The turnover of extracellular matrix is crucial for maintaining structural integrity of connective tissues, and aberrations in this process can lead to a variety of pathologic states. The activity of a group of neutral metalloproteinases, including interstitial collagenase, stromelysin, 72 kD type IV collagenase, and 92 kD gelatinase/type V collagenase is believed to control the turnover of extracellular matrix components. Tissue inhibitor of metalloproteinases, or TIMP, functions as the major, and probably, only, inhibitor of this entire family of neutral metalloproteinases within the interstitial spaces of connective tissues. The broad objective of this grant proposal is to study the biochemical mechanisms of action, molecular regulation, and in situ production of TIMP during normal matrix turnover and in disease states. Studies of the biochemical mechanisms of TIMP action will encompass several areas, including its capacity to complex zymogen versus active forms of the various metalloproteases, as assessed HPLC gel-filtration techniques. Furthermore, affinities between TIMP and the different enzymes will be determined using cross-linking reagents or by kinetic analyses against highly susceptible substrates. Localization of the structural sites in TIMP and metalloproteinases that are required for enzyme-inhibitor binding is a major priority and will be approached using covalent cross-linking reagents that can be iodinated, with transfer of the label to a second protein, and subsequent isolation/sequencing of the iodinated fragment(s). Studies of regulatory mechanisms will investigate the role of cell- matrix interactions in TIMP and collagenase biosynthesis and the consequences to matrix degradation. Cytokines and biologic factors such as gamma-interferon and LPS will be studied for capacity to regulate TIMP and collagenase expression at protein and mRNA levels. Retinoids will be further investigated with regards to potential transcriptional mechanisms of their effects. In situ hybridization will be performed on isolated cells and whole tissues specimens to gain insights in the cell types actively synthesizing TIMP and collagenase during normal connective tissue turnover and in disease processes. The cellular expression of these proteins during fetal development, actinic injury and in connective tissue diseases, and epidermolysis bullosa will be studied.
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