ACTIVATION OF COLLAGENASE IN RHEUMATOID ARTHRITIS

描述：（改编自申请人摘要）-风湿性关节炎 以慢性炎症和关节退化为特征。 这 关节的侵蚀可能是由于细胞外基质的不平衡 部分由基质成员诱导的合成和代谢 金属蛋白酶（MMP）家族。 MMPs是锌依赖性内肽酶 能够降解细胞外的大部分成分， 矩阵 已在滑液和关节液中发现了活性胶原酶 类风湿性关节炎患者的组织提取物以及 条件培养基来自于马槟榔碱刺激的滑膜成纤维细胞。 它有 有人认为，胶原酶的这种激活是通过 纤溶酶原级联和/或涉及溶基质素-1。 然而，激活 胶原酶途径是胶原降解的关键步骤， 仍然知之甚少。 本研究的总体目标是确定和 表征成纤维细胞型胶原酶的活化模式 （胶原酶-1）的滑膜成纤维细胞，并操纵其激活， 确定控制这一过程是否有利于治疗 类风湿关节炎 提出了以下具体目标， 具体目标1 -人滑膜成纤维细胞 能够介导重构基质破坏的细胞系， I型胶原原纤维将被鉴定和表征。 胶原 由这些细胞引发的降解将通过添加 胶原酶-1的抑制性抗体，以证明这种胶原 降解依赖于胶原酶-1。 激活的中间体 将通过α 2-巨球蛋白鉴定和表征胶原酶-1 以及TIMP（金属蛋白酶组织抑制剂）捕获技术 如通过它们在荧光马来酰亚胺中的反应性。 为了确定 处理位点，氨基末端测序将使用 通过抗体亲和色谱法或 免疫沉淀。 具体目标2 -调查人员建议区分 基质溶解素和其它MMPs在胶原酶-1的活化中起作用， 滑膜成纤维细胞，通过添加针对每个细胞制备的抑制性抗体， 酵素 可以包括TIMP-1的抑制性抗体以提供抗TIMP-1的抗体。 酶对抑制剂的不平衡，这可能导致 胶原酶-1。 将识别活化中间体， 表征了 具体目标3 -一旦鉴定出滑膜成纤维细胞系， 不依赖于基质溶解素-1和其他MMP激活胶原酶-1， 用于确定造成以下情况的机制或因素： CL-1激活。 这一具体目标的可行性有限， 取决于确定一个滑膜成纤维细胞系， 的搜索. 该项目的长期目标是确定药物或其他 可以阻断细胞激活机制的试剂（抗体） 胶原酶-1和其他MMPs，以防止持续或 关节的间歇性破坏，如在类风湿性关节炎中所见。

DESCRIPTION: (Adapted from the applicant's abstract) - Rheumatoid arthritis is characterized by chronic inflammation and joint degeneration. This erosion of the joint is likely due to an imbalance of extracellular matrix synthesis and metabolism induced, in part, by members of the matrix metalloproteinase (MMP) family. The MMPs are zinc-dependent endopeptidases that are capable of degrading most of the components of the extracellular matrix. Active collagenase has been identified in the synovial fluid and in tissue extracts from patients with rheumatoid arthritis as well as in conditioned media from cytokine-stimulated synovial fibroblasts. It has been suggested that this activation of collagenase was initiated via the plasminogen cascade and/or involves stromelysin-1. However, the activation pathway of collagenase is a pivotal step in collagen degradation that still remains poorly understood. The overall aim of this study is to identify and characterize the mode of activation of fibroblast-type collagenase (collagenase-1) by synovial fibroblasts and manipulate its activation to determine if control of this process could be beneficial in the treatment of rheumatoid arthritis. The following specific aims are proposed to accomplish these goals: Specific aim 1 - Human synovial fibroblast cell lines capable of mediating destruction of a reconstituted matrix consisting of type I collagen fibrils will be identified and characterized. Collagen degradation initiated by these cells will be blocked by addition of inhibitory antibodies to collagenase-1 to demonstrate that this collagen degradation is collagenase -1 dependent. Intermediates in the activation of collagenase-1 will be identified and characterized by alpha2-macroglobulin and TIMP (tissue inhibitor of metalloproteinases) capture techniques as well as by their reactivity in a fluorescent maleimide. In order to determine processing sites, amino-terminal sequencing will be carried out using activation intermediates purified by antibody affinity chromatography or by immunoprecipitation. Specific aim 2 - The investigators propose to distinguish the roles stromelysin and other MMPs play in the activation of collagenase-1 by synovial fibroblasts through addition of inhibitory antibodies made to each enzyme. Inhibitory antibodies to TIMP-1 may be included to provide an imbalance of enzymes to inhibitors which might lead to the activation of collagenase-1. Activation intermediates will be identified and characterized. Specific aim 3 - Once a synovial fibroblast cell line is identified that activates collagenase-1 independent of stromelysin-1 and other MMPs, it will be used to identify the mechanism(s) or factor(s) that are responsible for CL-1 activation. The feasibility of this specific aim is limited and depends on identifying a synovial fibroblast cell line that meets these criteria. The long-term goal of this project is to identify drugs or other reagents (antibodies) that can block the mechanism by which cells activate collagenase-1 and the other MMPs in order to prevent the continuous or intermittent destruction of the joints as seen in rheumatoid arthritis.