CSF-1 GENE EXPRESSION IN OSTEOCLAST BIOLOGY

骨髓基质细胞通过直接作用调节破骨细胞的形成 接触或通过释放可溶性细胞因子 集落刺激因子（CSF-1或M-CSF）。 CSF-1是一种关键的调节因子， 破骨细胞祖细胞增殖所必需的分子。 的 CSF-1在破骨细胞形成中的重要性已经通过其 对op/op小鼠（CSF-1缺陷的突变体）的治疗效果。 我们将 确定小鼠TC-1骨髓源性基质细胞的作用 在体外产生CSF-1对破骨细胞形成的影响。 CSF-1表达 在TC-1细胞中，血小板衍生生长因子（PDGF）和12- 0-十四酰基佛波醇-13-乙酸酯（TPA）。 我们在本提案中的重点将是 目的是确定CSF-1基因调控的分子机制 使用TC-1基质细胞作为模型，对PDGF、TPA和cAMP的响应。 具体目的是：1）确定TC-1和其他骨 骨髓来源的基质细胞对破骨细胞形成的影响; 2）确定， 在TC-1细胞中，PDGF、TPA和cAMP对CSF-1基因表达的影响 在CSF-1 mRNA的丰度、合成和稳定性水平上， 核转录延长试验和CSF-1 mRNA的分析 3）鉴定和表征顺式作用DNA元件 在CSF-1基因中介导其转录调节。 这些 研究将通过用嵌合抗体转染TC-1细胞来完成。 含有缺失CSF-1基因5 ′侧翼区的质粒 插入报告基因的上游。 我们还将识别反式作用 负责PDGF、TPA和cAMP介导的CSF-1基因的因子 4）确定CSF-1向op/op的骨的递送是否 通过基因工程改造的自体基质细胞， 产生CSF-1，治疗骨硬化缺陷。 拟议的实验 提供了识别新的调节机制的潜力， 控制精氨酸诱导的CSF-1表达。 这些研究也有 有可能扩大我们对基质细胞作用的理解， 在生理事件中调节破骨细胞的形成，以及 病理性疾病说。 增加对分子的理解 细胞因子通常调节CSF-1的机制可能导致 更有效的治疗方案，旨在增强或抑制 破骨细胞的增殖和功能活性。 战略 通过基质细胞将细胞因子靶向骨的方法， 具有潜在应用于各种 如骨质疏松症和骨折。

Bone marrow stromal cells modulate osteoclast formation through direct contact or via the release of soluble cytokines such as macrophage colony-stimulating factor (CSF-1 or M-CSF). CSF-1 is a key regulatory molecule essential for proliferation of osteoclast progenitors. The importance of CSF-1 in osteoclast formation has been emphasized by its curative effect on op/op mice, a mutant deficient in CSF-1. We will determine the effect of murine TC-1 bone marrow-derived stromal cells that produce CSF-1 on osteoclast formation in Vitro. CSF-1 expression in TC-1 cells is induced by platelet-derived growth factor (PDGF) and 12- 0-tetradecanoylphorbol-13-acetate (TPA). Our focus in this proposal will be to define the molecular mechanism involved in CSF-1 gene regulation in response to PDGF, TPA and cAMP using TC-1 stromal cells as a model. The specific aims are: 1) to determine the effect of TC-1 and other bone marrow-derived stromal cells on osteoclast formation; 2) to determine, in TC-1 cells, the effects of PDGF, TPA and cAMP on CSF-1 gene expression at the level of abundance, synthesis and stability of CSF-1 mRNA using nuclear transcript elongation assay and analysis of CSF-1 mRNA disappearance; 3) to identify and characterize cis-acting DNA elements in the CSF-1 gene that mediate its transcriptional regulation. These studies will be accomplished by transfecting TC-1 cells with chimeric plasmids which contain variably deleted 5'-flank region of the CSF-1 gene inserted upstream of a reporter gene. We will also identify trans-acting factors responsible for PDGF, TPA, and cAMP-mediated CSF-1 gene expression; 4) to determine if delivery of CSF-1 to the bone of op/op mice via autologous stromal cells that are genetically engineered to produce CSF-1, cures the osteopetrotic defect. The proposed experiments offer the potential for identifying novel regulatory mechanisms that control cytokine-induced expression of CSF-1. These studies also have the potential to expand our understanding of the role of stromal cells in regulating osteoclast formation during physiologic events, as well as pathological disease stated. Increased understanding of the molecular mechanisms by which cytokines, in general, regulate CSF-1 may lead to more effective treatment regimens designed to either enhance or inhibit the proliferation and functional activity of osteoclasts. The strategy of targeting cytokines to the bone via stromal cells represents a novel therapeutic approach with potential application to a variety of conditions such as osteoporosis and bone fracture.