HORMONE REGULATION OF BONE COLLAGEN SYNTHESIS

骨胶原合成的激素调节

• 批准号: 2457946
• 负责人: Alexander C Lichtler
• 金额: $ 21.2万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2457946
• 关键词: collagen; complementary DNA; gene induction /repression; gene mutation; genetic promoter element; genetically modified animals; hormone regulation /control mechanism; in situ hybridization; laboratory mouse; molecular cloning; nucleic acid probes; nucleic acid sequence; osteoblasts; protein biosynthesis; recombinant proteins; transcription factor; vitamin D; vitamin D receptors; yeasts

Type 1 collagen is the major structural protein in bone, and it's high level synthesis is an important characteristic of the osteoblast phenotype. We have evidence that transcription of the alpha1 (I) collagen gene is regulated differently in osteoblasts than in other collagen producing cell types. We believe that by investigating the DNA sequences and transcription factors that are involved in regulating the gene in osteoblasts that we will achieve a greater fundamental understanding of osteoblast differentiation, which is critical to our understanding of many bone disorders. The specific aims of this grant are: A. To functionally characterize the DNA sequences and clone the proteins which bind to a 49 bp region between -1719 and -1670 bp in the rat alpha1(1) collagen gene which has been shown to be necessary for significant expression of the gene in bone. This will be carried out by analysis of site specific mutations in transgenic animals, analysis of DNA-protein interactions using gel mobility shifts, cloning transcription factors, and analyzing their functions using expression vectors and antisense nucleic acids. B. To evaluate the possible role of the region between -2297 bp and -3521 bp of the alpha1(1) promoter in transcription during normal bone development using immunohistochemistry and in situ hybridization. If we find that this region plays a role in transcription of the gene during some phase of the osteoblast life cycle, we will analyze the DNA sequences and transcription factors responsible for this regulation by in vitro analysis of DNA-protein interactions, in vitro mutagenesis, transfection into cultured bone cells, and analysis of select mutations in transgenic mice. C. To determine the regions of the alpha1(1) gene necessary to direct high level transcription in bone and other collagen producing cells in transgenic animals and by transfection of cultured cells. D. The second basic goal of this grant and the fourth specific aim is to determine the mechanism of the 1,25 dihydroxyvitamin D (vitamin D) inhibition of the alpha1(1) gene in bone cells. This will be accomplished by testing mutated promoter constructs for vitamin D response in transfected cells and transgenic mice, by analyzing the binding of the vitamin D receptor to fragments of the alpha1(I) promoter, and by evaluating possible interactions of the vitamin D receptor with factors involved in basal transcription of the gene.

I型胶原是骨骼中的主要结构蛋白，它的含量很高 水平合成是成骨细胞的重要特征 表型。我们有证据表明α1(I)胶原的转录 基因在成骨细胞中的调控与在其他胶原中不同 产生细胞类型。我们相信通过研究DNA序列 以及参与调节该基因的转录因子 我们将对成骨细胞有更基本的了解 成骨细胞分化，这对我们理解许多 骨病。这笔赠款的具体目的是：a.在功能上 鉴定DNA序列并克隆与49结合的蛋白质 大鼠α1(1)胶原基因-1719~-1670bp之间的碱基区 它已被证明是有意义地表达 骨骼中的基因。这将通过分析现场具体情况来执行 转基因动物的突变，DNA-蛋白质相互作用的分析 利用凝胶迁移率变化，克隆转录因子，并分析 它们的功能使用表达载体和反义核酸。胡麻B. 以评估-2297 BP和-3521 BP之间区域的可能作用 α1(1)启动子在正常骨发育过程中转录的变化 采用免疫组织化学和原位杂交技术。如果我们发现 这一区域在基因转录的某些阶段起作用。 成骨细胞的生命周期，我们将分析DNA序列和 体外分析与此调控相关的转录因子 DNA-蛋白质相互作用，体外诱变，转染人 培养的骨细胞，以及转基因小鼠的选择突变分析。 C.确定alpha1(1)基因必需的区域，以指导高 在骨骼和其他胶原产生细胞中的转录水平 转基因动物和通过对培养细胞的转基因。D.第二次 这笔赠款的基本目标和第四个具体目标是确定 1，25-二羟基维生素D(维生素D)抑制小鼠视网膜色素变性的机制 骨细胞中的α1(1)基因。这将通过测试来实现 转基因细胞中维生素D反应的突变启动子构建 和转基因小鼠，通过分析维生素D受体与 α1(I)启动子片段，并通过评估可能的 维生素D受体与碱性磷酸酶相关因子的相互作用 基因的转录。