The epithelial response to inflammation is a fundamental component of several major diseases, including but not limited to gastrointestinal and renal disorders. Epithelial cell layers provide important barriers between compartments, regulating exchange of solutes and minimizing pathogen access to privileged spaces. This proposal seeks to elucidate the basic mechanisms underlying this epithelial barrier function through a combination of physiological and proteomic approaches. We propose to investigate the tumor necrosis factor-alpha (TNFalpha) and interferon- gamma (IFNgamma) signaling pathways that impair epithelial barrier function using the Madin Darby Canine Kidney (MDCK) renal cell line. Preliminary studies indicate that TNFalpha/ IFNgamma exposure increases epithelial permeability negatively impacting its barrier function. Multiple responses elicited by proinflammatory cytokines such as activation of specific kinases, oxidant damage, and increased proteolysis create a complex picture of potential mechanisms capable of altering epithelial permeability. Corresponding investigations will be conducted to examine global epithelial proteome modifications induced by inflammatory stress. Our general hypothesis is that different that proinflammatory signaling pathways act to modify distinct sets of tight junction proteins. Ultimately, elevated epithelial permeability is the result of independent modifications that destabilize the constituents of the tight junction complex. To test this hypothesis the following to specific aims are proposed: 1) correlate epithelial integrity to the expression pattern of TJ components following activation of proinflammatory signal pathways; 2) produce high resolution proteome maps of normal and TNFalpha/IFNgamma-treated MDCK cells using fuorescence two dimensional difference gel electrophoresis and peptide mass fmgerprinting. The proteome maps and publicly available database resulting from this study will serve as an important resource for subsequent investigations analyzing specific protein variations in epithelial tissue. Long-term goals for the laboratory are to identify proteins that interact with the TJ complex and identify specific mechanisms responsible for alterations in epithelial permeability during inflammation. In addition, this proposal will provide invaluable experience for a wide diversity of undergraduate students interested in research training and careers in the biosciences.