Calcium/Calmodulin-dependent Protein Kinase II Delta 6 (CaMKIIδ6) and RhoA Involvement in Thrombin-induced Endothelial Barrier Dysfunction

Abstract

Multiple Ca2؉ release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca 2؉ signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca 2؉ /calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKII␦ 6 isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKII␦ 6 activation assessed by Thr 287 autophosphorylation. Electroporation of siRNA targeting endogenous CaMKII␦ (siCaMKII␦) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nM thrombin-induced increases in monolayer permeability assessed by electrical cellsubstrate impedance sensing (ECIS). siCaMKII␦ inhibited 2.5 nM thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKII␦ 6 mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKII␦ 6 /RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.Endothelial cells line the luminal surface of blood vessels where they regulate the flux and/or transport of fluid, macromolecules and white blood cells from the vascular space to the interstitium. A loss of barrier function as found with inflammation leads to the accumulation of fluid that can disrupt tissue function. Thrombin, a key pro-coagulant serine-protease, is a well known inflammatory mediator that induces endothelial barrier dysfunction by activating endothelial expressed protease-activated receptors (PARs), resulting in activation of key signaling pathways, including increases in intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ), that mediate cytoskeletal reorganization through myosin light chain (MLC)-dependent EC contraction (1) and the disassembly of VE-cadherin containing adherent junctions (2). Multiple Ca 2ϩ release and entry mechanisms may contribute to agonist-dependent increases in [Ca 2ϩ ] i in endothelial cells including Oria1/STIM1-mediated pathways (3) and various TRP channels (4), including TRPC4 plasma membrane channels (5, 6). However, the immediate downstream effectors of Ca 2ϩ signaling pathways in the regulation of EC permeability still remain unclear.A numb...

Calcium Signaling Is Dispensable for Receptor Regulation of Endothelial Barrier Function

“…For instance, the endothelial CaMKII␦6 isoform was shown to play a role in thrombin-mediated endothelial barrier disruption. However, CaMKII␦6 contribution was through RhoA/ ROCK-dependent mechanism and was apparent only at low concentrations of thrombin (2.5 nM) where the Ca 2ϩ signal activated by thrombin was negligible, but not at higher concentrations of thrombin (56). Therefore, increased cytosolic Ca 2ϩ and MLCK are not absolutely required for triggering the acute and rapid disruption in barrier function in response to GPCR agonists; RhoA activation appears necessary and sufficient.…”

Dabigatran and Thrombin Modulate Electrophysiological Characteristics of Pulmonary Vein and Left Atrium

“…Thrombin has been shown to modulate CaMKII and activate NCX and L-type calcium current. 7,33,34 Therefore, dabigatran may reduce these calcium regulation proteins by inhibition of thrombin. Because CaMKII, NCX, and L-type calcium current play an important role in PV electric activity, 25,35 these findings may contribute to the slower beating rates in dabigatran-treated PVs.…”

Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells

“…The combined MS and bioinformatic analyses revealed altered phosphorylation levels of regulatory sites and predicted activity of many of the kinases which are the usual suspects in thrombin signaling, such as CAMK, PKA, and PKC, which regulate cytoskeletal protein reorganization 46 and rho kinase activity. 47 The kinase activity analysis also revealed a distinct temporal activation pattern, in which arginine-directed phosphorylations precede proline-directed ones. Interestingly, the latter ones, which include MAPK3/ERK1, MAPK1/ERK2, GSK3, and CDK5, were predicted with both increased (clusters 3 and 4) and decreased activity (cluster 6).…”