Monitoring of Active Notch Signaling in Mouse Bladder Urothelium
Notch signaling plays a crucial role in differentiation and homeostasis in a wide variety of epithelia. The tumor suppressor role of Notch in bladder urothelium is well accepted as the inactivation of this pathway due to damaging mutations in its componen
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Monitoring of Active Notch Signaling in Mouse Bladder Urothelium Panagiotis Karakaidos and Theodoros Rampias Abstract Notch signaling plays a crucial role in differentiation and homeostasis in a wide variety of epithelia. The tumor suppressor role of Notch in bladder urothelium is well accepted as the inactivation of this pathway due to damaging mutations in its components is associated with neoplastic transformation. Monitoring Notch signaling is therefore critical to understand how the deregulation of cell–cell communication can lead to differentiation loss and carcinogenesis. In this chapter, we provide a method to visualize active Notch signaling by the detection of the nuclear levels of Notch intracellular domain in mouse urothelium. The technique outlined below is characterized by high sensitivity and specificity and has been successfully applied to human tumor specimens. In this context, this technique could be used to characterize the molecular profile of Notch-deficient tumors and analyze the clonal expansion dynamics and the heterogeneity patterns of Notch inactivation. Key words Cancer, Immunofluorescence, Notch intracellular domain (NICD), Notch signaling, Tyramide signal amplification (TSA), Urothelium
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Introduction One of the most, if not the best, studied cell–cell communication signaling pathways is the canonical Notch signaling pathway. Notch is a highly conserved pathway across many species and it plays major roles in a diverge set of key cellular functions including cell proliferation, death, and differentiation with profound effects in metazoan development and disease. The uniqueness of Notch, as compared to other major intercellular signaling pathways, relies on features like the highly topological constraints (activation requires direct cell-to-cell contact), dose-dependent response (lack of signal amplification means), pleiotropic outcome (developmental stage and/or cell context dependent response), and its signal transduction mode of regulated proteolysis (Notch receptors are proteolytically cleaved upon ligand activation and a fragment, the Notch intracellular domain (NICD), is imported to the nucleus) [1]. In mammals, there are four Notch receptors (Notch1–4) all of which are single-pass type I transmembrane proteins that transduce
Panagiotis Karakaidos and Theodoros Rampias
signals by binding to membrane bound ligands (Jagged1–2, Deltalike 1, 3, and 4) on adjacent cells. Their extracellular domain contains several tandem EGF-like repeats (29–36 in humans), a negative regulatory region (NRR), a single transmembrane region (TM), and the NICD (Fig. 1a). In canonical Notch pathway, upon ligand binding, Notch receptors undergo a conformational change enabling access of a membrane-bound ADAM metalloprotease that cleaves receptors in the juxtamembrane extracellular domain proximal to the transmembrane region) [2]. ADAM-dependent cleavage, also termed S2 cleavage is followed by a cleavage in the helical transmembrane region (S3 cleavage site after valine residue 1754 in human Notch1 receptor
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