Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice

Abstract

Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects in KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting to generate a knockin mouse that replaces the Klk4 gene sequence, starting at the translation initiation site, with a lacZ reporter gene. Correct targeting of the transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) staining demonstrated expression of ␤-galactosidase in maturation stage ameloblasts. No X-gal staining was observed in secretory stage ameloblasts or in odontoblasts. Retained enamel proteins were observed in the maturation stage enamel of the Klk4 null mouse, but not in the Klk4 heterozygous or wild-type mice. The enamel layer in the Klk4 null mouse was normal in thickness and contained decussating enamel rods but was rapidly abraded following weaning, despite the mice being maintained on soft chow. In function the enamel readily fractured within the initial rod and interrod enamel above the parallel enamel covering the dentinoenamel junction. Despite the lack of Klk4 and the retention of enamel proteins, significant levels of crystal maturation occurred (although delayed), and the enamel achieved a mineral density in some places greater than that detected in bone and dentin. An important finding was that individual enamel crystallites of erupted teeth failed to grow together, interlock, and function as a unit. Instead, individual crystallites seemed to spill out of the enamel when fractured. These results demonstrate that Klk4 is essential for the removal of enamel proteins and the proper maturation of enamel crystals.Dental enamel is composed of highly ordered, very long crystals of calcium hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ). Mature enamel crystallites are about 70 nm wide and 30 nm thick, but are of unmeasurable length (1), probably extending all the way from the dentin layer to the surface of the tooth (2). Enamel crystallites are organized into bundles called rods, with about 10,000 parallel crystals in a rod (3). Each enamel rod is the product of a single ameloblast, the cell type that forms a continuous sheet over the developing enamel and orchestrates its formation. Dental enamel of erupted teeth is ϳ95% mineral (by weight) (4), with most of the non-mineral component being water. Protein comprises Ͻ1% of its weight. Forming enamel, however, is over 30% protein (5). Much of the protein is reabsorbed by ameloblasts and degraded in lysosomes (6, 7), but extracellular proteases also play a role in matrix protein removal (8 -10).Dental enamel formation is divided into secretory, transition, and maturation stages (11,12). During the secretory stage, enamel crystals grow primarily in length. As the crystals extend, the enamel layer expands. Enamel crystallites lengthen along a mineralization front at the secretory surface of the ameloblast cell membrane. There, mineral deposits rapidly on the crystallite tips, and very slowly on their sides...

Fluoride Affects Enamel Protein Content via TGF-β1-mediated KLK4 Inhibition

“…These data support the acid hypothesis, postulating that the acidic environment of the maturation stage makes ameloblasts more susceptible to the toxic effects of F -exposure. (Simmer et al, 2009). The knock-in LacZ has a nuclear localization signal to help distinguish its expression from endogenous lysosomal β-galactosidase.…”

“…KLK4 degrades enamel matrix proteins (Ryu et al, 2002) before their export from the hardening enamel . Ablation of Klk4 in mice results in abnormally soft enamel with a higher than normal protein content (Simmer et al, 2009), and fluorosed enamel displays the same characteristics. Previously, we showed that murine Klk4 transcript levels were reduced after F -treatment (Sharma et al, 2010), and here we propose a mechanism by which this occurs and demonstrate that protein levels are similarly affected.…”

“…Previously, we showed that murine Klk4 transcript levels were reduced after F -treatment (Sharma et al, 2010), and here we propose a mechanism by which this occurs and demonstrate that protein levels are similarly affected. +/lacZ mice are knock-in mice with LacZ inserted in frame into the Klk4 translation initiation site such that the endogenous upstream Klk4 promoter drives LacZ expression (Simmer et al, 2009). Six-week-old Sprague-Dawley rats and Klk4 +/lacZ mice were provided ad libitum water containing F -(0, 50, or 100 ppm) as NaF.…”

The Amelogenin Proteins and Enamel Development in Humans and Mice

“…Mouse models for AI have been developed to test the function of the amelogenin protein group, and to begin to define the roles of the individual amelogenin proteins. Mice have been generated to express an overabundance of one amelogenin, to express a mutated amelogenin or to express only one amelogenin, but none of the other amelogenins 24,25,26 .…”

Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

“…Similar to human KLK4, the mouse and pig homologues (enamel matrix serine proteinase 1) are required for proper tooth formation (Simmer et al, 2009;Hu et al, 2011). Murine mKLK4 possesses three sequons at Asn109, Asn159, and Asn202; it shares the latter two with porcine pKLK4 that has a third sequon at Asn120 (Figure 2).…”