Cftr Modulates Wnt/β-Catenin Signaling and Stem Cell Proliferation in Murine Intestine

• Published in: Cellular and molecular gastroenterology and hepatology Vol. 5; no. 3; pp. 253 - 271
• Main Authors: Strubberg, Ashlee M, Liu, Jinghua, Walker, Nancy M, Stefanski, Casey D, MacLeod, R. John, Magness, Scott T, Clarke, Lane L
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2018; Elsevier
• Subjects: Cystic Fibrosis; Dishevelled; Gastroenterology and Hepatology; Intracellular pH; Neoplasia; Organoids; Original Research; 5-ethynyl-2’-deoxyuridine; CBC; phosphate-buffered saline; EGFP; pHi; phospho-histone H3; Organoids; ISC; carbachol; Frizzled; WT; knockout; PBS; Cftr; GI; CF; Cystic Fibrosis; KO; PDZ; CCH; ROI; DEP; Intracellular pH; gastrointestinal; Dishevelled, Egl-10, and Pleckstrin; crypt-base columnar cell; Dishevelled; intestinal stem cell; enhanced green fluorescent protein; Fz; EdU; region of interest; cystic fibrosis transmembrane conductance regulator; Post synaptic density protein, Drosophila disc large tumor suppressor, and Zonula occludens-1 protein; leucine-rich G-protein–coupled receptor 5; Lgr5; PH3; wild type; Dvl; Neoplasia; CCH, carbachol; EGFP, enhanced green fluorescent protein; CBC, crypt-base columnar cell; Cftr, cystic fibrosis transmembrane conductance regulator; GI, gastrointestinal; PDZ, Post synaptic density protein, Drosophila disc large tumor suppressor, and Zonula occludens-1 protein; Fz, Frizzled; WT, wild type; CF, cystic fibrosis; DEP, Dishevelled, Egl-10, and Pleckstrin; pHi, intracellular pH; EdU, 5-ethynyl-2’-deoxyuridine; PBS, phosphate-buffered saline; KO, knockout; PH3, phospho-histone H3; ROI, region of interest; Lgr5, leucine-rich G-protein–coupled receptor 5; Dvl, Dishevelled; ISC, intestinal stem cell
• ISSN: 2352-345X; 2352-345X
• DOI: 10.1016/j.jcmgh.2017.11.013

Background & Aims Cystic fibrosis (CF) patients and CF mouse models have increased risk for gastrointestinal tumors. CF mice show augmented intestinal proliferation of unknown etiology and an altered intestinal environment. We examined the role of the cystic fibrosis transmembrane conductance regulator (Cftr) in Wnt/β-catenin signaling, stem cell proliferation, and its functional expression in the active intestinal stem cell (ISC) population. Dysregulation of intracellular pH (pHi ) in CF ISCs was investigated for facilitation of Wnt/β-catenin signaling. Methods Crypt epithelia from wild-type (WT) and CF mice were compared ex vivo and in intestinal organoids (enteroids) for proliferation and Wnt/β-catenin signaling by standard assays. Cftr in ISCs was assessed by immunoblot of sorted Sox9 enhanced green fluorescent protein (EGFP) intestinal epithelia and pHi  regulation by confocal microfluorimetry of leucine-rich G-protein–coupled receptor 5 ISCs. Plasma membrane association of the Wnt transducer Dishevelled 2 (Dvl2) was assessed by fluorescence imaging of live enteroids from WT and CF mice crossed with Dvl2-EGFP/ACTB-tdTomato,-EGFP)Luo/J (RosamT/mG ) mice. Results Relative to WT, CF intestinal crypts showed an ∼30% increase in epithelial and Lgr5+ ISC proliferation and increased Wnt/β-catenin signaling. Cftr was expressed in Sox9EGFPLo ISCs and loss of Cftr induced an alkaline pHi in ISCs. CF crypt-base columnar cells showed a generalized increase in plasma membrane Dvl2-EGFP association as compared with WT. Dvl2-EGFP membrane association was charge- and pH-dependent and increased in WT crypt-base columnar cells by Cftr inhibition. Conclusions CF intestine shows increased ISC proliferation and Wnt/β-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/β-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors.