ESTROGEN REGULATION OF THE OSTEOBLAST INSP3 RECEPTOR

这项拟议的研究将比较17β-雌二醇对 三磷酸肌醇受体表达与IL-6的分泌 来自G-292细胞。G-292细胞来自已建立的人类 成骨细胞性骨肉瘤细胞系。的原代培养研究 人成骨细胞来源于口腔(上颌骨结节)和非成骨细胞 还将进行口腔(海绵骨)骨移植以充实细胞。 文化模式。拟议研究的目的是测试 雌激素下调INSP3R表达的假说 成骨细胞，因此，降低了这一能力 分泌破骨细胞激活细胞因子的细胞群，包括 IL-6。一个积极的结果将具有重大意义，因为 它将确定成骨细胞中雌激素作用的位置，从而影响 信号转导和分泌能力。这些研究将实现 同时，比较雌激素反应性的目的 口腔和非口腔来源的人类成骨细胞，从而建立 骨质疏松症与口腔骨关系的研究基础 损失。衍生成骨细胞的生化和分子标记， 包括与生长和分化表型相关的基因以及 雌激素受体状态，将在特定的阶段进行评估和使用 雌激素反应性的评价。我们的初步观察 17β-雌二醇下调I型INSP3R基因 人G-292骨肉瘤细胞和大鼠原代细胞的表达 颅骨成骨细胞。这些观察结果将延伸到 研究其他INSP3R类型的潜在调控 检测InsP3R表达改变对IL-6分泌的影响。 雌激素对InsP3R表达和IL-6分泌的影响 使用核糖核酸酶保护分析对特定类型进行量化 MRNA水平、配体结合研究和Wester blotting技术 确定InsP3R密度，荧光Ca2plus释放研究以评估 功能状态和夹心ELISA法用于定量 IL-6分泌。InsP3敏感的分泌型钙离子耗竭 证实在诱导IL-6分泌中对这个池的需求。 对所陈述的假设的检验可以提供令人信服的 为广泛的未来研究和确定潜在地点提供的初步数据 在这一领域进行治疗干预。

The proposed study will compare the effects of 17 beta-estradiol on inositol trisphosphate receptor (InsP3R) expressioin and IL-6 secretion from G-292 cells . G-292 cells are from an established human osteoblastic osteosarcoma cell line. Studies of primary cultures of human osteoblasts derived from oral (maxillary tuberosities) and non- oral (spongy bone) bone will also be carried out to substantiate the cell culture model. The objective of the proposed study is to test the hypothesis that estrogens down regulate INSP3R expressioin in osteoblasts and, as a consequence, reduce the capacity of this population of cells to secrete osteoclst-activating cytokines, including IL-6. A positive outcome would be of substantial significance because it would identify a site of estrogen action in osteoblasts that affects signal transduction and secretory capacity. The studies will achieve simultaneously the objective of comparing the estrogen-responsiveness of oral and non-oral derived human osteoblasts and thus establish a basis for investigative relationships between osteoporosis and oral bone loss. Biochemical and molecular markers of the derived osteoblasts, including growth and differentiation phenotype related genes as well as estrogen receptor status, will be assessed and used in the stage specific evaluation of estrogen responsiveness. Our preliminary observations indicate that 17 beta-estradiol down regulates type I INSP3R gene expression in human G-292 osteosarcoma cells and rat primary calvarial osteoblasts. These observations will be extended by examining potential regulation of other INSP3R types and by determining the effects of altered InsP3R expression on IL-6 secretion. The effects of estrogen on InsP3R expression and IL-6 secretion will be determined using RNase protection assays to quantitate type-specific mRNA levels, ligand binding studies and wester blotting techniques to determine InsP3R density, fluorescent Ca2plus release studies to assess funcitonal status, and sandwich ELISA protocols for quantitation of IL-6 secrettion. Depletion of InsP3-sensitive secretory calcicum will substantiate the requirement for this pool in elicited IL-6 secretion. The testing of the stated hypothesis could provide compelling preliminary data for extensive future studies and identify potential sites for therapeutic intervention in this area.