Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease

Abstract

Intestinal fibrosis and stricture formation is an aggressive complication of Crohns disease (CD), linked to increased morbidity and costs. The present study investigates the contribution of Wingless-Int-1 (Wnt) signalling to intestinal fibrogenesis, considers potential cross-talk between Wnt and transforming growth factor β1 (TGFβ) signalling pathways, and assesses the therapeutic potential of small-molecule Wnt inhibitors. β-catenin expression was explored by immunohistochemistry (IHC) in formalin-fixed paraffin embedded (FFPE) tissue from patient-matched nonstrictured (NSCD) and strictured (SCD) intestine (n=6 pairs). Functional interactions between Wnt activation, TGFβ signalling, and type I collagen (Collagen-I) expression were explored in CCD-18Co cells and primary CD myofibroblast cultures established from surgical resection specimens (n=16) using small-molecule Wnt inhibitors and molecular techniques, including siRNA-mediated gene knockdown, immunofluorescence (IF), Wnt gene expression arrays, and western blotting. Fibrotic SCD tissue was marked by an increase in β-catenin-positive cells. In vitro, activation of Wnt-β-catenin signalling increased Collagen-I expression in CCD-18Co cells. Conversely, ICG-001, an inhibitor of β-catenin signalling, reduced Collagen-I expression in cell lines and primary CD myofibroblasts. TGFβ increased β-catenin protein levels but did not activate canonical Wnt signalling. Rather, TGFβ up-regulated WNT5B, a noncanonical Wnt ligand, and the Wnt receptor FZD8, which contributed directly to the up-regulation of Collagen-I through a β-catenin-independent mechanism. Treatment of CCD-18Co fibroblasts and patient-derived myofibroblasts with the FZD8 inhibitor 3235-0367 reduced extracellular matrix (ECM) expression. Our data highlight small-molecule Wnt inhibitors of both canonical and noncanonical Wnt signalling, as potential antifibrotic drugs to treat SCD intestinal fibrosis. They also highlight the importance of the cross-talk between Wnt and TGFβ signalling pathways in CD intestinal fibrosis.

Keywords: Crohns disease; Wnt proteins; fibrosis; inhibitors; intestine.

Figure 1. Altered β-catenin expression associated with intestinal fibrosis in SCD patients

(A) FFPE sections from a colorectal adenocarcinoma block were also used for a positive to control IHC and IF protein analysis, given the link between APC mutations in patients, altered Wnt signalling, and changes in β-catenin staining. Nuclear accumulation of β-catenin in epithelial cells (EC) within the tumour are shown by the yellow arrow. The IF further shows clear colocalisation (yellow) of β-catenin (488) and Vimentin (555) in the cytoplasm of fibroblastic cells (pink arrow); DAPI staining demarcates the cell nuclei. (B) Representative IHC and IF images of β-catenin in the mucosa (M) overlying SCD and patient-matched NSCD areas (n=6) and MP of SCD and NSCD intestine (n=5). In the images, positively stained EC, endothelial cells surrounding blood vessels (BV) and stromal cells, including cells that morphologically resembled fibroblasts (indicated by arrows) are observed. (C) Enlarged (Zoom) IHC sections from SCD and NSCD mucosa highlighting negatively (yellow arrow) and positively (green arrow) stained fibroblastic cells are highlighted. (D–G) Quantification of the total number and percentage of stromal β-catenin-positive cells in the mucosa and the percentage of β-catenin-positive cells in the MP, excluding endothelial cells associated with BV, demonstrates transmural increases in β-catenin throughout SCD tissues. Differences between SCD and NSCD samples were determined by a paired t-test. Significant results are indicated by * symbol (*<0.05, **<0.01, ***<0.001).

Figure 2. Activation of canonical Wnt β-catenin signalling promotes Collagen-I expression in intestinal fibroblasts in the absence of TGFβ

(A) Representative IF images of cells stained for DAPI (blue) and β-catenin (green) or Collagen-I (green) 48 h post-transfection with either a negative control siRNA NTC (Cat#1027281) or an APC-targeting siRNA (siAPC, Hs_APC_6 Flexitube, Qiagen, UK, n=3), or treatment with TGFβ (n=4). (B–D) The levels of nuclear β-catenin, cytoplasmic β-catenin levels, as well as the cytoplasmic:nuclear ratio of β-catenin were quantified from IF images. (E) Cytosolic Collagen-I protein levels were also calculated for all treatment groups. (F,G) Representative western blots for activated (dephosphorylated) β-catenin (ABC), total β-catenin, and the loading control β-Actin, along with graphical representation of active:total β-catenin ratio, in CCD-18Co cells treated with TGFβ. (H) Results of TOP/FOP assays in CCD-18Co cells treated with TGFβ or the positive control CHIR-99021 (CT99021), a GSK-3 inhibitor, are presented (n=3). FITC mean intensity for TOP- and FOP-transduced cells was determined and the TOP/FOP ratio given as a fold-change relative to vehicle control; DL = density levels. Differences between treatments were determined by a paired t-test to account for different cell passages. In general, data are presented as fold-changes with panels B–E, G, and H as box plots showing 25th to 75th percentiles, median (horizontal bar), and the smallest and largest value (whiskers). Significant results relative to control are indicated by * symbol (*<0.05, **<0.01, ***<0.001).

Figure 3. TGFβ increased the expression of FZD8, which is required for TGFβ-mediated up-regulation of Collagen-I

(A) Genes regulated by TGFβ in the Wnt signalling pathway were identified using a targeted qPCR profiling array (n=3) and the data are presented as a heatmap and P-values are presented in the figure. The relative fold-change in gene expression in TGFβ-treated cells is given for each of the three replicates. Differences between treatments were determined by t-test and corrected for multiple testing. (B–K) qPCR quantification of selected collagen and TGFβ-regulated genes in the Wnt-signalling pathway from RNA isolated from the mucosa overlying SCD and patient-matched NSCD intestine (n=6). The mRNA levels are normalised to the house-keeping gene RPLPO. Differences between treatments were determined by a paired t-test to account for different cell passages. In general, data are presented as fold-changes with panels C–J as box plots showing 25th to 75th percentiles, median (horizontal bar), and the smallest and largest value (whiskers). Significant results relative to control are indicated by * symbol (*<0.05, **<0.01, ***<0.001). A bar indicates specific statistical comparisons.

Figure 4. Small-molecule Wnt inhibitors of FZD8 and Wnt5B inhibit Collagen-I expression in intestinal fibroblasts

(A) β-catenin and Collagen-I protein levels were also determined by IF in treated CCD-18Co cells with Wnt-C59 (C59), 3235-0367 (C1), or the vehicle control (DMSO) in combination with TGFβ. Representative images are provided. (B) β-catenin levels are presented as a ratio of the nuclear:cytoplasmic levels of the protein (n=2). (C,D) Cytosolic Collagen-I levels are also provided (n=3), along with cell counts for each treatment (n=3). (E) Pro-Collagen-Iα1 levels in the media were determined by ELISA (n=3). (F) Representative western blot showing the effects of 48 h of treatments on Fibronectin, dephosphorylated ABC levels, and total β-catenin protein levels. (G) β-catenin protein levels are presented as a ratio of ABC:β-catenin as determined by quantification of the IF images (n=3). (H) Fibronectin levels are expressed normalised to the loading control (β-Actin); DL = density levels. Differences between treatments were determined by a paired t-test to account for different cell passages. In general, data are presented as fold-changes with panels B–E, G, and H as box plots showing 25th to 75th percentiles, median (horizontal bar), and the smallest and largest value (whiskers). Significant results relative to control are indicated by * symbol (*<0.05, **<0.01, ***<0.001). A bar indicates specific statistical comparisons.

Figure 5. Inhibition of β-catenin-dependent Wnt signalling by ICG-001 blocks both steady state and TGFβ-induced Collagen-I up-regulation

(A) Levels of β-catenin, Collagen-I, and cell numbers were evaluated by IF (n=4). (B) Representative western blots for β-catenin, FZD8, and Fibronectin; these data are normalised to the loading control β-Actin (n=4). (C–I) Protein quantifications from IF and western blots. (D) Pro-Collagen-Iα1 levels in the cell media, measured by ELISA, are also provided (n=4). (J) The ability of ICG-001 to suppress markers of TGFβ-induced myofibroblast activation in CCD-18Co cells was also assessed by qPCR and the data presented in a bar graph (n=4). (K) ICG-001 effects on gel remodelling and pro-Collagen-Iα1 were also confirmed in a 3D organotypic model in the absence of TGFβ (n=4); DL = density levels. Differences between treatments were determined by a paired t-test to account for different cell passages. In general, data are presented as fold-changes with panels C–I and K(i–ii) as box plots showing 25th to 75th percentiles, median (horizontal bar), and the smallest and largest value (whiskers). Panel J shows mean ± SEM. Significant results relative to control are indicated by * symbol (*<0.05, **<0.01, ***<0.001). A bar indicates specific statistical comparisons.

Figure 6. ICG-001, β-catenin/CBP inhibitor reduces Collagen-I expression in primary CD myofibroblast cultures

(A) Representative IF from primary CD cultures (n=7 and n=9 NSCD and SCD cultures, respectively) treated with and without TGFβ in combination with ICG-001 and C1. (B–G) Protein quantifications from IF images and ELISA (conditioned media) from ICG-001 and C1 treated myofibroblast cultures. *DL = density levels. Differences between treatments were determined by a paired t-test to account for different cell passages. Differences between SCD and NSCD cultures determined by an unpaired t-test assuming equal variance. Significant results relative to control are indicated by * symbol (*<0.05, **<0.01, ***<0.001). A bar indicates specific statistical comparisons.

Figure 7. Model for Wnt-mediated fibrosis in CD

Binding of Wnt ligands to FZD receptors leads to canonical activation, inhibition of the β-catenin destruction complex, and an increase in dephosphorylated active β-catenin. β-catenin is then free to translocate to the nucleus, where along with cofactors such as CBP, it activates TCF/LEF-dependent gene transcription and as shown in the present study up-regulates Collagen-I. Conversely, inhibitors of the β-catenin-dependent transcription such as ICG-001 reduce Collagen-I expression. ICG-001 can also disrupt interactions between Smad3 and β-catenin are CBP-dependent [34]. In intestinal fibroblasts, the profibrotic cytokine TGFβ does not directly activate the canonical Wnt pathway in intestinal fibroblasts. Instead, TGFβ promotes noncanonical Wnt signalling mediated by FZD8/Wnt5B. Inhibiting either the FZD8 receptor with a small-molecule inhibitor (C1; 3235-0367) or blocking Wnt ligand production in TGFβ-stimulated fibroblasts, which also results in reduced Collagen-I expression from intestinal fibroblasts. These two parallel pathways can regulate Collagen-I independently but there is also potential for cross-talk, given that the TGFβR complex can associate with the FZD8 receptor and TGFβ1 can promote the accumulation of β-catenin in fibroblasts, which may prime cells to be more Wnt-responsive.