THE BIOLOGICAL ROLE OF HUMAN SKIN COLLANGENASE INHIBITOR

胶原酶催化胶原降解的限速步骤(7)。 人皮肤成纤维细胞合成胶原酶和一种特异性抑制物 这种酶(人皮肤成纤维细胞胶原酶抑制剂，HSFCI；[17])。 HSFCI已提纯至均一状态，并进行了生化表征 发展了定量免疫学(ELISA法)和功能分析(18，19)。 这项资助侧重于作用、生物合成和调节的机制。 HSFCI在结缔组织周转中的作用，以及它作为一种 过度胶原酶溶解障碍的治疗剂(例如， 类风湿性关节炎、牙周病和隐性营养不良 大疱性表皮松解症)。 HSFCI的作用机理将用凝胶过滤进行研究 缓蚀剂滴定的层析和Lineweaver-Burk分析。 抑制物在细胞培养中的产生及其胞内生物合成和 细胞外分泌，以及药物对其的调节将是 采用免疫学(ELISA法、免疫沉淀法)和 功能分析。这样的实验应该描绘出胶原酶 和HSFCI是协调调节的，也就是说，由相同的调节器调节。 将进行HSFCI的细胞和染色体定位研究， 前者利用双抗体标记和免疫荧光 技术，以及后者的体细胞杂交。这些研究是 旨在确定1)同一成纤维细胞是否合成了这两种细胞 胶原酶和抑制物，以及2)结构基因是否对 胶原酶和抑制物位于同一染色体上。的作用 将对HSFCI在疾病中的纤维化障碍进行调查 (硬皮病、吗啡、特发性肺纤维化)、类风湿 关节炎和RDEB。可以在血清中测量抑制物水平(正常 [HSFCI]=1.03+0.25微克/毫升)，皮肤或类风湿滑膜细胞培养， 和肺灌洗液中。据推测，血清抑制物源于 来自结缔组织来源，但由细胞成分产生 血液也必须进行检查。 一种免疫上与HSFCI相同的抑制剂是由大多数人类产生的 结缔组织(19例)。此外，这种蛋白质还抑制其他 结缔组织金属蛋白酶，如明胶酶和 蛋白聚糖酶(35)。因此，HSFCI可能在调节连接蛋白中起关键作用。 大多数器官的组织周转。这些考虑因素及其物理意义 对极端温度和pH的稳定性都增强了它的潜力 对胶原蛋白过度分解的紊乱有帮助。治疗性的 干预可以实现：1)通过调节 内源性抑制物产生，2)局部或关节内使用 纯抑制物，或3)通过外源给予活性抑制物 碎片。事实上，如果HSFCI被化学物质(CNBr)或 蛋白分解(胰酶)意味着产生能够抑制的多肽 胶原酶、其用途或合成衍生的类似物可提供 实现这些治疗目标的最佳途径。

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.