REGULATION OF COLLAGENASE 3 GENE EXPRESSION BY CYTOKINES

这项建议的总体目标是研究细胞因子介导的 为了更好地了解胶原酶-3基因的表达 关节软骨退变的机制研究进展 风湿性疾病，如类风湿性关节炎(RA)和骨关节炎( 办公自动化)。骨性关节炎是最常见的关节疾病，仅次于 心血管疾病是导致提前退休和残疾的原因之一。这个 透明关节软骨破坏是骨性关节炎和骨性关节炎的特征。 禁用RA。虽然各种治疗方案都会引起症状 缓解，没有一个团被证明可以延缓关节的进展 软骨退化。在疾病中，要么是正常的抑制 软骨细胞的功能或在这些细胞的结构性能力 将修复速度与增加的降解率相匹配 矩阵。各种细胞因子和炎症介质已经被证明可以 要么破坏软骨细胞的合成功能，要么增加 调控多种基质降解作用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，已被证明 与其他胶原酶相比，有一个额外的裂解位点。它 还具有聚集葡聚糖酶和明胶酶活性。它的表达是 对IL-1和其他炎性细胞因子的反应。因此，它可能会扮演一个 在生理性骨骼重塑和破坏中的重要作用 疾病中的软骨。胶原酶-3基因最近被克隆，但 多种细胞因子在转录调控中的作用 基因还没有被阐明。我们克隆了胶原酶-3启动子 从人类基因组DNA文库中。我们将准备报告基因构建 含胶原酶-3启动子的(CAT)基因转染永生化人 并分析选定的细胞因子/配体的作用，例如IL-2 1β、肿瘤坏死因子-α和肿瘤坏死因子-1-β。顺式元件和反式作用因素 也将使用转基因和DNA结合分析对其进行表征。 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的表达及作用分析 胶原酶在体内关节炎发生发展中的作用。转基因小鼠将成为 IL-1ra、TNFR1-IgG1融合蛋白联合地塞米松治疗 关节炎的诱导和细胞因子/配体在控制关节炎中的作用 胶原酶的表达及其在关节炎发生发展中的作用 已澄清。这项提议将提供对这些机制的洞察 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提供靶点 关节疾病中的软骨破坏。

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.