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Cingulin (CGN) is a 140 kDa protein, which is localized to the cytoplasmic region of vertebrate tight junctions (TJ), and regulates gene expression and RhoA signaling in cultured cells. To investigate the function of CGN at the organism level, we generated CGN knockout (CGN-/-) mice by homologous recombination. CGN-/- mice are viable and fertile, and are born at the expected mendelian ratios. Immunohistochemistry, immunofluorescence, electron microscopy and permeability assays of epithelial tissues of CGN-/- mice show no cingulin labeling at junctions, a normal localization of TJ proteins, and normal TJ structure and barrier function. Microarray analysis of intestinal cells does not show significant changes in gene expression between CGN-/- and CGN+/+ mice, whereas immunoblotting analysis shows a twofold increase in the levels of claudin-2 protein in the duodenum and the kidney of CGN-/- mice, compared to CGN+/+ littermates. Furthermore, CGN-/- mice show an exacerbated response to the ulcerogenic action of cysteamine, whereas acute injury of the colon by dextran sodium sulfate elicits undistinguishable responses in CGN-/- and CGN+/+ mice. We conclude that at the organism level cingulin is dispensable for the structure and barrier function of TJ, and is embedded in signaling networks that control the expression of claudin-2, and the mucosal response to acute injury in the duodenum. [ABSTRACT FROM AUTHOR]

Claudins are critical components of epithelial and endothelial tight junction seals, but their post-transcriptional regulation remains poorly understood. Several studies have implicated phosphorylation in control of claudin localisation and/or function, but these have focused on single sites or pathways with differing results, so that it has been difficult to draw general functional conclusions. In this study, we used mass spectrometry (MS) analysis of purified claudin-2 from MDCK II cells and found that the cytoplasmic tail is multiply phosphorylated on serines, a threonine and tyrosines. Phos-tag SDS PAGE revealed that one site, S208, is heavily constitutively phosphorylated in MDCK II cells and in mouse kidney; this site was targeted for further study. Mutational analysis revealed that the phosphomimetic mutant of claudin-2, S208E, was preferentially localised to the plasma membrane while claudin-2 S208A, which could not be phosphorylated at this site, both immunolocalized and co-fractionated with lysosomal markers. Mutations at sites that were previously reported to interfere with plasma membrane targeting of claudin-2 reduced phosphorylation at S208, suggesting that membrane localisation is required for phosphorylation; however phosphorylation at S208 did not affect binding to ZO-1 or ZO-2 Administration of forskolin or PGE2 resulted in dephosphorylation at S208 and transient small increases in transepithelial electrical resistance (TER). Together these data are consistent with phosphorylation at S208 playing a major role in the retention of claudin-2 at the plasma membrane. [ABSTRACT FROM AUTHOR]

Whether or not significant amounts of water pass the tight junction (TJ) of leaky epithelia is still unresolved, because it is difficult to separate transcellular water flux from TJ-controlled paracellular water flux. Using an approach without differentiating technically between the transcellular and paracellular route, we measured transepithelial water flux with and without selective molecular perturbation of the TJ to unequivocally attribute changes to the paracellular pathway. To this end, MDCK C7 cells were stably transfected with either claudin-2 or claudin-10b, two paracellular cation-channel-forming TJ proteins that are not endogenously expressed in this cell line. Claudin-2 is typical of leaky, water-transporting epithelia, such as the kidney proximal tubule, whereas claudin-10b is present in numerous epithelia, including water-impermeable segments of the loop of Henle. Neither transfection altered the expression of endogenous claudins or aquaporins. Water flux was induced by an osmotic gradient, a Na+ gradient or both. Under all conditions, water flux in claudin-2-transfected cells was elevated compared with vector controls, indicating claudin-2-mediated paracellular water permeability. Na+-driven water transport in the absence of an osmotic gradient indicates a single-file mechanism. By contrast, claudin-10b transfection did not alter water flux. We conclude that claudin-2, but not claudin-10b, forms a paracellular water channel and thus mediates paracellular water transport in leaky epithelia. [ABSTRACT FROM AUTHOR]