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Original research
Tacrolimus-binding protein FKBP8 directs myosin light chain kinase-dependent barrier regulation and is a potential therapeutic target in Crohn’s disease
  1. Li Zuo1,2,3,
  2. Wei-Ting Kuo2,3,4,
  3. Feng Cao3,5,
  4. Sandra D Chanez-Paredes2,3,
  5. Daniel Zeve6,7,
  6. Prabhath Mannam6,
  7. Léa Jean-François3,
  8. Anne Day3,
  9. W Vallen Graham8,
  10. Yan Y Sweat2,3,
  11. Nitesh Shashikanth2,3,
  12. David T Breault6,7,
  13. Jerrold R Turner2,3,8
  1. 1Anhui Medical University, Hefei, Anhui, China
  2. 2Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA
  3. 3Laboratory of Mucosal Barrier Pathobiology, Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA
  4. 4Graduate Institute of Oral Biology, National Taiwan University, Taipei, Taiwan
  5. 5Department of Otorhinolaryngology Head and Neck Surgery, Second People's Hospital of Hefei, Hefei, Anhui, China
  6. 6Pediatrics, Boston Children's Hospital, Boston, Massachusetts, USA
  7. 7Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA
  8. 8Department of Pathology, The University of Chicago, Chicago, Illinois, USA
  1. Correspondence to Dr Jerrold R Turner, Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA; JRTURNER{at}BWH.HARVARD.EDU; Dr Li Zuo, Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA; lzuo{at}bwh.harvard.edu

Abstract

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
  • TNF
  • 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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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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Footnotes

  • 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.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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