Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells.

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.

Cytokine-dependent transcriptional down-regulation of epithelial sodium channel in ulcerative colitis.

BACKGROUND & AIMS: The main limiting factor for sodium absorption in distal colon is the amiloride-sensitive epithelial sodium channel (ENaC). This study aimed to characterize mechanisms involved in the dysregulation of ENaC expression in ulcerative colitis (UC). METHODS: Epithelial preparations from surgically removed inflamed and control sigmoid colons were used. Active electrogenic Na(+) transport (J(Na)) was determined after 8-hour aldosterone stimulation in Ussing-chambers (corrected for the altered epithelial/subepithelial resistance ratio). Subsequently, ENaC alpha-, beta-, and gamma-subunits were analyzed immunohistochemically and in Western and Northern blots (corrected for the inflammatory increase in subepithelial protein content). To study gene regulation, the promoters of beta- and gamma-ENaC were analyzed in reporter gene assays. RESULTS: In controls, aldosterone stimulated J(Na) and induced ENaC beta- and gamma-subunit expression, whereas this response was virtually abolished in UC. Preservation of surface epithelium in UC was indicated by unchanged ENaC alpha-subunit expression, which points also against a mere immaturity or epithelial cell loss. Inhibition of electrogenic sodium transport as well as beta- and gamma-ENaC mRNA expression could be mimicked in control colon by in vitro preexposure for 8 hours to tumor necrosis factor alpha and interferon gamma. Promoter analysis revealed that down-regulation of beta- and gamma-ENaC gene expression was primarily induced by tumor necrosis factor alpha. CONCLUSIONS: We conclude that, in UC, elevated proinflammatory cytokines selectively impair beta- and gamma-ENaC expression, which contributes to diarrhea by reducing colonic sodium absorption.

IL-1beta and TNFalpha regulate sodium absorption in rat distal colon.

The epithelial Na+ channel (ENaC) provides the main absorptive pathway of the distal large intestine. This study aimed to characterize regulatory influences of cytokines in rat late distal colon. After 6 h incubation with either IL1beta, TNFalpha, IFNgamma, or combinations of TNFalpha and IFNgamma, ENaC was measured as electrogenic Na+ transport after 8 h induction by 3 nM aldosterone (JNa) in totally stripped specimens in the Ussing chamber. Subsequently, alpha-, beta-, and gamma-ENaC subunit mRNAs were analyzed by Northern blotting. The gamma-ENaC promoter was cloned and characterized by reporter gene assays. IL-1beta and TNFalpha, but not interferon-gamma, decreased JNa. In parallel, beta- and gamma-ENaC transcription was inhibited, whereas alpha-ENaC was unaffected. gamma-ENaC promoter activity was inhibited by IL-1beta and TNFalpha but not by IFNgamma. We conclude that the pro-inflammatory cytokines IL-1beta and TNFalpha inhibit electrogenic sodium absorption in rat distal colon by mRNA expression regulation of the beta- and gamma-ENaC subunits.

Functional crosstalk between Wnt signaling and Cdx-related transcriptional activation in the regulation of the claudin-2 promoter activity.

Assembly of tight junctions at the most apical part of the lateral cell membrane is a key event in the differentiation of polarized epithelial cells. Claudin-2, a transmembrane protein involved in tight junction strand formation, has turned out to play a crucial role for the paracellular barrier function by opening pores for small cations. Physiological and pathological variations of epithelial barrier function are accompanied by differential expression of tight junction proteins. Therefore, we characterized molecular mechanisms regulating claudin-2 gene expression. Genomic DNA containing the transcription start point of human claudin-2 was isolated and functionally characterized by reporter gene assays. Activity of the claudin-2 promoter was elevated in mouse mammary epithelial C57 cells expressing Wnt-1. LEF-1, a nuclear effector of the Wnt signaling pathway which is involved in the regulation of cell differentiation and polarization, was found to bind directly to the claudin-2 promoter as revealed by electrophoretic mobility shift assays. Expression of LEF-1 and beta-catenin both enhanced claudin-2 promoter activity. This increase was reduced after mutation of LEF-1 binding sites within the claudin-2 promoter. Furthermore, claudin-2 promoter activity was found to be enhanced by the TCF-4/beta-catenin transcription complex. Therefore, we conclude that gene expression mediated by the promoter of the human tight junction protein claudin-2 is regulated by factors involved in Wnt signaling. Moreover, a functional crosstalk between Wnt signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins could be demonstrated in the regulation of claudin-2 promoter-mediated gene expression.

Gene expression of the tight junction protein occludin includes differential splicing and alternative promoter usage.

Occludin is an integral membrane protein located at the tight junctions of epithelial cells. Multiple domains of occludin are involved in the regulation of paracellular permeability as well as in the targeting of the protein to the tight junction. In this study, different occludin variants were identified on the mRNA level. Four differentially spliced occludin-specific mRNA transcripts were detected. Expression of the resulting proteins revealed an altered subcellular distribution and a loss of co-localization with zonula occludens protein ZO-1 in the tight junction for two of the four splice variants. Our findings demonstrate that the fourth transmembrane domain of occludin is important for targeting occludin to the tight junction. Loss of the fourth transmembrane domain leads to a relocation of the C-terminal domain to the extracellular space. The structural diversity of natural occludin variants is further increased by an additional promoter and transcription start giving rise to an alternative exon 1. Gene expression mediated by this promoter is influenced by the pro-inflammatory cytokine tumor necrosis factor alpha.