MOLECULAR MECHANISMS OF COLLAGEN DEGRADATION BY ORAL MUCOSAL FIBROBLASTS

项目3将研究口腔粘膜使用的酶机制。 成纤维细胞降解间质胶原纤维。新陈代谢 基质细胞对间质胶原纤维的降解可能涉及 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)、基质分解素-1(SL-1)和MR 72K 明胶酶(GL)和两种内源性抑制物TIMP-1和TIMP-2。F-CL 在这一过程中起着关键作用，因为它有能力分裂 天然胶原蛋白分子以催化有意义的速率 启动胶原纤维网络的溶解。我们建议 研究细胞内和细胞外的分子反应 人口腔黏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文库 克隆以确定胶原降解的天然差异 容量以下列特定组合的表达式进行编码 金属蛋白酶及其抑制基因。胶原蛋白降解能力将 通过监测重组纤维的溶解来确定 由活细胞产生的I型胶原。这些研究将延长到 选择性地诱导或取消靶基因的表达 生长因子和细胞因子，以及反义脱氧寡核苷酸。 此外，单个基因产物的作用将通过使用进行评估。 阻断酶或抑制作用的功能扰乱抗体 活动。主要的焦点将放在细胞周围的激活上。 ProCL，因为这一步骤在细胞介导的溶解中是限速的 胶原蛋白纤维。纤溶酶原、sL-1和TIMPs在心肌梗死中的作用 胶原的一般降解，特别是proCL的激活 将会被检查。我们推测，细胞周围的蛋白水解酶 通过与固相底物接触而种植的活细胞 三个不同的隔室，即培养液、细胞表面和基质 浮出水面。我们将推导出每个分子中的组成分子反应 基于细胞识别和跟踪的三个细胞周区 中间体和产品。在此过程中形成的反应产物 将通过Alpha2M和TIMP捕获技术分离并鉴定 通过NH2-末端序列分析或通过使用 序列特异性抗体。胶原蛋白碎片的命运 从纤维网络中释放的分子或阿尔法链将是 监测以确定是否符合构成因素的MR 72K GL 由细胞表达在这一过程中起作用，以及是否 降解过程涉及特定的表面结合、内化 以及胶原链片段的吞噬降解。

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.