Protein Kinase C-related Kinase and ROCK Are Required for Thrombin-induced Endothelial Cell Permeability Downstream from Gα12/13 and Gα11/q

Abstract

Increase in vascular permeability occurs under many physiological conditions such as wound repair, inflammation, and thrombotic reactions and is central in diverse human pathologies, including tumor-induced angiogenesis, ocular diseases, and septic shock. Thrombin is a pro-coagulant serine protease, which causes the local loss of endothelial barrier integrity thereby enabling the rapid extravasation of plasma proteins and the local formation of fibrin-containing clots. Available information suggests that thrombin induces endothelial permeability by promoting actomyosin contractility through the Rho/ROCK signaling pathway. Here we took advantage of pharmacological inhibitors, knockdown approaches, and the emerging knowledge on how permeability factors affect endothelial junctions to investigate in detail the mechanism underlying thrombin-induced endothelial permeability. We show that thrombin signals through PAR-1 and its coupled G proteins G␣ 12/13 and G␣ 11/q to induce RhoA activation and intracellular calcium elevation, and that these events are interrelated. In turn, this leads to the stimulation of ROCK, which causes actin stressfiber formation. However, this alone is not sufficient to account for thrombin-induced permeability. Instead, we found that protein kinase C-related kinase, a Rho-dependent serine/threonine kinase, is activated in endothelial cells upon thrombin stimulation and that its expression is required for endothelial permeability and the remodeling of cell-extracellular matrix and cellcell adhesions. Our results demonstrate that the signal initiated by thrombin bifurcates at the level of RhoA to promote changes in the cytoskeletal architecture through ROCK, and the remodeling of focal adhesion components through protein kinase C-related kinase. Ultimately, both pathways converge to cause cell-cell junction disruption and provoke vascular leakage.

Integrins stimulate E-cadherin-mediated intercellular adhesion by regulating Src-kinase activation and actomyosin contractility

“…Cadherin ligation also affects the activity of Rho family GTPases (Noren et al, 2001;Charasse et al, 2002;Braga and Yap, 2005), and several reports provide evidence that ROCK and acto-myosin contraction mediate opposite effects on intercellular adhesions. They can promote either the formation of cell-cell contacts (Conti et al, 2004;Shewan et al, 2005) or the disassembly of the cadherins at adhesion sites (Ivanov et al, 2009;Ivanov et al, 2004;Gavard and Gutkind, 2008;de Rooij et al, 2005;Avizienyte et al, 2004;Sahai and Marshall, 2002). Our results indicate that ROCK and myosin-based contraction of the actin cytoskeleton is required for cadherin-mediated adhesion and maintenance (Fig.…”

Uncoupling of the profibrotic and hemostatic effects of thrombin in lung fibrosis

“…Lastly, although we did not detect significant differences in BAL leukocytes or BAL total protein concentration with thrombin inhibition in this model, we did detect subtle decreases in BAL albumin and α-2 macroglobulin levels, suggesting an attenuation of vascular leak by thrombin inhibition. This is not surprising, since thrombin itself is known to induce endothelial barrier disruption (66). Therefore, it is also possible the protective effects of dabigatran on the development of fibrosis in this model may be partially due to the attenuation of vascular leak itself.…”

CCK activates RhoA and Rac1 differentially through Gα13 and Gαq in mouse pancreatic acini

“…A previous study showed that, in NIH 3T3 cells expressing the CCK1 receptor, CCK activates G␣ 12/13 and, thereby, the small GTP-binding protein RhoA, inducing actin cytoskeletal reorganization (23). In intestinal smooth muscle, CCK stimulates phospholipase D activity via RhoA through G␣ 13 activation (30).…”