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

In several gut inflammatory or cancer diseases, cell-cell interactions are compromised, and an increased cytoplasmic expression of ß-catenin is observed. Over the last decade, numerous studies provided compelling experimental evidence that the loss of cadherin-mediated cell adhesion can promote ß-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 ß-catenin signaling. Under these experimental conditions, a green fluorescent protein Wnt reporter plasmid called ΔTop_EGFP3a was successfully tested to screen ß-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 ß-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.

A new fluorescence-based reporter gene vector as a tool for analyzing and fishing cells with activated wnt signaling pathway.

The dysregulated Wnt pathway is a major cause for the activation of cell proliferation and reduced differentiation in tumor cells. Therefore the Wnt signaling pathway is the on-top target in searching for new anticancer drugs or therapeutic strategies. Although the key players of the pathway are known, no specific anti-Wnt drug entered a clinical trial by now. Several screening approaches for potential compounds have been performed with a reporter gene assay using multiple T-cell factor/lymphoid enhancer factor (TCF/LEF) binding motifs as promoters which control luciferase or ß -galactosidase as reporter genes. In our work, we designed a reporter gene construct which anchors the enhanced green fluorescent protein (eGFP) to the plasma membrane. HEK 293T cells, which were stably transfected with this construct, express eGFP on the outer membrane after activation with either LiCl or WNT3A protein. Thus, cells with activated Wnt pathway could be identified and fished out of a heterogeneous cell pool by the use of magnetic-labeled anti-GFP antibodies. In summary, we present a new tool to easily detect, quantify, and sort cells with activated Wnt signaling pathway in a simple, fast, and cost-effective way.