Role of ras-dependent ERK activation in phorbol ester-induced endothelial cell barrier dysfunction.
The treatment of endothelial cell monolayers with phorbol 12-myristate 13-acetate (PMA), a direct protein kinase C (PKC) activator, leads to disruption of endothelial cell monolayer integrity and intercellular gap formation. Selective inhibition of PKC (with bisindolylmaleimide) and extracellular signal-regulated kinases (ERKs; with PD-98059, olomoucine, or ERK antisense oligonucleotides) significantly attenuated PMA-induced reductions in transmonolayer electrical resistance consistent with PKC- and ERK-mediated endothelial cell barrier regulation. An inhibitor of the dual-specificity ERK kinase (MEK), PD-98059, completely abolished PMA-induced ERK activation. PMA also produced significant time-dependent increases in the activity of Raf-1, a Ser/Thr kinase known to activate MEK ( approximately 6-fold increase over basal level). Similarly, PMA increased the activity of Ras, which binds and activates Raf-1 ( approximately 80% increase over basal level). The Ras inhibitor farnesyltransferase inhibitor III (100 microM for 3 h) completely abolished PMA-induced Raf-1 activation. Taken together, these data suggest that the sequential activation of Ras, Raf-1, and MEK are involved in PKC-dependent endothelial cell barrier regulation.
Verin
A D
AD
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA. averin@welch.jhu.edu
Liu
F
F
Bogatcheva
N
N
Borbiev
T
T
Hershenson
M B
MB
Wang
P
P
Garcia
J G
JG
D000818:Animals; D015153:Blotting, Western; D002199:Capillary Permeability / Q000187:drug effects; D002417:Cattle; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000503:physiopathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate; D014652:Vascular Diseases / Q000139:chemically induced / Q000378:metabolism; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors
D004791:Enzyme Inhibitors; D018994:Myosin Light Chains; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins; D020741:rho GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate