Abstract 12898: Oxidative Stress and Rho-Associated Coiled-Coil Protein Kinase (ROCK)-Mediated Double Phosphorylation of Myosin Light Chain in the Submembranous Region Plays a Key Role in Thrombin-Induced Barrier Dysfunction in Vascular Endothelial Cells
Endothelial barrier dysfunction plays a fundamental role in the pathogenesis of various vascular diseases such as atherosclerosis. The phosphorylation of the myosin light chain (MLC) is thought to occur sequentially, first at S19 and then at T18, and the resultant actin stress fiber formation is thought to cause a disruption of the inter-endothelial junction. MLC phosphorylation thus plays a key role in barrier dysfunction. However, both the role of the mono- and di-phosphorylation of MLC (pMLC and ppMLC) and the mechanism for MLC phosphorylation during barrier disruption still remain unclear. This study addressed this question and investigated the mechanisms of thrombin-induced barrier dysfunction in porcine aortic endothelial cells. Thrombin (1 u/mL) decreased the transendothelial electrical resistance (TER) with a peak at 3-5 min. A new SDS-PAGE method using a Phos-tag compound, which specifically binds to phosphoprotein, was used to separately quantify the levels of pMLC and ppMLC. The confluent cells contained 25% pMLC and 2% ppMLC before stimulation. Thrombin slightly increased pMLC at 3 min, which became localized in the perinuclear region. In contrast, ppMLC transiently increased to 35% at 3min, and became localized in the submembranous region at the inter-endothelial junction. Thrombin also induced the formation of peripheral actin bundles in the submembranous region. The stress fiber formation was less prominent in the cells at confluency. ppMLC colocalized with the submembranous actin bundles. N-acetyl-L-cysteine inhibited the thrombin-induced increase in ppMLC and decrease in TER. ROCK inhibitors, Y27632 and H1152, abolished the thrombin-induced increase in ppMLC and decrease in TER, and the submembranous localization of ppMLC and formation of actin bundles. The activation of ROCK by thrombin was supported by an increase in the phosphorylation of MYPT1, a regulatory subunit of MLC phosphatase. This study revealed, for the first time, that thrombin-induced endothelial barrier disruption depends on the oxidative stress and ROCK-mediated double phosphorylation of MLC and peripheral actin bundle formation in the submembranous region. The results also suggest the differential, but not sequential, regulation of MLC phosphorylation.
- © 2012 by American Heart Association, Inc.