Matrix Metalloproteinase-20 Research Articles

Matrix Metalloproteinase-20 Over-Expression Is Detrimental to Enamel Development: A Mus musculus Model

BackgroundMatrix 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.

M180 Amelogenin Processed by MMP20 is Sufficient for Decussating Murine Enamel

Amelogenin (AMELX) and matrix metalloproteinase-20 (MMP20) are essential for proper enamel development. Amelx and Mmp20 mutations cause amelogenesis imperfecta. MMP20, a protease secreted by ameloblasts, is responsible for processing enamel proteins, including AMELX, during the secretory stage of enamel formation. Of at least 16 different amelogenin splice products, the most abundant isoform found in murine ameloblasts and developing enamel is the M180 protein. To understand the role of MMP20 processing of M180 AMELX, we generated AmelxKO/Mmp20KO (DKO) mice with an amelogenin (M180Tg) transgene. We analyzed the enamel phenotype by SEM to determine enamel structure and thickness, µCT, and by nanoindentation to quantify enamel mechanical properties. M180Tg/DKO mouse enamel had 37% of the hardness of M180Tg/AmelxKO teeth and demonstrated a complete lack of normal prismatic architecture. Although molar enamel of M180Tg/AmelxKO mice was thinner than WT, it had similar mechanical properties and decussating enamel prisms, which were abolished by the loss of MMP20 in the M180Tg/DKO mice. Retention of the C-terminus or complete lack of this domain is unable to rescue amelogenin null enamel. We conclude that among amelogenins, M180 alone is sufficient for normal enamel mechanical properties and prism patterns, but that additional amelogenin splice products are required to restore enamel thickness.

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.

Dental enamel development: proteinases and their enamel matrix substrates.

This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These proteins and their respective null mice and human mutations are also evaluated to shed light on the mechanisms that cause nonsyndromic enamel malformations termed amelogenesis imperfecta. Pertinent controversies are addressed. For example, do any of these proteins have a critical function in addition to their role in enamel development? Does amelogenin initiate crystallite growth, does it inhibit crystallite growth in width and thickness, or does it do neither? Detailed examination of the null mouse literature provides unmistakable clues and/or answers to these questions, and this data is thoroughly analyzed. Striking conclusions from this analysis reveal that widely held paradigms of enamel formation are inadequate. The final section of this review weaves the recent data into a plausible new mechanism by which these enamel matrix proteins support and promote enamel development.

Homozygous and Compound Heterozygous MMP20 Mutations in Amelogenesis Imperfecta

In this article, we focus on hypomaturation autosomal-recessive-type amelogenesis imperfecta (type IIA2) and describe 2 new causal Matrix metalloproteinase 20 (MMP20) mutations validated in two unrelated families: a missense mutation p.T130I at the expected homozygous state, and a compound heterozygous mutation having the same mutation combined with a nucleotide deletion, leading to a premature stop codon (p.N120fz*2). We characterized the enamel structure of the latter case using scanning electron microscopy analysis and microanalysis (Energy-dispersive X-ray Spectroscopy, EDX) and confirmed the hypomaturation-type amelogenesis imperfecta as identified in the clinical diagnosis. The mineralized content was slightly decreased, with magnesium substituting for calcium in the crystal structure. The anomalies affected enamel with minimal inter-rod enamel present and apatite crystals perpendicular to the enamel prisms, suggesting a possible new role for MMP20 in enamel formation.

Biophysical characterization of synthetic amelogenin C-terminal peptides.

Amelogenin plays a key role in the formation of the highly mineralized structure of tooth enamel. During the secretory stage, amelogenin is cleaved gradually by a protease, matrix metalloproteinase-20 (MMP-20), releasing hydrophilic C-terminal peptides. In this study, the biophysical properties of synthetic C-terminal peptides (of 28, 17, and 11 residues), mimicking native peptides, were explored in vitro. A sudden decrease was observed in the zeta (ζ)-potential upon the addition of calcium or phosphates, which was also accompanied by an increased aggregation propensity of the peptides. Under most of the experimental conditions, the particle size increased at a pH 2-3 units higher than the isoelectric point (pI) of the peptides, while the peptides existed as smaller particles (<2 nm) near their pI values and in the acidic range. They showed poor affinity for calcium and phosphates, comparable to full-length amelogenin and variants. The secondary structure determination showed that the 11-amino-acid peptide contained defined secondary structure comprising beta-sheets and turns. Atomic force microscopy analysis revealed the presence of thin, disk-like nanostructures of 54.4 nm diameter for the 28-amino-acid peptide and 54.9 nm diameter for the 11-amino acid peptide, whereas no definite structures were observed for the 17-amino-acid peptide. It is concluded that the amelogenin C-terminal peptides are capable of interacting with calcium and phosphate ions, of self-assembly into nanostructures, and may have some secondary structure, and hence may have some role in enamel synthesis.

Molecular evolution of matrix metalloproteinase 20

Dental enamel is a hypermineralized tissue, containing only trace amounts of organic components. During enamel formation, matrix metalloproteinase 20 (MMP20) processes proteins comprising enamel matrix and facilitates hypermineralization. In the human genome, 24 distinct MMP genes have been identified. Among these genes, MMP20 is clustered with eight other genes, including MMP13, and all these clustered genes show phylogenetically close relationships. In this study, we investigated MMP20 and closely related MMP genes in various tetrapods and in a teleost fish, fugu. In the genome of the chicken, a toothless tetrapod, we identified degraded exons of MMP20, which supports the previous proposition that MMP20 is important specifically for enamel formation. Nevertheless, for unknown reasons, we failed to identify MMP20 in the platypus genome. In the opossum, lizard, and frog genomes, MMP20 was found clustered with MMP13. Furthermore, in the fugu genome, we identified an MMP20-like gene located adjacent to MMP13, suggesting that MMP20 arose before the divergence of ray-finned fish and lobe-finned fish. The teleost tooth surface is covered with enameloid, a hypermineralized tissue different from enamel. Thus, we hypothesize that MMP20 could have been used in an ancient hypermineralized tissue, which evolved into enameloid in teleosts and into enamel in tetrapods.

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.

Fluoride incorporation into apatite crystals delays amelogenin hydrolysis

Enamel fluorosis has been related to an increase in the amount of amelogenin in fluorosed enamel compared with normal enamel in the maturation stage. In this study we tested the hypothesis that fluoride incorporated into carbonated apatite alters amelogenin hydrolysis. Recombinant human amelogenin (rh174) was allowed to bind to 0.15 mg of carbonated hydroxyapatite (CAP) or to fluoride-containing carbonated hydroxyapatite (F-CAP) synthesized to contain 100, 1,000, or 4,000 ppm F(-). After 3 h of digestion with recombinant human matrix metalloproteinase 20 (MMP20) or kallikrein-related peptidase 4 (KLK4), bound protein was characterized by reverse-phase high-performance liquid chromatography (HPLC). Proteolytic fragments of amelogenin formed after 24h of digestion with MMP20 of KLK 4 were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The hydrolysis, by both MMP20 and KLK4, of amelogenin bound to F100-CAP was significantly reduced in a dose-dependent manner compared with the hydrolysis of amelogenin bound to CAP. After 24 h of hydrolysis, a similar number of MMP20 cleavage sites was found for amelogenin bound to CAP and amelogenin bound to F100-CAP; however, 24 fewer KLK4 cleavage sites were identified for amelogenin bound to F100-CAP than for amelogenin bound to CAP. These results suggest that the reduced hydrolysis of amelogenins in fluorosed enamel may be partially caused by the increased fluoride content in fluoride-containing apatite, contributing to the hypomineralized enamel matrix phenotype observed in fluorosed enamel.

Mutation Screening of ENAM , KLK4, MMP20 and FAM83H Genes among the Members of Five Iranian Families Affected with Autosomal Recessive Hypoplastic Amelogenesis Imperfecta

Amelogenesis Imperfectas (AIs) are clinically and genetically heterogeneous conditions characterized by a wide range of clinical features. These abnormalities of enamel formation are categorized into three main groups, hypoplastic, hypomaturation and hypocalcified with different modes of inheritance such as autosomal recessive (AR), autosomal dominant (AD) and X-lined recessive (XLR). In spite of the fact that frequent studies have explained the histological features of AIs, our knowledge regarding the molecular etiology of the affected enamel is not adequate. Up to now, different loci have been suggested to associate with the causation of AIs. Several genetic mutations including enamelin ( ENAM ), amelogenin ( AMELX ), ameloblastin ( AMBN ), tuftelin (TUFT1), kallikrein 4 ( KLK4), matrix metalloproteinase 20 ( MMP20 ) and family with sequence similarity 83, member H (FAM83H) have been suggested to play critical roles in the pathogenesis of these disorders. Therefore, the aim of this investigation was to study of mutation screening in ENAM, KLK4, MMP20 and FAM83H genes, responsible for AIs development in five Iranian families in which the probands were diagnosed with autosomal recessive hypoplastic amelogenesis imperfecta (ARHAI). Genomic DNA was extracted from probands and exon/intron boundaries of afore-mentioned genes were amplified by PCR and subjected to direct sequencing. We could not detect any mutation in the studied genes however; two different novel polymorphisms including T18515C and G18504A were identified in the intron 4 of MMP20 in the probands of two families. Our findings support the notion that different genes may be involved in the development of amelogenesis imperfectas.

Relationships between protein and mineral during enamel development in normal and genetically altered mice

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.

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.

Excessive fluoride induces endoplasmic reticulum stress and interferes enamel proteinases secretion

Protein retention in the enamel layer during tooth formation is well known to be associated with dental fluorosis but the underlying mechanism is unclear. The functions of the endoplasmic reticulum (ER) correlate directly with secreted protein metabolism. We used an ameloblast-derived cell line to determine whether excessive amounts of fluoride cause ER stress, and whether this interferes with the secretion of enamel matrix proteinases. ER stress activates a signaling network called the unfolded protein response (UPR). Here, we used real-time RT-PCR and immunofluorescence to study the effect of fluoride on the expression, translation, and secretion of UPR transcription factors in ameloblast-like cells. Measurement of both the gene and protein expression of UPR transcription factors indicated that high-dose fluoride increases the expression of UPR transcription factors in a dose-dependent manner. We also used ELISA to detect and quantify the enamel proteinases secreted by ameloblasts. We found a corresponding decrease in extracellular secretion of the enamel proteinases matrix metalloproteinase-20 and kallikrein-4, after exposure to fluoride. Furthermore, correlation analysis indicated that the expression of UPR transcription factors showed a strong inverse correlation with that of enamel proteinases. The results suggest that high-dose fluoride initiates an ER stress response in ameloblasts and induces the UPR, which interferes with the synthesis and secretion of enamel proteinases. Taken together, these results suggest that excessive ingestion of fluoride during tooth formation can decrease the secretion of proteinases, thus causing protein retention in the enamel layer, indicating that the ER stress response may be responsible for dental fluorosis.

Prognostic significance of matrix metalloproteinase-20 overexpression in laryngeal squamous cell carcinoma

Conclusion: Matrix metalloproteinase (MMP)-20 is overexpressed in laryngeal squamous cell carcinoma (LSCC) compared with the adjacent normal laryngeal epithelium and MMP-20 plays a role in lymph node metastasis. Overexpression of MMP-20 may be used as a significant prognostic factor for lymph node metastasis. All the findings indicate that MMP-20 may play a role in the initiation and progression of LSCC. Objective: The MMPs are a gene family of zinc-dependent endopeptidases that have been implicated in tumor invasion and metastasis, and MMP-20 is a new member of the MMP family. The purpose of this study was to investigate whether MMP-20 is overexpressed in human LSCC and, if so, the significance of its overexpression in relation to clinical parameters. Methods: We analyzed 33 cases of LSCC with RT-PCR and 73 cases of LSCC with immunohistochemistry compared with normal laryngeal epithelium. Results: We found that MMP-20 is overexpresssed in LSCC compared with the adjacent normal laryngeal epithelium.

Fluoride Does Not Inhibit Enamel Protease Activity

Fluorosed enamel can be porous, mottled, discolored, hypomineralized, and protein-rich if the enamel matrix is not completely removed. Proteolytic processing by matrix metalloproteinase-20 (MMP20) and kallikrein-4 (KLK4) is critical for enamel formation, and homozygous mutation of either protease results in hypomineralized, protein-rich enamel. Herein, we demonstrate that the lysosomal proteinase cathepsin K is expressed in the enamel organ in a developmentally defined manner that suggests a role for cathepsin K in degrading re-absorbed enamel matrix proteins. We therefore asked if fluoride directly inhibits the activity of MMP20, KLK4, dipeptidyl peptidase I (DPPI) (an in vitro activator of KLK4), or cathepsin K. Enzyme kinetics were studied with quenched fluorescent peptides with purified enzyme in the presence of 0–10 mM NaF, and data were fit to Michaelis-Menten curves. Increasing concentrations of known inhibitors showed decreases in enzyme activity. However, concentrations of up to 10 mM NaF had no effect on KLK4, MMP20, DPPI, or cathepsin K activity. Our results show that fluoride does not directly inhibit enamel proteolytic activity.

Altered Ion-responsive Gene Expression in Mmp20 Null Mice

During enamel maturation, hydroxyapatite crystallites expand in volume, releasing protons that acidify the developing enamel. This acidity is neutralized by the buffering activity of carbonic anhydrases and ion transporters. Less hydroxyapatite forms in matrix metalloproteinase-20 null (Mmp20 -/-) mouse incisors, because enamel thickness is reduced by approximately 50%. We therefore asked if ion regulation was altered in Mmp20 -/- mouse enamel. Staining of wild-type and Mmp20 -/- incisors with pH indicators demonstrated that wild-type mice had pronounced changes in enamel pH as development progressed. These pH changes were greatly attenuated in Mmp20 -/- mice. Expression of 4 ion-regulatory genes (Atp2b4, Slc4a2, Car6, Cftr) was significantly decreased in enamel organs from Mmp20 -/- mice. Notably, expression of secreted carbonic anhydrase (Car6) was reduced to almost undetectable levels in the null enamel organ. In contrast, Odam and Klk4 expression was unaffected. We concluded that a feedback mechanism regulates ion-responsive gene expression during enamel development.

Serotonin and fluoxetine receptors are expressed in enamel organs and LS8 cells and modulate gene expression in LS8 cells

The selective serotonin re-uptake inhibitor (SSRI) fluoxetine is widely used in the treatment of depression in children and fertile women, but its effect on developing tissues has been sparsely investigated. The aim of this study was to investigate if enamel organs and ameloblast-derived cells express serotonin receptors that are affected by peripherally circulating serotonin or fluoxetine. Using RT-PCR and western blot analysis we found that enamel organs from 3-d-old mice and ameloblast-like cells (LS8 cells) express functional serotonin receptors, the rate-limiting enzyme in serotonin synthesis (Thp1), as well as the serotonin transporter (5HTT), indicating that enamel organs and ameloblasts are able to respond to serotonin and regulate serotonin availability. Fluoxetine and serotonin enhanced the alkaline phosphatase activity in the cell culture medium from cultured LS8 cells, whereas the expression of enamelin (Enam), amelogenin (Amel), and matrix metalloproteinase-20 (MMP-20) were all significantly down-regulated. The secretion of vascular endothelial growth factor (VEGF), monocyte chemotactic protein 1 (MCP-1), and interferon-inducible protein 10 (IP-10) was also reduced compared with controls. In conclusion, enamel organs and ameloblast-like cells express functional serotonin receptors. Reduced transcription of enamel proteins and secretion of vascular factors may indicate possible adverse effects of fluoxetine on amelogenesis.

The odontogenic ameloblast‐associated protein (ODAM) cooperates with RUNX2 and modulates enamel mineralization via regulation of MMP‐20

We have previously reported that the odontogenic ameloblast-associated protein (ODAM) plays important roles in enamel mineralization through the regulation of matrix metalloproteinase-20 (MMP-20). However, the precise function of ODAM in MMP-20 regulation remains largely unknown. The aim of the present study was to uncover the molecular mechanisms responsible for MMP-20 regulation. The subcellular localization of ODAM varies in a stage-specific fashion during ameloblast differentiation. During the secretory stage of amelogenesis ODAM was localized to both the nucleus and cytoplasm of ameloblasts. However, during the maturation stage of amelogenesis, ODAM was observed in the cytoplasm and at the interface between ameloblasts and the enamel layer, but not in the nucleus. Secreted ODAM was detected in the conditioned medium of ameloblast-lineage cell line (ALC) from days 14 to 21, which coincided with the maturation stage of amelogenesis. Interestingly, the expression of Runx2 and nuclear ODAM correlated with MMP-20 expression in ALC. We therefore examined whether ODAM cooperates with Runx2 to regulate MMP-20 and modulate enamel mineralization. Increased expression of ODAM and Runx2 augmented MMP-20 expression, and Runx2 expression enhanced expression of ODAM, although overexpression of ODAM did not influence Runx2 expression. Conversely, loss of Runx2 in ALC decreased ODAM expression, resulting in down-regulation of MMP-20 expression. Increased MMP-20 expression accelerated amelogenin processing during enamel mineralization. Our data suggest that Runx2 regulates the expression of ODAM and that nuclear ODAM serves an important regulatory function in the mineralization of enamel through the regulation of MMP-20 apart from a different, currently unidentified, function of extracellular ODAM.

Cellular Effects of Enamel Matrix Derivative Are Associated With Different Molecular Weight Fractions Following Separation by Size‐Exclusion Chromatography

Enamel matrix derivative (EMD) was shown to enhance soft tissue healing and regeneration of the periodontium; however, the mechanisms of this action are unknown. It is assumed that amelogenin, the most abundant protein in EMD, is the protein primarily responsible for the effects of EMD. The purpose of this study was to fractionate EMD and associate its specific cellular effects with different molecular weight fractions following size-exclusion chromatography. Freshly dissolved EMD was fractionated by gel filtration, and forty-five 7-ml fractions were collected, desalted, lyophilized, and resuspended. These fractions were analyzed for their effects on the differentiation of osteoprogenitor cells (C2C12) and the proliferation and differentiation of human microvascular endothelial cells (HMVECs). Alkaline phosphatase activity (C2C12) was measured as a marker for osteogenic differentiation before and after preincubation of the fractions with the bone morphogenetic protein (BMP) decoy receptor, noggin. Angiogenesis (HMVEC) was evaluated as a marker for endothelial cell differentiation. Enzymographic assays used polyacrylamide gels copolymerized with denatured type I collagen to determine gelatinolytic activities in each fraction. EMD fractionated into three major protein peaks following size exclusion chromatography with cross-linked dextran particle matrix. Peak I was associated with the column void volume, whereas peak III eluted near the salt volume. Peak II eluted between these two peaks. Proliferation and angiogenic activities were associated with peaks II and III for the microvascular cells. The differentiation of osteoprogenitor cells, indicated by alkaline phosphatase activity, was induced by EMD components present in peak I and the leading edge of peak II. The additional observation that this differentiation was inhibited by prior treatment of the fractions with noggin suggested the activity was induced by BMP rather than amelogenin or other unknown proteins. Gelatinolytic activities were detected in the early fractions of peaks I and II of gel-fractionated EMD. The cellular activities stimulated by EMD are not associated with a single molecular weight species. The fact that noggin abolishes C2C12 alkaline phosphatase activity suggests that effects on osteoprogenitor cell differentiation are the result of a BMP-like protein(s), whereas effects on proliferation and angiogenesis are associated with lower molecular weight species present in peaks II and III. Finally, unheated EMD displays gelatinolytic activities that are also detectable following size-exclusion separation of its constituents. The masses of these activities were consistent with those reported for latent and active matrix metalloproteinase-20.

Lysosomal Protease Expression in Mature Enamel

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.