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The Small Alternatively Spliced Amelogenin LRAP Modulates Early Stage Ameloblast Differentiation

  • Author(s): Stahl, Jonathan
  • Advisor(s): DenBesten, Pamela
  • et al.
Abstract

Amelogenesis is a regulated and sequential developmental cascade that results in expression of tissue specific gene products that form the enamel extracellular matrix. There remains considerable lack of knowledge regarding the precise mechanisms that control ameloblast differentiation and enamel matrix biomineralization. Amelogenins are the major protein product of ameloblasts and are comprised of multiple alternatively spliced isoforms that may function as structural molecules to regulate enamel crystal growth in addition to being signaling molecules that regulate cell differentiation. Hypothesis: The small alternatively spliced amelogenin known as leucine rich amelogenin peptide (LRAP) functions to modulate ameloblast differentiation from pre-ameloblasts to terminal differentiation. Methods: Transgenic mouse models that overexpressed LRAP in both a WT (TgLRAP) and an amelogenin null background (TgLRAP/AmelX Null) were examined to determine if this alternatively spliced protein had a direct effect in vivo on ameloblast differentiation by assaying histomorphology, gene expression, and protein expression patterns in comparison to wild-type and amelogenin null mice. Biomineralization was further assessed with microCT and von Kossa staining. In vitro primary ameloblast lineage cells were transfected with LRAP to study early developmental effects. Results: In vivo TgLRAP mice in the WT background showed a significant upregulation of enamel matrix gene products in pre-ameloblasts with earlier and greater amelogenin protein expression in pre-secretory and secretory ameloblasts. Apoptosis was increased in secretory and transitional TgLRAP ameloblasts. Earlier mineral formation was also associated with the increased amelogenin expression. Downregulation of the master gene regulator SATB1 was also detected in pre-secretory and secretory ameloblasts. In the AmelX Null background TgLRAP overexpression did not lead to an observable phenotype compared to AmelX Null mice. In Vitro primary cell culture demonstrated down regulation of developmental genes Notch1 and NfKappa&beta1 was found. Conclusions: LRAP overexpression specifically modulates the expression of enamel matrix associated genes and proteins suggesting an earlier differentiation of ameloblasts from pre-ameloblasts to pre-secretory ameloblasts and an important role in regulating the timing of enamel matrix biomineralization.

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