Calcium/Calmodulin-dependent protein kinase II delta 6 (CaMKIIdelta6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction.
Multiple Ca(2+) 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 CaMKIIdelta(6) isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKIIdelta(6) activation assessed by Thr(287) autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIdelta (siCaMKIIdelta) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nM thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIdelta 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, CaMKIIdelta(6) mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIdelta(6)/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.
Wang
Zhen
Z
Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208, USA.
Ginnan
Roman
R
Abdullaev
Iskandar F
IF
Trebak
Mohamed
M
Vincent
Peter A
PA
Singer
Harold A
HA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology; D004727:Endothelium / Q000378:metabolism / Q000473:pathology*; D004730:Endothelium, Vascular / Q000166:cytology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008297:Male; D008954:Models, Biological; D020033:Protein Isoforms; D051381:Rats; D017207:Rats, Sprague-Dawley; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D020033:Protein Isoforms; C066737:RHOA protein, human; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048049:Extracellular Signal-Regulated MAP Kinases; D013917:Thrombin; D020742:rhoA GTP-Binding Protein; D002118:Calcium