Change of thegene expression of bone sialoprotein and transcription factors in osteoblasts during periodontal tissue regeration.

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Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. In this study, we investigated the effects of prostaglandin E_2 (PGE_2) and static magnetic fields (SMF) on BSP gene expression. To determine the molecular mechanism of PGE_2 and SMF regulation of BSP, we analyzed the effects of the PGE_2 and SMF on the expression of BSP in UMR 106 cells. PGE_2 (3μM, 12h) and application of 300 and 800 Gauss SMF (24h) increased BSP mRNA levels detected by real-time PCR.From transient transfection assays using various BSP promoter-luciferase constructs, PGE_2 and SMF increased expression of the construct (pLUC3 ; -116 to +60). Further deletion analysis of the BSP promoter showed that a FGF response element (FRE) and a cAMP response element (CRE) were identified as a target of transcriptional I activation by PGE_2, the effects of which were inihibited by protein kinase A, src tyrosine kinase and MAP kinase inhibitors. FRE and a pituitary-specific transcription factor-1 regulatory element (Pit-1) were identified as a target for SMF, the effect of which was inihibited by tyrosine kinase inhibitor.Binding of nuclear proteins to a radiolabeled FRE and CRE were increased after stimulation by PGE_2. To further characterize the proteins in the complexes formed with the CRE and FRE, we used antibodies for several transcription factors. The addition of antibody to CREB disrupted the formation of the CRE DNA-protein complexes, while incubation of nuclear extracts with anti-phospho-CREB antibody produced a visible supershift complex. Binding of nuclear proteins to a radiolabeled FRE was increased and that to a Pit-1 was decreased in nuclear extracts prepared from SMF-stimulated UMR 106 cells.These studies, therefore, have identified response elements in the proximal promoter of the BSP gene that mediates PGE_2 and SMF-induced BSP transcription.