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. CONCLUSION: 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.
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 ratBSP 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. CONCLUSION: These studies have, therefore, identified EMD response elements in the ratBSP gene promoter that may mediates the effects of EMD on BSP gene transcription.
Authors: Daniel A W Oortgiesen; Na Yu; Antonius L J J Bronckers; Fang Yang; X Frank Walboomers; John A Jansen Journal: Tissue Eng Part C Methods Date: 2012-01-04 Impact factor: 3.056