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Myosin light chain kinase mediates intestinal barrier dysfunction via occludin endocytosis during anoxia/reoxygenation injury.
Am J Physiol Cell Physiol. 2016 Dec 01; 311(6):C996-C1004.AJ

Abstract

Intestinal anoxia/reoxygenation (A/R) injury induces loss of barrier function followed by epithelial repair. Myosin light chain kinase (MLCK) has been shown to alter barrier function via regulation of interepithelial tight junctions, but has not been studied in intestinal A/R injury. We hypothesized that A/R injury would disrupt tight junction barrier function via MLCK activation and myosin light chain (MLC) phosphorylation. Caco-2BBe1 monolayers were subjected to anoxia for 2 h followed by reoxygenation in 21% O2, after which barrier function was determined by measuring transepithelial electrical resistance (TER) and FITC-dextran flux. Tight junction proteins and MLCK signaling were assessed by Western blotting, real-time PCR, or immunofluorescence microscopy. The role of MLCK was further investigated with select inhibitors (ML-7 and peptide 18) by using in vitro and ex vivo models. Following A/R injury, there was a significant increase in paracellular permeability compared with control cells, as determined by TER and dextran fluxes (P < 0.05). The tight junction protein occludin was internalized during A/R injury and relocalized to the region of the tight junction after 4 h of recovery. MLC phosphorylation was significantly increased by A/R injury (P < 0.05), and treatment with the MLCK inhibitor peptide 18 attenuated the increased epithelial monolayer permeability and occludin endocytosis caused by A/R injury. Application of MLCK inhibitors to ischemia-injured porcine ileal mucosa induced significant increases in TER and reduced mucosal-to-serosal fluxes of 3H-labeled mannitol. These data suggest that MLCK-induced occludin endocytosis mediates intestinal epithelial barrier dysfunction during A/R injury. Our results also indicate that MLCK-dependent occludin regulation may be a target for the therapeutic treatment of ischemia/reperfusion injury.

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Publisher Full Text (DOI)

Authors+Show Affiliations

Jin Y
Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina.
Blikslager AT
Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina anthony_blikslager@ncsu.edu.

MeSH

AnimalsCaco-2 CellsCell Line, TumorDextransEndocytosisEpithelial CellsHumansHypoxiaIntestinal MucosaMyosin Light ChainsMyosin-Light-Chain KinaseOccludinPermeabilityPhosphorylationSignal TransductionSwineTight Junctions

Pub Type(s)

Journal Article

Language

eng

PubMed ID

27760753

Citation

Jin, Younggeon, and Anthony T. Blikslager. "Myosin Light Chain Kinase Mediates Intestinal Barrier Dysfunction Via Occludin Endocytosis During Anoxia/reoxygenation Injury." American Journal of Physiology. Cell Physiology, vol. 311, no. 6, 2016, pp. C996-C1004.
Jin Y, Blikslager AT. Myosin light chain kinase mediates intestinal barrier dysfunction via occludin endocytosis during anoxia/reoxygenation injury. Am J Physiol Cell Physiol. 2016;311(6):C996-C1004.
Jin, Y., & Blikslager, A. T. (2016). Myosin light chain kinase mediates intestinal barrier dysfunction via occludin endocytosis during anoxia/reoxygenation injury. American Journal of Physiology. Cell Physiology, 311(6), C996-C1004. https://doi.org/10.1152/ajpcell.00113.2016
Jin Y, Blikslager AT. Myosin Light Chain Kinase Mediates Intestinal Barrier Dysfunction Via Occludin Endocytosis During Anoxia/reoxygenation Injury. Am J Physiol Cell Physiol. 2016 Dec 1;311(6):C996-C1004. PubMed PMID: 27760753.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Myosin light chain kinase mediates intestinal barrier dysfunction via occludin endocytosis during anoxia/reoxygenation injury. AU - Jin,Younggeon, AU - Blikslager,Anthony T, Y1 - 2016/10/19/ PY - 2016/04/25/received PY - 2016/10/13/accepted PY - 2016/10/21/pubmed PY - 2017/6/6/medline PY - 2016/10/21/entrez KW - Caco-2BBe1 KW - intestinal anoxia/reoxygenation injury KW - intestinal barrier functions KW - myosin light chain kinase KW - tight junctions SP - C996 EP - C1004 JF - American journal of physiology. Cell physiology JO - Am J Physiol Cell Physiol VL - 311 IS - 6 N2 - Intestinal anoxia/reoxygenation (A/R) injury induces loss of barrier function followed by epithelial repair. Myosin light chain kinase (MLCK) has been shown to alter barrier function via regulation of interepithelial tight junctions, but has not been studied in intestinal A/R injury. We hypothesized that A/R injury would disrupt tight junction barrier function via MLCK activation and myosin light chain (MLC) phosphorylation. Caco-2BBe1 monolayers were subjected to anoxia for 2 h followed by reoxygenation in 21% O2, after which barrier function was determined by measuring transepithelial electrical resistance (TER) and FITC-dextran flux. Tight junction proteins and MLCK signaling were assessed by Western blotting, real-time PCR, or immunofluorescence microscopy. The role of MLCK was further investigated with select inhibitors (ML-7 and peptide 18) by using in vitro and ex vivo models. Following A/R injury, there was a significant increase in paracellular permeability compared with control cells, as determined by TER and dextran fluxes (P < 0.05). The tight junction protein occludin was internalized during A/R injury and relocalized to the region of the tight junction after 4 h of recovery. MLC phosphorylation was significantly increased by A/R injury (P < 0.05), and treatment with the MLCK inhibitor peptide 18 attenuated the increased epithelial monolayer permeability and occludin endocytosis caused by A/R injury. Application of MLCK inhibitors to ischemia-injured porcine ileal mucosa induced significant increases in TER and reduced mucosal-to-serosal fluxes of 3H-labeled mannitol. These data suggest that MLCK-induced occludin endocytosis mediates intestinal epithelial barrier dysfunction during A/R injury. Our results also indicate that MLCK-dependent occludin regulation may be a target for the therapeutic treatment of ischemia/reperfusion injury. SN - 1522-1563 UR - https://www.unboundmedicine.com/medline/citation/27760753/Myosin_light_chain_kinase_mediates_intestinal_barrier_dysfunction_via_occludin_endocytosis_during_anoxia/reoxygenation_injury_ DB - PRIME DP - Unbound Medicine ER -