Role of amelogenin phosphorylation in regulating dental enamel formation.

Amelogenin (AMELX), the predominant matrix protein in enamel formation, contains a singular phosphorylation site at Serine 16 (S16) that greatly enhances AMELX's capacity to stabilize amorphous calcium phosphate (ACP) and inhibit its transformation to apatitic enamel crystals. To explore the potential role of AMELX phosphorylation in vivo, we developed a knock-in (KI) mouse model in which AMELX phosphorylation is prevented by substituting S16 with Ala (A). As anticipated, AMELX KI mice displayed a severe phenotype characterized by weak hypoplastic enamel, absence of enamel rods, extensive ectopic calcifications, a greater rate of ACP transformation to apatitic crystals, and progressive cell pathology in enamel-forming cells (ameloblasts).

Identification of stages of amelogenesis in the continuously growing mandiblular incisor of C57BL/6J male mice throughout life using molar teeth as landmarks.

Continuously growing mouse incisors are widely used to study amelogenesis, since all stages of this process (., secretory, transition and maturation) are present in a spatially determined sequence at any given time. To study biological changes associated with enamel formation, it is important to develop reliable methods for collecting ameloblasts, the cells that regulate enamel formation, from different stages of amelogenesis.

The phosphorylation of serine in enamelin is essential for murine amelogenesis.

Amelogenesis imperfecta (AI) is an inherited developmental enamel defect affecting tooth masticatory function, esthetic appearance, and the well-being of patients. As one of the major enamel matrix proteins (EMPs), enamelin (ENAM) has three serines located in Ser-x-Glu (S-x-E) motifs, which are potential phosphorylation sites for the Golgi casein kinase FAM20C. Defects in FAM20C have similarly been associated with AI.

Loss of biological control of enamel mineralization in amelogenin-phosphorylation-deficient mice.

Amelogenin, the most abundant enamel matrix protein, plays several critical roles in enamel formation. Importantly, we previously found that the singular phosphorylation site at Ser16 in amelogenin plays an essential role in amelogenesis. Studies of genetically knock-in (KI) modified mice in which Ser16 in amelogenin is substituted with Ala that prevents amelogenin phosphorylation, and in vitro mineralization experiments, have shown that phosphorylated amelogenin transiently stabilizes amorphous calcium phosphate (ACP), the initial mineral phase in forming enamel.

Amelogenin phosphorylation regulates tooth enamel formation by stabilizing a transient amorphous mineral precursor.

Dental enamel comprises interwoven arrays of extremely long and narrow crystals of carbonated hydroxyapatite called enamel rods. Amelogenin (AMELX) is the predominant extracellular enamel matrix protein and plays an essential role in enamel formation (amelogenesis). Previously, we have demonstrated that full-length AMELX forms higher-order supramolecular assemblies that regulate ordered mineralization , as observed in enamel rods.

Potential function of TGF-β isoforms in maturation-stage ameloblasts.

Objectives: To investigate potential functions of transforming growth factor-beta (TGF-β) isoforms in maturation-stage ameloblasts during amelogenesis.

Methods: In vivo activation of TGF-β was characterized by using matrix metalloproteinase 20 null (Mmp20) and wild-type (Mmp20) mice. Using mHAT9d cells cultured in the presence of each TGF-β isoform, (1) cell proliferation was determined by MTS assay, (2) immunostaining with anti-cleaved caspase-3 monoclonal antibody was performed and apoptotic indices were measured, (3) gene expression was analyzed by RT-qPCR, and (4) the uptake of amelogenin into mHAT9d cells was directly observed using a fluorescence microscope.

The dynamics of TGF-β in dental pulp, odontoblasts and dentin.

Transforming growth factor-beta (TGF-β) is critical for cell proliferation and differentiation in dental pulp. Here, we show the dynamic mechanisms of TGF-β in porcine dental pulp, odontoblasts and dentin. The mRNA of latent TGF-β1 and TGF-β3 is predominantly expressed in odontoblasts, whereas the mRNA expression level of latent TGF-β2 is high in dental pulp.

Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide.

Previously, we have shown that serine-16 phosphorylation in native full-length porcine amelogenin (P173) and the Leucine-Rich Amelogenin Peptide (LRAP(+P)), an alternative amelogenin splice product, affects protein assembly and mineralization . Notably, P173 and LRAP(+P) stabilize amorphous calcium phosphate (ACP) and inhibit hydroxyapatite (HA) formation, while non-phosphorylated counterparts (rP172, LRAP(-P)) guide the growth of ordered bundles of HA crystals. Based on these findings, we hypothesize that the phosphorylation of full-length amelogenin and LRAP induces conformational changes that critically affect its capacity to interact with forming calcium phosphate mineral phases.

Dynamic interactions of amelogenin with hydroxyapatite surfaces are dependent on protein phosphorylation and solution pH.

Amelogenin, the predominant extracellular matrix protein secreted by ameloblasts, has been shown to be essential for proper tooth enamel formation. In this study, amelogenin adsorption to hydroxyapatite (HAP) surfaces, a prototype for enamel mineral, has been studied using a quartz crystal microbalance (QCM) to interrogate effects of protein phosphorylation and solution pH. Dynamic flow-based experiments were conducted at pH 7.

Comparative study on the resorbability and dissolution behavior of octacalcium phosphate, β-tricalcium phosphate, and hydroxyapatite under physiological conditions.

The dissolution behaviors of octacalcium phosphate (OCP), β-tricalcium phosphate (β-TCP), and hydroxyapatite (HA) were compared by implanting the materials in rat subcutaneous pouches for 8 weeks using a filter chamber or immersing them in simulated body fluid (SBF) or Tris-HCl buffer for 2 weeks at pH 7.4 and 37(o)C. X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, and chemical analysis were conducted on these materials.

TBX1 protein interactions and microRNA-96-5p regulation controls cell proliferation during craniofacial and dental development: implications for 22q11.2 deletion syndrome.

T-box transcription factor TBX1 is the major candidate gene for 22q11.2 deletion syndrome (22q11.2DS, DiGeorge syndrome/Velo-cardio-facial syndrome), whose phenotypes include craniofacial malformations such as dental defects and cleft palate.

Role of mineralization inhibitors in the regulation of hard tissue biomineralization: relevance to initial enamel formation and maturation.

Vertebrate mineralized tissues, i.e., enamel, dentin, cementum, and bone, have unique hierarchical structures and chemical compositions.

Regulation of calcium phosphate formation by native amelogenins in vitro.

Our previous in vitro studies have shown that recombinant full-length porcine amelogenin rP172 can transiently stabilize amorphous calcium phosphate (ACP) and uniquely guide the formation of well-aligned bundles of hydroxyapatite (HA) crystals, as seen in the secretory stage of amelogenesis. This functional capacity is dependent on the hydrophilic C-terminal domain of full-length amelogenin. However, we have also found that native phosphorylated (single S-16 site) forms of full-length (P173) and C-terminal cleaved (P148) amelogenins can stabilize ACP for > 2 d and prevent HA formation.

CryoTEM study of effects of phosphorylation on the hierarchical assembly of porcine amelogenin and its regulation of mineralization in vitro.

Amelogenin, the major extracellular enamel matrix protein, plays a critical role in regulating the growth and organization of enamel. Assembly and mineralization of full-length native (P173) and recombinant (rP172) porcine amelogenins were studied by cryogenic Transmission Electron Microscopy (cryoTEM). The cryoTEM revealed that both native and recombinant porcine amelogenins undergo step-wise self-assembly.

The LIM homeodomain transcription factor LHX6: a transcriptional repressor that interacts with pituitary homeobox 2 (PITX2) to regulate odontogenesis.

LHX6 is a LIM-homeobox transcription factor expressed during embryogenesis; however, the molecular mechanisms regulating LHX6 transcriptional activities are unknown. LHX6 and the PITX2 homeodomain transcription factor have overlapping expression patterns during tooth and craniofacial development, and in this report, we demonstrate new transcriptional mechanisms for these factors. PITX2 and LHX6 are co-expressed in the oral and dental epithelium and epithelial cell lines.

Nano-sized calcium phosphate particles for periodontal gene therapy.

Background: Growth factors such as platelet-derived growth factor (PDGF) have significantly enhanced periodontal therapy outcomes with a high degree of variability, mostly due to the lack of continual supply for a required period of time. One method to overcome this barrier is gene therapy. The aim of this in vitro study is to evaluate PDGF-B gene delivery in fibroblasts using nano-sized calcium phosphate particles (NCaPP) as vectors.

Effect of phosphorylation on the interaction of calcium with leucine-rich amelogenin peptide.

Amelogenin undergoes self-assembly and plays an essential role in guiding enamel mineral formation. The leucine-rich amelogenin peptide (LRAP) is an alternative splice product of the amelogenin gene and is composed of the N terminus (containing the only phosphate group) and the C terminus of full-length amelogenin. This study was conducted to investigate further the role of phosphorylation in LRAP self-assembly in the presence and absence of calcium using small angle X-ray scattering (SAXS).

Regulation of calcium phosphate formation by amelogenins under physiological conditions.

Amelogenin is essential for proper enamel formation. The present in vitro study extends our previous work at low (10 mM) ionic strength (IS) by examining the effect of amelogenin on mineralization under higher (162 mM) IS conditions found in developing enamel. Full-length phosphorylated (P173) and non-phosphorylated (rP172) amelogenins were examined, along with P148 and rP147 that lack the hydrophilic C-terminus.

Hierarchical self-assembly of amelogenin and the regulation of biomineralization at the nanoscale.

Enamel is a highly organized hierarchical nanocomposite, which consists of parallel arrays of elongated apatitic crystallites forming an intricate three-dimensional microstructure. Amelogenin, the major extracellular matrix protein of dental enamel, regulates the formation of these crystalline arrays via cooperative interactions with forming mineral phase. Using cryoelectron microscopy, we demonstrate that amelogenin undergoes stepwise hierarchical self-assembly.

Cryogenic transmission electron microscopy study of amelogenin self-assembly at different pH.

Cryogenic transmission electron microscopy (cryo-EM) was used to explore the self-assembly of recombinant murine amelogenin (rM179) in vitro. Our cryo-EM data showed that amelogenin self-assembly is a strongly pH-dependent process. At pH 4.

Effects of phosphorylation on the self-assembly of native full-length porcine amelogenin and its regulation of calcium phosphate formation in vitro.

The self-assembly of the predominant extracellular enamel matrix protein amelogenin plays an essential role in regulating the growth and organization of enamel mineral during early stages of dental enamel formation. The present study describes the effect of the phosphorylation of a single site on the full-length native porcine amelogenin P173 on self-assembly and on the regulation of spontaneous calcium phosphate formation in vitro. Studies were also conducted using recombinant non-phosphorylated (rP172) porcine amelogenin, along with the most abundant amelogenin cleavage product (P148) and its recombinant form (rP147).

Amelogenin-collagen interactions regulate calcium phosphate mineralization in vitro.

Collagen and amelogenin are two major extracellular organic matrix proteins of dentin and enamel, the mineralized tissues comprising a tooth crown. They both are present at the dentin-enamel boundary (DEB), a remarkably robust interface holding dentin and enamel together. It is believed that interactions of dentin and enamel protein assemblies regulate growth and structural organization of mineral crystals at the DEB, leading to a continuum at the molecular level between dentin and enamel organic and mineral phases.

Amelogenin nanoparticles in suspension: deviations from spherical shape and pH-dependent aggregation.

It is well-known that amelogenin self-assembles to form nanoparticles, usually referred to as amelogenin nanospheres, despite the fact that not much is known about their actual shape in solution. In the current paper, we combine SAXS and DLS to study the three-dimensional shape of the recombinant amelogenins rP172 and rM179. Our results show for the first time that amelogenins build oblate nanoparticles in suspension using experimental approaches that do not require the proteins to be in contact with a support material surface.

Role of 20-kDa amelogenin (P148) phosphorylation in calcium phosphate formation in vitro.

The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP).