Question: 

It has been shown recently that urinary bladder urothelial cells produce hydrogen peroxide (H₂O₂) through the calcium-dependent NADPH oxidase Duox1. Herein, we investigated if mechanical stretch due to different filling pressures influences Duox1 expression and H₂O₂ formation in these cells.

Methods: 

Whole bladders were isolated from C57BL/6 mice and used in an ex vivo organ culture model. Time-dependent effects of defined filling pressures (up to 6 hours at low [7 mmHg] or high pressure [30 mmHg], verified by a pressure transducer) on mRNA and protein expression/localization were evaluated using quantitative PCR, Western blot analysis and immunofluorescence microscopy. Decoy oligonucleotides were used to demonstrate a possible functional role of target transcription factors in Duox1 expression. H₂O₂ formation was determined by the Amplex Red method.

Results: 

Immunofluorescence and Western blot analyses demonstrated that Duox1 protein solely localises to the urothelial but not to the smooth muscle cell layer. Exposure to low filling pressure caused the Duox1 protein level to decrease in urothelial cells which was prevented by application of high filling pressure. PCR analysis revealed a transient pressure-dependent rise in Duox1 mRNA levels in the urothelial cell layer only. Expression of another H₂O₂-generating NADPH oxidase Nox4 was transiently increased in both cell layers whereas known stretch-inducible gene products such as COX2 and GROa (mouse IL-8 homolog) were up-regulated in the smooth muscle cell layer only. The selective TRPV4 antagonist HC-067047, which prevents the stretch-dependent rise in intracellular calcium, inhibited the rise in urothelial Duox1 mRNA. In silico analysis of the murine Duox1 promoter identified ATF2 as a potential transcriptional mediator of calcium-evoked p38 MAPK-mediated stretch signalling. Duox1 expression was in fact inhibited in pressurized bladders by neutralising ATF2 with a specific decoy oligonucleotide while translocation of ATF-2 to the nucleus of the stretched urothelial cells was demonstrated by immunofluorescence analysis. H₂O₂ levels in urothelium homogenates were significant elevated in bladders exposed to 30 mmHg as compared to 7 mmHg. By using Nox4-deficient mice, a contribution of this NADPH oxidase to this H₂O₂ formation could be excluded.

Conclusions: 

These results demonstrate that stretching of urothelial cells leads to a calcium-dependent ATF2-mediated rise in Duox1 expression and subsequent H₂O₂ production which may be advantageous in diminishing infections of the bladder and urinary tract.