Amelogenin binds to both heparan sulfate and bone morphogenetic protein 2 and pharmacologically suppresses the effect of noggin.
Enamel matrix derivative (EMD) is widely considered useful to promote tissue regeneration during periodontal treatment. It has been reported that the main constituent of EMD is amelogenin and that the BMP-like and TGF-beta-like activity of EMD promotes osteogenesis. However, it remains unclear whether those activities are dependent on amelogenin or another growth factor contained in EMD. We performed two-dimensional SDS-PAGE analysis of EMD, as well as Western blot analyses using anti-amelogenin, anti-BMP2/4, and anti-TGF-beta1 antibodies, and amino acid sequencing. Our results revealed that a large number of splicing forms of amelogenin, BMP2/4, and other unknown molecules were involved in EMD, though TGF-beta1 was not. In addition, we have evaluated intracellular signaling of ERK1/2 and Smad1/5/8, binding potential and alkaline phosphatase activity and have explored the potential regulatory relationship between amelogenin and BMP. Amelogenin bound to BMP2 as well as heparin/heparan sulfate. Thus, it was suggested that BMP2/4 carried over in EMD during processing promote binding activity and phosphorylate Smad1/5/8 in osteoblasts. On the other hand, amelogenin did not phosphorylate Smad1/5/8, but rather ERK1/2. Further, high-density amelogenin reduced the inhibition of alkaline phosphatase activity by noggin, though amelogenin did not have antagonistic properties against BMP. Together with the above findings, our findings suggest that the BMP2/4 contaminated during the purification process of EMD because of the avidity of amelogenin plays an important role in signaling pathway of calcification.
Saito
Kan
K
Division of Pediatric Dentistry, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.
Konishi
Ikuri
I
Division of Pediatric Dentistry, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.
Nishiguchi
Miyuki
M
Division of Pediatric Dentistry, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.
Hoshino
Tomonori
T
Division of Pediatric Dentistry, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan. Electronic address: thoshino@nagasaki-u.ac.jp.
Fujiwara
Taku
T
Division of Pediatric Dentistry, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.
D053523:Amelogenin / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D003746:Dental Enamel Proteins / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D020539:Sequence Analysis, Protein; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism; D013552:Swine; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D000099176:Noggin Protein
D053523:Amelogenin; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D002352:Carrier Proteins; D003746:Dental Enamel Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D006497:Heparan Sulfate; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D000099176:Noggin Protein; C525719:Bmp2 protein, mouse; C525810:Bmp4 protein, mouse; C109069:enamel matrix proteins