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.
Keywords: CBC, crypt-base columnar cell; CCH, carbachol; CF, cystic fibrosis; Cftr, cystic fibrosis transmembrane conductance regulator; Cystic Fibrosis; DEP, Dishevelled, Egl-10, and Pleckstrin; Dishevelled; Dvl, Dishevelled; EGFP, enhanced green fluorescent protein; EdU, 5-ethynyl-2’-deoxyuridine; Fz, Frizzled; GI, gastrointestinal; ISC, intestinal stem cell; Intracellular pH; KO, knockout; Lgr5, leucine-rich G-protein–coupled receptor 5; Neoplasia; Organoids; PBS, phosphate-buffered saline; PDZ, Post synaptic density protein, Drosophila disc large tumor suppressor, and Zonula occludens-1 protein; PH3, phospho-histone H3; ROI, region of interest; WT, wild type; pHi, intracellular pH.