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

Differential transcription factor expression by human epithelial cells of buccal and urothelial derivation

Identification of transcription factors expressed by differentiated cells is informative not only of tissue-specific pathways, but to help identify master regulators for cellular reprogramming. If applied, such an approach could generate healthy autologous tissue-specific cells for clinical use where cells from the homologous tissue are unavailable due to disease. Normal human epithelial cells of buccal and urothelial derivation maintained in identical culture conditions that lacked significant instructive or permissive signalling cues were found to display inherent similarities and differences of phenotype. Investigation of transcription factors implicated in driving urothelial-type differentiation revealed buccal epithelial cells to have minimal or absent expression of PPARG, GATA3 and FOXA1 genes. Retroviral overexpression of GATA3 or PPARG1 coding sequences in buccal epithelial cells resulted in nuclear immunolocalisation of the respective proteins, with both transductions also inducing expression of the urothelial differentiation-associated claudin 3 tight junction protein. PPARγ1 overexpression alone entrained expression of nuclear FOXA1 and GATA3 proteins, providing objective evidence of its upstream positioning in a transcription factor network and identifying it as a candidate factor for urothelial-type transdifferentiation or reprogramming

Hypoxic changes to the urothelium as a bystander of end-stage bladder disease

Introduction: Urothelial cells harvested from benign diseased bladders have a compromised capacity to propagate or differentiate in vitro, potentially limiting their application in autologous tissue engineering approaches. The causative pathways behind this altered phenotype are unknown. The hypothesis is that hypoxic damage to the urothelium occurs as a bystander to chronic or recurrent episodes of infection and inflammation. Objective: The aim of this study was to assess immunohistochemically detected nuclear hypoxia-inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor in the urothelium when exposed to hypoxia. Study design: Human bladder sections from a total of 29 adult and paediatric patients, representing a variety of different pathologies including neuropathy (n = 15), were analysed. Tissues from adults with bladder outlet obstruction secondary to prostatic disease (n = 1), urothelial carcinoma (n = 1) and tonsil (n = 1) were used as positive tissue controls for immunohistochemistry. Hypoxia-inducible factor 1 alpha–labelled sections were scanned using a Zeiss AxioScan Z1 slide scanner. Analysis of urothelial nuclear HIF-1α labelling was performed using HistoQuest image analysis software (TissueGnostics). Comparison of nuclear HIF-1α labelling between neuropathic and non-neuropathic sections was performed using one-way analysis of variance with the post hoc Tukey honestly significant difference (HSD) test. Patient urodynamic studies performed before tissue sample harvest were evaluated and correlated to the HIF-1α intensity using Spearman's rank correlation. Results: Hypoxia-inducible factor 1 alpha appeared more intense in the urothelial compartment from neuropathic bladder samples (n = 15) than in the control tissues, including non-obstructed samples (n = 9). Image analysis supported this; median nuclear HIF-1α labelling was 29.98 ± 3.10 (standard deviation [SD]) (n = 9) in controls and 74.29 ± 7.55 (SD) in neuropathic samples (n = 15). A statistically significant difference between the control and neuropathic tissue groups was shown (P < 0.05). Of the 15 neuropathic samples, 11 had traceable urodynamic studies. Both initial and maximum detrusor pressures indicated a positive relationship when plotted against HIF-1α labelling. Spearman's rank correlation, with no missing events, confirmed significant correlations between both initial or maximum detrusor pressure and nuclear HIF-1α labelling intensity (median score); P ≤ 0.046 and P ≤ 0.05, respectively. The null hypothesis was accordingly rejected. Conclusions: This study indicates that urothelial nuclear HIF-1α may be a biomarker of hypoxia and a common feature in end-stage bladder disease associated with high-pressure systems

Loss of Janus Associated Kinase1 alters urothelial cell functionand facilitates the development of bladder cancer

Inherited Primary Immunodeficiency (PID) disorders are associated with increased risk ofmalignancy that may relate to impaired antitumor immune responses or a direct role for PIDgermline mutations in tumorigenesis. We recently identified germline loss of function mutations inJanus Associated Kinase 1 (JAK1) causing primary immunodeficiency characterised by infectionsand associated with early onset, fatal high-grade bladder carcinoma. Somatic mutations in JAK1,required for immune cell signalling in response to interferon gamma (IFNγ), have been associatedwith several non-hematopoietic and hematopoietic cancer cell types but pathogenic mechanismsremain largely unexplored. Here we demonstrate that JAK1 is required for the intrinsic IFNγresponse of urothelial cells impacting immunogenicity and cell survival. Specifically, JAK1-deficient urothelial cells showed reduced surface expression of major histocompatibility complexclass II (MHC II), intercellular adhesion molecule-1 (ICAM-1) and programmed death-ligand-1(PD-L1) after IFNγ stimulation and were resistant to IFNγ-induced apoptosis and lymphocytemediatedkilling. In addition, we identify a previously unknown role for IFNγ signalling inmodulating urothelial differentiation. Together, our findings support a role for urothelial cell JAK1in immune surveillance and development of bladder cancer. Our results have implications forpatients with rare JAK1 PID and, more broadly, inform development of biomarker and targetedtherapies for urothelial carcinoma

Barrier forming potential of epithelial cells from the exstrophic bladder

Bladder exstrophy (BEX) is a rare developmental abnormality resulting in an open, exposed bladder plate. Although normal bladder urothelium is a mitotically quiescent barrier epithelium, histologic studies of BEX epithelia report squamous and proliferative changes that can persist beyond surgical closure. The current study examined whether patient-derived BEX epithelial cells in vitro were capable of generating a barrier-forming epithelium under permissive conditions. Epithelial cells isolated from 11 BEX samples, classified histologically as transitional (n = 6) or squamous (n = 5), were propagated in vitro. In conditions conducive to differentiated tight barrier formation by normal human urothelial cell cultures, 8 of 11 BEX lines developed transepithelial electrical resistances of more than 1000 Ω.cm(2), with 3 squamous lines failing to generate tight barriers. An inverse relationship was found between expression of squamous KRT14 transcript and barrier development. Transcriptional drivers of urothelial differentiation PPARG, GATA3, and FOXA1 showed reduced expression in squamous BEX cultures. These findings implicate developmental interruption of urothelial transcriptional programming in the spectrum of transitional to squamous epithelial phenotypes found in BEX. Assessment of BEX epithelial phenotype may inform management and treatment strategies, for which distinction between reversible versus intractably squamous epithelium could identify patients at risk of medical complications or those who are most appropriate for reconstructive tissue engineering strategies