ACTIVATION OF COLLAGENASE IN RHEUMATOID ARTHRITIS

描述：(改编自申请人的摘要)-类风湿性关节炎 以慢性炎症和关节退变为特征。这 关节的侵蚀可能是由于细胞外基质失衡所致。 合成和代谢，部分是由基质的成员诱导的 金属蛋白酶(MMPs)家族。MMPs是锌依赖的内肽酶 能够降解细胞外的大部分成分 矩阵。在滑液和滑液中都发现了活性胶原酶。 类风湿性关节炎患者的组织提取物以及 细胞因子刺激的滑膜成纤维细胞的条件培养液。它有 一直认为胶原酶的这种激活是通过 纤溶酶原级联和/或涉及基质分解酶-1。然而，激活 胶原酶途径是胶原蛋白降解的关键步骤，目前仍 人们对此仍然知之甚少。这项研究的总体目标是确定和 成纤维细胞型胶原酶激活方式的表征 (胶原酶-1)通过滑膜成纤维细胞并操纵其激活来 确定对这一过程的控制是否有助于治疗 类风湿关节炎。提出了以下具体目标： 实现这些目标：特定目标1-人滑膜成纤维细胞 能够调解破坏重构矩阵的线，包括 将对I型胶原纤维进行鉴定和表征。胶原蛋白 由这些细胞引发的降解将通过添加 胶原酶-1的抑制性抗体证明这种胶原 降解依赖于胶原酶-1。活化过程中的中间体 胶原酶-1将由α2-巨球蛋白鉴定和鉴定 和TIMP(金属蛋白酶组织抑制物)捕获技术 因为它们在荧光马来酰亚胺中的反应性。为了确定 加工地点，氨基末端测序将使用 通过抗体亲和层析或通过 免疫沉淀。 具体目标2--调查人员建议区分角色 基质分解素和其他MMPs通过以下途径参与胶原酶-1的激活 通过添加针对每个滑膜成纤维细胞的抑制性抗体 酵素。可以包括针对TIMP-1的抑制性抗体以提供 酶与抑制剂的失衡，可能导致酶的激活 胶原酶-1。将确定激活中间体，并 特色化的。 特定目标3-一旦鉴定出滑膜成纤维细胞系 激活胶原酶-1，不依赖于基质分解蛋白-1和其他MMPs，它将 用来确定负责的机制(S)或因素(S) CL-1激活。这一具体目标的可行性是有限的， 取决于确定符合这些条件的滑膜成纤维细胞系 标准。该项目的长期目标是识别药物或其他 能阻断细胞激活机制的试剂(抗体) 胶原酶-1和其他MMPs，以防止连续或 关节间歇性破坏，如类风湿性关节炎。

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.