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
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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 7

CTE-associated HAI-2 mutant proteins are less potent inhibitors of matriptase-mediated EpCAM cleavage and claudin degradation than wild-type HAI-2.

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CTE-associated HAI-2 mutant proteins are less potent inhibitors of matri...
(A) HEK293 cells were transiently transfected with fixed amounts of pcDNA3 encoding EpCAM and matriptase and varied amounts of pcDNA3 encoding wild-type or Y163C mutant SPINT2. After 48 hours, EpCAM, matriptase, and HAI-2 were detected by Western blotting. (B–D) Caco-2 cells were transfected with pcDNA3 or pcDNA3 encoding HA-tagged wild-type SPINT2, SPINT2 Y163C, or SPINT2 G168S and selected with G418 for 2 weeks. Surviving cells were pooled (B and D) or cloned (C), and HAI-2 expression was assessed via immunoblotting with anti-HA and anti-HAI-2. Pooled cells (B) or cloned cells (C) transfected with plasmids encoding wild-type HAI-2 or HAI-2 mutant proteins were transfected with control siRNA or SPINT2 siRNA (targeting the 5′ untranslated region of SPINT2 mRNA). Cell lysates were harvested 72 hours later and immunoblotted with anti-EpCAM, anti-claudin-7, anti-occludin, anti-HA, anti-HAI-2, or anti–β-actin. Representative data from 1 of 3 experiments is shown in A and B. Band intensities corresponding to claudin-7 and EpCAM in (C) were quantified and normalized to β-actin. Data are depicted as mean ± SEM of ratios of signals corresponding to full-length EpCAM, cleaved EpCAM, and claudin-7 relative to those in control siRNA transfections (n = 4). P values of comparisons between wild-type HASPINT2 and HASPINT2 Y163C transfected cells were determined as described in Supplemental Figure 4B. (D) Pooled Caco-2 cells transfected with vector, SPINT2, SPINT2 Y163C, or SPINT2 G168S plasmids were transiently transfected with control siRNA or SPINT2 siRNA. siRNA transfected cells were replated into Transwells the next day and cultured for 5 additional days to establish monolayers. Methanol-fixed monolayers were stained with anti-HA (green) or anti-claudin-7 (red) and analyzed using confocal microscopy. Scale bar: 20 μm. Representative images from 1 of 3 experiments are presented. For the experiment depicted, 10 images of SPINT2 siRNA transfected wild-type HASPINT2, HASPINT2 Y163C, or HASPINT2 G168S expressing cells were subjected to quantitative analysis. Values depicted represent mean ± SEM of claudin-7 pixels/HA-HAI-2 pixels (n = 10, each image including at least 50 cells, for each experimental condition). Two-tailed P values for the comparisons between effects of individual mutant (Y163C or G168S) and wild-type constructs were calculated via 1-way ANOVA using Dunnett’s method (*P < 0.0001).