Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress
Song Li, … , John Y.-J. Shyy, Shu Chien
Song Li, … , John Y.-J. Shyy, Shu Chien
Published April 15, 1999
Citation Information: J Clin Invest. 1999;103(8):1141-1150. https://doi.org/10.1172/JCI5367.
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Article

Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress

Abstract

Shear stress, the tangential component of hemodynamic forces, plays an important role in endothelial remodeling. In this study, we investigated the role of Rho family GTPases Cdc42 and Rho in shear stress–induced signal transduction and cytoskeleton reorganization. Our results showed that shear stress induced the translocation of Cdc42 and Rho from cytosol to membrane. Although both Cdc42 and Rho were involved in the shear stress–induced transcription factor AP-1 acting on the 12-O-tetradecanoyl-13-phorbol-acetate–responsive element (TRE), only Cdc42 was sufficient to activate AP-1/TRE. Dominant-negative mutants of Cdc42 and Rho, as well as recombinant C3 exoenzyme, attenuated the shear stress activation of c-Jun NH2-terminal kinases (JNKs), suggesting that Cdc42 and Rho regulate the shear stress induction of AP-1/TRE activity through JNKs. Shear stress–induced cell alignment and stress fiber formation were inhibited by the dominant-negative mutants of Rho and p160ROCK, but not by the dominant-negative mutant of Cdc42, indicating that the Rho-p160ROCK pathway regulates the cytoskeletal reorganization in response to shear stress.

Authors

Song Li, Benjamin P.C. Chen, Nobuyoshi Azuma, Ying-Li Hu, Steven Z. Wu, Bauer E. Sumpio, John Y.-J. Shyy, Shu Chien

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Cdc42 and RhoA regulate the shear stress activation of JNK. (a) HA-JNK1 ...
Cdc42 and RhoA regulate the shear stress activation of JNK. (a) HA-JNK1 (1 μg/slide) was cotransfected with pcDNA3, HA-Cdc42(N17), or HA-RhoA(N19) (2 μg/slide) into BAECs cultured on glass slides. The total amount of the transfected plasmid DNA in the various samples was kept constant by supplementing with pcDNA3. About 40 h after transfection, the confluent BAEC monolayers were either sheared at 12 dyn/cm2 for 30 min or kept under static conditions for 30 min. The cell lysates were immunoprecipitated with a polyclonal anti-JNK1 antibody, and GST-c-Jun(1-79) and (γ-32P)ATP were added to the immunoprecipitates for immunocomplex kinase assays. The bands indicated by the arrow represent the phosphorylated GST-c-Jun(1-79). Shown at bottom is the immunoblotting with anti-HA mAb indicating that the levels of HA-JNK1 were comparable among the various samples. The expression levels of transfected HA-Cdc42(N17) and HA-RhoA(N19) were also shown in the immunoblot. (b) Confluent monolayers of BAECs were treated with or without 6.5 μg/ml recombinant C3 for 48 h. The cells were then either sheared at 12 dyn/cm2 for 30 min or kept under static conditions, followed by JNK1 kinase activity assays. The bar graphs, representing mean ± SD from three separate experiments, show the JNK kinase activity of the various samples relative to that in the pcDNA-transfected cells and static controls in a, and relative to that in the static control without C3 pretreatment in b. Asterisks in a indicate significant difference (P < 0.05) compared with pcDNA3-transfected cells after shearing. Asterisks in b indicate significant difference (P < 0.05) compared with control cells after shearing.