Gastrointestinal mucosal wound healing requires the migration of sheets of intestinal epithelial cells across a mucosal defect, together with modulation of the differentiated phenotype of these cells. This process is critical to the response of the gut mucosa to surgical or traumatic injury, inflammatory bowel disease, and peptic or infectious mucosal ulceration. Preliminary investigations have validated a cell culture model for the study of intestinal epithelial cell sheet migration and have demonstrated modulation of the differentiation state of these cells during migration. We have hypothesized that enterocytic differentiation and epithelial sheet migration reflect opposing and mutually exclusive cell specializations. Preliminary data and analogy to studies of tyrosine kinase and phosphoprotein modulation during integrin engagement in other cell types suggest that migrating intestinal epithelial cells adapt to migration rather than nutrient absorption via a signal transduction cascade which begins with engagement of reorganized integrin receptors by matrix proteins and modulation of integrin-associated tyrosine phosphoproteins and has as one result the downregulation of digestive brush border expression. We therefore propose to investigate the mechanisms which regulate phenotype (using digestive brush border expression as a marker) in intestinal epithelial cells migrating over laminin, a physiologically relevant substrate, focusing specifically on initial integrin engagement and tyrosine phosphorylation events in order to begin to trace this signal transduction cascade. In particular, we will elucidate the manner in which phenotype is modulated by engagement of specific integrin receptors for laminin via modulation of integrin- associated tyrosine phosphoproteins. The relevance of these findings will be verified by comparison with protein tyrosine phosphorylation states during inhibition of migration by tyrosine kinase inhibitors, stimulation of migration and modulation of integrin expression and organization using EGF, and induction of differentiation by sodium butyrate, each of which we have previously characterized in this system. Specific antibodies and mRNA probes for proteins of interest will then be developed in order to confirm the biological relevance of these findings in vivo in healing mucosa. Such studies should not only illuminate the regulation of a process of fundamental biological significance (modulation of intestinal epithelial cell phenotype during mucosal wound healing) but may also provide a firm scientific basis for future investigation into and intervention in iatrogenic or pathological disturbances of gastrointestinal healing.
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