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 4

Characterization of calpain 14 overexpression.

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Characterization of calpain 14 overexpression. (A) Calpain 14 (CAPN14) (...
(A) Calpain 14 (CAPN14) (green) immunofluorescence in EPC2 cells transduced with either empty vector (EV) or CAPN14 overexpression vector (CAPN14). (B) Data shown are from calpain activity assays of lysates from EPC2 cells transduced and selected for EV and CAPN14 overexpression lentivirus uptake (n = 3). (C) A model of the overexpression system is shown. Cells were transduced with either EV or CAPN14 overexpression vector by lentiviral transduction. Differentiated EPC2 esophageal epithelial cells were grown for 11 days in high-calcium media starting at day 2. Cells were brought to the air-liquid interface (ALI) starting at day 7 in the presence or absence of IL-13 treatment (100 ng/ml). Images of H&E-stained cells correspond to the time points above. Original magnification, ×20. (D) Expression of CAPN14 mRNA relative to GAPDH mRNA by quantitative PCR in ALI-cultured EPC2 cells. IL-13 stimulation is a positive control (n = 3). (E) Western blot analysis of CAPN14 protein overexpression in ALI-cultured EPC2 cells. (F) Immunofluorescence of CAPN14 (green) overexpression in ALI-cultured EPC2 cells. DAPI is shown in blue. Original magnification, ×20 (top and middle); ×100 (bottom). Scale bar: 10 μm. Data are representative of 3 independent experiments. For B and D, data are expressed as the mean ± SEM; ***P < 0.001, ****P < 0.0001; statistical significance determined using a 2-tailed t test.