The Circadian expression of Piezo1, TRPV4, Connexin26, and VNUT, associated with the expression levels of the clock genes in mouse primary cultured urothelial cells

AimsTo investigate circadian gene expressions in the mouse bladder urothelium to establish an experimental model and study the functions of the circadian rhythm. MethodsThe gene expression rhythms of the clock genes, mechano-sensors such as Piezo1 and TRPV4, ATP release mediated molecules (ARMM) such as Cx26 and VNUT were investigated in mouse primary cultured urothelial cells (UCs) of wild-type (WT) and Clock mutant (Clock(19/19)) mice using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting analysis. The long-term oscillation of the clock genes in UC was investigated by measuring bioluminescence from UC isolated from Period2(luciferase) knock-in mice (Per2::luc) and Per2::luc with Clock(19/19) using a luminometer. The mRNA expression rhythms after treatment with Clock short interfering RNA (siRNA) were also measured to compare differences between Clock point mutations and Clock deficiency. ResultsThe UCs from WT mice showed the time-dependent gene expressions for clock genes, mechano-sensors, and ARMM. The abundances of the products of these genes also correlated with the mRNA expression rhythms in UCs. The bioluminescence of Per2::Luc in UCs showed a circadian rhythm. By contrast, all the gene expression rhythms observed in WT mice were abrogated in the Clock(19/19) mice. Transfection with Clock siRNA in UCs had the same effect as the Clock mutation. ConclusionsWe demonstrated that the time-dependent gene expressions, including clock genes, mechano-sensors, and ARMM, were reproducible in UCs. These findings demonstrated that UCs have the potential to progress research into the circadian functions of the lower urinary tract regulated by clock genes.