OSTEOBLAST AND PTH/PTHRP BONE RESORPTION

成骨细胞和 PTH/PTHRP 骨吸收

• 批准号: 2079587
• 负责人: KARL Leonard INSOGNA
• 金额: $ 19.9万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2079587
• 关键词: biological signal transduction; colony stimulating factor; genetic library; homeostasis; hormone binding protein; in situ hybridization; laboratory rat; molecular biology; newborn animals; northern blottings; nuclear runoff assay; nucleic acid sequence; osteoblasts; parathyroid hormones; pathologic bone resorption; receptor expression

The osteoblast or an osteoblast-like cell is the principal to which parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind in bone. We hypothesize that osteoblast-derived factors are therefore critically important local mediators of PTH-and PTHrP-induced bone resorption. A corollary to this hypothesis is that differences in the actions of PTH and PTHrP on osteoblasts may help explain the different in vivo effects of these two hormones. To pursue this hypothesis, the effects of these two agonists on osteoblasts-derived factors which may 1) activate mature osteoclasts or 2) stimulate recruitment of pre-osteoclasts will be examined. We have observed that PTH and PTHrP-treated osteoblasts release a soluble bone-resorbing activity. This activity has been partially purified and can directly stimulate resorption by isolated osteoclasts (under conditions where PTHrP is inactive). This material will be purified to homogeneity to obtain primary structural information. Amino-acid sequence information will be used to synthesize oligonucleotide probes for screening an Saos-2cDNA library. Based on our previous work, demonstrating PTH-induce GM-CSF release from osteoblasts, this molecule will be studied as an example of an osteoblast-derived factor which may regulate osteoclast recruitment. The molecular mechanisms underlying PTH/PTHrP-stimulated release of GM-CSF from osteoclast will be examining the effects of neutralizing antisera to GM-CSF on PTH/PTHrP-induced resorption in the fetal rat long-bone and mouse metatarsal assays. We have very recently identified CSF-1 receptors (c-fms) in mature rat osteoclasts. Further, in preliminary studies we have identified down- regulation of this receptor by GM-CSF suggesting an important interaction between these two colony-stimulating factors in regulating osteoclast biology. These observations will be pursued using immunocytochemical techniques and in situ hybridization. Specifically, we will study whether the effect of GM-CSF on c-fms occurs at the level of transcription or translation. Finally, a mammalian expression system employing rat insulinoma cells has been developed using the three available cDNA clones for PTHrP. The effects of amino-terminal purified recombinant PTHrP on signal transduction mechanisms in, and release of resorptive cytokines from, osteoblasts will be studied. In the aggregate, these studies should allow a more precise picture of the cellular mechanisms by which PTH and PTHrP induce resorption in bone and should lead to a clearer picture of how these compounds act under physiologic conditions as well as those of disturbed mineral homeostasis.

成骨细胞或成骨细胞样细胞是主要的， 甲状旁腺激素（PTH）和甲状旁腺相关蛋白（PTHrP） 在骨头上绑起来。 我们假设成骨细胞衍生因子是 因此，PTH和PTHrP诱导的 骨吸收 这一假设的一个推论是， PTH和PTHrP对成骨细胞的作用可能有助于解释 这两种激素在体内的不同作用。 追求这个 假设，这两种激动剂对成骨细胞衍生的 可能1）激活成熟破骨细胞或2）刺激 将检查前破骨细胞的募集。 我们已经观察到PTH和PTHrP处理的成骨细胞释放可溶性 骨吸收活性。 该活性已被部分纯化， 可以直接刺激分离的破骨细胞的吸收（在 PTHrP失活的条件）。 这些材料将被纯化， 同质性以获得初级结构信息。 氨基酸 序列信息将用于合成寡核苷酸探针 筛选Saos-2cDNA文库。 基于我们之前的工作， 证明PTH诱导GM-CSF从成骨细胞释放，这种分子 将作为成骨细胞衍生因子的一个例子进行研究， 调节破骨细胞募集。 的分子机制 将检查PTH/PTHrP刺激的破骨细胞释放GM-CSF GM-CSF中和抗血清对PTH/PTHrP诱导的 胎鼠长骨和小鼠跖骨测定中的吸收。 我们最近在成年大鼠中鉴定了CSF-1受体（c-fms）， 破骨细胞 此外，在初步研究中，我们已经确定下来- GM-CSF对该受体的调节表明了重要的相互作用 这两种集落刺激因子在调节破骨细胞中的作用 生物学 这些观察结果将采用免疫细胞化学 技术和原位杂交。 具体来说，我们将研究 GM-CSF对c-fms的影响是否发生在水平 转录或翻译。 最后，使用大鼠胰岛素瘤细胞的哺乳动物表达系统具有 使用PTHrP的三个可用cDNA克隆开发。 的 氨基端纯化的重组PTHrP对信号的影响 再吸收性细胞因子的转导机制和释放， 将研究成骨细胞。 总体而言，这些研究应 允许更精确地了解PTH和 PTHrP诱导骨吸收，并应导致更清晰的图片， 这些化合物如何在生理条件下起作用，以及 矿物质平衡紊乱