Murine matrix metalloproteinase-20 overexpression stimulates cell invasion into the enamel layer via enhanced Wnt signaling.

Matrix metalloproteinase-20 (MMP20) is expressed by ameloblasts in developing teeth and MMP20 mutations cause enamel malformation. We established a stably transfected Tet-Off Mmp20-inducible ameloblast-lineage cell line and found that MMP20 expression promoted cell invasion. Previously, we engineered transgenic mice (Tg) that drive Mmp20 expression and showed that Mmp20(+/+)Tg mice had soft enamel. Here we asked if Mmp20 overexpression disrupts ameloblast function. Incisors from Mmp20(+/+) mice expressing the Mmp20 Tg had a striking cell infiltrate which nearly replaced the entire enamel layer. A thin layer of enamel-like material remained over the dentin and at the outer tooth surface, but between these regions were invading fibroblasts and epithelial cells that surrounded ectopic bone-like calcifications. Mmp20(+/+)Tg mice had decreased enamel organ cadherin levels compared to the Mmp20 ablated and WT mice and, instead of predominantly locating adjacent to the ameloblast cell membrane, ß-catenin was predominantly present within the nuclei of invading cells. Our data suggest that increased cadherin cleavage by transgenic MMP20 in the WT background releases excess ß-catenin, which translocates to ameloblast nuclei to promote cell migration/invasion. Therefore, we conclude that MMP20 plays a role in normal ameloblast migration through tightly controlled Wnt signaling and that MMP20 overexpression disrupts this process.

High-Level Expression and Prognostic Significance of Matrix Metalloprotease-19 and Matrix Metalloprotease-20 in Human Pancreatic Ductal Adenocarcinoma.

OBJECTIVE: Matrix metalloproteinase (MMP)-19 and MMP-20 are important members of the MMP family, and their roles in tumor survivorship and progression are continually reported. This work aimed to determine the expression and prognostic significance of MMP-19 and MMP-20 in pancreatic ductal adenocarcinoma (PDAC). METHODS: Immunohistochemistry was used to investigate the levels of MMP-19 and MMP-20 expression in carcinoma tissues and paracancerous tissues from 102 PDAC patients. RESULTS: The MMP-19 and MMP-20 were, respectively, expressed in 71.6% (73/102) and 70.6% (72/102) of carcinoma tissues, and the expression was positively correlated (r = 0.643, P < 0.001). High-level expression of MMP-19 and MMP-20 was strongly correlated with aggressive clinicopathological characteristics. Kaplan-Meier analysis showed that high-level expression of MMP-19 and MMP-20 was significantly associated with decreased event-free survival (P < 0.001) and overall survival (P < 0.001). Multivariate analysis showed that high-level expression of MMP-19 could act as an independent predictive biomarker for poor event-free survival and overall survival. CONCLUSIONS: Levels of MMP-19 and MMP-20 expression are significantly increased in PDAC. High-level expression of MMP-19 and MMP-20 is closely correlated to progression and prognosis of PDAC, and these may be considered as promising markers for unfavorable prognoses.

Chymotrypsin C (caldecrin) is associated with enamel development.

Two main proteases cleave enamel extracellular matrix proteins during amelogenesis. Matrix metalloprotease-20 (Mmp20) is the predominant enzyme expressed during the secretory stage, while kallikrein-related peptidase-4 (Klk4) is predominantly expressed during maturation. Mutations to both Mmp20 and Klk4 result in abnormal enamel phenotypes. During a recent whole-genome microarray analysis of rat incisor enamel organ cells derived from the secretory and maturation stages of amelogenesis, the serine protease chymotrypsin C (caldecrin, Ctrc) was identified as significantly up-regulated (> 11-fold) during enamel maturation. Prior reports indicate that Ctrc expression is pancreas-specific, albeit low levels were also noted in brain. We here report on the expression of Ctrc in the enamel organ. Quantitative PCR (qPCR) and Western blot analysis were used to confirm the expression of Ctrc in the developing enamel organ. The expression profile of Ctrc is similar to that of Klk4, increasing markedly during the maturation stage relative to the secretory stage, although levels of Ctrc mRNA are lower than for Klk4. The discovery of a new serine protease possibly involved in enamel development has important implications for our understanding of the factors that regulate enamel biomineralization.

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.

Assessment of dental fluorosis in Mmp20 +/- mice.

The molecular mechanisms that underlie dental fluorosis are poorly understood. The retention of enamel proteins hallmarking fluorotic enamel may result from impaired hydrolysis and/or removal of enamel proteins. Previous studies have suggested that partial inhibition of Mmp20 expression is involved in the etiology of dental fluorosis. Here we ask if mice expressing only one functional Mmp20 allele are more susceptible to fluorosis. We demonstrate that Mmp20 (+/-) mice express approximately half the amount of MMP20 as do wild-type mice. The Mmp20 heterozygous mice have normal-appearing enamel, with Vickers microhardness values similar to those of wild-type control enamel. Therefore, reduced MMP20 expression is not solely responsible for dental fluorosis. With 50-ppm-fluoride (F(-)) treatment ad libitum, the Mmp20 (+/-) mice had F(-) tissue levels similar to those of Mmp20 (+/+) mice. No significant difference in enamel hardness was observed between the F(-)-treated heterozygous and wild-type mice. Interestingly, we did find a small but significant difference in quantitative fluorescence between these two groups, which may be attributable to slightly higher protein content in the Mmp20 (+/-) mouse enamel. We conclude that MMP20 plays a nominal role in dental enamel fluorosis.

Excess fluoride interferes with chloride-channel-dependent endocytosis in ameloblasts.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Both CF and dental fluorosis result in protein retention in mature enamel. We hypothesized that excess fluoride might cause protein retention by interfering with CFTR function, resulting in abnormal expression of proteases and pathological endocytosis. Millimolar concentrations of fluoride reduced uptake of Emdogain, an enamel matrix derivative, in ameloblast-like PABSo-E cells, while stimulating an acidic intracellular environment at the same time. When CFTR function was inhibited by either an siRNA or a chloride channel inhibitor, CFTRinh-172, fluoride's effect on Emdogain uptake was partially blocked. Treatment of cells with CFTR siRNA down-regulated expression of proteases MMP20 and KLK4 and increased intracellular pH. We conclude that excess fluoride inhibits endocytic activity of ameloblasts through the CFTR chloride channel or other chloride channels. The intracellular pH might be the key mechanism by which abnormal proteolytic activity and defective endocytosis cause the residual protein observed in enamel of patients with CF and dental fluorosis.

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.