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      ZO-1 protein is required for hydrogen peroxide to increase MDCK cell paracellular permeability in an ERK 1/2-dependent manner

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          Hydrogen peroxide (H 2O 2) increases paracellular permeability of Madin-Darby canine kidney (MDCK) cells, but the mechanism mediating this effect remains unclear. Treatment of MDCK cells with H 2O 2 activated ERK 1/2. Inhibition of ERK 1/2 activation blocked the ability of H 2O 2 to increase paracellular permeability. Knockdown of zonula occludens-1 (ZO-1) protein but not occludin eliminated the ability of H 2O 2 to increase paracellular permeability. H 2O 2 treatment did not, however, affect the total cell content or contents of the Triton X-100-soluble and -insoluble fractions for occludin, ZO-1, or ZO-2. H 2O 2 treatment decreased the number of F-actin stress fibers in the basal portion of the cells. Similar to wild-type MDCK cells, H 2O 2 increased ERK 1/2 activation in ZO-1 knockdown and occludin knockdown cells. Inhibition of ERK 1/2 activation blocked the increase in paracellular permeability in occludin knockdown cells. ZO-1 knockdown cell paracellular permeability was regulated by PP1, an src inhibitor, indicating that the loss of response to H 2O 2 was not a general loss of the ability to regulate the paracellular barrier. Inhibition of myosin ATPase activity with blebbistatin increased paracellular permeability in ZO-1 knockdown cells but not in wild-type MDCK cells. H 2O 2 treatment sensitized wild-type MDCK cells to inhibition of myosin ATPase. Knockdown of TOCA-1 protein, which promotes formation of local branched actin networks, reproduced the effects of ZO-1 protein knockdown. These results demonstrate that H 2O 2 increases MDCK cell paracellular permeability through activation of ERK 1/2. This H 2O 2 action requires ZO-1 protein and TOCA-1 protein, suggesting involvement of the actin cytoskeleton.

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          Author and article information

          Am J Physiol Cell Physiol
          Am. J. Physiol., Cell Physiol
          Am J Physiol Cell Physiol
          American Journal of Physiology - Cell Physiology
          American Physiological Society (Bethesda, MD )
          1 September 2018
          6 June 2018
          1 September 2019
          : 315
          : 3
          : C422-C431
          Department of Biomedical Sciences, New York Institute of Technology College of Osteopathic Medicine, Old Westbury, New York
          Author notes

          S. Bilal, S. Jaggi, D. Janosevic, N. Shah, S. Teymour, A. Voronina, and J. Watari contributed equally to this work.

          Address for reprint requests and other correspondence: K. Amsler, Rockefeller Building Room 314F, NYIT College of Osteopathic Medicine, Northern Boulevard, Old Westbury, NY 11568 (e-mail: kamsler@ 123456nyit.edu ).
          PMC6171043 PMC6171043 6171043 C-00185-2017 C-00185-2017
          Copyright © 2018 the American Physiological Society
          Funded by: HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) 10.13039/100000062
          Award ID: R15-DK-091749-01A1
          Research Article

          hydrogen peroxide, tight junction, permeability


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