REGULATION OF COLLAGENASE 3 GENE EXPRESSION BY CYTOKINES

本提案的总体目标是研究麻黄碱介导的 调节胶原酶-3基因表达，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（ OA）。OA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。的 透明关节软骨的破坏是OA的标志， 禁用RA。虽然各种治疗方案均可引起症状， 缓解，没有任何方案被证明可以延缓关节炎的进展。 软骨退化在疾病中，要么是正常的 软骨细胞的功能或在这些细胞的组成性无能， 使修复速度与 矩阵各种细胞因子和炎症介质已被证明是 要么扰乱软骨细胞的合成功能， 通过调节各种基质降解， 酶，包括胶原酶。胶原酶-3特异性地 在包括软骨细胞在内的骨骼细胞中表达， 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。它的表达方式是 对IL-1和其他炎性细胞因子的反应。它可以发挥一个 在生理性骨骼重塑和破坏中的重要作用 软骨病胶原酶-3基因最近已被克隆，但 各种细胞因子在转录调控中的作用， 基因尚待阐明。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体 (CAT)含有胶原酶-3启动子和转染的永生化人 细胞系，并分析所选细胞因子/配体（例如IL-2）的作用。 1 β、TNF-α和TGF-1 β。顺式元件和反式作用因子 也将使用转染和DNA结合测定来表征。 我们还将培育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和作用 胶原酶在体内关节炎发展中的作用。转基因小鼠将是 用IL-1 ra、TNFR 1-IgG 1融合蛋白和地塞米松治疗， 诱导关节炎和细胞因子/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展中的作用将是 阐明。这一建议将使人们深入了解 通过细胞因子参与胶原酶-3的表达，从而可以 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.