COLLAGEN GENE EXPRESSION IN CHONDROCYTES IN INFLAMMATION

软骨细胞能够参与自身的分解。 基质在炎症介质反应和修复中的作用 胶原蛋白网络受损。一种细胞培养模型已经被 人软骨细胞单层培养方法的建立 潜在细胞间介质作用的靶细胞 由可能调节软骨细胞的炎性细胞产生 合成软骨基质大分子等功能。 我们发现一种炎性介质，干扰素-伽马，是 T淋巴细胞，抑制II型胶原的合成以及 I型和III型胶原和纤维连接蛋白与细胞水平 软骨细胞中的前胶原mRNAs。白介素1，a 刺激PGE2合成的单核细胞产物和中性 蛋白水解酶，增加I型和III型胶原的合成 当前列腺素E_2合成时相关的前胶原mRNA水平 抑制胶原合成，被消炎痛阻断。在……里面 相反，IL-1抑制II型和III型前胶原水平 从而导致软骨特异性表型的丧失。这个 这项提案的目的是确定这些积极和 负信号控制胶原基因的表达。初步 有证据表明，这些效应中的一些在 转录水平。使用重组IL-1和IL-1制剂 干扰素-γ与培养的人软骨细胞及其他人 以组织细胞和小鼠细胞为靶点，控制胶原基因 表达的研究将通过：(1)核转录分析 前胶原mRNAs的体外及细胞内稳定性；(2)脱氧核糖核酸酶 用DNA进行超敏反应和S1核酸酶保护实验 含有胶原启动子和增强子序列的探针 基因；(3)顺式作用元件的瞬变分析 融合构建的5‘侧翼DNA的转染实验 (不同胶原基因的启动子增强子区偶联 报告基因，如氯霉素乙酰转移酶 (4)利用DNA分析潜在的反式作用因子- 蛋白质凝胶迁移率改变分析和/或DNA足迹 技巧。由于IL-1对II型和I型的作用相反 和III型胶原基因的表达，一个主要目标将是 确定相似或不同的反式作用因素和/或 其中涉及顺式作用因素。这些炎症介质可能 在体内诱导软骨基质的不适当修复 研究应该确定它们的影响是否是中介的 主要在转录或转录后水平。

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.