Gastroenterology

Gastroenterology

Volume 123, Issue 1, July 2002, Pages 163-172
Gastroenterology

Basic–Alimentary Tract
A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease,☆☆,

https://doi.org/10.1053/gast.2002.34235Get rights and content

Abstract

Background & Aims: Maintenance of the mucosal barrier is a critical function of intestinal epithelia. Myosin regulatory light chain (MLC) phosphorylation is a common intermediate in the pathophysiologic regulation of this barrier. The aim of this study was to determine whether a membranepermeantinhibitor of MLCkinase (PIK) could inhibit intracellular MLC kinase and regulate paracellular permeability. Methods: Recombinant MLC and Caco-2 MLC kinase were used for kinase assays. T84 and Caco-2 monolayers were treated with enteropathogenic Escherichia coli (EPEC) or tumor necrosis factor (TNF)-α and interferon (IFN)-γ to induce barrier dysfunction. Results: PIK inhibited MLC kinase in vitro and was able to cross cell membranes and concentrate at the perijunctional actomyosin ring. Consistent with these properties, apical addition of PIK reduced intracellular MLC phosphorylation by 22% ± 2%, increased transepithelial resistance (TER) by 50% ± 1%, and decreased paracellular mannitol flux rates by 5.2 ± 0.2-fold. EPEC infection induced TER decreases of 37% ± 6% that were limited to 16% ± 5% by PIK. TNF-α and IFN-γ induced TER decreases of 22% ± 3% that were associated with a 172% ± 1% increase in MLC phosphorylation. Subsequent PIK addition caused MLC phosphorylation to decrease by 25% ± 4% while TER increased to 97% ± 6% of control. Conclusions: PIK can prevent TER defects induced by EPEC and reverse MLC phosphorylation increases and TER decreases induced by TNF-α and IFN-γ. The data also suggest that TNF-α and IFN-γ regulate TER, at least in part, via the perijunctional cytoskeleton. Thus, PIK may be the prototype for a new class of targeted therapeutic agents that can restore barrier function in intestinal disease states.

GASTROENTEROLOGY 2002;123:163-172

Section snippets

Peptide synthesis

Peptides were synthesized using an automated Pioneer Peptide Synthesizer (Applied Biosystems, Foster City, CA) on Fmoc-PEG-PS-resin. D-Biotin (Sigma, St. Louis, MO) was incorporated into peptides at the amino terminus using HBTU-HOBt/DIPEA in Me2SO. Peptide resins were cleaved with a 1-hour exposure of a 95% trifluoroacetic acid/2.5% triisopropylsilane/2.5% H2O solvent mixture. Released peptides were purified by preparative reverse-phase C18 high-performance liquid chromatography, characterized

A linear oligopeptide can inhibit Caco-2 MLC kinase in vitro

In confluent Caco-2 cell monolayers, the 215-kilodalton isoform of MLC kinase27 accounts for more than 95% of MLC kinase activity.16 This activity is critical to the regulation of myosin, because phosphorylation of MLC at serine 19 leads to increased actomyosin contraction. The activity of smooth-muscle MLC kinase is regulated by intramolecular interactions between the catalytic domain and the calmodulin-binding inhibitory domain. In the presence of Ca++ and calmodulin, this intramolecular

Discussion

Intestinal paracellular permeability is precisely regulated by the epithelial TJ.31, 32 Although the mechanisms by which TJ permeability is regulated are incompletely defined, MLC within the perijunctional actomyosin ring does appear to be a critical regulator of TJ permeability.17, 33 To better understand the details of actomyosin-dependent TJ regulation, we sought to identify a specific agent capable of inhibiting epithelial MLC kinase. We chose an oligopeptide derived from the inhibitory

Acknowledgements

The authors thank Sean P. Colgan for helpful discussions.

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    Address requests for reprints to: Jerrold R. Turner, M.D., Ph.D., The University of Chicago, 5841 South Maryland Avenue, MC 1089, Chicago, Illinois 60637. e-mail: [email protected].

    ☆☆

    The current address for R. J. Mrsny is: Welsh School of Pharmacy, Cardiff University, Wales, United Kingdom.

    Supported by grants from the National Institutes of Health (DK61931 and DK56121 to J.R.T. and DK50694 to G.H.) and a merit award from the Department of Veterans Affairs (to G.H.).

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