Abstract WP438: Rho Kinase-mediated Di-phosphorylation of Myosin Light Chain in the Sub-membranous Regions and Circumferential Actomyosin Contraction Mediate Thrombin-induced Barrier Disruption in Vascular Endothelial Cells

Abstract

The disruption of blood-brain barrier plays a critical role in the pathophysiology of cerebrovascular diseases. Thrombin is one of the major factors which cause barrier disruption. The phosphorylation of myosin light chain (MLC) is a key signal of barrier disruption. MLC is thought to be di-phosphorylated sequentially at Ser19 and then Thr18, thereby inducing stress fiber formation to generate traction force to disrupt cell-cell contact. However, it is unclear how the phosphorylation at two sites contributes to barrier disruption. The present study investigated the role of mono- and di-phosphorylation of MLC (MLC-P and MLC-PP) in thrombin-induced barrier disruption. Thrombin (1 u/mL) decreased the transendothelial electrical resistance (TEER) with a peak at 3-5 min in porcine aortic endothelial cells (PAEC). A Phos-tag SDS-PAGE method was used to quantify the amount of MLC-P and MLC-PP. PAEC at confluence contained 25% MLC-P and 2% MLC-PP before stimulation. Upon thrombin stimulation, MLC-P marginally increased, while MLC-PP transiently increased to a peak of 35% at 3-5 min. MLC-P was localized mainly in the peri-nuclear region, while MLC-PP was localized mainly in the sub-membranous region of cell-cell contact. MLC-PP was also co-localized with the peripheral actin bundles. In contrast, thrombin induced stress fiber formation and localization of MLC-P and MLC-PP on the stress fibers when the cell-cell contact was loosed by removing extracellular Ca 2+ or in the cells at the growing phase with sparse cell-cell contact. Two different Rho kinase inhibitors, Y27632 and H1152, inhibited the thrombin-induced increase in MLC-PP, sub-membranous localization of MLC-PP and decrease in TEER, while having no effect on the level of MLC-P. Inhibition of myosin ATPase activity by 100 μmol/L blebbistatin inhibited the thrombin-induced decrease in TEER. The present study suggests that MLC-P and MLC-PP are independently regulated in endothelial cells, and that Rho kinase-mediated MLC-PP in the sub-membranous regions and the circumferential, but not radial, contraction plays a critical role in the thrombin-induced barrier disruption. Inhibition of MLC-PP thus provides a crucial strategy for restoring normal function of the blood-brain barrier.