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ABSTRACT: The crowns of matrix metalloproteinase 20 (Mmp20) null mice fracture at the dentino-enamel junction (DEJ), whereas the crowns of kallikrein-related peptidase 4 (Klk4) null mice fracture in the deep enamel just above the DEJ. We used backscatter scanning electron microscopy to assess enamel mineralization in incisors from 9-wk-old wild-type, Klk4 null, and Mmp20 null mice, and in developing pig molars. We observed a line of hypermineralization along the DEJ in developing wild-type mouse and pig teeth. This line was discernible from the early secretory stage until the enamel in the maturation stage reached a similar density. The line was apparent in Klk4 null mice, but absent in Mmp20 null mice. Enamel in the Klk4 null mice matured normally at the surface, but was progressively less mineralized with depth. Enamel in the Mmp20 null mice formed as a mineral bilayer, with neither layer looking like true enamel. The most superficial mineral layer expanded during the maturation stage and formed irregular surface nodules. A surprising finding was the observation of electron backscatter from mid-maturation wild-type ameloblasts, which we attributed to the accumulation and release of iron. We conclude that enamel breaks in the deep enamel of Klk4 null mice because of decreasing enamel maturation with depth, and at the DEJ in Mmp20 null mice because of hypomineralization at the DEJ.
European Journal Of Oral Sciences 12/2011; 119 Suppl 1:217-25. · 1.88 Impact Factor
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ABSTRACT: The purpose of this study was to quantify and compare the amounts of volatiles (mostly protein) and mineral present in developing incisor enamel in normal mice and in those genetically engineered for absence of intact enamelin, ameloblastin, matrix metalloproteinase 20 (MMP20) or kallikrein-related peptidase 4 (KLK4). Data indicated that all mice showed peaks in the gross weight of volatiles and a similar weight of mineral at locations on incisors normally associated with early maturation. Thereafter, the content of volatiles on normal incisors declined rapidly by as much as 62%, but not by 100%, over 2 mm, accompanied by increases of ≈ threefold in mineral weights. Enamelin heterozygous mice (lower incisors) showed a decrease in volatile content across the maturation stage, yet mineral failed to increase significantly. Mmp20 null mice showed no significant loss of volatiles from maturing enamel, yet the amount of mineral increased. Klk4 null mice showed normal mineral acquisition up to early maturation, but the input of new volatiles in mid to late maturation caused the final mineralization to slow below normal levels. These results suggest that it is not only the amount of protein but also the nature or type of protein or fragments present in the local crystallite environment that affects their volumetric expansion as they mature.
European Journal Of Oral Sciences 12/2011; 119 Suppl 1:125-35. · 1.88 Impact Factor
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ABSTRACT: Mutations of the matrix metalloproteinase 20 (MMP20, enamelysin) gene cause autosomal-recessive amelogenesis imperfecta, and Mmp20 ablated mice also have malformed dental enamel. Here we showed that Mmp20 null mouse secretory-stage ameloblasts maintain a columnar shape and are present as a single layer of cells. However, the maturation-stage ameloblasts from null mouse cover extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin, indicative of a faulty dentino-enamel junction (DEJ). The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The enamel rods in Mmp20 null mice were grossly malformed or absent, indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ.
European Journal Of Oral Sciences 12/2011; 119 Suppl 1:199-205. · 1.88 Impact Factor
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ABSTRACT: Matrix metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are thought to be necessary to clear proteins from the enamel matrix of developing teeth. We characterized Mmp20 and Klk4 null mice to better understand their roles in matrix degradation and removal. Histological examination showed retained organic matrix in Mmp20, Klk4, and Mmp20/Klk4 double-null mouse enamel matrix, but not in the wild-type. X-gal histostaining of Mmp20 null mice heterozygous for the Klk4 knockout/lacZ knockin showed that Klk4 is expressed normally in the Mmp20 null background. This finding was corroborated by zymogram and western blotting, which discovered a 40-kDa protease induced in the maturation stage of Mmp20 null mice. Proteins were extracted from secretory-stage or maturation-stage maxillary first molars from wild-type, Mmp20 null, Klk4 null, and Mmp20/Klk4 double-null mice and were analyzed by SDS-PAGE and western blotting. Only intact amelogenins and ameloblastin were observed in secretory-stage enamel of Mmp20 null mice, whereas the secretory-stage matrix from Klk4 null mice was identical to the matrix from wild-type mice. More residual matrix was observed in the double-null mice compared with either of the single-null mice. These results support the importance of MMP20 during the secretory stage and of KLK4 during the maturation stage and show there is only limited functional redundancy for these enzymes.
European Journal Of Oral Sciences 12/2011; 119 Suppl 1:206-16. · 1.88 Impact Factor
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ABSTRACT: Enamel formation depends on a triad of tissue-specific matrix proteins (amelogenin, ameloblastin, and enamelin) to help initiate
and stabilize progressively elongating, thin mineral ribbons of hydroxyapatite formed during an appositional growth phase.
Subsequently, these proteins are eradicated to facilitate lateral expansion of the hydroxyapatite crystallites. The purpose
of this study was to investigate changes in enamel mineralization occurring in mice unable to produce kallikrein 4 (Klk4),
a proteinase associated with terminal extracellular degradation of matrix proteins during the maturation stage. Mice lacking
functional matrix metalloproteinase 20 (Mmp20), a proteinase associated with early cleavage of matrix proteins during the
secretory stage, were also analyzed as a frame of reference. The results indicated that mice lacking Klk4 produce enamel that
is normal in thickness and overall organization in terms of layers and rod/inter-rod structure, but there is a developmental
defect in enamel rods where they first form near the dentinoenamel junction. Mineralization is normal up to early maturation
after which the enamel both retains and gains additional proteins and is unable to mature beyond 85% mineral by weight. The
outmost enamel is hard, but inner regions are soft and contain much more protein than normal. The rate of mineral acquisition
overall is lower by 25%. Mice lacking functional Mmp20 produce enamel that is thin and structurally abnormal. Relatively high
amounts of protein remain throughout maturation, but the enamel is able to change from 67 to 75% mineral by weight during
maturation. These findings reaffirm the importance of secreted proteinases to enamel mineral acquisition.
Journal of Biological Chemistry 05/2011; 286(20):18149-18160. · 4.77 Impact Factor
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ABSTRACT: The enamel layerof kallikrein 4 (Klk4)-null mice has a normal thickness and a decussating pattern of enamel rods, but it contains residual enamel proteins, is less highly mineralized, and fractures in its deepest part just above the dentino-enamel junction (DEJ). The plane of fracture is puzzling because the deepest enamel is deposited earliest and, through the action of the secretory stage enamel protease (Mmp20), is the most mature part of the enamel layer at the time of the onset of Klk4 expression. Objectives: To characterize the planes of fracture in Mmp20- and Klk4-null mice and to localize Klk4 expression in developing teeth. Methods:Klk4- and Mmp20-null mice were sacrificed at 7 weeks and their mandibular incisors were characterized by scanning electron microscopy. Klk4(+/)(lac)(Z) mice were mated with Klk4(+/)(lac)(Z) mice. Offspring were genotyped by polymerase chain reaction. Klk4(+/)(+), Klk4(+/)(lac)(Z), and Klk4(lac)(Z/)(lac)(Z) (null) littermates on postnatal days 5, 8, 11, and 14 were processed for β-galactosidase histochemistry.
The enamel layer fractures at the DEJ in Mmp20-null mice, and fractures occur in enamel above the DEJ in Klk4-null mice. Klk4 is not expressed by secretory-stage ameloblasts, murine odontoblasts beneath the secretory stage, or maturation-stage ameloblasts. Klk4 is specifically expressed by transition and maturation-stage ameloblasts.
The breakage of enamel near the DEJ in Klk4-null mice is not due to a failure of odontoblasts to express Klk4, but it relates to a progressive hypomineralization of enamel with depth.
Cells Tissues Organs 05/2011; 194(2-4):211-5. · 2.20 Impact Factor
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ABSTRACT: Dental enamel development occurs in stages as observed by the changing morphology of the ameloblasts that are responsible for enamel formation. During the secretory stage of development, proteins including MMP20 are secreted into the enamel matrix. MMP20 is required for proper enamel formation as mutation of the Mmp20 gene causes autosomal recessive amelogenesis imperfecta. Here, we examined in detail the morphology of the Mmp20-null ameloblast cell layer. Intriguingly, we found that the Mmp20-null mouse secretory stage ameloblasts retract their Tomes' processes as if preparing to enter the maturation stage but later reextend their Tomes' processes as if resuming the secretory stage. We also demonstrated that MMP20 cleaves epithelial cadherin, i.e. E-cadherin. Cadherins are transmembrane proteins with extracellular domains that provide adhesive contacts between neighboring cells. Their intracellular domains bind to the cell cytoskeleton through catenins, including β-catenin. When specific MMPs cleave the cadherin extracellular domain, β-catenin is released and may locate to the cell nucleus as a transcription factor. Therefore, MMP20 may influence ameloblast developmental progression through hydrolysis of cadherin extracellular domains with associated release of transcription factor(s).
Cells Tissues Organs 04/2011; 194(2-4):222-6. · 2.20 Impact Factor
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ABSTRACT: Enamel formation depends on a triad of tissue-specific matrix proteins (amelogenin, ameloblastin, and enamelin) to help initiate and stabilize progressively elongating, thin mineral ribbons of hydroxyapatite formed during an appositional growth phase. Subsequently, these proteins are eradicated to facilitate lateral expansion of the hydroxyapatite crystallites. The purpose of this study was to investigate changes in enamel mineralization occurring in mice unable to produce kallikrein 4 (Klk4), a proteinase associated with terminal extracellular degradation of matrix proteins during the maturation stage. Mice lacking functional matrix metalloproteinase 20 (Mmp20), a proteinase associated with early cleavage of matrix proteins during the secretory stage, were also analyzed as a frame of reference. The results indicated that mice lacking Klk4 produce enamel that is normal in thickness and overall organization in terms of layers and rod/inter-rod structure, but there is a developmental defect in enamel rods where they first form near the dentinoenamel junction. Mineralization is normal up to early maturation after which the enamel both retains and gains additional proteins and is unable to mature beyond 85% mineral by weight. The outmost enamel is hard, but inner regions are soft and contain much more protein than normal. The rate of mineral acquisition overall is lower by 25%. Mice lacking functional Mmp20 produce enamel that is thin and structurally abnormal. Relatively high amounts of protein remain throughout maturation, but the enamel is able to change from 67 to 75% mineral by weight during maturation. These findings reaffirm the importance of secreted proteinases to enamel mineral acquisition.
Journal of Biological Chemistry 03/2011; 286(20):18149-60. · 4.77 Impact Factor
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ABSTRACT: Endoplasmic reticulum (ER) stress and its consequent activation of the unfolded protein response (UPR) signaling pathway have been implicated in several pathophysiologic disorders as well as in drug resistance to treatment of tumors. Several techniques have been devised that qualitatively and quantitatively demonstrate the presence of ER stress and the activation of the UPR; however, most of these methods cannot be used to measure ER stress in real time. Here we describe the use of cells stably transduced with a secreted reporter, Gaussia luciferase (Gluc), to measure fluoride-induced ER stress. Factors that affect ER homeostasis, such as high-dose fluoride, will cause decreased Gluc secretion that can be measured as a decrease in Gluc activity in the culture medium supernatant. Gluc catalyzes the oxidative decarboxylation of coelenterazine (CTZ) to coeleneteramide, resulting in blue bioluminescence (λ(max) 485 nm). Therefore, Gluc activity can be easily quantified by mixing a small aliquot of the medium supernatant with CTZ and measuring the resulting bioluminescence in a luminometer. Among the various reporters used so far, Gluc is regarded as the most sensitive indicator of ER stress. A second advantage for using Gluc is its ability to function in a wide pH range. This is especially useful for studying fluoride-mediated toxicity as fluoride-induced stress is enhanced under acidic conditions. Since Gluc can be measured in a noninvasive manner, it has been used in several in vitro and in vivo applications. In this chapter, we detail our methodology for using Gluc to monitor fluoride-induced ER stress.
Methods in enzymology 01/2011; 491:111-25. · 1.90 Impact Factor
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ABSTRACT: Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows.
PLoS ONE 01/2010; 5(9). · 4.09 Impact Factor
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ABSTRACT: It is not known why the ameloblasts responsible for dental enamel formation are uniquely sensitive to fluoride (F(-)). Herein, we present a novel theory with supporting data to show that the low pH environment of maturating stage ameloblasts enhances their sensitivity to a given dose of F(-). Enamel formation is initiated in a neutral pH environment (secretory stage); however, the pH can fall to below 6.0 as most of the mineral precipitates (maturation stage). Low pH can facilitate entry of F(-) into cells. Here, we asked if F(-) was more toxic at low pH, as measured by increased cell stress and decreased cell function.
Treatment of ameloblast-derived LS8 cells with F(-) at low pH reduced the threshold dose of F(-) required to phosphorylate stress-related proteins, PERK, eIF2alpha, JNK and c-jun. To assess protein secretion, LS8 cells were stably transduced with a secreted reporter, Gaussia luciferase, and secretion was quantified as a function of F(-) dose and pH. Luciferase secretion significantly decreased within 2 hr of F(-) treatment at low pH versus neutral pH, indicating increased functional toxicity. Rats given 100 ppm F(-) in their drinking water exhibited increased stress-mediated phosphorylation of eIF2alpha in maturation stage ameloblasts (pH<6.0) as compared to secretory stage ameloblasts (pH approximately 7.2). Intriguingly, F(-)-treated rats demonstrated a striking decrease in transcripts expressed during the maturation stage of enamel development (Klk4 and Amtn). In contrast, the expression of secretory stage genes, AmelX, Ambn, Enam and Mmp20, was unaffected.
The low pH environment of maturation stage ameloblasts facilitates the uptake of F(-), causing increased cell stress that compromises ameloblast function, resulting in dental fluorosis.
PLoS ONE 01/2010; 5(5):e10895. · 4.09 Impact Factor
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ABSTRACT: Kallikrein-4 (KLK4) is a serine protease expressed during enamel maturation, which is critical for proper enamel formation.
KLK4 is secreted as an inactive zymogen and identification of its activator remains elusive. Herein we discuss what is currently
known about the activation of pro-KLK4.
Key wordsenamel-kallikrein-4-matrix metalloproteinase-20-dipeptidyl peptidase I
12/2009: pages 413-415;
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ABSTRACT: Transforming growth factor-beta1 (TGF-beta1) regulates a variety of cellular responses that are dependent on the developmental stage and on the origins of the cell or the tissue. In mature tissues, and especially in tissues of epithelial origin, TGF-beta1 is generally considered to be a growth inhibitor that may also promote apoptosis. The ameloblast cells of the enamel organ epithelium are adjacent to and responsible for the developing enamel layer on unerupted teeth. Once the enamel layer reaches its full thickness, the tall columnar secretory-stage ameloblasts shorten, and a portion of these maturation-stage ameloblasts become apoptotic. Here we investigate whether TGF-beta1 plays a role in apoptosis of the maturation-stage ameloblasts. We demonstrate in vitro that ameloblast lineage cells are highly susceptible to TGF-beta1-mediated growth arrest and are prone to TGF-beta1-mediated cell death/apoptosis. We also demonstrate in vivo that TGF-beta1 is expressed in the maturation-stage enamel organ at significantly higher levels than in the earlier secretory-stage enamel organ. This increased expression of TGF-beta1 correlates with an increase in expression of the enamel organ immediate-early stress-response gene and with a decrease in the anti-apoptotic Bcl2 : Bax expression ratio. We conclude that TGF-beta1 may play an important role in ameloblast apoptosis during the maturation stage of enamel development.
European Journal Of Oral Sciences 05/2009; 117(2):105-12. · 1.88 Impact Factor
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ABSTRACT: Exposure to excessive amounts of fluoride (F(-)) causes dental fluorosis in susceptible individuals; however, the mechanism of F(-)-induced toxicity is unclear. Previously, we have shown that high-dose F(-) activates the unfolded protein response (UPR) in ameloblasts that are responsible for dental enamel formation. The UPR is a signaling pathway responsible for either alleviating endoplasmic reticulum (ER) stress or for inducing apoptosis of the stressed cells.
In this study we determined if low-dose F(-) causes ER stress and activates the UPR, and we also determined whether F(-) interferes with the secretion of proteins from the ER.
We stably transfected the ameloblast-derived LS8 cell line with secreted alkaline phosphatase (SEAP) and determined activity and localization of SEAP and F(-)-mediated induction of UPR proteins. Also, incisors from mice given drinking water containing various concentrations of F(-) were examined for eucaryotic initiation factor-2, subunit alpha (eIF2alpha) phosphorylation.
We found that F(-) decreases the extracellular secretion of SEAP in a linear, dose-dependent manner. We also found a corresponding increase in the intracellular accumulation of SEAP after exposure to F(-). These changes are associated with the induction of UPR proteins such as the molecular chaperone BiP and phosphorylation of the UPR sensor PKR-like ER kinase, and its substrate, eIF2alpha. Importantly, F(-)-induced phosphorylation of eIF2alphawas confirmed in vivo.
These data suggest that F(-) initiates an ER stress response in ameloblasts that interferes with protein synthesis and secretion. Consequently, ameloblast function during enamel development may be impaired, and this may culminate in dental fluorosis.
Environmental Health Perspectives 10/2008; 116(9):1142-6. · 7.04 Impact Factor
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J Timothy Wright,
Thomas C Hart,
P Suzanne Hart,
Darrin Simmons,
Cynthia Suggs,
Bill Daley,
Jim Simmer,
Jan Hu, John D Bartlett,
Yong Li,
Zhi-An Yuan,
W Kim Seow,
Carolyn W Gibson
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ABSTRACT: Amelogenesis imperfecta (AI) is caused by AMEL, ENAM, MMP20 and KLK4 gene mutations. Mice lacking expression of the AmelX, Enam and Mmp20 genes have been generated. These mouse models provide tools for understanding enamel formation and AI pathogenesis. This study describes the AI phenotypes and relates them to their mouse model counterparts. Human AI phenotypes were determined in a clinical population of AI families and published cases. Human and murine teeth were evaluated using light and electron microscopy. A total of 463 individuals from 54 families were evaluated and mutations in the AMEL, ENAM and KLK4 genes were identified. The majority of human mutations for genes coding enamel nonproteinase proteins (AMEL and ENAM) resulted in variable hypoplasia ranging from local pitting to a marked, generalized enamel thinning. Specific AMEL mutations were associated with abnormal mineralization and maturation defects. Amel and Enam null murine models displayed marked enamel hypoplasia and a complete loss of prism structure. Human mutations in genes coding for the enamel proteinases (MMP20 and KLK4) cause variable degrees of hypomineralization. The murine Mmp20 null mouse exhibits both hypoplastic and hypomineralized defects. The currently available Amel and Enam mouse models for AI exhibit enamel phenotypes (hypoplastic) that are generally similar to those seen in humans. Mmp20 null mice have a greater degree of hypoplasia than humans with MMP20 mutations. Mice lacking expression of the currently known genes associated with the human AI conditions provide useful models for understanding the pathogenesis of these conditions.
Cells Tissues Organs 09/2008; 189(1-4):224-9. · 2.20 Impact Factor
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ABSTRACT: The enamel matrix proteins (amelogenin, enamelin and ameloblastin) are degraded by matrix metalloproteinase-20 and kallikrein-4 during enamel development and mature enamel is virtually protein free. The precise mechanism of removal and degradation of the enamel protein cleavage products from the matrix, however, remains poorly understood. It has been proposed that receptor-mediated endocytosis allows for the cleaved proteins to be removed from the matrix during enamel formation and then transported to the lysosome for further degradation. This study aims to identify lysosomal proteases that are present in maturation-stage enamel organ. RNA from first molars of 11-day-old mice was collected and expression was initially assessed by RT-PCR and then quantified by qPCR. The pattern of expression of selected proteases was assessed by immunohistochemical staining of demineralized mouse incisors. With the exception of cathepsin G, all lysosomal proteases assessed were expressed in maturation-stage enamel organ. Identified proteases included cathepsins B, D, F, H, K, L, O, S and Z. Tripeptidyl peptidases I and II as well as dipeptidyl peptidases I, II, III and IV were also found to be expressed. Immunohistochemical staining confirmed that the maturation-stage ameloblasts express cathepsins L and S and tripeptidyl peptidase II. Our results suggest that the ameloblasts are enriched by a large number of lysosomal proteases at maturation that are likely involved in the degradation of the organic matrix.
Cells Tissues Organs 09/2008; 189(1-4):111-4. · 2.20 Impact Factor
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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.
Biological Chemistry 06/2008; 389(6):695-700. · 2.96 Impact Factor
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ABSTRACT: Dental enamel forms through the concerted activities of specialized extracellular matrix proteins, including amelogenin, enamelin, MMP20, and KLK4. Defects in the genes encoding these proteins cause non-syndromic inherited enamel malformations collectively designated as amelogenesis imperfecta (AI). These genes, however, account for only about a quarter of all AI cases. Recently we identified mutations in FAM83H that caused autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI). Unlike other genes that cause AI, FAM83 H does not encode an extracellular matrix protein. Its location inside the cell is completely unknown, as is its function. We here report novel FAM83H mutations in four kindreds with ADHCAI. All are nonsense mutations in the last exon (c.1243G>T, p.E415X; c.891T>A, p.Y297X; c.1380G>A, p.W460X; and c.2029C>T, p.Q677X). These mutations delete between 503 and 883 amino acids from the C-terminus of a protein normally comprised of 1179 residues. The reason these mutations cause such extreme defects in the enamel layer without affecting other parts of the body is not known yet. However it seems evident that the large C-terminal part of the protein is essential for proper enamel calcification. © 2008 Wiley-Liss, Inc.
Human Mutation 05/2008; 29(8):E95 - E99. · 5.69 Impact Factor
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ABSTRACT: Dental enamel is the most mineralized tissue of vertebrate organisms. Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. The formation of an ordered enamel organic extracellular matrix (ECM) seems be a crucial step for the proper formation of mineral phase. Polarizing microscopy demonstrates that the ordered supramolecular structure of the secretory-stage enamel organic ECM is strongly birefringent. In the present work we analyzed the birefringence of secretory-stage enamel organic ECM in amelogenin (Amelx)- and enamelysin (Mmp20)-deficient mice. Female Amelx+/- animals showed significant reduction in optical retardation values when compared with the Amelx+/+ subgroup (p=0.0029). The secretory-stage enamel organic ECM of the Amelx-/- subgroup did not exhibit birefringence. The secretory-stage enamel organic ECM of Mmp20-/- mice showed a significant decrease in optical retardation as compared with Mmp20+/+ and Mmp20+/- mice (p=0.0000). Mmp20+/- and Mmp20+/+ mice exhibited similar birefringence (p=1.0000). The results presented here support growing evidence for the idea that the birefringence of secretory-stage enamel organic ECM is influenced by the ordered supramolecular organization of its components.
Connective Tissue Research 02/2007; 48(1):39-45. · 1.20 Impact Factor
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ABSTRACT: Mutations in both the human amelogenin and human matrix metalloproteinase-20 (MMP20, enamelysin) genes cause amelogenesis imperfecta. Both genes have also been individually deleted from the mouse and each deletion results in defective dental enamel. Here, we compare the stage-specific progression of enamel development in continuously erupting mouse incisors from amelogenin null and MMP-20 null mice. Our goal was to closely examine differences in enamel and enamel organ structure between these mice that would allow a better understanding of each protein's function. The predominant feature of the amelogenin null incisors was the late onset of mineral deposition, with little or no protein present within the forming mineral. Conversely, the developing MMP-20 null incisors had a layer of protein between the apical surface of the ameloblasts and the forming enamel. Furthermore, the protein present within the enamel matrix was disorganized. An analysis of crystal structure demonstrated that the thin amelogenin null enamel was plate-like, while the MMP-20 null enamel had a disrupted prism pattern. These results suggest that amelogenin is essential for appositional crystal growth during the early to mid-secretory stage and for the maintenance of the crystal ribbon structure. They also suggest that MMP-20 is responsible for enamel matrix organization and for subsequent efficient reabsorption of enamel matrix proteins. Both genes are essential for the generation of full-thickness enamel containing the characteristic decussating prism pattern.
European Journal Of Oral Sciences 06/2006; 114 Suppl 1:18-23; discussion 39-41, 379. · 1.88 Impact Factor
