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    Experimental Cell Research 2The intestinal epithelium performs an important barrier

    function, selectively restricting the permeation of ions and

    nonelectrolytes. It also prevents macromolecules from

    accessing the internal milieu as well as losing cells and

    extracellular proteins into the intestinal lumen. Macromo-

    lecules have been reported to permeate the intestinal

    epithelium mainly via the paracellular pathway regulated

    by intercellular tight junctions between adjacent cells [1

    3]. Previous studies in experimental animals and clinical

    studies of human disease have demonstrated an association

    intestinal mucosal inflammation [48]. Although tight

    junctions have been shown to consist of at least a dozen

    molecular species, including occludin, claudins, cingulin,

    ZO-1, ZO-2, ZO-3 etc., that extend from their lips to the

    cytoskeleton [9,10], the mechanism by which the perme-

    ability of tight junctions is regulated has yet to be fully

    elucidated [1118].

    Gap junctional intercellular communication (GJIC)

    channels allow rapid exchange of ions and metabolites

    up to approximately 1 kDa in size, including second

    messengers such as cyclic AMP, IP3, and Ca2+ betweenIntroduction between increased epithelial paracellular permeability andIn some cell types, gap junctional intercellular communication (GJIC) is associated with tight junctions. The present study was performed

    to determine the roles of GJIC in regulation of the barrier function of tight junctions. Caco-2 human colonic cells were used as a monolayer

    model, and barrier function was monitored by measuring mannitol permeability and transepithelial electrical resistance (TER). The

    monolayers were chemically disrupted by treatment with oleic acid and taurocholic acid. Western blotting analyses were performed to

    evaluate the protein levels of connexins, which are components of gap junctional intercellular channels. Cx26 expression was detected in

    preconfluent Caco-2 cells, and its level increased gradually after the monolayer reached confluency. These results prompted us to examine

    whether overexpression of Cx26 affects barrier function. Monolayers of Caco-2 cells stably expressing Cx26 showed significantly lower

    mannitol permeability and higher TER than mock transfectants when the monolayers were chemically disrupted. The levels of claudin-4, an

    important component of tight junctions, were significantly increased in the stable Cx26 transfectant. These results suggest that Cx26-

    mediated GJIC may play a crucial role in enhancing the barrier function of Caco-2 cell monolayers.

    D 2004 Elsevier Inc. All rights reserved.

    Keywords: Gap junctional intercellular communication (GJIC); Tight junctions; Caco-2 cells; Paracellular permeability; Connexin 26; Claudin-4Received 10 November 2003, revised version received 16 March 2004

    Available online 6 May 2004

    AbstractConnexin 26-mediated gap junc

    suppresses paracellular permeab

    cell mo

    Hidekazu Morita, Tatsuro KatsunoYasushi Saito,

    Clinical Cell Biology (F5), Graduate School of0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved.


    * Corresponding author. Division of Clinical Cell Biology (F5),

    Department of Internal Medicine, Graduate School of Medicine, Chiba

    University, 1-8-1 Inohana, Chuo-Ward, Chiba 260-8670, Japan. Fax: +81-


    E-mail address: (T. Katsuno).nal intercellular communication

    y of human intestinal epithelial


    ihiro Hoshimoto, Noriaki Hirano,Yasuo Suzuki

    ine, Chiba University, Chiba 260-8670, Japan

    98 (2004) 18adjacent cells. Gap junctions are plasma membrane spa-

    tial microdomains constructed of assemblies of channel

    proteins called connexins. Approximately 20 types of

    connexins have been identified in the human and mouse

    genomes. Most cell types express multiple connexin

    isoforms providing a structural basis for the charge and

    size selectivity of these intercellular channels. However,

  • #71-7800), anti-claudin-4, anti-occludin (Zymed Labora-

    tories, Inc., San Francisco, CA), anti-Cx45 (Santa Cruz

    Aliquots of 1 Ag of total RNA were reverse-transcribedinto cDNA using a mixture of oligo(dT) and AMV reverse

    transcriptase under the recommended conditions. cDNA

    synthesis was performed in a total volume of 20 Al for 20min at 50jC and terminated by incubation for 5 min at99jC. PCR was performed in mixtures containing 20 pMof the appropriate primer pair and 2.5 U of Taq DNA

    polymerase (Takara Bio Inc., Tokyo, Japan). Reaction

    mixtures (total volume, 100 Al) were subjected to 40 cyclesof PCR, with a profile of 30 s at 94jC, 30 s at 58jC, and60 s at 72jC with a final elongation step of 7 min at 72jC,using a GeneAmp PCR system 9700 (Applied Biosystems,

    H. Morita et al. / Experimental Cell Research 298 (2004) 182Biotechnology, Inc., Santa Cruz, CA), or anti-h actin(Sigma-Aldrich Corp., St. Louis, MO) antibodies at room

    temperature overnight. The membranes were incubated

    with horseradish peroxidase (HRP)-conjugated anti-rabbit

    or mouse IgG (Vector Laboratories, Burlingame, CA) at

    room temperature for 45 min, and detection was carried

    out using an enhanced chemiluminescence (ECL) West-

    ern blotting system (Bio-Rad Laboratories, Hercules,


    RNA isolation and reverse transcription-PCR (RT-PCR)

    RT-PCR was performed on total RNA extracted from thethe precise nature of the GJIC channel remains unclear


    Tight junction strands as well as the integral tight

    junction proteins have been shown to be induced in Cx32-

    transfected hepatocytes [21,22]. In fibroblasts and cardiac

    myocytes, Cx43 was shown to interact with ZO-1 [23,24].

    In the present study, we examined the roles of GJIC in the

    regulation of protein expression and the function of tight

    junctions using human intestinal epithelial cells (Caco-2

    cells) overexpressing human Cx26 protein. Our results

    indicated that GJIC regulates claudin-4 protein expression

    and the paracellular permeability of Caco-2 human intestinal

    epithelial cells.

    Materials and methods

    Cell culture

    Caco-2 cells were cultured at 37jC in an atmosphere of5% CO2/95% air. The cells were maintained in DMEM with

    4.5 g/l glucose, 2 mM L-glutamine, 50 units/ml penicillin,

    50 Ag/ml streptomycin, 10 mM HEPES, 1% essential andnonessential amino acids, and 15% FBS, unless otherwise


    Western blotting

    Parental and transfected Caco-2 cells were lysed by

    boiling in PBS and 1% SDS containing 100 Ag/mlphenylmethylsulfonyl fluoride and 1 mM sodium ortho-

    vanadate at the indicated times after plating. Proteins

    were assayed with bicinchoninic acid. Aliquots of 20 Agof proteins were separated by SDS-PAGE (1020%

    resolving gels) and electroblotted onto nitrocellulose

    membranes (NENk Life Science Products, Inc., Boston,MA). The membranes were saturated for 30 min at room

    temperature with blocking buffer (0.6% Tween 20, 1%

    bovine serum albumin (BSA), 10% skimmed milk) and

    incubated with anti-Cx26, anti-Cx32, anti-claudin-1 (Catcells using RNAzol (Tel-Test Inc., Friendswood, TX).Foster City, CA). Aliquots of 3 Al of the PCR productswere analyzed by 1% agarose gel electrophoresis with

    staining with ethidium bromide. The primers used to detect

    Cx26 by RT-PCR were sense, 5V-CCG CCC AGA GTAGAA GA-3V; and anti-sense, 5V-CGG GTT GCC TCA TCC-3V.

    cDNA construction and transfection

    Human Cx26 cDNA [51] was subcloned into the

    HindIIIXbaI restriction sites of the expression vector

    pcDNA3.1. For transient transfection, parental cells were

    transfected with 4 Ag of Cx26 cDNA using Lipofect-AMINE (Invitrogen Corp., Carlsbad, CA). After 5 h of

    incubation, the cells were transferred to DMEM containing

    15% FBS. All cell cultures were maintained for 48 h after

    transfection and then examined by immunocytochemistry.

    For stable transfection, parental cells were transfected with

    4 Ag of Cx26 cDNA using LipofectAMINE. After 48 h,the cells were transferred to selection medium containing

    400 Ag/dl G418 (Sigma). When surviving colonies hadgrown sufficiently to allow detection visually, they were

    individually picked and propagated separately. Following

    initial screening of 20 clones for Cx26 by Western

    blotting, we chose one clone (termed #1) for further


    Immunofluorescence microscopy

    Cells grown on glass coverslips were fixed with cold

    50% acetone/50% ethanol for 5 min. Immunocytochemis-

    Fig. 1. Western blotting analysis for Cx26 and h-actin in Caco-2 cells. Celllysates were prepared from parental Caco-2 cells on days 1, 0, 3, and 6post-confluency. Western blotting analysis was performed using anti-Cx26

    and anti-h-actin antibodies. Data shown are from one representative of five

    independent experiments.

  • of polycarbonate with 6.5 mm wells and a pore size of

    5.0 Am, with 600 Al of medium in the basal compart-ment. Twenty-four hours before addition of agents that

    damage the monolayer, 100 Al of 106 M 18a-glycyr-rhetinic acid (AGA) (ICN Biomedicals Inc., Costa Mesa,

    CA), which interferes with gap junctional intercellular

    communication (GJIC), was added to the apical well

    [2528]. Subsequently, solutions (100 Al/well) contain-ing 3 103 M oleic acid plus 4.5 103 Mtaurocholic acid,