title: Fluorescence-based gene reporter plasmid to track canonical Wnt signaling in ENS inflammation.
abstract: In several gut inflammatory or cancer diseases, cell-cell interactions are compromised, and an increased cytoplasmic expression of β-catenin is observed. Over the last decade, numerous studies provided compelling experimental evidence that the loss of cadherin-mediated cell adhesion can promote β-catenin release and signaling without any specific activation of the canonical Wnt pathway. In the present work, we took advantage of the ability of lipofectamine-like reagent to cause a synchronous dissociation of adherent junctions in cells isolated from the rat enteric nervous system (ENS) for obtaining an in vitro model of deregulated β-catenin signaling. Under these experimental conditions, a green fluorescent protein Wnt reporter plasmid called ΔTop_EGFP3a was successfully tested to screen β-catenin stabilization at resting and primed conditions with exogenous Wnt3a or lipopolysaccharide (LPS). ΔTop_EGFP3a provided a reliable and strong fluorescent signal that was easily measurable and at the same time highly sensitive to modulations of Wnt signaling following Wnt3a and LPS stimulation. The reporter gene was useful to demonstrate that Wnt3a exerts a protective activity in the ENS from overstimulated Wnt signaling by promoting a downregulation of the total β-catenin level. Based on this evidence, the use of ΔTop_EGFP3a reporter plasmid could represent a more reliable tool for the investigation of Wnt and cross-talking pathways in ENS inflammation.
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy; rosa.diliddo@unipd.it.
authors.forename: Rosa
authors.identifier: http://orcid.org/0000-0001-7794-6804
authors.initials: R
authors.lastname: Di Liddo
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy;
authors.forename: Thomas
authors.initials: T
authors.lastname: Bertalot
authors.affiliation: Department of Biotechnology, University of Applied Sciences Kaiserslautern/Zweibrücken, Germany;
authors.forename: Anne
authors.initials: A
authors.lastname: Schuster
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy;
authors.forename: Sandra
authors.initials: S
authors.lastname: Schrenk
authors.affiliation: Department of Biochemistry, University of Applied Sciences Kaiserslautern, Kaiserslautern, Germany;
authors.forename: Oliver
authors.initials: O
authors.lastname: Müller
authors.affiliation: Department of Biochemistry, University of Applied Sciences Kaiserslautern, Kaiserslautern, Germany;
authors.forename: Johanna
authors.initials: J
authors.lastname: Apfel
authors.affiliation: Department of Biochemistry, University of Applied Sciences Kaiserslautern, Kaiserslautern, Germany;
authors.forename: Patricia
authors.initials: P
authors.lastname: Reischmann
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy;
authors.forename: Senthilkumar
authors.initials: S
authors.lastname: Rajendran
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy;
authors.forename: Riccardo
authors.initials: R
authors.lastname: Sfriso
authors.affiliation: Department of Woman and Child Health, University of Padova, Padova, Italy;
authors.forename: Marco
authors.initials: M
authors.lastname: Gasparella
authors.affiliation: Tissue Engineering and Signaling-Onlus, Caselle di Selvazzano Dentro, Padova, Italy; and.
authors.forename: Pier Paolo
authors.initials: PP
authors.lastname: Parnigotto
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy;
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Conconi
authors.affiliation: Department of Biotechnology, University of Applied Sciences Kaiserslautern/Zweibrücken, Germany; Medical Faculty Mannheim, Department of Pediatric Surgery, University of Heidelberg, Mannheim, Germany.
authors.forename: Karl Herbert
authors.initials: KH
authors.lastname: Schäfer
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002462:Cell Membrane / Q000473:pathology; D015536:Down-Regulation / Q000187:drug effects; D017615:Enteric Nervous System; D005453:Fluorescence; D005759:Gastroenteritis / Q000235:genetics* / Q000503:physiopathology; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008055:Lipids; D008070:Lipopolysaccharides / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D060449:Wnt Signaling Pathway / Q000235:genetics*; D060509:Wnt3A Protein / Q000494:pharmacology; D051176:beta Catenin / Q000378:metabolism
chemical_list: D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D008070:Lipopolysaccharides; D060509:Wnt3A Protein; D051176:beta Catenin; D049452:Green Fluorescent Proteins
keywords: cell adhesions; enteric nervous system; green fluorescent protein Wnt reporter plasmid; lipopolysaccharide