Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress
Song Li, Benjamin P.C. Chen, Nobuyoshi Azuma, Ying-Li Hu, Steven Z. Wu, Bauer E. Sumpio, John Y.-J. Shyy, Shu Chien
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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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 Rho regulate the shear stress activation of AP-1/TRE transcrip...
Cdc42 and Rho regulate the shear stress activation of AP-1/TRE transcriptional activity. (a) BAECs cultured on glass slides were transfected with pcDNA3, HA-Cdc42(N17), or HA-RhoA(N19) (2 μg/slide), together with 4XTRE-Pl-Luc (1 μg/slide) and β-gal (0.5 μg/slide). Approximately 40 h after transfection, the confluent BAEC monolayers were either sheared at 12 dyn/cm2 for eight hours or kept under static conditions for eight hours, followed by luciferase and β-gal assays. The luminometer readings for luciferase were normalized by β-gal activities. (b) 4XTRE-Pl-Luc (1 μg/slide) and β-gal (0.5 μg/slide) were cotransfected with pcDNA3, myc-Cdc42(V12), myc-RhoA(V14), myc-Cdc42(V12) together with HA-RhoA(N19), or myc-RhoA(V14) together with HA-Cdc42(N17) (2 μg/slide for each plasmids). 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 cells were lysed for luciferase and β-gal activities assays. Bar graphs, representing mean ± SD from three separate experiments, show the normalized luciferase activity of the various samples relative to that in the pcDNA3-transfected, static controls. 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 pcDNA3-transfected cells. The bottom of a shows immunoblotting with anti-HA mAb; the bottom of b shows immunoblotting with polyclonal anti-Cdc42, polyclonal anti-RhoA, or anti-HA mAb indicating that the levels of the expressed exogenous proteins were comparable among the various samples.