title: Role of the N-terminal peptide of amelogenin on osteoblastic differentiation of human mesenchymal stem cells.
abstract: Porcine enamel matrix derivative (pEMD), a complex mixture of proteins and peptides including full-length amelogenin protein, splice variants, and proteolytic peptides, is used clinically with a carrier to regenerate supportive tissue around teeth. During application, pEMD self-assembles as nanospheres and precipitates as a three-dimensional matrix to facilitate cell migration and differentiation. Amelogenin, the primary constituent of pEMD, stimulates osteoblast differentiation, but it is unclear what specific roles other components of pEMD play in determining biological response. This study examined the potential of one constituent of pEMD, the N-terminal amelogenin peptide (NTAP), to promote osteoblastic differentiation of human mesenchymal stem cells (MSCs) and to elucidate possible signaling pathways involved. Effects of porcine NTAP on MSC cultures were compared to those of recombinant human amelogenin. While amelogenin induced MSC osteoblastic differentiation, a more robust osteoblastic response was seen after NTAP treatment. A phospho-kinase proteasome array measuring phosphorylation of 35 proteins indicated that protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and β-catenin were highly phosphorylated by NTAP. This was confirmed by measuring PKC activity and levels of phospho-ERK1/2 and β-catenin. Both amelogenin and NTAP increased PKC, but NTAP induced higher phosho-ERK1/2 and phospho-β-catenin than amelogenin. ERK1/2 inhibition blocked both amelogenin- and NTAP-induced increases in RUNX2, ALP, OCN, COL1, and BMP2. The results demonstrate that NTAP induces osteogenic differentiation of MSCs via PKC and ERK1/2 activation and β-catenin degradation. NTAP may be an active bone regeneration component of amelogenin, and may play this role in pEMD-stimulated periodontal regeneration.
authors.affiliation: Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, 601 West Main Street, Suite 396, Richmond, VA 23284, USA.ronavarrete@vcu.edu.
authors.forename: R
authors.initials: R
authors.lastname: Olivares-Navarrete
authors.forename: K
authors.initials: K
authors.lastname: Vesper
authors.forename: S L
authors.initials: SL
authors.lastname: Hyzy
authors.forename: A
authors.initials: A
authors.lastname: Almaguer-Flores
authors.forename: B D
authors.initials: BD
authors.lastname: Boyan
authors.forename: Z
authors.initials: Z
authors.lastname: Schwartz
mesh_terms: D053523:Amelogenin / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D006801:Humans; D020935:MAP Kinase Signaling System; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects*; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D013552:Swine; D051176:beta Catenin / Q000378:metabolism
chemical_list: D053523:Amelogenin; D010446:Peptide Fragments; D051176:beta Catenin; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3