EpCAM is essential for maintenance of the small intestinal epithelium architecture via regulation of the expression and localization of proteins that compose adherens junctions

Abstract

Epithelial cell adhesion molecule (EpCAM) is highly expressed in mammalian intestines, and is essential for maintaining the homeostasis of the intestinal epithelium. EpCAM protein is localized at tight junctions and the basolateral membrane of the intestinal epithelium, where it interacts with many cell adhesion molecules. To explore the molecular functions of EpCAM in regulating adherens junctions in the intestinal epithelium, EpCAM knockout embryos and newborn pups were analyzed. Hematoxylin and eosin staining was used to assess the histology of the duodenum, jejunum, ileum and colon from wild-type and EpCAM‑/‑ mice at E18.5, P0 and P3. The expression and localization of adherens junction‑associated genes and genes that encode the proteins that participate in the assembly of adherens junctions were measured at the mRNA and protein levels using qPCR, western blot analysis and immunofluorescence staining. The results showed that although there was no significant damage to the intestines of EpCAM‑/‑ mice at E18.5 and P0, they were significantly damaged at P3 in mutant mice. The expression of adherens junction‑associated genes in EpCAM mutant mice was normal at the mRNA level from E18.5 to P3, but their protein levels were gradually reduced and mislocalized from E18.5 to P3. The expression of nectin 1, which can regulate the assembly and adhesion activity of E‑cadherin, was also gradually reduced at both the mRNA and protein levels in the intestinal epithelium of EpCAM mutant mice from E18.5 to P3. In summary, the loss of EpCAM may cause the reduction and mislocalization of proteins that compose adherens junctions partly via the downregulation of nectin 1 in the intestines.

Keywords: EpCAM; intestinal epithelium; adherens junction; nectin 1.

Figure 1

The phenotype and intestinal defects of EpCAM^−/− mice. (A) The body size and body weight of WT and EpCAM^−/− mice at P0 (n=20) and P3 (from 5 WT pups and 6 EpCAM^−/− pups) stages. (B) Images of immunofluorescent staining of EpCAM in the jejunum and colon of WT and EpCAM^−/− mice at E18.5 stage. Nuclei were also stained with DAPI. Bar, 50 µm. (C) The morphology and length of intestines from WT and EpCAM^−/− mice at E18.5 (n=8) and P3 (n=6) stages. (D) Images of H&E staining of the duodenum, jejunum, ileum and colon from WT and EpCAM^−/− mice at P3 stage. Scale bar, 20 µm. ^***P<0.001. WT, wild-type; EpCAM, epithelial cell adhesion molecule; H&E, hematoxylin and eosin.

Figure 2

The E-cadherin protein gradually mislocalized in the small intestinal epithelial cells of postnatal EpCAM mutant mice during development. Images of immunofluorescent staining of E-cadherin in the duodenum, jejunum, ileum and colon from WT and EpCAM^−/− mice at E18.5 (A), P0 (B), and P3 (C) stages. Scale bar, 20 µm. White arrowhead, the mislocalization of E-cadherin. WT, wild-type; EpCAM, epithelial cell adhesion molecule.

Figure 3

The β-catenin protein gradually mislocalized in the small intestinal epithelial cells of postnatal EpCAM mutant mice during development. Images of immunofluorescent staining of β-catenin in the duodenum, jejunum, ileum and colon from WT and EpCAM^−/− mice at E18.5 (A), P0 (B), and P3 (C) stages. Scale bar, 20 µm. White arrowhead, the mislocalization of β-catenin. WT, wild-type; EpCAM, epithelial cell adhesion molecule.

Figure 4

The p120 protein gradually mislocalized in the small intestinal epithelial cells of postnatal EpCAM mutant mice during development. Images of immunofluorescent staining of p120 on the duodenum, jejunum, ileum and colon from WT and EpCAM^−/− mice at E18.5 (A), P0 (B), and P3 (C) stages. Scale bar, 20 µm. White arrowhead, the mislocalization of p120. WT, wild-type; EpCAM, epithelial cell adhesion molecule.

Figure 5

Proteins that compose adherens junctions were gradually reduced in the small intestinal epithelial cells of EpCAM mutant mice during development. Western blot results of E-cadherin, β-catenin, and p120 in the small intestines of WT and EpCAM^−/− mice at E18.5 (n=4) (A), P0 (n=4) (B) and P3 (n=4) (C) stages respectively. ^**P<0.01, and ^***P<0.001. WT, wild-type; EpCAM, epithelial cell adhesion molecule.

Figure 6

The mRNA expression of proteins that compose adherens junctions was normal in the small intestines of EpCAM mutant mice during development. qPCR results of E-cadherin, β-catenin, and p120 in the small intestines of WT and EpCAM^−/− mice at E18.5 (A), P0 (B) and P3 (C) stages respectively. Eight samples from each group were used at E18.5 and P3 stages. At P0 stage, 4 samples from each group were used to test the mRNA levels of E-cadherin and β-catenin, and 7 samples from each group were used to test the expression level of p120. WT, wild-type; EpCAM, epithelial cell adhesion molecule.

Figure 7

The expression of nectin 1 gradually decreased in the intestines of postnatal EpCAM mutant mice during development. (A) qPCR results of nectin 1 in the small intestines of WT and EpCAM^−/− mice at E18.5 (n=6), P0 (n=7) and P3 (n=8) stages. (B) Western blot results of nectin 1 in the small intestines of WT and EpCAM^−/− mice at E18.5, P0 and P3 (n=4). ^*P<0.05, ^**P<0.01, and ^***P<0.001. WT, wild-type; EpCAM, epithelial cell adhesion molecule.