Eosinophilic esophagitis–linked calpain 14 is an IL-13–induced protease that mediates esophageal epithelial barrier impairment
Benjamin P. Davis, … , Leah C. Kottyan, Marc E. Rothenberg
Benjamin P. Davis, … , Leah C. Kottyan, Marc E. Rothenberg
Published April 7, 2016
Citation Information: JCI Insight. 2016;1(4):e86355. https://doi.org/10.1172/jci.insight.86355.
View: Text | PDF
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

×

Figure 6

Effect of calpain 14 gene silencing on epithelial cell responses to IL-13.

Options: View larger image (or click on image) Download as PowerPoint
Effect of calpain 14 gene silencing on epithelial cell responses to IL-1...
EPC2 cells transduced with either a nonsilencing control (NSC) or calpain 14 (CAPN14) gene silencing (KD-1 and KD-2) vector were grown at the air-liquid interface (ALI) with and without IL-13 and analyzed by (A) quantitative PCR of CAPN14 mRNA relative to GAPDH mRNA and normalized to NSC without IL-13 (n = 3). (B) Western blot analysis of CAPN14 protein in ALI culture (n = 3) with HSP-90 as a loading control. (C) H&E staining of ALI-cultured EPC2 cells. (D) Quantification of the percentage of total area of dilated intercellular spaces. (E) Quantification of nuclei of basal cells in contact with basolateral edge of ALI epithelium. (F) Transepithelial resistance (Rt, n = 3) and (G) FITC-dextran flux (n = 3) of ALI system. Data are representative of 3 independent experiments. Scale bar: 10 μm. For A and D–G, data are represented as the mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; statistical significance determined using a 2-tailed t test.