Intestinal barrier function is reduced in inflammatory bowel disease (IBD) and other intestinal disorders. Alterations of tight junctions, which form the major paracellular barrier, contribute to barrier loss by allowing increased paracellular flux of ions and molecules. Published reports and my preliminary data, which assess the role of different inflammatory cytokines in the lamina propria of IBD patients, support the hypothesis that at least two mechanisms of tight junction barrier regulation are activated by inflammatory cytokines. TNF induces tight junction dysfunction via a mechanism involving occludin internalization that alters tight junction size selectivity and permits increased macromolecular flux. In contrast IL-13 activates a functionally distinct pathway involving expression of claudin proteins that affects ion selectivity but does not increase macromolecular flux. Despite advances in understanding the processes involved in cytokine-induced barrier regulation, the molecular events that define size- or ion-selective changes are unclear. This, in part, represents the technical obstacle posed by traditional time and spatially averaged measurements of barrier function. I hypothesize that the tight junction barrier is highly dynamic at the local, sub-micron level, and that regulation of paracellular flux occurs through modulation of opening and closing "events" involving more than one class of tight junction pore or other conductance pathway. Because the tight junction spans two cells, it has not been amenable to molecular and biophysical analyses and development of specific pharmacologic modulators, such as those that exist for transmembrane ion channels. In order to eliminate this gap in available methods and molecular understanding, I have developed a novel approach to analyze tight junction barrier function at the local sub-micron level, using a high resolution single electrode patch clamp technique. While this approach is extensively used in the study of transmembrane ion channels and transporters, it has not been applied to the study of tight junction function. My preliminary recordings using this approach show previously unrecognized barrier dynamics, and I hypothesize that this behavior underlies normal tight junction function as a selectively-permeable barrier and is also necessary for understanding the molecular mechanisms and basis for multiple pathways of barrier dysfunction in disease. These local measurements will be performed alongside traditional assays of tight junction function (Aim 1) to study tight junction dynamics at steady state (Aim 2) and after exposure to inflammatory cytokines IL-13 and TNF (Aim 3). The data will have significant positive effects on human health by improving the understanding of barrier function and dysfunction, which may provide better therapeutic approaches for intestinal disorders such as IBD, celiac disease, and infectious colitis. Furthermore, both the knowledge gained and the technical approaches developed will be easily adapted to the study of tight junctions in other organs, including integument, central nervous system, vasculature, lung, kidney, and skin, and, therefore, may have broad impact beyond gastrointestinal disease.
Narrative Intestinal barrier function is reduced in inflammatory bowel disease and other intestinal disorders. Alterations of tight junctions, which form the major paracellular barrier, contribute to barrier loss by allowing increased paracellular flux of ions and molecules. Barrier dysfunction involves separate mechanisms for ionic and macromolecular flux, but the molecular basis for these different pathways is unclear. The research outlined will study these mechanisms using high resolution patch clamp recordings of tight junction barrier dynamics.
|Liang, Guo Hua; Weber, Christopher R (2014) Molecular aspects of tight junction barrier function. Curr Opin Pharmacol 19:84-9|
|Weber, Christopher R; Rubin, David T (2013) Chronic pouchitis versus recurrent Crohn's disease: a diagnostic challenge. Dig Dis Sci 58:2748-50|
|Buschmann, Mary M; Shen, Le; Rajapakse, Harsha et al. (2013) Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux. Mol Biol Cell 24:3056-68|
|Qi, Wentao; Fitchev, Philip S; Cornwell, Mona L et al. (2013) FOXO3 growth inhibition of colonic cells is dependent on intraepithelial lipid droplet density. J Biol Chem 288:16274-81|
|Edelblum, Karen L; Shen, Le; Weber, Christopher R et al. (2012) Dynamic migration of ýýýý intraepithelial lymphocytes requires occludin. Proc Natl Acad Sci U S A 109:7097-102|
|Raleigh, David R; Boe, Devin M; Yu, Dan et al. (2011) Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function. J Cell Biol 193:565-82|
|Shen, Le; Weber, Christopher R; Raleigh, David R et al. (2011) Tight junction pore and leak pathways: a dynamic duo. Annu Rev Physiol 73:283-309|