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The process of connective tissue remodeling is an important mechanism contributing to tissue morphogenesis in development and homeostasis. Although it has long been known that remodeling tissues actively mediate collagenolysis, little is understood about the molecular mechanisms controlling this cell-regulated process. In this study, we examined the biosynthesis of collagenase and the related metalloproteinase, stromelysin, during remodeling of repair tissue deposited after mechanical injury to the rabbit cornea. Neither enzyme was synthesized by uninjured corneas; however, synthesis and secretion was detectable within one day after injury. Collagenase accumulated in its latent form while stromelysin appeared to be partially activated. Enzymes were synthesized by cells having a fibroblast phenotype. These cells were found within the stroma. New synthesis was correlated with accumulation of enzyme-specific mRNA. Highest levels of enzyme synthesis were observed in the repair tissue. However, stromal cells outside of the repairing area also synthesized both enzymes. The level of synthesis decreased in a gradient radiating from the repair tissue. Total synthetic levels in a given area of cornea were dependent on both the number of cells expressing enzyme and the rate of enzyme synthesis. Synthesis of collagenase was detected in repair tissue as long as nine months after injury. Our findings provide direct support for the hypothesis that new collagenase synthesis by cells in repair tissue is the first step in collagen degradation during long-term tissue remodeling.

We have examined the pattern of expression of four different matrix metalloproteinases (MMPs), collagenase, stromelysin, 92 kD gelatinase, and 72 kD gelatinase, by primary and passaged cultures of rabbit corneal fibroblasts. Primary cultures of this cell type have previously been shown to reproduce the normal tissue regulation of collagenase expression. We demonstrate qualitative and quantitative changes in the pattern of MMP expression as the cells are passaged in culture. Only a single MMP, 72 kD gelatinase, is constitutively expressed by primary fibroblast cultures. Phorbol myristate acetate (PMA) treatment upregulates expression of 72 kD gelatinase and turns on the expression of collagenase and stromelysin, as well as 92 kD gelatinase. However, the degree to which MMP expression is induced is minimal. Cells subcultured but a single time constitutively produce not only 72 kD gelatinase, but also collagenase and stromelysin. In addition, PMA treatment upregulates expression of collagenase, stromelysin and 92 kD gelatinase to high levels. In contrast, the expression of 72 kD gelatinase is repressed by treatment of passaged cell cultures with PMA. Our data indicate that the cell does not simply turn the MMP genes on or off, as a group, in response to various agents, but that it has the capacity for fine control over which MMPs are expressed and the degree to which each is expressed. Changes in MMP protein expression induced by PMA treatment are correlated with changes in specific mRNA levels in passaged cultures. The kinetics of mRNA accumulation suggest that the MMP genes can respond to multiple intracellular signals initiated in a temporal cascade by PMA. It is the combined effects of the individual signals on the accumulation of specific mRNAs that must determine the ultimate pattern of MMP protein expression. The distinct patterns of MMP expression produced by primary and passaged cell cultures may be analogous to patterns of expression that might occur under particular in vivo conditions.

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