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

ORIGINAL RESEARCH Cftr Modulates Wnt/b-Catenin Signaling and Stem Cell Proliferation in Murine Intestine Ashlee M. Strubberg,1 Jinghua Liu,2 Nancy M. Walker,2 Casey D. Stefanski,1 R. John MacLeod,3 Scott T. Magness,4 and Lane L. Clarke1,2 1 Department of Biomedical Sciences, 2Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri; Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canada; 4Department of Medicine, Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 3 In vivo Cftr pHi Wnt Fz7 Y Dvl Stem Cell Proliferation In vitro β -cat Cftr KO stem cell SUMMARY This study documents the functional activity of cystic fibrosis transmembrane conductance regulator (Cftr) in the active intestinal stem cell population of murine intestine. In the absence of Cftr, the intracellular pH, inner membrane localization of the Wnt-transducer Dishevelled, Wnt/b-catenin signaling, and stem cell proliferation are all increased, which may contribute to increased gastrointestinal cancer risk in cystic fibrosis. 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/b-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/b-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/b-catenin signaling by standard assays. Cftr in ISCs was assessed by immunoblot of sorted Sox9enhanced 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 w30% increase in epithelial and Lgr5þ ISC proliferation and increased Wnt/b-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/b-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/b-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors. (Cell Mol Gastroenterol Hepatol 2018;5:253–271; https://doi.org/10.1016/j.jcmgh.2017.11.013) Keywords: Cystic Fibrosis; Dishevelled; Organoids; Intracellular pH; Neoplasia. 254 Strubberg et al See editorial on page 418. C ystic fibrosis (CF) is a heritable genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The gene product (CFTR) is a major anion channel of fluid transporting epithelia where it functions in transepithelial Cl- and HCO3- secretion.1,2 CF affects multiple organs, in particular the airway epithelia where failure of mucociliary clearance results in bacterial colonization of the lung. However, intestinal disease is one of the earliest manifestations of CF and presents with life-long conditions, including small intestinal bacterial overgrowth,3 low-grade small-bowel inflammation,4,5 obstructive bowel disease,6,7 and an increased incidence of gastrointestinal (GI) cancer.8,9 Cftr knockout (KO) mice recapitulate CF intestinal disease without significant manifestations of pancreatic, liver, or lung disease. The disease phenotype includes a high incidence of bowel obstruction,10 low-grade bowel inflammation,11 small intestinal bacterial overgrowth,3 dysbiosis,12 and the spontaneous development of intestinal tumors with age.13 Previous in vivo studies have shown that Cftr KO mice show increased intestinal epithelial proliferation without a corresponding increase in apoptosis,14 a condition that may predispose to intestinal neoplasia.15 Recent epidemiologic studies have shown strong correlations between the rate of stem cell division and the incidence of cancer.16 Because the intestine has one of the highest rates of epithelial turnover in the body, pathologic manifestations of CF that enhance the rate of epithelial turnover and contribute to intestinal inflammation are predicted to increase the risk of GI cancer. However, a mechanistic understanding linking the absence of Cftr with enhanced proliferation of the intestinal epithelium, particularly the stem cell population, has not been advanced. Cftr is highly expressed in intestinal crypts,17,18 the proliferative compartment of the intestine, and by providing apical membrane Cl- and HCO3- ion permeability has an impact on the regulation of epithelial intracellular pH (pHi). Loss of Cftr function by acute channel blockade or in Cftr KO enteroids results in an incompletely compensated alkaline pHi in the crypt epithelium.19 Compensation of the alkaline pHi is impaired by a corresponding increase in intracellular Cl- concentration, which reduces cellular anion exchange activity.20 Several aspects of cell proliferation are known to be facilitated by an alkaline pHi, including cell-cycle phase progression at G2/M,21 optimization of DNA replication,22 cytoskeleton remodeling and cell migration,23,24 and membrane biogenesis.25 Cell alkalinity also has been shown to facilitate Wnt signaling,26,27 which may directly affect stem cell proliferation. Wnt/b-catenin signaling is essential for homeostasis and proliferation of intestinal stem cells28 and often aberrantly is activated in intestinal cancer. In Drosophila species, pHi changes can alter Wnt signaling by modulating the interaction of the initial signal mediator Dishevelled (Dsh) with the Wnt receptor Frizzled (Fz) at the plasma membrane.26 The critical binding of Dvl’s post synaptic density protein, Drosophila disc large tumor suppressor and Zonula Cellular and Molecular Gastroenterology and Hepatology Vol. 5, No. 3 occludens-1 protein (PDZ) domain with the PDZ binding domain of Fz is facilitated by a stable interaction of Dvl’s polybasic Dishevelled, Egl-10, and Pleckstrin (DEP) domain to negatively charged phospholipids (phosphatidic acid, phosphatidylglycerol) at the inner leaflet of th (...truncated)