ESTROGEN REGULATION OF THE OSTEOBLAST INSP3 RECEPTOR

雌激素对成骨细胞 INSP3 受体的调节

• 批准号: 2407875
• 负责人: PETER G BRADFORD
• 金额: $ 3.65万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 1999-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2407875
• 关键词: RNase protection assay; biological signal transduction; calcium channel; cell line; cell type; enzyme linked immunosorbent assay; estradiol; estrogen receptors; hormone regulation /control mechanism; human subject; human tissue; inositol phosphates; interleukin 6; messenger RNA; osteoblasts; osteoclast activating factor; osteosarcoma; receptor expression; thapsigargin; tooth extraction; western blottings

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.

这项拟议的研究将比较17 β-雌二醇对 三磷酸肌醇受体（InsP 3R）表达与IL-6分泌 G-292细胞 G-292细胞来自已建立的人类 成骨细胞骨肉瘤细胞系。 原代培养物的研究 人成骨细胞来源于口腔（上颌结节）和非 口腔（松质骨）骨也将进行充实的细胞 文化模式 拟议研究的目的是测试 雌激素下调INSP 3R表达假说 成骨细胞，因此，减少这种能力， 分泌破骨细胞活化细胞因子的细胞群，包括 IL-6。 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部位， 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成骨细胞，从而建立一个 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志物， 包括生长和分化表型相关基因以及 雌激素受体状态，将被评估，并在特定阶段使用 评价雌激素反应性。 我们的初步观察 表明17 β-雌二醇下调I型INSP 3R基因 在人G-292骨肉瘤细胞和大鼠原代骨肉瘤细胞中的表达 颅骨成骨细胞 这些观察将通过以下方式加以扩展： 检查其他INSP 3R类型的潜在监管， 确定改变的InsP 3R表达对IL-6分泌的影响。 雌激素对InsP 3R表达和IL-6分泌的影响 使用RNA酶保护测定来定量类型特异性 mRNA水平，配体结合研究和wester印迹技术， 确定InsP 3R密度，荧光Ca 2+释放研究，以评估 功能状态和夹心ELISA方案定量 IL-6分泌。 InsP 3敏感性分泌钙的消耗将 证实了在引发的IL-6分泌中对该库的需要。 对所述假设的检验可以提供令人信服的 为未来广泛研究提供初步数据，并确定潜在地点 在这个领域进行治疗干预。