Regulation of rat bone sialoprotein gene transcription by enamel matrix derivative.
BACKGROUND
Enamel matrix derivative (EMD) has recently been developed for use as a periodontal regenerative treatment. While EMD is believed to induce regeneration of periodontal tissue, the precise mechanism is not known. Bone sialoprotein (BSP), an early phenotypic marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation. In this study, we examined the ability of EMD to regulate BSP gene transcription in osteoblast-like cells.


METHODS
To determine the molecular basis of the transcriptional regulation of BSP gene transcription by EMD, we conducted Northern hybridization, transient transfection analyses with chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene, and gel mobility shift assays.


RESULTS
Using the osteoblastic cell line ROS 17/2.8, we determined that BSP mRNA levels increased approximately 2.8-fold by EMD. In transient transfection analyses, EMD (50 microg/ml, 12 hours) increased luciferase activities of pLUC4 (nt -425 to +60) and pLUC5 (nt -801 to +60), transfected into ROS 17/2.8 cells. Within the pLUC4 and 5, a homeodomain binding element (HOX) and a transforming growth factor (TGF)-beta activation element (TAE) are present. Gel mobility shift assays with radiolabeled HOX and TAE ds-oligonucleotides revealed increased binding of nuclear proteins from EMD stimulated ROS 17/2.8 cells.


CONCLUSIONS
These studies have, therefore, identified EMD response elements in the rat BSP gene promoter that may mediates the effects of EMD on BSP gene transcription.
Shimizu
Emi
E
Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Nakajima
Yu
Y
Kato
Naoko
N
Nakayama
Youhei
Y
Saito
Ryoichiro
R
Samoto
Hiroshi
H
Ogata
Yorimasa
Y
D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects; D002460:Cell Line; D002678:Chimera / Q000235:genetics; D002999:Clone Cells; D003746:Dental Enamel Proteins / Q000494:pharmacology*; D004742:Enhancer Elements, Genetic / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D005801:Genes, Homeobox / Q000187:drug effects / Q000235:genetics; D058574:Integrin-Binding Sialoprotein; D008156:Luciferases / Q000187:drug effects / Q000235:genetics; D010006:Osteoblasts / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000187:drug effects; D051381:Rats; D020218:Response Elements / Q000187:drug effects / Q000235:genetics; D012795:Sialoglycoproteins / Q000187:drug effects* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000187:drug effects / Q000235:genetics
D003746:Dental Enamel Proteins; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D012795:Sialoglycoproteins; D016212:Transforming Growth Factor beta; C109069:enamel matrix proteins; D008156:Luciferases