Maintenance of the epithelial barrier in a bronchial epithelial cell line is dependent on functional E-cadherin local to the tight junctions.
Tight junctions (TJ) are essential components of polarized epithelia, and E-cadherin is important for their formation and maintenance. The bronchial epithelial cell line, 16HBE14o-expresses E- and P-cadherin, but not N-cadherin. E- and P-cadherin levels changed during culture, the former increasing after confluence, and the latter were markedly reduced. All detectable E-cadherin was bound to beta- and gamma-catenins. We investigated involvement of E-cadherin with epithelial integrity using an E-cadherin specific, function-blocking antibody, SHE78-7. Surprisingly, apical SHE78-7 exposure caused a prompt fall in transepithelial resistance (TER), while TER remained unchanged for 8 hrs after basal exposure then dropped. SHE78-7 exposure increased epithelial permeability to mannitol, inulin, and 9.5 kDa and 77 kDa dextrans and caused fragmentation and loss of the tight junction protein, ZO-1, from the cell borders in some areas. Ultrastructural studies showed that all junctional intercellular contact was lost in the center of SHE78-7 induced lesions. Near the lesion periphery, epithelial structure was maintained, but TJs were dysfunctional as shown by ruthenium red penetration. Analysis of epithelial penetration by SHE78-7 revealed discrete, local defects in the apical barrier at the top of some cell hills that permitted rapid access of the antibody to E-cadherin near the apical surface. In contrast, after basal exposure, antibody initially engaged with E-cadherin nearer the basal surface and only accessed apical E-cadherin later. Taken together with the TER measurements, these data suggest compartmentalization of E-cadherin function within 16HBE14o-cells, with only the apical E-cadherin adjacent to the tight junctions contributing to the function of the latter.
GlaxoSmithkline, Gunnelswood Rd., Stevenage, SG1 2NY, UK. mrw4779@gsk.com
M R
MR
West
D J P
DJ
Ferguson
V J
VJ
Hart
S
S
Sanjar
Y
Y
Man
D000818:Animals; D015153:Blotting, Western; D001980:Bronchi / Q000166:cytology*; D015820:Cadherins / Q000378:metabolism*; D002460:Cell Line; D003598:Cytoskeletal Proteins; D003911:Dextrans / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007150:Immunohistochemistry; D007444:Inulin / Q000378:metabolism; D008353:Mannitol / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D012430:Ruthenium Red / Q000494:pharmacology; D019108:Tight Junctions; D013997:Time Factors; D062826:Zonula Occludens-1 Protein
D015820:Cadherins; D003598:Cytoskeletal Proteins; D003911:Dextrans; D007074:Immunoglobulin G; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568986:Ocln protein, mouse; C568987:Ocln protein, rat; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D012430:Ruthenium Red; D008353:Mannitol; D007444:Inulin