COLLAGEN GENE EXPRESSION IN CHONDROCYTES IN INFLAMMATION

炎症中软骨细胞中胶原蛋白基因的表达

• 批准号: 3156827
• 负责人: MARY B GOLDRING
• 金额: $ 15.25万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-08-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3156827
• 关键词: DNA binding protein; DNA footprinting; articular cartilage; cartilage metabolism; chondrocytes; collagen; connective tissue metabolism; deoxyribonuclease I; epidermal growth factor; genetic manipulation; genetic promoter element; genetic transcription; human tissue; immunomodulators; indomethacin; inflammation; interferons; interleukin 1; laboratory mouse; messenger RNA; nucleic acid probes; plasmids; procollagen; prostaglandin E; protein biosynthesis; tissue /cell culture; transcription factor; transfection

Chondrocytes are capable of participating in breakdown of their own matrix in response to inflammatory mediators and in repair of the damaged collagen network. A cell culture model has been established employing human chondrocytes in monolayer culture as target cells for the effects of potential intercellular mediators produced by inflammatory cells which might modulate chondrocyte functions such as synthesis of a cartilage matrix macromolecules. We found that one inflammatory mediator, IFN-gamma, a product of T-lymphocytes, suppresses synthesis of type II collagen as well as types I and III collagens and fibronectin and levels of cellular procollagen mRNAs in chondrocytes. Interleukin 1 (IL-1), a monocyte product which stimulates the synthesis of PGE2 and neutral proteases, increases synthesis of types I and III collagens and associated procollagen mRNA levels when the synthesis of PGE2 which inhibits collagen synthesis, is blocked by indomethacin. In contrast, IL-1 suppresses levels of type II and type IX procollagen mRNAs thereby inducing loss of cartilage-specific phenotype. The aim of this proposal is to determine how these positive and negative signals control collagen gene expression. Preliminary evidence indicates that some of these effects are modulated at the transcriptional level. Using recombinant preparations of IL-1 and IFN- gamma and cultured human chondrocytes as well as other human tissue cells and mouse cells as targets, control of collagen gene expression will be studied by: (1) assays of nuclear transcription in vitro and stability in cellulo of procollagen mRNAs; (2) DNAase I hypersensitivity and S1 nuclease protection experiments using DNA probes containing promoter and enhancer sequences of collagen genes; (3) analysis of cis-acting elements by transient transfection experiments using fusion construct of 5'flanking DNA (promoter + enhancer regions of different collagen genes coupled to a reporter gene such as chloramphenicol acetyl transferase (CAT); (4) analysis of potential trans-acting factors by DNA- protein gel mobility shift assay and/or DNA footprinting techniques. Since IL-1 has opposite effects on type II vs type I and type III collagen gene expression, a major goal will be to determine whether similar or different trans-acting factors and/or cis-acting elements are involved. These inflammatory mediators may induce inappropriate repair of cartilage matrix in vivo and these studies should determine whether their effects are mediated primarily at the transcriptional or post-transcriptional level.

软骨细胞能够参与自身的分解， 基质对炎症介质的反应和修复 胶原蛋白网络受损。 一种细胞培养模型， 采用单层培养的人软骨细胞建立， 潜在细胞间介质作用的靶细胞 由炎症细胞产生，可能调节软骨细胞 功能如软骨基质大分子的合成。 我们发现一种炎症介质IFN-γ， T淋巴细胞，抑制II型胶原蛋白的合成， I型和III型胶原蛋白和纤连蛋白以及细胞水平 软骨细胞中的前胶原mRNA。 白细胞介素1（IL-1），a 单核细胞产物，其刺激PGE 2和中性粒细胞的合成。 蛋白酶，增加I型和III型胶原蛋白的合成， 当PGE 2合成时， 抑制胶原蛋白的合成，被吲哚美辛阻断。 在 相反，IL-1抑制II型和IX型前胶原的水平， mRNA，从而诱导软骨特异性表型的丧失。 的 本提案的目的是确定这些积极和 负信号控制胶原基因表达。 初步 有证据表明，这些影响中的一些是在 转录水平。 使用IL-1的重组制剂和 IFN-γ和培养的人软骨细胞以及其他人软骨细胞 组织细胞和小鼠细胞为靶点，控制胶原基因 表达将通过以下方法研究：（1）核转录测定 前胶原mRNA体外和细胞内稳定性研究（2）DNA酶 使用DNA的I超敏反应和S1核酸酶保护实验 含有胶原蛋白启动子和增强子序列的探针 （3）顺式作用元件的瞬时分析 使用5 ′侧翼DNA的融合构建体的转染实验 （不同胶原蛋白基因的启动子+增强子区偶联， 与报告基因如氯霉素乙酰转移酶 (CAT)（4）利用DNA-PCR技术分析潜在的反式作用因子。 蛋白质凝胶迁移率变动分析和/或DNA足迹法 技术. 由于IL-1对II型与I型具有相反的作用， 和III型胶原基因表达，一个主要目标将是 确定是否相似或不同的反式作用因子和/或 涉及顺式作用元件。 这些炎症介质可能 诱导体内软骨基质不适当修复， 研究应该确定它们的影响是否是通过 主要是在转录或转录后水平。