A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease.
OBJECTIVE
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 membrane permeant inhibitor of MLC kinase (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)-alpha and interferon (IFN)-gamma 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-alpha and IFN-gamma 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-alpha and IFN-gamma. The data also suggest that TNF-alpha and IFN-gamma 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.
Zolotarevsky
Yevgeny
Y
Department of Pathology, Wayne State University, Detroit, Michigan, USA.
Hecht
Gail
G
Koutsouris
Athanasia
A
Gonzalez
Deborah E
DE
Quan
Cliff
C
Tom
Jeffrey
J
Mrsny
Randall J
RJ
Turner
Jerrold R
JR
D000205:Actomyosin / Q000378:metabolism; D000595:Amino Acid Sequence; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology*; D004927:Escherichia coli Infections / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007410:Intestinal Diseases / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D007425:Intracellular Membranes / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors*; D009842:Oligopeptides / Q000493:pharmacokinetics / Q000494:pharmacology*; D010539:Permeability; D010766:Phosphorylation / Q000187:drug effects; D019108:Tight Junctions / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
C118153:Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D000205:Actomyosin; D009219:Myosin-Light-Chain Kinase