Multipotent mesenchymal stem cells (MSCs) are promising candidates for regenerative cell-based therapy. The mechanisms underlying MSC differentiation and other functions relevant to therapeutic avenues remain however a matter of debate. Recent reports imply a critical role for intercellular contacts in MSC differentiation. We studied MSC differentiation to vascular smooth muscle cells (VSMCs) in a coculture model using human primary MSCs and VSMCs. We observed that under these conditions, MSCs did not undergo the expected differentiation process. Instead, they revealed an increased proliferation rate. The upregulated MSC proliferation was initiated by direct contacts of MSCs with VSMCs; indirect coculture of both cell types in transwells was ineffective. Intercellular contacts affected cell growth in a unidirectional fashion, since VSMC proliferation was not changed. We observed formation of so-called tunneling nanotubes (TNTs) between MSCs and VSMCs that revealed an intercellular exchange of a fluorescent cell tracker dye. Disruption of TNTs using cytochalasin D or latrunculin B abolished increased proliferation of MSCs initiated by contacts with VSMCs. Using specific fluorescent markers, we identified exchange of mitochondria via TNTs. By generation of VSMCs with mitochondrial dysfunction, we show that mitochondrial transfer from VSMCs to MSCs was required to regulate MSC proliferation in coculture. Our data suggest that MSC interaction with other cell types does not necessarily result in the differentiation process, but rather may initiate a proliferative response. They further point to complex machinery of intercellular communications at the place of vascular injury and to an unrecognized role of mitochondria in these processes.

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-type plasminogen activator (uPA) participates in diverse (patho)physiological processes through intracellular signaling events that affect cell adhesion, migration, and proliferation, although the mechanisms by which these occur are only partially understood. Here we report that upon cell binding and internalization, single-chain uPA (scuPA) translocates to the nucleus within minutes. Nuclear translocation does not involve proteolytic activation or degradation of scuPA. Neither the urokinase receptor (uPAR) nor the low-density lipoprotein-related receptor (LRP) is required for nuclear targeting. Rather, translocation involves the binding of scuPA to the nucleocytoplasmic shuttle protein nucleolin through a region containing the kringle domain. RNA interference and mutational analysis demonstrate that nucleolin is required for the nuclear transport of scuPA. Furthermore, nucleolin is required for the induction smooth muscle ␣-actin (␣-SMA) by scuPA. These data reveal a novel pathway by which uPA is rapidly translocated to the nucleus where it might participate in regulating gene expression. (Blood. 2008;112: 100-110) IntroductionUrokinase-type plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes, including cell proliferation and migration during angiogenesis, tissue regeneration, inflammatory responses, and tumor growth/metastases. These complex processes all involve intracellular signal transduction and regulation of gene transcription in addition to proteolysis (see Alfano et al 1 for review). uPA is secreted as a single-chain protein (scuPA) that consists of an N-terminal EGF-like domain (GFD), a kringle domain (KD), and a serine protease domain. Binding of uPA to its high-affinity receptor CD87 (uPAR) is mediated by the GFD. 2 Plasmin converts scuPA into a proteolytically active 2-chain enzyme (tcuPA) 3 that is rapidly inhibited primarily by plasminogen activator inhibitor-1 (PAI-1). tcuPA-PAI-1 complexes are internalized with the aid of lipoprotein receptor-related protein (LRP) 4 by clathrin-mediated endocytosis. The tcuPA-PAl-1 complexes traffic to lysosomes and are degraded, while unoccupied uPAR and LRP recycle back to the cell surface. 5 uPA-induced signal transduction occurs via uPAR-dependent and uPAR-independent pathways (reviewed in Alfano et al 1 ; Kjoller 6 ; Blasi and Carmeliet 7 ). Among the latter, we have shown that cleavage of scuPA by plasmin releases the GFD fragment, generating a form of uPA unable to bind to uPAR, 8 but that stimulates migration of smooth muscle cells (SMCs). 9 Signal transduction by this scuPA fragment may be mediated in part by LRP 10 and certain integrins. 11 However, there is limited information as to the mechanism by which uPA modifies gene transcription, [12][13][14][15] and our previous studies have provided reason to hypothesize that cells express additional uPA-binding proteins that possess distinct signal-transducing activities involved in cell contractility, migration, an...

Urokinase (uPA)-induced signaling in human vascular smooth muscle cells (VSMC) elicits important cellular functional responses, such as cell migration and proliferation. However, how intracellular signaling is linked to glycolipid-anchored uPA receptor (uPAR) is unknown. We provide evidence that uPAR activation by uPA induces its association with platelet-derived growth factor receptor (PDGFR)-beta. The interaction results in PDGF-independent PDGFR-beta activation by phosphorylation of cytoplasmic tyrosine kinase domains and receptor dimerization. Association of the receptors as well as the tyrosine kinase activity of PDGFR-beta are decisive in mediating uPA-induced downstream signaling that regulates VSMC migration and proliferation. These findings provide a molecular basis for mechanisms VSMC use to induce uPAR- and PDGFR-directed signaling. The processes may be relevant to VSMC function and vascular remodeling.

We conclude that in human vascular smooth muscle cells, uPA induces the formation and activation of a newly identified signaling complex comprising uPAR, nucleolin, and casein kinase 2, that is responsible for the uPA-related mitogenic response. The complex is not a unique feature of vascular smooth muscle cells, as it was also found in other uPAR-expressing cell types.

Janus kinases Jak1 and Tyk2 play an important role in urokinase-type plasminogen activator (uPA)-dependent signaling. We have recently demonstrated that both kinases are associated with the uPA receptor (uPAR) and mediate uPA-induced activation of signal transducers and activators of transcription (Stat1, Stat2, and Stat4) in human vascular smooth muscle cells (VSMC). Janus kinases are not only required for Stat activation but may also interfere with other intracellular signaling pathways. Here we report that in VSMC, Tyk2 interacts with a downstream signaling cascade involving phosphatidylinositol 3-kinase (PI3-K). We demonstrate that uPA induces PI3-K activation, which is abolished in VSMC expressing the dominant negative form of Tyk2. The regulatory subunit p85 of PI3-K co-immunoprecipitates with Tyk2 but not with Jak1, Jak2, or Jak3, and uPA stimulation increases the PI3-K activity in Tyk2 immunoprecipitates. Tyk2 directly binds to either of the two Src homology 2(SH2)p85 domains in a uPA-dependent fashion. We provide evidence that the Tyk2-mediated PI3-K activation in response to uPA is required for VSMC migration. Thus, two unrelated structurally distinct specific inhibitors of PI3-K, wortmannin and LY294002, prevent VSMC migration induced by uPA. No migratory effect of uPA was observed in VSMC expressing the dominant negative form of Tyk2. Our results underscore the versatile function of Tyk2 in uPA-related intracellular signaling and indicate that PI3-K plays a selective role in the regulation of VSMC migration.

We demonstrate by immunopreoipitation that u-PAR is associated with a 38 kDa protein that is phosphorylated on tyrosine after u-PA treatment of cells. As tyrosine phosphorylation is the hallmark of many signal transduction pathways that promote growth and differentiation, these data suggest that u-PA, besides its role as a regulatory protease, might act as a para-or autocrine hormone.

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