Tacrolimus-binding protein FKBP8 directs myosin light chain kinase-dependent barrier regulation and is a potential therapeutic target in Crohn’s disease

• Published in: Gut Vol. 72; no. 5; pp. 870 - 881
• Main Authors: Zuo, Li, Kuo, Wei-Ting, Cao, Feng, Chanez-Paredes, Sandra D, Zeve, Daniel, Mannam, Prabhath, Jean-François, Léa, Day, Anne, Vallen Graham, W, Sweat, Yan Y, Shashikanth, Nitesh, Breault, David T, Turner, Jerrold R
• Format: Journal Article
• Language: English
• Published: England BMJ Publishing Group Ltd and British Society of Gastroenterology 01.05.2023; BMJ Publishing Group LTD
• Subjects: actin cytoskeleton; Animals; Biopsy; Caco-2 Cells; Cell junctions; Crohn Disease - drug therapy; Crohn Disease - metabolism; Crohn's disease; Epithelial cells; Humans; IBD basic research; Inflammatory bowel disease; Inflammatory bowel diseases; intestinal barrier function; Intestinal Mucosa - metabolism; intestinal stem cell; Intestine; Kinases; Light; Mice; Mice, Knockout; Myosin-light-chain kinase; Myosin-Light-Chain Kinase - metabolism; Organoids; Patients; Permeability; Phosphorylation; Protein interaction; Proteins; Recruitment; Remission (Medicine); Risk factors; Tacrolimus; Tacrolimus - pharmacology; Tacrolimus Binding Proteins - metabolism; Tacrolimus-binding protein; Therapeutic targets; Tight Junctions - physiology; TNF; Tumor necrosis factor; Tumor Necrosis Factor-alpha - metabolism; Tumor necrosis factor-TNF; Tumors; Womens health; United States > US; Massachusetts; IBD basic research; actin cytoskeleton; intestinal stem cell; intestinal barrier function; TNF
• ISSN: 0017-5749; 1468-3288; 1468-3288
• DOI: 10.1136/gutjnl-2021-326534

ObjectiveIntestinal barrier loss is a Crohn’s disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically.DesignProtein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects.ResultsMLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects.ConclusionBinding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions.