Depletion of MOB1A/B causes intestinal epithelial degeneration by suppressing Wnt activity and activating BMP/TGF-β signaling

Abstract

The Hippo pathway is involved in intestinal epithelial homeostasis with Wnt, BMP, Notch, and EGF signaling. We investigated the relationship between Hippo and other signaling pathways and the role of MOB kinase activator 1A/1B (MOB1A/B) in intestinal homeostasis. Mice with intestinal epithelial cell (IEC)-specific depletion of MOB1A/B showed hyperproliferation in IECs, defects in secretory lineage differentiation and loss of intestinal stem cells and eventually died at 10-12 days after tamoxifen treatment. In MOB1A/B-depleted IECs, expression of Wnt target genes were downregulated but Bmp2 and Tgfbr2 were transcriptionally activated with enhanced YAP activity. In in vivo and in vitro experiments with several signaling inhibitors, it has been shown that the BMP inhibitor LDN193189 or TGF-β inhibitor SB431542 had effects on partial restoration of the intestinal degenerative phenotype. Treatment with these inhibitors restored differentiation of secretory lineage cells in MOB1A/B-deficient mice, but not ISC pools in the crypt region. These studies reveal that IEC-specific depletion of MOB1A/B induced overexpression of Bmp2 and Tgfbr2 and inhibited Wnt activity, finally leading to loss of ISCs and functional epithelia in the mouse intestine. These results suggest that MOB1A/B has an essential function for intestinal epithelial homeostasis by regulating YAP, Wnt activity, and BMP/TGF-β signaling.

Fig. 1. MOB1A/B is essential for homeostasis of the intestinal epithelium.

a Change in body weight of wild type (n = 10) and MOB1A/B iKO mice (n = 17) after tamoxifen treatment. b Representative hematoxylin and eosin (H&E) staining in control and MOB1A/B-depleted intestines sampled 10 days after tamoxifen treatment (80 mg/kg per day, during 2 days). Scale bars, 100 μm. c Staining for secretory cell lineage in the intestine seven days after tamoxifen treatment. From left to right; alkaline phosphatase, Alcian blue staining, IHC staining of intestines with anti-Chromogranin A and anti-Lysozyme antibody. Scale bars, 50 μm. d, e The number of Alcian blue- and Chromogranin A-positive cells in three individual control and MOB1A/B-depleted mice. f Relative mRNA expression levels of Atoh1 in isolated IECs of control and MOB1A/B iKO mice (n = 3). Data are presented as the mean ± SEM. ^*P < 0.05, ^***P < 0.001

Fig. 2. Loss of MOB1A/B leads to YAP activation and hyperplasia in the crypt and villus.

a Western blot analysis of IECs isolated seven days after tamoxifen treatment in three individual control and MOB1A/B iKO mice. b Relative expression levels of Yap and Taz mRNA in 5 or 10 days after tamoxifen treatment in three individual control and MOB1A/B iKO mice. c Representative IHC staining of intestines with anti-YAP antibody at the indicated times after tamoxifen treatment. Scale bars, 20 μm. d Representative IHC staining of intestines with anti-Ki67 antibody at 4 days after tamoxifen treatment. Scale bars, 50 μm. e, f H&E staining (e) and TUNEL staining (f) of control and MOB1A/B-depleted intestines seven days after tamoxifen treatment. The upper regions of the yellow dotted lines indicate IECs. Scale bars, 200 μm (e) and 500 μm (f), respectively. Data are presented as the mean ± SEM. ^**P < 0.01, ^***P < 0.001

Fig. 3. Depletion of MOB1A/B induces changes in stem niche factors leading to degeneration of ISCs/crypt.

a Heatmap of IECs isolated from tamoxifen-treated mice (n = 2) for the indicated periods showing 13,700 downregulated genes and 5912 upregulated genes in rank order of fold change value at 7 days. Labeled genes are examples known to be involved in Wnt, Notch, EGF, TGF-β, and BMP signaling and ISCs markers. b Representative IHC staining with anti-CD44 antibody at the 7 days after tamoxifen treatment in three individual control and MOB1A/B-depleted mice. Scale bars, 20 μm. c Relative mRNA expression levels of ISCs marker genes (Lgr5, Olfm4, and Ascl2) in isolated IECs of control and MOB1A/B iKO mice (n = 3). d Representative bright-field images of intestinal organoids and the percentage of organoids showing 0–3 or ≥4 de novo crypt formation. MOB1A/B-depleted ISCs were isolated from control or tamoxifen-treated MOB1A/B iKO mice and analysis of crypt formation was performed 4 days after ISCs isolation (n = 3, n represents the number of separate cultures). Data are presented as the mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 4. MOB1A/B depletion reduces β-catenin activity.

a Representative IHC staining with anti-β-catenin antibodies in control and MOB1A/B-depleted intestines sampled 7 days after tamoxifen treatment. Scale bars, 20 μm. b Western blot analysis of IECs isolated at 7 days after tamoxifen treatment in three individual control and MOB1A/B iKO mice. c, d Western blot analysis (c) and semiquantitative PCR analysis (d) of IECs isolated 2 cm long segments duodenum—jejunum region at 7 days after tamoxifen treatment in three individual control and MOB1A/B iKO mice

Fig. 5. Inhibition of TGF-β or BMP signaling delays the decrease in body weight of MOB1A/B-depleted mice.

a Percent changes in body weight of vehicle-treated wild type and vehicle- or inhibitor-treated MOB1A/B iKO mice after tamoxifen treatment. Vehicle-treated wild-type mice, n = 6; vehicle-treated MOB1A/B iKO mice, n = 8; dibenzazepine (DBZ)-treated MOB1A/B iKO mice, n = 6; pyrvinium-treated MOB1A/B iKO mice, n = 6; LDN-treated MOB1A/B iKO mice, n = 7; SB-treated MOB1A/B iKO mice, n = 6; combination treatment with LDN and SB in MOB1A/B iKO mice, n = 6. b Western blot analysis of IECs isolated 6 days after treatment with tamoxifen and indicated inhibitors in control and MOB1A/B iKO mice. c Quantification of protein levels for pSmad1/5 and pSmad2 from (b). d Quantitative results showing expression levels of Bmp2 and Tgfbr2 mRNA isolated from IECs in (b). Data are presented as the mean ± SEM. ^**P < 0.01, ^***P < 0.001

Fig. 6. Inhibition of TGF-β or BMP signaling restores defects in secretory lineage differentiation in MOB1A/B-depleted IECs.

a Representative IHC staining with anti-CD44 antibodies in three individual control and MOB1A/B-depleted intestines sampled 7 days after treatment with tamoxifen and/or indicated inhibitors. The lower images are an enlarged view of red-dotted box in the upper images. Scale bars, 100 μm (upper) and 20 μm (lower). b Representative IHC staining with anti-CD44 antibodies and Alcian blue staining in three individual control and MOB1A/B-depleted intestines sampled 7 days after treatment with tamoxifen and/or indicated inhibitors. Scale bars, 20 μm. c Quantitative results showing expression levels of Atoh1 mRNA from IECs isolated from LDN- or SB-treated control and MOB1A/B iKO mice (n = 3). d Representative immunofluorescence image of intestines of indicated mice (n = 3). Dashed lines depict the crypt structure. Scale bars, 20 μm. e Quantitative results showing expression levels of Lgr5, Olfm4, and Ascl2 mRNA from IECs isolated from LDN- or SB-treated control and MOB1A/B iKO mice (n = 3). Data are presented as the mean ± SEM. ^**P < 0.01, ^***P < 0.001