RhoA/Rho-Associated Kinase Pathway Selectively Regulates Thrombin-Induced Intercellular Adhesion Molecule-1 Expression in Endothelial Cells via Activation of I B Kinase and Phosphorylation of RelA/p65

Department of Pediatrics, University of Rochester School of Medicine, Rochester, NY 14642, USA.
The Journal of Immunology (Impact Factor: 4.92). 01/2005; 173(11):6965-72. DOI: 10.4049/jimmunol.173.11.6965
Source: PubMed


We investigated the involvement of the RhoA/Rho-associated kinase (ROCK) pathway in regulating ICAM-1 expression in endothelial cells by the procoagulant, thrombin. Exposure of HUVECs to C3 exoenzyme, a selective inhibitor of Rho, markedly reduced thrombin-induced ICAM-1 expression. Inhibition of ROCK, the downstream effector of Rho, also prevented thrombin-induced ICAM-1 expression. Blockade of thrombin-induced ICAM-1 expression was secondary to inhibition of NF-kappaB activity, the key regulator of ICAM-1 expression in endothelial cells. In parallel studies we observed that inhibition of the RhoA/ROCK pathway by the same pharmacological and genetic approaches failed to inhibit TNF-alpha-induced NF-kappaB activation and ICAM-1 expression. The effect of RhoA/ROCK inhibition on thrombin-induced NF-kappaB activation was secondary to inhibition of IkappaB kinase activation and subsequent IkappaBalpha degradation and nuclear uptake and the DNA binding of NF-kappaB. Inhibition of the RhoA/ROCK pathway also prevented phosphorylation of Ser(536) within the transactivation domain 1 of NF-kappaB p65/RelA, a critical event conferring transcriptional competency to the bound NF-kappaB. Thus, the RhoA/ROCK pathway signals thrombin-induced ICAM-1 expression through the activation of IkappaB kinase, which promotes NF-kappaB binding to ICAM-1 promoter and phosphorylation of RelA/p65, thus mediating the transcriptional activation of bound NF-kappaB.

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Available from: Asrar Malik, Aug 04, 2015
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    • "Moreover, thrombin and interleukin 1β (IL-1β) were shown to increase ROCK activity, the transcriptional activation of NF-κB and then adhesion molecule expression (20,29,30). However, in parallel studies, researchers failed to observe the inhibitory effects on NF-κB activation in response to tumor necrosis factor α (TNF-α) and lipopolysaccharide (LPS) (29,30). By contrast, our findings revealed that ROCK was involved in AGE-induced NF-κB activation and the expression of adhesion molecules. "
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    • "Total RNA was isolated using RNeasy kit (QIAGEN Inc.) according to manufacturer’s recommendations. Quantification and purity of RNA were assessed by A260/A280 absorption and an aliquot of RNA (20 µg) from samples with ratio above 1.6 was subjected to Northern blot analysis as described [34]. "
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    • "This concept is supported by a number of observations in other cell types. For instance, RhoA is important for thrombin-induced ICAM-1 expression, but not for the induction of ICAM-1 expression by TNFα, in endothelial cells [30]. In chondrocytes, RhoA regulates TGF-β- but not IL-1-induced actin cytoskeleton reorganization [31]. "
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