Adaptor protein complex 2–mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis

Authors

  • Rodrigo S Lacruz,

    1. Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA
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  • Steven J Brookes,

    1. Department of Oral Biology, Leeds Dental Institute, University of Leeds, Leeds, UK
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  • Xin Wen,

    1. Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA
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  • Jaime M Jimenez,

    1. Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA
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  • Susanna Vikman,

    1. Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
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  • Ping Hu,

    1. Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA
    2. Center of Stomatology, Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology, Wuhan, People's Republic of China
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  • Shane N White,

    1. School of Dentistry, University of California at Los Angeles, Los Angeles, CA, USA
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  • S Petter Lyngstadaas,

    1. Department of Biomaterials, Faculty of Dentistry, University of Oslo, Oslo, Norway
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  • Curtis T Okamoto,

    1. University of Southern California School of Pharmacy, Department of Pharmacology and Pharmaceutical Sciences, Los Angeles, CA, USA
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  • Charles E Smith,

    1. Facility for Electron Microscopy Research, Department of Anatomy & Cell Biology, and Faculty of Dentistry, McGill University, Montreal, Canada
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  • Michael L Paine

    Corresponding author
    1. Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA
    • Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, 2250 Alcazar Street, CSA103, Los Angeles, CA 90605, USA
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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. © 2013 American Society for Bone and Mineral Research.

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