Kevin Tompkins

Specific Amelogenin Gene Splice Products Have Signaling Effects on Cells in Culture and in Implants in Vivo

Low molecular mass amelogenin-related polypeptides extracted from mineralized dentin have the ability to affect the differentiation pathway of embryonic muscle fibroblasts in culture and lead to the formation of mineralized matrix in in vivo implants. The objective of the present study was to determine whether the bioactive peptides could have been amelogenin protein degradation products or specific amelogenin gene splice products. Thus, the splice products were prepared, and their activities were determined in vitro and in vivo. A rat incisor tooth odontoblast pulp cDNA library was screened using probes based on the peptide amino acid sequencing data. Two specific cDNAs comprised from amelogenin gene exons 2,3,4,5,6d,7 and 2,3,5,6d,7 were identified. The corresponding recombinant proteins, designated r[A+4] (8.1 kDa) and r[A−4] (6.9 kDa), were produced. Both peptides enhanced in vitrosulfate incorporation into proteoglycan, the induction of type II collagen, and Sox9 or Cbfa1 mRNA expression. In vivoimplant assays demonstrated implant mineralization accompanied by vascularization and the presence of the bone matrix proteins, BSP and BAG-75. We postulate that during tooth development these specific amelogenin gene splice products, [A+4] and [A−4], may have a role in preodontoblast maturation. The [A+4] and [A−4] may thus be tissue-specific epithelial mesenchymal signaling molecules.

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

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.

Molecular Mechanisms of Cytodifferentiation in Mammalian Tooth Development

The morphological stages of tooth development—bud, cap, bell, and terminal differentiation—have been known for decades. The past 10 years have seen the elucidation of many of the molecular events driving these morphogenetic stages. Signaling via the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), hedgehog, and wingless protein families and their downstream transcription factors have been identified as key players in the epithelial-mesenchymal signaling loops driving tooth development. Currently the most complete description of the mechanisms in tooth development extends only through the cap stage. The body of work concerning the mechanisms directing the bell and cytodifferentiation stages is growing. This has mainly, but not exclusively, focused on the expression and effects of FGFs and BMPs in these latter stages, and is reviewed here. Additionally, recent results suggest that phenotypic proteins of both ameloblasts and odontoblasts, such as amelogenin and dentin matrix protein 2 may act as the final instructive signals in cytodifferentiation.

Polypeptides translated from alternatively spliced transcripts of the amelogenin gene, devoid of the exon 6a, b, c region, have specific effects on tooth germ development in culture.

Recombinant proteins have been produced from cDNAs corresponding to alternatively spliced transcripts comprised from exons 2,3,4,5,6d,7 and 2,3,5,6d,7 of the rat amelogenin gene. These peptides, designated as [A + 4] and [A m 4], respectively, induce embryonic muscle fibroblasts in culture in vitro to express proteins characteristic of the chondrogenic and osteogenic phenotypes, and in matrix-supported implants into rat muscle, in vivo, induce typical bone matrix proteins. The aim of the present work was to examine the potential role of these proteins on the development of odontogenic tissue. The lower first molars were collected from Charles River CD-1 mice at postnatal days 1 and 2 and were grown on semisolid, serum-free medium supplemented with ascorbic and retinoic acids and transferrin. The peptides were added to the serum-free media at 10 ng/ml. As controls, the medium was either 20% fetal bovine serum or the supplemented serum-free medium without either amelogenin peptide. The tooth germs were cultured for 6 days, then fixed and paraffin embedded by standard procedures. The tissue blocks were serially sectioned and stained with hematoxylin-eosin (H&E), or antibodies to collagen 1 (Col1), phosphophoryn (DMP2), or cementum attachment protein (CAP). CAP, DMP2, and Col1 expression was enhanced by the addition of the amelogenin peptides, as compared to the 0% fetal bovine serum (FBS) controls, but the peptides showed different effects. Expression of DMP2, characteristic of dentin matrix, was upregulated by [A + 4], whereas CAP, characteristic of cementum, was upregulated by [A m 4]. Since the recombinant peptides are active, their corresponding tissue forms may be important in the stimulation of mesenchymal tissue differentiation. Thus, these specific amelogenin proteins may be involved in tooth morphogenesis.