title: Adaptor protein complex 2-mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis.
abstract: Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real-time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP-2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin-dependent endocytosis, thus implying the likelihood of specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also upregulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H(+) transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H(+) transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage-sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor-regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation.
authors.affiliation: Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA 90605, USA.
authors.forename: Rodrigo S
authors.initials: RS
authors.lastname: Lacruz
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Brookes
authors.forename: Xin
authors.initials: X
authors.lastname: Wen
authors.forename: Jaime M
authors.initials: JM
authors.lastname: Jimenez
authors.forename: Susanna
authors.initials: S
authors.lastname: Vikman
authors.forename: Ping
authors.initials: P
authors.lastname: Hu
authors.forename: Shane N
authors.initials: SN
authors.lastname: White
authors.forename: S Petter
authors.initials: SP
authors.lastname: Lyngstadaas
authors.forename: Curtis T
authors.initials: CT
authors.lastname: Okamoto
authors.forename: Charles E
authors.initials: CE
authors.lastname: Smith
authors.forename: Michael L
authors.initials: ML
authors.lastname: Paine
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000502:physiology*; D000566:Amelogenesis; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology*; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D060149:Tetraspanin 30 / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D033962:Adaptor Protein Complex 2; C554587:Cd63 protein, mouse; D060149:Tetraspanin 30