The objective of the research project is to investigate the role played by TIMP, a tissue inhibitor of matrix metalloproteinases, in the regulation of collagen breakdown by fibroblasts. Evidence suggests that TIMP plays a pivotal role in the regulation of collagen degradation in normal and pathologic tissue remodeling. Since tissue remodeling is a major limiting factor in prosthodontic treatment, it is important to the future development of the discipline to understand the molecular mechanisms which regulate this process. Blocking polyclonal (pAb) and monoclonal (mAb) antibodies to native or recombinant human TIMP which neutralize the inhibitory effect of TIMP on matrix metalloproteinases will be produced. IN addition, antibodies will be raised against synthetic peptides modeled after various domains of the TIMP molecule. In order to study the role played by TIMP in the regulation of collagen breakdown, mutant human gingival fibroblast lines will be developed which either constitutively express, or fail to express, interstitial, procollagenase and TIMP (pColl+/TIMP, pColl+/TIMP-, pColl-/TIMP+, and p/Coll-/TIMP-). The role of procollagenase and TIMP expression in the cell-mediated degradation of reconstituted collagen fibrils will be investigated. Cells will be seeded directly on a film of reconstituted collagen fibrils and the rate of collagen breakdown will be measured. At the same time, the rate of accumulation in the medium of procollagenase, collagenase, and TIMP will be monitored. The specific role played by TIMP will be examined by use of blocking antibodies to TIMP which are capable of neutralizing the effect of the inhibitor on the extracellular environment. Specifically, we shall determine whether TIMP acts primarily by blocking the activity of collagenase against collagen or by preventing the activation of the collagenase precursor, procollagenase.
|Sang, Q A; Bodden, M K; Windsor, L J (1996) Activation of human progelatinase A by collagenase and matrilysin: activation of procollagenase by matrilysin. J Protein Chem 15:243-53|