scholarly journals PALS1 Regulates E-Cadherin Trafficking in Mammalian Epithelial Cells

2007 ◽  
Vol 18 (3) ◽  
pp. 874-885 ◽  
Author(s):  
Qian Wang ◽  
Xiao-Wei Chen ◽  
Ben Margolis
Keyword(s):  
Tight Junction ◽  
Adherens Junction ◽  
Cell Periphery ◽  
Madin Darby Canine Kidney ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Evolutionarily Conserved ◽  
Junction Formation ◽  
Darby Canine Kidney ◽  
E Cadherin

Protein Associated with Lin Seven 1 (PALS1) is an evolutionarily conserved scaffold protein that targets to the tight junction in mammalian epithelia. Prior work in our laboratory demonstrated that the knockdown of PALS1 in Madin Darby canine kidney cells leads to tight junction and polarity defects. We have created new PALS1 stable knockdown cell lines with more profound reduction of PALS1 expression, and a more severe defect in tight junction formation was observed. Unexpectedly, we also observed a severe adherens junction defect, and both defects were corrected when PALS1 wild type and certain PALS1 mutants were expressed in the knockdown cells. We found that the adherens junction structural component E-cadherin was not effectively delivered to the cell surface in the PALS1 knockdown cells, and E-cadherin puncta accumulated in the cell periphery. The exocyst complex was also found to be mislocalized in PALS1 knockdown cells, potentially explaining why E-cadherin trafficking is disrupted. Our results suggest a broad and evolutionarily conserved role for the tight junction protein PALS1 in the biogenesis of adherens junction.

scholarly journals Glucocorticoid-Induced Degradation of Glycogen Synthase Kinase-3 Protein Is Triggered by Serum- and Glucocorticoid-Induced Protein Kinase and Akt Signaling and Controls β-Catenin Dynamics and Tight Junction Formation in Mammary Epithelial Tumor Cells

2007 ◽  
Vol 21 (10) ◽  
pp. 2403-2415 ◽  
Author(s):  
Kim L. Failor ◽  
Yelena Desyatnikov ◽  
Lindsay A. Finger ◽  
Gary L. Firestone
Keyword(s):  
Protein Kinase ◽  
Tight Junction ◽  
Glycogen Synthase ◽  
Adherens Junction ◽  
Mammary Epithelial ◽  
Dominant Negative ◽  
Cell Periphery ◽  
Epithelial Tumor ◽  
Tight Junction Formation ◽  
Junction Formation

Abstract Glucocorticoid hormones stimulate adherens junction and tight junction formation in Con8 mammary epithelial tumor cells and induce the production of a stable nonphosphorylated β-catenin protein localized exclusively to the cell periphery. Glycogen synthase kinase-3 (GSK3) phosphorylation of β-catenin is known to trigger the degradation of this adherens junction protein, suggesting that steroid-activated cascades may be targeting this protein kinase. We now demonstrate that treatment with the synthetic glucocorticoid dexamethasone induces the ubiquitin-26S proteasome-mediated degradation of GSK3 protein with no change in GSK3 transcript levels. In transfected cells, deletion of the N-terminal nine amino acids or mutation of the serine-9 phosphorylation site on GSK3-β prevented its glucocorticoid-induced degradation. Expression of stabilized GSK3 mutant proteins ablated the glucocorticoid-induced tight junction sealing and resulted in production of a nonphosphorylated β-catenin that localizes to both the nucleus and the cell periphery in steroid-treated cells. Serine-9 on GSK3 can be phosphorylated by Sgk (serum- and glucocorticoid-induced protein kinase) and by Akt. Expression of dominant-negative forms of either Sgk- or Akt-inhibited glucocorticoid induced GSK3 ubiquitination and degradation and disrupted the dexamethasone-induced effects on β-catenin dynamics. Furthermore, the steroid-induced tight junction sealing is attenuated in cells expressing dominant-negative forms of either Sgk or Akt, although the effect of blunting Sgk signaling was significantly greater. Taken together, we have uncovered a new cellular cascade in which Sgk and Akt trigger the glucocorticoid-regulated phosphorylation, ubiquitination, and degradation of GSK3, which alters β-catenin dynamics, leading to the formation of adherens junctions and tight junction sealing.

Tight junction protein cingulin is expressed by maternal and embryonic genomes during early mouse development

Development ◽  
1993 ◽  
Vol 117 (3) ◽  
pp. 1145-1151 ◽  
Author(s):  
Q. Javed ◽  
T.P. Fleming ◽  
M. Hay ◽  
S. Citi
Keyword(s):  
Tight Junction ◽  
Cell Mass ◽  
Preimplantation Embryos ◽  
Cell Contact ◽  
Mouse Development ◽  
Early Cleavage ◽  
Cell Stage ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Junction Formation

The expression of the tight junction peripheral membrane protein, cingulin (140 × 10(3) M(r), was investigated in mouse eggs and staged preimplantation embryos by immunoblotting and immunoprecipitation. Polyclonal antibody to chicken brush cingulin detected a single 140 × 10(3) M(r) protein in immunoblots of unfertilised eggs and all preimplantation stages. Relative protein levels were high in eggs and early cleavage stages, declined during later cleavage and increased again in expanding blastocysts. Quantitative immunoprecipitation of metabolically labelled eggs and staged embryos also revealed a biphasic pattern for cingulin synthesis with relative net levels being high in unfertilised eggs, minimal during early cleavage, rising 2.3-fold specifically at the onset of compaction (8-cell stage, when tight junction formation begins), and increasing further at a linear rate during morula and blastocyst stages. Cingulin synthesis in eggs is not influenced by fertilisation (or aging, if unfertilised), but this level declines sharply after first cleavage. These results indicate that cingulin is expressed by both maternal and embryonic genomes. The turnover of maternal cingulin (unfertilised eggs) and embryonic cingulin at a stage before tight junction formation begins (4-cell stage) is higher (t1/2 approximately 4 hours) than cingulin synthesised after tight junction formation (blastocysts; t1/2 approximately 10 hours). This increase in cingulin stability is reversed in the absence of extracellular calcium. Cingulin synthesis is also tissue-specific in blastocysts, being up-regulated in trophectoderm and down-regulated in the inner cell mass. Taken together, the results suggest that (i) cingulin may have a role during oogenesis and (ii) cell-cell contact patterns regulate cingulin biosynthesis during early morphogenesis, contributing to lineage-specific epithelial maturation.

scholarly journals Restoration of Tight Junction Structure and Barrier Function by Down-Regulation of the Mitogen-activated Protein Kinase Pathway in Ras-transformed Madin-Darby Canine Kidney Cells

2000 ◽  
Vol 11 (3) ◽  
pp. 849-862 ◽  
Author(s):  
Yan-hua Chen ◽  
Qun Lu ◽  
Eveline E. Schneeberger ◽  
Daniel A. Goodenough
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Tight Junctions ◽  
Mitogen Activated Protein Kinase ◽  
Madin Darby Canine Kidney ◽  
Mitogen Activated Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell–cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin–mediated cell–cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell–cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell–cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.

scholarly journals Junctional Adhesion Molecule-A Is Critical for the Formation of Pseudocanaliculi and Modulates E-cadherin Expression in Hepatic Cells

2007 ◽  
Vol 282 (38) ◽  
pp. 28137-28148 ◽  
Author(s):  
Genevieve Konopka ◽  
Jackie Tekiela ◽  
Moriah Iverson ◽  
Clive Wells ◽  
Stephen A. Duncan
Keyword(s):  
Tight Junction ◽  
Adhesion Molecule ◽  
Cell Morphology ◽  
Adherens Junction ◽  
Tight Junction Proteins ◽  
Hepatic Cells ◽  
Junction Protein ◽  
Striking Change ◽  
Junction Proteins ◽  
E Cadherin

Hepatocytes are polarized epithelial cells whose function depends upon their ability to distinguish between the apical and basolateral surfaces that are located at intercellular tight junctions. It has been proposed that the signaling cascades originating at these junctions influence cellular activity by controlling gene expression in the cell nucleus. To assess the validity of this proposal with regard to hepatocytes, we depleted expression of the tight junction protein junctional adhesion molecule-A (JAM-A) in the HepG2 human hepatocellular carcinoma cell line. Reduction of JAM-A resulted in a striking change in cell morphology, with cells forming sheets 1-2 cells thick instead of the normal multilayered clusters. In the absence of JAM-A, other tight junction proteins were mislocalized, and pseudocanaliculi, which form the apical face of the hepatocyte, were consequently absent. There was a strong transcriptional induction of the adherens junction protein E-cadherin in cells with reduced levels of JAM-A. This increase in E-cadherin was partially responsible for the observed alterations in cell morphology and mislocalization of tight junction proteins. We therefore propose the existence of a novel mechanism of cross-talk between specific components of tight and adherens junctions that can be utilized to regulate adhesion between hepatic cells.

scholarly journals Functional characterization and localization of a gill-specific claudin isoform in Atlantic salmon

2012 ◽  
Vol 302 (2) ◽  
pp. R300-R311 ◽  
Author(s):  
M. B. Engelund ◽  
A. S. L. Yu ◽  
J. Li ◽  
S. S. Madsen ◽  
N. J. Færgeman ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Atlantic Salmon ◽  
Protein Expression ◽  
Tight Junction ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Junction Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Cell Cell

Claudins are the major determinants of paracellular epithelial permeability in multicellular organisms. In Atlantic salmon ( Salmo salar L.), we previously found that mRNA expression of the abundant gill-specific claudin 30 decreases during seawater (SW) acclimation, suggesting that this claudin is associated with remodeling of the epithelium during salinity change. This study investigated localization, protein expression, and function of claudin 30. Confocal microscopy showed that claudin 30 protein was located at cell-cell interfaces in the gill filament in SW- and fresh water (FW)-acclimated salmon, with the same distribution, overall, as the tight junction protein ZO-1. Claudin 30 was located at the apical tight junction interface and in cell membranes deeper in the epithelia. Colocalization with the α-subunit of the Na+-K+-ATPase was negligible, suggesting limited association with mitochondria-rich cells. Immunoblotting of gill samples showed lower claudin 30 protein expression in SW than FW fish. Retroviral transduction of claudin 30 into Madin-Darby canine kidney cells resulted in a decreased conductance of 19%. The decreased conductance correlated with a decreased permeability of the cell monolayer to monovalent cations, whereas permeability to chloride was unaffected. Confocal microscopy revealed that claudin 30 was expressed in the lateral membrane, as well as in tight junctions of Madin-Darby canine kidney cells, thereby paralleling the findings in the native gill. This study suggests that claudin 30 functions as a cation barrier between pavement cells in the gill and also has a general role in cell-cell adhesion in deeper layers of the epithelium.

scholarly journals Phosphorylation of the tight-junction protein ZO-1 in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance

1989 ◽  
Vol 263 (2) ◽  
pp. 597-599 ◽  
Author(s):  
B R Stevenson ◽  
J M Anderson ◽  
I D Braun ◽  
M S Mooseker
Keyword(s):  
Tight Junction ◽  
Specific Protein ◽  
Resistance Strain ◽  
Transepithelial Resistance ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Junction Protein ◽  
Junctional Permeability ◽  
Darby Canine Kidney ◽  
Canine Kidney

A comparison was made of the phosphate content of the tight-junction-specific protein ZO-1 in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance, a parameter reflective of tight-junctional permeability. Analysis revealed that the ZO-1 from the low-resistance strain contained approximately twice as much phosphate as that from the high-resistance strain.

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 PATJ regulates tight junction formation and polarity in mammalian epithelial cells

2005 ◽  
Vol 168 (5) ◽  
pp. 705-711 ◽  
Author(s):  
Kunyoo Shin ◽  
Sam Straight ◽  
Ben Margolis
Keyword(s):  
Tight Junction ◽  
Protein Complexes ◽  
Transmembrane Protein ◽  
Complex Function ◽  
Cell Polarization ◽  
Tight Junction Protein ◽  
Apical Region ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Junction Formation

Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs–PALS1–PATJ complex function in epithelial polarity.

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