Leucine-Rich Amelogenin Peptide (LRAP) Uptake by Cementoblast Requires Flotillin-1 Mediated Endocytosis.
Basic, pre-clinical, and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine-Rich Amelogenin Peptide-LRAP), which is supposed to act as a signaling molecule in epithelial-mesenchymal interactions. LRAP-binding protein complexes from immortalized murine cementoblasts (OCCM-30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin-1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co-precipitation and co-localization assays as a protein-binding partner for LRAP in OCCM-30 cells. In addition, we found that exogenously added GST-LRAP recombinant protein was internalized by OCCM-30 cells, predominantly localized in the perinuclear region and, that inhibition of flotillin1-dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM-30 cells, including LRAP effect on the expression of genes encoding osteocalcin (Ocn), bone sialoprotein (Bsp), and runt-related transcription factor 2 (RunX2). In conclusion, LRAP uptake by cementoblast involves flotillin-assisted endocytosis, which suggests an involvement of LRAP in lipid-raft-dependent signaling pathways which are mediated by flotillin-1. J. Cell. Physiol. 232: 556-565, 2017. © 2016 Wiley Periodicals, Inc.
Division of Periodontics, Department of Prosthodontics and Periodontics, Piracicaba Dental School, University of Campinas-UNICAMP, Piracicaba, Sao Paulo, Brazil.
Luciane
L
Martins
Brazilian Biosciences National Laboratory (LNBio), CNPEM, Campinas, Brazil.
Adriana Franco Paes
AF
Leme
Department of Pediatric Dentistry, Piracicaba Dental School, University of Campinas-UNICAMP, Piracicaba, Sao Paulo, Brazil.
Kamila Rosamilia
KR
Kantovitz
Department of Dental Materials, São Leopoldo Mandic Research Center, Campinas, Sao Paulo, Brazil.
Em Nome
EN
de Luciane Martins
Division of Periodontics, Department of Prosthodontics and Periodontics, Piracicaba Dental School, University of Campinas-UNICAMP, Piracicaba, Sao Paulo, Brazil.
Enilson Antonio
EA
Sallum
Division of Periodontics, Department of Prosthodontics and Periodontics, Piracicaba Dental School, University of Campinas-UNICAMP, Piracicaba, Sao Paulo, Brazil.
Márcio Zaffalon
MZ
Casati
Division of Periodontics, Department of Prosthodontics and Periodontics, Piracicaba Dental School, University of Campinas-UNICAMP, Piracicaba, Sao Paulo, Brazil.
Francisco Humberto
FH
Nociti
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003739:Dental Cementum / Q000166:cytology* / Q000378:metabolism*; D003746:Dental Enamel Proteins / Q000378:metabolism*; D004705:Endocytosis; D020868:Gene Silencing; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice
D003746:Dental Enamel Proteins; D008565:Membrane Proteins; C106354:flotillins; C067432:leucine-rich amelogenin peptide