scholarly journals AMPK Activation Promotes Tight Junction Assembly in Intestinal Epithelial Caco-2 Cells

2019 ◽  
Vol 20 (20) ◽  
pp. 5171 ◽  
Author(s):  
Séverine Olivier ◽  
Jocelyne Leclerc ◽  
Adrien Grenier ◽  
Marc Foretz ◽  
Jérôme Tamburini ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Tight Junctions ◽  
Paracellular Permeability ◽  
Cell Junctions ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Ampk Activation ◽  
Energy Status ◽  
The Impact

The AMP-activated protein kinase (AMPK) is principally known as a major regulator of cellular energy status, but it has been recently shown to play a key structural role in cell-cell junctions. The aim of this study was to evaluate the impact of AMPK activation on the reassembly of tight junctions in intestinal epithelial Caco-2 cells. We generated Caco-2 cells invalidated for AMPK α1/α2 (AMPK dKO) by CRISPR/Cas9 technology and evaluated the effect of the direct AMPK activator 991 on the reassembly of tight junctions following a calcium switch assay. We analyzed the integrity of the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), the paracellular permeability, and quantification of zonula occludens 1 (ZO-1) deposit at plasma membrane by immunofluorescence. Here, we demonstrated that AMPK deletion induced a delay in tight junction reassembly and relocalization at the plasma membrane during calcium switch, leading to impairments in the establishment of TEER and paracellular permeability. We also showed that 991-induced AMPK activation accelerated the reassembly and reorganization of tight junctions, improved the development of TEER and paracellular permeability after calcium switch. Thus, our results show that AMPK activation ensures a better recovery of epithelial barrier function following injury.

Human junction adhesion molecule regulates tight junction resealing in epithelia

2000 ◽  
Vol 113 (13) ◽  
pp. 2363-2374 ◽  
Author(s):  
Y. Liu ◽  
A. Nusrat ◽  
F.J. Schnell ◽  
T.A. Reaves ◽  
S. Walsh ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Adhesion Molecule ◽  
Transmembrane Protein ◽  
Extracellular Domain ◽  
Cell Junctions ◽  
Human Neutrophils ◽  
Epithelial Barrier ◽  
Cell Cell

Epithelial cells form a highly selective barrier and line many organs. The epithelial barrier is maintained by closely apposed cell-cell contacts containing tight junctions, the regulation of which is incompletely understood. Here we report the cloning, tissue localization and evidence for a role in epithelial barrier regulation of an immunoglobulin superfamily member that likely represents the human homolog of murine junction adhesion molecule (JAM). Analysis of the primary structure of human JAM, cloned from T84 epithelial cells, predicts a transmembrane protein with an extracellular domain that contains two IgV loops. Monoclonal antibodies generated against the putative extracellular domain were reactive with a 35–39 kDa protein from both T84 epithelial cells and human neutrophils. By immunofluorescence, JAM mAbs labeled epithelial cells from intestine, lung, and kidney, prominently in the region of tight junctions (co-localization with occludin) and also along lateral cell membranes below the tight junctions. Flow cytometric studies confirmed predominant JAM expression in epithelial cells but also revealed expression on endothelial and hematopoietic cells of all lineages. Functional studies demonstrated that JAM specific mAbs markedly inhibited transepithelial resistance recovery of T84 monolayers after disruption of intercellular junctions (including tight junctions) by transient calcium depletion. Morphologic analysis revealed that, after disassembly of cell-cell junctions, anti-JAM inhibition of barrier function recovery correlated with a loss of both occludin and JAM, but not ZO-1, in reassembling tight junction structure. Reassembly of the major adherens junction component E-cadherin was not affected by JAM specific mAbs. Our findings suggest that JAM plays an important role in the regulation of tight junction assembly in epithelia. Furthermore, these JAM-mediated effects may occur by either direct, or indirect interactions with occludin.

GENETIC SUSCEPTIBILITY TO IBD OPERATES VIA CONTROL OF APICAL JUNCTIONAL COMPLEXES

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S30-S30
Author(s):  
Isabelle Hébert-Milette ◽  
Chloé Lévesque ◽  
Guy Charron ◽  
John Rioux
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Intestinal Inflammation ◽  
Protein Localization ◽  
Junctional Complex ◽  
Epithelial Permeability ◽  
3D Cultures ◽  
Apical Junctional Complex ◽  
Junction Protein ◽  
The Impact

Abstract Introduction Intestinal permeability is increased in unaffected 1st degree relatives of patients with inflammatory bowel disease (IBD), and is considered a risk factor for the development of IBD, likely increasing the interactions between intestinal microorganisms and the immune system. We recently reported that C1orf106, a gene located within a genomic region associated with IBD, regulates epithelial permeability. We further demonstrated that a rare coding variant within C1orf106 (p.Y333F) decreases protein stability and that lower levels of C1orf106 protein leads altered stability of adherens junctions (AJ) and to an increase in epithelial permeability. Hypothesis In addition to altering AJ, we believe that C1orf106 is also involved in the regulation of tight junction (TJ) formation, which also impacts epithelial permeability. Objectives The objectives of the project are to (a) validate the impact of C1orf106 on tight junctions and (b) verify the impact of C1orf106 IBD-associated variants on intestinal barrier integrity. Results We observed that knocking down the expression of C1orf106 in Caco-2 cells leads to a number of phenotypes in human epithelial monolayer (2D) and spheroid (3D) cultures that are associated with alterations in TJs. Specifically, when studying the dynamic reformation of TJ in 2D cultures after transient withdrawal of calcium, which is required for TJ stability, we observed that lower levels of C1orf106 resulted in (1) decreased recovery of barrier function as measured by transepithelial electrical resistance (TEER); (2) an alteration of tight junction protein localization; and (3) thickening of the circumferential actin belt. Moreover, in 3D cultures, we observed an altered spheroid formation associated with impaired epithelial polarization. In addition, our preliminary studies of human induced pluripotent stem cell (hiPSC)-derived epithelial cultures support that Y333F heterozygotes also have altered structure and function of their tight junctions. Conclusion Our observations indicate an important role of C1orf106 in apical junctional complex (AJC) formation likely mediated by a regulation of the circumferential actin belt. This can affect other functions of AJC, like the establishment of cell polarity. AJC formation is important for epithelial repair after an injury and its dysregulation impairs the formation of an impermeable epithelial barrier, which likely facilitates the passage of microorganisms and the induction and maintenance of intestinal inflammation.

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.

Increased Tyr phosphorylation of ZO-1 during modification of tight junctions between glomerular foot processes

1995 ◽  
Vol 268 (3) ◽  
pp. F514-F524 ◽  
Author(s):  
H. Kurihara ◽  
J. M. Anderson ◽  
M. G. Farquhar
Keyword(s):  
Tight Junction ◽  
Tyrosine Phosphorylation ◽  
Tight Junctions ◽  
Mesangial Cells ◽  
Basal Membrane ◽  
Cell Junctions ◽  
Phosphorylated Proteins ◽  
Cell Matrix ◽  
Rapid Changes ◽  
And Control

The slit diaphragms between the glomerular epithelial foot processes represent a variant of the tight junction that are rapidly replaced by typical tight junctions after perfusion with protamine sulfate (PS). To investigate the mechanism of signaling involved, tyrosine phosphorylation of glomerular proteins was analyzed in newborn, PS-treated, and control rats using antiphosphotyrosine immunoglobulin G. In glomeruli of normal adults, phosphotyrosine (Ptyr) staining was confined largely to mesangial cells by immunofluorescence, whereas in newborn and PS-treated rats, the Ptyr signal was dramatically increased in the glomerular epithelium. By immunogold labeling, it was found that newly phosphorylated proteins were concentrated along the newly formed tight junctions (cell-cell junctions) and the basal membrane of the foot processes (cell-matrix junctions). By immunoblotting, several prominent bands were detected with anti-Ptyr in glomerular lysates of controls; in PS-treated rats, additional bands were detected at 225, 180, and 100 kDa. The 225-kDa protein was identified as ZO-1 by immunoprecipitation with anti-ZO-1 followed by immunoblotting with anti-Ptyr. These findings indicate that ZO-1 is one of the targets for tyrosine phosphorylation after PS treatment. They indicate that phosphorylation of tight junction and other proteins occurs during the formation of tight junctions in glomeruli under circumstances where there are rapid changes in epithelial cell shape.

scholarly journals Rab13 regulates PKA signaling during tight junction assembly

2004 ◽  
Vol 165 (2) ◽  
pp. 175-180 ◽  
Author(s):  
Katja Köhler ◽  
Daniel Louvard ◽  
Ahmed Zahraoui
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Inhibitory Effect ◽  
Cell Junctions ◽  
Unknown Mechanism ◽  
Epithelial Tight Junctions ◽  
Actin Remodelling ◽  
Cell Cell

The GTPase Rab13 regulates the assembly of functional epithelial tight junctions (TJs) through a yet unknown mechanism. Here, we show that expression of the GTP-bound form of Rab13 inhibits PKA-dependent phosphorylation and TJ recruitment of the vasodilator-stimulated phosphoprotein, an actin remodelling protein. We demonstrate that Rab13GTP directly binds to PKA and inhibits its activity. Interestingly, activation of PKA abrogates the inhibitory effect of Rab13 on the recruitment of vasodilator-stimulated phosphoprotein, ZO-1, and claudin1 to cell–cell junctions. Rab13 is, therefore, the first GTPase that controls PKA activity and provides an unexpected link between PKA signaling and the dynamics of TJ assembly.

scholarly journals Translocation of Pseudomonas aeruginosa from the Intestinal Tract Is Mediated by the Binding of ExoS to an Na,K-ATPase Regulator, FXYD3

2010 ◽  
Vol 78 (11) ◽  
pp. 4511-4522 ◽  
Author(s):  
Jun Okuda ◽  
Naoki Hayashi ◽  
Masashi Okamoto ◽  
Shinji Sawada ◽  
Shu Minagawa ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Tight Junctions ◽  
Intestinal Tract ◽  
Transmembrane Domain ◽  
Epithelial Barrier ◽  
Comparative Genomic ◽  
Intestinal Epithelial ◽  
Intestinal Epithelial Barrier ◽  
Gut Colonization ◽  
Bacterial Penetration

ABSTRACT The intestinal tract is considered the most important reservoir of Pseudomonas aeruginosa in intensive care units (ICUs). Gut colonization by P. aeruginosa underlies the development of invasive infections such as gut-derived sepsis. Intestinal colonization by P. aeruginosa is associated with higher ICU mortality rates. The translocation of endogenous P. aeruginosa from the colonized intestinal tract is an important pathogenic phenomenon. Here we identify bacterial and host proteins associated with bacterial penetration through the intestinal epithelial barrier. We first show by comparative genomic hybridization analysis that the exoS gene, encoding the type III effector protein, ExoS, was specifically detected in a clinical isolate that showed higher virulence in silkworms following midgut injection. We further show using a silkworm oral infection model that exoS is required both for virulence and for bacterial translocation from the midgut to the hemolymph. Using a bacterial two-hybrid screen, we show that the mammalian factor FXYD3, which colocalizes with and regulates the function of Na,K-ATPase, directly binds ExoS. A pulldown assay revealed that ExoS binds to the transmembrane domain of FXYD3, which also interacts with Na,K-ATPase. Na,K-ATPase controls the structure and barrier function of tight junctions in epithelial cells. Collectively, our results suggest that ExoS facilitates P. aeruginosa penetration through the intestinal epithelial barrier by binding to FXYD3 and thereby impairing the defense function of tight junctions against bacterial penetration.

scholarly journals The HIF target ATG9A is essential for epithelial barrier function and tight junction biogenesis

2020 ◽  
Vol 31 (20) ◽  
pp. 2249-2258
Author(s):  
Alexander S. Dowdell ◽  
Ian M. Cartwright ◽  
Matthew S. Goldberg ◽  
Rachael Kostelecky ◽  
Tyler Ross ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Tight Junction ◽  
Barrier Function ◽  
Intestinal Epithelial Cells ◽  
Hypoxia Inducible Factor ◽  
Epithelial Barrier ◽  
Adaptation To Hypoxia ◽  
Intestinal Epithelial ◽  
Hypoxia Response ◽  
Epithelial Barrier Function

The transcription factor hypoxia-inducible factor (HIF) mediates adaptation to hypoxia. We found that HIF regulates the autophagy protein ATG9A in intestinal epithelial cells. Subsequent knockdown of ATG9A resulted in tight junction mislocalization and cytoskeletal defects. These results suggest a link among the hypoxia response, autophagy, and junctional biogenesis.

scholarly journals In VitroPrevention ofSalmonellaLipopolysaccharide-Induced Damages in Epithelial Barrier Function by VariousLactobacillusStrains

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Chun-Yan Yeung ◽  
Jen-Shiu Chiang Chiau ◽  
Wai-Tao Chan ◽  
Chun-Bin Jiang ◽  
Mei-Lien Cheng ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Mononuclear Cells ◽  
Intestinal Epithelial Cells ◽  
Barrier Properties ◽  
Bacterial Invasion ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Peripheral Blood Mononuclear

Background.Lactobacillusshows beneficial anti-inflammatory effects onSalmonellainfection. The maintenance of the tight junction (TJ) integrity plays an importance role in avoiding bacterial invasion. WhetherLactobacilluscould be used to regulate the TJ protein expression and distribution in inflamed intestinal epithelial cells was determined.Methods. Using the transwell coculture model,Salmonellalipopolysaccharide (LPS) was apically added to polarized Caco-2 cells cocultured with peripheral blood mononuclear cells in the basolateral compartment. LPS-stimulated Caco-2 cells were incubated with variousLactobacillusstrains. TJ integrity was determined by measuring transepithelial electrical resistance across Caco-2 monolayer. Expression and localization of TJ proteins (zonula-occludens- (ZO-) 1) were determined by Western blot and immunofluorescence microscopy.Results. Various strains ofLactobacilluswere responsible for the different modulations of cell layer integrity. LPS was specifically able to disrupt epithelial barrier and change the location of ZO-1. Our data demonstrate thatLactobacilluscould attenuate the barrier disruption of intestinal epithelial cells caused bySalmonellaLPS administration. We showed thatLactobacillusstrains are associated with the maintenance of the tight junction integrity and appearance.Conclusion. In this study we provide insight that live probiotics could improve epithelial barrier properties and this may explain the potential mechanism behind their beneficial effectin vivo.

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