title: Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro.
abstract: Mineralization is a process of deposition of calcium phosphate crystals within a fibrous extracellular matrix (ECM). In mineralizing tissues, such as dentin, bone and hypertrophic cartilage, this process is initiated by a specific population of extracellular vesicles (EV), called matrix vesicles (MV). Although it has been proposed that MV are formed by shedding of the plasma membrane, the cellular and molecular mechanisms regulating formation of mineralization-competent MV are not fully elucidated. In these studies, 17IIA11, ST2, and MC3T3-E1 osteogenic cell lines were used to determine how formation of MV is regulated during initiation of the mineralization process. In addition, the molecular composition of MV secreted by 17IIA11 cells and exosomes from blood and B16-F10 melanoma cell line was compared to identify the molecular characteristics distinguishing MV from other EV. Western blot analyses demonstrated that MV released from 17IIA11 cells are characterized by high levels of proteins engaged in calcium and phosphate regulation, but do not express the exosomal markers CD81 and HSP70. Furthermore, we uncovered that the molecular composition of MV released by 17IIA11 cells changes upon exposure to the classical inducers of osteogenic differentiation, namely ascorbic acid and phosphate. Specifically, lysosomal proteins Lamp1 and Lamp2a were only detected in MV secreted by cells stimulated with osteogenic factors. Quantitative nanoparticle tracking analyses of MV secreted by osteogenic cells determined that standard osteogenic factors stimulate MV secretion and that phosphate is the main driver of their secretion. On the molecular level, phosphate-induced MV secretion is mediated through activation of extracellular signal-regulated kinases Erk1/2 and is accompanied by re-organization of filamentous actin. In summary, we determined that mineralization-competent MV are distinct from exosomes, and we identified a new role of phosphate in the process of ECM mineralization. These data provide novel insights into the mechanisms of MV formation during initiation of the mineralization process.
authors.affiliation: Department of Oral and Maxillofacial Surgery, Institute of Oral Health Research, School of Dentistry, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: Sandeep C
authors.initials: SC
authors.lastname: Chaudhary
authors.affiliation: Department of Oral and Maxillofacial Surgery, Institute of Oral Health Research, School of Dentistry, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Kuzynski
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy; Inflammatory and Infectious Disease Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
authors.forename: Massimo
authors.initials: M
authors.lastname: Bottini
authors.affiliation: Department of Oral Biology, University of Pittsburgh School of Dental Medicine, Pittsburgh, PA, USA.
authors.forename: Elia
authors.initials: E
authors.lastname: Beniash
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: Terje
authors.initials: T
authors.lastname: Dokland
authors.affiliation: Department of Oral and Maxillofacial Surgery, Institute of Oral Health Research, School of Dentistry, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: Callie G
authors.initials: CG
authors.lastname: Mobley
authors.affiliation: Sanford Children's Health Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
authors.forename: Manisha C
authors.initials: MC
authors.lastname: Yadav
authors.affiliation: EA2496 UFR d'Odontologie, Université Paris Descartes, Montrouge, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Poliard
authors.affiliation: INSERM UMR-S 1124, Université René Descartes Paris 5, Centre Universitaire des Saints-Pères, Paris, France.
authors.forename: Odile
authors.initials: O
authors.lastname: Kellermann
authors.affiliation: Sanford Children's Health Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Millán
authors.affiliation: Department of Oral and Maxillofacial Surgery, Institute of Oral Health Research, School of Dentistry, University of Alabama at Birmingham, Birmingham, AL, USA. Electronic address: dobrawan@uab.edu.
authors.forename: Dobrawa
authors.initials: D
authors.lastname: Napierala
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002113:Calcification, Physiologic; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005109:Extracellular Matrix / Q000378:metabolism; D000067128:Extracellular Vesicles / Q000187:drug effects / Q000378:metabolism*; D052119:Lysosomal-Associated Membrane Protein 2 / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D051379:Mice; D009804:Odontoblasts / Q000502:physiology*; D010012:Osteogenesis; D010710:Phosphates / Q000378:metabolism*; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D015415:Biomarkers; D002118:Calcium; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; D010710:Phosphates; C052255:Lamp1 protein, mouse; D052118:Lysosomal-Associated Membrane Protein 1
keywords: Erk1/2; Matrix vesicles; Mineralization; Odontoblast; Phosphate