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

Sahar Bilal, Shirin Jaggi, Danielle Janosevic, Nikita Shah, Shereen Teymour, Angelina Voronina, Jessica Watari, Josephine Axis, Kurt Amsler
American Journal of Physiology. Cell Physiology 2018 September 1, 315 (3): C422-C431
Hydrogen peroxide (H2 O2 ) increases paracellular permeability of Madin-Darby canine kidney (MDCK) cells, but the mechanism mediating this effect remains unclear. Treatment of MDCK cells with H2 O2 activated ERK 1/2. Inhibition of ERK 1/2 activation blocked the ability of H2 O2 to increase paracellular permeability. Knockdown of zonula occludens-1 (ZO-1) protein but not occludin eliminated the ability of H2 O2 to increase paracellular permeability. H2 O2 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. H2 O2 treatment decreased the number of F-actin stress fibers in the basal portion of the cells. Similar to wild-type MDCK cells, H2 O2 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 H2 O2 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. H2 O2 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 H2 O2 increases MDCK cell paracellular permeability through activation of ERK 1/2. This H2 O2 action requires ZO-1 protein and TOCA-1 protein, suggesting involvement of the actin cytoskeleton.

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