Insulin-like growth factor-I (IGF-I) promotes bone formation by stimulating proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP), is thought to function in the initial mineralization of bone, is selectively expressed by differentiated osteoblast. To determine the molecular mechanism of IGF-I regulation of osteogenesis, we analyzed the effects of IGF-I on the expression of BSP in osteoblast-like Saos2 and in rat stromal bone marrow (RBMC-D8) cells. IGF-I (50 ng/ml) increased BSP mRNA levels at 12 h in Saos2 cells. In RBMC-D8 cells, IGF-I increased BSP mRNA levels at 3 h. From transient transfection assays, a twofold increase in transcription by IGF-I was observed at 12 h in pLUC3 construct that included the promoter sequence from -116 to +60. Effect of IGF-I was abrogated by 2-bp mutations in either the FGF2 response element (FRE) or homeodomain protein-binding site (HOX). Gel shift analyses showed that IGF-I increased binding of nuclear proteins to the FRE and HOX elements. Notably, the HOX-protein complex was supershifted by Smad1 antibody, while the FRE-protein complex was shifted by Smad1 and Cbfa1 antibodies. Dlx2 and Dlx5 antibodies disrupted the formation of the FRE- and HOX-protein complexes. The IGF-I effects on the formation of FRE-protein complexes were abolished by tyrosine kinase inhibitor herbimycin A (HA), PI3-kinase/Akt inhibitor LY249002, and MAP kinase kinase inhibitor U0126, while IGF-I effects on HOX-protein complexes were abolished by HA and LY249002. These studies demonstrate that IGF-I stimulates BSP transcription by targeting the FRE and HOX elements in the proximal promoter of BSP gene.

This study aimed to analyze the enzyme activity in gingival crevicular fluid (GCF) and its association with clinical parameters, especially bleeding on probing (BOP), and thus reconsider the significance and accuracy of recording BOP. A total of 184 patients who had entered supportive periodontal therapy were selected and GCF was collected from 401 sites before recording the clinical parameters, probing pocket depth (PPD), BOP, clinical attachment level, gingival index and plaque index. The enzyme activity of neutrophil elastase and aspartate aminotransferase and amount of protein in GCF were also analyzed. In the clinical parameters for biochemical data, amount of GCF showed the most correlation. A cut-off value for BOP and PPD were determined by the ROC curve and Youden index. Analysis was performed with all clinical parameters and biochemical data. Of the 401 sites, 51 were less than the cut-off value and were BOP-negative. On the other hand, 29 sites had values more than the cut-off value, with 14 BOP-negative sites and 15 BOP-positive sites. A conclusion is as follows: twenty-nine sites with values more than the cut-off value were diagnosed as sites requiring periodontal management, however, 14 of these were BOP-negative. These results suggest that combining other biochemical tests with examination of BOP and PPD may improve the validity of periodontal disease diagnosis. In future studies, it will be essential to find a marker that can precisely detect periodontal disease activity, and to develop a diagnostic tool for chair-side use.

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ObjectiveMicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we have analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF).Materials and methodsTotal RNA and protein were extracted from HGF after stimulation by interleukin-1β (IL-1β; 1 ng/ml) or tumor necrosis factor-α (TNF-α; 10 ng/ml) and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1β, inhibitor of nuclear factor kappa-B kinaseβ (IKKβ), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot.ResultsIL-1β and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKβ and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKβ and ZEB1 and increased E-cadherin mRNA and protein levels in HGF.ConclusionsThese results suggest that miR-200b attenuates inflammatory response via IKKβ and ZEB1 in periodontal tissue.

This study indicates that nicotine suppresses BSP transcription mediated through CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene.