Format

Send to

Choose Destination
Am J Physiol Gastrointest Liver Physiol. 2016 Mar 15;310(6):G337-46. doi: 10.1152/ajpgi.00191.2015. Epub 2016 Jan 14.

Fluorescence-based gene reporter plasmid to track canonical Wnt signaling in ENS inflammation.

Author information

1
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy; rosa.diliddo@unipd.it.
2
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy;
3
Department of Biotechnology, University of Applied Sciences Kaiserslautern/Zweibrücken, Germany;
4
Department of Biochemistry, University of Applied Sciences Kaiserslautern, Kaiserslautern, Germany;
5
Department of Woman and Child Health, University of Padova, Padova, Italy;
6
Tissue Engineering and Signaling-Onlus, Caselle di Selvazzano Dentro, Padova, Italy; and.
7
Department of Biotechnology, University of Applied Sciences Kaiserslautern/Zweibrücken, Germany; Medical Faculty Mannheim, Department of Pediatric Surgery, University of Heidelberg, Mannheim, Germany.

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.

KEYWORDS:

cell adhesions; enteric nervous system; green fluorescent protein Wnt reporter plasmid; lipopolysaccharide

PMID:
26767983
DOI:
10.1152/ajpgi.00191.2015
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for Atypon
Loading ...
Support Center