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.

Synaptopodin is upregulated by IL-13 in eosinophilic esophagitis and regulates esophageal epithelial cell motility and barrier integrity

“…Accordingly, SYNPO expression is highly correlated with a subset of esophageal-enriched genes involved in epithelial differentiation. Notably, the majority of these genes are downregulated in EoE, except CAPN14, which is considered to be a genetic driver of the disease and is linked to epithelial differentiation (14,53,54). These data further support that SYNPO dysregulation is a part of a broader alteration in esophageal epithelial differentiation program in response to IL-13.…”

“…Most findings that underscore the critical role of IBF in pathogenesis of EoE have focused on structural epithelial proteins, such as DSG1, or proteases, such as CAPN14 (11,14,53). Our findings -that actin-associated proteins are significantly upregulated in EoE, correlate with EoE severity, and alter esophageal epithelial integrity in cultured cells -signify the potential involvement of SYNPO and other actin-binding proteins in the pathogenesis of EoE.…”

“…EPC2 cells were subjected to ALI on 12-well and 24-well filter inserts as previously described (53). TEER and 3-to 5-kDa FITC-dextran (Sigma-Aldrich) paracellular flux were measured using an EVOM (World Precision Instruments) and a fluorescence plate reader (BioTek), respectively.…”

Novel immunologic mechanisms in eosinophilic esophagitis

“…Remarkably, certain genetic loci associated with increased EoE risk (e.g., FLG , CAPN14 ) [17,20 •• ,21 • ] encode proteins that mediate epithelial barrier function [22,23]. Furthermore, certain rare, damaging genetic variants observed in affected individuals exhibiting a familial pattern of EoE inheritance occur in esophagus-specific genes [19 •• ].…”

The antiprotease SPINK7 serves as an inhibitory checkpoint for esophageal epithelial inflammatory responses

“…Multiple lines of emerging evidence substantiate a key role for the epithelium in the propagation and pathoetiology of EoE as the disease is mediated in part by loss of epithelial DSG1 ( 9 ). In addition, linkage of disease susceptibility with genetic variants in the epithelial genes TSLP (5q22) and CAPN14 (2p23) has been established ( 10 , 11 ). Notably, IL-13 induces not only impaired barrier function in esophageal epithelium but also the gene product of CAPN14 , calpain 14, an intracellular protease that is a key regulator of barrier function ( 11 ).…”