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.

STW 5 Herbal Preparation Modulates Wnt3a and Claudin 1 Gene Expression in Zebrafish IBS-like Model

“…Moreover, as reported by Manicassamy et al [32], the activation of β-Catenin in dendritic cells contributes to the switch from immunity to tolerance, stimulating the expression of the anti-inflammatory NF-kβ target gene IL-10. As previously reported by our group, the Wnt receptor Frizzled-9 and its ligand Wnt3a are expressed in the rat myenteric plexus of the enteric nervous system (ENS) [25], and the alteration of adherent junctions in ENS cells in vitro induces an aberrant accumulation of β-Catenin [33]. In contrast, the activation of pro-inflammatory mediators seems to be dependent on the non-canonical Wnt pathway [34].…”

Role of Inflammation and the NF-κB Signaling Pathway in Hirschsprung Disease

“…In addition, the inflammatory environment specific immune cells (macrophages, dendritic cells) are a significant source of pro-inflammatory cytokines including IFN, IL-1, and TNF, which induce inflammation through the NF-κB pathway [33,34]. Moreover, the inflammation itself plays a role in neurostimulation and enteric neuronal migration [35][36][37][38][39], as well as in neuroregeneration through the NF-κB pathway [40].…”