Inna Dumler

The Jak/Stat pathway and urokinase receptor signaling in human aortic vascular smooth muscle cells.

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 p55 fgr . 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.

Urokinase-induced mitogenesis is mediated by casein kinase 2 and nucleolin

BACKGROUND Urokinase (uPA) and the urokinase receptor (uPAR) form a multifunctional system capable of concurrently regulating pericellular proteolysis, cell-surface adhesion, and mitogenesis. The role of uPA and uPAR in directed proteolysis is well established and its function in cellular adhesiveness has recently been clarified by numerous studies. The molecular mechanisms underlying the mitogenic effects of uPA and uPAR are still unclear, however. RESULTS We identified mechanisms that might participate in uPA-related mitogenesis in human vascular smooth muscle cells and demonstrated that uPA induces activation of a unique signaling complex. This complex contains uPAR and two additional proteins, nucleolin and casein kinase 2, which are implicated in cell proliferation. Both proteins were isolated by affinity chromatography on uPA-conjugated cyanogen-bromide-activated Sepharose 4B and were identified using nano-electrospray mass spectrometry and immunoblotting. We used laser scanning and immunoelectron microscopy studies to further demonstrate that nucleolin and casein kinase 2 are located on the cell surface where they colocalize with the uPAR. Moreover, the proteins were co-internalized into the cell as an entire complex. Immunoprecipitation experiments in combination with an in vitro kinase assay demonstrated a specific association of uPAR with nucleolin and casein kinase 2 and revealed a uPA-induced activation of casein kinase 2, which presumably led to phosphorylation of nucleolin. Blockade of nucleolin and casein kinase 2 with specific modulators led to the inhibition of uPA-induced cell proliferation. CONCLUSIONS 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.

Vascular Smooth Muscle Cells Initiate Proliferation of Mesenchymal Stem Cells by Mitochondrial Transfer via Tunneling Nanotubes

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.

Urokinase Stimulates Human Vascular Smooth Muscle Cell Migration via a Phosphatidylinositol 3-Kinase-Tyk2 Interaction

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.

Urokinase‐induced signaling in human vascular smooth muscle cells is mediated by PDGFR‐β

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)‐β. The interaction results in PDGF‐independent PDGFR‐β activation by phosphorylation of cytoplasmic tyrosine kinase domains and receptor dimerization. Association of the receptors as well as the tyrosine kinase activity of PDGFR‐β 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.

Urokinase Activates the Jak/Stat Signal Transduction Pathway in Human Vascular Endothelial Cells

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-gamma 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.

Nuclear translocation of urokinase-type plasminogen activator.

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 alpha-actin (alpha-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.

Urokinase Induces Activation and Formation of Stat4 and Stat1-Stat2 Complexes in Human Vascular Smooth Muscle Cells

Urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) act in concert to stimulate cytoplasmic signaling machinery and transcription factors responsible for cell migration and proliferation. Recently we demonstrated that uPA activates the Janus kinase/signal transducers and activators of transcription (Stat1) signaling in human vascular smooth muscle and endothelial cells. However, the important question whether other transcription factors of the Stat family, in addition to Stat1, are involved in the uPAR-related signaling has not been addressed. In this study, we demonstrate that Stat4 and Stat2, but not Stat3, Stat5, or Stat6, are rapidly activated in response to uPA. We demonstrate further that Stat4 and Stat2 rapidly and transiently translocate to the cell nucleus where they bind specifically to the regulatory DNA elements. Analysis of Stat complexes formed in response to uPA revealed a Stat2-Stat1 heterodimer, which lacks p48, a DNA-binding protein known to combine with Stat1-Stat2. This new uPA-induced Stat2-Stat1 heterodimer binds to GAS (the interferon-γ activation site) distinct from the interferon-stimulated response element to which the p48 protein containing complexes generally bind. We conclude that uPA activates a specific and unusual subset of latent cytoplasmic transcription factors in human vascular smooth muscle cells that suggests a critical role of uPA in these cells.

Urokinase-induced activation of the gp130/Tyk2/Stat3 pathway mediates a pro-inflammatory effect in human mesangial cells via expression of the anaphylatoxin C5a receptor

Glomerular mesangial cells (MCs) are central to the pathogenesis of progressive glomeruli-associated renal diseases. However, molecular mechanisms underlying changes in MC functions still remain poorly understood. Here, we show that in MCs, the urokinase-type plasminogen activator (uPA) induces, via its specific receptor (uPAR, CD87), upregulated expression of the complement anaphylatoxin C5a receptor (C5aR, CD88), and modulates C5a-dependent functional responses. This effect is mediated via the interaction of the uPA-specific receptor (uPAR, CD87) and gp130, a signal transducing subunit of the receptor complexes for the IL-6 cytokine family. The Janus kinase Tyk2 and the transcription factor Stat3 serve as downstream components in the signaling cascade resulting in upregulation of C5aR expression. In vivo, expression of C5aR and uPAR was increased in the mesangium of wild-type mice in a lipopolysaccharide (LPS)-induced model of inflammation, whereas in uPAR–/– animals C5aR expression remained unchanged. This is the first demonstration in vitro and in vivo that uPA acts in MCs as a modulator of immune responses via control of immune-competent receptors. The data suggest a novel role for uPA/uPAR in glomeruli-associated renal failure via a signaling cross-talk between the fibrinolytic and immune systems.

oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

The pathogenesis of atherosclerosis involves an imbalanced lipid metabolism and a deregulated immune response culminating in chronic inflammation of the arterial wall. Recent studies show that endogenous ligands, such as modified plasma lipoproteins, can trigger pattern recognition receptors (PRR) of innate immunity for cellular and humoral reactions. The underlying molecular pathways remain less explored. In this study, we investigated the mechanisms of inflammatory effects of oxidized low-density lipoproteins (oxLDL) on human primary coronary artery smooth muscle cells (VSMC). We show that already low concentration of oxLDL initiated atherogenic signals triggering VSMC transition to proinflammatory phenotype. oxLDL impaired the expression of contractile proteins and myocardin in VSMC and initiated changes in cell functional responses, including expression of proinflammatory molecules. The effects of oxLDL were abolished by downregulation of the multifunctional urokinase receptor (uPAR). In response to oxLDL uPAR associated with CD36 and TLR4, the two main PRR for both pathogen and endogenous ligands. We demonstrate that uPAR association with CD36 and TLR4 mediated oxLDL-induced and NF-κB-dependent G-CSF and GM-CSF expression and changes in VSMC contractile proteins. uPAR-mediated release of G-CSF and GM-CSF by VSMC affected macrophage behavior and production of MCP-1. We provide evidence for functional relevance of our in vitro findings to in vivo human atherosclerotic tissues. Our data imply uPAR as a part of a PRR cluster interfering structurally and functionally with CD36 and TLR4 and responding to endogenous atherogenic ligands. They further point to specific function of each component of this cluster in mediating the ultimate signaling pattern.