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.

scholarly journals CRB3 Binds Directly to Par6 and Regulates the Morphogenesis of the Tight Junctions in Mammalian Epithelial Cells

2004 ◽  
Vol 15 (3) ◽  
pp. 1324-1333 ◽  
Author(s):  
Céline Lemmers ◽  
Didier Michel ◽  
Lydie Lane-Guermonprez ◽  
Marie-Hélène Delgrossi ◽  
Emmanuelle Médina ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Transmembrane Protein ◽  
Direct Interaction ◽  
Extracellular Domain ◽  
Cytoplasmic Domain ◽  
Epithelial Morphogenesis ◽  
Neurotrophin Receptor ◽  
Tight Junction Formation

Crumbs is an apical transmembrane protein crucial for epithelial morphogenesis in Drosophila melanogaster embryos. A protein with all the characteristics for a Crumbs homologue has been identified from patients suffering from retinitis pigmentosa group 12, but this protein (CRB1) is only expressed in retina and some parts of the brain, both in human and mouse. Here, we describe CRB3, another Crumbs homologue that is preferentially expressed in epithelial tissues and skeletal muscles in human. CRB3 shares the conserved cytoplasmic domain with other Crumbs but exhibits a very short extracellular domain without the EGF- and laminin A-like G repeats present in the other Crumbs. CRB3 is localized to the apical and subapical area of epithelial cells from the mouse and human intestine, suggesting that it could play a role in epithelial morphogenesis. Indeed, expression of CRB3 or of a chimera containing the extracellular domain of the neurotrophin receptor p75NTR and the transmembrane and cytoplasmic domains of CRB3 led to a slower development of functional tight junctions in Madin-Darby canine kidney cells. This phenotype relied on the presence of CRB3 four last amino acids (ERLI) that are involved in a direct interaction with Par6, a regulator of epithelial polarity and tight junction formation. Thus, CRB3, through its cytoplasmic domain and its interactors, plays a role in apical membrane morphogenesis and tight junction regulation.

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 JAM4, a Junctional Cell Adhesion Molecule Interacting with a Tight Junction Protein, MAGI-1

2003 ◽  
Vol 23 (12) ◽  
pp. 4267-4282 ◽  
Author(s):  
Susumu Hirabayashi ◽  
Makiko Tajima ◽  
Ikuko Yao ◽  
Wataru Nishimura ◽  
Hiroki Mori ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Tight Junctions ◽  
Adhesion Molecule ◽  
Intestinal Epithelial Cells ◽  
Cell Junctions ◽  
Intestinal Epithelial ◽  
Cell Contacts ◽  
L Cells ◽  
Junction Protein

ABSTRACT MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1-binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bind to MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells.

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.

scholarly journals The Ras Target AF-6 Interacts with ZO-1 and Serves as a Peripheral Component of Tight Junctions in Epithelial Cells

1997 ◽  
Vol 139 (3) ◽  
pp. 785-795 ◽  
Author(s):  
Takaharu Yamamoto ◽  
Naozumi Harada ◽  
Kyoko Kano ◽  
Shin-ichiro Taya ◽  
Eli Canaani ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junctions ◽  
Adherens Junctions ◽  
Pdz Domain ◽  
Cell Junctions ◽  
Cell Contact ◽  
Fusion Partner ◽  
Cell Contacts ◽  
Contact Sites ◽  
Cell Cell

The dynamic rearrangement of cell–cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell–cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell–cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell–cell contacts and found that AF-6 accumulated at the cell–cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell–cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell–cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell–cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell–cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.

Microtubule dynamics and Rac-1 signaling independently regulate barrier function in lung epithelial cells

2007 ◽  
Vol 293 (5) ◽  
pp. L1321-L1331 ◽  
Author(s):  
Magdalena J. Lorenowicz ◽  
Mar Fernandez-Borja ◽  
Anne-Marieke D. van Stalborch ◽  
Marian A. J. A. van Sterkenburg ◽  
Pieter S. Hiemstra ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Barrier Function ◽  
Lung Epithelial Cells ◽  
Cell Junctions ◽  
Epithelial Barrier ◽  
Cell Contact ◽  
Epithelial Barrier Function ◽  
Lung Epithelial ◽  
Cell Cell

Cadherin-mediated cell-cell adhesion controls the morphology and function of epithelial cells and is a critical component of the pathology of chronic inflammatory disorders. Dynamic interactions between cadherins and the actin cytoskeleton are required for stable cell-cell contact. Besides actin, microtubules also target intercellular, cadherin-based junctions and contribute to their formation and stability. Here, we studied the role of microtubules in conjunction with Rho-like GTPases in the regulation of lung epithelial barrier function using real-time monitoring of transepithelial electrical resistance. Unexpectedly, we found that disruption of microtubules promotes epithelial cell-cell adhesion. This increase in epithelial barrier function is accompanied by the accumulation of β-catenin at cell-cell junctions, as detected by immunofluorescence. Moreover, we found that the increase in cell-cell contact, induced by microtubule depolymerization, requires signaling through a RhoA/Rho kinase pathway. The Rac-1 GTPase counteracts this pathway, because inhibition of Rac-1 signaling rapidly promotes epithelial barrier function, in a microtubule- and RhoA-independent fashion. Together, our data suggest that microtubule-RhoA-mediated signaling and Rac-1 control lung epithelial integrity through counteracting independent pathways.

Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation

2002 ◽  
Vol 115 (12) ◽  
pp. 2485-2495 ◽  
Author(s):  
Tomonori Hirose ◽  
Yasushi Izumi ◽  
Yoji Nagashima ◽  
Yoko Tamai-Nagai ◽  
Hidetake Kurihara ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Mdck Cells ◽  
C Elegans ◽  
Tight Junction Formation ◽  
Junction Formation ◽  
Epithelial Tight Junction ◽  
Binding Sequence ◽  
Cell Cell

The mammalian protein ASIP/PAR-3 interacts with atypical protein kinase C isotypes (aPKC) and shows overall sequence similarity to the invertebrate proteins C. elegans PAR-3 and Drosophila Bazooka, which are crucial for the establishment of polarity in various cells. The physical interaction between ASIP/PAR-3 and aPKC is also conserved in C. elegans PAR-3 and PKC-3 and in Drosophila Bazooka and DaPKC. In mammals, ASIP/PAR-3 colocalizes with aPKC and concentrates at the tight junctions of epithelial cells, but the biological meaning of ASIP/PAR-3 in tight junctions remains to be clarified. In the present study, we show that ASIP/PAR-3 staining distributes to the subapical domain of epithelial cell-cell junctions, including epithelial cells with less-developed tight junctions, in clear contrast with ZO-1, another tight-junction-associated protein, the staining of which is stronger in cells with well-developed tight junctions. Consistently, immunogold electron microscopy revealed that ASIP/PAR-3 concentrates at the apical edge of tight junctions, whereas ZO-1 distributes alongside tight junctions. To clarify the meaning of this characteristic localization of ASIP, we analyzed the effects of overexpressed ASIP/PAR-3 on tight junction formation in cultured epithelial MDCK cells. The induced overexpression of ASIP/PAR-3, but not its deletion mutant lacking the aPKC-binding sequence, promotes cell-cell contact-induced tight junction formation in MDCK cells when evaluated on the basis of transepithelial electrical resistance and occludin insolubilization. The significance of the aPKC-binding sequence in tight junction formation is also supported by the finding that the conserved PKC-phosphorylation site within this sequence,ASIP-Ser827, is phosphorylated at the most apical tip of cell-cell contacts during the initial phase of tight junction formation in MDCK cells. Together,our present data suggest that ASIP/PAR-3 regulates epithelial tight junction formation positively through interaction with aPKC.

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 Mechanosensitive calcium signaling promotes epithelial tight junction remodeling by activating RhoA

2021 ◽  
Author(s):  
Saranyaraajan Varadarajan ◽  
Rachel E. Stephenson ◽  
Eileen R. Misterovich ◽  
Jessica L. Wu ◽  
Ivan S. Erofeev ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intracellular Calcium ◽  
Tight Junctions ◽  
Calcium Influx ◽  
Cell Junctions ◽  
Mechanical Stimuli ◽  
Local Repair ◽  
Transient Activation ◽  
Epithelial Tight Junction ◽  
Sense And Respond

Epithelia maintain an effective barrier by remodeling cell-cell junctions in response to mechanical stimuli. Cells often respond to mechanical stress through activating RhoA and remodeling actomyosin. Previously, we found that local leaks in the barrier are rapidly repaired by localized, transient activation of RhoA – ″Rho flares″ – but how Rho flares are initiated remains unknown. Here, we discovered that intracellular calcium flashes occur in Xenopus laevis epithelial cells undergoing Rho flare-mediated remodeling of tight junctions. Calcium flashes originate at the site of barrier leaks and propagate into the cell. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSC) reduced the amplitude of calcium flashes and diminished the activation of Rho flares. Furthermore, MSC-dependent calcium influx was necessary to maintain global barrier function by regulating local repair of tight junctions through efficient junction contraction. We propose that MSC-dependent calcium flashes are an important mechanism allowing epithelial cells to sense and respond to local leaks induced by mechanical stimuli.

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