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Review
. 2020 Feb 3;21(3):993.
doi: 10.3390/ijms21030993.

Contributions of Myosin Light Chain Kinase to Regulation of Epithelial Paracellular Permeability and Mucosal Homeostasis

Wei-Qi He  1 , Jing Wang  1 , Jian-Ying Sheng  1 , Juan-Min Zha  1 , W Vallen Graham  2   3 , Jerrold R Turner  4
Affiliations
Review

Contributions of Myosin Light Chain Kinase to Regulation of Epithelial Paracellular Permeability and Mucosal Homeostasis

Wei-Qi He et al. Int J Mol Sci. .

Abstract

Intestinal barrier function is required for the maintenance of mucosal homeostasis. Barrier dysfunction is thought to promote progression of both intestinal and systemic diseases. In many cases, this barrier loss reflects increased permeability of the paracellular tight junction as a consequence of myosin light chain kinase (MLCK) activation and myosin II regulatory light chain (MLC) phosphorylation. Although some details about MLCK activation remain to be defined, it is clear that this triggers perijunctional actomyosin ring (PAMR) contraction that leads to molecular reorganization of tight junction structure and composition, including occludin endocytosis. In disease states, this process can be triggered by pro-inflammatory cytokines including tumor necrosis factor-α (TNF), interleukin-1β (IL-1β), and several related molecules. Of these, TNF has been studied in the greatest detail and is known to activate long MLCK transcription, expression, enzymatic activity, and recruitment to the PAMR. Unfortunately, toxicities associated with inhibition of MLCK expression or enzymatic activity make these unsuitable as therapeutic targets. Recent work has, however, identified a small molecule that prevents MLCK1 recruitment to the PAMR without inhibiting enzymatic function. This small molecule, termed Divertin, restores barrier function after TNF-induced barrier loss and prevents disease progression in experimental chronic inflammatory bowel disease.

Keywords: ZO-1; actomyosin; barrier function; claudin; cytokines; drug development; inflammatory bowel disease; mucosal immunology; occludin; tight junction.

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Conflict of interest statement

W.V.G. is an employee of Thelium Therapeutics, Inc. W.V.G. and J.R.T. are co-founders of Thelium Therapeutics, Inc.

Figures

Figure 1
Figure 1
The structure of epithelial intercellular junctions. (A) Schematic showing interactions between the perijunctional actomyosin ring (PAMR), zonula occludens-1 (ZO-1), occludin, claudins at the tight junction; E-cadherin, α-catenin, and β-catenin at the adherens junction; and desmogelin, desmocollin, and intermediate filaments at the desmosome. (B) Transmission electron micrograph showing the tight junction (TJ), adherens junction (AJ), desmosome (D), and microvilli (Mv). From Turner. Nat Rev Immunol 2009. (C) Freeze-fracture electron micrograph of intramembranous tight junction strands. From Shen et al. Annu. Rev. Physiol. 2011.
Figure 2
Figure 2
Roles of myosin light chain kinase (MLCK) in physiological and pathophysiological tight junction regulation. PAMR: perijunctional actomyosin ring; TNF: tumor necrosis factor.
Figure 3
Figure 3
Specific targeting of long MLCK isoform 1 (MLCK1) prevents TNF-induced barrier loss in vivo. (A) Protein domain structure of long MLCK isoforms 1 and 2. Immunoglobulin-cell adhesion molecule (IgCAM) domains are numbered from the amino terminus. (B) Long MLCK1 (green), total MLCK (red), and nuclei (blue) in normal human jejunum. MLCK1 is preferentially-localized to the perijunctional actomyosin ring. (C) Virtually screened compounds docked to a binding pocket within the unique IgCAM3 domain of MLCK1. (D) Mice were injected with vehicle or TNF, and jejunal loops were perfused with either saline-containing vehicle or Divertin. TNF-induced increases in albumin flux (from bloodstream into the gut lumen) were blocked by Divertin. Divertin also blocked TNF-induced myosin II regulatory light chain (MLC) phosphorylation and MLCK1 recruitment to the PAMR. Notably, Divertin does not inhibit MLCK enzymatic activity. ** p < 0.01 by ANOVA with Dunn’s multiple comparison test. From Graham et al. Nat. Med. 2019.
Figure 4
Figure 4
Inhibition of MLCK1 recruitment to the perijunctional actomyosin ring attenuates immune-mediated colitis. (A) Proposed mechanism of Divertin action. (B) Immunodeficient mice received naïve CD4+ effector T cells. Therapy with saline (vehicle), divertin, anti-TNF, or combined Divertin and anti-TNF was initiated after definitive features of disease developed (day 19). (C) Divertin limited weight loss after T cell transfer and was superior to anti-TNF antibody treatment. ** p < 0.01 by two-tailed t test for no transfer vs. all other mice at day 18. *** p < 0.001 by ANOVA with Tukey’s multiple comparison test over the interval from 19−35 days. (D) Divertin enhanced survival after T cell transfer. * p < 0.05, versus saline-treated mice, by Gehan–Breslow–Wilcoxon test. (E) Divertin limited intestinal barrier loss. * p < 0.05, versus saline-treated mice, by ANOVA with Newman–Keuls multiple comparison test. (F) Divertin was superior to anti-TNF antibody treatment in limiting mucosal cytokine production and histopathology after T cell transfer. * p < 0.05; *** p < 0.001 by ANOVA with Bonferroni correction. From Graham et al. Nat. Med. 2019.
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