Evidence for Altered Non‐Canonical Wnt Signaling and Increased Tight Junction Remodeling in Cftr Knockout (KO) Mouse Small Intestine

Abstract

Recent investigations have shown Cftr expression and its functional activity in the Lgr5+ intestinal stem cell (ISC) population of mice where it acts as a negative regulator of proliferation (Strubberg et al., CMGH 2017). In freshly‐isolated small intestinal crypts and crypts of cultured enteroids from Cftr KO mice, loss of Cftr activity results in increased Wnt/β‐catenin signaling and ISC hyperproliferation. A persistent alkaline intracellular pHi in Cftr KO ISC increases plasma membrane association of the Wnt transducer Disheveled (Dvl) thereby facilitating binding to the Wnt receptor Frizzled 7. Dvl also acts a transducer of non‐canonical Wnt signaling involved in directed cell migration and cell polarity, a necessary accompaniment to crypt proliferation. It is known that both cystic fibrosis (CF) patients and Cftr KO mice exhibit increased intestinal permeability. We hypothesized that increased pHi in progenitor cells of the crypt transit‐amplifying (TA) zone in Cftr KO mice increases plasma membrane Dvl association, potentially driving non‐canonical Wnt signaling for active tight junction (TJ) remodeling and increased permeability. Dvl2 association at the apical membrane was measured in TA cells of live WT and Cftr KO enteroids from mice expressing Dvl2‐EGFP (Dvl2 KO/Dvl2‐EGFP rescue) and membrane Tomato (RosamT/mG) using fluorescence confocal microscopy. Activity of the noncanonical Wnt receptor Frizzled 6 (Fz6) and the Rho GTPase Cdc42, an important regulator of intestinal polarity and migration, assessed TJ remodeling. Enteroid permeability was measured by FITC‐dextran (3 kD) diffusion into the lumen. Dvl2‐EGFP intensity at the apical membrane in TA zone crypt cells was increased ~2‐fold in Cftr KO crypts as compared to WT. Increased Dvl2‐EGFP apical membrane localization could be normalized in Cftr KO crypts by two days exposure to reduced medium pH (pH 6.6). Immunolocalization indicated the Fz6 was well expressed on the basolateral membranes near TJ of both WT and Cftr KO TA zone cells. Immunolocalization revealed increased Cdc42 activity at the TJ and immunoblots showed increased Cdc42 protein amounts in both fresh and enteroid crypts in Cftr KO intestine as compared to WT. Intestinal permeability was significantly increased in Cftr KO vs. WT enteroids. We conclude that cell alkalinity in the Cftr KO crypt epithelium affects Dvl transduction of both canonical and non‐canonical Wnt signaling thereby abnormally increasing proliferation and TJ remodeling. Increased TJ remodeling may contribute to increased intestinal permeability in CF patients.Support or Funding InformationSupported by NIDDK 5R01DK048816 and the Cystic Fibrosis Foundation (CLARKE11G0 and LIU13Q0).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.