Amelogenins make up over 90% of the secretory enamel proteins. One of the most studied alternatively spliced amelogenins is leucine-rich amelogenin peptide (LRAP), which functions as a cell-signaling molecule to regulate mesenchymal-derived cell proliferation and/or differentiation. However, the function of LRAP in enamel formation is unclear. Objective: In this study, we investigated the biological activity of LRAP on ameloblast-lineage cell (ALC) differentiation. Methods: Human recombinant LRAP was synthesized, purified from E. coli. After characterization using SDS-PAGE, Western blot and mass spectrometry, LRAP was exogenously added to human primary ALC cultures to detect the evidence of cell differentiation using immunohistochemistry. Results: LRAP-treated cells appeared to be rounded, larger and non-proliferating as compared to the smaller, cobblestone-like morphology of the untreated control cells. Immunofluorescent results showed that LRAP promoted cell differentiation by increased amelogenin synthesis and downregulation of Notch1. LAMP-1, a membrane receptor reported as a LRAP receptor in mesenchymal cells, was identified and was upregulated with the addition of LRAP. Conclusion: These results show that LRAP promotes differentiation of ameloblast-lineage cells, suggesting that LRAP functions to regulate enamel tooth formation. Supported: NIH/NIDCR T32-DE07306-09 (to T.L.), P01-DE009859 (to P.D.B.) and R01-DE015821 (to W.L.).

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