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.

Congenital tufting enteropathy (CTE) is a rare chronic diarrheal disease of infancy caused by mutations in epithelial cell adhesion molecule (EpCAM). Previously, a murine CTE model showed mis-localization of EpCAM away from the basolateral cell surface in the intestine. Here we demonstrate that mutant EpCAM accumulated in the endoplasmic reticulum (ER) where it co-localized with ER chaperone, GRP78/BiP, revealing potential involvement of ER stress-induced unfolded protein response (UPR) pathway in CTE. To investigate the significance of ER-localized mutant EpCAM in CTE, activation of the three UPR signaling branches initiated by the ER transmembrane protein components IRE1, PERK, and ATF6 was tested. A significant reduction in BLOS1 and SCARA3 mRNA levels in EpCAM mutant intestinal cells demonstrated that regulated IRE1-dependent decay (RIDD) was activated. However, IRE1 dependent XBP1 mRNA splicing was not induced. Furthermore, an increase in nuclear-localized ATF6 in mutant intestinal tissues revealed activation of the ATF6-signaling arm. Finally, an increase in both the phosphorylated form of the translation initiation factor, eIF2α, and ATF4 expression in the mutant intestine provided support for activation of the PERK-mediated pathway. Our results are consistent with a significant role for UPR in gastrointestinal homeostasis and provide a working model for CTE pathophysiology.

Background Foeniculum vulgare, F. vulgare, commonly known as fennel, is believed to be one of the world’s oldest medicinal herbs and has been exploited by people for centuries as a nutritional aid for digestive disorders. In many southeast Asian countries, it is ingested as an after-meal snack, mukhvas, due to its breath-freshening and digestive aid properties. F. vulgare is used in some countries, such as Iran, as a complementary and alternative treatment for inflammatory bowel disease (IBD). Methods This study investigated the effects of fennel seed extract on intestinal epithelium barrier function and the Signal Transducer and Activator of Transcription (STAT) pathway. This pathway is active in inflammatory bowel disease. To study the protective effects of fennel seed extract in vitro, monolayers derived from the T84 colonic cell line were challenged with interferon-gamma (IFN-γ) and monitored with and without fennel seed extract. To complement our in vitro studies, the dextran sodium sulfate induced murine colitis model was employed to ascertain whether the protective effect of fennel seed extract can be recapitulated in vivo. Results Fennel seed extract was shown to exert a protective effect on transepithelial electrical resistance (TEER) in both T84 and murine models and showed increases in tight junction-associated mRNA in T84 cell monolayers. Both models demonstrated significant decreases in phosphorylated STAT1 (pSTAT1), indicating reduced activation of the STAT pathway. Additionally, mice treated with fennel seed showed significantly lower ulcer indices than control mice. Conclusions We conclude barrier function of the gastrointestinal tract is improved by fennel seed extract, suggesting the potential utility of this agent as an alternative or adjunctive therapy in IBD.

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Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein which is expressed in the basolateral cell surface of most of the epithelial tissues with the highest expression in the gut. Apart from its role in homotypic cellular adhesion and tight junctions, EpCAM is known to be involved in cellular signaling that promotes proliferation, migration, and maintenance of the undifferentiated state of pluripotent stem cells. Several tumors and carcinomas have been linked with EpCAM overexpression, while the mutations in EpCAM is directly associated with a severe diarrheal disease of infancy known as Congenital Tufting Enteropathy (CTE). Alterations in intestinal cell types and barrier permeability have been observed in the patient and the murine model with CTE. We hypothesize that EpCAM plays a pivotal role in intestinal cell type differentiation and intestinal barrier function. In the current study, we evaluate whether the mutation of EpCAM has an effect on the differentiation and permeability of the intestinal epithelial cells (IECs).We have developed an inducible EpCAM mutant enteroid model based on mutations found in CTE patients (deletion of EpCAM exon 4) to allow for ex vivo study of the consequences of EpCAM mutation on all types of IECs. We first established the mutant enteroid model by confirming the deletion of EpCAM exon 4. Immuno‐fluorescence study demonstrates the mislocalization of mutant EpCAM expression away from the cell surface. The different IECs markers were studied by RNA and protein. The gene expression of different transcription factors that are involved in the secretory pathway of intestinal cell differentiation were analyzed. We observe that mutant enteroids have significantly decreased (n=3; p<0.0001) gene expression of the differentiated goblet and paneth cells markers (Muc2, Lysozyme respectively) than the control enteroids. The expression of other cell types and differentiation markers (enteroendocrine cell marker ChgA, intestinal stem cell marker Lgr5, intestinal differentiation marker Krt20) are also decreased in the mutant model (n=3) as shown by qPCR. Moreover, the gene expression of Hnf1b and ATOH1, two early transcription factors for secretory pathway differentiation, are significantly decreased (n=3; p< 0.05) in mutant enteroids compared with controls. The enteroids were polarized onto monolayers to check the trans‐epithelial resistance and barrier integrity with FITC‐dextran flux assay. Trans‐epithelial resistance values remained intact between the control and mutant enteroid derived monolayer, while the epithelial permeability was significantly impaired in the EpCAM mutant enteroid model compared to the control (n=3; p<0.0001).In this study, we develop the first EpCAM mutant enteroid model. With this model, we note the role of EpCAM in the differentiation of IECs from intestinal crypt progenitor/stem cells. The involvement of EpCAM in barrier permeability is also confirmed. This study further establishes a new facet of EpCAM biology which will help in understanding the pathophysiology CTE and other EpCAM associated diseases.Support or Funding InformationNIH R01 DK107764This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.