Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan; Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan.
Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan.
Gene. 2014 Apr 10;539(1):50-7. doi: 10.1016/j.gene.2014.01.063. Epub 2014 Feb 2.
Estrogen is one of the steroid hormones essential for skeletal development. The estrogen receptor (ER) is a transcription factor and a member of the steroid receptor superfamily. There are two different forms of the ER, usually referred to as α and β, each encoded by a separate gene. Hormone-activated ERs form dimers, since the two forms are coexpressed in many cell types. Bone sialoprotein (BSP) is a tissue-specific acidic glycoprotein that is expressed by differentiated osteoblasts, odontoblasts and cementoblasts during the initial formation of mineralized tissue. To determine the molecular basis of the tissue-specific expression of BSP and its regulation by estrogen and the ER, we have analyzed the effects of β-estradiol and ERα on BSP gene transcription. ERα protein levels were increased after ERα overexpression in ROS17/2.8 cells. While BSP mRNA levels were increased by ERα overexpression, the endogenous and overexpressed BSP mRNA levels were not changed by β-estradiol (10(-8)M, 24 h). Luciferase activities of different sized BSP promoter constructs (pLUC3~6) were increased by ERα overexpression, whereas basal and induced luciferase activities by ERα overexpression were not influenced by β-estradiol. Effects of ERα overexpression were abrogated by 2 bp mutations in either the cAMP response element (CRE) or activator protein 1 (AP1)/glucocorticoid response element (GRE). Gel shift analyses showed that ERα overexpression increased binding to the CRE and AP1/GRE elements. Notably, the CRE-protein complexes were disrupted by ERα, CREB and phospho-CREB antibodies. The AP1/GRE-protein complexes were supershifted by the c-Fos antibody. These studies demonstrate that ERα stimulates BSP gene transcription in a ligand-independent manner by targeting the CRE and AP1/GRE elements in the rat BSP gene promoter.
雌激素是骨骼发育所必需的甾体激素之一。雌激素受体 (ER) 是一种转录因子，属于甾体受体超家族成员。ER 有两种不同的形式，通常称为 α 和 β，每种形式都由单独的基因编码。激素激活的 ER 形成二聚体，因为这两种形式在许多细胞类型中都共同表达。骨涎蛋白 (BSP) 是一种组织特异性酸性糖蛋白，在矿化组织初始形成过程中，由分化的成骨细胞、成牙本质细胞和成牙骨质细胞表达。为了确定 BSP 的组织特异性表达的分子基础及其受雌激素和 ER 的调节，我们分析了 β-雌二醇和 ERα 对 BSP 基因转录的影响。在 ROS17/2.8 细胞中过表达 ERα 后，ERα 蛋白水平增加。虽然 BSP mRNA 水平因 ERα 过表达而增加，但内源性和过表达的 BSP mRNA 水平不受 β-雌二醇 (10(-8)M，24 h) 的影响。不同大小的 BSP 启动子构建体 (pLUC3~6) 的荧光素酶活性因 ERα 过表达而增加，而 ERα 过表达诱导的基础和诱导的荧光素酶活性不受 β-雌二醇的影响。在 cAMP 反应元件 (CRE) 或激活蛋白 1 (AP1)/糖皮质激素反应元件 (GRE) 中的 2 个 bp 突变后，ERα 过表达的作用被阻断。凝胶迁移分析显示，ERα 过表达增加了对 CRE 和 AP1/GRE 元件的结合。值得注意的是，CRE-蛋白复合物被 ERα、CREB 和磷酸化-CREB 抗体破坏。AP1/GRE-蛋白复合物被 c-Fos 抗体超迁移。这些研究表明，ERα 通过靶向大鼠 BSP 基因启动子中的 CRE 和 AP1/GRE 元件，以非配体依赖的方式刺激 BSP 基因转录。
