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 2

IL-13 induces calpain 14 in esophageal epithelial cells.

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IL-13 induces calpain 14 in esophageal epithelial cells. (A) Expression ...
(A) Expression of calpain 14 (CAPN14) mRNA relative to GAPDH mRNA by quantitative PCR reaction in EPC2 cells exposed to IL-13 (100 ng/ml) for the indicated times. Eotaxin-3 is a positive control for IL-13 stimulation. (B) Western blot analysis of CAPN14 protein in air-liquid interface–cultured (ALI–cultured) EPC2 cells with or without (control) IL-13 for 96 hours; data are representative of 3 independent experiments, and HSP-90 is a loading control. (C) mRNA levels of calpain family members in ALI-cultured EPC2 cells with and without IL-13 treatment; data are derived from RNA-sequencing analysis, expressed as fragments per kilobase of exon per million, and representative of 3 independent experiments. For A and C, data are expressed as the mean or mean ± SEM; ***P < 0.001; statistical significance determined using a 2-tailed t test. CAST, calpastatin; CAPNS1, calpain small subunit 1.