Role of CaM kinase II and ERK activation in thrombin-induced endothelial cell barrier dysfunction.
We have previously shown that thrombin-induced endothelial cell barrier dysfunction involves cytoskeletal rearrangement and contraction, and we have elucidated the important role of endothelial cell myosin light chain kinase and the actin- and myosin-binding protein caldesmon. We evaluated the contribution of calmodulin (CaM) kinase II and extracellular signal-regulated kinase (ERK) activation in thrombin-mediated bovine pulmonary artery endothelial cell contraction and barrier dysfunction. Similar to thrombin, infection with a constitutively active adenoviral alpha-CaM kinase II construct induced significant ERK activation, indicating that CaM kinase II activation lies upstream of ERK. Thrombin-induced ERK-dependent caldesmon phosphorylation (Ser789) was inhibited by either KN-93, a specific CaM kinase II inhibitor, or U0126, an inhibitor of MEK activation. Immunofluorescence microscopy studies revealed phosphocaldesmon colocalization within thrombin-induced actin stress fibers. Pretreatment with either U0126 or KN-93 attenuated thrombin-mediated cytoskeletal rearrangement and evoked declines in transendothelial electrical resistance while reversing thrombin-induced dissociation of myosin from nondenaturing caldesmon immunoprecipitates. These results strongly suggest the involvement of CaM kinase II and ERK activities in thrombin-mediated caldesmon phosphorylation and both contractile and barrier regulation.
Borbiev
Talaibek
T
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21224, USA.
Verin
Alexander D
AD
Birukova
Anna
A
Liu
Feng
F
Crow
Michael T
MT
Garcia
Joe G N
JG
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D006490:Hemostatics / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D011651:Pulmonary Artery / Q000166:cytology / Q000201:enzymology*; D022502:Stress Fibers / Q000201:enzymology; D013917:Thrombin / Q000494:pharmacology*
D002148:Calmodulin-Binding Proteins; D006490:Hemostatics; D018994:Myosin Light Chains; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin