Normal eye function depends on corneal transparency which, in turn, depends on maintenance of a normal corneal stroma. The stromal keratocyte, normally quiescent, can be stimulated to synthesize and secrete neutral proteinases and extracellular matrix. In stromal wound healing and remodeling, degradation of existing extracellular matrix and synthesis of new extracellular matrix must be controlled spatially and temporally. Excess collagenase secretion causes corneal ulceration or melting. Cytokines and cytoskeletal disruptors like cytochalasin B and phorbol induce collagenase. Collagenase activity is regulated transcriptionally and by extracellular inhibitors (TIMP) or activators. We present preliminary data that keratocyte collagenase is induced via a fibronectin signal transduction pathway. We hypothesize that keratocyte fibronectin receptors (integrins) distinguish intact fibronectin from fibronectin fragments: one or more recognition sequences in the fibronectin fragment, sensed as degradation products, activate the keratocyte collagenase pathway via fibronectin receptors.
Our specific aims are to: 1. Define more precisely the extracellular matrix signals for collagenase induction. Are there domain(s) of fibronectin or other extracellular matrix components which regulate metalloproteinase secretion? 2. Identify the integrins of corneal keratocytes and determine whether their relative number is regulated by endocytosis of fibronectin or by stimuli which induce collagenase. 3. Determine morphologically, and by kinetic and quantitative assays, whether a strict correlation exists between procollagenase induction and changes in the actin cytoskeleton. 4. Determine differential regulation through the fibronectin receptor of new extracellular matrix synthesis and proteinase synthesis/activity. Specifically, are collagenase, collagen, fibronectin, and TIMP induced in patterns which promote new synthesis or degradation? Can this be demonstrated in an individual cell? These studies will aid understanding of corneal wound healing, in which keratocytes degrade and re-synthesize extracellular matrix. Understanding mechanisms of collagenase regulation will help prevent damage from uninhibited collagenase secretion, as in keratoconus. Our long term goal is to determine at the cellular, subcellular and molecular level how the extracellular matrix and the individual keratocyte interact temporally and spatially in normal and pathological situations such as wound healing or keratoconus.
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