Connexin 26 expression prevents down-regulation of barrier and fence functions of tight junctions by Na+/K+-ATPase inhibitor ouabain in human airway epithelial cell line Calu-3.
Gap junctions are considered to play a crucial role in differentiation of epithelial cells and to be associated with tight junction proteins. In this study, to investigate the role of gap junctions in regulation of the barrier function and fence function on the tight junctions, we introduced the Cx26 gene into human airway epithelial cell line Clau-3 and used a disruption model of tight junctions employing the Na(+)/K(+)-ATPase inhibitor ouabain. In parental Calu-3 cells, gap junction proteins Cx32 and Cx43, but not Cx26, and tight junction proteins occludin, JAM-1, ZO-1, claudin-1, -2, -3, -4, -5, -6, -7, -8, -9, and -14 were detected by RT-PCR. The barrier function and fence function of tight junctions were well maintained, whereas the GJIC was low level. Treatment with ouabain caused disruption of the barrier function and fence function of tight junctions together with down-regulation of occludin, JAM-1, claudin-2, and -4 and up-regulation of ZO-1 and claudin-14. In Cx26 transfectants, Cx26 protein was detected by Western blotting and immunocytochemistry, and many gap junction plaques were observed with well-developed tight junction strands. Expression of claudin-14 was significantly increased in Cx26 transfectants compared to parental cells, and in some cells, Cx26 was co-localized with claudin-14. Interestingly, transfection with Cx26 prevented disruption of both functions of tight junctions by treatment with ouabain without changes in the tight junction proteins. Pretreatment with the GJIC blockers 18beta-glycyrrhetinic acid and oleamide did not affect the changes induced by Cx26 transfection. These results suggest that Cx26 expression, but not the mediated intercellular communication, may regulate tight junction barrier and fence functions in human airway epithelial cell line Calu-3.
Go
Mitsuru
M
Department of Pathology, Sapporo Medical University School of Medicine, S1. W17. Sapporo 060-8556, Japan.
Kojima
Takashi
T
Takano
Ken-ichi
K
Murata
Masaki
M
Koizumi
Junichi
J
Kurose
Makoto
M
Kamekura
Ryuta
R
Osanai
Makoto
M
Chiba
Hideki
H
Spray
David C
DC
Himi
Tetsuo
T
Sawada
Norimasa
N
D001483:Base Sequence; D001980:Bronchi / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D010042:Ouabain / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism*; D014162:Transfection
D017630:Connexins; D017931:DNA Primers; D004791:Enzyme Inhibitors; C000607042:GJB2 protein, human; D011994:Recombinant Proteins; D000072259:Connexin 26; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase