ZO-1 Stabilizes the Tight Junction Solute Barrier through Coupling to the Perijunctional Cytoskeleton

ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. 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

Inflammatory processes have differential effects on claudins 2, 3 and 4 in colonic epithelial cells

Claudin proteins comprise a recently described family of tight junction proteins that differentially regulate paracellular permeability. Since other tight junction proteins show alterations in distribution or expression in inflammatory bowel disease (IBD) we assessed expression of claudins (CL) 2, 3 and 4 in IBD. CL 2 was strongly expressed along the inflamed crypt epithelium, whilst absent or barely detectable in normal colon. In contrast, CL 3 and 4 were present throughout normal colonic epithelium and were reduced or redistributed in the diseased surface epithelium. In a T84-cell culture model of the gut barrier, paracellular permeability decreased with time after plating and correlated with a marked decrease in the expression of CL 2. Addition of IFN/TNF led to further decreases in CL 2 and 3, the redistribution of CL 4 and a marked increase in paracellular permeability. Conversely, IL-13 dramatically increased CL 2, with little effect on CL 3 or 4, but also resulted in increased paracellular permeability. Expression of CL 2 did not correlate with proliferation or junctional reorganisation after calcium ion depletion. Re-expression of CL 2 in response to IL-13 was inhibited by phophatidylinositol 3 kinase inhibitor, LY294002, which also restored the ion permeability to previous levels. CL 2 expression could be stimulated in the absence of IL-13 by activation of phospho-Akt in the phophatidylinositol 3 kinase pathway. These results suggest that INF/TNF and IL-13 have differential effects on CL 2, 3 and 4 in tight junctions, which may lead to increased permeability via different mechanisms