NADPH oxidase 1 modulates WNT and NOTCH1 signaling to control the fate of proliferative progenitor cells in the colon

Abstract

The homeostatic self-renewal of the colonic epithelium requires coordinated regulation of the canonical Wnt/beta-catenin and Notch signaling pathways to control proliferation and lineage commitment of multipotent stem cells. However, the molecular mechanisms by which the Wnt/beta-catenin and Notch1 pathways interplay in controlling cell proliferation and fate in the colon are poorly understood. Here we show that NADPH oxidase 1 (NOX1), a reactive oxygen species (ROS)-producing oxidase that is highly expressed in colonic epithelial cells, is a pivotal determinant of cell proliferation and fate that integrates Wnt/beta-catenin and Notch1 signals. NOX1-deficient mice reveal a massive conversion of progenitor cells into postmitotic goblet cells at the cost of colonocytes due to the concerted repression of phosphatidylinositol 3-kinase (PI3K)/AKT/Wnt/beta-catenin and Notch1 signaling. This conversion correlates with the following: (i) the redox-dependent activation of the dual phosphatase PTEN, causing the inactivation of the Wnt pathway effector beta-catenin, and (ii) the downregulation of Notch1 signaling that provokes derepression of mouse atonal homolog 1 (Math1) expression. We conclude that NOX1 controls the balance between goblet and absorptive cell types in the colon by coordinately modulating PI3K/AKT/Wnt/beta-catenin and Notch1 signaling. This finding provides the molecular basis for the role of NOX1 in cell proliferation and postmitotic differentiation.

FIG. 1.

NOX1^KO mice exhibit upregulation of goblet cells. Representative sections of distal colon from wild-type (WT) (A and C) or NOX1^KO (D and F) mice stained with periodic acid-Schiff and Alcian blue (A and D) or Alcian blue alone (C and F) show marked accumulation of goblet cells in NOX1^KO mice. Ultrastructural examination of colonic sections processed for transmission electron microscopy reveals a higher density of goblet cells in NOX1^KO mice (E) than in WT mice (B). (G) Eight crypts per section with 2 individual sections from each animal were counted. The percentage of goblet cells and colonocytes per crypt is represented as mean ± SEM (n = 15 for WT and n = 13 for NOX1^KO mice); *, significantly different from the WT (P < 0.001), Student's t test. (H) Confocal microscopy with anti-MUC2 (green) and antivillin (green) in sections from the distal colon shows accumulation of goblet cells at the expense of colonocytes in NOX1^KO mice and is consistent with the data presented in panel G. Nuclei (blue) were stained with DAPI. Photomicrographs shown here are representative sections from 10 animals per group. Scale bar, 100 μm (A and D), 50 μm (C and F) or 5 μm (B and E). Original magnification, ×60 (H).

FIG. 2.

Differentiation-dependent expression of NOX1 and regulatory partners in different mucus-secreting or enterocyte-like clones derived from parental HT-29 or Caco-2 cells. Semiquantitative RT-PCR shows the expression of NOX1 and its regulatory partners: NOXO1, NOXA1, p22^phox, and rac1 mRNA levels at days 4 and 5 (exponential phase of growth) and days 19 to 22 (confluence) in parental, 5-fluorouracil-resistant (5F31, 5F12, and 5F7) (31, 62), methotrexate-resistant (5M21) (31, 62), or nutrient deprivation (Glc⁻) HT-29 cells and in Caco-2 cells (TC7 and PF11) (31, 62). These clones display an undifferentiated (A), mucus-secreting (B), or enterocyte-like phenotype (C). The GAPDH mRNA level was used as a loading control. The ratio of each mRNA level to that of GAPDH was quantified by scanning densitometry. Values are representative of 3 individual experiments performed in triplicate; means ± SEM are given. *, P < 0.001; **, P < 0.01 (Student's t test).

FIG. 3.

Overexpression or silencing of NOX1 modulates the expression of transcription factors involved in cell lineage differentiation. HT-29Cl.16E and Caco-2 cells were transfected with pcDNA3-NOX1 or pEBV-shRNA-NOX1, respectively, or with the corresponding empty vectors. Semiquantitative RT-PCR analysis of NOX1, NOXO1, NOXA1, p22^phox, and rac1 mRNA levels (A) or of transcription factors involved in cell fate (C) during cell proliferation (day 3) and differentiation (days 13 to 23) is shown. GAPDH and β-actin mRNA levels were used as controls for HT-29Cl.16E and Caco-2 cells, respectively. The ratio between each mRNA level and that for GAPDH or β-actin was quantified by scanning densitometry. Values are representative of 3 individual experiments performed in triplicate; means ± SEM are shown. *, P < 0.001 relative to empty vector. (B) PMA-induced ROS production in transfected HT-29Cl.16E and Caco-2 cells was measured by luminol-enhanced chemiluminescence in the presence or absence of DPI, an inhibitor of flavoproteins. Values are means ± SEM for three independent experiments. *, P < 0.001 relative to results with empty vector.

FIG. 4.

NOX1 deficiency reduces the population of proliferating progenitors and modifies their spatial distribution. Immunohistochemical analysis of sections of distal colon from WT and NOX1^KO mice stained with anti-BrdU antibody 1 h or 24 h after administration of BrdU (A) or with an antibody against the proliferating antigen phospho-histone 3 (B). (A) The number of BrdU-positive cells (red) 1 h after administration of BrdU is reduced in the colons of NOX1^KO mice, and the proliferating cells, which are mostly confined to the bottom of the crypt domain in WT mice, are scattered along the first third of the crypt in NOX1^KO mice. BrdU-positive cells migrated to the tops of crypts in NOX1^KO mice 24 h after administration of BrdU, whereas they did not exceed two-thirds of the crypt length in WT mice (A). Scale bars, 80 μm. (B) Phospho-histone-3 staining (brown) confirms the decrease in the number of proliferating cells and their altered location in the crypt (arrows) in NOX1^KO mice. Scale bar, 80 μm (top panels) or 50 μm (bottom panels). (C) Electron microscopy observation of colonic crypt sections reveals the presence of mature goblet cells (arrows) in an area of the crypt where proliferating cells normally reside. Scale bar, 5 μm.

FIG. 5.

Expression and localization of caudal-related homeobox Cdx proteins in the colon. (A) Immunohistological analysis of Cdx1 and Cdx2 in distal colon sections reveals similar staining for Cdx1 along the crypt (brown) in WT and NOX1^KO mice; scale bar, 80 μm. In contrast, Cdx2 expression was upregulated in NOX1^KO mice; scale bar, 80 μm. Magnification of the micrographs shows that nuclear staining of Cdx2 can be ectopically found at the bottom of the crypts in NOX1^KO mice (arrows); scale bar, 50 μm. (B) RT-PCR from the colon reveals similar expression levels of Cdx1 in both groups of mice and significantly increased levels of Cdx2 in NOX1^KO mice. Cdx1/β-actin and Cdx2/β-actin mRNAs were quantified by scanning densitometry. Values are representative of 2 individual experiments (means ± SEM); n = 9 for WT and NOX1^KO mice. *, P < 0.001 relative to results for control (Student's t test).

FIG. 6.

NOX1 fine-tunes the activity of Notch1 signaling pathway. (A) Representative immunoblot analysis of NICD expression in colonic tissues from three individual WT and NOX1^KO mice. β-Actin was used as a loading control. Note that NICD expression was severely depressed in NOX1^KO mice compared to that in WT mice. NICD/β-actin was quantified by scanning densitometry. Data obtained in two independent experiments are expressed as means ± SEM; n = 6 for WT and NOX1^KO mice; *, P < 0.001 relative to results for control (Student's t test). (B) Semiquantitative RT-PCR from the colon shows decreased expression of Hes1 and Hes5 associated with derepression of Math1, KLF4, and ElF3 transcription. Values are representative of two individual experiments, means ± SEM; n = 9 for WT and NOX1^KO mice. *, P < 0.001 relative to results for control (Student's t test). (C) Expression of γ-secretase (presenilin 1 [PS1]), proteases (matrix-metalloproteinases 2/9 [MMP2/9] and ADAM10), and TIMP1 (an inhibitor of MMPs) directly or indirectly involved in Notch1 activation, measured by semiquantitative RT-PCR for WT and NOX1^KO mice. The GAPDH mRNA level was used as a loading control. The ratio between each mRNA level and GAPDH was quantified by scanning densitometry. Values are means ± SEM of two independent experiments (n = 9 for WT and NOX1^KO mice); *, P < 0.001; **, P < 0.0001 (relative to results for control).

FIG. 7.

NOX1 modulates NF-kB activation, MMP9/2 expression, and NICD production. (A) Assessment of NF-κB activity by Western blot analysis in WT and NOX1^KO mice. β-Actin was used as a loading control. Values are means ± SEM of results for 3 mice per group. *, P < 0.001 relative to results for the control. (B) Western blot analysis of NICD in HT-29Cl.16E cells transfected with pcDNA3-NOX1 or empty vector in proliferating (day 3) or differentiated (days 16 and 23) cells. β-Actin was used as a loading control. Values are representative of 3 individual experiments performed in triplicate. (C) Expression of matrix-metalloproteinases 9/2 (MMP9 and -2) in HT-29Cl.16E cells transfected with pcDNA3-NOX1 or empty vector in proliferating (day 3) or differentiated (days 13 to 23) cells. The GAPDH mRNA level was used as a loading control. The ratio of each mRNA level to that for GAPDH was quantified by scanning densitometry. Values are representative of 3 individual experiments performed in triplicate (means ± SEM)l *, P < 0.001; **, P < 0.005 (relative to results with empty vector).

FIG. 8.

NOX1 deficiency attenuates Wnt/β-catenin signaling pathway. (A) Immunofluorescence of colon sections using anti-β-catenin (green) and anti-E-cadherin (red) reveals that β-catenin associated with the plasma membrane as part of the β-catenin-E-cadherin complex was essentially detected at the upper part of the crypts in WT mice whereas this complex was also present at the bottom of the crypts in NOX1^KO mice. Original magnification, ×60. The illustration in the left-hand micrograph schematizes the distribution of membrane β-catenin and E-cadherin in colonic crypt regions. Immunohistochemistry of colon sections using anti-β-catenin shows that nuclear β-catenin staining (brown) can be observed in many cells at basal positions of colon crypts only in WT mice (arrows). Scale bars, 80 μm or 50 μm. (B) Representative immunoblot analysis of phospho-β-catenin expression in colonic tissues from three individual WT and NOX1^KO mice. Phospho-β-catenin (Ser^33/37/Thr⁴¹)/β-catenin was quantified by scanning densitometry. Data obtained in two independent experiments are expressed as means ± SEM; n = 6 for WT and NOX1^KO mice; *, P < 0.001 relative to results for the control (Student's t test). (C) Accordingly, c-Myc and Cyclin D1 transcription was repressed in NOX1^KO mice. Values are representative of 2 individual experiments (means ± SEM); n = 9 for WT and NOX1^KO mice. *, P < 0.001 relative to results for control (Student's t test).

FIG. 9.

Effect of NOX1 on goblet cell differentiation and cell proliferation implicates PTEN/AKT signaling cascade. Representative immunoblot analysis of phospho-AKT (A), phospho-GSK3 (B), or phospho-PTEN (C) expression in colonic tissues from three individual WT and NOX1^KO mice. Phospho-AKT (Ser⁴⁷³)/AKT and phospho-AKT (Thr³⁰⁸)/AKT (A), phospho-GSK3α (Ser²¹)/GSK3α and phospho-GSK3β (Ser⁹)/GSK3β (B), and phospho-PTEN (Ser³⁸⁰)/pan-PTEN (C) were quantified by scanning densitometry. Data obtained in two independent experiments are expressed as means ± SEM; n = 9 for WT and NOX1^KO mice; *, P < 0.001; **, P < 0.05 relative to results for the control (Student's t test). (D) Detection of active (reduced) and inactive (oxidized) forms of PTEN on the basis of an electrophoretic mobility shift. Colonic crypt extracts from WT and NOX1^KO mice were subjected to alkylation in the presence or absence of β-mercaptoethanol (β-M) and fractionated by nonreducing SDS/PAGE. The percentage of the active form of PTEN was determined by scanning densitometry. Data obtained in two independent experiments are expressed as means ± SEM; n = 6 for WT and NOX1^KO mice; *, P < 0.0001 relative to results for the control (Student's t test). (E) Representative immunoblot analysis of Phospho-AKT (Ser⁴⁷³)/AKT expression in colonic tissues from three individual NOX1^KO mice treated twice daily with 2 μg/g Long-Arg³-IGF1 (IGF1) for 4 days or with vehicle (HCl, 2 mM in PBS). Phospho-AKT/AKT was quantified by scanning densitometry. Data are expressed as means ± SEM; n = 3 for untreated and IGF1-treated NOX1^KO mice; *, P < 0.001 relative to results for the control (Student's t test). (F) Representative sections of distal colon from NOX1^KO mice treated (IGF1) or not (Ctrl) with IGF1 stained with periodic acid-Schiff and Alcian blue. Eight crypts per section with 2 individual sections from each animal were counted. Percentages of goblet cells and colonocytes per crypt are represented as means ± SEM (n = 3 for untreated and IGF1-treated NOX1^KO mice); *, significantly different from Ctrl (P < 0.001) (Student's t test). (G) Immunohistochemical analysis of sections of distal colon from NOX1^KO mice treated (IGF1) or not (Ctrl) with IGF1 stained with anti-BrdU antibody 1 h after administration of BrdU. The number of BrdU-positive cells per gland section was reported as detailed in Materials and Methods. *, P < 0.01 relative to results for Ctrl (Student's t test). Scale bars, 80 μm.

FIG. 10.

PTEN activation facilitates β-catenin/E-cadherin association in NOX1^KO mice. Colonic crypt lysates from WT and NOX1^KO mice were subjected to immunoprecipitation (IP) with anti-β-catenin or E-cadherin antibodies. The IP material was then subjected to immunoblot analysis (IB) with antibodies recognizing β-catenin, E-cadherin, PTEN, or phosphotyrosine (PY). Data are means ± SEM; n = 3 for WT and NOX1^KO mice; *, P < 0.005; **, P < 0.001 relative to results for control.

FIG. 11.

Silencing of PTEN impairs β-catenin-E-cadherin complex formation in sh-NOX1 RNA-transfected Caco-2 cells. (A) Semiquantitative RT-PCR shows the expression of PTEN in stably transfected Caco-2 cells with the sh-NOX1 RNA construct (sh-NOX1) treated with increased concentrations of PTEN siRNA (siPTEN). the GAPDH mRNA level was used as a loading control. The ratio between PTEN and GAPDH mRNA levels was quantified by scanning densitometry. Values are representative of two individual experiments performed in triplicate. (B) The efficiency of PTEN siRNA was confirmed by Western blotting in sh-NOX1 Caco-2 cells transfected with siPTEN or scrambled (Scr) siRNA. β-Actin served as the loading control. PTEN/β-actin was quantified by scanning densitometry. Values are representative of two individual experiments performed in triplicate. *, P < 0.001 relative to results for scrambled siRNA. (C) Cell lysates from sh-NOX1 Caco-2 cells transfected with siPTEN or scrambled (Scr) siRNA were subjected to immunoprecipitation (IP) with anti-β-catenin or anti-E-cadherin antibodies. The IP material was then subjected to immunoblot analysis (IB) with antibodies recognizing β-catenin, E-cadherin, PTEN, or phosphotyrosine (PY). Values are representative of two individual experiments performed in triplicate. *, P < 0.001 relative to results for scrambled siRNA.

FIG. 12.

Schematic representation of NOX1-induced regulatory networks controlling progenitor cell fate in the colon.