Matriptase cleaves EpCAM and TROP2 in keratinocytes, destabilizing both proteins and associated claudins

The homologs EpCAM and TROP2, which both interact with claudin-1 and claudin-7, are frequently coexpressed in epithelia including skin. Intestine uniquely expresses high levels of EpCAM but not TROP2. We previously identified EpCAM as a substrate of the membrane-anchored protease matriptase and linked HAI-2, matriptase, EpCAM and claudin-7 in a pathway that is pivotal for intestinal epithelial cells (IEC) homeostasis. Herein, we reveal that TROP2 is also a matriptase substrate. Matriptase cleaved TROP2 when purified recombinant proteins were mixed in vitro. TROP2, like EpCAM, was also cleaved after co-transfection of matriptase in 293T cells. Neither EpCAM nor TROP2 cleavage was promoted by protease-disabled matriptase or matriptase that harbored the ichthyosis-associated G827R mutation. We confirmed that EpCAM and TROP2 are both expressed in skin and detected cleavage of these proteins in human keratinocytes (HaCaT cells) after the physiologic inhibition of matriptase by HAI proteins was relieved by siRNA knockdown. Knockdown of EpCAM or TROP2 individually had only small effects on claudin-1 and claudin-7 levels, whereas elimination of both markedly diminished claudin levels. HAI-1 knockdown promoted EpCAM and TROP2 cleavage accompanied by reductions in claudins, whereas HAI-2 knockdown had little impact. Double knockdown of HAI-1 and HAI-2 induced nearly complete cleavage of EpCAM and TROP2 and drastic reductions of claudins. These effects were eliminated by concurrent matriptase knockdown. Decreases in claudin levels were also diminished by the lysosomal inhibitor chloroquine and cleaved EpCAM/TROP2 fragments accumulated preferentially. We demonstrate that TROP2 and EpCAM exhibit redundancies with regard to regulation of claudin metabolism and that an HAI, matriptase, EpCAM and claudin pathway analogous to what we described in IECs exists in keratinocytes. This study may offer insights into the mechanistic basis for matriptase dysregulation-induced ichthyosis

Distinct microRNA expression profiles in follicle-associated epithelium and villous epithelium

M cells in follicle-associated epithelium (FAE) covering intestinal lymphoid follicles serve as a portal of entry for particulate antigens (Kanaya and Ohno, 2014 [1]). Despite their biological significance, molecular mechanisms that govern M-cell differentiation and function have not been fully elucidated. MicroRNAs (miRNAs) have a role to control host gene expression profiles that modulate cellular physiology and characteristic. Many studies have shown that miRNAs regulate diverse biological processes including developmental timing, differentiation and growth control of cells and tissues (Ivey and Srivastava, 2010 [2]). miRNAs are also relevant to differentiation and function of intestinal epithelium (McKenna et al., 2010 [3]; Runtsch et al., 2014 [4]). Expression profiles and functions of miRNAs in the intestinal epithelium have been examined in jejunal and colonic mucosa [3]. In contrast, those in FAE remain uncharacterized. To address this deficiency, we isolated Peyer's Patch (PP) FAE and villous epithelium (VE) surrounding the FAE, and compared the miRNA expression profiles of FAE and VE by microarray analysis. This revealed that 43 miRNAs were up-regulated, whereas 9 miRNAs were down-regulated, in FAE compared to VE. A unique pattern of miRNA expression by FAE may reflect important diversity in cellular phenotypes and/or functional features of FAE. All microarray data has been deposited at GEO under accession number GSE46264

Amelioration of congenital tufting enteropathy in EpCAM (TROP1)-deficient mice via heterotopic expression of TROP2 in intestinal epithelial cells

TROP1 (EpCAM) and TROP2 are homologous cell surface proteins that are widely expressed, and often co-expressed, in developing and adult epithelia. Various functions have been ascribed to EpCAM and TROP2, but responsible mechanisms are incompletely characterized and functional equivalence has not been examined. Adult intestinal epithelial cells (IEC) express high levels of EpCAM, while TROP2 is not expressed. EpCAM deficiency causes congenital tufting enteropathy (CTE) in humans and a corresponding lethal condition in mice. We expressed TROP2 and EpCAM in the IEC of EpCAM-deficient mice utilizing a villin promoter to assess EpCAM and TROP2 function. Expression of EpCAM or TROP2 in the IEC of EpCAM knockout mice prevented CTE. TROP2 rescue (T2R) mice were smaller than controls, while EpCAM rescue (EpR) mice were not. Abnormalities were observed in the diameters and histology of T2R small intestine, and Paneth and stem cell markers were decreased. T2R mice also exhibited enlarged mesenteric lymph nodes, enhanced permeability to 4 kDa FITC-dextran and increased sensitivity to detergent-induced colitis, consistent with compromised barrier function. Studies of IEC organoids and spheroids revealed that stem cell function was also compromised in T2R mice. We conclude that EpCAM and TROP2 exhibit functional redundancy, but they are not equivalent

Epithelium-Intrinsic MicroRNAs Contribute to Mucosal Immune Homeostasis by Promoting M-Cell Maturation.

M cells in the follicle-associated epithelium (FAE) of Peyer's patches (PPs) serve as a main portal for external antigens and function as a sentinel in mucosal immune responses. The scarcity of these cells has hampered identification of M cell-specific molecules. Recent efforts have begun to provide insight into antigen transcytosis and differentiation of M cells; however, the molecular mechanisms underlying these processes are not fully elucidated. Small non-coding RNAs including microRNA (miRNA) have been reported to regulate gene expression and control various biological processes such as cellular differentiation and function. To evaluate the expression of miRNAs in FAE, including M cells, we previously performed microarray analysis comparing intestinal villous epithelium (VE) and PP FAE. Here we confirmed FAE specific miRNA expression levels by quantitative PCR. To gain insight into miRNA function, we generated mice with intestinal epithelial cell-specific deletion of Dicer1 (DicerΔIEC) and analyzed intestinal phenotypes, including M-cell differentiation, morphology and function. DicerΔIEC mice had a marked decrease in M cells compared to control floxed Dicer mice, suggesting an essential role of miRNAs in maturation of these cells. Furthermore, transmission electron microscopic analysis revealed that depletion of miRNA caused the loss of endosomal structures in M cells. In addition, antigen uptake by M cells was impaired in DicerΔIEC mice. These results suggest that miRNAs play a significant role in M cell differentiation and help secure mucosal immune homeostasis

Morphology of M cells in Dicer^ΔIEC.

<p>Electron micrographs of Dicer^F/F and Dicer^ΔIEC PP. (A) Surface of FAE by scanning electron microscopy. Arrowheads indicate M cells. Scale bars: 10 μm (B) Transmission electron micrographs of an M cell in FAE and enterocyte in VE. Scale bars: 1.8 μm (C) High magnification image of (B). Arrow indicate the endosomes in M cell. Scale bars: 500 nm.</p

miRNA expression profiles in intestinal epithelium.

<p>Q-PCR analysis was performed for miRNA expression in FAE and VE. The relative levels of each miRNA relative to the small nucleolar RNA <i>Sno202</i> are shown. Values are mean ± SE of three samples from different mice. *P<0.05.</p