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 mineraliz ed dentin have the ability to affect the differentiation pathway of embryon ic muscle fibroblasts in culture and lead to the formation of mineralized m atrix in in vivo implants. The objective of the present study was to determ ine whether the bioactive peptides could have been amelogenin protein degra dation products or specific amelogenin gene splice products. Thus, the spli ce products were prepared, and their activities were determined in vitro an d in vivo, A rat incisor tooth odontoblast pulp cDNA library was screened u sing probes based on the peptide amino acid sequencing data. Two specific c DNAs 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 vitro s ulfate incorporation into proteoglycan, the induction of type II collagen, and Sox9 or Cbfa1 mRNA expression. In vivo implant assays demonstrated impl ant mineralization accompanied by vascularization and the presence of the b one matrix proteins, BSP and BAG-75, We postulate that during tooth develop ment these specific amelogenin gene splice products, [A+4] and [A-4], may h ave a role in preodontoblast maturation. The [A+4] and [A-4] may thus be ti ssue-specific epithelial mesenchymal signaling molecules.