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

Julie Gavard,J Silvio Gutkind

doi:10.1074/jbc.m803880200

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 Galpha(12/13) and Galpha(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 stress-fiber 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 cell-cell 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.

Highlights

Endothelial homeostasis and vascular integrity are tightly regulated during normal angiogenesis, wound repair, and thrombotic and inflammatory reactions [1]
We show that thrombin signals through protease-activated receptor (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
Thrombin Induces Endothelial Permeability Mainly through PAR-1 and G␣12/13/11/q Coupling—Because multiple receptors for thrombin (PAR) might co-exist at the surface of endothelial cells, we first assessed which PAR was involved during the endothelial permeability response using competitive antagonists

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Transfections—Immortalized human vascular endothelial cells were obtained as indicated in Ref. 27 and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Sigma). Permeability Assays—In vitro permeability assays were conducted as described in Ref. 30, using 3-day-old endothelial monolayers on collagen-coated 3-␮m porous membranes (Transwell, Corning Costar, Acton, MA) and 40-kDa fluorescein-conjugated dextran (Invitrogen). Alexa fluorescent dye-conjugated secondary antibodies were purchased from Invitrogen. VE-cadherin Internalization Assay and Immunofluorescence— VE-cadherin antibody uptake and immunofluorescence staining protocols were described in Refs. Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA) and Alexa546-conjugated phalloidin was from Invitrogen. Statistical analysis was performed with the Prism software (ANOVA test, GraphPad), ***, p Ͻ 0.001, **, p Ͻ 0.01, *, p Ͻ 0.05

RESULTS

ROCK and PRK Are Both

DISCUSSION

Detailed genetic studies have revealed that thrombin activates