Eosinophilic esophagitis–linked calpain 14 is an IL-13–induced protease that mediates esophageal epithelial barrier impairment
Benjamin P. Davis, Emily M. Stucke, M. Eyad Khorki, Vladislav A. Litosh, Jeffrey K. Rymer, Mark Rochman, Jared Travers, Leah C. Kottyan, Marc E. Rothenberg
Benjamin P. Davis, Emily M. Stucke, M. Eyad Khorki, Vladislav A. Litosh, Jeffrey K. Rymer, Mark Rochman, Jared Travers, Leah C. Kottyan, Marc E. Rothenberg
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Research Article Immunology

Eosinophilic esophagitis–linked calpain 14 is an IL-13–induced protease that mediates esophageal epithelial barrier impairment

Abstract

We recently identified a genome-wide genetic association of eosinophilic esophagitis (EoE) at 2p23 spanning the calpain 14 (CAPN14) gene, yet the causal mechanism has not been elucidated. We now show that recombinant CAPN14 cleaves a calpain-specific substrate and is inhibited by 4 classical calpain inhibitors: MDL-28170, acetyl-calpastatin, E-64, and PD151746. CAPN14 is specifically induced (>100-fold) in esophageal epithelium after IL-13 treatment. Epithelial cells overexpressing CAPN14 display impaired epithelial architecture, characterized by acantholysis, epidermal clefting, and epidermolysis. CAPN14 overexpression impairs epithelial barrier function, as demonstrated by decreased transepithelial resistance (2.1-fold) and increased FITC-dextran flux (2.6-fold). Epithelium with gene-silenced CAPN14 demonstrates increased dilated intercellular spaces (5.5-fold) and less organized basal cell layering (1.5-fold) following IL-13 treatment. Finally, CAPN14 overexpression results in loss of desmoglein 1 (DSG1) expression, whereas the IL-13–induced loss of DSG1 is normalized by CAPN14 gene silencing. Importantly, these findings were specific to CAPN14, as they were not observed with modulation of CAPN1 expression. These results, along with the potent induction of CAPN14 by IL-13 and genetic linkage of EoE to the CAPN14 gene locus, demonstrate a molecular and cellular pathway that contributes to T helper type 2 responses in mucosal epithelium.

Authors

Benjamin P. Davis, Emily M. Stucke, M. Eyad Khorki, Vladislav A. Litosh, Jeffrey K. Rymer, Mark Rochman, Jared Travers, Leah C. Kottyan, Marc E. Rothenberg

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Figure 7

Effect of calpain 14 overexpression on desmoglein 1 expression.

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Effect of calpain 14 overexpression on desmoglein 1 expression. EPC2 cel...
EPC2 cells transduced with empty vector (EV), calpain 14 overexpression vector (CAPN14), nonsilencing control (NSC), or CAPN14 gene-silencing (KD-1 and KD-2) vectors were grown at the air-liquid interface (ALI) and analyzed by (A) Western blot analysis for desmoglein 1 (DSG1). Quantitation of the 50-kDa DSG1 band intensity normalized to the full-length DSG1 band intensity is shown. (B) Immunofluorescence of DSG1 (green) in ALI culture; DAPI is shown in blue. (C) Western blot analysis of the CAPN14 gene-silencing effect on IL-13–mediated appearance of a DSG1 immunoreactive molecular species (50-kDa band) and quantitation of the 50-kDa DSG1 band intensity normalized to full-length DSG1 band intensity. (D) Immunofluorescence of DSG1 (green) in ALI-cultured EPC2 cells with and without IL-13 stimulation and CAPN14 gene silencing; DAPI is shown in blue. (E) ALI immunofluorescence intensity relationship of CAPN14 (green) and DSG1 (purple) and (F) colocalization of CAPN14 (green) and DSG1 (purple); DAPI is shown in blue. Data are representative of 3 independent experiments performed with replicates (n = 3–6). For A and C, data are expressed as the mean ± SEM; ***P < 0.001; statistical significance determined using a 2-tailed t test. Original magnification, ×20. Scale bar: 10 μm.