Loss of Spatial Constraints Induces Motility in Corneal Epithelial Cells through Heparin–binding Epidermal Growth Factor–like Growth Factor Signaling

Abstract

Abstract: : Purpose: The purpose of this study was to assess whether cell damage is important for healing of wounds in sheets of corneal epithelial cells, and to determine the initial events that lead to induction of cell movement. Methods: Primary and secondary corneal epithelial cells were grown in culture. The rates of wound healing were compared when wounds were inflicted either by mechanical means or after removal of a physical block. A novel procedure was devised such that a large fraction of the cells in a culture were presented with cell–free tissue culture area. Results:We devised an alternative procedure to introduce gaps in layers of corneal epithelial cells by casting agarose strips on tissue culture plates. In contrast to mechanical wounding, removal of the strips did not lead to detectable membrane leakage or to activation of the stress activated kinase, JNK. Nonetheless, cells at the edge underwent the typical morphological transition to a highly motile phenotype, and the gaps closed at speeds that were similar to those seen upon mechanical wounding, suggesting that the triggering event for movement is related to the sudden accessibility of permissive surface area rather than to mechanical damage. In order to perform biochemical analysis of cell extracts, procedures were devised such that cell–free surface area was acutely made available to a large proportion of the cells in cultures. Rapid activation of the epidermal growth factor receptor was detected, and addition of an epidermal growth factor receptor blocking antibody completely abolished wound healing. Wound healing was inhibited by antibodies to heparin–binding epidermal growth factor–like growth factor, and by a metalloprotease inhibitor, galardin. In addition, a very marked activation of phospholipase D which is a down–stream target of the epidermal growth factor receptor was noted, after presenting cells with cell–free surface area. Conclusions: These studies suggest that cell damage is not necessary for initiation of cell movement in corneal epithelial cells. Initiation of movement is strictly dependent on activation of the epidermal growth factor receptor through heparin–binding epidermal growth factor–like growth factor signaling.

Keywords: wound healing • cornea: epithelium • chaperones