Inducible expression of claudin-1-myc but not occludin-VSV-G results in aberrant tight junction strand formation in MDCK cells

Occludin and 18 distinct members of the claudin family are tetra-span transmembrane proteins that are localized in cell-specific tight junctions (TJs). A previous study showed that expression of chick occludin in Madin-Darby canine kidney (MDCK) cells raised transepithelial electrical resistance (TER) and, paradoxically, increased mannitol flux. In the present study, we employed epitope tagged canine occludin expression, under the control of the tetracycline repressible transactivator, to determine the extent to which the unexpected parallel increase in TER and mannitol flux was related to a structural mismatch between avian and canine occludins, which are only 50% identical. To determine whether the paradoxical changes in permeability was specific to occludin, we assessed the effect of over-expressing epitope tagged murine claudin-1. Our data revealed that over-expression of either of the epitope tagged mammalian tight junction proteins increased TER, mannitol and FITC-dextran flux. We observed a 2- and up to 5.6-fold over-expression of occludin-VSV-G and claudin-1-myc, respectively, with no change in ZO-1, endogenous occludin or claudin-1 expression. Confocal microscopy revealed that occludin-VSV-G, claudin-1-myc and ZO-1 co-localized at the TJ. In addition, claudin-1-myc formed aberrant strands along the lateral cell surface without an underlying ZO-1 scaffold. In fracture labeled replicas these strands consisted of claudin-1-myc with little accompanying occludin. These observations suggest that in epithelial cells claudin-1 can assemble into TJ strands without the participation of either ZO-1 or occludin. The proximity of the myc tag to the COOH-terminal YV sequence of claudin-1 appeared to interfere with its interaction with ZO-1, since over-expression of non-tagged claudin-1 increased TER but had a minimal effect on solute flux and no aberrant strands formed. From our data we conclude that differences in structure between avian and mammalian occludin do not account for the observed paradoxical increase in mannitol flux. Levels of ZO-1 remained unchanged despite substantial increases in induced TJ integral protein expression, suggesting that an imbalance between levels of ZO-1 and occludin or claudin-1 leads to altered regulation of pores through which non-charged solute flux occurs. We suggest that ion and solute flux are differentially regulated at the TJ.

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Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells.

The Journal of Cell Biology ◽

10.1083/jcb.106.4.1141 ◽

1988 ◽

Vol 106 (4) ◽

pp. 1141-1149 ◽

Cited By ~ 235

Author(s):

J M Anderson ◽

B R Stevenson ◽

L A Jesaitis ◽

D A Goodenough ◽

M S Mooseker

Keyword(s):

Tight Junction ◽

Mdck Cell ◽

Mouse Liver ◽

Mdck Cells ◽

Cell Protein ◽

Scatchard Analysis ◽

Madin Darby Canine Kidney ◽

Mouse Tissues ◽

Darby Canine Kidney ◽

Canine Kidney

ZO-1, originally identified by mAb techniques, is the first protein shown to be specifically associated with the tight junction. Here we describe and compare the physical characteristics of ZO-1 from mouse liver and the Madin-Darby canine kidney (MDCK) epithelial cell line. The ZO-1 polypeptide has an apparent size of 225 kD in mouse tissues and 210 kD in canine-derived MDCK cells as determined by SDS-PAGE/immunoblot analysis. ZO-1 from both sources is optimally solubilized from isolated plasma membranes by either 6 M urea or high pH conditions; partial solubilization occurs with 0.3 M KCl. The nonionic detergents, Triton X-100 and octyl-beta-D-glucopyranoside, do not solubilize ZO-1. These solubility properties indicate that ZO-1 is a peripherally associated membrane protein. ZO-1 was purified to electrophoretic homogeneity from [35S]methionine metabolically labeled MDCK cells by a combination of gel filtration and immunoaffinity chromatography. Purified ZO-1 has an s20,w of 5.3 and Stokes radius of 8.6 nm. These values suggest that purified ZO-1 is an asymmetric monomeric molecule. Corresponding values for mouse liver ZO-1, characterized in impure protein extracts, were 6 s20,w and 9 nm. ZO-1 was shown to be a phosphoprotein in MDCK cells metabolically labeled with [32P]orthophosphate; analysis of phosphoamino acids from purified ZO-1 revealed only phosphoserine. ZO-1 epitope number was determined by Scatchard analysis of competitive and saturable binding of two different 125I-mAbs to SDS-solubilized proteins from liver and MDCK cells immobilized on nitrocellulose. Saturation binding occurs at 26 ng mAb/mg liver and 63 ng/mg of MDCK cell protein. This is equivalent to 30,000 ZO-1 molecules per MDCK cell assuming a single epitope/ZO-1 molecule.

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Inducible expression of Snail selectively increases paracellular ion permeability and differentially modulates tight junction proteins

AJP Cell Physiology ◽

10.1152/ajpcell.00175.2005 ◽

2005 ◽

Vol 289 (4) ◽

pp. C1002-C1014 ◽

Cited By ~ 61

Author(s):

Fabio Carrozzino ◽

Priscilla Soulié ◽

Denise Huber ◽

Noury Mensi ◽

Lelio Orci ◽

...

Keyword(s):

Tight Junction ◽

Epithelial Mesenchymal Transition ◽

Mdck Cells ◽

Constitutive Expression ◽

Inducible Expression ◽

Epithelial Barrier Function ◽

Mesenchymal Transition ◽

Ionic Permeability ◽

Cell Monolayers ◽

Darby Canine Kidney

Constitutive expression of the transcription factor Snail was previously shown to trigger complete epithelial-mesenchymal transition (EMT). The aim of this study was to determine whether inducible expression of Snail could modify epithelial properties without eliciting full mesenchymal conversion. For this purpose, we expressed mouse Snail (mSnail) cDNA in Madin-Darby canine kidney (MDCK) cells under the control of a doxycycline-repressible transactivator. Inducible expression of Snail did not result in overt EMT but induced a number of phenotypic alterations of MDCK cells, the most significant of which was the absence of fluid-filled blisterlike structures called “domes.” To understand the mechanisms responsible for dome suppression, we assessed the effect of mSnail expression on epithelial barrier function. Although mSnail did not alter tight junction (TJ) organization and permeability to uncharged solutes, it markedly decreased transepithelial electrical resistance. In light of these findings, we evaluated the ability of MDCK cell monolayers to maintain ionic gradients and found that expression of mSnail selectively increases Na+ and Cl− permeability. Analysis of the expression of claudins, transmembrane proteins that regulate TJ ionic permeability, showed that mSnail induces a moderate decrease in claudin-2 and a substantial decrease in claudin-4 and -7 expression. Together, these results suggest that induction of mSnail selectively increases the ionic permeability of TJs by differentially modulating the expression of specific claudins.

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The PIKfyve Inhibitor YM201636 Blocks the Continuous Recycling of the Tight Junction Proteins Claudin-1 and Claudin-2 in MDCK cells

PLoS ONE ◽

10.1371/journal.pone.0028659 ◽

2012 ◽

Vol 7 (3) ◽

pp. e28659 ◽

Cited By ~ 31

Author(s):

Joseph D. Dukes ◽

Paul Whitley ◽

Andrew D. Chalmers

Keyword(s):

Tight Junction ◽

Mdck Cells ◽

Tight Junction Proteins ◽

Junction Proteins

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Juxtacrine activation of EGFR regulates claudin expression and increases transepithelial resistance

AJP Cell Physiology ◽

10.1152/ajpcell.00274.2007 ◽

2007 ◽

Vol 293 (5) ◽

pp. C1660-C1668 ◽

Cited By ~ 37

Author(s):

Amar B. Singh ◽

Keisuke Sugimoto ◽

Punita Dhawan ◽

Raymond C. Harris

Keyword(s):

Egf Receptor ◽

Mdck Cells ◽

Transepithelial Resistance ◽

Stable Expression ◽

Heparin Binding ◽

Egf Receptors ◽

Physiological Importance ◽

Ion Substitution ◽

Darby Canine Kidney ◽

Junction Proteins

Heparin-binding (HB)-EGF, a ligand for EGF receptors, is synthesized as a membrane-anchored precursor that is potentially capable of juxtacrine activation of EGF receptors. However, the physiological importance of such juxtacrine signaling remains poorly described, due to frequent inability to distinguish effects mediated by membrane-anchored HB-EGF vs. mature “secreted HB-EGF.” In our studies, using stable expression of a noncleavable, membrane-anchored rat HB-EGF isoform (MDCKrat5aa cells) in Madin-Darby canine kidney (MDCK) II cells, we observed a significant increase in transepithelial resistance (TER). Similar significant increases in TER were observed on stable expression of an analogous, noncleavable, membrane-anchored human HB-EGF construct (MDCKhuman5aa cells). The presence of noncleavable, membrane-anchored HB-EGF led to alterations in the expression of selected claudin family members, including a marked decrease in claudin-2 in MDCKrat5aa cells compared with the control MDCK cells. Reexpression of claudin-2 in MDCKrat5aa cells largely prevented the increases in TER. Ion substitution studies indicated decreased paracellular ionic permeability of Na+ in MDCKrat5aa cells, further indicating that the altered claudin-2 expression mediated the increased TER seen in these cells. In a Ca2+-switch model, increased phosphorylation of EGF receptor and Akt was observed in MDCKrat5aa cells compared with the control MDCK cells, and inhibition of these pathways inhibited TER changes specifically in MDCKrat5aa cells. Therefore, we hypothesize that juxtacrine activation of EGFR by membrane-anchored HB-EGF may play an important role in the regulation of tight junction proteins and TER.

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Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance.

The Journal of Cell Biology ◽

10.1083/jcb.107.6.2401 ◽

1988 ◽

Vol 107 (6) ◽

pp. 2401-2408 ◽

Cited By ~ 166

Author(s):

B R Stevenson ◽

J M Anderson ◽

D A Goodenough ◽

M S Mooseker

Keyword(s):

Tight Junction ◽

Mdck Cells ◽

Freeze Fracture ◽

Transepithelial Resistance ◽

Madin Darby Canine Kidney ◽

Junctional Permeability ◽

Tight Junction Permeability ◽

Junction Structure ◽

Darby Canine Kidney ◽

Canine Kidney

The relationship of tight junction permeability to junction structure and composition was examined using two strains of Madin-Darby canine kidney (MDCK) cells (I and II) which differ greater than 30-fold in transepithelial resistance. This parameter is largely determined by paracellular, and hence junctional, permeability under most conditions. When these two strains of cells were grown on permeable filter supports, they formed monolayers with equivalent linear amounts of junction/area of monolayer. Ultrastructural analysis of these monolayers by thin section EM revealed no differences in overall cellular morphology or in tight junction organization. Morphometric analysis of freeze-fractured preparations indicated that the tight junctions of these two cell strains were similar in both number and density of junctional fibrils. Prediction of transepithelial resistance for the two strains from this freeze-fracture data and a published structure-function formulation (Claude, P. 1978, J. Memb. Biol. 39:219-232) yielded values (I = 26.5 omega/cm2, II = 35.7 omega/cm2) that were significantly lower than those observed (I = 2,500-5,000 omega/cm2, II = 50-70 omega/cm2). Consistent with these structural studies, a comparison of the distribution and cellular content of ZO-1, a polypeptide localized exclusively to the tight junction, revealed no significant differences in either the localization of ZO-1 or the amount of ZO-1 per micron of junction (I = 1,415 +/- 101 molecules/micron, II = 1,514 +/- 215 molecules/micron).

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Bidirectional regulation of tonicity-responsive enhancer binding protein in response to changes in tonicity

AJP Renal Physiology ◽

10.1152/ajprenal.2000.278.6.f1006 ◽

2000 ◽

Vol 278 (6) ◽

pp. F1006-F1012 ◽

Cited By ~ 99

Author(s):

Seung Kyoon Woo ◽

Stephen C. Dahl ◽

Joseph S. Handler ◽

H. Moo Kwon

Keyword(s):

Culture Medium ◽

Mdck Cells ◽

Binding Protein ◽

Nuclear Distribution ◽

Bidirectional Regulation ◽

Parallel Increase ◽

Enhancer Binding Protein ◽

Activated State ◽

The Stability ◽

Darby Canine Kidney

Tonicity-responsive enhancer binding protein (TonEBP) regulates transcription of tonicity responsive genes such as the sodium- myo-inositol cotransporter (SMIT), the sodium-chloride-betaine cotransporter (BGT1), and aldose reductase (AR). To characterize signals that activate TonEBP in Madin-Darby canine kidney (MDCK) cells, the abundance and nuclear distribution of TonEBP were studied after the osmolality of the culture medium was changed. Hypertonicity but not hyperosmolality is effective in activation of TonEBP as expected. Surprisingly, exposure to hypotonic medium leads to a dramatic downregulation of TonEBP both in abundance and nuclear distribution, indicating that under isotonic conditions, TonEBP is at a low-level activated state and can respond to both increase and decrease in tonicity. Additional experiments suggest that cellular ionic strength is the signal that initiates regulation of TonEBP. The increase in abundance of TonEBP is mediated by an increase in mRNA abundance and a parallel increase in synthesis of TonEBP. The stability of TonEBP mRNA is not affected by hypertonicity indicating that transcription plays a major role in the induction of TonEBP by hypertonicity.

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Nucleofection disrupts tight junction fence function to alter membrane polarity of renal epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00152.2010 ◽

2010 ◽

Vol 299 (5) ◽

pp. F1178-F1184 ◽

Cited By ~ 13

Author(s):

Di Mo ◽

Beth A. Potter ◽

Carol A. Bertrand ◽

Jeffrey D. Hildebrand ◽

Jennifer R. Bruns ◽

...

Keyword(s):

Tight Junction ◽

Mdck Cells ◽

Subsequent Development ◽

Initial Surface ◽

Apical Surface ◽

Surface Polarity ◽

Madin Darby Canine Kidney ◽

Polarized Secretion ◽

Fence Function ◽

Darby Canine Kidney

Here, we compared the effects of nucleofection and lipid-based approaches to introduce siRNA duplexes on the subsequent development of membrane polarity in kidney cells. Nucleofection of Madin-Darby canine kidney (MDCK) cells, even with control siRNA duplexes, disrupted the initial surface polarity as well as the steady-state distribution of membrane proteins. Transfection using lipofectamine yielded slightly less efficient knockdown but did not disrupt membrane polarity. Polarized secretion was unaffected by nucleofection, suggesting a selective defect in the development of membrane polarity. Cilia frequency and length were not altered by nucleofection. However, the basolateral appearance of a fluorescent lipid tracer added to the apical surface of nucleofected cells was dramatically enhanced relative to untransfected controls or lipofectamine-treated cells. In contrast, [3H]inulin diffusion and transepithelial electrical resistance were not altered in nucleofected cells compared with untransfected ones. We conclude that nucleofection selectively hinders development of the tight junction fence function in MDCK cells.

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