The human urothelial tight junction: claudin 3 and the ZO-1α+switch.
OBJECTIVE
Tight junctions are multicomponent structures, with claudin proteins defining paracellular permeability. Claudin 3 is a candidate for the exceptional "tightness" of human urothelium, being localised to the terminal tight junction (TJ) of superficial cells. Our aim was to determine whether claudin 3 plays an instigating and/or a functional role in the urothelial TJ.


METHODS
Normal human urothelial (NHU) cells maintained as non-immortalised cell lines were retrovirally-transduced to over-express or silence claudin 3 expression. Stable sublines induced to stratify or differentiate were assessed for TJ formation by immunocytochemistry and transepithelial electrical resistance (TER). Expression of claudin 3, ZO-1 and ZO-1α+was examined in native urothelium by immunohistochemistry.


RESULTS
Claudin 3 expression was associated with differentiation and development of a tight barrier and along with ZO-1 and ZO-1α+was localised to the apical tight junction in native urothelium. Knockdown of claudin 3 inhibited formation of a tight barrier in three independent cell lines, however, overexpression of claudin 3 was not sufficient to induce tight barrier development in the absence of differentiation. A differentiation-dependent induction of the ZO-1α+isoform was found to coincide with barrier formation. Whereas claudin 3 overexpression did not induce the switch to co-expression of ZO-1α-/ZO-1α+, claudin 3 knockdown decreased localisation of ZO-1 to the TJ and resulted in compromised barrier function.


CONCLUSIONS
Urothelial cytodifferentiation is accompanied by induction of claudin 3 which is essential for the development of a terminal TJ. A coordinated switch to the ZO-1α+isotype was also observed and for the first time may indicate that ZO-1α+is involved in the structural assembly and function of the urothelial terminal TJ.
Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5DD, United Kingdom ; Pyrah Department of Urology, St James's University Hospital, Leeds LS9 7TF, United Kingdom.
Nicholas J
NJ
Smith
Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5DD, United Kingdom.
Jennifer
J
Hinley
Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5DD, United Kingdom.
Claire L
CL
Varley
Pyrah Department of Urology, St James's University Hospital, Leeds LS9 7TF, United Kingdom.
Ian
I
Eardley
Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5DD, United Kingdom.
Ludwik K
LK
Trejdosiewicz
Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5DD, United Kingdom.
Jennifer
J
Southgate
claudin; differentiation; tight junction; urothelium; zonula occludens