NEGATIVE REGULATION OF IGFBP-4 EXPRESSION IN BONE CELLS

描述(来自应用程序)： 细胞更新对于骨重建和修复过程是必不可少的， 在一生中都会发生。细胞更新的损害被认为是一种 老年(II型)发病的分子机制 骨质疏松。胰岛素样生长因子(IGF)在糖尿病中的重要性 以下主要发现强调了骨骼细胞的更新：1)IGFS 是由骨细胞产生的最丰富的有丝分裂原，2)IGF是重要的 骨细胞增殖的刺激物，以及3)IGFS 40%对体外培养的基底骨细胞增殖有抑制作用。尽管政府间金融服务机构的行动 在骨骼细胞更新中可以受到许多IGF系统的调节 组件，本提案侧重于一个IGF系统组件，即IGF 结合蛋白4(IGFBP-4)。我们最近的研究结果强调， IGFBP-4的表达可能部分参与了受损的骨骼细胞 骨质疏松等疾病的更新：第一，IGFBP-4水平在 骨细胞条件培养液与骨细胞呈负相关 增殖率。第二，抑制内源性IGFBP-4的产生有 已被证明能刺激骨细胞的增殖。第三，血清IGFBP-4 在II型骨质疏松症合并髋关节的患者中，水平随着年龄的增长而升高 骨折。基于这样一个前提，即调控的分子机制 IGFBP-4的表达对我们理解IGFBP-4的作用至关重要 在骨骼细胞更新方面，我们提出了一项研究，以表征一种 一种涉及特定负面监管的负面监管机制 IGFBP-4基因第一外显子中存在NRE元件。在建议的 研究，IGFBP-4 NRE与之相互作用的精确序列(S) 蛋白质(S)以细胞特有的方式将由足迹决定 分析、凝胶迁移率漂移分析和西南分析。 随后，NRE中DNA-蛋白质相互作用的位置将是 系统突变并包含在IGFBP-4启动子-报告基因中 用于功能研究的结构。IGFBP-4启动子的差异 使用含有启动子的野生型和突变型NRE构建物的活性将 在瞬时转染骨细胞后进行评估 不同水平的IGFBP-4mRNA和蛋白的表达。预计 证实了NRE与特定基因之间相互作用的假设 结合蛋白参与负转录调控 IGFBP-4基因在不同细胞类型中的表达可以导致更好的 对老年性骨质疏松症发病机制的认识 通过抑制促进细胞更新的新疗法的开发 IGFBP-4在骨质疏松等疾病中的表达。

DESCRIPTION (from the application): Cell renewal is essential for bone remodeling and repair processes which occur throughout life. Impairment in cell renewal has been identified as a molecular mechanism responsible for the pathogenesis of senile (type II) osteoporosis. The importance of insulin-like growth factors (IGFs) in skeletal cell renewal is emphasized by the following key findings: 1) IGFs are the most abundant mitogens produced by bone cells, 2) IGFs are important stimulaters of bone cell proliferation, and 3) IGFs contribute approximately 40% to basal bone cell proliferation in vitro. Although the actions of IGFs in skeletal cell renewal can be modulated by a number of IGF system components, this proposal is focused on one IGF system component, namely IGF binding protein-4 (IGFBP-4). Our recent findings emphasize that changes in IGFBP-4 expression may in part contribute to the impaired skeletal cell renewal in diseases such as osteoporosis: First, IGFBP-4 levels in the conditioned medium of bone cells correlate negatively with the bone cell proliferation rate. Second, inhibition of endogenous IGFBP-4 production has been shown to stimulate bone cell proliferation. Third, serum IGFBP-4 levels are elevated with age and in type II osteoporosis patients with hip fracture. Based on the premise that molecular mechanisms which regulate IGFBP-4 expression are crucial to our understanding of the role of IGFBP-4 in skeletal cell renewal, we have proposed studies to characterize a negative regulatory mechanism which involves a specific negative regulatory element (NRE) present in the first exon of IGFBP-4 gene. In the proposed studies, the precise sequence(s) in the IGFBP-4 NRE which interact with protein(s) in a cell specific manner will be determined by footprint analysis, electrophoretic mobility shift analysis and Southwestern analysis. Subsequently, the sites of DNA-protein interaction in the NRE will be systematically mutated and included in the IGFBP-4- promoter-reporter constructs used for functional studies. The differences in IGFBP-4 promoter activity using wild type and mutant NRE containing promoter constructs will be evaluated after transient transfection of bone cells which express varying levels of IGFBP-4 mRNA and protein. It is anticipated that confirmation of the hypothesis that interaction between the NRE and specific binding proteins contribute to negative transcriptional regulation of IGFBP-4 gene expression in various cell types could lead to a better understanding of the pathogenesis of senile osteoporosis and to the development of novel therapies to increase cell renewal by suppressing IGFBP-4 expression in diseases such as osteoporosis.