Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo

Abstract

Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double transgene and M180Tg, CTRNCTg, LRAPTg triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene (p < 0.05). The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.

Mapping the Tooth Enamel Proteome and Amelogenin Phosphorylation Onto Mineralizing Porcine Tooth Crowns

“…LRAP phosphorylation appears to affect mineralization activity of ameloblast cell lines and cultured tooth germs (Wiedemann-Bidlack et al, 2011; Le Norcy et al, 2018). Studies in transgenic mouse models have shown that the presence and relative abundance of amelogenin N-terminal, C-terminal, and hydrophilic core domains are important for the generation of proper tooth enamel (Xia et al, 2016; Bidlack et al, 2017). AMELX, ENAM and AMBN are cleaved by MMP20 upon secretion and by KLK4 during later stages of enamel formation (Yamakoshi et al, 2006a, 2011; Nagano et al, 2009; Chun et al, 2010).…”

Impact of TNFR1 pro-inflammatory receptor ablation in amelogenesis and dental enamel biomineralization in mice

“…Studies have shown that in most cases where there are molecular changes in the signaling of enamel formation, there will be a failure in the protein removal of enamel organic matrix, which leads to a reduction of mineral content and an increase of proteins (28,32,33). Amelogenin is the main protein in the enamel organic matrix during amelogenesis, also serving as a scaffold that will guide the mineralization of hydroxyapatite crystals (27).…”

Enhancing Collagen Mineralization with Amelogenin Peptide: Toward the Restoration of Dentin

“…The selection of the major enamel matrix protein; amelogenin, as an archetype for biomineralization has been well established through several in vitro , and animal studies. − Amelogenin in enamel and noncollagenous matrix proteins (NCPs) in dentin influence the organization and growth of HAP crystals during the biomineralization processes. Proteomic analysis of the DEJ interface in human molars has revealed high biological activity, predominantly rich in collagen (type I) and enamel-specific proteins such as amelogenin and ameloblastin. , …”