Tooth enamel forms through appositional growth in an organic matrix that is secreted in daily increments until the full thickness of the crown is reached. During this first, secretory, stage of enamel formation, enamel crystallites grow primarily in length. Once the final enamel thickness is attained, the mineral content increases as crystallites grow in thickness, while the organic phase is removed in a highly controlled way. The enamel matrix facilitates mineralization and organization, and is transient in abundance as well as composition. Starting out with a ratio of 70 wt% organic matter and water, and 30 wt% mineral (Smith, 1998) the ratio of organic matrix to mineral changes over the course of enamel development and reaches 95% mineral content, with about 1–2% organic matter retained in completed enamel. During the secretory stage, the full-length enamel matrix molecules amelogenin, ameloblastin, and enamelin are cleaved upon their secretion by matrix metalloproteinase 20 (MMP20). After the secretory stage, a second enzyme, kallikrein 4 (KLK-4) is active to further cleave the matrix proteins and allow for the removal of matrix until only a very small amount remains, which is important for the mechanical properties through the control of crack propagation. The three structural enamel matrix proteins and the alternative splice product of amelogenin, leucine rich amelogenin protein (LRAP) have been described (Smith, 1998; Bartlett, 2013; Tarasevich et al., 2015; Lacruz et al., 2017). Yet, is not resolved what the in vivo function of these matrix components is, what role the full-length molecules, their alternative splice products as well as their cleavage products play for the control of mineral phase, crystallite shape and orientation, pH regulation, and potentially feed-back to ameloblasts (Lacruz et al., 2017).

The organic enamel matrix comprises the three structural matrix proteins amelogenin, enamelin, and ameloblastin, with amelogenin accounting for 90 wt% of the composition. However, the amelogenin primary RNA transcript is extensively alternatively spliced to produce 16 amelogenin isoforms reported (Bartlett et al., 2006). It is not clear, whether these isoforms are critical for enamel formation, or what their roles are in amelogenesis. The most abundant of these isoforms are the full-length molecule of 180 amino acids and the 59-amino acid long LRAP, leucine-rich amelogenin protein, which consist of the 33 N-terminal and 26 C-terminal amino acids, but lacks the hydrophobic core of the full-length molecule.

Ameloblasts secrete the full-length 180 amino acid sized amelogenin, which shortly thereafter is cleaved by the metalloproteinase MMP20, beginning from the C-terminus. The most abundant cleavage product is 167 amino acids long, and referred here to as CTRNC. All three structural matrix proteins are required for proper enamel formation and the AmelxKO enamel is hypoplastic, with no prismatic architecture. In order to determine the roles of the most abundant amelogenin isoforms, transgenic mice have been developed that overexpress (a) the full-length amelogenin M180, (b) the major amelogenin cleavage product CTRNC, and (c) LRAP in both C57BL6/J wild-type and AmelxKO genetic backgrounds (see Table 1 for abbreviations).

Addition of LRAPTg to M180Tg in the amelogenin KO model improves the thickness and structure of enamel, suggesting that transgenes have a complementary function (Gibson et al., 2011). Among amelogenin isoforms, M180 by itself is sufficient for the formation of normal mechanical properties and prism patterns in enamel. Yet, additional amelogenin splice products are required to restore enamel thickness (Gibson et al., 2011; Snead et al., 2011; Pugach et al., 2013). The overexpression of CTRNCTg and LRAPTg together improved significantly the enamel phenotype of LRAPTg/KO and CTRNCTg/KO mouse enamel, however enamel microhardness was recovered only when M180Tg was expressed, alone or with LRAPTg. Expression of LRAP and CTRNC together provides all three regions of the amelogenin protein N-terminus, C-terminus and hydrophobic core further improved the phenotype to reach normal WT enamel thickness and prism organization in the reported mouse model (Chen et al., 2003; Pugach et al., 2010; Xia et al., 2016). Enamel phenotypes in M180Tg/KO, CTRNCTg/KO, LRAPTg/KO, and double transgenic mice have been heterogeneous. This is due to varying transgene dosages and suggests a cumulative effect on improving the AmelxKO enamel phenotype (Xia et al., 2016).

The importance of the ratio between cleaved and uncleaved amelogenin has been previously reported (Shin et al., 2014). In order to determine the optimal ratio of amelogenin major cleavage products and splice variants required for normal enamel structure, thickness, and mechanical properties, we generated double and triple transgenic mice with two different transgene dosages in KO backgrounds.

It has been shown previously that enamel prism decussation and 83% of thickness is recovered in the double hemizygous transgenic mouse M180Tg/LRAPTg/KO model (Gibson et al., 2011). To better understand the in vivo role of full-length amelogenin vs. cleavage and alternative splice products, we generated four genotypes that differ from each other in their relative abundance of M180, its most abundant cleavage product CTRNC that lacks the C-terminus, and the most abundant alternative splice product LRAP, which contains only the N- and C-terminus of the full-length molecule, but not the hydrophobic core region. However, in the present study, enamel thickness did not rescue by more than 58%, and prism organization was not fully achieved in incisors (Figure 3; Table 3).

In normal enamel development, M180 is cleaved shortly after secretion. Mutations in either the C-terminus or N-terminus compromise enamel formation. There are five reported mutations in the AMELX C-terminal region, and six mutations in the N-terminal region, leading to thin, discolored, and hypoplastic enamel (Lagerström et al., 1991; Lagerström-Fermer et al., 1995; Lench and Winter, 1995; Kindelan et al., 2000; Sekiguchi et al., 2001; Greene et al., 2002; Hart et al., 2002; Kim et al., 2004; Cho et al., 2014). Mutations in the N-terminus cause self-assembly defects in vitro (Buchko et al., 2013). Amelogenin lacking the N-terminus does not form nanospheres in vitro, and in vivo, the enamel is thin with short crystallites and irregular enamel prisms, indicating the key role of the N-terminus in amelogenin self-assembly and crystallite elongation (Zhu et al., 2006).

Both recent in vitro and in vivo evidence suggest that CTRNC plays an important role in enamel mineralization (Kwak et al., 2009, 2011; Martinez-Avila et al., 2011; Xia et al., 2016). In vitro studies have suggested a role of full-length amelogenin in the control of mineral phase, specifically the stabilization of amorphous mineral phases such as amorphous calcium phosphate (ACP), and alignment of forming crystallites (Kwak et al., 2011). With cleavage of the full-length molecule, one would expect the transformation of ACP to hydroxyapatite to proceed. In vivo, amelogenin transcription and MMP20 activity regulate the ratio of M180 to its cleavage product. The choreographed interplay between amelogenin secretion, MMP20 activity, and the resulting abundance and ratio of M180 to CTRNC can provide a mechanism to regulate mineralization rate.

Our data show that the dose of transgene expression has a major effect on enamel formation as is seen in the comparison between homozygous double transgene of M180Tg++/CTRNCTg++/KO and hemizygous transgene of M180 and CTRNC. This hemizygous transgene best rescues the structural organization and prism decussation of enamel. However, the produced enamel is softer than in any other transgene model used in this study. In contrast, the homozygous transgene provides excess protein that disrupts the process of both mineral phase regulation and crystallite alignment, as seen in the small platelets compared to the elongated crystals (Figure 3I) in the hemizygous enamel. The excess organic matrix in the homozygous M180Tg++/CTRNCTg++/KO transgene remains in the extracellular space, covers enamel prisms and seems to prevent proper decussation resulting in diminished hardness (Figures 3G,H; Table 3). In addition, matrix secretion is disrupted in the homozygous transgene, resulting in ectopic depositions (Figures 2H,I, 3G) and decreased enamel thickness. This finding highlights the importance of sheer abundance of matrix components at a given time, and in relation to MMP20 activity, and is paralleled in the results for the homozygous transgene M180Tg++/CTRNCTg++/LRAPTg++, where also ectopic depositions are observed, excess organic material covering prisms and diminished enamel thickness (Figures 2, 3). The rate of enamel apposition and crown extension is higher in incisors compared to molars, which could contribute to the observed differences in structural organization, matrix deposition, and matrix removal between molars and incisors (Smith and Warshawsky, 1977).

Interestingly, enamel organization and prism decussation, as well as hardness is better rescued than in the homozygous transgene without LRAP, M180Tg++/CTRNCTg++. The capacity of LRAP to regulate mineral phase in vitro, specifically stabilize ACP, has been shown by Le Norcy et al. (2011). In contrast to the homozygous transgene model, a comparison between the hemizygous transgenic mice M180Tg+/CTRNCTg+/LRAPTg+ and M180Tg+/CTRNCTg+ shows that the structural organization is much better without LRAP than when it is expressed uniformly with M180 and CTRNC. Our data further suggest that LRAPTg overexpressed with M180Tg and CTRNCTg does not affect enamel thickness (Table 3). These findings underscore the importance of other factors besides absolute abundance of matrix molecules, namely, the timing of transgene expression and the relative abundance of cleavage product and alternative splice products. While LRAP uses the same promoter in vivo as M180, it is not known how LRAP expression varies between stages of enamel development. In addition, the pH buffering effect of matrix components is relevant for mineralization rate and produced mineral phase.

A possible role of the central core domain of amelogenin is the buffering of pH during enamel mineralization and it has been suggested previously that amelogenins may act as a buffer to neutralize protons generated during enamel crystal formation in the secretory stage (Smith, 1998; Smith et al., 2005). Although the role of the central hydrophobic core remains elusive, seven amelogenesis imperfecta-causing mutations in this region are published to date (Aldred et al., 1992; Lench et al., 1994; Lench and Winter, 1995; Collier et al., 1997; Hart et al., 2000, 2002; Ravassipour et al., 2000; Sekiguchi et al., 2001; Greene et al., 2002). In all the transgene models presented here, enamel thickness is decreased, indicating abridged matrix secretion compared to normal enamel development in the WT, where the ending of amelogenin secretion coincides with both the attainment of the full enamel thickness and the enamel matrix becoming acidic (Smith, 1998). Amelogenins including the central hydrophobic core contain 14 histidine residues, which can bind protons such that a single amelogenin molecule can bind up to 15 protons in vitro (Ryu et al., 1998). Further support for a suggested role of amelogenin in pH regulation comes from data comparing ion channel expression in ameloblasts during secretory and maturation stage between WT and AmelxKO mice. In WT, secretory ameloblasts do not express the anion exchanger Ae2 basolaterally (Lyaruu et al., 2008; Bronckers et al., 2009). However, the expression of Ae2 in secretory ameloblasts of amelogenin KO mice indicate a mechanism to compensate for the lack of buffering in the absence of amelogenin through upregulation of Ae2 expression to secrete bicarbonate (Guo et al., 2015).

Taken together, our data suggest that all three domains of amelogenin play key roles in enamel formation and that the relative abundance over time is critical. The N- and C-termini of amelogenin, which are present in both the most abundant amelogenin (M180) and LRAP, are highly conserved and believed to have different but critical roles in enamel formation (Delgado et al., 2007). Our results support the hypothesis that the core domain affects enamel formation, in particular the aspects of enamel thickness in vivo through crystal elongation. This study highlights the need to appreciate the relative abundance of enamel matrix molecules and their role for pH regulation as a key factor of enamel formation. In conclusion, we have shown that excess amelogenin transgenes disrupted the process of enamel formation, likely through the disproportionate presence of amelogenin splice products and disruption of matrix removal. The presence of excess retained organic matrix, layering within enamel, and ectopic depositions in the mouse models studied, suggest that an optimal ratio between M180, CTRNC, and LRAP is critical for normal enamel structure, thickness, and hardness.

This study was carried out in accordance with the recommendations of Institute, Federal and Institutional Animal Care and Use Committee (IACUC) guidelines. The protocol was approved by the IACUC at Forsyth.

MP and FB wrote the manuscript with contributions from all authors. MP, FB, and YX contributed to the design of the experiments. MP, FB, and YX performed and analyzed experiments. MP and FB supervised the project. All authors reviewed and approved the final version of the manuscript.