The alimentary tract is continuously exposed to substances which result in epithelial injury. Restitution,, which is seen throughout the gastrointestinal tract, is a process that rapidly repairs epithelial defects by cell flattening, lamellapodia extension, and active migration. Parallels with other wounding models suggests that gastrointestinal epithelial injury results in a Ras-dependent expression of early response genes, increased TGF-Beta1 expression and enhanced cellular motility. The signaling pathways responsible for these events remain unknown. Our preliminary data and the work of others supports the hypothesis that restitution involves the activation of the mitogen-activated protein kinase (MAP kinase) pathways, leading to early gene expression and increased bioactive TGF-Beta1. The MAP kinase pathway sits at the point of convergence of extracellular matrix and growth factor signals and allows integration of these signals into an appropriate cellular response. We have developed two models to test this hypothesis. The first is an in vitro model of colonic mucosal injury and restitution using strips of mucosa from human colonic surgical resections. The second model uses human intestinal epithelial cell lines in monolayer cultures to study restitution after injury. To gain a better understanding of the early signaling events that regulate restitution,, we propose to use these two models to address the following specific aims: 1) To characterize the activation of the MAP kinase pathways (ERK, JNK, and p38), in response to wounding and exogenous cytokines. 2) To identify the signaling steps necessary for immediate early gene expression, TGF-Beta1 expression and enhanced cell motility by the use of inhibitors and dominant negative mutations to target specific pathways, and to determine if causal relationships exist between MAP kinase pathways and early response genes (c-Fos, c-Jun), TGF-Beta1 expression and motility changes. 3) To characterize the effect of extracellular matrix composition on the activation of MAP kinase pathways in response to wounding and cytokine administration. A better understanding of the molecular mechanisms of epithelial repair have broad relevance for multiple gastrointestinal diseases including inflammatory bowel disease, ischemic injury, and multiple infectious causes of injury.
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