Cx32 Gap Junctions in Human Urothelial Barrier Generation and Restitution

The ability of the urothelium to act as a urinary barrier is afforded by two key features: 1) It has the tightest barrier function of any epithelium, generated by tight junctions which assemble upon differentiation; 2) In response to damage, the quiescent urothelium can rapidly switch to a regenerative phenotype to enable regeneration and restitution of barrier function. Central to repair is the ability for the urothelium to sense damage; a process hypothesised to involve cell-cell communication. Direct cell-cell communication occurs through gap junctions, channels comprising connexin (Cx) proteins which allow for the passage of signalling molecules. Cxs have been linked to wound healing, as well as to maintenance of polarity and homeostasis in other epithelia, by both communication-dependent and independent mechanisms. A systematic characterisation of Cx expression has not been performed in urothelium and the significance of specific Cxs to urothelial physiology is not understood. This study aimed to investigate whether specific Cx proteins contribute to the regulation of barrier maintenance and restitution in human urothelium. Analysis of Cx transcripts identified Cx32, which was expressed in situ and highly induced upon in vitro differentiation of normal human urothelial (NHU) cells using two independent methods. In differentiated NHU cells, Cx32 proteins assembled into functional gap junction channels at cell borders, co-localising with the barrier defining tight junction proteins occludin and ZO-2. shRNA studies demonstrated that normal tight junction development and barrier function were dependent on the presence of intercellular Cx32, but not gap junction communication. In wound-healing experiments, intercellular communication through Cx32 channels inhibited urothelial cell migration and proliferation, in a process which involved suppression of activated SMAD3. Together the evidence presented here supports an unanticipated central role for Cx32 in orchestrating the homeostasis between barrier function and repair in human urothelium.