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 cytokines such as tumor necrosis factor (TNF)alpha and epidermal growth (EGF). In spite a clear role for TNFalpha in the pathogenesis of inflammatory bowel disease, surprisingly little information is known about its role in the process of restitution. This proposal has evolved out of our long term studies on the mechanisms of growth factor regulated intestinal growth and development. Based on the synthesis of our preliminary and reported data, evidence from animal models of inflammatory bowel disease and on human clinical trials, the hypothesis being tested in this proposal is that TNFalpha specifically regulates intestinal epithelial restitution through signal transduction pathways required for cell migration and proliferation. We have established specific aims to address the following questions: (l) What are the principal mechanisms of TNFalpha regulation of intestinal epithelial cell migration? (2) What are the principal mechanisms of TNFalpha regulation of intestinal epithelial cell proliferation? and (3) How does TNFalpha interfere with EGF receptor signal transduction? To answer these questions we will use cellular transfection of the two human TNFalpha receptors (TNFalphaR1 and TNFalphaR2) and a series of constructs into the young adult mouse colon (YAMC) or mouse small intestinal epithelial (MSIE) cell lines. Using species-specific reagents we will investigate the function of TNFalphaR1 and TNFalphaR1 in the migration, proliferation and EGF receptor signal transduction of intestinal epithelial cells. Mutational analysis of the respective receptors will be used to determine regulatory domains effecting these processes. Once identified, regulatory domains will be placed in """"""""bait"""""""" plasmid constructs for screening in the yeast two-hybrid assay for interacting proteins. To enhance the likelihood of recovering functional interacting proteins, we will develop GST-fusion proteins using the domains to screen intestinal cell-derived cDNA libraries and for purification of proteins, from YAMC and MSIE cell lysates, for amino acid sequence analysis. Antibodies will be generated against novel proteins to characterize the nature and function of these protein-protein interactions. The proposed studies should enhance our understanding of mechanisms of cytokine regulation of epithelial restitution and the prioritization of signal transduction when multipleligands, such as TNFalpha and EGF, are simultaneously activating their cognate receptors. The findings will have broad implications on intestinal biology, from normal growth and development, to tumorigenesis and metastasis, to the chronic mucosal ulceration state of inflammatory bowel disease.
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