THE BIOLOGICAL ROLE OF HUMAN SKIN COLLAGENASE INHIBITOR

人类皮肤胶原酶抑制剂的生物学作用

• 批准号: 3157366
• 负责人: HOWARD G WELGUS
• 金额: $ 8.64万
• 依托单位: JEWISH HOSPITAL OF SAINT LOUIS
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1989-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3157366
• 关键词: autosomal recessive trait; chemical structure function; collagenase; enzyme inhibitors; enzyme linked immunosorbent assay; epidermolysis bullosa; fibroblasts; gel filtration chromatography; laboratory rabbit; periodontium disorder; pulmonary fibrosis /granuloma; rheumatoid arthritis; scleroderma; skin; synovial membrane; tissue /cell culture

Collagenase catalyzes the rate-limiting step of collagen degradation. Human skin fibroblasts synthesize both collagenase and a specific inhibitor of this enzyme (human skin fibroblast collagenase inhibitor, HSFCI). HSFCI has been purified to homogeneity, biochemically characterized, and quantitative immunologic (ELISA) and functional assays developed. 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 epidermoloysis 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 plus and minus 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. Furthermore, this protein also inhibits other connective tissue metalloproteases, such as gelatinases and proteoglycanases. 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.

胶原酶催化胶原降解的限速步骤。 人皮肤成纤维细胞合成胶原酶和一种特异性抑制剂 这种酶（人皮肤成纤维细胞胶原酶抑制剂，HSFCI）。 HSFCI 已被纯化至同质，生物化学特征， 开发了定量免疫学（ELISA）和功能测定。 这 格兰特的重点是作用机制，生物合成和调节， HSFCI，其在结缔组织转换中的作用，及其作为一种免疫调节剂的潜力， 过度胶原溶解病症的治疗剂（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[RDEB]）。 HSFCI的作用机制将使用凝胶过滤进行研究 抑制剂滴定的色谱和Lineweaver-Burk分析。 细胞培养中抑制剂的产生、其细胞内生物合成和 细胞外分泌，以及药理学试剂对其的调节将是 采用免疫学（ELISA，免疫沉淀）和 功能测定 这样的实验应该能确定胶原酶 和HSFCI是协调调节的，即，由相同的调节剂调节。 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 设计来确定1）如果相同的成纤维细胞合成两种 胶原酶和抑制剂，和2）是否结构基因， 胶原酶和抑制剂位于同一染色体上。 的作用 将研究疾病中的HSFCI的纤维化疾病 （硬皮病、硬斑病、特发性肺纤维化）、类风湿 关节炎和RDEB。 可在血清中测量抑制剂水平（正常 [HSFCI] = 1.03 ± 0.25 μ g/ml），在皮肤或 类风湿性滑膜和肺灌洗液中。 据推测，血清 抑制剂来源于结缔组织来源，但由 还必须检查血液的细胞成分。 HSFCI的免疫学上相同的抑制剂由大多数人产生。 结缔组织 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 因此，HSFCI可能在调节连接蛋白中起关键作用。 在大多数器官中的组织更新。 这些考虑及其物理 对极端温度和pH值的稳定性都增强了其潜力 可用于过度胶原分解的疾病。 治疗 可以实现干预：1）通过调节 内源性抑制剂产生，2）通过局部或关节内使用 纯抑制剂，或3）通过外源性施用活性抑制剂 碎片 事实上，如果HSFCI通过化学（CNBr）或 蛋白水解（胰蛋白酶）手段产生能够抑制 胶原酶、它们的用途或合成衍生的类似物可以提供 实现这些治疗目标的最佳方法。