Regulation of Tight Junction Permeability and Occludin Phosphorylation by RhoA-p160ROCK-dependent and -independent Mechanisms

Abstract

In epithelial and endothelial cells, tight junctions regulate the paracellular permeability of ions and proteins. Disruption of tight junctions by inflammation is often associated with tissue edema, but regulatory mechanisms are not fully understood. Using ECV304 cells as a model system, lysophosphatidic acid and histamine were found to increase the paracellular permeability of the tracer horseradish peroxidase. Cytoskeletal changes induced by these agents included stimulation of stress fiber formation and myosin light chain phosphorylation. Additionally, occludin, a tight junction protein, was a target for signaling events triggered by lysophosphatidic acid and histamine, events that resulted in its phosphorylation. A dominant-negative mutant of RhoA, RhoA T19N, or a specific inhibitor of Rho-activated kinases, Y-27632, prevented stress fiber formation, myosin light chain phosphorylation, 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 stimulation of occludin phosphorylation or increased tracer flux, indicating that occludin phosphorylation may regulate tight junction permeability independently of cytoskeletal events. Thus, occludin is a target for receptor-initiated signaling events regulating its phosphorylation, and this phosphorylation may be a key regulator of tight junction permeability.

VEGF-mediated disruption of endothelial CLN-5 promotes blood-brain barrier breakdown

“…Like claudins, OCLN is a component of tight junction strands (7), but strands also form in OCLN-deficient cells (8), and the complex phenotype of OCLN -/-mice is not clearly related to tight junction abnormalities (9). However, phosphorylation of the cytoplasmic domain of OCLN correlates with tight junction formation (33) and has been shown to regulate junction permeability (34). Both external loops, as well as the transmembrane and the cytoplasmic domains of OCLN, are important for regulation of paracellular permeability (35), and studies have also shown that OCLN is responsible for sealing of tight junctions (36).…”

Angiotensin II induces microtubule reorganization mediated by a deacetylase SIRT2 in endothelial cells

“…The membranes were subjected to immunoblotting as described previously. 12 Immunofluorescence and confocal microscopy Cells cultured on coverslips were fixed with 3% paraformaldehyde for 15 min and then rinsed with phosphate buffered saline. The cells were permeabilized for 15 min with phosphate buffered saline containing 0.5% Triton-X.…”

Evaluation of Vitamin C for Adjuvant Sepsis Therapy

“…Occludin dephosphorylation may increase tight junction permeability independently of the changes in surface charge and actin cytoskeleton that also are associated with endothelial barrier dysfunction (28,54).…”