Effects of porcine 25 kDa amelogenin and its proteolytic derivatives on bone sialoprotein expression.
OBJECTIVE
Amelogenins are hydrophobic proteins that are the major component of developing enamel. Enamel matrix derivative has been used for periodontal regeneration. Bone sialoprotein is an early phenotypic marker of osteoblast differentiation. In this study, we examined the ability of porcine amelogenins to regulate bone sialoprotein transcription.


METHODS
To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene by amelogenins, we conducted northern hybridization, transient transfection analyses and gel mobility shift assays using the osteoblast-like ROS 17/2.8 cells.


RESULTS
Amelogenins (100 ng/mL) up-regulated bone sialoprotein mRNA at 3 h, with maximal mRNA expression occurring at 12 h (25 and 20 kDa) and 6 h (13 and 6 kDa). Amelogenins (100 ng/mL, 12 h) increased luciferase activities in pLUC3 (nucleotides -116 to +60), and 6 kDa amelogenin up-regulated pLUC4 (nucleotides -425 to +60) activity. The tyrosine kinase inhibitor inhibited amelogenin-induced luciferase activities, whereas the protein kinase A inhibitor abolished 25 kDa amelogenin-induced bone sialoprotein transcription. The effects of amelogenins were abrogated by 2-bp mutations in the fibroblast growth factor 2 response element (FRE). Gel-shift assays with radiolabeled FRE, homeodomain-protein binding site (HOX) and transforming growth factor-beta1 activation element (TAE) double-strand oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells at 3 h (25 and 13 kDa) and 6 h (20 and 6 kDa).


CONCLUSIONS
These results demonstrate that porcine 25 kDa amelogenin and its proteolytic derivatives stimulate bone sialoprotein transcription by targeting FRE, HOX and TAE in the bone sialoprotein gene promoter, and that full-length amelogenin and amelogenin cleavage products are able to regulate bone sialoprotein transcription via different signaling pathways.
Nakayama
Y
Y
Department of Periodontology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
Yang
L
L
Mezawa
M
M
Araki
S
S
Li
Z
Z
Wang
Z
Z
Sasaki
Y
Y
Takai
H
H
Nakao
S
S
Fukae
M
M
Ogata
Y
Y
D053523:Amelogenin / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D002460:Cell Line; D015342:DNA Probes; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics; D058574:Integrin-Binding Sialoprotein / Q000096:biosynthesis* / Q000235:genetics; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D051381:Rats; D020218:Response Elements / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects*; D013552:Swine; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics; D015854:Up-Regulation
D053523:Amelogenin; D015342:DNA Probes; D018398:Homeodomain Proteins; D058574:Integrin-Binding Sialoprotein; D053773:Transforming Growth Factor beta1; D016222:Fibroblast Growth Factor 2; D008156:Luciferases