Collagenase catalyzes the rate-limiting step of collagen degradation (7). Human skin fibroblasts synthesize both collagenase and a specific inhibitor of this enzyme (human skin fibroblast collagenase inhibitor, HSFCI;[17]). HSFCI has been purified to homogeneity, biochemically characterized, and quantitative immunologic (ELISA) and functional assays developed (18, 19). This grant focuses on the mechanism of action, biosynthesis, and regulation of HSFCI, its role in connective tissue turnover, and its potential as a therapeutic agent in disorders of excessive collagenolysis (e.g., rheumatoid arthritis, periodontal disease, and recessive dystrophic epidermolysis bullosa [RDEB]). The mechanism of HSFCI's action will be studied using gel filtration chromatography and Lineweaver-Burk analysis of inhibitor titrations. Inhibitor production in cell culture, its intracellular biosynthesis and extracellular secretion, and its regulation by pharmacologic agents will be examined by employing both immunologic (ELISA, immunoprecipitation) and functional assays. Such experiments should delineate whether collagenase and HSFCI are regulated coordinately, that is, by the same modulators. Cellular and chromosomal localization studies of HSFCI will be performed, the former utilizing double antibody labeling and immunofluorescent techniques, and the latter somatic cell hybridizations. These studies are designed to determine 1) if the same fibroblast cell synthesizes both collagenase and inhibitor, and 2) whether the structural genes for collagenase and inhibitor are located on the same chromosome. The role of HSFCI in disease will be investigated for the fibrosing disorders (scleroderma, morphea, idiopathic pulmonary fibrosis), rheumatoid arthritis, and RDEB. Inhibitor levels can be measured in serum (normal [HSFCI]=1.03+0.25Mug/ml), in cell cultures of skin or rheumatoid synovium, and in pulmonary lavage fluids. Presumably, serum inhibitor is derived from connective tissue sources, but production by a cellular constituent of blood must also be examined. An immunologically identical inhibitor to HSFCI is produced by most human connective tissues (19). Furthermore, this protein also inhibits other connective tissue metalloproteases, such as gelatinases and proteoglycanases (35). Thus, HSFCI may be crucial in regulating connective tissue turnover in most organs. These considerations and its physical stability to extremes of temperature and pH all enhance its potential usefulness in disorders of excessive collagen breakdown. Therapeutic intervention could be achieved: 1) by pharmacologic agents which modulate endogeneous inhibitor production, 2) by topical or intra-articular use of pure inhibitor, or 3) by exogenous administration of an active inhibitory fragment. Indeed, if fragmentation of HSFCI by chemical (CNBr) or proteolytic (trypsin) means yields peptides capable of inhibiting collagenase, their use or synthetically-derived analogues may provide the best approach to realize these therapeutic goals.
