Band intensities corresponding to claudin-7 and EpCAM in (C) were quantified and normalized to -actin. Rabbit polyclonal to DYKDDDDK Tag EpCAM, and claudin-7 inside a functionally important pathway that causes disease when it is dysregulated. Intro Truncating and selected missense mutations in (encoding epithelial cell adhesion molecule [EpCAM; CD326]) cause a severe autosomal recessive child years diarrheal syndrome termed congenital tufting enteropathy (CTE) (1, 2). CTE is definitely characterized by common small intestinal epithelial dysplasia, and intestinal mucosal biopsies demonstrate Olcegepant unique tufts of epithelial cells in the suggestions of blunted villi (1, 3). EpCAM is definitely a cell surface glycoprotein that is present in many developing epithelia, some adult epithelia (including intestine), carcinomas, tumor-initiating cells, circulating tumor cells, and cells and embryonic stem cells (4, 5). Although Olcegepant EpCAM was initially reported to mediate intercellular Olcegepant adhesion directly via homotypic relationships (6), subsequent studies have suggested that EpCAM modulates epithelial cell physiology via several seemingly nonoverlapping mechanisms (7C9). Definitive insights into EpCAM function may come from studies of individuals and mice with mutant alleles. Despite the wide cells distribution of EpCAM, individuals with CTE do not show prominent extraintestinal features (1). Mice with germline null mutations in develop the murine equivalent of CTE and pass away within 2 weeks after birth (10, 11). Consistent with EpCAMs claudin-stabilizing effects (12), intestinal manifestation of selected claudins, including claudin-7, is definitely markedly decreased in mice and humans with mutations (3, 10). The strong similarities between the phenotypes of and knockout mice suggest that EpCAM-claudin relationships are extremely important in the intestine (8, 13, 14). Recent studies of CTE individuals revealed that a significant Olcegepant minority of individuals harbor mutations in and not in (2). encodes a cell membraneCassociated Kunitz type 2 serine protease inhibitor, HAI-2, that can regulate the activity of a variety of proteases (15). The cell surface serine protease matriptase is probably the enzymes that can be inhibited by HAI-2 indirectly, and possibly directly (15, 16). Matriptase is definitely produced like a zymogen, and it becomes fully active only after control by prostasin, another membrane-associated serine protease, or by matriptase itself (16C18). Both HAI-2 and the closely related protease inhibitor HAI-1 are regulators of the proteolytic cascade that includes prostasin and matriptase (16, 19, 20). Matriptase influences tight junction composition and regulates intestinal epithelial cell (IEC) monolayer permeability in vitro (21) and in vivo (22), and loss of matriptase in IECs promotes intestinal carcinogenesis in vivo (23, 24). However, detailed mechanisms by which matriptase regulates intestinal epithelial physiology have not been elucidated, and it is not certain that previously recognized matriptase substrates (urokinase plasminogen activator [uPA], EGF receptor, protease-activated receptor-2 [PAR2], and HGF/scatter element) are involved (21, 25). We hypothesized that there might be a direct link between (HAI-2), matriptase, EpCAM, and claudin-7 that relates to IEC homeostasis and CTE. In the present study, we demonstrate that EpCAM is definitely a physiologically relevant substrate of matriptase. We also identified that loss of HAI-2 in IECs results in matriptase activation that in turn leads to efficient but limited proteolysis of EpCAM at cell surfaces followed by lysosomal degradation of both EpCAM and claudin-7. This pathway is an important determinant of intestinal cells and cell homeostasis, and it provides a platform for understanding why mutations in any of 3 genes (= 8). The 2-tailed value (* 0.0001) for the assessment of abundances of full-length EpCAM and cleaved EpCAM in chloroquine-treated and untreated cells was determined using a paired test. (B) Caco-2 cells were labeled with sulfo-NHS-SS-biotin for 30 minutes at 4C, followed by incubation at 37C Olcegepant for the indicated occasions to allow cell surface proteins to be internalized. Cell surface biotin was stripped via treatment with MESNA, cell lysates were prepared, and biotin-labeled proteins were recovered as explained in Methods. Proteins were resolved using SDS-PAGE and immunoblotted with anti-EpCAM or antiCtransferrin receptor (TFR). Representative data from 1 of 3 experiments are demonstrated. (C).