The unique biomineralization transcriptome and proteome of Lytechinus variegatus teeth.

BACKGROUND: Matrix-regulated biomineralization involves the specific nucleation and growth of mineral phases within or upon preformed structured organic matrices. We hypothesized that there might be a general mechanism whereby anionic, phosphorylated mineral ion-binding proteins assist in specifically locating the mineral ions with respect to the mineralizing structural organic matrix. Here we extended these studies to invertebrate mineralization in Lytechinus variegatus (Lv) teeth. MATERIALS AND METHODS: The tooth proteins were extracted and the phosphoproteins occluded in the mineral were enriched by passage through a ProQ Diamond phosphoprotein enrichment column, and subjected to MS/MS analysis. A Lv RNA-seq derived transcriptome database was generated. The MS/MS data found 25 proteins previously classified as "Predicted uncharacterized proteins" and many of the spicule matrix proteins. As these 25 proteins were also identified with the transcriptome analysis, and were thus no longer "hypothetical" but real proteins in the Lv tooth. Each protein was analyzed for the presence of a signal peptide, an acidic pI≤4, and the ability to be phosphorylated. RESULTS: Four new Lv tooth specific Pro-Ala-rich proteins were found, representing a new class of proteins. CONCLUSION: The tooth is different from the spicules and other urchin skeletal elements in that only the tooth contains both "high" and "very high" magnesium calcite, [Ca(1-X) Mg(X) CO3], where X is the mole fraction of Mg. We speculate that our newly discovered proline-alanine rich proteins, also containing sequences of acidic amino acids, may be involved in the formation of high magnesium and very high magnesium calcite.

Expression of the invertebrate sea urchin P16 protein into mammalian MC3T3 osteoblasts transforms and reprograms them into "osteocyte-like" cells.

P16 is an acidic phosphoprotein important in both sea urchin embryonic spicule development and transient mineralization during embryogenesis, syncytium formation, and mineralization in mature urchin tooth. Anti-P16 has been used to localize P16 to the syncytial membranes and the calcite mineral. Specific amino acid sequence motifs in P16 are similar to sequences in DSPP, a protein common to all vertebrate teeth, and crucial for their mineralization. Here, we examine the effect of P16 on vertebrate fibroblastic NIH3T3 cells and osteoblastic MC3T3 cells. Transfection of NIH3T3 cells with P16 cDNA resulted in profound changes in the morphology of the cells. In culture, the transfected cells sent out long processes that contacted processes from neighboring cells forming networks or syncytia. There was a similar change in morphology in cultured osteoblastic MC3T3 cells. In addition, the MC3T3 developed numerous dendrites as found in osteocytes. Importantly, there was also a change in the expression of the osteoblast and osteocyte specific genes. MC3T3 cells transfected with P16 showed an 18-fold increase in expression of the osteocyte specific Dentin matrix protein (DMP1) gene, accompanied by decreased expression of osteoblast specific genes: Bone sialoprotein (BSP), osteocalcin (OCN), and ß-catenin decreased by 70%, 64%, and 68 %, respectively. Thus, invertebrate urchin P16 with no previously known analog in vertebrates was able to induce changes in both cell morphology and gene expression, converting vertebrate-derived osteoblast-like precursor cells to an "osteocyte-like" phenotype, an important process in bone biology. The mechanisms involved are presently under study.

Dentin phosphoprotein binds annexin 2 and is involved in calcium transport in rat kidney ureteric bud cells.

Dentin phosphoprotein (DPP) is the most abundant noncollagenous protein in the dentin, where it plays a major role in the mineralization of dentin. However, we and others have shown that in addition to being present in the dentin, DPP is also present in nonmineralizing tissues like the kidney, lung, and salivary glands, where it conceivably has other functions such as in calcium transport. Because annexins have been implicated as calcium transporters, we examined the relationships between DPP and annexins. In this report, we show that DPP binds to annexin 2 and 6 present in a rat ureteric bud cell line (RUB1). Immunofluorescence studies show that annexin 2 and DPP colocalize in these cells. In addition, DPP and annexin 2 colocalize in the ureteric bud branches of embryonic metanephric kidney. In the RUB1 cells and ureteric bud branches of embryonic kidney, colocalization was restricted to the cell membrane. Studies on calcium influx into RUB cells show that in the presence of anti-DPP, there was a 40% reduction of calcium influx into these cells. We postulate that DPP has different functions in the kidney as compared with the odontoblasts. In the odontoblasts, its primary function is in the extracellular mineralization of dentin, whereas in the kidney it may participate in calcium transport.

Peritubular dentin, a highly mineralized, non-collagenous, component of dentin: isolation and capture by laser microdissection.

We demonstrate the capability and technique to perform microdissection and isolation of select regions of untreated, mineralized dentin using laser capture. Dentin is a complex, non-homogeneous tissue comprised of a mineralized collagenous matrix (intertubular dentin [ITD]), odontoblastic processes (ODPs), a void space (tubules) that forms within the ITD left behind by the retraction of ODPs during dentin maturation, and a highly mineralized non-collagenous component that exists at the interface between the tubules and ITD known as peritubular dentin (PTD). PTD forms as the dentin matures. The ODPs retract toward the direction of the pulp; leaving very little PTD at either the DEJ or near the pulp. Statistical analysis of thin cross-sections of coronal bovine dentin imaged by light microscopy reveal that the area occupied by PTD >50%. To examine the nature of PTD and its relation to both the tubules and ITD, we devised a series of steps to carefully prepare sections of coronal bovine dentin so that areas of the dentin tissue could be cut and isolated for further analysis. We demonstrate that it is possible to selectively isolate targeted regions of dentin for analysis and that high resolution analysis of such sections can be performed using electron microscopy. Results show that the mineralized PTD has a different texture than mineralized ITD and that there is a distinct boundary between the PTD and the ITD. Selective isolation of mineralized tissue components for further analytical study opens the door for the investigation of similar enigmatic mineralized structures.

Water in the formation of biogenic minerals: peeling away the hydration layers.

Minerals of biogenic origin form and crystallize from aqueous environments at ambient temperatures and pressures. The in vivo environment either intracellular or intercellular, contains many components that modulate both the activity of the ions which associate to form the mineral, as well as the activity and structure of the crowded water. Most of the studies about the mechanism of mineralization, that is, the detailed pathways by which the mineral ions proceed from solution to crystal state, have been carried out in relatively dilute solutions and clean solutions. These studies have considered both thermodynamic and kinetic controls. Most have not considered the water itself. Is the water a passive bystander, or is it intimately a participant in the mineral ion densification reaction? A wide range of experiments show that the mineralization pathways proceed through a series of densification stages with intermediates, such as a "dense liquid" phase and the prenucleation clusters that form within it. This is in contrast to the idea of a single step phase transition, but consistent with the Gibbs concept of discontinuous phase transitions from supersaturated mother liquor to crystal. Further changes in the water structure at every surface and interface during densification guides the free energy trajectory leading to the crystalline state. In vertebrates, mineralization takes place in a hydrated collagen matrix, thus water must be considered as a direct participant. Although different in detail, the crystallization of calcium phosphates, as apatite, and calcium carbonates, as calcite, are mechanistically identical from the viewpoint of water.

Biomineralization mechanisms: a new paradigm for crystal nucleation in organic matrices.

There is substantial practical interest in the mechanism by which the carbonated apatite of bone mineral can be initiated specifically in a matrix. The current literature is replete with studies aimed at mimicking the properties of vertebrate bone, teeth, and other hard tissues by creating organic matrices that can be mineralized in vitro and either functionally substitute for bone on a permanent basis or serve as a temporary structure that can be replaced by normal remodeling processes. A key element in this is mineralization of an implant with the matrix and mineral arranged in the proper orientations and relationships. This review examines the pathway to crystallization from a supersaturated calcium phosphate solution in vitro, focusing on the basic mechanistic questions concerning mineral nucleation and growth. Since bone and dentin mineral forms within collagenous matrices, we consider how the in vitro crystallization mechanisms might or might not be applicable to understanding the in vivo processes of biomineralization in bone and dentin. We propose that the pathway to crystallization from the calcium phosphate-supersaturated tissue fluids involves the formation of a dense liquid phase of first-layer bound-water hydrated calcium and phosphate ions in which the crystallization is nucleated. SIBLING proteins and their in vitro analogs, such as polyaspartic acids, have similar dense liquid first-layer bound-water surfaces which interact with the dense liquid calcium phosphate nucleation clusters and modulate the rate of crystallization within the bone and dentin collagen fibril matrix.

Sea urchin tooth mineralization: calcite present early in the aboral plumula.

In both vertebrate bone, containing carbonated hydroxyapatite as the mineral phase, and in invertebrate hard tissue comprised of calcium carbonate, a popular view is that the mineral phase develops from a long-lived amorphous precursor which later transforms into crystal form. Important questions linked to this popular view are: when and where is the crystallized material formed, and is amorphous solid added subsequently to the crystalline substrate? Sea urchin teeth, in which the earliest mineral forms within isolated compartments, in a time and position dependent manner, allow direct investigation of the timing of crystallization of the calcite primary plates. Living teeth of the sea urchin Lytechinus variegatus, in their native coelomic fluid, were examined by high-energy synchrotron X-ray diffraction. The diffraction data show that calcite is present in the most aboral portions of the plumula, representing the very earliest stages of mineralization, and that this calcite has the same crystal orientation as in the more mature adoral portions of the same tooth. Raman spectroscopy of the aboral plumula confirms the initial primary plate mineral material is calcite and does not detect amorphous calcium carbonate; in the more mature adoral incisal flange, it does detect a broader calcite peak, consistent with two or more magnesium compositions. We hypothesize that some portion of each syncytial membrane in the plumula provides the information for nucleation of identically oriented calcite crystals that subsequently develop to form the complex geometry of the single crystal sea urchin tooth.

On the formation and functions of high and very high magnesium calcites in the continuously growing teeth of the echinoderm Lytechinus variegatus: development of crystallinity and protein involvement.

Sea urchin teeth grow continuously and develop a complex mineralized structure consisting of spatially separate but crystallographically aligned first stage calcitic elements of high Mg content (5-15 mol% mineral). These become cemented together by epitaxially oriented second stage very high Mg calcite (30-40 mol% mineral). In the tooth plumula, ingressing preodontoblasts create layered cellular syncytia. Mineral deposits develop within membrane-bound compartments between cellular syncytial layers. We seek to understand how this complex tooth architecture is developed, how individual crystalline calcitic elements become crystallographically aligned, and how their Mg composition is regulated. Synchrotron microbeam X-ray scattering was performed on live, freshly dissected teeth. We observed that the initial diffracting crystals lie within independent syncytial spaces in the plumula. These diffraction patterns match those of mature tooth calcite. Thus, the spatially separate crystallites grow with the same crystallographic orientation seen in the mature tooth. Mineral-related proteins from regions with differing Mg contents were isolated, sequenced, and characterized. A tooth cDNA library was constructed, and selected matrix-related proteins were cloned. Antibodies were prepared and used for immunolocaliztion. Matrix-related proteins are acidic, phosphorylated, and associated with the syncytial membranes. Time-of-flight secondary ion mass spectroscopy of various crystal elements shows unique amino acid, Mg, and Ca ion distributions. High and very high Mg calcites differ in Asp content. Matrix-related proteins are phosphorylated. Very high Mg calcite is associated with Asp-rich protein, and it is restricted to the second stage mineral. Thus, the composition at each part of the tooth is related to architecture and function.

Echinoderm phosphorylated matrix proteins UTMP16 and UTMP19 have different functions in sea urchin tooth mineralization.

Studies of mineralization of embryonic spicules and of the sea urchin genome have identified several putative mineralization-related proteins. These predicted proteins have not been isolated or confirmed in mature mineralized tissues. Mature Lytechinus variegatus teeth were demineralized with 0.6 N HCl after prior removal of non-mineralized constituents with 4.0 M guanidinium HCl. The HCl-extracted proteins were fractionated on ceramic hydroxyapatite and separated into bound and unbound pools. Gel electrophoresis compared the protein distributions. The differentially present bands were purified and digested with trypsin, and the tryptic peptides were separated by high pressure liquid chromatography. NH2-terminal sequences were determined by Edman degradation and compared with the genomic sequence bank data. Two of the putative mineralization-related proteins were found. Their complete amino acid sequences were cloned from our L. variegatus cDNA library. Apatite-binding UTMP16 was found to be present in two isoforms; both isoforms had a signal sequence, a Ser-Asp-rich extracellular matrix domain, and a transmembrane and cytosolic insertion sequence. UTMP19, although rich in Glu and Thr did not bind to apatite. It had neither signal peptide nor transmembrane domain but did have typical nuclear localization and nuclear exit signal sequences. Both proteins were phosphorylated and good substrates for phosphatase. Immunolocalization studies with anti-UTMP16 show it to concentrate at the syncytial membranes in contact with the mineral. On the basis of our TOF-SIMS analyses of magnesium ion and Asp mapping of the mineral phase composition, we speculate that UTMP16 may be important in establishing the high magnesium columns that fuse the calcite plates together to enhance the mechanical strength of the mineralized tooth.

The composition of bovine peritubular dentin: matching TOF-SIMS, scanning electron microscopy and biochemical component distributions. New light on peritubular dentin function.

Peritubular dentin (PTD) is a hypermineralized phase within the dentinal tubules in some vertebrate teeth as an interface between the intertubular dentin (ITD) and the cell processes. Our aim has been to understand the composition, structure and role of PTD as a mineralized tissue. We have utilized the technique of time of flight secondary ion mass spectrometry (TOF-SIMS) to map the distribution of positive and negative inorganic ions as well as organic components in the fully mineralized, intact PTD structure in bovine tooth cross-sections, and correlated these with scanning electron microscopy (SEM) in standard and backscatter modes. In recent work, we developed a procedure to freeze fracture the teeth and separate PTD from the less dense ITD by the use of aqueous sodium phosphotungstate step density gradients, after degrading the ITD collagen with NaOCl. Here, PTD-containing fragments were characterized by SEM and TOF-SIMS surface structure analysis. The TOF-SIMS data show that the isolated PTD does not contain collagen, but its surface is rich in glutamic acid-containing protein(s). The TOF-SIMS spectra also indicated that the intact PTD fragments contain phospholipids, and chemical analyses showed phosphatidylserine, phosphatidylinositol and phosphatidylcholine as the principal lipid components. In SEM sections, untreated PTD shows as a smooth collar around the tubule, but after digestion with ethylenediamine to remove all organic components, the porous nature of the mineral phase of small, thin platy apatite crystals becomes evident. Thus, the organic matrix of PTD appears to be a proteolipid-phospholipid complex.

Identification of the functional activity of the [A-4] amelogenin gene splice product in newborn mouse ameloblasts.

In the mouse tooth organ, shortly after birth, ameloblasts acquire their secretory phenotype, which is characterized by the prominent expression and subsequent secretion of two isoforms of amelogenin, M180 and M59 (LRAP, [A-4]). Amelogenin deposition into the ameloblast extracellular matrix promotes enamel biomineralization. A complex set of intercellular signaling events, reciprocal communications between the developing oral epithelium and its underlying dental mesenchyme, guide the expression of amelogenin mRNA, and limit it to a defined period of tooth development. In tooth germ organ culture, addition of the [A-4] isoform, lacking amelogenin exon 4 and exon 6 segments a, b, c, was shown to affect ameloblast development. To understand the basis for this regulatory activity, we have studied the effects of r[A-4] on ameloblast-like LS8 cells, and the role of the putative [A-4] cell surface receptor, LAMP1, as well as the related receptor LAMP3. In the LS8 cells, the expression of the spliced isoforms of amelogenin, LAMP1, and LAMP3 were identified by RT-PCR, and real-time PCR semi-quantitative analysis assessed the modulation of M180 message. M180 mRNA was up-regulated by exogenous [A-4], and this was further increased by blockade of LAMP1, suggesting additive effects between the intracellular signaling pathways activated by the discrete agonists. Immunofluorescence staining identified the patterns of [A-4] and LAMP1 localization in LS8 cells. Internalized r[A-4] was co-localized with LAMP1 in late endosomal/lysosomal compartments. Thus, the LAMP1 and [A-4] intracellular sorting pathways are interrelated. The nitric oxide (NO) signaling pathway was activated by exogenous [A-4]. [A-4] modulated inducible nitric oxide synthase (iNOS, NOS2) and endothelial nitric oxide synthase (eNOS, NOS3) expression, albeit, to different extents. NOS2 was significantly up-regulated after 4 h, while NOS3 increased slightly after 24 h. Co-treatment of LS8 cells with r[A-4] and anti-LAMP1 antibodies further enhanced NOS2 expression. Anti-LAMP1 antibodies did not abrogate NO production in LS8 cells treated for 4 h with r[A-4], but the iNOS inhibitor, l-Nil, down-regulated both NO production and the expression of M180 mRNA. These data suggest that [A-4] modulates M180 mRNA expression, partly, via the NO signaling pathway.

Characterization of a mouse amelogenin [A-4]/M59 cell surface receptor.

Amelogenin proteins comprise up to 90% of the organic matrix of developing enamel in the vertebrate tooth. Alternative splicing of mouse amelogenin pre-mRNA leads to the production of more than 14 protein isoforms, the functions of which are not totally understood. The smaller splice products, [A + 4] or M73 and [A - 4] or M59, have been shown to act differently as signaling molecules affecting odontogenic and other cell types. The mechanisms of these signaling processes, beginning with receptor identification, are not well understood. Utilizing radiolabeled [A - 4], we show here that 3H[A - 4] binds in a saturable fashion to the cell surface of C2C12 mouse fetal myoblasts at 4 degrees C, and not only binds at the surface but is internalized at 37 degrees C. "Far Western" immunohistochemistry performed on sections of E18 mouse incisors and molars with biotin-labeled [A - 4] as the primary ligand demonstrates [A - 4]-biotin binding to polarizing ameloblasts and odontoblasts, cells of the dental follicle, and along the stratum intermedium. Using [A - 4] affinity column chromatography and [A - 4]-biotin label transfer reaction, we have identified a 95 kDa C2C12 cell surface protein which bound [A - 4]. Utilizing Tandem MS (MS/MS) sequencing, we report the novel finding of the 95 kDa murine transmembrane protein, LAMP-1, originally identified as a lysosomal membrane protein that is also found at the cell surface, as an [A - 4] cell binding protein.

The impact of bioactive molecules to stimulate tooth repair and regeneration as part of restorative dentistry.

After implantation in the exposed pulp, some molecules of the den-tin extracellular matrix induce the formation of a reparative dentinal bridge in the coronal pulp. In some cases, total occlusion of the root canal also is observed. This is the case for bone sialoprotein, bone morphogenetic protein-7, Dentonin (a fragment from matrix extracellular phosphoglycoprotein), and two small amelogenin gene splice products (A+4 and A-4). Cells implicated in the reparative process are recruited, proliferate, and differentiate into osteoblast-like and odontoblast-like cells. The same results may be obtained by direct implantation of odontoblast progenitor cell into the pulp.

Chondroitin sulfate is involved in the hypercalcification of the organic matrix of bovine peritubular dentin.

Apatitic mineral of dentin forms within the collagenous matrix (intertubular dentin, ITD) secreted from the odontoblastic processes (OP). Highly calcified mineral (peritubular dentin, PTD) is deposited at the interface between the ITD and each process membrane, creating a tubular system penetrating the dentin that extends from the dentino-enamel junction to the predentin-dentin junction. We focus on determining the composition of the PTD both with regard to its organic matrix and the inorganic phase. A laser capture technique has been adapted for the isolation of the mineralized PTD free from the ITD, and for the analysis of the PTD by SEM, TEM, and energy dispersive spectrometry (EDS), these data were subsequently compared with similar analyses of intact dentin slices containing ITD bounded-PTD annuli. Elemental line scans reveal clearly marked boundaries between ITD, PTD, and OP components, and illustrate the differences in composition, and topographical surface roughness. The organic matrix of the PTD was shown to be sulfur rich, and further antibody labeling showed the sulfated organic component to be chondroitin sulfate [corrected]. In this PTD organic matrix the S/Ca and Ca/P ratios were distinctly higher than in the ITD, indicating that polysaccharide bound S supplies the anionic counterion facilitating the formation of the apatitic PTD mineral.

Two related low molecular mass polypeptide isoforms of amelogenin have distinct activities in mouse tooth germ differentiation in vitro.

UNLABELLED: Embryonic mouse tooth germs were cultured in vitro in the presence of two related amelogenin isoforms to determine their effects on tooth development. Our results show that these individual proteins have specific but quite different effects on epithelial-derived ameloblasts versus mesenchymal-derived odontoblasts. INTRODUCTION: Amelogenins, the main protein components of enamel matrix, have been shown to have signaling activity. Amelogenin isoforms differing only by the presence or exclusion of exon 4, designated [A+4] (composed of exons 2, 3, 4, 5, 6d, and 7) and [A-4] (composed of exons 2, 3, 5, 6d, and 7), showed similar, but different, effects both in vitro and in vivo on postnatal teeth. MATERIALS AND METHODS: Lower first molar tooth germs of E15/16 CD1 mice were microdissected and cultured in vitro in a semisolid media containing either 20% FBS, 2% FBS, or 2% FBS with either 1.5 nM [A+4], [A-4], or both for 6 days. Tooth germs were analyzed by H&E staining and immunohistochemistry for collagen I, dentin matrix protein 2, and DAPI nuclear staining. RESULTS: Teeth cultured in media containing 20% FBS showed normal development with polarized ameloblasts, and odontoblasts producing dentin matrix, and DMP2 expression in odontoblasts and pre-ameloblasts. Culture in 2% FBS media resulted in no ameloblast polarization and modest odontoblast differentiation with scant dentin matrix. Tooth germs cultured with [A+4] in 2% FBS media had well-polarized odontoblasts with robust dentin production and concomitant ameloblast polarization. DMP2 expression was equal to or greater than seen in the 20% FBS culture condition. In cultures with [A-4] in 2% FBS media, odontoblast polarization and dentin production was reduced compared with [A+4]. However, the pre-ameloblast layer was disorganized, with no ameloblast polarization occurring along the dentin surface. DMP2 expression was reduced in the odontoblasts compared with the 20% FBS and [A+4] conditions and was almost completely abrogated in the pre-ameloblasts. CONCLUSION: These data show different signaling activities of these closely related amelogenin isoforms on tooth development. Here we make the novel observation that [A-4] has an inhibitory effect on ameloblast development, whereas [A+4] strongly stimulates odontoblast development. We show for the first time that specific amelogenin isoforms have effects on embryonic tooth development in vitro and also hypothesize that DMP2 may play a role in the terminal differentiation of both ameloblasts and odontoblasts.