Enamel matrix derivative stimulates core binding factor alpha1/Runt-related transcription factor-2 expression via activation of Smad1 in C2C12 cells.
BACKGROUND
Although enamel matrix derivative (EMD) can promote osteogenic differentiation of the pluripotent mesenchymal precursor cell line, C2C12, the molecular mechanism that underlies this phenomenon is unclear. The purpose of this study was to determine which molecules in EMD stimulate osteogenic differentiation.


METHODS
C2C12 cells were cultured in 5% serum-containing medium to induce differentiation, either with or without the addition of EMD. The expression of core binding factor alpha1/runtrelated transcription factor-2 (Cbfa1/Runx2) was measured using Northern blot, Western blot, and/or real-time polymerase chain reaction (R-PCR) analysis. Phosphorylation of mothers against decapentaplegic homolog 1 (Smad1) and bone morphogenetic protein (BMP)-like molecules in EMD was determined by Western blot.


RESULTS
EMD increased Cbfa1/Runx2 mRNA and protein expression substantially. EMD also induced phosphorylation of Smad1. Noggin inhibited the EMD-induced phosphorylation of Smad1 markedly, and also partially blocked EMD-induced Cbfa1/ Runx2 mRNA expression. In the Western blot analysis, single bands that corresponded to approximately 15 and approximately 17.5 kDa proteins were recognized in EMD by anti-BMP-2/4 and anti-BMP-7 antibodies, respectively.


CONCLUSIONS
Our study demonstrates that EMD stimulates Cbfa1/Runx2 expression and the phosphorylation of Smad1, and that both of these processes can be blocked by noggin. Therefore, the osteogenic activity of EMD may be mediated by BMPlike molecules in EMD.
Department of Periodontology, Nihon University School of Dentistry, Tokyo, Japan.
Tadahiro
T
Takayama
Naoto
N
Suzuki
Masataka
M
Narukawa
Tsuyoshi
T
Tokunaga
Kichibee
K
Otsuka
Koichi
K
Ito
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D002352:Carrier Proteins; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit; D050660:Core Binding Factors; D004268:DNA-Binding Proteins / Q000378:metabolism; D003746:Dental Enamel Proteins / Q000737:chemistry* / Q000494:pharmacology*; D059630:Mesenchymal Stem Cells / Q000187:drug effects; D051379:Mice; D008809:Mice, Inbred C3H; D009363:Neoplasm Proteins / Q000096:biosynthesis; D010012:Osteogenesis / Q000187:drug effects*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D051785:Smad Proteins; D051898:Smad1 Protein; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000037:antagonists & inhibitors; D000099176:Noggin Protein
D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D002352:Carrier Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D050660:Core Binding Factors; D004268:DNA-Binding Proteins; D003746:Dental Enamel Proteins; D009363:Neoplasm Proteins; D051785:Smad Proteins; D051898:Smad1 Protein; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D000099176:Noggin Protein; C525719:Bmp2 protein, mouse; C525810:Bmp4 protein, mouse; C497401:Smad1 protein, mouse; C109069:enamel matrix proteins