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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.

ORGANISM(S): Mus musculus

PROVIDER: GSE140859 | GEO | 2020/04/07

REPOSITORIES: GEO

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Project description:The pathogenesis of interstitial cystitis/bladder pain syndrome (IC/BPS) remains incompletely understood. Bladder fibrosis is a significant histopathological feature of IC/BPS, particularly in non-Hunner-type IC (NHIC). Transient receptor potential cation channel subfamily C member 3 (TRPC3) is known to play a crucial role in myocardial and renal fibrosis. This study investigates the involvement of TRPC3 in bladder fibrosis associated with IC/BPS. A rat model of IC/BPS was established using cyclophosphamide (CYP, 50 mg/kg, intraperitoneally, every 3 days for 3 doses). Bulk RNA sequencing was used to identify differentially expressed transient receptor potential (TRP) channel genes in CYP-induced cystitis rats, whereas single-cell RNA sequencing was utilized to pinpoint the cell type predominantly expressing transient receptor potential cation channel subfamily C member 3 (TRPC3) . TRPC3 inhibition was achieved through intraperitoneal injection of Pyrazole 3 (Pyr3, 0.1 mg/kg or 1 mg/kg). Suprapubic mechanical allodynia was assessed using up-down method with von Frey filaments and micturition frequency was assessed by cystometry. The expression of TRPC3 and components of the transforming growth factor beta (TGF-β)/Smad signaling pathway (TGF-β, p-Smad2, and p-Smad3) in the bladder was analyzed by Western blot. Bladder fibrosis was assessed through Masson’s staining and detection of fibrosis markers (Vimentin, Collagen I, and Collagen III) using Western blot. The RNA and protein expression of TRPC3 was up-regulated in CYP-induced cystitis rats. TRPC3 inhibition led to significant improvements in suprapubic mechanical allodynia and reduced micturition frequency in CYP-induced cystitis rats. Western blot analyses revealed that markers of bladder fibrosis (Vimentin, Collagen I, and Collagen III) were markedly upregulated in CYP-induced cystitis rats. Masson’s staining showed an increased area of collagen fibers. Additionally, inhibition of TRPC3 resulted in significant downregulation of the TGF-β/Smad signaling pathway (TGF-β, p-Smad2, and p-Smad3) in CYP-induced cystitis rats. TRPC3 activation contributes to bladder fibrosis in IC/BPS. Inhibition of TRPC3 ameliorates suprapubic mechanical allodynia and micturition frequency in CYP-induced cystitis rats by downregulating the TGF-β/Smad pathway. TRPC3 represents a potential therapeutic target for managing bladder fibrosis in IC/BPS.

Project description:Changes in human bladder epithelial cell gene expression associated with interstitial cystitis or antiproliferative factor treatment. Explanted bladder epithelial cells from patients with interstitial cystitis (IC) have been shown to differ from explanted control cells in several ways, including production of an antiproliferative factor (APF), altered production of certain epithelial growth factors, and rate of proliferation. To better understand the role of the APF in abnormal bladder epithelial cell proliferation in IC, we studied gene expression patterns in normal bladder epithelial cells treated with APF vs. mock APF and compared them to expression patterns in IC vs. normal cells using microarray analysis. Oligo-dT-primed total cellular RNA was labeled with [33P]dCTP and hybridized to GeneFilter GF211 microarray membranes (Research Genetics) containing cDNA for 3,964 human genes. Thirteen genes that function in epithelial cell proliferation or differentiation were consistently differentially expressed in both IC (compared with control) and APF-treated (compared with mock APF-treated) normal bladder epithelial cells. The general pattern of gene expression in IC and APF-treated cells suggested a less proliferative phenotype, with increased expression of E-cadherin, phosphoribosylpyrophosphate synthetase-associated protein 39, and SWI/SNF complex 170-kDa subunit, and decreased expression of vimentin, {alpha}2-integrin, {alpha}1-catenin, cyclin D1, and jun N-terminal kinase 1; these findings were confirmed for the structural gene products (E-cadherin, vimentin, {alpha}2-integrin, and {alpha}-catenin) by immunohistochemistry. These results are compatible with the previously noted decreased proliferation rate of IC and APF-treated normal cells, and indicate that the mechanism whereby APF inhibits cell proliferation may involve both downregulation of genes that stimulate cell proliferation along with upregulation of genes that inhibit cell growth.

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Notch controls urothelial integrity in the mouse bladder.

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