scholarly journals Proteinases in Developing Dental Enamel

1999 ◽  
Vol 10 (4) ◽  
pp. 425-441 ◽  
Author(s):  
J.D. Bartlett ◽  
J.P. Simmer
Keyword(s):  
Proteinase Inhibitors ◽  
Dental Enamel ◽  
Serine Proteinase ◽  
Organic Matrix ◽  
Enamel Organ ◽  
Maturation Stage ◽  
Enamel Matrix ◽  
Enamel Matrix Proteins ◽  
Early Maturation

For almost three decades, proteinases have been known to reside within developing dental enamel. However, identification and characterization of these proteinases have been slow and difficult, because they are present in very small quantities and they are difficult to purify directly from the mineralizing enamel. Enamel matrix proteins such as amelogenin, ameloblastin, and enamelin are cleaved by proteinases soon after they are secreted, and their cleavage products accumulate in the deeper, more mature enamel layers, while the full-length proteins are observed only at the surface. These results suggest that proteinases are necessary for "activating" enamel proteins so the parent proteins and their cleavage products may perform different functions. A novel matrix metalloproteinase named enamelysin (MMP-20) was recently cloned from tooth tissues and was later shown to localize primarily within the most recently formed enamel. Furthermore, recombinant porcine enamelysin was demonstrated to cleave recombinant porcine amelogenin at virtually all of the sites that have previously been described in vivo. Therefore, enamelysin is at least one enzyme that may be important during early enamel development. As enamel development progresses to the later stages, a profound decrease in the enamel protein content is observed. Proteinases have traditionally been assumed to degrade the organic matrix prior to its removal from the enamel. Recently, a novel serine proteinase named enamel matrix serine proteinase-1 (EMSP1) was cloned from enamel organ epithelia. EMSP1 localizes primarily to the early maturation stage enamel and may, therefore, be involved in the degradation of proteins prior to their removal from the maturing enamel. Other, as yet unidentified, proteinases and proteinase inhibitors are almost certainly present within the forming enamel and await discovery.

scholarly journals Enamelysin (Matrix Metalloproteinase 20)-deficient Mice Display an Amelogenesis Imperfecta Phenotype

2002 ◽  
Vol 277 (51) ◽  
pp. 49598-49604 ◽  
Author(s):  
John J. Caterina ◽  
Ziedonis Skobe ◽  
Joanne Shi ◽  
Yanli Ding ◽  
James P. Simmer ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Tooth Development ◽  
Developmental Time ◽  
Enamel Organ ◽  
Null Mouse ◽  
Enamel Matrix ◽  
Enamel Matrix Proteins ◽  
Time Period

Enamelysin is a tooth-specific matrix metalloproteinase that is expressed during the early through middle stages of enamel development. The enamel matrix proteins amelogenin, ameloblastin, and enamelin are also expressed during this same approximate developmental time period, suggesting that enamelysin may play a role in their hydrolysis. In support of this interpretation, recombinant enamelysin was previously demonstrated to cleave recombinant amelogenin at virtually all of the precise sites known to occurin vivo. Thus, enamelysin is likely an important amelogenin-processing enzyme. To characterize thein vivobiological role of enamelysin during tooth development, we generated an enamelysin-deficient mouse by gene targeting. Although mice heterozygous for the mutation have no apparent phenotype, the enamelysin null mouse has a severe and profound tooth phenotype. Specifically, the null mouse does not process amelogenin properly, possesses an altered enamel matrix and rod pattern, has hypoplastic enamel that delaminates from the dentin, and has a deteriorating enamel organ morphology as development progresses. Our findings demonstrate that enamelysin activity is essential for proper enamel development.

scholarly journals Synthesis and degradation in vivo of a phosphoprotein from rat dental enamel. Identification of a phosphorylated precursor protein in the extracellular organic matrix

1985 ◽  
Vol 230 (2) ◽  
pp. 423-433 ◽  
Author(s):  
E Strawich ◽  
M J Glimcher
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dental Enamel ◽  
Organic Matrix ◽  
Precursor Protein ◽  
Female Rats ◽  
Enamel Organ ◽  
Major Species ◽  
Tail Tendon

The cellular enamel organ and the cell-free organic matrix of developing enamel of female rats injected intravascularly with [3H]serine and [3H]proline were extracted in a number of solvents and examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and h.p.l.c. in 6M-guanidinium chloride at intervals varying from 5 min to 1 week after injection. Three major species soluble in NH4HCO3 with Mr values of approx. 100 000, 25 000 and 11 000 were identified in the cellular enamel organ. The Mr 100 000 and 11 000 components were not secreted but remained intracellular for periods of up to 1 week after injection of the radioactively labelled amino acids. In contrast, the Mr 25 000 species was secreted from the cells and was first detected in the extracellular organic matrix approx. 15-30 min after injection. With time, labelled components, first of Mr approx. 11 000 and subsequently approx. 6500, were detected in the organic matrix concomitant with a relative decrease in the Mr 25 000 component, demonstrating that the lower Mr species were derived from degradation of the putative extracellular precursor protein (Mr 25 000). All of the extracellular components were found to contain O-phosphoserine. No radioactively labelled component with an Mr greater than approx. 25 000, either an amelogenin or an enamelin, was observed in the extracellular organic matrix or in an intracellular component which subsequently was lost from the intracellular pool. The Mr of the highest Mr protein or class of proteins is calculated to be approx. 22 000-26 000 when standard proteins are used as markers, but only 15 000-18 000 when using the CNBr peptides of alpha 1 chains of rat tail tendon collagen as markers.

Studies on the Changes in Developing Enamel Caused By Ingestion of High Levels of Fluoride in the Rat

1987 ◽  
Vol 1 (2) ◽  
pp. 176-180 ◽  
Author(s):  
P.K. Denbesten ◽  
M.A. Crenshaw
Keyword(s):  
Enamel Organ ◽  
Maturation Stage ◽  
Careful Study ◽  
Enamel Matrix ◽  
Rat Incisor ◽  
Early Maturation ◽  
Enamel Proteins ◽  
And Control ◽  
Water Fluoride

Exposure to chronic high levels of fluoride results in the formation of fluorosed enamel. Although enamel may be more susceptible to fluorotic effects at certain stages of development, fluoride at sufficiently high levels may affect enamel at all stages of formation. Careful study of the changes in enamel caused by chronic fluoride ingestion is needed to understand more fully the mechanisms involved in the formation of fluorotic enamel. This paper discusses the various studies we have completed to define the changes, in developing enamel of the rat incisor, caused by long-term ingestion of fluoride in drinking water. Fluoride has been found to inhibit secretion of enamel proteins. Changes in the maturation stage of enamel formation include the retention of amelogenin proteins during early maturation. The various mechanisms which have been investigated in the formation of fluorosed enamel include a direct effect of fluoride on the enamel organ, and specific interactions of fluoride with the extracellular enamel matrix. Although the same amount of protease appears to be secreted in fluorosed and control enamel, a delay in the digestion of amelogenin protein occurs. This suggests that fluoride may directly or indirectly inhibit the protease present in fluorosed enamel to slow the proteolysis of amelogenins.

scholarly journals MMP20 Overexpression Disrupts Molar Ameloblast Polarity and Migration

2018 ◽  
Vol 97 (7) ◽  
pp. 820-827 ◽  
Author(s):  
M. Shin ◽  
M.B. Chavez ◽  
A. Ikeda ◽  
B.L. Foster ◽  
J.D. Bartlett
Keyword(s):  
Cell Migration ◽  
Enamel Organ ◽  
Maturation Stage ◽  
Micro Computed Tomography ◽  
Mineral Density ◽  
Enamel Matrix Proteins ◽  
Enamel Mineralization ◽  
Enamel Layer

Ameloblasts responsible for enamel formation express matrix metalloproteinase 20 (MMP20), an enzyme that cleaves enamel matrix proteins, including amelogenin (AMELX) and ameloblastin (AMBN). Previously, we showed that continuously erupting incisors from transgenic mice overexpressing active MMP20 had a massive cell infiltrate present within their enamel space, leading to enamel mineralization defects. However, effects of MMP20 overexpression on mouse molars were not analyzed, although these teeth more accurately represent human odontogenesis. Therefore, MMP20-overexpressing mice ( Mmp20+/+Tg+) were assessed by multiscale analyses, combining several approaches from high-resolution micro–computed tomography to enamel organ immunoblots. During the secretory stage at postnatal day 6 (P6), Mmp20+/+Tg+ mice had a discontinuous ameloblast layer and, unlike incisors, molar P12 maturation stage ameloblasts abnormally migrated away from the enamel layer into the stratum intermedium/stellate reticulum. TOPflash assays performed in vitro demonstrated that MMP20 expression promoted β-catenin nuclear localization and that MMP20 expression promoted invasion through Matrigel-coated filters. However, for both assays, significant differences were eliminated in the presence of the β-catenin inhibitor ICG-001. This suggests that MMP20 activity promotes cell migration via the Wnt pathway. In vivo, the unique molar migration of amelogenin-expressing ameloblasts was associated with abnormal deposition of ectopic calcified nodules surrounding the adherent enamel layer. Enamel content was assessed just prior to eruption at P15. Compared to wild-type, Mmp20+/+Tg+ molars exhibited significant reductions in enamel thickness (70%), volume (60%), and mineral density (40%), and MMP20 overexpression resulted in premature cleavage of AMBN, which likely contributed to the severe defects in enamel mineralization. In addition, Mmp20+/+Tg+ mouse molar enamel organs had increased levels of inactive p-cofilin, a protein that regulates cell polarity. These data demonstrate that increased MMP20 activity in molars causes premature degradation of ameloblastin and inactivation of cofilin, which may contribute to pathological Wnt-mediated cell migration away from the enamel layer.

Matrix and Mineral Changes in Developing Enamel

1979 ◽  
Vol 58 (2_suppl) ◽  
pp. 871-882 ◽  
Author(s):  
C. Robinson ◽  
H.D. Briggs ◽  
P.J. Atkinson ◽  
J.A. Weatherell
Keyword(s):  
Mineral Content ◽  
Inorganic Ions ◽  
Organic Matrix ◽  
Small Samples ◽  
Enamel Organ ◽  
Enamel Matrix ◽  
Subsequent Loss ◽  
Matrix Components ◽  
Significant Concentration ◽  
Mineral Changes

An investigation of the changes taking place in the enamel and the enamel organ during enamel development has been carried out by analyzing small samples of tissue dissected from developing incisors of rat and bovine incisors. Observations showed that the synthesis of the enamel matrix and its subsequent loss were associated chiefly with a change in the major matrix components. This consisted of a selective loss of amelogenin components prior to secondary mineralization. Before this loss, some increase in the proportion of smaller molecular weight components suggested the possibility of limited breakdown. Even at the earliest stages examined, significant concentration of mineral ions was present. This increased steeply after most of the organic matrix had been removed. The Ca/P ratio of this mineral was constant throughout development. The concentration of minor inorganic ions (F, Mg and CO3) decreased as the tissue developed and a tendency was observed for certain ions (F, 32PO4) to penetrate and concentrate in the enamel, apparently as a consequence of the lost matrix being replaced by water, just prior to the steep increase in mineral content of the tissue.

scholarly journals Functions of KLK4 and MMP-20 in dental enamel formation

2008 ◽  
Vol 389 (6) ◽  
Author(s):  
Yuhe Lu ◽  
Petros Papagerakis ◽  
Yasuo Yamakoshi ◽  
Jan C.-C. Hu ◽  
John D. Bartlett ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Dental Enamel ◽  
Organic Matrix ◽  
Maturation Stage ◽  
Protein Cleavage ◽  
Enamel Formation ◽  
Residual Protein ◽  
Kallikrein 4 ◽  
Enamel Protein ◽  
Enamel Proteins

Abstract Two proteases are secreted into the enamel matrix of developing teeth. The early protease is enamelysin (MMP-20). The late protease is kallikrein 4 (KLK4). Mutations in MMP20 and KLK4 both cause autosomal recessive amelogenesis imperfecta, a condition featuring soft, porous enamel containing residual protein. MMP-20 is secreted along with enamel proteins by secretory-stage ameloblasts. Enamel protein-cleavage products accumulate in the space between the crystal ribbons, helping to support them. MMP-20 steadily cleaves accumulated enamel proteins, so their concentration decreases with depth. KLK4 is secreted by transition- and maturation-stage ameloblasts. KLK4 aggressively degrades the retained organic matrix following the termination of enamel protein secretion. The principle functions of MMP-20 and KLK4 in dental enamel formation are to facilitate the orderly replacement of organic matrix with mineral, generating an enamel layer that is harder, less porous, and unstained by retained enamel proteins.

Correlation of Apical and Lateral Membrane Modulations of Maturation Ameloblasts

1985 ◽  
Vol 64 (8) ◽  
pp. 1055-1061 ◽  
Author(s):  
Z. Skobe ◽  
F. LaFrazia ◽  
K. Prostak
Keyword(s):  
Apical Membrane ◽  
Apical Cell ◽  
Matrix Proteins ◽  
Maturation Stage ◽  
Enamel Matrix ◽  
Enamel Matrix Proteins ◽  
Rat Incisor ◽  
Lateral Membrane ◽  
Apical Junctions ◽  
Scanning Electron

Maturation ameloblasts of rat incisor teeth have smooth-ended and ruffle-ended apical membrane configurations. It has also been reported that maturation ameloblasts have several lateral membrane configurations. The purpose of this study was to determine the correlation between the modulations of lateral and apical cell membranes of murine incisor ameloblasts in the maturation stage of amelogenesis. Maxillary and mandibular incisors were dissected, demineralized, embedded in paraffin, sectioned and then de-paraffinized, and the enamel organs were prepared for scanning electron microscopy. Additional mouse and rat incisor enamel organs were fixed and teased apart during dehydration, then observed in the SEM. The lengths of smooth- and ruffle-ended ameloblast segments were measured, and the site, length, and frequency of each lateral membrane configuration were determined within each segment. The lateral membrane configuration with folds forming from 12 to 14 channels around the periphery of the cells was most predominant in both smooth- and ruffle-ended cells. Cells surrounded by from six to eight channels were the only other lateral membrane configuration observed in ruffle-ended ameloblasts. Smooth-ended ameloblasts had lateral membrane configurations with either dense or sparse microvillous projections in addition to both types of channel cells. The observation that channelled extracellular spaces are always associated with ruffle-ended cells suggests that channels somehow function in conjunction with the ruffled apical membrane in resorption and removal of enamel matrix proteins. The smooth-ended ameloblasts lack tight apical junctions, and their microvillous lateral membranes permit the passage of plasma fluids around cells to the maturing enamel surface. Analysis of our data indicates that specific lateral membrane configurations are related to the type of apical membrane present.

scholarly journals Protein nanoribbons template enamel mineralization

2020 ◽  
Vol 117 (32) ◽  
pp. 19201-19208 ◽  
Author(s):  
Yushi Bai ◽  
Zanlin Yu ◽  
Larry Ackerman ◽  
Yan Zhang ◽  
Johan Bonde ◽  
...  
Keyword(s):  
Supramolecular Assembly ◽  
Organic Content ◽  
Matrix Proteins ◽  
Maturation Stage ◽  
Enamel Matrix ◽  
Structural Protein ◽  
Enamel Matrix Proteins ◽  
Tissue Hardness ◽  
Enamel Mineralization

As the hardest tissue formed by vertebrates, enamel represents nature’s engineering masterpiece with complex organizations of fibrous apatite crystals at the nanometer scale. Supramolecular assemblies of enamel matrix proteins (EMPs) play a key role as the structural scaffolds for regulating mineral morphology during enamel development. However, to achieve maximum tissue hardness, most organic content in enamel is digested and removed at the maturation stage, and thus knowledge of a structural protein template that could guide enamel mineralization is limited at this date. Herein, by examining a gene-modified mouse that lacked enzymatic degradation of EMPs, we demonstrate the presence of protein nanoribbons as the structural scaffolds in developing enamel matrix. Using in vitro mineralization assays we showed that both recombinant and enamel-tissue–based amelogenin nanoribbons are capable of guiding fibrous apatite nanocrystal formation. In accordance with our understanding of the natural process of enamel formation, templated crystal growth was achieved by interaction of amelogenin scaffolds with acidic macromolecules that facilitate the formation of an amorphous calcium phosphate precursor which gradually transforms into oriented apatite fibers along the protein nanoribbons. Furthermore, this study elucidated that matrix metalloproteinase-20 is a critical regulator of the enamel mineralization as only a recombinant analog of a MMP20-cleavage product of amelogenin was capable of guiding apatite mineralization. This study highlights that supramolecular assembly of the scaffold protein, its enzymatic processing, and its ability to interact with acidic carrier proteins are critical steps for proper enamel development.

Gene Expression and Localization of Amelogenin in the Rat Incisor

1996 ◽  
Vol 10 (2) ◽  
pp. 201-207 ◽  
Author(s):  
T. Inage ◽  
H. Shimokawa ◽  
K. Wakao ◽  
S. Sasaki
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Maturation Stage ◽  
Enamel Matrix ◽  
Rat Incisor ◽  
Early Maturation ◽  
Incisal Edge ◽  
Enamel Epithelium ◽  
Developing Rat

Gene expression and localization of amelogenin were studied in the developing rat incisor by the methods of in situ hybridization and immunohistochemistry. ISH revealed the first expression of amelogenin mRNA in the inner enamel epithelium of the cervical loop. The signals were clearly observed in pre-ameloblasts in the region bordering on predentin formation and became more intense toward the cells on the initial enamel matrix secretion. The maximal signals were found in the cytoplasm of secretory ameloblasts. From the terminal secretion zone, the signals then became gradually weaker toward the incisal edge but were still evident in the cytoplasm of shortening, transitional ameloblasts and those at the early maturation stage. No signals were found in the cells of the stratum intermedium and stellate reticulum throughout amelogenesis. Immunohistochemistry by means of an antibody against amelogenin C-telopeptide consisting of 12 amino acids revealed immunoreaction in the secretory ameloblasts reacting to the ISH. When a polyclonal antibody against amelogenin was used, immunoreaction was found in the distal ends of ruffle-ended ameloblasts (RA) in the maturation zone. Those results indicated that amelogenin is synthesized by ameloblastic cells from the inner enamel epithelium to the early maturation stage and is then resorbed by the RA.

The Role of Albumin in Developing Rodent Dental Enamel: A Possible Explanation for White Spot Hypoplasia

1992 ◽  
Vol 71 (6) ◽  
pp. 1270-1274 ◽  
Author(s):  
C. Robinson ◽  
J. Kirkham ◽  
S.J. Brookes ◽  
R.C. Shore
Keyword(s):  
Monoclonal Antibodies ◽  
Calcium Phosphate ◽  
Gel Electrophoresis ◽  
Crystal Size ◽  
Dental Enamel ◽  
Maturation Stage ◽  
Crystallite Growth

The uptake of serum albumin by maturation-stage rodent enamel and the resulting effects on the growth of enamel crystallites were investigated in vitro. Albumin uptake was demonstrated by means of gel electrophoresis and confirmed by Western blotting with use of monoclonal antibodies. Measurement of crystal size was carried out by direct TEM measurement of enamel crystallite outlines after incubations in metastable solutions of calcium phosphate. The ability of endogenous enamel enzymes to degrade albumin was investigated by substrate-specific zymography. The results showed that albumin could be taken up by maturation-stage enamel and produce inhibition of crystallite growth. There was no detectable proteolytic activity in the enamel against albumin substrate, which suggests that albumin entering enamel by extravasation in vivo may produce incomplete tissue maturation, resulting in a white, opaque appearance on eruption.

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