MMP20-generated amelogenin cleavage products prevent formation of fan-shaped enamel malformations.

Dental enamel forms extracellularly as thin ribbons of amorphous calcium phosphate (ACP) that initiate on dentin mineral in close proximity to the ameloblast distal membrane. Secreted proteins are critical for this process. Enam-/- and Ambn-/- mice fail to form enamel. We characterize enamel ribbon formation in wild-type (WT), Amelx-/- and Mmp20-/- mouse mandibular incisors using focused ion beam scanning electron microscopy (FIB-SEM) in inverted backscatter mode. In Amelx-/- mice, initial enamel mineral ribbons extending from dentin are similar in form to those of WT mice. As early enamel development progresses, the Amelx-/- mineral ribbons develop multiple branches, resembling the staves of a Japanese fan. These striking fan-shaped structures cease growing after attaining ~ 20 µm of enamel thickness (WT is ~ 120 µm). The initial enamel mineral ribbons in Mmp20-/- mice, like those of the Amelx-/- and WT, extend from the dentin surface to the ameloblast membrane, but appear to be fewer in number and coated on their sides with organic material. Remarkably, Mmp20-/- mineral ribbons also form fan-like structures that extend to ~ 20 µm from the dentin surface. However, these fans are subsequently capped with a hard, disorganized outer mineral layer. Amelogenin cleavage products are the only matrix components absent in both Amelx-/- and Mmp20-/- mice. We conclude that MMP20 and amelogenin are not critical for enamel mineral ribbon initiation, orientation, or initial shape. The pathological fan-like plates in these mice may form from the lack of amelogenin cleavage products, which appear necessary to form ordered hydroxyapatite.

Evaluation of genetic polymorphisms in MMP2, MMP9 and MMP20 in Brazilian children with dental fluorosis.

Recent studies suggested that genetics contribute to differences in dental fluorosis (DF) susceptibility among individuals having the same environmental exposure. This study evaluated if MMP2, MMP9 and MMP20 are expressed during enamel development and assessed the association between polymorphisms in these genes with DF. Mice susceptible and resistant to DF were used to evaluate if MMPs were candidate genes for DF. The animals received fluoride and their enamels were used for immunohistochemistry. Additionally, 481 subjects from a city with fluoridation of public water supplies were recruited. Genotyping was performed using real time PCR. Allele/genotype frequencies were compared between groups. MMP2, MMP9 and MMP20 immunostaining was detected in both animal groups. DF was observed in 22.4% of the subjects. A borderline association was observed in MMP2 (rs243865), MMP9 (rs17576) and in MMP20 (rs1784418) (p = 0.06, p = 0.08 and p = 0.06 respectively). Briefly, MMPs were expressed during enamel maturation and genetic polymorphisms were not associated with DF.

Expression Pattern of Matrix Metalloproteinase 20 (MMP20) in Human Tumors.

BACKGROUND/AIM: Matrix metalloproteinase 20 (MMP20) is a member of the family of matrix metalloproteinases. Under normal conditions the expression of MMP20 is restricted to ameloblasts and odontoblasts. In order to identify a possible expression of MMP20 under pathological conditions, we investigated three major human tumor entities, i.e. colon, breast and lung tumors, on the mRNA and protein level. MATERIALS AND METHODS: Real-time RT-PCR and immunocytochemical analyses of established human tumor cell lines were employed for our study; immunohistochemical analysis was performed on both primary tumors and normal control tissues. RESULTS: MMP20 was identified on both the mRNA and the protein level in breast MCF-7, colon HT-29, and lung A549 cell lines. MMP20 was also detected in primary tumor tissue by immunohistochemistry. CONCLUSION: MMP20 is a new potential candidate for tumor diagnosis or therapy.

Matrix metalloproteinase-20 over-expression is detrimental to enamel development: a Mus musculus model.

BACKGROUND: Matrix metalloproteinase-20 (Mmp20) ablated mice have enamel that is thin and soft with an abnormal rod pattern that abrades from the underlying dentin. We asked if introduction of transgenes expressing Mmp20 would revert this Mmp20 null phenotype back to normal. Unexpectedly, for transgenes expressing medium or high levels of Mmp20, we found opposite enamel phenotypes depending on the genetic background (Mmp20(-/-) or Mmp20(+/+) ) in which the transgenes were expressed. METHODOLOGY/PRINCIPAL FINDINGS: Amelx-promoter-Mmp20 transgenic founder mouse lines were assessed for transgene expression and those expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Regardless of expression level, each transgene brought the null enamel back to full thickness. However, the high and medium expressing Mmp20 transgenes in the Mmp20 null background had significantly harder more mineralized enamel than did the low transgene expresser. Strikingly, when the high and medium expressing Mmp20 transgenes were present in the wild-type background, the enamel was significantly less well mineralized than normal. Protein gel analysis of enamel matrix proteins from the high and medium expressing transgenes present in the wild-type background demonstrated that greater than normal amounts of cleavage products and smaller quantities of higher molecular weight proteins were present within their enamel matrices. CONCLUSIONS/SIGNIFICANCE: Mmp20 expression levels must be within a specific range for normal enamel development to occur. Creation of a normally thick enamel layer may occur over a wider range of Mmp20 expression levels, but acquisition of normal enamel hardness has a narrower range. Since over-expression of Mmp20 results in decreased enamel hardness, this suggests that a balance exists between cleaved and full-length enamel matrix proteins that are essential for formation of a properly hardened enamel layer. It also suggests that few feedback controls are present in the enamel matrix to prevent excessive MMP20 activity.

MMP20 modulates cadherin expression in ameloblasts as enamel develops.

Matrix metalloproteinase-20 (enamelysin, MMP20) is essential for dental enamel development. Seven different MMP20 mutations in humans cause non-syndromic enamel malformations, termed amelogenesis imperfecta, and ablation of Mmp20 in mice results in thin brittle enamel with a dysplastic rod pattern. Healthy enamel formation requires the sliding movement of ameloblasts in rows during the secretory stage of development. This is essential for formation of the characteristic decussating enamel rod pattern observed in rodents, and this is also when MMP20 is secreted into the enamel matrix. Therefore, we propose that MMP20 facilitates ameloblast movement by cleaving ameloblast cell-cell contacts. Here we show that MMP20 cleaves the extracellular domains of the E- and N-cadherin adherens junction proteins, that both E- and N-cadherin transcripts are expressed at significantly higher levels in Mmp20 null vs. wild-type (WT) mice, and that in Mmp20 ablated mice, high-level ameloblast N-cadherin expression persists during the maturation stage of development. Furthermore, we show that E-cadherin gene expression is down-regulated from the pre-secretory to the secretory stage, while N-cadherin levels are up-regulated. This E- to N-cadherin switch supports epithelial migration in other tissues and may be an important event necessary for the ameloblasts to start moving in rows that slide by one another.

Modulation of cell-cell junctional complexes by matrix metalloproteinases.

The ameloblast cell layer of the enamel organ is in contact with the forming enamel as it develops into the hardest substance in the body. Ameloblasts move in groups that slide by one another as the enamel layer thickens. Each ameloblast is responsible for the formation of one enamel rod, and the rods are the mineralized trail that moving ameloblasts leave behind. Matrix metalloproteinases (MMPs) facilitate cell movement in various tissues during development, and in this review we suggest that the tooth-specific MMP, enamelysin (MMP20), facilitates ameloblast movements during enamel development. Mmp20 null mice have thin brittle enamel with disrupted rod patterns that easily abrades from the underlying dentin. Strikingly, the Mmp20 null mouse enamel organ morphology is noticeably dysplastic during late-stage development, when MMP20 is no longer expressed. We suggest that in addition to its role of cleaving enamel matrix proteins, MMP20 also cleaves junctional complexes present on ameloblasts to foster the cell movement necessary for formation of the decussating enamel rod pattern. Therefore, inactivation of MMP20 would result in tight ameloblast cell-cell attachments that may cause maturation-stage enamel organ dysplasia. The tight ameloblast attachments would also preclude the ameloblast movement necessary to form decussating enamel rod patterns.

The use of mouse models to investigate shear bond strength in amelogenesis imperfecta.

Patients with amelogenesis imperfecta (AI) have defective enamel; therefore, bonded restorations of patients with AI have variable success rates. To distinguish which cases of AI may have good clinical outcomes with bonded materials, we evaluated etching characteristics and bond strength of enamel in mouse models, comparing wild-type (WT) with those having mutations in amelogenin (Amelx) and matrix metalloproteinase-20 (Mmp20), which mimic 2 forms of human AI. Etched enamel surfaces were compared for roughness by scanning electron microscopy (SEM) images. Bonding was compared through shear bond strength (SBS) studies with 2 different systems (etch-and-rinse and self-etch). Etched enamel surfaces of incisors from Amelx knock-out (AmelxKO) mice appeared randomly organized and non-uniform compared with WT. Etching of Mmp20KO surfaces left little enamel, and the etching pattern was indistinguishable from unetched surfaces. SBS results were significantly different when AmelxKO and Mmp20KO enamel surfaces were compared. A significant increase in SBS was measured for all samples when the self-etch system was compared with the etch-and-rinse system. We have developed a novel system for testing shear bond strength of mouse incisors with AI variants, and analysis of these data may have important clinical implications for the treatment of patients with AI.

Matrix metalloproteinase 20 promotes a smooth enamel surface, a strong dentino-enamel junction, and a decussating enamel rod pattern.

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.

Enamel proteins and proteases in Mmp20 and Klk4 null and double-null mice.

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.

Kallikrein-related peptidase 4, matrix metalloproteinase 20, and the maturation of murine and porcine enamel.

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.