MOLECULAR MECHANISMS OF COLLAGEN DEGRADATION BY ORAL MUCOSAL FIBROBLASTS

口腔粘膜成纤维细胞降解胶原蛋白的分子机制

• 批准号: 6296242
• 负责人: HENNING BIRKEDAL-HANSEN
• 金额: $ 4.21万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6296242
• 关键词: antisense nucleic acid; blocking antibody; collagen; collagenase; enzyme activity; enzyme induction /repression; enzyme mechanism; fibroblasts; gene expression; human tissue; in situ hybridization; laboratory mouse; oral mucosa; phenotype; polymerase chain reaction; protein degradation; protein sequence; stromelysin; tissue inhibitor of metalloproteinases; western blottings; zymogens

Project 3 will investigate the enzymatic mechanisms used by oral mucosal fibroblasts to degrade interstitial collagen fibrils. The metabolic degradation of interstitial collagen fibrils by stromal cells may involve as many as five different gene products, three matrix metalloproteinases, fibroblast-type collagenase (F-CL), stromelysin-1 (SL-1) and Mr 72K gelatinase (GL), and two endogenous inhibitors, TIMP-1 and TIMP-2. F-CL plays a pivotal role in this process because of its ability to cleave native collagen molecules at catalytically meaningful rates and thereby initiate the dissolution of the collagen fibril network. We propose to investigate the peri- and extracellular molecular reactions that enable human oral mucosal fibroblasts to degrade interstitial collagen fibrils. We shall initially generate a library of human oral mucosal fibroblast clones to determine whether natural differences in collagen-degrading capacity are encoded in expression of particular combinations of metalloproteinase and inhibitor genes. Collagen-degrading ability will be determined by monitoring the dissolution of reconstituted fibrils of type I collagen by live cells. These studies will be extended by selective induction or abrogation of expression of targeted genes by growth factors and cytokines, and by antisense deoxyoligonucleotides. In addition, the role of individual gene products will be assessed by use of function-perturbing antibodies that block enzymatic or inhibitory activities. The primary focus will be on the pericellular activation of proCL because this step is rate-limiting in the cell-mediated dissolution of collagen fibrils. The roles played by plasminogen, SL-1 and TIMPs in collagen degradation in general and in activation of proCL in particular will be examined. We speculate that pericellular proteolysis mediated by live cells seeded in contact with a solid phase substrate involves three distinct compartments, the medium, the cell surface and the matrix surface. We will deduce the component molecular reactions in each of the three peri-cellular compartments based on identification and tracking of intermediates and products. Reaction products formed during the process will be isolated by alpha2M- and TIMP-capture techniques and identified either by NH2-terminal sequence analysis or by Western analysis using sequence-specific antibodies. the fate of fragments of collagen molecules or alpha-chains released from the fibril network will be monitored to determine whether the Mr 72K GL which is constitutively expressed by the cells plays a role in this process and whether the degradative process involves specific surface binding, internalization and phagolysosomal degradation of collagen chain fragments.

项目3将研究口腔粘膜的酶机制， 成纤维细胞降解间质胶原纤维。 代谢 基质细胞降解间质胶原纤维可能涉及 多达五种不同的基因产物，三种基质金属蛋白酶， 成纤维细胞型胶原酶（F-CL）、基质溶解素-1（SL-1）和Mr 72 K 明胶酶（GL）和两种内源性抑制剂TIMP-1和TIMP-2。 F-CL 在这一过程中起着关键作用，因为它能够切割 天然胶原蛋白分子在催化有意义的速率，从而 引发胶原纤维网络的溶解。 我们建议 研究细胞内和细胞外分子反应， 人口腔粘膜成纤维细胞降解间质胶原纤维。 我们将首先建立一个人口腔粘膜成纤维细胞库 克隆，以确定胶原降解的天然差异是否 能力被编码在特定组合的表达中， 金属蛋白酶和抑制剂基因。 胶原蛋白降解能力将 通过监测以下物质的重构原纤维的溶解来确定： I型胶原蛋白。 这些研究将扩展到 选择性诱导或消除靶基因的表达， 生长因子和细胞因子以及反义脱氧寡核苷酸。 此外，个别基因产物的作用将通过使用 功能干扰抗体阻断酶或抑制 活动 主要的焦点将是细胞周围的激活， proCL，因为该步骤在细胞介导的溶出中是限速的 胶原蛋白纤维。 纤溶酶原、SL-1和TIMPs在血管内皮细胞凋亡中的作用 胶原蛋白降解，特别是在proCL活化中 将被审查。 我们推测细胞周围蛋白水解介导的 通过与固相基质接触接种的活细胞， 三个不同的隔室，培养基，细胞表面和基质 面 我们将推导出每一个分子反应的组成部分， 基于识别和跟踪的三个细胞周围区室 中间体和产品。 在该过程中形成的反应产物 将通过α 2 M-和TIMP-捕获技术分离并鉴定 通过NH 2-末端序列分析或通过Western分析， 序列特异性抗体。 胶原蛋白碎片的命运 从原纤维网络释放的分子或α-链将 监察，以确定72 K GL先生是否符合宪法 细胞表达的蛋白质在这一过程中发挥作用， 降解过程涉及特异性表面结合、内化 和胶原蛋白链片段的吞噬溶酶体降解。