Kim R H, Li J J, Ogata Y, Yamauchi M, Freedman L P, Sodek J
Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Canada.
Biochem J. 1996 Aug 15;318 ( Pt 1)(Pt 1):219-26. doi: 10.1042/bj3180219.
Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation de novo. Our studies, using the osteoblastic cell line ROS 17/2.8, have revealed that rat BSP gene expression is suppressed by 1,25-dihydroxyvitamin D3[1,25(OH)2D3], which is a powerful regulator of bone formation and resorption. To determine the molecular basis of the transcriptional suppression of BSP gene transcription by 1,25(OH)2D3, we have conducted transient transfection analyses with chimaeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. 1,25(OH)2D3 suppressed expression in all constructs, including a short construct (pLUC 3; nt -116 to +60) that contained a putative vitamin D3-response element (VDRE; AGGGTTTATAGGTCA; nt -28 to -14) that overlaps a unique inverted TATA (TTTATA) box. Mobility shift assays demonstrated strong binding of recombinant human vitamin D3 receptor protein (hVDR) to the VDRE. Point mutations introduced into each half-site and analysed for 1,25(OH)2D3-mediated suppression of transcription and for hVDR binding either decreased or increased both transcriptional suppression and binding. In comparison with activating VDREs, the rat BSP VDRE bound VDR-VDR homodimers more avidly than VDR-RXR alpha heterodimers (where RXR is retinoid X receptor). These studies have therefore identified a novel 1,25(OH)2D3 suppressor element that overlaps the inverted TATA box in the rat BSP gene and indicate that transcriptional suppression of the rat BSP gene by 1,25(OH)2D3 might involve competition between the VDR and the TATA binding protein (TBP).
骨唾液蛋白（BSP）是成骨细胞分化的早期标志物，在骨从头形成过程中与羟基磷灰石的成核有关。我们使用成骨细胞系ROS 17/2.8进行的研究表明，大鼠BSP基因表达受到1,25 - 二羟基维生素D3 [1,25(OH)2D3]的抑制，1,25(OH)2D3是骨形成和吸收的有力调节因子。为了确定1,25(OH)2D3对BSP基因转录进行转录抑制的分子基础，我们用与荧光素酶报告基因相连的大鼠BSP基因启动子的嵌合构建体进行了瞬时转染分析。1,25(OH)2D3抑制了所有构建体中的表达，包括一个短构建体（pLUC 3；核苷酸 -116至 +60），该构建体包含一个假定的维生素D3反应元件（VDRE；AGGGTTTATAGGTCA；核苷酸 -28至 -14），它与一个独特的反向TATA（TTTATA）框重叠。凝胶迁移试验表明重组人维生素D3受体蛋白（hVDR）与VDRE有强结合。引入每个半位点的点突变，并分析其对1,25(OH)2D3介导的转录抑制和hVDR结合的影响，结果显示转录抑制和结合均降低或增加。与激活型VDRE相比，大鼠BSP VDRE与VDR - VDR同二聚体的结合比VDR - RXRα异二聚体（其中RXR是视黄酸X受体）更紧密。因此，这些研究确定了一个与大鼠BSP基因中的反向TATA框重叠的新型二羟基维生素D3抑制元件，并表明1,25(OH)2D3对大鼠BSP基因的转录抑制可能涉及VDR和TATA结合蛋白（TBP）之间的竞争。
