MMP20 Overexpression Disrupts Molar Ameloblast Polarity and Migration

Abstract

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 ( Mmp20Tg) 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), Mmp20Tg 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, Mmp20Tg 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, Mmp20Tg 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.

Guidelines for Micro–Computed Tomography Analysis of Rodent Dentoalveolar Tissues

“…These also have their own capabilities and limitations that dictate to some degree the ease and efficiency of analysis. All analyses and images included in this report were accomplished using Scanco proprietary software for scan reconstructions (default settings) and AnalyzePro 1.0 medical imaging software (Analyze Direct, Overland Park, KS), a software system used in our studies (23,(44)(45)(46)(47)(48)(49).…”

“…In studies analyzing the functions of MMP20, mice over-expressing this metalloproteinase (Mmp20 +/+ Tg + ) exhibited severe enamel defects marked by a patchy, thin, poorly defined enamel layer that delaminated from dentin (Fig. 5A) (46). Molar enamel volume was decreased ~ 60% and mineral density reduced ~40%.…”

“…All analyses and images included in this report were accomplished using Scanco proprietary software for scan reconstructions (default settings) and AnalyzePro 1.0 medical imaging software (Analyze Direct, Overland Park, KS), a software system used in our studies. (23,(44)(45)(46)(47)(48)(49) CT images are created using X-rays directed and rotated around a sample (or a sample rotating in relation to the X-ray source), creating a series of projections. X-rays are a form of electromagnetic radiation with high photon energy, and image generation relies on the partial transmission of X-rays; ie, differential absorption of X-rays by tissues.…”

“…Based on anatomical landmarks examined over several studies, 1600 mg HA/cm 3 was determined to most accurately label enamel in our scans. (44)(45)(46)(47)(48)(49)69,81) Qualitatively, a lower threshold (1400 mg HA/cm 3 ) resulted in areas of dentin being identified as enamel (visualized as speckling in dentin areas), whereas a higher threshold (1800 mg HA/cm 3 ) resulted in enamel volume (EV) underestimation (seen as visible gaps in 3D renderings, Fig. 4A3 red arrow).…”

Canine models of human amelogenesis imperfecta: identification of novel recessive ENAM and ACP4 variants

“…Enamelysin (Mmp20)-deficient mice fail to process amelogenin resulting in serious enamel defects (Caterina et al 2002 ). Mice overexpressing Mmp20 exhibit significant reductions in enamel thickness, volume, and mineral density in their molars (Shin et al 2018 ). Enamelin-deficient mice present with defects in enamel mineralization (Hu et al 2008 ), while an enamelin overexpression model suggests that an adequate quantity of enamelin is essential for normal enamel formation (Hu et al 2014 ).…”