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, 5 (3), 253-271
eCollection

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

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

Ashlee M Strubberg et al. Cell Mol Gastroenterol Hepatol.

Abstract

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.

Figures

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Figure 1
Figure 1
Increased proliferation in the Cftr KO intestine ex vivo and early passage enteroids. (A) Left: Isolated crypts from WT and Cftr KO mice labeled for PH3 (green), a marker of mitosis. Nuclei are labeled with TO-PRO 3 nuclear stain (red). Right: Cumulative data showing differences in proliferation rates between freshly isolated WT and Cftr KO crypts as measured by PH3 immunofluorescence. Data are expressed as the number of PH3+ cells/cryptbase optical cross-section. *P < .001; n = 4 WT/Cftr KO pairs (32–38 crypts/genotype). (B) Cumulative data showing the average Lgr5-EGFP+ stem cell numbers/Lgr5-EGFP+ crypt in freshly isolated crypts from WT/Lgr5-EGFP and Cftr KO/Lgr5-EGFP small intestine. *P < .02; n = 3 WT/Cftr KO pairs (81–113 Lgr5+ crypts/genotype). (C) Left: Enteroid crypts from WT and Cftr KO mice labeled with EdU for S-phase nuclei (green). Nuclei are labeled with 4′,6-diamidino-2-phenylindole nuclear stain (blue). Right: Cumulative data showing differences in proliferation rates between WT and Cftr KO enteroid crypts as measured by EdU+ nuclei and the percentage of EdU+ (%EdU+ nuclei) per crypt optical cross-section. *P < .03; n = 5 WT/Cftr KO pairs (48–52 crypts/genotype). (D) Cumulative data showing the average Lgr5-EGFP+ stem cell numbers/Lgr5-EGFP+ crypt in enteroid crypts from WT and Cftr KO small intestine. *P < .05; n = 5 WT/Cftr KO pairs (156–171 Lgr5+ crypts/genotype).
Figure 2
Figure 2
Increased Wnt/β-catenin signaling in the Cftr KO intestine ex vivo and early passage enteroids. (A) Immunoblot for active β-catenin (dephosphorylated at Ser37/Thr41) and downstream Wnt target gene Lef1 using freshly isolated crypts (Fresh crypts) and passage 1 enteroids (Passaged enteroids) from a matched pair of WT and Cftr KO mice. Right: WT enteroids treated with Wnt3a conditioned medium for 24 hours before collection. (B) Left: Densitometric analysis for immunoblots of active β-catenin from WT and Cftr KO freshly isolated crypts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin were used as loading controls. Data are presented as % WT; n = 11 WT/Cftr KO pairs. *P < .03. Right: Densitometric analysis for immunoblots of Lef1 protein expression from WT and Cftr KO isolated crypts. GAPDH or β-actin were used as loading control. Data are presented as % WT; n = 7 WT/Cftr KO pairs. (C) Left: Densitometric analysis for immunoblots of active β-catenin from WT and Cftr KO enteroids. GAPDH or β-actin were used as loading controls. Data are presented as % WT; n = 5 WT/Cftr KO pairs. *P < .02. Right: Densitometric analysis for immunoblots of Lef1 protein expression in WT and Cftr KO enteroids. GAPDH or β-actin were used as loading controls. Data are presented as % WT; n = 4 WT/Cftr KO pairs. *P < .03.
Figure 3
Figure 3
Expression and function of Cftr in small intestinal stem cells. (A) Immunoblot for Cftr in flow cytometry cell fractions of small intestinal epithelium isolated from Sox9EGFP mice. SubLo, sublow Sox9EGFP-expressing cell fraction (transit-amplifying progenitors); Lo, low Sox9EGFP-expressing cell fraction (crypt-base stem cells, Lgr5+); Neg, negative for Sox9EGFP expression cell fraction (enterocyte, goblet, Paneth); Total, total small intestinal epithelium. Cftr KO total epithelium, negative control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), loading control. Representative of 3 Sox9EGFP mice. (B) Confocal micrograph of Lgr5-EGFP–expressing intestinal stem cells in crypt epithelium (white arrows). White dashed line outlines crypt. (C) Intracellular pH of Lgr5-EGFP+ cells in WT/Lgr5–EGFP enteroids treated with either dimethyl sulfoxide (vehicle, small circles) or Cftrinh-172 (10 μmol/L for 1 h, small squares). Treatment average, large circle or square. *P < .02 vs vehicle; n = 10–13 Lgr5–EGFP cells from 7–8 enteroids cultured from 6 WT/Lgr5–EGFP mice. (D) Intracellular pH of Lgr5–EGFP+ cells in enteroids from matched WT/Lgr5–EGFP (open small circles) and Cftr KO/Lgr5–EGFP (filled small circles) mice. Average shown as large circles. *P < .04; n = 10–12 Lgr5–EGFP cells from 6 enteroids from 3 WT/Lgr5–EGFP and Cftr KO/Lgr5–EGFP matched mice. (E) Left: ISC-enriched enterospheres from WT and Cftr KO matched mice and a photomicrograph showing EdU-positive nuclei (green) and nuclei (red) in an optical cross-section of WT and Cftr KO enterospheres. Right: Percentage of EdU-positive nuclei relative to total nuclei in optical cross-sections of WT and Cftr KO enterospheres. (Total number of nuclei/enterosphere was WT = 36.1 ± 3.8; Cftr KO = 46.7 ± 2.0, n = 32 enterospheres/genotype; P < .009). *P < .05 vs WT; n = 4 WT and Cftr KO matched mice (8 enterospheres/mouse).
Figure 4
Figure 4
Increased Dvl2–EGFP localization at the plasma membrane in Cftr KO crypt-base columnar cells. (A) Representative images of WT/Dvl2-EGFP/RosamT/mG (top) and Cftr KO/Dvl2-EGFP/RosamT/mG (bottom) enteroid crypts showing Dvl2-EGFP (green), RosamT/mG (red), and merged images. Magnified merged and differential interference contrast merged with Dvl2–EGFP (DIC+Dvl2–EGFP) images of crypt base (from white boxes), were magnified (×3.6). P, Paneth cell; L, crypt lumen. White dashed lines outline apical membrane orient toward the crypt lumen. (B) Cumulative pHi data for the supranuclear region of CBCs in WT and Cftr KO enteroids. *P < .03 vs WT; n = 4–8 crypts from 4 WT and Cftr KO matched mice. (C) Crypt base model depicting the measurement by confocal microfluorimetry of Dvl2–EGFP intensity 2 pixels interior to the lateral-Lateral Membrane (lateral to central crypt axis) and medial-Lateral membrane (medial to central crypt axis) plasma membrane of CBCs. Green dashed line, central crypt axis. Brackets indicate sites of measurement on the supranuclear lateral plasma membranes. Paneth, Paneth cell. (D) Left: Cumulative data showing the average Dvl2–EGFP intensity ratio within 1.15 μm of the CBC supranuclear plasma membrane of individual mice (small circles) and overall average (large circles) for WT/Dvl2-EGFP/RosamT/mG (WT) and Cftr KO/Dvl2-EGFP/RosamT/mG (Cftr KO) sex-matched mouse pairs (n = 7 and 6 mice, respectively). Dashed line indicates the average Dvl2–EGFP pixel intensity for the entire supranuclear region of CBC, which has been set to 1.0 (average intensity = 59.5 ± 8.8 for WT and 88.5 ± 15.9 for Cftr KO; ns; n = 7–6 mice, respectively; average of 1–3 CBCs/crypt from 2 to 4 crypts of passage 1 and 2 enteroids). *P < .01 vs WT plasma membrane. Right: Cumulative data showing the average Dvl2–EGFP intensity ratio at the individual supranuclear lateral-Lateral and medial-Lateral plasma membranes of individual mice (small circles) and overall average (large circles) for WT/Dvl2-EGFP/RosamT/mG (WT) and Cftr KO/Dvl2-EGFP/RosamT/mG (Cftr KO) sex-matched mouse pairs (n = 7 and 6 mice, respectively). Dashed line indicates the average Dvl2–EGFP pixel intensity for the entire supranuclear region of CBC, which has been set to 1.0. +P < .002 vs WT lateral-Lateral cell membrane; *P < .001 vs WT medial-Lateral cell membrane. (E) Immunofluorescence image of Fz7 (cyan), plasma membrane (red), and merged (light yellow) of CBCs in WT and Cftr KO enteroid crypts. Representative of 3 WT–Cftr KO mouse pairs. L, lumen. White arrowheads denote lack of Fz7 staining at apical membrane (red).
Figure 5
Figure 5
Dvl2–EGFP proximity to the plasma membrane is charge- and pHi–dependent in Cftr KO crypt-base columnar cells. (A) Left: Representative images of ETOH vehicle- (Veh, top) and sphingosine-treated (Sphingosine, bottom) Cftr KO/Dvl2–EGFP/RosamT/mG enteroid crypts showing Dvl2–EGFP (green), RosamT/mG (red), and merged images. Magnified merged image (×2.5) of crypt base (from white boxes). L, crypt lumen; lateral cell membrane for comparison (white arrows). Right: Cumulative data showing the average Dvl2–EGFP intensity ratio within 1.15 μm of the CBC supranuclear plasma membrane of individual mice (small circles) and overall average (large circles) for vehicle- and sphingosine-treated Cftr KO/Dvl2–EGFP/RosamT/mG (Cftr KO) mice (n = 6). Dashed line indicates the average Dvl2–EGFP pixel intensity for the entire supranuclear region of CBC, which has been set to 1.0. *P < .001 vs vehicle. Averaged data for each mouse represents measurements from 1 to 3 CBCs/crypt from 2 to 4 crypts from passage 1 and 2 enteroids. (B) Left: Representative images of Cftr KO enteroids cultured for 48 hours in medium, pH 7.1 (top), and medium, pH 6.6 (bottom), showing Dvl2–EGFP (green), RosamT/mG (red), and merged images. Magnified merged image (×4.1) of crypt base (from white boxes). L, crypt lumen; lateral cell membrane for comparison (white arrows). Right: Cumulative data showing the average Dvl2–EGFP intensity ratio within 1.15 μm of the CBC supranuclear plasma membrane of individual mice (small circles) and overall average (large circles) for pH 7.1- and pH 6.6-treated enteroids from Cftr KO/Dvl2–EGFP/RosamT/mG (Cftr KO) mice (n = 4). Dashed line indicates the average Dvl2–EGFP pixel intensity for the entire supranuclear region of CBC, which has been set to 1.0. *P < .001 vs pH 7.1 medium. Averaged data for each mouse represents measurements from 1 to 3 CBCs/crypt from 2 to 4 crypt from passage 1 and 2 enteroids.
Figure 6
Figure 6
Pharmacologic manipulation of Dvl2–EGFP plasma membrane association. (A) Left: Representative images of dimethyl sulfoxide vehicle- (Veh, top) and bumetanide + carbachol–treated (Bumet-CCH, bottom) Cftr KO/Dvl2–EGFP/RosamT/mG enteroid crypts showing Dvl2–EGFP (green), RosamT/mG (red), and merged images. Magnified merged image (×3.6) of crypt base (from white boxes). L, crypt lumen; lateral cell membrane for comparison (white arrows). Right: Cumulative data showing the average Dvl2–EGFP intensity ratio within 1.15 μm of the CBC supranuclear lateral plasma membrane of individual mice (small circles) and overall average (large circles). Cftr KO/Dvl2–EGFP/RosamT/mG (Cftr KO) mice were treated with vehicle (Veh, dimethyl sulfoxide) or sequential exposure to 50 μmol/L bumetanide (Bumet, 15 min) followed by 100 μmol/L CCH (15 min), n = 4 mice. Averaged data for each mouse represents measurements from 1 to 3 CBCs/crypt from 2 to 4 crypts from passage 1 and 2 enteroids. Dashed line indicates the average Dvl2–EGFP pixel intensity for the entire supranuclear region of CBC, which has been set to 1.0. *P < .005 vs vehicle. (B) Left: Representative images of dimethyl sulfoxide vehicle- (Veh, top) and Cftrinh-172+GlyH-101–treated (Cftr Inhib., bottom) WT/Dvl2–EGFP/RosamT/mG enteroid crypts showing Dvl2–EGFP (green), RosamT/mG (red), and merged images. Magnified merged image (×3.8) of crypt base (from white boxes). L, crypt lumen; lateral cell membrane for comparison (white arrows). Right: Cumulative data showing the average Dvl2–EGFP intensity ratio within 1.15 μm of the CBC supranuclear lateral plasma membrane of individual mice (small circles) and overall average (large circles). WT/Dvl2–EGFP/RosamT/mG (WT) mice were treated with vehicle (Veh, dimethyl sulfoxide) or 10 μmol/L Cftrinh-172 plus 20 μmol/L GlyH-101 for 1 hour, n = 3 mice. Averaged data for each mouse represents measurements from 1 to 3 CBCs/crypt from 2 to 4 crypts from passage 1 and 2 enteroids. Dashed line indicates the average Dvl2–EGFP pixel intensity for the entire supranuclear region of CBC, which has been set to 1.0. *P < .013 vs vehicle.

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