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

Abstract

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.

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

“…ER stress is also reported to be responsible for loss of intestinal epithelial stemness [40] through activation of the unfolded protein response (UPR). Our prior finding of ER stress and UPR in CTE mice [41] may be the cause of of the loss of Lgr5-positive ISCs in these mice. Further studies are needed to explain the direct mechanism by which mutant EpCAM affects Lgr5-positive ISCs but not other stem cells, transient amplifying cells or other progenitor cells.…”

“…Thus, the observation that NHE3 protein, but not at mRNA, expression may imply post transcriptional regulation via altered interactions with the PDZ domain protein, NHERF [43]. Moreover, we recently reported that endoplasmic reticulum stress and the unfolded protein response (UPR) play a role in pathogenesis in mutant EPCAM mice [41]. Activation of UPR might lead to alterations in polarized absorptive enterocytes, which normally depend on intracellular transport of various components of the brush border for proper functioning.…”

“…Proteins were resolved on a 4-12% SDS-PAGE gel, followed by transfer to a PVDF membrane (Immobilon-PSQ PVDF membrane, Millipore-Sigma; Burlington, MA). Immunoblotting was performed as described earlier [41], using rabbit polyclonal antibodies to HES1 (Thermo Fisher Scientific), MATH1 (ATOH1) (Developmental Studies Hybridoma Bank), NICD (Abcam, Cambridge, UK), NHE3 (GTX41967, GeneTex), SGLT1…”

Current View on EpCAM Structural Biology

“…Mutant EpCAM (deletion of exon 4) is not present on the cell surface, it rather accumulates in the ER. This activates the ER stress-induced mechanism unfolded protein response (UPR) [ 96 ]. There are also two reports of homozygous full EPCAM gene knockouts [ 80 , 87 ]; however, they are lethal in mice [ 97 ].…”

“…However, the connection between structural and functional consequences of mutations is not immediately obvious in case of extracellular single amino acid substitutions, far from the identified interaction surfaces with the abovementioned proteins (for an extensive review please see [ 83 ]). We hypothesize that these mutations affect the stability of EpCAM protein as a whole and lead to either its increased internalization or proteolytic degradation or accumulation in ER, as in the case of exon 4 deletion mutant [ 96 ].…”