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

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Abstract
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.