REGULATION OF COLLAGENASE 3 GENE EXPRESSION BY CYTOKINES

细胞因子对胶原酶 3 基因表达的调节

• 批准号: 6137295
• 负责人: MAHBOOB U RAHMAN
• 金额: $ 12.46万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-14 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6137295
• 关键词: DNA footprinting; articular cartilage; collagenase; computer assisted sequence analysis; cytokine; enzyme activity; gel mobility shift assay; gene expression; genetic regulation; genetic regulatory element; genetically modified animals; human genetic material tag; human tissue; interleukin 1; laboratory mouse; molecular cloning; osteoarthritis; pathologic process; rheumatoid arthritis; southern blotting; transcription factor; transfection; transforming growth factors; tumor necrosis factor alpha; western blottings; yeast two hybrid system

The overall goal of this proposal is to study the cytokine-mediated regulation of collagenase-3 gene expression in order to better understand the mechanisms involved in the degradation of articular cartilage in rheumatic diseases such as rheumatoid arthritis (RA) and osteoarthritis ( OA). OA is the most common form of joint disease and is second only to cardiovascular disease as a cause of early retirement and disability. The destruction of hyaline articular cartilage is the hallmark of OA and disabling RA. Although various therapeutic regiment can cause symptom relief, no regiment has been proven to retard progression of articular cartilage degradation. In disease there is either a suppression of normal chondrocyte functions or in the constitutive inability of these cells to match the rate of repair with the increased rate of degradation of the matrix. Various cytokines and inflammatory mediators have been shown to either derange the synthetic functions of the chondrocytes of increase cartilage matrix catabolism by regulating various matrix-degrading enzymes, including the collagenases. Collagenase-3 is specifically expressed in skeletal cells including chondrocytes and has been shown to have an additional cleavage site when compared to other collagenases. It has aggrecanase and gelatinase activity as well. It's expression is response to IL-1 and other inflammatory cytokines. Thus it may play a significant role in physiological skeletal remodeling and destruction of cartilage in disease. The collagenase-3 gene has been recently cloned, but the role of various cytokines in the transcriptional regulation of this gene is yet to be elucidated. We have cloned the collagenase-3 promoter from a human genomic DNA library. We will prepare reporter gene constructs (CAT) containing collagenase-3 promoter and transfect immortalized human cell lines and analyze the effects of selected cytokines/ligands e.g. IL- 1beta, TNF-alpha and TGF1-beta. The cis-elements and trans-acting factors will also be characterized employing transfection and DNA-binding assays. We will also develop transgenic mice containing the collagenase promoter- beta-galactosidase fusion gene to analyze the expression and role of collagenase in development of arthritis in vivo. Transgenic mice will be treated with IL-1ra, TNFR1-IgG1 fusion protein, and dexamethasone after induction of arthritis and the role of cytokines/ligands in the control of expression of collagenase and its role in development of arthritis will be elucidated. This proposal will provide insight into the mechanisms involved in the expression of collagenase-3 by cytokines and thereby may provide targets for developing novel therapeutic measures to inhibit cartilage destruction in joint disease.

该提案的总体目标是研究细胞因子介导的 胶原酶 3 基因表达的调控，以便更好地了解 关节软骨退化的机制 风湿性疾病，如类风湿性关节炎 (RA) 和骨关节炎 ( 办公自动化）。 OA是最常见的关节疾病，仅次于 心血管疾病是提前退休和残疾的一个原因。这 透明关节软骨的破坏是 OA 的标志 禁用 RA。尽管各种治疗方案都会引起症状 缓解，没有任何药物被证明可以延缓关节病的进展 软骨退化。在疾病中，要么正常的抑制 软骨细胞功能或这些细胞的组成性无能力 将修复率与降解率的增加相匹配 矩阵。多种细胞因子和炎症介质已被证明 要么扰乱增加的软骨细胞的合成功能 通过调节各种基质降解来促进软骨基质分解代谢 酶，包括胶原酶。胶原酶-3 特别是 在包括软骨细胞在内的骨骼细胞中表达，并已被证明 与其他胶原酶相比，具有额外的切割位点。它 还具有聚集蛋白聚糖酶和明胶酶活性。其表达式为 对 IL-1 和其他炎症细胞因子的反应。因此它可以起到 在生理性骨骼重塑和破坏中发挥重要作用 软骨处于疾病状态。胶原酶 3 基因最近已被克隆，但是 各种细胞因子在转录调控中的作用 基因尚未阐明。我们克隆了胶原酶3启动子 来自人类基因组 DNA 文库。我们将准备报告基因构建体 (CAT)含有胶原酶3启动子并转染永生化人类 细胞系并分析所选细胞因子/配体的影响，例如白细胞介素- 1β、TNF-α 和 TGF1-β。顺式作用因子和反式作用因子 还将采用转染和 DNA 结合测定来表征。 我们还将开发含有胶原酶启动子的转基因小鼠- β-半乳糖苷酶融合基因分析表达及作用 体内关节炎发展中的胶原酶。转基因小鼠将 治疗后用IL-1ra、TNFR1-IgG1融合蛋白和地塞米松治疗 关节炎的诱导以及细胞因子/配体在控制中的作用 胶原酶的表达及其在关节炎发展中的作用 阐明了。该提案将提供对机制的深入了解 通过细胞因子参与胶原酶 3 的表达，从而可能 为开发新的治疗措施来抑制 关节疾病中的软骨破坏。