Truncated amelogenin and LRAP transgenes improve Amelx null mouse enamel

Abstract

Amelogenin is the most abundant enamel protein involved in enamel mineralization. Our goal was to determine whether all three regions of amelogenin (N-terminus, C-terminus, central core) are required for enamel formation. Amelogenin RNA is alternatively spliced, resulting in at least 16 different amelogenin isoforms in mice, with M180 and LRAP expressed most abundantly. Soon after secretion by ameloblasts, M180 is cleaved by MMP20 resulting in C-terminal truncated (CTRNC) amelogenin. We aimed to determine whether the 2 transgenes (Tg), LRAP and CTRNC together, can improve LRAPTg/Amelx-/- and CTRNCTg/Amelx-/- enamel thickness and prism organization, which were not rescued in Amelx-/- enamel. We generated CTRNCTg/LRAPTg/Amelx-/- mice and analyzed developing and mature incisor and molar enamel histologically, by microCT, SEM and microhardness testing. CTRNCTg and LRAPTg overexpression together significantly improved the enamel phenotype of LRAPTg/Amelx-/- and CTRNCTg/Amelx-/- mouse enamel, however enamel microhardness was recovered only when M180Tg was expressed, alone or with LRAPTg. We determined that both LRAP and CTRNC, which together express all three regions of the amelogenin protein (N-terminus, C-terminus and hydrophobic core) contribute to the final enamel thickness and prism organization in mice.

Keywords: Ameloblasts; Amelogenin; Enamel; MMP20; Secretory stage.

Fig.. Schematic of amelogenin transgenes used in this study

M180Tg and LRAPTg represent the major amelogenin isoforms, while CTRNCTg represents the major amelogenin cleavage product. M180Tg contains exons 2, 3, 5, 6a–6d, 7; CTRNCTg contains exons 2, 3, 5, 6a-part of 6d; LRAPTg contains exons 2, 3, 5, 6d, 7. Amino acid numbers and sequences corresponding to transgene exons are shown. Orange arrowhead indicates the site of the first MMP20 cleavage.

Fig. 2. Amelx protein expression in WT, Amelx−/− and transgenic 5 day-old developing molars

WT (lane 1) molars had a smear of amelogenin protein between 17–20 kD representing most of the splice variants and cleavage products expressed during the secretory stage. In this immunoblot, the LRAP band (~7 kD) is not visible (although it was at longer exposure times), but other immunoblots of different WT mice show faint bands around 7 kD representing LRAP. Lanes 2–5 are from Tg mice in Amelx−/− backgrounds. Lane 6 is a Amelx−/− molar showing no amelogenin protein. LRAPTg expression in double transgenic/Amelx−/− mice (lanes 3 & 4) is noticeably higher than that in WT molars. M180Tg (lanes 4 & 5) and CTRNCTg (lane 3) expression was slightly less than to endogenous WT levels (lane 1), presumably due to the presence of less cleavage products.

Fig. 3. Histological analysis of developing enamel in 5 day-old 1^st molars

Images of H&E stained molars were obtained within the distal cusp of the developing 1^st molar at 40× and showed noticeably more enamel in WT (A) and CTRNCTg/LRAPTg/Amelx−/− (B) compared to LRAPTg/Amelx−/− (C) which showed a thin layer of enamel, and Amelx−/− (D), which had no enamel presence. Loss of endogenous amelogenin (B, C, D) and the addition of transgene overexpression (B & C) appeared to have no effect on ameloblasts, odontoblasts or dentin.

Fig. 4. MicroCT analysis of adult molar and incisor enamel

MicroCT scanned sections were selected in the same location developmentally in each mouse mandible: within the distal root of the first molar, which is adjacent to early maturation-stage of the incisor. Arrowheads point to mineralized enamel layers visible in molars and incisors. Little to no enamel was visible in Amelx−/− and LRAPTg/Amelx−/− molars and incisors (B & C), and CTRNCTg/Amelx−/− incisors (D). Thin enamel layers were visible in CTRNCTg/Amelx−/− molars (D), and M180Tg/Amelx−/− (E), M180Tg/LRAPTg/Amelx−/− (F) and CTRNCTg/LRAPTg/Amelx−/− (G) incisors. Thicker enamel layers were visible in WT molars and incisors (A), M180Tg/Amelx−/− (E), M180Tg/LRAPTg/Amelx−/− (F), and CTRNCTg/LRAPTg/Amelx−/− molars (G). Interestingly, incisor enamel in CTRNCTg/LRAPTg/Amelx−/− (G) appeared much less mineralized than that in the other genotypes with visible incisor enamel (A, E, F).

Fig. 5. MicroCT analysis of adult incisor maturation stage enamel

MicroCT scanned sections were selected in the same location developmentally in each mouse mandible: prior to eruption in the maturation-stage of the incisor in the final section that shows the incisors completely surrounded by alveolar bone. Arrows point to mineralized enamel layers visible in incisors. Little to no enamel was visible in Amelx−/−, LRAPTg/Amelx−/−, CTRNCTg/Amelx−/− incisors (B, C, D). Thin mineralized enamel layers were visible in M180Tg/Amelx−/− (E), M180Tg/LRAPTg/Amelx−/− (F) and CTRNCTg/LRAPTg/Amelx−/− (G) incisors. Thicker mineralized enamel layers were only visible in WT incisors (A).

Fig. 6. Molar enamel thickness

Enamel thickness was measured from SEM images of longitudinal sections through distal first molars. All of the transgenic molar enamel was significantly thicker than Amelx−/− molar enamel (p<0.05), and not as thick as WT enamel (p<0.05). However, mice with M180Tg overexpressed alone or with LRAPTg had thicker enamel than those mice without M180Tg (CTRNCTg/Amelx−/−, LRAPTg/Amelx−/− and CTRNCTg/LRAPTg/Amelx−/−). CTRNCTg/LRAPTg/Amelx−/− mice had thicker molar enamel than both CTRNCTg/Amelx−/− and LRAPTg/Amelx−/− mice (p<0.05). * = significantly different from WT (p<0.05), ** = significantly different from Amelx−/− (p<0.05). Boxes represent a range of values between first and third quartile, the line in the center being the median, while the vertical lines represent the maximum and minimum values.

Fig. 7. Incisor enamel structure imaged by SEM

Polished and etched longitudinal sections through mandibular incisor enamel were imaged by scanning electron microscopy in secondary mode at 2000×. While WT (A) exhibits the classic decussating prismatic organization of incisor enamel, Amelx−/− (B), LRAPTg/Amelx−/− (C), and CTRNCTg/Amelx−/− (D) incisor enamel was non-prismatic, with CTRNCTg/Amelx−/− enamel exhibiting slightly more organized mineral. CTRNCTg/LRAPTg/Amelx−/− (F) incisor enamel had noticeably more mineral organization with some decussating prisms, although not as much decussating prisms as appeared in M180Tg/LRAPTg/Amelx−/− incisor enamel (E). p = plastic embedding material, e = enamel, d = dentin

Fig. 8. Vickers Microhardness Testing

Polished longitudinal sections through mandibular molar enamel were used for microhardness testing. If possible, 20 indents per mouse were obtained in adult molar enamel. M180Tg overexpression either alone or with LRAPTg significantly improved enamel hardness over Amelx−/− or transgenic mice without M180Tg. * = significantly different from WT (p<0.05), ** = significantly different from Amelx−/− (p<0.05). Boxes represent a range of values between first and third quartile, the line in the center being the median, while the vertical lines represent the maximum and minimum values.