Dataset Information

The Regulation of Intestinal Mucosal Barrier by Myosin Light Chain Kinase/Rho Kinases.

ABSTRACT: The intestinal epithelial apical junctional complex, which includes tight and adherens junctions, contributes to the intestinal barrier function via their role in regulating paracellular permeability. Myosin light chain II (MLC-2), has been shown to be a critical regulatory protein in altering paracellular permeability during gastrointestinal disorders. Previous studies have demonstrated that phosphorylation of MLC-2 is a biochemical marker for perijunctional actomyosin ring contraction, which increases paracellular permeability by regulating the apical junctional complex. The phosphorylation of MLC-2 is dominantly regulated by myosin light chain kinase- (MLCK-) and Rho-associated coiled-coil containing protein kinase- (ROCK-) mediated pathways. In this review, we aim to summarize the current state of knowledge regarding the role of MLCK- and ROCK-mediated pathways in the regulation of the intestinal barrier during normal homeostasis and digestive diseases. Additionally, we will also suggest potential therapeutic targeting of MLCK- and ROCK-associated pathways in gastrointestinal disorders that compromise the intestinal barrier.

SUBMITTER: Jin Y

PROVIDER: S-EPMC7278945 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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The Regulation of Intestinal Mucosal Barrier by Myosin Light Chain Kinase/Rho Kinases.

Jin Younggeon Y Blikslager Anthony T AT

International journal of molecular sciences 20200518 10

The intestinal epithelial apical junctional complex, which includes tight and adherens junctions, contributes to the intestinal barrier function via their role in regulating paracellular permeability. Myosin light chain II (MLC-2), has been shown to be a critical regulatory protein in altering paracellular permeability during gastrointestinal disorders. Previous studies have demonstrated that phosphorylation of MLC-2 is a biochemical marker for perijunctional actomyosin ring contraction, which i ...[more]

PMID: 32443411

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Project description:The myosin light chain kinase (MLCK) pathway controls intestinal epithelial barrier permeability by regulating the tight junction. 1,25-dihydroxyvitamin D (1,25(OH)2D3)-vitamin D receptor (VDR) signaling protects the epithelial barrier, but the molecular mechanism is incompletely understood.MLCK activation and barrier permeability were studied using monolayers of HCT116, Caco-2, and SW480 cells treated with tissue necrosis factor ? with or without 1,25(OH)2D3. The MLCK pathway was analyzed in normal and inflamed colonic biopsies from patients with ulcerative colitis. Colonic mucosal barrier permeability and MLCK activation were also investigated using trinitrobenzene sulfonic acid-induced colitis models in vitamin D analog paricalcitol-treated wild-type mice and mice carrying VDR deletion in colonic epithelial cells.Tissue necrosis factor ? increased cell monolayer permeability and induced long isoform of MLCK expression and myosin II regulatory light chain (MLC) phosphorylation, and 1,25(OH)2D3 blocked tissue necrosis factor ?-induced increases in monolayer permeability and MLCK-MLC pathway activation by a VDR-dependent fashion. 1,25(OH)2D3 directly suppressed long MLCK expression by attenuating NF-?B activation, and chromatin immunoprecipitation assays confirmed that 1,25(OH)2D3 disrupted p65 binding to 3 ?B sites in long MLCK gene promoter. In human ulcerative colitis biopsies, VDR reduction was associated with increases in long MLCK expression and MLC phosphorylation. In trinitrobenzene sulfonic acid colitis models, paricalcitol ameliorated colitis, attenuated the increase in mucosal barrier permeability, and inhibited long MLCK induction and MLC phosphorylation. In contrast, mice with colonic epithelial VDR deletion exhibited more robust increases in mucosal barrier permeability and MLCK activation compared with wild-type mice.These data demonstrate that 1,25(OH)2D3-VDR signaling preserves the mucosal barrier integrity by abrogating MLCK-dependent tight junction dysregulation during colonic inflammation.

Project description:Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30-120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.

Dataset Information

The Regulation of Intestinal Mucosal Barrier by Myosin Light Chain Kinase/Rho Kinases.

Publications

The Regulation of Intestinal Mucosal Barrier by Myosin Light Chain Kinase/Rho Kinases.

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