Synaptopodin is upregulated by IL-13 in eosinophilic esophagitis and regulates esophageal epithelial cell motility and barrier integrity
Mark Rochman, Jared Travers, J. Pablo Abonia, Julie M. Caldwell, Marc E. Rothenberg
Mark Rochman, Jared Travers, J. Pablo Abonia, Julie M. Caldwell, Marc E. Rothenberg
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Research Article Gastroenterology Inflammation

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

Abstract

Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of the esophagus mediated by an IL-13–driven epithelial cell transcriptional program. Herein, we show that the cytoskeletal protein synaptopodin (SYNPO), previously associated with podocytes, is constitutively expressed in esophageal epithelium and induced during allergic inflammation. In addition, we show that the SYNPO gene is transcriptionally and epigenetically regulated by IL-13 in esophageal epithelial cells. SYNPO was expressed in the basal layer of homeostatic esophageal epithelium, colocalized with actin filaments, and expanded into the suprabasal epithelium in EoE patients, where expression was elevated 25-fold compared with control individuals. The expression level of SYNPO in esophageal biopsies correlated with esophageal eosinophil density and was improved following anti–IL-13 treatment in EoE patients. In esophageal epithelial cells, SYNPO gene silencing reduced epithelial motility in a wound healing model, whereas SYNPO overexpression impaired epithelial barrier integrity and reduced esophageal differentiation. Taken together, we demonstrate that SYNPO is induced by IL-13 in vitro and in vivo, is a nonredundant regulator of epithelial cell barrier function and motility, and is likely involved in EoE pathogenesis.

Authors

Mark Rochman, Jared Travers, J. Pablo Abonia, Julie M. Caldwell, Marc E. Rothenberg

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Figure 4

Expression of SYNPO in esophageal biopsies and correlation with EoE transcriptome.

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Expression of SYNPO in esophageal biopsies and correlation with EoE tran...
(A) The normalized expression level for SYNPO in esophageal biopsy tissue from patients with active EoE (n = 10) and control patients (n = 6) (8). ***P < 0.001, t test. (B) The relative expression level of SYNPO isoforms (see Figure 2A) in esophageal biopsies from patients with active EoE and without EoE (Ctrl) was quantified by measuring expression of SYNPO PCR products. For A–C, each dot represents an esophageal biopsy from independent patients. (A and B) **P < 0.01, ***P < 0.001, Holm-Sidak method. (C) The correlation between the expression of SYNPO (PCR product 1–2) and the number of eosinophils per high-power field (Eos/hpf) in an EoE esophageal biopsies. The Spearman correlation (r) and P value are indicated. (D) The graph shows correlation of SYNPO expression with EoE molecular signature genes from RNA-sequencing analysis of EoE patients (8). Gene categories were based on those used for the molecular diagnostics panel for EoE (27). The significance of the correlation is plotted as a negative log(10) P value. The horizontal dashed line represents P = 0.05. The genes probed in each category are indicated; significantly correlated genes are in blue font. Arrows indicate positive (up) and negative (down) correlation. (E) Representative immunofluorescence of SYNPO in esophageal biopsies from independent patients. The white dotted line demarcates the basement membrane. Scale bar: 100 μM. (F) A representative Western blot for SYNPO in control and EoE biopsies; ERK 1/2 is used as a loading control. Quantification of SYNPO expression was performed in control and EoE biopsies from independent patients (n = 11 per each group). ***P < 0.001, Holm-Sidak method. (G) Relative expression level of SYNPO was assessed in the EoE patients either treated with anti–IL-13 antibody or placebo, as described in ref. 29. “Before” corresponds to the beginning of the study, and “after” corresponds to day 85 of the study. *P < 0.05, t test. For A, B, and G expression level was normalized to GAPDH. For box-and-whisker plots, the box represents 50th percentile of the data, whiskers show minimum and maximum values, and the line in the box represents the median.