Amelogenin signaling: Effects and mechanisms

Amelogenin proteins make up over 90% of the organic matrix of developing enamel in the vertebrate tooth. Alternative splicing of the amelogenin premRNA leads to the production of more than 11 protein isoforms, the functions of which are not totally understood. The larger forms in the mouse, M180, M170, and M156, participate in the formation of the enamel. Smaller splice products, [A+4] or M73 and [A-4] or M59, have demonstrated signaling ability. To investigate the role of amelogenin signaling in tooth development, embryonic mouse tooth germs cultured in media containing 20% FBS showed normal development with polarized ameloblasts, and odontoblasts producing dentin matrix, and DMP2 expression in odontoblast and preameloblasts. Culture in 2% FBS media resulted in no ameloblast polarization and modest odontoblast differentiation with scant dentin matrix. Tooth germs cultured in 2% FBS/[A+4] 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 2% FBS/[A-4] media odontoblast polarization and dentin production was reduced compared to [A+4]. There was a disorganized preameloblast layer without ameloblast polarization. DMP2 expression was reduced in the odontoblast compared to the 20% FBS and [A+4] conditions and almost completely abrogated in the preameloblasts.;The mechanisms of these signaling processes beginning with receptor identification, has not been well understood. Utilizing radiolabeled [A-4] we show that 3H[A-4] binds in a saturatable fashion to the cell surface of C2C12 mouse fetal myoblasts at 4°C, and both binds at the surface and is internalized at 37°C. Immunohistochemistry performed on sections of E18 mouse teeth with Biotin labeled [A-4] as the primary ligand demonstrates binding to polarizing ameloblasts and odontoblasts, stratum intermedium cells, and cells of the dental follicle. Using [A-4] affinity column chromatography and [A-4]-Biotin label transfer reaction we have identified a 95 KDa C2C12 cell surface protein which bound [A-4]. Utilizing Tandem MS (MS/MS) sequencing revealed the novel finding of the 95 KDa murine transmembrane protein, LAMP-1, a lysosomal membrane protein that is also found at the cell surface, as an [A-4] cell binding protein.