ZO-1 protein is required for hydrogen peroxide to increase MDCK cell paracellular permeability in an ERK...

Hydrogen peroxide (H₂O₂) increases paracellular permeability of Madin-Darby canine kidney (MDCK) cells, but the mechanism mediating this effect remains unclear. Treatment of MDCK cells with H₂O₂ activated ERK 1/2. Inhibition of ERK 1/2 activation blocked the ability of H₂O₂ to increase paracellular permeability. Knockdown of zonula occludens-1 (ZO-1) protein but not occludin eliminated the ability of H₂O₂ to increase paracellular permeability. H₂O₂ 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. H₂O₂ treatment decreased the number of F-actin stress fibers in the basal portion of the cells. Similar to wild-type MDCK cells, H₂O₂ 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 H₂O₂ 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. H₂O₂ 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 H₂O₂ increases MDCK cell paracellular permeability through activation of ERK 1/2. This H₂O₂ action requires ZO-1 protein and TOCA-1 protein, suggesting involvement of the actin cytoskeleton.