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

Abstract

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.

Distinct steady-state properties and TNF responses of epithelial long myosin light chain kinase (MLCK) splice variants

“…Our previous work using small molecules and dominant negative constructs has demonstrated that protein-protein interactions involving the IgCAM3 domain that differentiates MLCK1 from MLCK2 are viable therapeutic targets (6, 18). Specifically, we identified small molecules that bind directly to IgCAM3 or interfere with binding of an MLCK1 region that includes IgCAM3 to a chaperone protein and demonstrated that these molecules are able to reverse inflammation-induced barrier loss in vitro and in vivo (6, 18). While they emphasize the central importance of the MLCK1 IgCAM3 domain, these data do not demonstrate that IgCAM3 alone is sufficient to direct steady-state or inflammation-induced MLCK1 recruitment to the perijunctional actomyosin ring.…”

“…We recently reported that, similar to well-differentiated Caco-2 BBe monolayers, TNF increased MLCK1 expression and perijunctional recruitment in human duodenal enteroids (18). To determine if this also occurs in human disease, we analyzed ileal biopsies from Crohn's disease patients and healthy controls by quantitative immunofluorescence using MLCK1-specific or panlong MLCK antibodies.…”

“…The subcellular distributions of these splice variants also differed, with MLCK1 preferentially localized to the perijunctional actomyosin ring (6, 17). More recently, we demonstrated that inflammatory stimuli including tumor necrosis factor (TNF) trigger further MLCK1 recruitment to the perijunctional actomyosin ring in vitro and in vivo (6, 18).…”

“…IgCAM3 is, therefore, incomplete in MLCK2. Our recent studies indicate that IgCAM3 is required for steady-state and inflammation-induced MLCK1 recruitment (6, 18) and that disruption of interactions between IgCAM3 and FKBP8 displaces perijunctional MLCK1 and prevents TNF-induced MLC phosphorylation and barrier loss in vitro and in vivo (6).…”

Finding a mate for MLCK: improving the potential for therapeutic targeting of gut permeability

“…In GUT , Zuo et al present an innovative study identifying that the tacrolimus-binding protein FKBP8 is a specific binding partner for the tight junction regulatory mediator, myosin light-chain kinase 1 (MLCK1) 5. MLCK1 is one of two splice variants of the canonical tight junction regulatory protein, MLCK.…”