Calcium/calmodulin-dependent protein kinase II delta 6 (CaMKIIδ 6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction

Zhen Wang, Roman Ginnan, Iskandar F. Abdullaev, Mohamed Trebak, Peter A. Vincent, Harold A. Singer

Research output: Contribution to journalArticle

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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 Ca2+ 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 CaMKIIδ6 isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKIIδ6 activation assessed by Thr287 autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIδ(siCaMKIIδ) 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). siCaMKIIδ 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, CaMKIIδ6 mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIδ6/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.

Original languageEnglish (US)
Pages (from-to)21303-21312
Number of pages10
JournalJournal of Biological Chemistry
Volume285
Issue number28
DOIs
StatePublished - Jul 9 2010

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Calcium-Calmodulin-Dependent Protein Kinase Type 2
Thrombin
Endothelial cells
Chemical activation
Human Umbilical Vein Endothelial Cells
Permeability
rho-Associated Kinases
Small Interfering RNA
Monolayers
Protein Isoforms
Calcium-Calmodulin-Dependent Protein Kinases
Acoustic impedance
Electroporation
Electric Impedance
Endothelium
Blood Vessels
Phosphotransferases
Endothelial Cells

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Wang, Zhen ; Ginnan, Roman ; Abdullaev, Iskandar F. ; Trebak, Mohamed ; Vincent, Peter A. ; Singer, Harold A. / Calcium/calmodulin-dependent protein kinase II delta 6 (CaMKIIδ 6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 28. pp. 21303-21312.
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Calcium/calmodulin-dependent protein kinase II delta 6 (CaMKIIδ 6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction. / Wang, Zhen; Ginnan, Roman; Abdullaev, Iskandar F.; Trebak, Mohamed; Vincent, Peter A.; Singer, Harold A.

In: Journal of Biological Chemistry, Vol. 285, No. 28, 09.07.2010, p. 21303-21312.

Research output: Contribution to journalArticle

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AU - Wang, Zhen

AU - Ginnan, Roman

AU - Abdullaev, Iskandar F.

AU - Trebak, Mohamed

AU - Vincent, Peter A.

AU - Singer, Harold A.

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AB - Multiple Ca2+ release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca2+ 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 CaMKIIδ6 isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKIIδ6 activation assessed by Thr287 autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIδ(siCaMKIIδ) 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). siCaMKIIδ 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, CaMKIIδ6 mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIδ6/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.

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