Amelogenin peptides have unique milieu-dependent roles in morphogenic path determination

Abstract

In early studies of the bone morphogenetic proteins from various mineralized tissues, Urist [1,2] reported that the proteins from dentin matrix had a higher bone inductive activity in implants than did bone matrix, and had different inductive effects than demineralized enamel matrix. We set out to determine the source of the activity in dentin, expecting to find a high concentration of BMP. Our strategy was to identify the active fraction from dentin extracts using an in vitro assay related to the presumed first step in ectopic implant bone induction, chondrogenesis. Thus, we developed anin vitrosystem in which embryonic muscle fibroblasts were cultured in monolayers, under conditions not conducive to the formation of chondrogenic nodules (as compared with suspension cultures), and then exposed to the various extract fractions [3]. “Activity” was defined as the enhanced incorporation of ³⁵SSO₄ into cetyl pyridinium chloride-precipitable proteoglycan. A fraction with activity was finally isolated and shown by amino acid composition studies to be unrelated to the BMP family of proteins. Specifically, the active peptides had compositions devoid of Cys residues, a hallmark of the BMP-TGFβ family of proteins. Moreover, the active peptides had molecular masses in the range from 6,000 to 10,000, smaller than any of the BMPs [4]. After extensive fractionation and purification, an active peptide was finally isolated and subjected to amino terminal and internal sequencing following trypsin hydrolysis. To our amazement, the peptide sequence proved to be related to amelogenins, proteins produced by ameloblasts, specialized epithelial cells not thought to be present in dentin, which is produced by mesenchyme-derived odontoblasts [5].