Regulation of tight junction permeability and occludin phosphorylation by RhoA-p160ROCK-dependent and -independent mechanisms

In epithelial and endothelial cells, tight junctions regulate the paracellu lar permeability of ions and proteins. Disruption of tight junctions by inf lammation is often associated with tissue edema, but regulatory mechanisms are not fully understood. Using ECV304 cells as a model system, lysophospha tidic acid and histamine were found to increase the paracellular permeabili ty of the tracer horseradish peroxidase. Cytoskeletal changes induced by th ese agents included stimulation of stress fiber formation and myosin light chain phosphorylation, Additionally, occludin, a tight junction protein, wa s a target for signaling events triggered by lysophosphatidic acid and hist amine, events that resulted in its phosphorylation, A dominant-negative mut ant of RhoA, RhoA T19N, or a specific inhibitor of Rho-activated kinases, Y -27632, prevented stress fiber formation, myosin light chain phosphorylatio n, occludin phosphorylation, and the increase in tracer flux in response to lysophosphatidic acid. In contrast, although RhoA T19N and Y-27632 blocked the cytoskeletal events induced by histamine, they had no effect on the st imulation of occludin phosphorylation or increased tracer flux, indicating that occludin phosphorylation may regulate tight junction permeability inde pendently of cytoskeletal events. Thus, occludin is a target for receptor-i nitiated signaling events regulating its phosphorylation, and this phosphor ylation may be a key regulator of tight junction permeability.