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

Luciane Martins, Bruna Rabelo Amorim, Cristiane Ribeiro Salmon, Adriana Franco Paes Leme, Kamila Rosamilia Kantovitz, Francisco Humberto Nociti

Journal of cellular physiology 2020 May

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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 < .05). Together, our findings shed new light on the potential role of plasminogen signaling pathway in the control of the amelogenin isoform-mediated response in cementoblasts and provide new insights into the development of targeted therapies. © 2019 Wiley Periodicals, Inc.

Citation

Luciane Martins, Bruna Rabelo Amorim, Cristiane Ribeiro Salmon, Adriana Franco Paes Leme, Kamila Rosamilia Kantovitz, Francisco Humberto Nociti. Novel LRAP-binding partner revealing the plasminogen activation system as a regulator of cementoblast differentiation and mineral nodule formation in vitro. Journal of cellular physiology. 2020 May;235(5):4545-4558