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Wang Z.,Nihon University | Wang Z.,Tianjin Stomatology Hospital | Li X.,Nihon University | Li X.,Tianjin Stomatology Hospital | And 12 more authors.
Gene | Year: 2010

Inorganic polyphosphate (poly(P)) is a biopolymer existing in almost all cells and tissues. The biological functions of poly(P) in micro-organisms have been extensively investigated in studies of poly(P) in eukaryotic cells, especially osteoblasts, and are increasing. Bone sialoprotein (BSP) is thought to function in bone mineralization, and is selectively expressed by differentiated osteoblasts. In this study, application of sodium phosphate glass type 25 (SPG25, 12.5 and 125 μM) increased BSP mRNA levels at 12 h in osteoblast-like ROS 17/2.8 cells. In transient transfection assay, 12.5 and 125 μM SPG25 increased luciferase activities of the constructs pLUC3 (-116 to +60), pLUC4 (-425 to +60), pLUC5 (-801 to +60) and pLUC6 (-938 to +60). Introduction of 2 bp mutations to the luciferase constructs showed that the effects of SPG25 were mediated by a FGF2 response element (FRE) and a homeodomain protein binding site (HOX). Luciferase activities induced by SPG25 were blocked by tyrosine kinase inhibitor herbimycine A, MAP kinase kinase inhibitor U0126, PI3-kinase/Akt inhibitor LY249002 and inorganic phosphate transport inhibitor foscarnet. Gel shift analyses showed that both 12.5 and 125 μM SPG25 increased nuclear protein binding to FRE and HOX elements. These studies demonstrate that SPG25 stimulates BSP transcription by targeting FRE and HOX elements in the proximal promoter of the rat BSP gene. © 2010 Elsevier B.V.


Wang S.,Nihon University | Sasaki Y.,Nihon University | Zhou L.,Nihon University | Zhou L.,Anhui Province Stomatology Hospital | And 5 more authors.
Gene | Year: 2011

Interleukin-11 (IL-11) is a stromal cell-derived cytokine that belongs to the interleukin-6 family of cytokines. IL-11 has many biological activities and has roles in hematopoiesis, immune responses, the nervous system and bone metabolism. Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed in differentiated osteoblasts that appears to function in the initial mineralization of bone. IL-11 (20. ng/ml) increased BSP mRNA and protein levels at 12. h in osteoblast-like ROS 17/2.8 cells. In a transient transfection assay, IL-11 (20. ng/ml) increased luciferase activity of the construct (-116 to +. 60) in ROS 17/2.8 cells and rat bone marrow stromal cells. Introduction of 2. bp mutations to the luciferase constructs showed that the effects of IL-11 were mediated by a cAMP response element (CRE), a fibroblast growth factor 2 response element (FRE) and a homeodomain protein-binding site (HOX). Luciferase activities induced by IL-11 were blocked by protein kinase A inhibitor, tyrosine kinase inhibitor and ERK1/2 inhibitor. Gel shift analyses showed that IL-11 (20. ng/ml) increased nuclear protein binding to CRE, FRE and HOX. CREB1, phospho-CREB1, c-Fos, c-Jun, JunD and Fra2 antibodies disrupted the formation of CRE-protein complexes. Dlx5, Msx2, Runx2 and Smad1 antibodies disrupted FRE- and HOX-protein complex formations. These studies demonstrate that IL-11 stimulates BSP transcription by targeting CRE, FRE and HOX sites in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB1, c-Fos, c-Jun, JunD, Fra2, Dlx5, Msx2, Runx2 and Smadl transcription factors appear to be key regulators of IL-11 effects on BSP transcription. © 2011 Elsevier B.V.


Li Z.,Nihon University | Li Z.,Tianjin Stomatology Hospital | Sasaki Y.,Nihon University | Mezawa M.,Nihon University | And 14 more authors.
Gene | Year: 2011

Bone sialoprotein (BSP) is a noncollagenous protein of the extracellular matrix in mineralized connective tissues that has been implicated in the nucleation of hydroxyapatite. Forskolin (FSK), an activator of adenylate cyclase, increased the intracellular cAMP level, which stimulates the proliferation and differentiation of osteoblasts. Fibroblast growth factor 2 (FGF2) is a potent mitogen in many cell types, including osteoblasts. In human prostate cancer DU145 cells, FSK (1μM) and FGF2 (10. ng/ml) increased BSP and Runx2 mRNA and protein levels at 3 and 12. h, respectively. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of DU145 cells with FSK (1μM) and FGF2 (10. ng/ml) increased the luciferase activities of constructs between -60LUC to -927LUC and -108LUC to -927LUC, including the human BSP gene promoter. Effects of FSK and FGF2 abrogated in constructs included 2. bp mutations in the two cAMP response elements (CRE1 and CRE2). Luciferase activities induced by FSK and FGF2 were blocked by protein kinase A and tyrosine kinase inhibitors. Gel mobility shift analyses showed that FSK and FGF2 increased the binding of CRE1 and CRE2. CRE1-protein complexes were supershifted by phospho-CREB1 and c-Fos antibodies, and disrupted by CREB1, c-Jun, JunD, Fra2, p300, Runx2, Dlx5 and Smad1 antibodies. CRE2-protein complexes were disrupted by CREB1, phospho-CREB1, c-Fos, c-Jun, JunD, Fra2, p300, Runx2, Dlx5 and Smad1 antibodies. These studies demonstrate that FSK and FGF2 stimulate BSP transcription in DU145 human prostate cancer cells by targeting the CRE1 and CRE2 elements in the human BSP gene promoter. © 2010 Elsevier B.V.

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