CELLULAR MECHANISMS FOR HUMAN PROCOLLAGENASE-3 (MMP-13) ACTIVATION - EVIDENCE THAT MT1-MMP (MMP-14) AND GELATINASE-A (MMP-2) ARE ABLE TO GENERATE ACTIVE ENZYME

Gelatinase A and membrane-type metalloproteinase (MT1-MMP) were able t o process human procollagenase 3 (M(r) 60,000) to the fully active enz yme (Tyr(85) N terminus; M(r) 48,000). MT1-MMP activated procollagenas e-3 via a M(r) 56,000 intermediate (Ile(36) N terminus) to 48,000 whic h was the result of the cleavage of the Glu(84)-Tyr(85) peptide bond. We have established that the activation rate of procollagenase-3 by MT 1-MMP was enhanced in the presence of progelatinase A, thereby demonst rating a unique new activation cascade consisting of three members of the matrix metalloproteinase family. In addition, procollagenase-3 can be activated by plasmin, which cleaved the Lys(38)-GlU(39) and Arg(76 )-Cys(77) peptide bonds in the propeptide domain, Autoproteolysis then resulted in the release of the rest of She propeptide domain generati ng Tyr(85) N-terminal active collagenase 3. However, plasmin cleaved t he C-terminal domain of collagenase-3 which results in the loss of its collagenolytic activity. Concanavalin A-stimulated fibroblasts expres sing MT1-MMP and fibroblast-derived plasma membranes were able to proc ess human procollagenase-3 via a M, 56,000 intermediate form to the fi nal M(r) 48,000 active enzyme which, by analogy with progelatinase A a ctivation, may represent a model system for in vivo activation. Inhibi tion experiments using tissue inhibitor of metalloproteinases, plasmin ogen activator inhibitor-a, or aprotinin demonstrated that activation in the cellular model system was due to MT1-MMP/gelatinase A and exclu ded the participation of serine proteinases such as plasmin during pro collagenase-3 activation, We have established that progelatinase A can considerably potentiate the activation rate of procollagenase-3 by cr ude plasma membrane preparations from concanavalin A-stimulated fibrob lasts, thus confirming our results using purified progelatinase A and MT1-MMP. This new activation cascade may be significant in human breas t cancer pathology, where all three enzymes have been implicated as pl aying important roles.