Notch controls urothelial integrity in the mouse bladder
Varvara Paraskevopoulou, … , Ioannis S. Pateras, Apostolos Klinakis
Varvara Paraskevopoulou, … , Ioannis S. Pateras, Apostolos Klinakis
Published February 13, 2020
Citation Information: JCI Insight. 2020;5(3):e133232. https://doi.org/10.1172/jci.insight.133232.
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Research Article Cell biology

Notch controls urothelial integrity in the mouse bladder

Abstract

The Notch signaling pathway mediates cell-cell communication regulating cell differentiation and proliferation and cell fate decisions in various tissues. In the urinary bladder, Notch acts as a tumor suppressor in mice, while mutations in Notch pathway components have been identified in human bladder cancer as well. Here we report that the genetic inactivation of Notch in mice leads to downregulation of cell-cell and cell-ECM interaction components, including proteins previously implicated in interstitial cystitis/bladder pain syndrome (IC/BPS), structural defects and mucosal sloughing, inflammation, and leaky urine-blood barrier. Molecular profiling of ailing mouse bladders showed similarities with IC/BPS patient tissue, which also presented low Notch pathway activity as indicated by reduced expression of canonical Notch targets. Urothelial integrity was reconstituted upon exogenous reactivation of the Notch pathway, implying a direct involvement of Notch. Despite damage and inflammation, urothelial cells failed to proliferate, uncovering a possible role for α4 integrin in urothelial homeostasis. Our data uncover a broad role for Notch in bladder homeostasis involving urothelial cell crosstalk with the microenvironment.

Authors

Varvara Paraskevopoulou, Vangelis Bonis, Vasilis S. Dionellis, Nikolaos Paschalidis, Pelagia Melissa, Evangelia Chavdoula, Eleni Vasilaki, Ioannis S. Pateras, Apostolos Klinakis

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Figure 4

ITGA4 controls proliferation of bladder cells.

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ITGA4 controls proliferation of bladder cells. (A) Immunofluorescence on...
(A) Immunofluorescence on Krt5CreERT2 Ncstnfl/fl mouse bladders against the proliferation marker Ki-67. Scale bar: 100 μm. Arrowheads indicate Ki-67+ cells. (B) Scatter dot plot indicating residual Itga4 expression levels in primary mouse bladder cells upon shRNA-mediated (sh1 and 2) knockdown of Itga4 expression. shRNAs were introduced through lentiviral infection. Expression is presented as ratio over control (cells infected with a scrambled shRNA sequence). In all cases n = 3 experiments were analyzed. *P < 0.05. (C) Representative images and scatter dot plot indicating clonogenic potential of primary bladder cell cultures on Matrigel isolated from C57BL/6 mice and infected with lentiviral vectors expressing control (Scr) or shRNAs against Itga4. On the y axis is presented the number of colonies obtained from 10,000 cells. Results from 3 independent experiments are shown. ****P < 0.0001. Scale bar: 1 mm. (D) Representative images and scatter dot plot indicating clonogenic potential of primary bladder cell cultures (n = 4) on Matrigel isolated from C57BL/6 mice treated with increasing concentration of the FAK1 inhibitor GSK2256098. On the y axis is presented the number of colonies obtained from 10,000 cells. ****P < 0.0001. Scale bar: 1 mm. (E) Representative immunofluorescence images with antibodies against the proliferation marker Ki-67 and KRT14 in WT C57BL/6 mice challenged with CPP having received vehicle or an ITGA4 blocking antibody before the CPP administration. Scatter dot plot from n = 3 mice for each group indicating proliferation index (percentage of Ki-67+ cells over total number of cells). *P < 0.05. Scale bar: 100 μm. (F) Representative images and scatter dot plot indicating clonogenic potential of primary bladder cell cultures (n = 4) on Matrigel isolated from C57BL/6 mice and treated with either vehicle (untreated) or an ITGA4 blocking antibody. **P < 0.01. Scale bar: 1 mm. (G) Representative whole-mount bright-field and fluorescence images (Tomato) of tissue explants from WT or Krt5CreERT2 Ncstnfl/fl mice with or without constitutive exogenous expression of a mouse Itga4 cDNA through lentiviral infection of the tissue explants. Scale bars: 1 mm. Explants were scored on day 3. For B–F, data presented are mean ± SEM and Student’s t test was used. P values were corrected for multiple testing using the Benjamini-Hochberg method (C and D). For C, D, and F, urospheres were photographed and scored on day 10. DAPI was used as nuclear counterstain in A and E.