INJURY OF STROMAL FIBROBLASTS INDUCES PHOSPHORYLATION OF FOCAL ADHESION PROTEINS

Purpose. The extracellular matrix serves as a structural support for t he corneal stroma and mediates signaling events that regulate the intr acellular environment of stromal keratocytes. We hypothesize that adhe sion and injury mediate signal transduction events causing the phospho rylation of tyrosine residues of specific adhesion proteins and that p hosphorylation is required for cellular adhesion and migration. Method s. For the adhesion experiments, primary rabbit stromal fibroblasts we re seeded and phosphorylation of tyrosine residues was followed from 1 min to 24 h, For the injury experiments, , confluent primary cultures were rendered quiescent, wounded, and tyrosine phosphorylation was fo llowed from 30 s to 6 h. The antibody (py-20) was used to detect prote ins phosphorylated on tyrosine residues. We examined changes in the ph osphorylation of focal adhesion kinase (FAK), paxillin and cortactin, using immunoprecipitation and Western blot analysis. Results. In the a dhesion experiments, the phosphorylation of a 68-kDa protein was detec ted after 1 min, and the phosphorylation of a 125-kDa protein was not detected until 15 min. These proteins were identified in re-probed blo ts as paxillin and FAK, In the injury experiments, FAK phosphorylation was detected within 30 s and remained elevated for 6 h when cells wer e cultured on fibronectin. Both FAK and paxillin phosphorylation were prominent after injury, but unlike FAK phosphorylation, paxillin phosp horylation decreased over time. Phosphorylation was prominent at the w ound margin. After wound closure, it returned to background levels. Ty rosine kinase inhibitors, genistein and herbimycin, decreased the numb er of adherent cells and altered the rate of cell migration after inju ry, compared to control (DMSO alone). Conclusion. The results indicate that injury and cell-matrix interaction mediate the phosphorylation o f specific adhesion proteins and that phosphorylation is required for wound repair.