Two Related Low Molecular Mass Polypeptide Isoforms of Amelogenin Have Distinct Activities in Mouse Tooth Germ Differentiation In Vitro

Abstract

Embryonic mouse tooth germs were cultured in vitro in the presence of two related amelogenin isoforms to determine their effects on tooth development. Our results show that these individual proteins have specific but quite different effects on epithelial-derived ameloblasts versus mesenchymal-derived odontoblasts.Introduction: Amelogenins, the main protein components of enamel matrix, have been shown to have signaling activity. Amelogenin isoforms differing only by the presence or exclusion of exon 4, designated [A+4] (composed of exons 2, 3, 4, 5, 6d, and 7) and [A-4] (composed of exons 2, 3, 5, 6d, and 7), showed similar, but different, effects both in vitro and in vivo on postnatal teeth. Materials and Methods: Lower first molar tooth germs of E15/16 CD1 mice were microdissected and cultured in vitro in a semisolid media containing either 20% FBS, 2% FBS, or 2% FBS with either 1.5 nM [A+4], [A-4], or both for 6 days. Tooth germs were analyzed by H&E staining and immunohistochemistry for collagen I, dentin matrix protein 2, and DAPI nuclear staining. Results: Teeth cultured in media containing 20% FBS showed normal development with polarized ameloblasts, and odontoblasts producing dentin matrix, and DMP2 expression in odontoblasts and pre-ameloblasts. Culture in 2% FBS media resulted in no ameloblast polarization and modest odontoblast differentiation with scant dentin matrix. Tooth germs cultured with [A+4] in 2% FBS media had well-polarized odontoblasts with robust dentin production and concomitant ameloblast polarization. DMP2 expression was equal to or greater than seen in the 20% FBS culture condition. In cultures with [A-4] in 2% FBS media, odontoblast polarization and dentin production was reduced compared with [A+4]. However, the pre-ameloblast layer was disorganized, with no ameloblast polarization occurring along the dentin surface. DMP2 expression was reduced in the odontoblasts compared with the 20% FBS and [A+4] conditions and was almost completely abrogated in the pre-ameloblasts. Conclusion: These data show different signaling activities of these closely related amelogenin isoforms on tooth development. Here we make the novel observation that [A-4] has an inhibitory effect on ameloblast development, whereas [A+4] strongly stimulates odontoblast development. We show for the first time that specific amelogenin isoforms have effects on embryonic tooth development in vitro and also hypothesize that DMP2 may play a role in the terminal differentiation of both ameloblasts and odontoblasts.

Identification of the functional activity of the [A-4] amelogenin gene splice product in newborn mouse ameloblasts

“…However, as maturation proceeds, cell specific factors, that is factors uniquely produced by the cells themselves, come into play. We demonstrated that particular amelogenin isoforms, which are translated from distinct amelogenin gene splice products, do have signaling properties in fibroblasts and tooth germs in culture (12,13,14,15). Furthermore, we demonstrated that in tooth organ cultures, the M59/ [A−4] isoform, secreted by maturing mouse odontoblasts before the onset of dentin mineralization, had a direct affect on ameloblast terminal differentiation (14).…”

“…We demonstrated that particular amelogenin isoforms, which are translated from distinct amelogenin gene splice products, do have signaling properties in fibroblasts and tooth germs in culture (12,13,14,15). Furthermore, we demonstrated that in tooth organ cultures, the M59/ [A−4] isoform, secreted by maturing mouse odontoblasts before the onset of dentin mineralization, had a direct affect on ameloblast terminal differentiation (14). The present study was undertaken to examine whether r[A−4] binding and uptake in newborn ameloblasts involves LAMP1, as previously observed by us in an in vitro model of fibroblasts (15) In fibroblasts, r[A−4] was shown to bind to cell surface LAMP1 (15).…”

“…Both of these peptides demonstrated chondrogenic-inducing potential at the ectopic site, when implanted intramuscular, or in vitro, when added to embryonic muscle fibroblasts (12,13). Further, the in vitro addition of r[A−4] and r[A+4] to the organ culture medium of developing tooth germs showed them to have distinct regulatory effects on tooth development (14). Hence, [A−4] and [A+4] might participate in epithelial-mesenchymal communication events that regulate the late stages of ameloblast and odontoblast maturation.…”

“…The developmentally-restricted expression of alternatively spliced transcripts translates into proteins with dissimilar function, regulated by discrete signaling pathways. The M59/[A−4] peptide demonstrated signaling properties exclusive to ameloblast differentiation in embryonic mouse molars (14,15). In contrast, in the postnatal mouse molar, [A−4] changed the expression of molecules important for matrix biomineralization (22).…”

The Exon 6ABC Region of Amelogenin mRNA Contribute to Increased Levels of Amelogenin mRNA through Amelogenin Protein-enhanced mRNA Stabilization

“…amelogenin and LRAP are capable of modulating expression of cementoblast-associated genes (12)(13)(14)(15)(16). In particular, LRAPs have been shown to act differently, as signaling molecules affecting odontogenic and other cell types (12)(13)(14)(15).…”