The Effect of Capsaicin Derivatives on Tight-Junction Integrity and Permeability of Madin-Darby Canine Kidney Cells

Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.

Download Full-text

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

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00286.2011 ◽

2012 ◽

Vol 302 (2) ◽

pp. R300-R311 ◽

Cited By ~ 15

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.

Download Full-text

aPKC-PAR complex dysfunction and tight junction disassembly in renal epithelial cells during ATP depletion

AJP Cell Physiology ◽

10.1152/ajpcell.00099.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. C1094-C1102 ◽

Cited By ~ 30

Author(s):

Shobha Gopalakrishnan ◽

Mark A. Hallett ◽

Simon J. Atkinson ◽

James A. Marrs

Keyword(s):

Tight Junction ◽

Atp Depletion ◽

Cell Junctions ◽

Cell Contact ◽

Kidney Cells ◽

Madin Darby Canine Kidney ◽

Rac Gtpase ◽

Signaling Complex ◽

Darby Canine Kidney ◽

Canine Kidney

Renal ischemia and in vitro ATP depletion result in disruption of the epithelial tight junction barrier, which is accompanied by breakdown of plasma membrane polarity. Tight junction formation is regulated by evolutionarily conserved complexes, including that of atypical protein kinase C (aPKC), Par3, and Par6. The aPKC signaling complex is activated by Rac and regulated by protein phosphorylation and associations with other tight junction regulatory proteins, for example, mLgl. In this study, we examined the role of aPKC signaling complex during ATP depletion and recovery in Madin-Darby canine kidney cells. ATP depletion reduced Rac GTPase activity and induced Par3, aPKCζ, and mLgl-1 redistribution from sites of cell-cell contact, which was restored following recovery from ATP depletion. Zonula occludens (ZO)-1 and Par3 phosphorylation was reduced and association of aPKCζ with its substrates Par3 and mLgl-1 was stabilized in ATP-depleted Madin-Darby canine kidney cells. ATP depletion also induced a stable association of Par3 with Tiam-1, a Rac GTPase exchange factor, which explains how aPKCζ and Rac activities were suppressed. Experimental inhibition of aPKCζ during recovery from ATP depletion interfered with reassembly of ZO-1 and Par3 at cell junctions. These data indicate that aPKC signaling is impaired during ATP depletion, participates in tight junction disassembly during cell injury and is important for tight junction reassembly during recovery.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj2630597 ◽

1989 ◽

Vol 263 (2) ◽

pp. 597-599 ◽

Cited By ~ 68

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.

Download Full-text

Sec61β Regulates Barrier Functions of Tight Junction through Expression of Claudin-4 in Madin–Darby Canine Kidney Cells

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.b13-00936 ◽

2014 ◽

Vol 37 (6) ◽

pp. 979-986 ◽

Cited By ~ 1

Author(s):

Makiko Washiyama ◽

Naoya Koizumi ◽

Makiko Fujii ◽

Yoshiteru Watanabe

Keyword(s):

Tight Junction ◽

Kidney Cells ◽

Madin Darby Canine Kidney ◽

Barrier Functions ◽

Darby Canine Kidney ◽

Canine Kidney

Download Full-text

NZO-3 Expression Causes Global Changes to Actin Cytoskeleton in Madin-Darby Canine Kidney Cells: Linking a Tight Junction Protein to Rho GTPases

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0486 ◽

2003 ◽

Vol 14 (5) ◽

pp. 1757-1768 ◽

Cited By ~ 42

Author(s):

Erika S. Wittchen ◽

Julie Haskins ◽

Bruce R. Stevenson

Keyword(s):

Tight Junction ◽

Actin Cytoskeleton ◽

Tight Junction Protein ◽

Junctional Complex ◽

Kidney Cells ◽

Madin Darby Canine Kidney ◽

P120 Catenin ◽

Junction Protein ◽

Darby Canine Kidney ◽

Canine Kidney

We previously demonstrated that exogenous expression of a truncated form of the tight junction protein ZO-3 affected junctional complex assembly and function. Current results indicate that this ZO-3 construct influences actin cytoskeleton dynamics more globally. We show that expression of the amino-terminal half of ZO-3 (NZO-3) in Madin-Darby canine kidney cells results in a decreased number of stress fibers and focal adhesions and causes an increased rate of cell migration in a wound healing assay. We also demonstrate that RhoA activity is reduced in NZO-3–expressing cells. We determined that ZO-3 interacts with p120 catenin and AF-6, proteins localized to the junctional complex and implicated in signaling pathways important for cytoskeleton regulation and cell motility. We also provide evidence that NZO-3 interacts directly with the C terminus of ZO-3, and we propose a model where altered interactions between ZO-3 and p120 catenin in NZO-3–expressing cells affect RhoA GTPase activity. This study reveals a potential link between ZO-3 and RhoA-related signaling events.

Download Full-text