THE JAK STAT PATHWAY AND UROKINASE RECEPTOR SIGNALING INHUMAN AORTIC VASCULAR SMOOTH-MUSCLE CELLS/

The binding of urokinase plasminogen activator (uPA) to its specific r eceptor (uPAR) facilitates migration of vascular smooth muscle cells ( VSMC). However, the signaling cascade utilized by the urokinase recept or is only incompletely understood, We investigated intracellular uPA/ uPAR signaling in human aortic VSMC from the cell membrane to the nucl eus. uPA binding to VSMC induced a rapid and pronounced increase in ty rosine phosphorylation of several proteins with molecular masses of 53 -60, 85-90, and 130-140 kDa, By using co-immunoprecipitation technique s and in vitro kinase assays, the uPAR-associated proteins were identi fied as Janus (Jab) and Src non-receptor protein-tyrosine kinases (PTK ) Jak1, Tyk2, and p59(fyn), p53/56(lyn), p53/59(hck), and p55(fgr). Fu rthermore, uPA induced a time-dependent reversible translocation of th e Stat1 (signal transducer and activator of transcription) protein to the VSMC nuclei, as shown by confocal microscopy studies, Using an ele ctrophoretic 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-gamma activation site) and ISRE (interferon-stimulated response element), Mobility sup ershift experiments confirmed DNA-protein complexes containing Stat1 p rotein, Migration experiments with double immunofluorescence staining revealed polarization of uPAR, and colocalization with Jak1 and Tyk2 t o the leading edge of the migrating cells, Under the same conditions, Jak2, Jak3, and the Src-PTKs remained randomly distributed over the en tire body of the cells, Our studies therefore suggest that, in VSMC, t he 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 regu lation of cell migration, while the functional purpose of the uPA asso ciated Src-PTR activation remains to be elucidated.