Regulation of tight junction permeability and occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms.
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.
Hirase
T
T
First Department of Internal Medicine, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. hirase@med.kobe-u.ac.jp
Kawashima
S
S
Wong
E Y
EY
Ueyama
T
T
Rikitake
Y
Y
Tsukita
S
S
Yokoyama
M
M
Staddon
J M
JM
D000199:Actins / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D018014:Gene Transfer Techniques; D005799:Genes, Dominant; D006632:Histamine / Q000494:pharmacology; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D062793:Occludin; D010539:Permeability; D010766:Phosphorylation; D011233:Precipitin Tests; D015398:Signal Transduction; D022502:Stress Fibers / Q000378:metabolism; D019108:Tight Junctions / Q000502:physiology*; D013997:Time Factors; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D008246:Lysophospholipids; D008565:Membrane Proteins; D019384:Nucleic Acid Synthesis Inhibitors; C568985:OCLN protein, human; D062793:Occludin; D015638:Cytochalasin D; D006632:Histamine; D006735:Horseradish Peroxidase; D020742:rhoA GTP-Binding Protein