave tight junction or desmosome proteins. Ameloblasts had not previously been screened for membrane anchored proteases that could contribute to cell movement. Here we performed a PCR screen for proteolyticlly active A Disintegrin And Metalloproteinase (ADAM) family members. These proteinases are termed sheddases because they have a transmembrane domain and their catalytic domain on the cell surface can function to release anchored proteins. Significantly, ADAMs can be targeted to specific substrates on the cell membrane through their interaction with tetraspanins. Six ADAMs (ADAM8, 9, 10, 15, 17, 19) were expressed in mouse enamel organs. We show that Adam10 expression begins in the apical loop, continues through the secretory stage and abruptly ends at the transition stage when ameloblast migration ceases. ADAM10 cleaves cadherins and tight junction plus desmosome proteins and is well characterized for its role in cell movement. ADAM10 facilitated LS8 cell migration/invasion through a Matrigel coated membrane and we demonstrate that ADAM10, but not ADAM17 cleaves the RELT extracellular domain. This striking result is significant because RELT mutations cause amelogenesis imperfecta (AI) and this directly links ADAM10 to an important role in enamel development.

The influence of fillers and protease inhibitors in experimental resins in the protein profile of the acquired pellicle formed in situ on enamel-resin specimens.

OBJECTIVE: This study evaluated the influence of the addition of fillers and/or protease inhibitors [(epigallocatechin gallate - EGCG) or (chlorhexidine - CHX)] in experimental resins in the protein profile of the acquired pellicle (AP) formed in situ on enamel-resin specimens. DESIGN: 324 samples of bovine enamel were prepared (6 × 6 × 2 mm). The center of each sample was added with one of the following experimental resins (Bis-GMA+TEGDMA): no filler, no inhibitor (NF-NI); filler no inhibitor (F-NI); no filler plus CHX (NF-CHX); filler plus CHX (F-CHX); no filler plus EGCG (NF-EGCG); filler plus EGCG (F-EGCG). Nine subjects used a removable jaw appliance (BISPM - Bauru in situ pellicle model) with 2 slabs from each group. The AP was formed for 120 min, in 9 days and collected with electrode filter paper soaked in 3% citric acid. The pellicles collected were processed for analysis by LC-ESI-MS/MS. RESULTS: A total of 140 proteins were found in the AP collected from all the substrates. Among them, 16 proteins were found in common in all the groups: 2 isoforms of Basic salivary proline-rich protein, Cystatin-S, Cystatin-AS, Cystatin-SN, Histatin-1, Ig alpha-1 chain C region, Lysozyme C, Mucin-7, Proline-rich protein 4, Protein S100-A9, Salivary acidic proline-rich phosphoprotein ½ and Statherin. Proteins with other functions, such as metabolism and transport, were also identified. CONCLUSION: The composition of the experimental resins influenced the protein profile of the AP. This opens a new avenue for the development of new materials able to guide for AP engineering, thus conferring protection to the adjacent teeth.

RUNX2 contributes to TGF-ß1-induced expression of Wdr72 in ameloblasts during enamel mineralization.

The elaborate modulation of the transforming growth factor ß (TGF-ß) superfamily signaling network plays an essential role in tooth morphogenesis and differentiation. In our previous studies, we have demonstrated that TGF-ß1 promotes enamel mineralization and maturation using TGF-ß1 gene conditional knockout (TGF-ß1-cKO) mice. However, the specific regulatory mechanisms of TGF-ß1 during enamel development remain unclear. Furthermore, we have previously found that the expression of WD repeat-containing protein 72(WDR72)in mouse enamel epithelium is decreased significantly in the absence of TGF-ß1. Therefore, the aim of the present study was to investigate how TGF-ß1 affects amelogenesis by regulating the expression of Wdr72. Histological examination showed that the absence of TGF-ß1 in ameloblastic epithelial cells resulted in a reduction in enamel mineralization and a delay in enamel matrix protein absorption. TGF-ß1, Runt-related transcription factor 2(RUNX2) and WDR72 were revealed to be colocalized in ameloblasts by immunohistochemistry, and it was also found that the expression of Runx2 and Wdr72 was markedly different between TGF-ß1-cKO mice and wild type(TGF-ß1-WT)mice. In addition, the effect of exogenous TGF-ß1 on Wdr72 was more significant when RUNX2 was present than when RUNX2 was absent. Furthermore, we found that there were binding sites for RUNX2 on the promoter of Wdr72 and that Wdr72 expression was regulated by RUNX2. Collectively, our results suggest that TGF-ß1 affects enamel mineralization by modulating RUNX2 and thus affecting the expression of Wdr72.