REGULATION OF OSTEOBLAST GENE EXPRESSION BY CYTOKINES

细胞因子对成骨细胞基因表达的调节

• 批准号: 6055586
• 负责人: JOHN R HARRISON
• 金额: $ 18.44万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-15 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6055586
• 关键词: 3T3 cells; DNA footprinting; collagen; gel mobility shift assay; gene deletion mutation; gene expression; genetic mapping; genetic promoter element; genetic regulatory element; genetically modified animals; interleukin 1; interleukin 6; laboratory mouse; molecular cloning; nuclear factor kappa beta; osteoblasts; prostaglandin endoperoxide synthase; site directed mutagenesis; transcription factor; western blottings

Recent evidence suggests that the inflammatory cytokine interleukin-J (IL-I) plays a key role in the pathogenesis of osteoporosis and in the bone loss which accompanies chronic inflammatory disease. We propose to determine the molecular mechanism by which this cytokine regulates transcription of three physiologically important target genes in osteoblastic cells, namely prostaglandin G/H synthase-2 (PGHS-2) and interleukin-6 (IL-6), which are up- regulated, and type I collagen (COL1Al), which is downregulated by IL-I. PGHS-2 is the key regulated enzyme in the conversion of arachidonic acid to biologically active prostanoids. IL-6 is a cytokine produced by osteoblasts which stimulates osteoclast differentiation and may play a role in post-menopausal osteoporosis. Type I collagen is the major structural component of bone matrix. The following specific aims are proposed: l)To examine the basal expression and regulation of NF-IL6, NF-kB and related transcription factors by IL-I in osteoblastic MC3T3-EI cells. Northern analysis will be performed in MC3T3-El cells and neonatal mouse calvariae to determine basal expression of individual members of the NF-IL6 and NF-kB multigene families, and the effects of IL-l on transcription factor mRNA levels will be determined. Protein levels will be assessed by Western blotting, Electrophoretic mobility shift analysis will be used to examine DNA- protein interactions at NF-IL6 and NP-kB sites in the PGHS-2, IL-6 and COLIAI promoters, 2) The function of NF-IL6 and NP-kB elements in the regulation of the PGHS-2, IL-6 and COLIAI genes will be analyzed by 5' deletion mapping, site directed mutagenesis of cis-acting elements, overexpression of trans-acting factors and dominant-negative regulators, and antisense approaches to inhibit the expression of specific transcription factors. 3) The mechanisms of COLIAI gene regulation by cytokines will be examined in the Co1CAT transgenic mouse model. Mapping of negative cytokine response elements in the COLIAI promoter will be performed by 5' deletion analysis, and by analysis of heterologous promoter constructs in which the osteoblast basal enhancer element is placed upstream of a basal enhancerless promoter. Elucidation of the mechanisms by which IL-I stimulates IL-6 and PGHS-2 expression while inhibiting type I collagen synthesis m osteoblasts should lead to a better understanding of the molecular basis for the bone loss which occurs in postmenopausal osteoporosis and chronic inflammatory disease states. In addition, these studies will contribute to our basic understanding of the molecular mechanisms involved in the transcriptional regulation of gene expression by cytokines via the NF-kB and NF-IL6 transcription factor families.

最近的证据表明，炎性细胞因子白细胞介素-J （IL-I）在骨质疏松的发病机理和 伴随慢性炎症性疾病的骨质流失。我们 提议确定该分子机制 细胞因子调节三种生理上重要的转录 成骨细胞中的靶基因，即前列腺素G/H 合成酶-2（PGHS-2）和白介素6（IL-6），它们是向上的 受调节和I型胶原蛋白（COL1AL），该胶原蛋白被下调 il-i。 PGHS-2是转化的关键调节酶 蛛网膜酸到生物活性前列腺素。 IL-6是一个 刺激破骨细胞的成骨细胞产生的细胞因子 分化并可能在绝经后骨质疏松症中发挥作用。 I型胶原蛋白是骨基质的主要结构成分。 提出了以下具体目标： l）检查NF-IL6，NF-KB的基础表达和调节 IL-1与成骨细胞MC3T3-EI中的相关转录因子 细胞。北方分析将在MC3T3-EL细胞中进行， 新生小鼠钙钙确定个体的基础表达 NF-IL6和NF-KB多基因家庭的成员，以及 IL-L对转录因子mRNA水平的影响将是 决定。蛋白质水平将通过蛋白质印迹评估， 电泳迁移率转移分析将用于检查DNA- PGHS-2，IL-6中NF-IL6和NP-KB位点的蛋白质相互作用 和Coliai启动子，2）NF-IL6和NP-KB的功能 PGHS-2，IL-6和COLIAI基因调节中的元素 将通过5'删除映射分析，位于站点的诱变 顺式作用元素，反式因子的过表达和 主导性调节剂和反义方法抑制 特定转录因子的表达。 3）机制 细胞因子调节的coliai基因将在 CO1CAT转基因小鼠模型。 阴性细胞因子的映射 Coliai启动子中的响应元素将通过5'执行 删除分析，并通过异源启动子进行分析 放置成骨细胞基础增强子元件的构造 基础增强子启动子的上游。阐明 IL-I刺激IL-6和PGHS-2表达的机制 虽然抑制I型胶原蛋白合成M成骨细胞应率领 更好地了解骨质流失的分子基础 这发生在绝经后骨质疏松症和慢性 炎症性疾病状态。此外，这些研究将有助于 为了我们对涉及的分子机制的基本理解 通过细胞因子对基因表达的转录调节 NF-KB和NF-IL6转录因子家族。