Targeting palmitoyl acyltransferase ZDHHC21 improves gut epithelial barrier dysfunction resulting from burn-induced systemic inflammation.

Clinical studies in burn patients demonstrate a close association between leaky guts and increased incidence or severity of sepsis and other complications. Severe thermal injury triggers intestinal inflammation that contributes to intestinal epithelial hyperpermeability, which exacerbates systemic response leading to multiple organ failure and sepsis. In this study, we identified a significant function of a particular palmitoyl acyltransferase, zinc finger DHHC domain-containing protein-21 (ZDHHC21), in mediating signaling events required for gut hyperpermeability induced by inflammation. Using quantitative PCR, we show that ZDHHC21 mRNA production was enhanced twofold when intestinal epithelial cells were treated with TNF-α-IFN-γ in vitro. In addition, pharmacological targeting of palmitoyl acyltransferases with 2-bromopalmitate (2-BP) showed significant improvement in TNF-α-IFN-γ-mediated epithelial barrier dysfunction by using electric cell-substrate impedance-sensing assays, as well as FITC-labeled dextran permeability assays. Using acyl-biotin exchange assay and click chemistry, we show that TNF-α-IFN-γ treatment of intestinal epithelial cells results in enhanced detection of total palmitoylated proteins and this response is inhibited by 2-BP. Using ZDHHC21-deficient mice or wild-type mice treated with 2-BP, we showed that mice with impaired ZDHHC21 expression or pharmacological inhibition resulted in attenuated intestinal barrier dysfunction caused by thermal injury. Moreover, hematoxylin and eosin staining of the small intestine, as well as transmission electron microscopy, showed that mice with genetic interruption of ZDHHC21 had attenuated villus structure disorganization associated with thermal injury-induced intestinal barrier damage. Taken together, these results suggest an important role of ZDHHC21 in mediating gut hyperpermeability resulting from thermal injury.NEW & NOTEWORTHY Increased mucosal permeability in the gut is one of the major complications following severe burn. Here we report the novel finding that zinc finger DHHC domain-containing protein-21 (ZDHHC21) mediates gut epithelial hyperpermeability resulting from an experimental model of thermal injury. The hyperpermeability response was significantly attenuated with a pharmacological inhibitor of palmitoyl acyltransferases and in mice with genetic ablation of ZDHHC21. These findings suggest that ZDHHC21 may serve as a novel therapeutic target for treating burn-induced intestinal barrier dysfunction.

Interleukin-1ß Mediates ß-Catenin-Driven Downregulation of Claudin-3 and Barrier Dysfunction in Caco2 Cells.

BACKGROUND: IL-1ß is a cytokine involved in mediating epithelial barrier dysfunction in the gut. It is known that IL-1ß mediates activation of non-muscle myosin light chain kinase in epithelial cells, but the precise mechanism by which epithelial barrier dysfunction is induced by IL-1ß is not understood. METHODS AND RESULTS: Using a Caco2 cell model, we show that the expression of the tight junction protein, claudin-3, is transcriptionally downregulated by IL-1ß treatment. In addition, after assessing protein and mRNA expression, and protein localization, we show that inhibition of nmMLCK rescues IL-1ß-mediated decrease in claudin-3 expression as well as junction protein redistribution. Using chromatin immunoprecipitation assays, we also show that ß-catenin targeting of the claudin-3 promoter occurs as a consequence of IL-1ß-mediated epithelial barrier dysfunction, and inhibition of nmMLCK interferes with this interaction. CONCLUSIONS: Taken together, these data represent the first line of evidence demonstrating nmMLCK regulation of claudin-3 expression in response to IL-1ß-treated epithelial cells.