The Jak/Stat Pathway and Urokinase Receptor Signaling in Human Aortic Vascular Smooth Muscle Cells*
- Inna Dumler‡§,
- Angelika Weis‡,
- Oleg A. Mayboroda¶,
- Christian Maasch‡,
- Uwe Jerke‡,
- Hermann Haller‡ and
- Dietrich C. Gulba‡
- From the ‡Franz Volhard Clinic and Max-Delbrück Center for Molecular Medicine, Virchow Klinikum, Humboldt University of Berlin, 13125 Berlin, Germany and the ¶Institute for Medical Neurobiology, Otto-Von-Guericke University Magdeburg, 39116 Magdeburg, Germany
Abstract
The binding of urokinase plasminogen activator (uPA) to its specific receptor (uPAR) facilitates migration of vascular smooth muscle cells (VSMC). However, the signaling cascade utilized by the urokinase receptor is only incompletely understood. We investigated intracellular uPA/uPAR signaling in human aortic VSMC from the cell membrane to the nucleus. uPA binding to VSMC induced a rapid and pronounced increase in tyrosine phosphorylation of several proteins with molecular masses of 53–60, 85–90, and 130–140 kDa. By using co-immunoprecipitation techniques and in vitro kinase assays, the uPAR-associated proteins were identified as Janus (Jak) and Src non-receptor protein-tyrosine kinases (PTK) Jak1, Tyk2, and p59fyn, p53/56lyn, p53/59hck, and p55fgr. Furthermore, uPA induced a time-dependent reversible translocation of the Stat1 (signal transducer and activator of transcription) protein to the VSMC nuclei, as shown by confocal microscopy studies. Using an electrophoretic mobility shift assay, we then demonstrated that Stat1 is rapidly activated in response to stimulation with uPA and specifically binds to the DNA regulatory elements GAS (interferon-γ activation site) and ISRE (interferon-stimulated response element). Mobility supershift experiments confirmed DNA-protein complexes containing Stat1 protein. Migration experiments with double immunofluorescence staining revealed polarization of uPAR, and colocalization with Jak1 and Tyk2 to the leading edge of the migrating cells. Under the same conditions, Jak2, Jak3, and the Src-PTKs remained randomly distributed over the entire body of the cells. Our studies therefore suggest that, in VSMC, the uPAR-signaling complex utilizes at least two different mechanisms, a direct signaling pathway utilizing the Jak/Stat cascade and a second signal transduction mechanism via Src-like protein-tyrosine kinases. uPA-induced signaling via Jak/Stat is most likely involved in the regulation of cell migration, while the functional purpose of the uPA-associated Src-PTK activation remains to be elucidated.
Footnotes
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↵* This work was supported by a grant from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom all correspondence should be addressed: Franz Volhard Clinic, Wiltbergstraße 50, 13125 Berlin-Buch, Germany. Tel.: 49-30-9417-2451; Fax: 49-30-9417-2453; E-mail:dumler{at}fvk-berlin.de.
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↵1 The abbreviations used are: VSMC, vascular smooth muscle cells; EMSA, electrophoretic mobility shift assay; GPI, glycosylphosphatidylinositol; Jak, Janus kinase; PTK, protein-tyrosine kinase; Stat, signal transducers and activators of transcription; uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; mAb, monoclonal antibody; GAS, interferon-γ activation site; ISRE, interferon-stimulated response element.
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- Received February 18, 1997.
- Revision received October 17, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
