EpCAM (CD326) Regulates Intestinal Epithelial Integrity and Stem Cells via Rho-Associated Kinase.

Takeshi Ouchi,Lukas E Dow,Hiroyuki Miyoshi,Mark C Udey,Sohshi Morimura

doi:10.3390/cells10020256

Abstract

Humans with biallelic inactivating mutations in Epithelial Cell Adhesion Molecule (EpCAM) develop congenital tufting enteropathy (CTE). To gain mechanistic insights regarding EpCAM function in this disorder, we prepared intestinal epithelial cell (IEC) organoids and spheroids. IEC organoids and spheroids were generated from ROSA-CreERT2 EpCAMfl/fl mice. Proliferation, tight junctions, cell polarity and epithelial integrity were assessed in tamoxifen-induced EpCAM-deficient organoids via confocal immunofluorescence microscopy and Western blotting. Olfm4-expressing stem cells were assessed in IEC cells in vitro and in vivo via fluorescence in situ hybridization. To determine if existing drugs could ameliorate effects of EpCAM deficiency in IEC cells, a variety of pharmacologic inhibitors were screened. Deletion of EpCAM resulted in increased apoptosis and attenuated growth of organoids and spheroids. Selected claudins were destabilized and epithelial integrity was severely compromised. Epithelial integrity was improved by treatment with Rho-associated coiled-coil kinase (ROCK) inhibitors without restoration of claudin expression. Correspondingly, enhanced phosphorylation of myosin light chain, a serine/threonine ROCK substrate, was observed in EpCAM-deficient organoids. Strikingly, frequencies of Olfm4-expressing stem cells in EpCAM-deficient IEC cells in vitro and in vivo were decreased. Treatment with ROCK inhibitors increased numbers of stem cells in EpCAM-deficient organoids and spheroids. Thus, EpCAM regulates intestinal epithelial homeostasis via a signaling pathway that includes ROCK.

Highlights

EpCAM (Epithelial Cell Adhesion Molecule; CD326) is a surface glycoprotein expressed in developing and adult epithelia, and selected carcinomas
Confocal immunofluorescence microscopy demonstrated that EpCAM was present on all intestinal epithelial cell (IEC) in organoids and that it accumulated at lateral intercellular interfaces (Figure 1A), as is seen in murine small intestine in vivo (Figure 1A)
EpCAM KO intestinal epithelium did not support formation of IEC organoids or spheroids (Figure S6A–D). These results suggest that IEC stem cells must express EpCAM to survive and proliferate both in vitro and in vivo

Summary

Introduction

EpCAM (Epithelial Cell Adhesion Molecule; CD326) is a surface glycoprotein expressed in developing and adult epithelia, and selected carcinomas. EpCAM modulates cell motility and migration and it regulates cell mixing at epithelial–dermal interfaces [4]. EpCAM modulates tight junction composition and function [5]. EpCAM and TROP2 are homologous cell surface proteins and exhibit functional redundancy, but they are not equivalent [6]. The most prominent feature of mutations in EpCAM is development of congenital tufting enteropathy (CTE) [7,8,9,10,11]. CTE is a severe diarrheal syndrome that presents shortly after birth and features severe epithelial dysplasia [7,8]

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Results

Conclusion