Objective Intestinal 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.
Design Protein 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.
Results MLCK1 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.
Conclusion Binding 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.
- intestinal barrier function
- IBD basic research
- actin cytoskeleton
- intestinal stem cell
Data availability statement
Data are available on reasonable request. Data, analytic methods and study materials will be made available to academic investigators on reasonable request.
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LZ and W-TK contributed equally.
Contributors Conceptualisation: LZ, W-TK, WVG, JRT. Experimentation: LZ, W-TK, FC, SDC-P, WVG, JRT. Data analysis: LZ, W-TK, FC, SDC-P, DZ, WVG, JRT. Manuscript preparation and revision: LZ, WTK, FC, SCP, DZ, WVG, PM, LJ-F, AD, YVS, NS, DB, JRT.
Funding This work was supported by the National Natural Science Foundation of China grants 82070548 (LZ) and 81800464 (LZ) and U.S. National Institutes of Health grants R01DK61931 (JRT), R01DK68271 (JRT) and P30DK034854 (the Harvard Digestive Disease Center).
Competing interests WVG is a founder, shareholder and employee of Thelium Therapeutics. JRT is a founder and shareholder of Thelium Therapeutics and has served as a consultant for Entrinsic, Immunic, and Kallyope.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.
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