Maturation stage enamel malformations in Amtn and Klk4 null mice

Abstract

Amelotin (AMTN) and kallikrein-4 (KLK4) are secreted proteins specialized for enamel biomineralization. We characterized enamel from wild-type, Amtn−/−, Klk4−/−, Amtn+/−Klk4+/− and Amtn−/−Klk4−/− mice to gain insights into AMTN and KLK4 functions during amelogenesis. All of the null mice were healthy and fertile. The mandibular incisors in Amtn−/−, Klk4−/− and Amtn−/−Klk4−/− mice were chalky-white and chipped. No abnormalities except in enamel were observed, and no significant differences were detected in enamel thickness or volume, or in rod decussation. Micro-computed tomography (µCT) maximum intensity projections localized the onset of enamel maturation in wild-type incisors distal to the first molar, but mesial to this position in Amtn−/−, Klk4−/− and Amtn−/−Klk4−/− mice, demonstrating a delay in enamel maturation in Amtn−/− incisors. Micro-CT detected significantly reduced enamel mineral density (2.5 and 2.4 gHA/cm3) in the Klk4−/− and Amtn−/−Klk4−/− mice respectively, compared with wild-type enamel (3.1 gHA/cm3). Backscatter scanning electron microscopy showed that mineral density progressively diminished with enamel depth in the Klk4−/− and Amtn−/−Klk4−/− mice. Knoop hardness of Amtn−/− outer enamel was significantly reduced relative to the wild-type and was not as hard as the middle or inner enamel. Klk4−/− enamel hardness was significantly reduced at all levels, but the outer enamel was significantly harder than the inner and middle enamel. Thus the hardness patterns of the Amtn−/− and Klk4−/− mice were distinctly different, while the Amtn−/−Klk4−/− outer enamel was not as hard as in the Amtn−/− and Klk4−/− mice. We conclude that AMTN and KLK4 function independently, but are both necessary for proper enamel maturation.

Deletion of amelotin exons 3–6 is associated with amelogenesis imperfecta

“…Our findings on teeth from a patient heterozygous for the AMTN deletion showed a 18.8–24.6% reduction in enamel mineral density compared to control teeth, in contrast, the final mean enamel mineral density achieved across the full enamel thickness in Amtn -/- teeth was reduced by 2.1% and was not significantly different to WT (32,47). Microhardness values for the inner and middle enamel layers in null mice were also no different to WT but the microhardness of the surface layer in null mice was significantly reduced by approximately 23% (47). It was concluded that AMTN plays a crucial role in the production of a normally mineralised enamel surface in rodent mandibular incisors.…”

“…In Amtn -/- mice (32,47), the effect on enamel phenotype varied depending on tooth type, with the continually erupting mandibular incisors most affected while molars appeared unaffected and maxillary incisors were merely stained. These differences may be related to the rate of formation/eruption of the different teeth; mandibular incisor enamel has less time in which to fully mineralise prior to eruption and AMTN may be required to ensure that the finished enamel surface is fully mineralised prior to enamel eruption.…”

“…Enamel thickness (at eruption) and the characteristic decussating prismatic architecture were also apparently unaffected in Amtn -/- teeth, perhaps reflecting the fact that AMTN is not expressed during the secretory stage in WT animals (enamel thickness and rod architecture being determined during the secretory stage (48)). In the ensuing transition/maturation stages, where AMTN is maximally expressed and the enamel accrues the majority of its final mineral content, secondary mineralization was delayed; especially in the deeper enamel layers (32,47). Our findings on teeth from a patient heterozygous for the AMTN deletion showed a 18.8–24.6% reduction in enamel mineral density compared to control teeth, in contrast, the final mean enamel mineral density achieved across the full enamel thickness in Amtn -/- teeth was reduced by 2.1% and was not significantly different to WT (32,47).…”

“…In the ensuing transition/maturation stages, where AMTN is maximally expressed and the enamel accrues the majority of its final mineral content, secondary mineralization was delayed; especially in the deeper enamel layers (32,47). Our findings on teeth from a patient heterozygous for the AMTN deletion showed a 18.8–24.6% reduction in enamel mineral density compared to control teeth, in contrast, the final mean enamel mineral density achieved across the full enamel thickness in Amtn -/- teeth was reduced by 2.1% and was not significantly different to WT (32,47). Microhardness values for the inner and middle enamel layers in null mice were also no different to WT but the microhardness of the surface layer in null mice was significantly reduced by approximately 23% (47).…”

“…This may reflect the type of mutation present, since the human patients have an in frame deletion, predicted to produce a mutant transcript and possibly also a mutant protein whereas the mice have been shown not to produce any protein and to exhibit a 300 fold reduction in transcript levels (32). Núñez et al reported that heterozygous Amtn +/- mice had no enamel defects and suggested that murine amelogenesis is unaffected even in the presence of half of the normal amount of amelotin (47). This suggests that the AMTN deletion identified here may have a dominant-negative (toxic gain of function) effect due to the presence of a mutated protein, rather than acting through haploinsufficiency.…”