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 The MAGUK Protein MPP7 Binds to the Polarity Protein hDlg1 and Facilitates Epithelial Tight Junction Formation

2007 ◽  
Vol 18 (5) ◽  
pp. 1744-1755 ◽  
Author(s):  
Volker M. Stucke ◽  
Evy Timmerman ◽  
Joel Vandekerckhove ◽  
Kris Gevaert ◽  
Alan Hall
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Tight Junctions ◽  
Cell Polarity ◽  
Functional Organization ◽  
Epithelial Cell Polarity ◽  
Tight Junction Formation ◽  
Evolutionarily Conserved ◽  
Junction Formation ◽  
Epithelial Tight Junction

Three groups of evolutionarily conserved proteins have been implicated in the establishment of epithelial cell polarity: the apically-localized proteins of the Par (Par3-Par6-aPKC-Cdc42) and Crumbs groups (Crb3-PALS1-PATJ) and the basolaterally localized proteins of the Dlg group (Dlg1-Scribble-Lgl). During epithelial morphogenesis, these proteins participate in a complex network of interdependent interactions that define the position and functional organization of adherens junctions and tight junctions. However, the biochemical pathways through which they control polarity are poorly understood. In this study, we identify an interaction between endogenous hDlg1 and MPP7, a previously uncharacterized MAGUK-p55 subfamily member. We find that MPP7 targets to the lateral surface of epithelial cells via its L27N domain, through an interaction with hDlg1. Loss of either hDlg1 or MPP7 from epithelial Caco-2 cells results in a significant defect in the assembly and maintenance of functional tight junctions. We conclude that the formation of a complex between hDlg1 and MPP7 promotes epithelial cell polarity and tight junction formation.

scholarly journals The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy.

1993 ◽  
Vol 121 (3) ◽  
pp. 491-502 ◽  
Author(s):  
M Itoh ◽  
A Nagafuchi ◽  
S Yonemura ◽  
T Kitani-Yasuda ◽  
S Tsukita ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Immunoelectron Microscopy ◽  
Intestinal Epithelial Cells ◽  
Microscopic Level ◽  
Junction Zone ◽  
Intestinal Epithelial ◽  
Tight Junction Formation ◽  
Junction Formation

We previously identified a 220-kD constitutive protein of the plasma membrane undercoat which colocalizes at the immunofluorescence microscopic level with cadherins and occurs not only in epithelial M., S. Yonemura, A. Nagafuchi, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). To clarify the nature and possible functions of this protein, we cloned its full-length cDNA and sequenced it. Unexpectedly, we found mouse 220-kD protein to be highly homologous to rat protein ZO-1, only a part of which had been already sequenced. This relationship was confirmed by immunoblotting with anti-ZO-1 antibody. As protein ZO-1 was originally identified as a component exclusively underlying tight junctions in epithelial cells, where cadherins are not believed to be localized, we analyzed the distribution of cadherins and the 220-kD protein by ultrathin cryosection immunoelectron microscopy. We found that in non-epithelial cells lacking tight junctions cadherins and the 220-kD protein colocalize, whereas in epithelial cells (e.g., intestinal epithelial cells) bearing well-developed tight junctions cadherins and the 220-kD protein are clearly segregated into adherens and tight junctions, respectively. Interestingly, in epithelial cells such as hepatocytes, which tight junctions are not so well developed, the 220-kD protein is detected not only in the tight junction zone but also at adherens junctions. Furthermore, we show in mouse L cells transfected with cDNAs encoding N-, P-, E-cadherins that cadherins interact directly or indirectly with the 220-kD protein. Possible functions of the 220-kD protein (ZO-1) are discussed with special reference to the molecular mechanism for adherens and tight junction formation.

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 P059 GB004 drives protective effects on immune cells and epithelial cells using human ex vivo monolayer and co-culture systems

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S165-S166
Author(s):  
K Taylor Meadows ◽  
S Murphy ◽  
B G Levesque ◽  
L L Carter ◽  
L Salter-Cid
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Immune Cells ◽  
Target Genes ◽  
Protective Effects ◽  
Barrier Integrity ◽  
Culture Systems ◽  
Tight Junction Formation ◽  
Junction Formation

Abstract Background GB004 is a small molecule that stabilizes hypoxia inducible factor (HIF-1α), a key transcription factor involved in the cellular responses at the intersection of hypoxia and inflammation. GB004 induction of HIF-1a target genes improves barrier integrity and reduces barrier permeability in rodent models of colitis. Here we investigated gene expression and secretion of soluble factors elicited by GB004 treatment on immune cells and epithelial cells, human monolayer assays and immune co-culture studies to further explore its protective mechanism. Methods In the BioMAP human co-culture assay, cells were treated with GB004 one hour prior to stimulation and incubated for 24–168 hours. Biomarkers, cell viability and proliferation were assessed. Repligut human stem-cell-derived monolayer platforms were used to assess GB004 effects under unstimulated or TNFa stimulation conditions. Barrier integrity was assessed by Transepithelial Endothelial Electric Resistance (TEER), HIF-1a target genes were assessed in cell lysates, and tight junction formation and epithelial monolayer viability were investigated by immunofluorescence staining. Results GB004 demonstrated activity in 11 of 12 systems in the BioMAP human co-culture assay, which covers a range of disease-relevant immune and non-immune mechanisms under stimulation conditions. Collectively, GB004 treatment led to changes in biomarkers associated with inflammation (sTNFa, IL-8, MCP-1, CXCL11), immunomodulation (sIL-17F, sIL-17A), and tissue remodeling (collagen I, collagen IV). GB004 treatment also reduced the proliferative activity in the in vitro lymphocyte assay systems. In the human Repligut intestinal epithelium assay, GB004 significantly reduced cell death and improved barrier integrity in response to pro-inflammatory cytokines. The protective role of GB004 on barrier integrity was further supported by immunostaining experiments demonstrating that GB004 treatment protected epithelial cells from TNFa-induced cell apoptosis, improving both epithelial cell number and tight junction protein ZO-1 staining. Conclusion GB004 modulates key anti-inflammatory and immunomodulatory activities in human co-culture systems. Furthermore, GB004 demonstrates protective effects on human derived monolayer cultures by reducing cell death, promoting tight junction formation, and improving barrier integrity. Targeting both barrier function and local colonic inflammation represents a multi-faceted approach to treatment of inflammatory bowel disease. A phase 2 clinical study of GB004 is ongoing in patients with ulcerative colitis (NCT04556383). Sponsored by GB004, Inc., a wholly owned subsidiary of Gossamer Bio, Inc.

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.

Effect of temperature on the assembly of tight junctions and on the mobility of lipids in membranes of HT29 cells

1990 ◽  
Vol 97 (1) ◽  
pp. 119-125
Author(s):  
E. Cohen ◽  
I. Ophir ◽  
Y.I. Henis ◽  
A. Bacher ◽  
Y. Ben Shaul
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Temperature Dependence ◽  
Tight Junctions ◽  
Membrane Lipids ◽  
Effect Of Temperature ◽  
Proteolytic Activation ◽  
Tight Junction Formation ◽  
Threshold Temperatures ◽  
Junction Formation

In the human colon adenocarcinoma cell line HT29, tight junctions can be induced by treatment with appropriate proteases or salt solutions. The temperature dependence of induced tight junction formation is characterized by a marked sigmoidal behavior. The different methods of induction used in this study were characterized by threshold temperatures ranging from 15 to 32 degrees C. Fluorescence photobleaching recovery measurements of the lateral diffusion of a fluorescent phospholipid probe in the cellular plasma membrane gave no evidence for a phase transition or for alteration in the organization of membrane lipids in lateral domains in the temperature range between 0 and 37 degrees C. Moreover, dynamic parameters of the probe in the plasma membrane did not change substantially on mild treatment with trypsin. Thus, the temperature dependence of tight junction formation is not dictated by the bulk properties of the cytoplasmic membrane lipids. The observed temperature dependence suggests that the assembly of tight junctions is a cooperative process, which may involve conformational rearrangement in a protein precursor subsequent to its proteolytic activation.

scholarly journals Possible Involvement of Phosphorylation of Occludin in Tight Junction Formation

1997 ◽  
Vol 137 (6) ◽  
pp. 1393-1401 ◽  
Author(s):  
Akira Sakakibara ◽  
Mikio Furuse ◽  
Mitinori Saitou ◽  
Yuhko Ando-Akatsuka ◽  
Shoichiro Tsukita
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Immunofluorescence Microscopy ◽  
Insoluble Fraction ◽  
Band Shift ◽  
Basolateral Membranes ◽  
Tight Junction Formation ◽  
Phosphorylated Form ◽  
Upward Shift ◽  
Junction Formation

Occludin is an integral membrane protein localizing at tight junctions in epithelial and endothelial cells. Occludin from confluent culture MDCK I cells resolved as several (>10) bands between 62 and 82 kD in SDS-PAGE, of which two or three bands of the lowest Mr were predominant. Among these bands, the lower predominant bands were essentially extracted with 1% NP-40, whereas the other higher Mr bands were selectively recovered in the NP-40–insoluble fraction. Alkaline phosphatase treatment converged these bands of occludin both in NP-40–soluble and -insoluble fractions into the lowest Mr band, and phosphoamino acid analyses identified phosphoserine (and phosphothreonine weakly) in the higher Mr bands of occludin. These findings indicated that phosphorylation causes an upward shift of occludin bands and that highly phosphorylated occludin resists NP-40 extraction. When cells were grown in low Ca medium, almost all occludin was NP-40 soluble. Switching from low to normal Ca medium increased the amount of NP-40–insoluble occludin within 10 min, followed by gradual upward shift of bands. This insolubilization and the band shift correlated temporally with tight junction formation detected by immunofluorescence microscopy. Furthermore, we found that the anti–chicken occludin mAb, Oc-3, did not recognize the predominant lower Mr bands of occludin (non- or less phosphorylated form) but was specific to the higher Mr bands (phosphorylated form) on immunoblotting. Immunofluorescence microscopy revealed that this mAb mainly stained the tight junction proper of intestinal epithelial cells, whereas other anti-occludin mAbs, which can recognize the predominant lower Mr bands, labeled their basolateral membranes (and the cytoplasm) as well as tight junctions. Therefore, we conclude that non- or less phosphorylated occludin is distributed on the basolateral membranes and that highly phosphorylated occludin is selectively concentrated at tight juctions as the NP-40–insoluble form. These findings suggest that the phosphorylation of occludin is a key step in tight junction assembly.

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