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 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 ∼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.
Post synaptic density protein, Drosophila disc large tumor suppressor, and Zonula occludens-1 protein
pHi
intracellular pH
PH3
phospho-histone H3
ROI
region of interest
WT
wild type
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Author contributions Ashlee M. Strubberg acquired data, analyzed and interpreted data, drafted the manuscript, and critically revised the manuscript; Jinghua Liu acquired data, analyzed and interpreted data, and critically revised the manuscript; Nancy M. Walker acquired data, analyzed and interpreted data, performed the statistical analysis, and provided technical support; Casey D. Stefanski acquired data, analyzed and interpreted data, and provided technical support; R. John MacLeod critically revised the manuscript; Scott T. Magness acquired, analyzed, and interpreted data; and Lane L. Clarke was responsible for the study concept and design, analyzed and interpreted data, critically revised the manuscript, obtained funding, provided administrative support, and supervised the study.
Conflicts of interest The authors disclose no conflicts.
Funding The research was supported by grants from the National Institute of Diabetes and Digestive and Kidney Disease (5R01DK048816 to L.L.C.) and the Cystic Fibrosis Foundation (CLARKE11G0 and CLARKE15G0 to L.L.C., and LIU13Q0 to J.L.).