Abstract
Background & Aims Cystic fibrosis (CF) patients and CF mouse models have increased risk for gastrointestinal tumors. CF mice exhibit augmented intestinal proliferation of unknown etiology and an altered intestinal environment. We examined the role of 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 Sox9EGFP intestinal epithelia and pHi regulation by confocal microfluorimetry of Lgr5+-EGFP ISCs. Plasma membrane association of the Wnt transducer Disheveled 2 (Dvl2) was assessed by fluorescence imaging of live enteroids from WT and CF mice crossed with Dvl2-EGFP/RosamT/mG mice.
Results Relative to WT, CF intestinal crypts showed a ~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 Lgr5+-EGFP ISCs. CF crypt-base columnar cells (CBCs) demonstrated a generalized increase in plasma membrane Dvl2-EGFP association as compared to WT. Dvl2-EGFP membrane association was charge- and pH-dependent and increased in WT CBCs by Cftr inhibition.
Conclusions CF intestine exhibits 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.
Footnotes
Synopsis: This study documents the functional activity of Cftr in the active intestinal stem cell population of murine intestine. In the absence of Cftr, stem cell proliferation and Wnt/β-catenin signaling are increased, which is associated with changes in intracellular pH-dependent plasma membrane localization of the Wnt-transducer Disheveled. Increased stem cell proliferation may contribute to gast rointestinal cancer risk in cystic fibrosis.
The authors have no conflicts of interest.
The research was supported by grants from the National Institute of Diabetes and Digestive and Kidney Disease (LLC: NIDDK 5R01DK048816) and the Cystic Fibrosis Foundation (LLC: CLARKE11G0; CLARKE15G0; JL: LIU13Q0).
