© 1999 American Heart Association, Inc.
Original Contributions |
Urokinase Activates the Jak/Stat Signal Transduction Pathway in Human Vascular Endothelial Cells
From the Franz Volhard Clinic and Max-Delbrück Center for Molecular Medicine, Virchow Klinikum-Charite, Humboldt University of Berlin, Berlin (I.D., A.K., A.W., K.W., D.C.G., H.H.), and the Institute for Medical Neurobiology, Otto-Von-Guericke University Magdeburg, Magdeburg (O.A.M.), Germany.
Correspondence to Inna Dumler, PhD, Franz Volhard Clinic, Wiltbergstrasse 50, 13125 Berlin-Buch, Germany. E-mail dumler{at}fvk-berlin.de
Abstract—Endothelial
cells demonstrate high urokinase expression and upregulation of
urokinase receptors in response to vascular injury. Urokinase receptor
binding facilitates endothelial cell migration into an
arterial wound; however, the signaling cascade induced by
the urokinase receptor in this cell type is incompletely understood.
Because the Janus kinase (Jak)/signal transducer and
activator of transcription (Stat) pathway seems to be
important for vessel function, we investigated the hypothesis that
urokinase receptor binding activates Jak/Stat signaling in
human vascular endothelial cells. Incubation of
endothelial cells with urokinase-type
plasminogen activator (uPA,1 nmol/L) induced a
rapid and pronounced increase in tyrosine
phosphorylation of several proteins with a molecular
weight between 80 to 90 and 130 to 140 kDa. The same pattern of
tyrosine phosphorylation was found after treatment with
1 nmol/L ATF, the urokinase amino-terminal fragment, which is devoid of
proteolytic activity but still binds to the urokinase receptor. Using
coimmunoprecipitation techniques, we demonstrated that the
activated urokinase receptor is associated with 2 cytoplasmic
tyrosine kinases of the Jak family, viz, Jak1 and Tyk2. uPA and ATF
induced a time-dependent activation of both kinases, as shown by
immunoprecipitation and Western blot analysis. Using
electrophoretic mobility shift and supershift assays, we then
demonstrated that Stat1 is rapidly activated in
endothelial cells in response to uPA and ATF.
Furthermore, Stat1 specifically binds to the regulatory elements
interferon-
activation site/interferon-stimulated response element.
The uPA-induced, time-dependent translocation of Stat1 to cell nuclei
was confirmed by confocal microscopy study and
immunoblotting of nuclear extracts with an anti-Stat1
antibody. This study provides evidence for a novel signaling pathway
for uPA in human vascular endothelial cells. Direct
activation of the Jak/Stat system via the uPA-receptor complex may be
an important mechanism for endothelial cell migration
and/or proliferation during angiogenesis and after vascular
injury.
Key Words: endothelial cells • urokinase receptor • signal transduction • Jak/Stat pathway
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