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. 2020 Aug 6;9(8):1847.
doi: 10.3390/cells9081847.

Amelioration of Congenital Tufting Enteropathy in EpCAM (TROP1)-Deficient Mice via Heterotopic Expression of TROP2 in Intestinal Epithelial Cells

Gaku Nakato  1 , Sohshi Morimura  2 , Michael Lu  3 , Xu Feng  4 , Chuanjin Wu  5 , Mark C Udey  6
Affiliations

Amelioration of Congenital Tufting Enteropathy in EpCAM (TROP1)-Deficient Mice via Heterotopic Expression of TROP2 in Intestinal Epithelial Cells

Gaku Nakato et al. Cells. .

Abstract

TROP1 (EpCAM) and TROP2 are homologous cell surface proteins that are widely expressed, and often co-expressed, in developing and adult epithelia. Various functions have been ascribed to EpCAM and TROP2, but responsible mechanisms are incompletely characterized and functional equivalence has not been examined. Adult intestinal epithelial cells (IEC) express high levels of EpCAM, while TROP2 is not expressed. EpCAM deficiency causes congenital tufting enteropathy (CTE) in humans and a corresponding lethal condition in mice. We expressed TROP2 and EpCAM in the IEC of EpCAM-deficient mice utilizing a villin promoter to assess EpCAM and TROP2 function. Expression of EpCAM or TROP2 in the IEC of EpCAM knockout mice prevented CTE. TROP2 rescue (T2R) mice were smaller than controls, while EpCAM rescue (EpR) mice were not. Abnormalities were observed in the diameters and histology of T2R small intestine, and Paneth and stem cell markers were decreased. T2R mice also exhibited enlarged mesenteric lymph nodes, enhanced permeability to 4 kDa FITC-dextran and increased sensitivity to detergent-induced colitis, consistent with compromised barrier function. Studies of IEC organoids and spheroids revealed that stem cell function was also compromised in T2R mice. We conclude that EpCAM and TROP2 exhibit functional redundancy, but they are not equivalent.

Keywords: CTE; TROP1(EpCAM); TROP2; intestinal epithelial cells.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Expression of EpCAM and TROP2 in intestinal epithelial cells (IEC) of transgenic and control mice. (a) Absolute numbers of murine EpCAM, murine TROP2, and human EpCAM transcripts in 1 ng intestinal total RNA were determined using quantitative RT-PCR. Data represent means ± SD in samples from 4 mice in each group (n = 4). ** p < 0.01, **** p < 0.0001 using a one-way ANOVA with Tukey’s multiple comparison test. n.d. means not detected. (b) Immunofluorescence microscopy verifying expression of murine EpCAM (green) and murine TROP2 (red) in the small intestines of wild type (WT) and murine TROP2 rescue (T2R) mice. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 100 μm. (c) Immunofluorescence microscopy verifying expression of murine EpCAM (green) and human EpCAM (red) in the small intestines of WT and human EpCAM rescue (hEpR) mice. Nuclei were stained with DAPI (blue). Scale bars, 100 μm.
Figure 2
Figure 2
TROP2 expression in IEC prevents congenital tufting enteropathy in EpCAM knockout mice. (a) Representative photographs of littermate control mice (EpCAM+/− TROP2Tg; Ctrl (T2)) and T2R (EpCAM−/− TROP2Tg) mice at 8 weeks of age. Scale bar, 1 cm. (b) Serial body weights of littermate Ctrl and T2R mice over 1 year. Data depicted represent means ± SD for 32 Ctrl mice and 24 T2R mice at each time point. * p < 0.05, **** p < 0.0001 using a multiple t test and the Holm–Sidak method. (c) Representative H & E-stained histologic sections of small intestine from relevant mouse strains. AcEp KO designates tissue obtained from 8-week-old EpCAMfl/fl ROSACreER transgenic mice 7 days after initiation of tamoxifen treatment. Scale bars, 100 μm. (d) Intestinal diameters from relevant mice. Data represent means ± SD for 5 mice in WT, Ctrl, and T2R groups and for 4 mice in the hEpR group. * p < 0.05, *** p < 0.001 using a one-way ANOVA with Tukey’s multiple comparison test. (e) Intestinal microvillus length determined in transmission electron micrographs of tissue of indicated mice. Data depicted represent means ± SD of 50 microvilli for each genotype.
Figure 3
Figure 3
Abnormal intestinal barrier function in TROP2 rescue mice. (a) Sizes (maximal dimensions) of mesenteric lymph nodes from relevant mouse strains were determined. Data depicted represent means ± SD of dimensions obtained from 9 mice for each group (n = 9). **** p < 0.0001 as determined via one-way ANOVA with Tukey’s multiple comparison test. (b) FITC-dextran concentrations in sera from adult WT, T2R, and hEpR mice measured 4 h after administration of 4-kDa FITC-Dextran by gavage. * p < 0.05, ** p < 0.01 as determined via one-way ANOVA with Tukey’s multiple comparison test. (c) Serial body weights of adult female WT, T2R, and hEpR mice treated with 2.5% dextran sodium sulfate (DSS) in their drinking water. * p < 0.05, ** p < 0.01, **** p < 0.0001 as determined via one-way ANOVA with Tukey’s multiple comparison test. (d) Representative H & E-stained histologic sections of colon obtained on day 5 of DSS treatment. Scale bar, 100 μm.
Figure 4
Figure 4
Characterization of IEC in TROP2 rescue mice and relevant controls. (a) Detection of proliferating cells in intestinal crypts by immunostaining. Ki-67 (green), F-actin (magenta), nuclei/DAPI (blue). Scale bars, 50 μm. (b) Goblet cells are visualized in fixed tissue sections using Alcian-PAS staining and light microscopy. Scale bar, 100 μm. (c) Identification of lysozyme-expressing Paneth cells in ileal tissue by immunostaining. Lysozyme (green), F-actin (magenta), and nuclei/DAPI (blue). Upper panel scale bars, 50 μm. Lower panel scale bars, 50 μm.
Figure 5
Figure 5
Diminished paneth cells and stem cells in TROP2 rescue mice. (a) Paneth cells (Defa1+, green) and stem cells (Olfm4+, red) were visualized in jejunum by RNA-Scope in situ hybridization and fluorescence microscopy. Scale bars, 100 μm. (b,c) qPCR analysis was performed to determine relative levels of mRNA expression in ileal tissue normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. Values depicted represent means ± SE of samples from 3 different mice (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 as determined using one-way ANOVA with Tukey’s multiple comparison test.
Figure 6
Figure 6
Characterization of organoids and spheroids from TROP2 rescue mice and relevant controls. (a) Representative serial phase contrast photomicrographs of IEC organoids obtained from WT, Ctrl (T2), T2R, and hEpR intestinal crypts in vitro. Scale bars, 100 μm. (b) Forming and passaging efficiencies of organoids from relevant mice. Organoid-forming efficiencies represent ratios of organoid numbers per well at day 9 to crypt numbers per well at day 1 (left panel). Aggregate data (means ± SD) from four independent experiments are presented. Ten wells were examined in each experiment. Organoid passage efficiencies represent ratios of organoid numbers/well at day 9 of secondary cultures to organoid numbers/well at day 9 of primary cultures (right panel). Aggregate data (means ± SD) from four independent experiments are presented. Six wells were examined in each experiment. ** p < 0.01, **** p < 0.0001 as determined via one-way ANOVA with Tukey’s multiple comparison test. (c) Representative phase contrast photomicrographs of spheroids from WT, Ctrl(T2), T2R, and hEpR mice on day 2 after initial passage and fourth passage as indicated. Scale bars, 100 μm.
Figure 7
Figure 7
Decreased expression and mislocalization of claudin-1 and claudin-7 in TROP2 rescue mice. (a) Normalized amounts of intestinal tissue lysate proteins were resolved using SDS-PAGE and immunoblotted with anti-hEpCAM, anti-mEpCAM, anti-mTROP2, anti-claudin-1, anti-claudin-7, and anti-β-actin. Specific proteins were detected with HRP-labeled secondary Ab and chemiluminescence. D: Duodenum, I: Ileum, P: Proximal colon, Di: Distal colon, and TAM: tamoxifen treated. (b) Claudin-1 was detected and localized via immunostaining in ileum villi and crypts of relevant mice. Claudin-1, green. Scale bars, 50 μm. (c) Claudin-7 was detected and localized via immunostaining in ileum villi and crypts of relevant mice. Claudin-7, green. Scale bars, 50 μm.
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