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

DPPI May Activate KLK4 during Enamel Formation

Kallikrein-4 (KLK4) is a serine protease expressed during enamel maturation, and proteolytic processing of the enamel matrix by KLK4 is critical for proper enamel formation. KLK4 is secreted as an inactive zymogen (pro-KLK4), and identification of its activator remains elusive. Dipeptidyl peptidase I (DPPI) is a cysteine aminopeptidase that can activate several serine proteases. In this study, we sought to examine DPPI expression in mouse enamel organ and determine if DPPI could activate KLK4. Real-time PCR showed DPPI expression throughout amelogenesis, with highest expression at maturation, and immunohistochemical staining of mouse incisors confirmed DPPI expression by ameloblasts. We demonstrate in vitro that DPPI activates pro-KLK4 to cleave a fluorogenic peptide containing a KLK4 cleavage site. Examination of mature enamel from DPPI null mice by FTIR showed no significant accumulation of protein; however, microhardness testing revealed that loss of DPPI expression significantly reduced enamel hardness

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

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

Nuclear and mitochondrial sequences confirm complex colonization patterns and clear species boundaries for flightless weevils in the Galápagos archipelago

Nuclear sequence data were collected from endemic Galápagos species and an introduced close relative, and contrasted with mitochondrial DNA sequences, continuing investigation into the colonization history and modes of diversification in the weevil genus Galapaganus. The current combined phylogeny together with previously published penalized likelihood age estimates builds a complex picture of the archipelago's colonization history. The present reconstruction relies on submerged platforms to explain the early divergence of the young southern Isabela endemics or the Española or San Cristobal populations. Diversity is later built through inter-island divergence starting on older islands and continuing on two simultaneous tracks towards younger islands. The amount of diversity generated through intra-island processes is skewed towards older islands, suggesting that island age significantly influences diversity. Phylogenetic concordance between nuclear and mitochondrial datasets and well-supported monophyletic species in mitochondrial derived topologies appear to reject the possibility of inter-species hybridization. These clear species boundaries might be related to the tight host associations of adult weevils in discrete ecological zones. If shared hosts facilitate hybridization, then host- or habitat-promoted divergences could prevent it, even in the case of species that share islands, since the altitudinal partitioning of habitats minimizes range overlap