IL-13 was identified as an important effector cytokine in UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity.

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Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.

The polarized morphology of epithelial cells depends on the establishment and maintenance of characteristic intercellular junctions. The dramatic morphological changes observed in apoptotic epithelial cells were ascribed at least in part to the specific fragmentation of components of adherens junctions and desmosomes. Little, however, is known about tight junctions during apoptosis. We have found that after induction of apoptosis in epithelial cells, tight junction proteins undergo proteolytic cleavage in a distinctive manner correlated with a disruption of tight junctions. The transmembrane protein occludin and, likewise, the cytoplasmic adaptor proteins ZO-1 and ZO-2 are fragmented by caspase cleavage. In addition, occludin is cleaved at an extracellular site by a metalloproteinase. The caspase cleavage site in occludin was mapped C-terminally to Asp320 within the C-terminal cytoplasmic domain. Mutagenesis of this site efficiently blocked fragmentation. In the presence of caspase and/or metalloproteinase inhibitors, fragmentation of occludin, ZO-1 and ZO-2 was blocked and cellular morphology was almost fully preserved. Interestingly, two members of the claudin family of transmembrane tight junction proteins exhibited a different behavior. While the amount of claudin-2 protein was reduced similarly to occludin, ZO-1 and ZO-2, claudin-1 was either fully preserved or was even increased in apoptotic cells.

Background & Aims-Small epithelial wounds heal by purse-string contraction of an actomyosin ring that is regulated by myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK). These studies aimed to define the roles of these kinases in purse-string wound closure.

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Restitution of single‐cell defects, a frequent event in epithelia with high turnover, is poorly understood. Morphological and functional changes were recorded, using intravital time‐lapse video microscopy, confocal fluorescence microscopy, and conductance scanning techniques. After artificial single‐cell loss from an HT‐29/B6 colonic cell monolayer, the basal ends of adjacent cells extended. Concurrently, the local conductive leak associated with the defect sealed with an exponential time course (from 0.48 ± 0.05 μS 2 min post lesion to 0.17 ± 0.02 μS 8 min post lesion, n= 17). Between 3 and 10 min post lesion, a band of actin arose around the gap, which colocalized with a ring of ZO‐1 and occludin. Hence, tight junction proteins bound to the actin band facing the gap, and competent tight junctions assembled in the adjoining cell membranes. Closure and sealing were inhibited when actin polymerization was blocked by cytochalasin D, delayed following decrease of myosin‐ATPase activity by butanedione monoxime, and blocked after myosin light chain kinase inhibition by ML‐7. The Rho‐associated protein kinase inhibitor Y‐27632 did not affect restitution. After loosening of intercellular contacts in low Ca2+ Ringer solution, the time course of restitution was not significantly altered. Albeit epithelial conductivity was 12‐fold higher in low Ca2+ Ringer solution than in controls, under both conditions the repaired epithelium assumed the same conductivity as distant intact epithelium. In conclusion, epithelial restitution of single‐cell defects comprises rapid closure by an actinomyosin ‘purse‐string’ mechanism and simultaneous formation of a functional barrier from tight junction proteins also associated with the purse string.

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