The Jak/Stat Pathway and Urokinase Receptor Signaling in Human Aortic Vascular Smooth Muscle Cells

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 p59 fyn , p53/56 lyn , p53/59 hck , 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-␥ 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 SrcPTKs 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.

Urokinase Induces Expression of Its Own Receptor in Beas2B Lung Epithelial Cells

“…uPAR also appears to associate with ␤ 2 integrins and members of the Src family of kinases (44,45). In vascular smooth muscle cells, uPAR was associated with JAK1, Tyk2, and Src kinases (46), whereas in kidney epithelial tumor cells, uPAR associates with gp130 and JAK1 (47).…”

Functions of the fibrinolytic system in human ito cells and its control by basic fibroblast and platelet-derived growth factor

“…However, some data suggest that signaling and activation of a chemotactic and proliferative program may take place upon interaction of u-PAR with a truncated form of u-PA, lacking the catalytic site, or in the presence of inhibitors of plasmin generation. 19,27,42,51,52 Furthermore, it has been shown that u-PAR, which is spatially located in focal contacts and at the leading edge of migrating cells, 53,54 may directly interact via its extracellular domain with activated integrins promoting adhesion and migration toward specific matrix proteins even in the absence of u-PA. 55 Thus, the potential biological interactions of the u-PA/u-PAR system appear more complex than expected and require further studies.…”

Bacterial endotoxin enhances colorectal cancer cell adhesion and invasion through TLR-4 and NF-κB-dependent activation of the urokinase plasminogen activator system

“…u-PA/u-PAR promotes cell/cell and cell/ECM proteolysis by regulating localised plasminogen activation . Independent of proteolysis, u-PA enhances cell invasion through activation of several migration-associated signalling molecules such as extracellular signal-regulated kinases (Nguyen et al, 1998), focal adhesion kinases (Tang et al, 1998) and signal transducers and activators of transcription 1 (STAT1) (Dumler et al, 1998). Such intercellular signal transduction is apparently facilitated by the interaction of u-PAR with integrins (Wei et al, 1996;Degryse et al, 2001) and cytoskeletal components (Xue et al, 1997).…”