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Molecular decipherment of Rho effector pathways regulating tight-junction permeability
H Fujita1, H Katoh, H Hasegawa
1Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
The Biochemical Journal
|March 4, 2000
Summary
RhoA regulates tight-junction permeability via Rho-associated kinase and other effectors. Mutations in RhoA
Area of Science:
- Cell biology
- Molecular biology
- Physiology
Background:
- RhoA activation increases transepithelial electrical resistance (TER).
- Understanding RhoA's effectors is crucial for regulating tight-junction permeability.
Purpose of the Study:
- To identify RhoA effectors involved in regulating tight-junction permeability.
- To investigate the role of Rho-associated kinase (ROCK) in RhoA-mediated TER changes.
Main Methods:
- Used a tetracycline-inducible expression system in Madin-Darby canine kidney cells.
- Introduced constitutively active RhoA(V14) and its effector-loop mutants (RhoA(V14/L40), RhoA(V14/C42)).
- Assessed stress-fiber formation and TER elevation, and tested the effect of ROCK inhibitor Y-27632.
Main Results:
- RhoA(V14/C42) induced stress-fiber formation, but RhoA(V14/L40) did not.
- Neither mutant induced TER elevation.
- Y-27632 inhibited RhoA(V14)-induced stress-fiber formation and TER elevation.
- Mutations at positions 40 or 42 disrupted RhoA's coupling to at least one effector.
Conclusions:
- RhoA-induced regulation of tight-junction permeability is mediated by Rho-associated kinase and an additional unidentified effector.
- Specific mutations in RhoA's effector loop affect its interaction with downstream targets.
- RhoA-associated kinase is a key mediator of RhoA's effects on cell junctions.