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.
Showing the most recent 10 out of 144 publications
