Genomes and Genes
Tight Junction Barriers in the Gastrointestinal Tract
Principal Investigator: James Anderson
Affiliation: University of North Carolina
Abstract: Loss of selectivity and increased permeability of the intestinal tight junction barrier contributes to inflammation and diarrhea. An impediment to interpreting and correcting these pathologic changes is that we lack an understanding of the molecular structure of the barrier and the basis for its size- and charge- selectivity and permeability. Recent data support the existence of two distinct pathways through the junction: one is formed by small size- and charge-selective pores; the second allows bulk size-independent flow of larger solutes and is likely to be an important component of pathologic "leakiness". Our long range goal is to understand how specific protein components of the tight junction define selectivity and permeability of these distinct pathways so that the intestinal barrier can be manipulated for therapeutic purposes, with the central hypothesis that specific transmembrane tight junction and adhesive proteins control different aspects of selectivity and permeability. Strong Preliminary Studies support this hypothesis, which will be tested in three Specific Aims. (1) Do claudins, occludin and tricellulin determine the number and size of the small paracellular pores? Based on our published and Preliminary Studies we hypothesize that claudins create the pores and may confer different pore sizes and numbers; other adhesion proteins like occludin may control porosity. The role of these proteins in regulating permselectivity will be tested in overexpression, mutational and siRNA knockdown studies in cultured Caco-2 and other epithelial monolayers. (2) Is the size-independent pathway influenced by non-claudin adhesion molecules at baseline and during physiologic changes? Our working hypothesis is that effectiveness of the size-independent pathway is controlled by adhesion molecules like occludin, tricellulin, JAM-A and other members of the Ig- super family. The role of these proteins will be tested after knockdown and after permeability enhancement by physiologic stimuli in cultured epithelial models (i.e. activating myosin phosphorylation pathways) and in mouse intestine (in JAM-A null mice). (3) Determine the structural basis for claudin interactions. Our working hypothesis is that claudin monomers interact both within one cell membrane and across the paracellular space to create the pores and the continuous barrier. Experiments will define the amino acid sequence rules for these interactions; and the composition of a 650kDa claudin-containing complex isolated in Blue-Native PAGE. The overall approach is innovative because of a profiling assay which allows quantification of the size and number of pores and level of permeability of the non-restrictive path. The significance of this work is that it will define the contribution of each protein to controlling barrier selectivity and permeability. This information can be used to interpret the role of specific protein changes in disease and to begin to design selective targeted strategies to preserve and restore the intestinal barrier or enhance transmucosal drug delivery. PUBLIC HEALTH RELEVANCE: The proposed studies are aimed at understanding at a molecular level how the intestinal epithelium creates a selective barrier between the bowel contents, which include both nutrients and potentially harmful bacteria and antigens, and the rest of the body. The public health significance of this project is that loss of selectivity and increased permeability contribute to a number of intestinal diseases, including diarrhea and inflammation. The findings will guide strategies to preserve and restore the intestinal barrier or enhance permeability for transmucosal drug delivery.
Funding Period: 1992-02-01 - 2013-06-30
more information: NIH RePORT
- Claudin profiling in the mouse during postnatal intestinal development and along the gastrointestinal tract reveals complex expression patternsJennifer L Holmes
Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Gene Expr Patterns 6:581-8. 2006..This database provides a resource for investigating regional and developmental differences in permselectivity, crypt to villus/surface differentiation and neoplastic changes along the gut and during postnatal development...
- Claudins and epithelial paracellular transportChristina M Van Itallie
Department of Medicine, Division of Gastroenterology and Hepatology, University of North Carolina, Chapel Hill, North Carolina 27599 7545, USA
Annu Rev Physiol 68:403-29. 2006....
- Two splice variants of claudin-10 in the kidney create paracellular pores with different ion selectivitiesChristina M Van Itallie
Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 7545, USA
Am J Physiol Renal Physiol 291:F1288-99. 2006..We conclude that alternative splicing of claudin-10 generates unique permselectivities and might contribute to the variable paracellular transport observed along the nephron...
- Compositional and stoichiometric analysis of Clostridium perfringens enterotoxin complexes in Caco-2 cells and claudin 4 fibroblast transfectantsSusan L Robertson
Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, USA
Cell Microbiol 9:2734-55. 2007..These results identify CPE as a unique toxin that combines with tight junction proteins to form high-molecular-mass hexameric pores and alter membrane permeability...
- Claudin-18: a dominant tight junction protein in Barrett's esophagus and likely contributor to its acid resistanceBiljana Jovov
Dept of Medicine, Univ of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Am J Physiol Gastrointest Liver Physiol 293:G1106-13. 2007..We conclude that Cldn-18 is the dominant claudin in the TJ of SCE and propose that the change from a Cldn-18-deficient TJ in SqE to a Cldn-18-rich TJ in SCE contributes to the greater acid resistance of BE...
- Structure of the claudin-binding domain of Clostridium perfringens enterotoxinChristina M Van Itallie
Department of Medicine, University of North Carolina at Chapel Hill School of Medicine, 103 Mason Farm Road, Chapel Hill, NC 27599, USA
J Biol Chem 283:268-74. 2008..Our results provide a structural framework to advance therapeutic applications of the toxin and suggest a common ancestor for several receptor-binding domains of bacterial toxins...
- The density of small tight junction pores varies among cell types and is increased by expression of claudin-2Christina M Van Itallie
Department of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
J Cell Sci 121:298-305. 2008..Although claudins are likely to be components of the small pores, other factors might regulate pore number...
- Claudin-2-dependent changes in noncharged solute flux are mediated by the extracellular domains and require attachment to the PDZ-scaffoldChristina M Van Itallie
Department Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA
Ann N Y Acad Sci 1165:82-7. 2009..4 A. They leave unanswered why both claudin-2 and -4 can influence electrical properties while only -2 can selectively increase permeability for small PEGs...
- ZO-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeletonChristina M Van Itallie
Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Mol Biol Cell 20:3930-40. 2009..These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin...