Novel LRAP‐binding partner revealing the plasminogen activation system as a regulator of cementoblast differentiation and mineral nodule formation in vitro

• Published in: Journal of cellular physiology 2020-05, Vol.235 (5), p.4545-4558
• Main Authors: Martins, Luciane, Amorim, Bruna Rabelo, Salmon, Cristiane Ribeiro, Leme, Adriana Franco Paes, Kantovitz, Kamila Rosamilia, Nociti, Francisco Humberto
• Format: Article
• Language: English
• Subjects: Alkaline phosphatase; Amelogenin; Amelogenin - genetics; Amelogenin - metabolism; Animals; Binding; Biomedical materials; Bone sialoprotein; Cbfa-1 protein; Cell Differentiation; Cell Line; Cell morphology; Cementogenesis; Collagen (type I); Cytology; Dental cement; dental cementum; Dental Cementum - metabolism; Dental enamel; Dental Enamel Proteins - metabolism; Differentiation; Enzyme Activation; Gene expression; Gene Expression Regulation; Gene Regulatory Networks; Immunoblotting; Isoforms; Leucine; Mass spectrometry; Mass spectroscopy; Mesenchyme; Mice; Molecular modelling; Morphology; Nodules; Osteocalcin; Osteopontin; periodontal regeneration; Plasmin; plasminogen; Plasminogen - metabolism; Protein Binding; Protein Interaction Maps; Protein purification; Proteins; protein–protein interaction (PPI) network; Regeneration; Signal Transduction; Signaling; Teeth
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.29331

Amelogenin isoforms, including full‐length amelogenin (AMEL) and leucine‐rich amelogenin peptide (LRAP), are major components of the enamel matrix, and are considered as signaling molecules in epithelial–mesenchymal interactions regulating tooth development and periodontal regeneration. Nevertheless, the molecular mechanisms involved are still poorly understood. The aim of the present study was to identify novel binding partners for amelogenin isoforms in the cementoblast (OCCM‐30), using an affinity purification assay (GST pull‐down) followed by mass spectrometry and immunoblotting. Protein‐protein interaction analysis for AMEL and LRAP evidenced the plasminogen activation system (PAS) as a potential player regulating OCCM‐30 response to amelogenin isoforms. For functional assays, PAS was either activated (plasmin) or inhibited (ε‐aminocaproic acid [aminocaproic]) in OCCM‐30 cells and the cell morphology, mineral nodule formation, and gene expression were assessed. PAS inhibition (EACA 100 mM) dramatically decreased mineral nodule formation and expression of OCCM‐30 differentiation markers, including osteocalcin (Bglap), bone sialoprotein (Ibsp), osteopontin (Spp1), tissue‐nonspecific alkaline phosphatase (Alpl) and collagen type I (Col1a1), and had no effect on runt‐related transcription factor 2 (Runx2) and Osterix (Osx) mRNA levels. PAS activation (plasmin 5 µg/µl) significantly increased Col1a1 and decreased Bglap mRNA levels (p