scholarly journals Claudin-2-dependent paracellular channels are dynamically gated

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Christopher R Weber ◽  
Guo Hua Liang ◽  
Yitang Wang ◽  
Sudipto Das ◽  
Le Shen ◽  
...  
Keyword(s):  
Tight Junction ◽  
Site Directed Mutagenesis ◽  
Intestinal Epithelial ◽  
Chemical Derivatization ◽  
Patch Clamp Technique ◽  
Epithelial Monolayers ◽  
Charge Selectivity ◽  
Permeable Barriers ◽  
Barrier Regulation ◽  
Reduced Temperature

Intercellular tight junctions form selectively permeable barriers that seal the paracellular space. Trans-tight junction flux has been measured across large epithelial surfaces, but conductance across individual channels has never been measured. We report a novel trans-tight junction patch clamp technique that detects flux across individual claudin-2 channels within the tight junction of cultured canine renal tubule or human intestinal epithelial monolayers. In both cells, claudin-2 channels display conductances of ~90 pS. The channels are gated, strictly dependent on claudin-2 expression, and display size- and charge-selectivity typical of claudin-2. Kinetic analyses indicate one open and two distinct closed states. Conductance is symmetrical and reversible, characteristic of a passive, paracellular process, and blocked by reduced temperature or site-directed mutagenesis and chemical derivatization of the claudin-2 pore. We conclude that claudin-2 forms gated paracellular channels and speculate that modulation of tight junction channel gating kinetics may be an unappreciated mechanism of barrier regulation.

scholarly journals Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase

2000 ◽  
Vol 279 (5) ◽  
pp. G875-G885 ◽  
Author(s):  
Thomas Y. Ma ◽  
Neil T. Hoa ◽  
Daniel D. Tran ◽  
Vuong Bui ◽  
Ali Pedram ◽  
...  
Keyword(s):  
Tight Junction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Cytochalasin B ◽  
Late Phase ◽  
Metabolic Inhibitors ◽  
Intestinal Epithelial ◽  
Atpase Inhibitor ◽  
Epithelial Monolayers

The intracellular mechanisms that mediate cytochalasin-induced increase in intestinal epithelial tight junction (TJ) permeability are unclear. In this study, we examined the involvement of myosin light chain kinase (MLCK) in this process, using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin B (Cyto B) (5 μg/ml) produced an increase in Caco-2 MLCK activity, which correlated with the increase in Caco-2 TJ permeability. The inhibition of Cyto B-induced MLCK activation prevented the increase in Caco-2 TJ permeability. Additionally, myosin-Mg2+-ATPase inhibitor and metabolic inhibitors (which inhibit MLCK induced actin-myosin contraction) also prevented the Cyto B-induced increase in Caco-2 TJ permeability. Cyto B caused a late-phase (15–30 min) aggregation of actin fragments into large actin clumps, which was also inhibited by MLCK inhibitors. Cyto B produced a morphological disturbance of the ZO-1 TJ proteins, visually correlating with the functional increase in Caco-2 TJ permeability. The MLCK and myosin-Mg2+-ATPase inhibitors prevented both the functional increase in TJ permeability and disruption of ZO-1 proteins. These findings suggested that Cyto B-induced increase in Caco-2 TJ permeability is regulated by MLCK activation.

Human ENS regulates the intestinal epithelial barrier permeability and a tight junction-associated protein ZO-1 via VIPergic pathways

2003 ◽  
Vol 285 (5) ◽  
pp. G1028-G1036 ◽  
Author(s):  
Michel Neunlist ◽  
Férial Toumi ◽  
Tsvetelina Oreschkova ◽  
Marc Denis ◽  
Joel Leborgne ◽  
...  
Keyword(s):  
Tight Junction ◽  
Electrical Field Stimulation ◽  
Immunoblot Analysis ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Intestinal Epithelial Barrier ◽  
Physiological Control ◽  
Barrier Permeability ◽  
Epithelial Monolayers ◽  
Neuron Activation

Although the enteric nervous system (ENS) has been shown to regulate various mucosal functions, its role in the physiological control of the human intestinal epithelial barrier is unknown. The aim of this study was to investigate whether the ENS is able to modulate epithelial barrier permeability and a key tight junction-associated protein, zonula occludens-1 (ZO-1). Therefore, we developed a co-culture model, consisting of human submucosa containing the submucosal neuronal network and human polarized colonic epithelial monolayers (HT29-Cl.16E or Caco-2). Submucosal neurons were activated by electrical field stimulation (EFS). Permeability was assessed by measuring the flux of paracellular permeability markers (FITC-dextran or FITC-inulin) across epithelial monolayers. Expression of ZO-1 was determined by immunofluorescence, quantitative immunoblot analysis, and real time RT-PCR. Using the coculture model, we showed that EFS of submucosal neurons resulted in a reduction in FITC-dextran or FITC-inulin fluxes, which was blocked by TTX. In HT29-Cl.16E, the effect of submucosal neuron activation was blocked by a VIP receptor antagonist (VIPra) and reproduced by VIP. Furthermore, ZO-1 expression (mRNA, protein) assessed in HT29-Cl.16E, was significantly increased after submucosal neuron activation by EFS. These effects on ZO-1 expression were blocked by TTX and VIPra and reproduced by VIP. In conclusion, our results strongly suggest a modulatory role of VIPergic submucosal neuronal pathways on intestinal epithelial barrier permeability and ZO-1 expression.

scholarly journals HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity

2015 ◽  
Vol 26 (12) ◽  
pp. 2252-2262 ◽  
Author(s):  
Bejan J. Saeedi ◽  
Daniel J. Kao ◽  
David A. Kitzenberg ◽  
Evgenia Dobrinskikh ◽  
Kayla D. Schwisow ◽  
...  
Keyword(s):  
Tight Junction ◽  
Barrier Function ◽  
Experimental Colitis ◽  
Site Directed Mutagenesis ◽  
Intestinal Epithelial ◽  
Low Oxygen ◽  
T84 Cells ◽  
Transcriptional Responses ◽  
Epithelial Tight Junction

Intestinal epithelial cells (IECs) are exposed to profound fluctuations in oxygen tension and have evolved adaptive transcriptional responses to a low-oxygen environment. These adaptations are mediated primarily through the hypoxia-inducible factor (HIF) complex. Given the central role of the IEC in barrier function, we sought to determine whether HIF influenced epithelial tight junction (TJ) structure and function. Initial studies revealed that short hairpin RNA–mediated depletion of the HIF1β in T84 cells resulted in profound defects in barrier and nonuniform, undulating TJ morphology. Global HIF1α chromatin immunoprecipitation (ChIP) analysis identified claudin-1 (CLDN1) as a prominent HIF target gene. Analysis of HIF1β-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1β-deficient cells resulted in resolution of morphological abnormalities and restoration of barrier function. ChIP and site-directed mutagenesis revealed prominent hypoxia response elements in the CLDN1 promoter region. Subsequent in vivo analysis revealed the importance of HIF-mediated CLDN1 expression during experimental colitis. These results identify a critical link between HIF and specific tight junction function, providing important insight into mechanisms of HIF-regulated epithelial homeostasis.

Desmoglein 2-mediated adhesion is required for intestinal epithelial barrier integrity

2010 ◽  
Vol 298 (5) ◽  
pp. G774-G783 ◽  
Author(s):  
Nicolas Schlegel ◽  
Michael Meir ◽  
Wolfgang-Moritz Heupel ◽  
Bastian Holthöfer ◽  
Rudolf E. Leube ◽  
...  
Keyword(s):  
Tight Junction ◽  
Intestinal Barrier ◽  
Intercellular Junctions ◽  
Barrier Properties ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Intestinal Epithelial Barrier ◽  
Barrier Functions ◽  
Epithelial Monolayers

The integrity of intercellular junctions that form the “terminal bar” in intestinal epithelium is crucial for sealing the intestinal barrier. Whereas specific roles of tight and adherens junctions are well known, the contribution of desmosomal adhesion for maintaining the intestinal epithelial barrier has not been specifically addressed. For the present study, we generated a desmoglein 2 antibody directed against the extracellular domain (Dsg2 ED) to test whether impaired Dsg2-mediated adhesion affects intestinal epithelial barrier functions in vitro. This antibody was able to specifically block Dsg2 interaction in cell-free atomic-force microscopy experiments. For in vitro studies of the intestinal barrier we used Caco2 cells following differentiation into tight enterocyte-like epithelial monolayers. Application of Dsg2 ED to Caco2 monolayers resulted in increased cell dissociation compared with controls in a dispase-based enterocyte dissociation assay. Under similar conditions, Dsg2 antibody significantly decreased transepithelial electrical resistance and increased FITC-dextran flux, indicating that Dsg2 interaction is critically involved in the maintenance of epithelial intestinal barrier functions. As revealed by immunostaining, this was due to Dsg2 ED antibody-induced rupture of tight junctions because tight junction proteins claudins 1, 4, and 5, occludin, and tight junction-associated protein zonula occludens-1 were partially removed from cell borders by Dsg2 ED treatment. Similar results were obtained by application of a commercial monoclonal antibody directed against the ED of Dsg2. Antibody-induced effects were blocked by absorption experiments using Dsg2-Fc-coated beads. Our data indicate that Dsg2-mediated adhesion affects tight junction integrity and is required to maintain intestinal epithelial barrier properties.

scholarly journals JunD Represses Transcription and Translation of the Tight Junction Protein Zona Occludens-1 Modulating Intestinal Epithelial Barrier Function

2008 ◽  
Vol 19 (9) ◽  
pp. 3701-3712 ◽  
Author(s):  
Jie Chen ◽  
Lan Xiao ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Liu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Barrier Function ◽  
Intestinal Epithelial Cells ◽  
Binding Protein ◽  
Mrna Translation ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Intestinal Epithelial Barrier ◽  
Epithelial Barrier Function

The AP-1 transcription factor JunD is highly expressed in intestinal epithelial cells, but its exact role in maintaining the integrity of intestinal epithelial barrier remains unknown. The tight junction (TJ) protein zonula occludens (ZO)-1 links the intracellular domain of TJ-transmembrane proteins occludin, claudins, and junctional adhesion molecules to many cytoplasmic proteins and the actin cytoskeleton and is crucial for assembly of the TJ complex. Here, we show that JunD negatively regulates expression of ZO-1 and is implicated in the regulation of intestinal epithelial barrier function. Increased JunD levels by ectopic overexpression of the junD gene or by depleting cellular polyamines repressed ZO-1 expression and increased epithelial paracellular permeability. JunD regulated ZO-1 expression at the levels of transcription and translation. Transcriptional repression of ZO-1 by JunD was mediated through cAMP response element-binding protein-binding site within its proximal region of the ZO-1-promoter, whereas induced JunD inhibited ZO-1 mRNA translation by enhancing the interaction of the ZO-1 3′-untranslated region with RNA-binding protein T cell-restricted intracellular antigen 1-related protein. These results indicate that JunD is a biological suppressor of ZO-1 expression in intestinal epithelial cells and plays a critical role in maintaining epithelial barrier function.

Digestion of epithelial tight junction proteins by the commensal Clostridium perfringens

2013 ◽  
Vol 305 (10) ◽  
pp. G740-G748 ◽  
Author(s):  
Mihaela Pruteanu ◽  
Fergus Shanahan
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Clostridium Perfringens ◽  
Culture Supernatant ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Tight Junction Proteins ◽  
Epithelial Tight Junction ◽  
Junction Proteins ◽  
E Cadherin

The enteric microbiota contributes to the pathogenesis of inflammatory bowel disease, but the pathways involved and bacterial participants may vary in different hosts. We previously reported that some components of the human commensal microbiota, particularly Clostridium perfringens ( C. perfringens), have the proteolytic capacity for host matrix degradation and reduce transepithelial resistance. Here, we examined the C. perfringens-derived proteolytic activity against epithelial tight junction proteins using human intestinal epithelial cell lines. We showed that the protein levels of E-cadherin, occludin, and junctional adhesion molecule 1 decrease in colonic cells treated with C. perfringens culture supernatant. E-cadherin ectodomain shedding in C. perfringens-stimulated intestinal epithelial cells was detected with antibodies against the extracellular domain of E-cadherin, and we demonstrate that this process occurs in a time- and dose-dependent manner. In addition, we showed that the filtered sterile culture supernatant of C. perfringens has no cytotoxic activity on the human intestinal cells at the concentrations used in this study. The direct cleavage of E-cadherin by the proteases from the C. perfringens culture supernatant was confirmed by C. perfringens supernatant-induced in vitro degradation of the human recombinant E-cadherin. We conclude that C. perfringens culture supernatant mediates digestion of epithelial cell junctional proteins, which is likely to enable access to the extracellular matrix components by the paracellular pathway.

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