Rho kinase regulates tight junction function and is necessary for tight junction assembly in polarized intestinal epithelia.
OBJECTIVE
Tight junctions are crucial determinants of barrier function in polarized intestinal epithelia and are regulated by Rho guanosine triphosphatase. Rho kinase (ROCK) is a downstream effector of Rho.


METHODS
A specific inhibitor of ROCK, Y-27632, was used to examine the role of ROCK in the regulation of tight junctions in model intestinal (T84) cells by electrophysiologic, biochemical, morphologic, and molecular biologic approaches.


RESULTS
ROCK inhibition induced reorganization of apical F-actin structures and enhanced paracellular permeability but did not alter the distribution or detergent solubility of tight junction proteins. Confocal microscopy showed colocalization of a subpool of ROCK with the tight junction protein zonula occludens 1. Inhibition of ROCK function by a dominant negative mutant of ROCK also produced reorganization of apical F-actin structures without disruption of tight junctions. ROCK inhibition in calcium switch assays showed that ROCK is necessary for the assembly of tight and adherens junctions. Upon calcium repletion, occludin, zonula occludens 1, and E-cadherin failed to redistribute to the intercellular junctions; assembly of the apical F-actin cytoskeleton was prevented; and barrier function failed to recover.


CONCLUSIONS
We suggest that ROCK regulates intact tight junctions via its effects on the F-actin cytoskeleton. ROCK is also critical for assembly of the apical junctional proteins and the F-actin cytoskeleton organization during junctional formation.
Walsh
S V
SV
Epithelial Pathobiology Research Unit, Department of Pathology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Hopkins
A M
AM
Chen
J
J
Narumiya
S
S
Parkos
C A
CA
Nusrat
A
A
D000199:Actins / Q000378:metabolism; D022005:Adherens Junctions / Q000737:chemistry / Q000201:enzymology; D000577:Amides / Q000494:pharmacology; D018938:Caco-2 Cells; D015820:Cadherins / Q000032:analysis; D002463:Cell Membrane Permeability / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology; D002478:Cells, Cultured; D003902:Detergents; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D007408:Intestinal Absorption / Q000502:physiology; D007413:Intestinal Mucosa / Q000166:cytology* / Q000201:enzymology*; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000032:analysis; D016296:Mutagenesis / Q000502:physiology; D062793:Occludin; D017830:Octoxynol; D010750:Phosphoproteins / Q000032:analysis; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D012995:Solubility; D019108:Tight Junctions / Q000737:chemistry / Q000201:enzymology*; D014162:Transfection; D062826:Zonula Occludens-1 Protein; D054460:rho-Associated Kinases
D000199:Actins; D000577:Amides; D015820:Cadherins; D003902:Detergents; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D011725:Pyridines; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C108830:Y 27632; D017830:Octoxynol; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases