- 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.
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