Enteroids expressing a disease-associated mutant of EpCAM are a model for congenital tufting enteropathy

Abstract

Congenital tufting enteropathy (CTE) is an autosomal recessive disease characterized by severe intestinal failure in infancy and mutations in the epithelial cell adhesion molecule ( EPCAM) gene. Previous studies of CTE in mice expressing mutant EpCAM show neonatal lethality. Hence, to study the cellular, molecular, and physiological alterations that result from EpCAM mutation, a tamoxifen-inducible mutant EpCAM enteroid model has been generated. The presence of mutant EpCAM in the model was confirmed at both mRNA and protein levels. Immunofluorescence microscopy demonstrated the reduced expression of mutant EpCAM. Mutant enteroids had reduced budding potential as well as significantly decreased mRNA expression for epithelial lineage markers ( Mucin 2, lysozyme, sucrase-isomaltase), proliferation marker Ki67, and secretory pathway transcription factors ( Atoh1, Hnf1b). Significantly decreased numbers of Paneth and goblet cells were confirmed by staining. These findings were correlated with intestinal tissue from CTE patients and the mutant mice model that had significantly fewer Paneth and goblet cells than in healthy counterparts. FITC-dextran studies demonstrated significantly impaired barrier function in monolayers derived from mutant enteroids compared with control monolayers. In conclusion, we have established an ex vivo CTE model. The role of EpCAM in the budding potential, differentiation, and barrier function of enteroids is noted. Our study establishes new facets of EpCAM biology that will aid in understanding the pathophysiology of CTE and role of EpCAM in health and disease. NEW & NOTEWORTHY Here, we develop a novel ex vivo enteroid model for congenital tufting enteropathy (CTE) based on epithelial cell adhesion molecule ( EPCAM) gene mutations found in patients. With this model we demonstrate the role of EpCAM in maintaining the functional homeostasis of the intestinal epithelium, including differentiation, proliferation, and barrier integrity. This study further establishes a new direction in EpCAM biology that will help in understanding the detailed pathophysiology of CTE and role of EpCAM.

Aberrant Epithelial Differentiation Contributes to Pathogenesis in a Murine Model of Congenital Tufting Enteropathy

“…A significant decrease in Paneth and goblet cell numbers in CTE patients and CTE mice has previously been reported [8]. In order to assess whether other secretory cells types are affected in CTE, we evaluated numbers of enteroendocrine cells in the CTE mice.…”

“…Another study of adult mice bearing the same mutation revealed growth retardation, CTE-like histopathology, impaired intestinal barrier function and decreases in the tight junction proteins zonula occludens-1 (ZO-1) and occludin [7]. CTE intestinal organoids from mice reveal alterations in differentiation in addition to barrier dysfunction [8]. Moreover, data suggest that deficiency of EpCAM in a colonic cell line is accompanied by ion transport and barrier defects [7].…”

“…The NOTCH and ATOH1 (MATH1) pathways play a crucial role in intestinal function by regulating the choice between absorptive and secretory lineages [14][15][16][17]. Previous studies in CTE patients, CTE mice and CTE enteroids revealed decreases in Paneth and goblet cell numbers [8], which may indicate an alteration in intestinal epithelial cell differentiation. Thus, detailed investigation of the intestinal epithelial cell differentiation pathway and its contributions to CTE pathogenesis was of interest.…”

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

“…Reductions in the frequency and/or function of Paneth cells and intestinal epithelial cells in T2R mice were detected in vivo. CTE patients and mutant mice model had significantly fewer Paneth cells and goblet cells than their healthy counterparts [28]. However, forced expression of murine TROP2 in intestine was sufficient to support adequate Paneth cell numbers for survival in the absence of endogenous EpCAM.…”

Congenital Tufting Enteropathy-Associated Mutant of Epithelial Cell Adhesion Molecule Activates the Unfolded Protein Response in a Murine Model of the Disease

“…However, for specialized secretory cells, challenged with mutated secretory proteins, UPR activation alone may not meet such demands. Indeed, the vulnerability of secretory cells to ER stress may account for the decreased numbers of Paneth and goblet cells seen in CTE patients and the CTE murine model [40]. Thus, alterations in mucosal homeostasis in CTE and models of the disease are likely due, at least in part, to aberrant expression of mutant EpCAM.…”