Molecular basis for three levels of competence to express genes required for corneal repair

Purpose. To understand the molecular basis for differences in the relative competence for expression of specific repair genes among fibroblast phenotypes found in the quiescent or repairing cornea. Methods. Freshly-isolated or subcultured fibroblasls from normal or wounded rabbit cornea were used. Myofibroblast cultures were created by plating fibroblasls at a 1:100 split. Results. Freshly-isolated fibroblasts from the uninjured cornea are incompetent for collagenase expression in response lo actin cytoskeletal disruption by cytochalasin B (CB), and respond poorly to phorbol myristate acetate (PMA). In contrast, subcultured fibroblasts, or fibroblasts freshly-isolated from corneal repair tissue, can respond to both agents. A third phenoiype is exhibited by myofibroblasts which can respond (o CB, but not to PMA. Each of these phenotypes results from relative competence to express the cytokine, 1Lla. which acts as an intermediate for routing of signals to the collagenase gene. IL-la controls expression of its own gene in an autocrine feed-back loop. PMA and CB act differently to activate the feed-back loop: PMA increases IL-la message half-life while CB enhances the rate of IL-la gene transcription. Therefore, myofibroblast incompetence for collagenase expression in response to PMA results from the inability of PMA lo increase IL-la half-life. Agents which interfere with activity of either protein kinase C or tyrosine kinases inactivate the autocrine loop in fibroblasts. Antioxidants also inactivate the loop, suggesting that reactive oxygen species (ROS) act as signalling intermediates. Antioxidants further disrupt activation of the transcription factor NF-icB. Importantly, NF-icB cannot be efficiently activated by IL1 treatment in fibroblasts freshly-isolated from the uninjured cornea. Conclusions. The results suggest that quiescent corneal stromal cells produce ROS inefficiently in comparison to wound fibroblasts. Therefore, the capacity to generate ROS might be key to determining whether fibroblasi activation occurs after corneal injury.