Fluoride Enhances Intracellular Degradation of Amelogenins During Secretory Phase of Amelogenesis of Hamster Teeth in Organ Culture

Abstract

Amelogenins are the major protein species synthesized by secretory ameloblasts and are believed to be involved in enamel mineralization. During enamel formation, amelogenins are progressively degraded into smaller fragments by protease activity. These amelogenin fragments are removed from the enamel extracellular space, thereby enabling full mineralization of the dental enamel. Enamel from fluorotic teeth is porous and contains more proteins and less mineral than sound enamel. In this study we examined the hypothesis that fluoride (F m ) is capable of inhibiting the proteolysis of amelogenins in enamel being formed in organ culture. Hamster molar tooth germs in stages of secretory amelogenesis were pulse labeled in vitro with [ 3 H]- or [ 14 C] proline and subsequently pulse chased. The explants were exposed to F m at different days of chase (i.e., during secretory amelogenesis early after labeling, later after labeling or at stages just beyond secretory amelogenesis). Exposure of secretory stage explants to F m enhanced the release of radiolabeled fragments when F m was applied early after labeling but progressively less if applied later. In contrast, F m had no such effect in stages beyond secretion. The enhanced release of radiolabeled fragments in secretory stages was associated with a reduction of radioactivity in the soft tissue enamel organ indicating that fragmentation of enamel matrix proteins (mainly amelogenins) occurred intracellularly. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the fluorotic enamel contained less radiolabeled parent amelogenins ( M r 28 kD and 26 kD) but more low-molecular-mass fragments than enamel from control explants. Our data indicate that F m promotes intracellular degradation of the newly synthesized parent amelogenins during secretory stage. Our in vitro data do not support the concept that F m impairs extracellular proteolysis of amelogenins, either in the secretory phase or in the stage just beyond the secretory phase.