Intravital imaging of mouse urothelium reveals activation of extracellular signal‐regulated kinase by stretch‐induced intravesical release of ATP

Abstract

To better understand the roles played by signaling molecules in the bladder, we established a protocol of intravital imaging of the bladder of mice expressing a Förster/fluorescence resonance energy transfer (FRET) biosensor for extracellular signal‐regulated kinase (ERK), which plays critical roles not only in cell growth but also stress responses. With an upright two‐photon excitation microscope and a vacuum‐stabilized imaging window, cellular ERK activity was visualized in the whole bladder wall, from adventitia to urothelium. We found that bladder distention caused by elevated intravesical pressure (IVP) activated ERK in the urothelium, but not in the detrusor smooth muscle. When bladder distension was prevented, high IVP failed to activate ERK, suggesting that mechanical stretch, but not the high IVP, caused ERK activation. To delineate its molecular mechanism, the stretch‐induced ERK activation was reproduced in an hTERT‐immortalized human urothelial cell line (TRT‐HU1) in vitro. We found that uniaxial stretch raised the ATP concentration in the culture medium and that inhibition of ATP signaling by apyrase or suramin suppressed the stretch‐induced ERK activation in TRT‐HU1 cells. In agreement with this in vitro observation, pretreatment with apyrase or suramin suppressed the high IVP‐induced urothelial ERK activation in vivo. Thus, we propose that mechanical stretch induces intravesical secretion of ATP and thereby activates ERK in the urothelium. Our method of intravital imaging of the bladder of FRET biosensor‐expressing mice should open a pathway for the future association of physiological stimuli with the activities of intracellular signaling networks.

Dying under pressure: cellular characterisation and in vivo functions of cell death induced by compaction

“…The sensing of deformations is triggered through the modulation of ERK activity which will impact cell survival via the inhibition of the pro-apoptotic protein Hid [Moreno et al, 2018]. Those results are in agreement with the previous reports of ERK activation by cellular tension in cell culture [Tschumperlin et al, 2004, Aoki et al, 2013, Hirata et al, 2015, Kawabata and Matsuda, 2016, Aoki et al, 2017 and in vivo [Sano et al, 2016]. Finally, even if they have not been formally associated with compaction-driven death yet, other pathways which regulate cell survival and can be modulated by mechanical forces may also be relevant for compaction-driven cell death.…”

Regulatory T Cells in Skin Facilitate Epithelial Stem Cell Differentiation

“…This technique was adapted from approaches utilized in other tissues (Boldajipour et al, 2016; Broz et al, 2014; Headley et al, 2016; Lin et al, 2007; Lindsay et al, 2015; Looney et al, 2011; Sano et al, 2016; Vinegoni et al, 2012). Evans blue dye was utilized to ensure all intravascular Tregs were excluded while all cells located in the extravascular space were included for analysis.…”

High-resolution bladder morphology in the intact mouse bladder: an intravital two-photon study

“…Intravital two-photon microscopy (TPM) is an excellent tool for real-time assessment of the physiological and metabolic state of the rodent bladder in its natural state. Sano and colleagues performed intravital FRET TPM on the bladder of transgenic mice, while inducing mechanical stretch to the tissue during the experiment (12). Since not all labs have access to expensive transgenic mice and special objectives and equipment, approaches for a simple, reproducible intravital bladder TPM protocol are needed.…”