Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells.
Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.
Amasheh
Salah
S
Department of Clinical Physiology, Charité-Universitary Medicine Berlin, Campus Benjamin Franklin, 12200 Berlin, Germany.
Schmidt
Thomas
T
Mahn
Michaela
M
Florian
Peter
P
Mankertz
Joachim
J
Tavalali
Shida
S
Gitter
Alfred H
AH
Schulzke
Jörg-Dieter
JD
Fromm
Michael
M
D015153:Blotting, Western; D018938:Caco-2 Cells; D002450:Cell Communication; D002463:Cell Membrane Permeability; D062507:Claudin-5; D017097:Electric Impedance; D004847:Epithelial Cells / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D019073:HT29 Cells; D006801:Humans; D008353:Mannitol / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D062793:Occludin; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000502:physiology*; D014162:Transfection
C107466:CLDN5 protein, human; D062507:Claudin-5; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D008353:Mannitol