Gliding Basal Cell Migration of the Urothelium during Wound Healing
2018; Elsevier BV; Volume: 188; Issue: 11 Linguagem: Inglês
10.1016/j.ajpath.2018.07.010
ISSN1525-2191
AutoresTakeshi Sano, Takashi Kobayashi, Osamu Ogawa, Michiyuki Matsuda,
Tópico(s)Urinary Bladder and Prostate Research
ResumoCollective cell migration during wound healing has been extensively studied in the epidermis. However, it remains unknown whether the urothelium repairs wounds in a manner similar to the epidermis. By in vivo two-photon excitation microscopy of transgenic mice that express fluorescent biosensors, we studied the collective cell migration of the urothelium in comparison with that of the epidermis. In vivo time-lapse imaging revealed that, even in the absence of a wound, urothelial cells continuously moved and sometimes glided as a sheet over the underlying lamina propria. On abrasion of the epithelium, the migration speed of each epidermal cell was inversely correlated with the distance to the wound edge. Repetitive activation waves of extracellular signal–regulated kinase (ERK) were generated at and propagated away from the wound edge. In contrast, urothelial cells glided as a sheet over the lamina propria without any ERK activation waves. Accordingly, the mitogen-activated protein kinase/ERK kinase inhibitor PD0325901 decreased the migration velocity of the epidermis but not the urothelium. Interestingly, the tyrosine kinase inhibitor dasatinib inhibited migration of the urothelium as well as the epidermis, suggesting that the gliding migration of the urothelium is an active, not a passive, migration. In conclusion, the urothelium glides over the lamina propria to fill wounds in an ERK-independent manner, whereas the epidermis crawls to cover wounds in an ERK-dependent manner. Collective cell migration during wound healing has been extensively studied in the epidermis. However, it remains unknown whether the urothelium repairs wounds in a manner similar to the epidermis. By in vivo two-photon excitation microscopy of transgenic mice that express fluorescent biosensors, we studied the collective cell migration of the urothelium in comparison with that of the epidermis. In vivo time-lapse imaging revealed that, even in the absence of a wound, urothelial cells continuously moved and sometimes glided as a sheet over the underlying lamina propria. On abrasion of the epithelium, the migration speed of each epidermal cell was inversely correlated with the distance to the wound edge. Repetitive activation waves of extracellular signal–regulated kinase (ERK) were generated at and propagated away from the wound edge. In contrast, urothelial cells glided as a sheet over the lamina propria without any ERK activation waves. Accordingly, the mitogen-activated protein kinase/ERK kinase inhibitor PD0325901 decreased the migration velocity of the epidermis but not the urothelium. Interestingly, the tyrosine kinase inhibitor dasatinib inhibited migration of the urothelium as well as the epidermis, suggesting that the gliding migration of the urothelium is an active, not a passive, migration. 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Propagating wave of ERK activation orients collective cell migration.Dev Cell. 2017; 43: 305-317Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar Tidal waves of ERK activation were previously found to be propagated from the wound edge by immunohistochemistry10Matsubayashi Y. Ebisuya M. Honjoh S. Nishida E. ERK activation propagates in epithelial cell sheets and regulates their migration during wound healing.Curr Biol. 2004; 14: 731-735Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 11Nikolic D.L. Boettiger A.N. Bar-Sagi D. Carbeck J.D. Shvartsman S.Y. Role of boundary conditions in an experimental model of epithelial wound healing.Am J Physiol Cell Physiol. 2006; 291: C68-C75Crossref PubMed Scopus (116) Google Scholar; however, the repetitive waves of ERK activation from the wound edge17Hiratsuka T. Fujita Y. Naoki H. Aoki K. Kamioka Y. Matsuda M. Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin.Elife. 2015; 4: e05178Crossref PubMed Scopus (127) Google Scholar or spontaneous wavelets in the regions apart from the wound edge could only be visualized by time-lapse imaging of ERK activity with FRET biosensors.18Aoki K. Kondo Y. Naoki H. Hiratsuka T. Itoh R.E. Matsuda M. Propagating wave of ERK activation orients collective cell migration.Dev Cell. 2017; 43: 305-317Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar More important, cells migrate against the direction of the ERK activation wave in both the mouse epidermis and the MDCK monolayer sheet.18Aoki K. Kondo Y. Naoki H. Hiratsuka T. Itoh R.E. Matsuda M. Propagating wave of ERK activation orients collective cell migration.Dev Cell. 2017; 43: 305-317Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar A substantial part of our knowledge about wound healing in vivo comes from studies of epidermal wound healing, and the basic mechanism underlying wound healing is assumed to be conserved among different animals and tissues.5Sonnemann K.J. Bement W.M. Wound repair: toward understanding and integration of single-cell and multicellular wound responses.Annu Rev Cell Dev Biol. 2011; 27: 237-263Crossref PubMed Scopus (213) Google Scholar, 19Gurtner G.C. Werner S. Barrandon Y. Longaker M.T. Wound repair and regeneration.Nature (London). 2008; 453: 314-321Crossref PubMed Scopus (3821) Google Scholar Meanwhile, although there have been several studies on the wound healing of the urothelium,20Dalal E. Medalia O. Harari O. Aronson M. 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Herein, we demonstrate that the collective migration of the urothelium is significantly different from that of the epidermis, not only regarding the mode of migration but also in terms of the requirement for ERK activity. The animal protocols were reviewed and approved by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine (Kyoto, Japan; numbers 12064, 13074, 14079, and 15064). Transgenic mice expressing ERK FRET biosensors have been described previously.26Kamioka Y. Sumiyama K. Mizuno R. Sakai Y. Hirata E. Kiyokawa E. Matsuda M. Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors.Cell Struct Funct. 2012; 37: 65-73Crossref PubMed Scopus (99) Google Scholar ERK FRET biosensors, EKAREV–nuclear export signal and EKAREV–nuclear localization signal, are localized in the cytoplasm and the nucleus, respectively.26Kamioka Y. Sumiyama K. Mizuno R. Sakai Y. Hirata E. Kiyokawa E. Matsuda M. Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors.Cell Struct Funct. 2012; 37: 65-73Crossref PubMed Scopus (99) Google Scholar EKAREV–nuclear export signal and EKAREV–nuclear localization signal that were backcrossed more than five generations to C57BL/6N Jcl (CLEA Japan, Tokyo, Japan) were used for analysis. The Fucci mice, which express mAG-hGeminin (1/110) and mKO2-hCdt1 (30/120), were obtained from the Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology.27Sakaue-Sawano A. Kurokawa H. Morimura T. Hanyu A. Hama H. Osawa H. Kashiwagi S. Fukami K. Miyata T. Miyoshi H. Imamura T. Ogawa M. Masai H. Miyawaki A. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.Cell. 2008; 132: 487-498Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar Mice were housed in a specific pathogen-free facility in temperature-controlled rooms with a 14-hour light/10-hour dark cycle and received a routine chow diet and water ad libitum. For intravital imaging of the skin and the bladder, 12- to 25-week–old mice were used. At the end of the experiments, mice were euthanized by anesthetic overdose. We used an FV1000MPE-BX61WI upright microscope (Olympus, Tokyo, Japan) equipped with a 25×/1.05 water-immersion objective lens (XLPLN 25XWMP; Olympus) and an InSight DeepSee Ultrafast laser (0.95 W at 900 nm; Spectra Physics, Mountain View, CA). The excitation wavelength for cyan fluorescent protein (CFP) was 840 nm. An infrared light–cut filter, BA685RIF-3 (Olympus); two dichroic mirrors, DM505 and DM570 (Olympus); and four emission filters, FF01-425/30 (Semrock, Rochester, NY) for the second harmonic generation, BA460-500 (Olympus) for CFP, BA520-560 (Olympus) for yellow fluorescent protein, and 645/60 (Chroma Technology, Bellows Falls, VT) for Qtracker 655 (Life Technologies, Carlsbad, CA), were used. Qtracker 655 is intravenously administered with other reagents to confirm drug delivery to target organs. For Fucci mouse imaging, an IR-cut filter, RDM690 (Olympus); two dichroic mirrors, DM505 and DM570; and three emission filters, FF01-472/30 (Semrock) for second harmonic generation images, BA495-540 (Olympus) for mAG, and BA575-630 (Olympus) for mKO2, were used. The microscope was equipped with a two-channel GaAsP detector unit and two built-in photomultiplier tubes. FluoView software version 4.1a (Olympus) was used to control the microscope and to acquire images, which were saved in the multilayer 16-bit tagged image file format. Intravital imaging of the bladder was performed, as described previously.25Sano T. Kobayashi T. Negoro H. Sengiku A. Hiratsuka T. Kamioka Y. Liou L.S. Ogawa O. Matsuda M. Intravital imaging of mouse urothelium reveals activation of extracellular signal-regulated kinase by stretch-induced intravesical release of ATP.Physiol Rep. 2016; 4: e13033Crossref PubMed Scopus (22) Google Scholar Briefly, female mice were anesthetized by inhalation of 1% to 1.5% isoflurane (Abbott Laboratories, North Chicago, IL) and placed in the supine position on an electric heat pad maintained at 37°C. A 24-gauge ethylene tetrafluoroethylene catheter (Terumo, Tokyo, Japan) connected to a 50-mL bottle of normal saline (Otsuka Pharmaceutical Factory, Tokushima, Japan) was inserted transurethrally into the bladder. The intravesical pressure was controlled by the bottle's height and kept at 15 to 20 cm H2O for 30 minutes. Then, the catheter, which caused mechanical irritation and intensified the rhythmic muscle contraction of the bladder, was removed for stable long-term imaging. The bladder was pulled out of the abdominal cavity, and the bladder wall was immobilized on a custom-made vacuum-stabilized imaging window (Olympus). For multidimensional imaging of the urothelium and the underlying lamina propria, Z-stack images were acquired using a 2.4 digital zoom at 0.5-μm intervals and at a scan speed of 8 microseconds/pixel. CFP, Qtracker 655, and second harmonic generation were imaged to show cells, blood vessels, and collagen fibers, respectively. Time-lapse images were acquired every 5 or 6 minutes using a 1.2 to 2.4 digital zoom at a scan speed of 4 microseconds/pixel. For the wound healing analysis of the urothelium, a square 100 μm on each side was set under the two-photon excitation microscope. After increasing the laser power to 80% to 100%, the area was repeatedly scanned until the CFP fluorescence signal became undetectable, even with the highest sensitivity of the GaAsP detector. Intravital imaging of the ear skin was performed, as described previously.17Hiratsuka T. Fujita Y. Naoki H. Aoki K. Kamioka Y. Matsuda M. Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin.Elife. 2015; 4: e05178Crossref PubMed Scopus (127) Google Scholar Hair was removed from an ear by using depilation cream 24 hours before experiments. An ear of an anesthetized mouse was sandwiched between a cover glass and a thermal conductive silicon gum sheet. For multidimensional imaging of the epidermis and underlying lamina propria, Z-stack images were acquired using a 3.0 digital zoom at 0.5-μm intervals and at a scan speed of 8 microseconds/pixel. Time-lapse images were acquired every 10 or 12 minutes. An epithelial wound was generated at the ear skin with a 29-gauge needle (Terumo) 2 hours before imaging. PD0325901 (5 mg/kg), a mitogen-activated protein kinase (MAPK)/ERK kinase inhibitor (EMD Millipore, Billerica, MA), was dissolved in 0.2 mL phosphate-buffered saline supplemented with 4 μL Qtracker 655 and injected via the tail vein at a dose of 5 mg/kg. Dasatinib, a tyrosine kinase inhibitor (AdooQ BioScience, Irvine, CA), was dissolved in 0.15 mL propylene glycol supplemented with 4 μL Qtracker 655 and injected via the tail vein at a dose of 10 mg/kg. Microscopic images were analyzed, as described previously, with MetaMorph software version 7.10.1.161 (Molecular Devices, Sunnyvale, CA).28Mizuno R. Kamioka Y. Kabashima K. Imajo M. Sumiyama K. Nakasho E. Ito T. Hamazaki Y. Okuchi Y. Sakai Y. Kiyokawa E. Matsuda M. In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines.J Exp Med. 2014; 211: 1123-1136Crossref PubMed Scopus (76) Google Scholar In brief, yellow fluorescent protein images obtained by the excitation of CFP were used as FRET images. The FRET level is evaluated by the FRET/CFP ratio and represented as an intensity-modulated display or golden pseudocolor images. In the intensity-modulated display mode, eight colors from red to blue represent the FRET/CFP ratio, and the 32 grades of intensity represent the signal intensity in each pixel of the CFP image. The warm and cold colors were assigned to high and low FRET levels, respectively. The FRET/CFP ratio of each cell was quantified as follows. For the biosensor located in the nucleus, a region of interest was generated to include each nucleus. For the biosensor located in the cytoplasm, nuclear signals were first subtracted by the H-basin filter of MetaMorph. By using autothreshold, a region of interest was set onto the cytoplasm. Then, the region was expanded three pixels outward. The average fluorescence intensity of the region of interest was used to calculate the FRET/CFP ratio of each cell. Cell cycle analysis was performed with Fucci mice, according to the method reported previously.17Hiratsuka T. Fujita Y. Naoki H. Aoki K. Kamioka Y. Matsuda M. Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin.Elife. 2015; 4: e05178Crossref PubMed Scopus (127) Google Scholar The Fucci biosensor system consisted of two fluorescence reporters, the mKO2-hCdt1 (30/120) G1 marker and mAG-hGeminin (1/110) S/G2M marker, which emanate orange and green colors, respectively. For the identification of the nuclei of S/G2/M cells, images of mKO2-hCdt1 (30/120) were subtracted from images of mAG-hGeminin (1/110). The resulting images were processed with the segmentation function of the multidimensional motion analysis module of MetaMorph. The parameters used for the segmentation were as follows: segmentation method, adaptive threshold; XY diameter, 4 to 20; local intensity above background, 100. The nuclei of G0/G1 cells were identified in a similar manner. To track cell migration, the FRET images were analyzed by using the Fiji TrackMate plugin.29Jaqaman K. Loerke D. Mettlen M. Kuwata H. Grinstein S. Schmid S.L. Danuser G. Robust single-particle tracking in live-cell time-lapse sequences.Nat Methods. 2008; 5: 695-702Crossref PubMed Scopus (1191) Google Scholar, 30Applegate K.T. Besson S. Matov A. Bagonis M.H. Jaqaman K. Danuser G. plusTipTracker: quantitative image analysis software for the measurement of microtubule dynamics.J Struct Biol. 2011; 176: 168-184Crossref PubMed Scopus (170) Google Scholar The tracking data were further processed by the Chemotaxis & Migration Tool version 1.01 (ibidi GmbH, Martinsried, Germany). All statistical analyses were performed using Prism5 software version 6 (GraphPad Software, La Jolla, CA). A paired t-test was used to evaluate statistically significant differences. P < 0.05 was considered statistically significant. Both skin and bladder are covered by stratified epithelium. The remarkable difference between these two epithelia in the mobility over the underlying lamina propria is first shown. Two transgenic mouse strains expressing a nuclear FRET biosensor for ERK, EKAREV–nuclear localization signal, or a cytoplasmic FRET biosensor for ERK, EKAREV–nuclear export signal, were used.25Sano T. Kobayashi T. Negoro H. Sengiku A. Hiratsuka T. Kamioka Y. Liou L.S. Ogawa O. Matsuda M. Intravital imaging of mouse urothelium reveals activation of extracellular signal-regulated kinase by stretch-induced intravesical release of ATP.Physiol Rep. 2016; 4: e13033Crossref PubMed Scopus (22) Google Scholar, 26Kamioka Y. Sumiyama K. Mizuno R. Sakai Y. Hirata E. Kiyokawa E. Matsuda M. Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors.Cell Struct Funct. 2012; 37: 65-73Crossref PubMed Scopus (99) Google Scholar Both the epidermis and the urothelium are supported by dense collagen fibers in the lamina propria (Figure 1, A–D ). A peculiar anatomic feature of the bladder is the presence of suburothelial capillary plexus and interstitial cells beneath the urothelium (Figure 1, B and D). During the 2-hour observation of mice expressing EKAREV–nuclear localization signal, the nuclei of epidermal basal cells did not move significantly (Figure 1, E and F, and Supplemental Video S1). In stark contrast, the nuclei of urothelial cells were frequently moving (Figure 1, E and F, and Supplemental Video S1). Consequently, the displacement during the 1-hour imaging was larger in the urothelium than the epidermis (Figure 1G). Notably, the urothelial cell sheet occasionally glided over the suburothelial capillary plexus (Figure 1H and Supplemental Video S2). These observations may suggest that the adhesion to the underlying lamina propria appears markedly weaker in the urothelium than the epidermis. A significant difference was not observed in the mobility between the basal layer cells and the umbrella cells. The seemingly loose adhesion of the urothelium to the underlying lamina propria prompted us to examine the mode of collective cell migration. The epidermis and the urothelium were examined during wound healing by TPEM. In the skin, a microscopic injury of 150- to 300-μm diameter was generated with a fine needle, followed by time-lapse imaging (Figure 2A and Supplemental Video S3). Cells were tracked by the TrackMate add-in program in Fiji to calculate their mean velocity and distance from the wound center (Figure 2, B and C). Epidermal cells of two to three rows from the wound edge rapidly migrated toward the wound center, whereas cells behind the front rows migrated rather slowly. This observation agrees with the previously reported results of an in vitro wound healing assay with MDCK cells.31Farooqui R. Fenteany G. Multiple rows of cells behind an epithelial wound edge extend cryptic lamellipodia to collectively drive cell-sheet movement.J Cell Sci. 2005; 118: 51-63Crossref PubMed Scopus (308) Google Scholar FRET/CFP ratio videos were generated to analyze the dynamics of ERK activity. As reported earlier,17Hiratsuka T. Fujita Y. Naoki H. Aoki K. Kamioka Y. Matsuda M. Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin.Elife. 2015; 4: e05178Crossref PubMed Scopus (127) Google Scholar, 18Aoki K. Kondo Y. Naoki H. Hiratsuka T. Itoh R.E. Matsuda M. Propagating wave of ERK activation orients collective cell migration.Dev Cell. 2017; 43: 305-317Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar ERK activation waves were propagated from the wound edge (Supplemental Video S3). Similar experiments were set up for the urothelium, although the imaging period of the urothelium could not be as long as that of the ear skin because of the invasiveness of the imaging procedure. Because a mechanical wound could not be generated with a fine needle through the urethral catheter, intensive laser radiation was applied to thermally ablate the urothelium. A square 100 μm on each side was set on the urothelium and scanned repeatedly under the microscope with 80% to 100% laser power until the fluorescence disappeared completely. In this condition, tissue damage of the lamina propria was not detected (Supplemental Figure S1). The wound healing process was initiated soon after the laser ablation (Figure 2, D–F, and Supplemental Video S4). In contrast to the epidermal cells, all urothelial cells within the imaged area glided at similar speeds to fill the defect, indicating that the mode of wound healing is significantly different between the epidermis and the urothelium. In addition, an ERK activation wave was not generated or propagated from the wound edge, suggesting that the biochemical mechanism underlying the cell migration may also be different between the epidermis and the urothelium. The experiments were repeated three times to confirm our observations (Figure 2F). Although the velocity of collective migration changed slightly in each experiment, the mode of collective migration did not change. To examine the role of ERK activation in collective cell migration, the MAPK/ERK kinase inhibitor PD0325901 was intravenously administered during time-lapse imaging. By the immunoblotting of the tissue samples, it was confirmed that ERK phosphorylation is markedly suppressed under this condition.25Sano T. Kobayashi T. Negoro H. Sengiku A. Hiratsuka T. Kamioka Y. Liou L.S. Ogawa O. Matsuda M. Intravital imaging of mouse urothelium reveals activation of extracellular signal-regulated kinase by stretch-induced intravesical release of ATP.Physiol Rep. 2016; 4: e13033Crossref PubMed Scopus (22) Google Scholar ERK activity and the migration velocity of cells within 20 μm of the wound edge were quantitated before and after the inhibitor administration (Figure 3, A–D ). In the wounded epidermis, both the basal activity in each cell and the propagation of activation waves of ERK were suppressed by the MAPK/ERK kinase inhibitor (Figure 3, A and C). Epidermal cell migration was also significantly inhibited, albeit not completely, suggesting the requirement of ERK activity for migration (Figure 3D). In the urothelium as well, PD0325901 inhibited ERK activity (Figure 3, B and C). However, the migration velocity of urothelial cells was not decreased to a statistically significant level, indicating that the ERK activ
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