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RABBIT COLLAGENASE GENE REGULATION BY IL-1

IL-1 对兔胶原酶基因的调控

基本信息

批准号：
2732804
负责人：
ULRIKE BENBOW
金额：
$ 3.05万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-07-01 至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/2732804
关键词：
collagenase crosslink fibroblasts gene expression genetic regulation interleukin 1 laboratory rabbit synovial membrane tissue /cell culture

项目摘要

The long term goal of this research is to gain a detailed understanding of the molecular mechanisms that regulate collagenase (matrix metalloproteinase 1, MMP-1) expression in response to IL- 1. IL-1 is a potent physiological inducer of collagenase mRNA, and under pathological conditions collagenase, the only enzyme capable to degrade interstitial collagens at neutral pH, is instrumental in cartilage destruction in rheumatoid arthritis. Because of the central role IL-1 plays in the pathophysiology of connective tissue breakdown it is an important area of investigation. An understanding of the transcriptional and post-transcriptional mechanisms may influence the development of therapies designed to abrogate IL-1 induced collagenase gene expression. Therefore, the transcriptional and post- transcriptional mechanisms that regulate collagenase gene expression in response to IL-1 will be investigated using a rabbit model of synovial fibroblast cultures. Both experimentally and on the nucleotide sequence level this model mimics primary cultures of human cells from patients with rheumatoid arthritis. IL-1 responsive elements in the rabbit collagenase gene will be identified by transient transfection reporter constructs and deletional/mutational analysis. The dependency of these fragments on sequences in the proximal promoter, including AP-1 sites will be investigated. Specific DNA/protein interactions occurring on these elements will be investigated using electrophoretic mobility shift analysis, antibodies for specific transcription factors and UV crosslinking. RNA/protein interaction occurring at the 3' untranslated region of the collagenase mRNA will be determined by the ability of native or mutated AUUUA sequences to mediate binding of proteins from IL-1 treated and untreated cells.

这项研究的长期目标是获得一个详细的对胶原酶调控分子机制的认识 (基质金属蛋白酶1，MMP-1)表达对IL-1的反应。 IL-1是胶原酶信使核糖核酸的有效生理诱导剂。病理条件下，胶原酶是唯一能够在中性pH下降解间质胶原蛋白，有助于类风湿性关节炎的软骨破坏。因为中央 IL-1在结缔组织破裂的病理生理学中的作用这是一个重要的调查领域。对这一概念的理解转录和转录后机制可能会影响旨在消除IL-1诱导的治疗方法的发展胶原酶基因的表达。因此，转录和后转录- 胶原酶基因表达调控的转录机制对IL-1的反应将使用兔模型进行研究滑膜成纤维细胞培养。无论是在实验上还是在核苷酸序列水平这个模型模拟了人类的原代培养来自类风湿性关节炎患者的细胞。IL-1应答兔胶原酶基因中的元件将通过瞬时转染报告基因的构建与缺失/突变分析。这些片段对序列的依赖性近端启动子，包括AP-1位点将被调查。在这些元素上发生的特定DNA/蛋白质相互作用将是使用电泳迁移率变化分析进行研究，针对特定转录因子和紫外线交联物的抗体。 RNA3‘端非翻译区与蛋白质的相互作用胶原酶的mR-NA将由天然的或天然的突变的AUUA序列介导IL-1蛋白的结合处理过的和未处理过的细胞。