Matriptase-mediated cleavage of EpCAM destabilizes claudins and dysregulates intestinal epithelial homeostasis
Chuan-Jin Wu, Xu Feng, Michael Lu, Sohshi Morimura, Mark C. Udey
Chuan-Jin Wu, Xu Feng, Michael Lu, Sohshi Morimura, Mark C. Udey
View: Text | PDF
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

×

Figure 6

HAI-2 prevents EpCAM cleavage and subsequent claudin degradation in lysosomes by inhibiting matriptase.

Options: View larger image (or click on image) Download as PowerPoint
HAI-2 prevents EpCAM cleavage and subsequent claudin degradation in lyso...
(A) HEK293 cells were transiently transfected with fixed amounts of pcDNA3 encoding EpCAM and matriptase and varied amounts of SPINT2 expression plasmids as indicated. Proteins of interest were quantified in cell lysates via immunoblotting using relevant Ab. (B–D) Caco-2 cells (B and C) and T84 cells (D) were transfected with control siRNA, EpCAM siRNA, matriptase siRNA, or SPINT2 siRNAs via electroporation. Transfected cells were replated the next day and cultured for 2 more days. Cell lysate proteins were resolved using SDS-PAGE and immunoblotted with anti-matriptase, anti–HAI-2, anti-EpCAM, anti–claudin-7, anti–claudin-1, or anti-occludin as indicated. Claudin-7 and EpCAM band intensities in C were quantified and normalized to β-actin signals. Data are depicted as ratios (mean ± SEM) relative to corresponding siControls (n = 5). A repeated-measures 2-ANOVA Dunnett’s method was used to calculate 2-tailed P values corrected for multiple comparisons to assess mean differences between groups (*P < 0.0001, **P < 0.005, ***P < 0.05). (E) Caco-2 cells were transfected with control siRNA, EpCAM siRNA, matriptase siRNA, or SPINT2 siRNA, and treated with or without 100 μM chloroquine for 20 hours. RIPA lysates were subjected to SDS-PAGE and analyzed for EpCAM, HAI-2, claudin-7, and occludin via Western blotting. β-Actin was used as a loading control. Representative data from 1 of 3 (A, D, and E) or 1 of 5 (B and C) experiments are shown.