Matriptase-mediated cleavage of EpCAM destabilizes claudins and dysregulates intestinal epithelial homeostasis
Chuan-Jin Wu, … , Sohshi Morimura, Mark C. Udey
Chuan-Jin Wu, … , Sohshi Morimura, Mark C. Udey
Published February 1, 2017; First published January 17, 2017
Citation Information: J Clin Invest. 2017;127(2):623-634. https://doi.org/10.1172/JCI88428.
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Categories: Research Article Cell biology Gastroenterology

Matriptase-mediated cleavage of EpCAM destabilizes claudins and dysregulates intestinal epithelial homeostasis

Abstract

Congenital tufting enteropathy (CTE) is a severe autosomal recessive human diarrheal disorder with characteristic intestinal epithelial dysplasia. CTE can be caused by mutations in genes encoding EpCAM, a putative adhesion molecule, and HAI-2, a cell surface protease inhibitor. A similar phenotype occurs in mice whose intestinal epithelial cells (IECs) fail to express the tight junction–associated protein claudin-7. EpCAM stabilizes claudin-7 in IECs, and HAI-2 regulates the cell surface serine protease matriptase, a known modifier of intestinal epithelial physiology. Therefore, we hypothesized that HAI-2, matriptase, EpCAM, and claudin-7 were functionally linked. Herein we have demonstrated that active matriptase cleaves EpCAM after Arg80 and that loss of HAI-2 in IECs led to unrestrained matriptase activity and efficient cleavage of EpCAM. Cleavage of EpCAM decreased its ability to associate with claudin-7 and targeted it for internalization and lysosomal degradation in conjunction with claudin-7. CTE-associated HAI-2 mutant proteins exhibited reduced ability to inhibit matriptase and also failed to efficiently stabilize claudin-7 in IECs. These results identify EpCAM as a substrate of matriptase and link HAI-2, matriptase, EpCAM, and claudin-7 in a functionally important pathway that causes disease when it is dysregulated.

Authors

Chuan-Jin Wu, Xu Feng, Michael Lu, Sohshi Morimura, Mark C. Udey

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Figure 1

Multiple EpCAM species in IEC lysates reflect proteolytic processing.

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Multiple EpCAM species in IEC lysates reflect proteolytic processing. (A...
(A) Nonionic detergent lysates of Caco-2 and T84 cells were resolved via SDS-PAGE and immunoblotted with polyclonal Ab raised against the entire EpCAM extracellular domain. Representative data from 1 of more than 5 experiments are shown. (B) Stable vector– or EpCAM shRNA–transduced Caco-2 cells were lysed, and SDS-PAGE–resolved proteins were immunoblotted with anti-EpCAM polyclonal Ab raised against a C-terminal peptide. Representative data from 1 of 3 experiments are shown. (C) RIPA buffer lysates of Caco-2 cells were immunoprecipitated with control IgG or anti-EpCAM mAb. Immunoprecipitates were fractionated using SDS-PAGE and stained with Coomassie blue. Representative data from 1 of 3 experiments are shown. Two EpCAM-reactive species (CE1 and CE2) were recovered, and N-terminal amino acid sequences were determined. H and L correspond to IgG heavy and light chains, respectively. (D) X represents an indeterminate amino acid; A/L indicates that both A and L were detected at this position. (E and F) RIPA lysates of small intestine organoids (E) and ileum and colon tissues (F) from three 8- to 10-week-old mice (C57BL/6; Charles River Laboratories) were resolved using reduced SDS-PAGE and immunoblotted with the anti-EpCAM polyclonal Ab used in A.