Maintenance of an intact intestinal epithelium is critical for intestinal growth, development, wound healing and prevention of disease. Restitution, the initial phase of epithelial wound repair, is regulated by a number of peptides, including epidermal growth factor (EGF). Altered mucosal reepithelialization is a significant component of a number of diseases primarily affecting the gastrointestinal tract, such as ulcerative colitis and peptic ulcer disease. Surprisingly, little information is known about the initial signaling events in the process of basal or stimulated intestinal cell migration. Preliminary data in intestinal epithelial cells from our laboratory and reports on fibroblast migration have permitted the development of the proposed hypothesis that: Epidermal growth factor receptor regulates intestinal epithelial cell migration via integrated signaling pathways requiring tyrosine kinase and the enhanced activities of specific target molecules during the earliest phases of cellular movement. The objective of this proposal is to understand the cellular and molecular mechanisms of growth factor regulated intestinal cell migration through a series of questions comprising the following Specific Aims: 1) What are the principal mechanisms of EGF receptor regulation of intestinal epithelial cell migration? 2) What are the roles of phosphatidylinositol phospholipase C (PLC) gamma 1, protein kinase (PK) C epsilon, and other signal transduction molecules in EGF receptor-regulated cellular migration? and 3) How does the EGF receptor mediate cytoskeletal rearrangement during intestinal cell migration? To address the mechanisms of EGF receptor regulation of migration in aim 1, we will express various EGF receptor constructs in a mouse colon cell line derived from the EGF receptor null mouse.
In aims 2 and 3, we will express constitutively active and dominant negative forms of PLC gamma 1, PKC epsilon and the Rho small GTPase proteins to establish a molecular ordering of signal transduction from the EGF receptor to cytoskeletal rearrangement regulating motility. We will use expression based cloning and coprecipitation assays to identify interacting proteins in the pathway leading to cellular migration. These studies should enhance our understanding of the initial signaling events required for growth factor-regulated intestinal epithelial restitution and provide a mechanistic basis for potential development of novel therapeutic targets.
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