Heinrichs A A, Bortell R, Rahman S, Stein J L, Alnemri E S, Litwack G, Lian J B, Stein G S
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.
Biochemistry. 1993 Oct 26;32(42):11436-44. doi: 10.1021/bi00093a022.
The biosynthesis of osteocalcin (OC), a bone-specific, noncollagenous protein, is stringently regulated during differentiation of the osteoblast phenotype. Glucocorticoids, and also 1,25(OH)2D3, mediate the developmental regulation of OC gene transcription. In this study, we established that the -1097 to +23 promoter (pOCZCat) of the rat OC gene confers glucocorticoid responsiveness to both basal and vitamin D-induced OC expression. The presence of multiple glucocorticoid receptor (GR) binding sites in the proximal rat OC gene promoter was determined by the combined use of DNase I footprinting, dimethyl sulfate fingerprinting, and gel mobility shift analysis with glucocorticoid receptor protein. One glucocorticoid receptor binding element (GRE) resides immediately downstream of the TATA box (-16 to -1). In vivo activity was established by cotransfection of ROS 17/2.8 osteosarcoma cells with an OC-CAT construct in the presence of cloned GRE sequences (wild type or mutant) as competitors. A putative second, less protected GR binding site is located further upstream in the OC gene basal promoter within the region overlapping the TATA box. This is in direct contrast to the organization of GREs in the human OC proximal promoter wherein GR binding at the upstream GRE overlapping the TATA is stronger than at the downstream GRE. In addition, we detected sequence-specific binding of GR protein to another basal promoter element, the OC box (-99 to -76), which contains a central CCAAT motif. The presence of multiple GR binding sites in the rat OC gene proximal promoter indicates that regulation of basal and vitamin D-enhanced transcription by glucocorticoids may involve the integrated activities of multiple, independent GREs.
骨钙素（OC）是一种骨特异性非胶原蛋白，其生物合成在成骨细胞表型分化过程中受到严格调控。糖皮质激素以及1,25(OH)₂D₃介导OC基因转录的发育调控。在本研究中，我们确定大鼠OC基因的-1097至+23启动子（pOCZCat）赋予糖皮质激素对基础和维生素D诱导的OC表达的反应性。通过联合使用DNA酶I足迹法、硫酸二甲酯指纹法以及糖皮质激素受体蛋白凝胶迁移率变动分析，确定了大鼠OC基因近端启动子中存在多个糖皮质激素受体（GR）结合位点。一个糖皮质激素受体结合元件（GRE）位于TATA框下游紧邻处（-16至-1）。通过在存在克隆的GRE序列（野生型或突变型）作为竞争者的情况下，将OC-CAT构建体与ROS 17/2.8骨肉瘤细胞共转染，确定了体内活性。在OC基因基础启动子中，一个假定的第二个、保护较弱的GR结合位点位于与TATA框重叠区域的更上游。这与人类OC近端启动子中GRE的组织方式形成直接对比，在人类OC近端启动子中，与TATA重叠的上游GRE处的GR结合比下游GRE处更强。此外，我们检测到GR蛋白与另一个基础启动子元件OC框（-99至-76）的序列特异性结合，该元件包含一个中心CCAAT基序。大鼠OC基因近端启动子中存在多个GR结合位点表明，糖皮质激素对基础和维生素D增强转录的调控可能涉及多个独立GRE的综合活性。
