Loss of CFTR Function Increases Wnt/Planar Cell Polarity (PCP) Signaling and Tight Junction Permeability in Intestinal Epithelium

Abstract

Cystic fibrosis (CF) patients and cystic fibrosis transmembrane conductance regulator (Cftr) knockout (KO) mice exhibit increased intestinal paracellular permeability. This can lead to the translocation of bacterial toxins from the gut lumen into the circulation, adversely affecting liver and lung function. However, the molecular mechanism(s) underlying increased paracellular permeability in CF is unknown. Loss of Cftr Cl− and HCO3− channel function increased intracellular pH (pHi) in crypt epithelium, causing intestinal stem cell hyperproliferation via increased Wnt/β‐catenin signaling. An alkaline pHi increases plasma membrane binding of the Wnt transducer Dishevelled (Dvl), which is postulated to stabilize the association of Dvl with the Wnt receptor Frizzled. We hypothesized that this mechanism would also increase Dvl‐mediated transduction of Wnt/PCP signaling, which regulates cell migration, cell polarity and tight junction (TJ) remodeling. In this study, Dvl2 apical membrane association was measured by fluorescence confocal microscopy in transit‐amplifying cells of enteroid crypts from wild‐type (WT) and Cftr KO mice expressing Dvl2‐EGFP and membrane Tomato (RosamT/mG). Immunofluorescence was used to assess TJ localization of the Wnt/PCP receptor Frizzled 6 (Fz6) and the Rho GTPase Cdc42, a downstream target of Wnt/PCP signaling that regulates intestinal polarity and migration. Paracellular permeability was estimated as the influx rate of Cascade Blue®‐labeled dextran into enteroid lumens using confocal microscopy. Western blot analysis compared Cdc42 protein expression and active (GTP‐bound) Cdc42 was measured via a colorimetric assay in WT and CFTR KO Caco2 cell lines. Quantitative RT‐PCR was used in evaluating the mRNA expression of multiple TJ‐associated proteins in freshly isolated murine crypts. Cftr KO enteroids had increased paracellular permeability relative to WT. Dvl2‐EGFP apical membrane association was increased ~2‐fold in Cftr KO enteroid crypts, as compared to that in WT, and co‐localized with Fz6. Cdc42 was increased in TJ localization, protein expression and activation in CF intestinal epithelium, as compared to WT. mRNA expression of Par3 and Par6b, which are involved in the remodeling of TJ proteins, was significantly decreased in Cftr KO crypts, as compared to WT. In contrast, mRNA expression in Cftr KO crypts of TJ structural protein claudin‐2 was increased, which correlates with greater epithelial Na+ permeability. These findings are consistent with the hypothesis that the alkaline pHi of CF crypt epithelium increases Dvl‐dependent transduction of Wnt/PCP signaling, leading to increased paracellular permeability via TJ remodeling. Wnt/PCP signaling may be a potential target to normalize intestinal paracellular permeability in CF patients.Support or Funding InformationSupported by NIH DK048816 and the Cystic Fibrosis Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.