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

  • Angelika Kusch
    Affiliations
    Charité-Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Humboldt University Berlin, Wiltbergstraße 50, 13125 Berlin-Buch, Germany
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  • Sergey Tkachuk
    Affiliations
    Charité-Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Humboldt University Berlin, Wiltbergstraße 50, 13125 Berlin-Buch, Germany
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  • Hermann Haller
    Affiliations
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  • Rainer Dietz
    Affiliations
    Charité-Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Humboldt University Berlin, Wiltbergstraße 50, 13125 Berlin-Buch, Germany
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  • Dietrich C. Gulba
    Affiliations
    Charité-Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Humboldt University Berlin, Wiltbergstraße 50, 13125 Berlin-Buch, Germany
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  • Martin Lipp
    Affiliations
    Charité-Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Humboldt University Berlin, Wiltbergstraße 50, 13125 Berlin-Buch, Germany
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  • Inna Dumler
    Footnotes
    Affiliations
    Charité-Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Humboldt University Berlin, Wiltbergstraße 50, 13125 Berlin-Buch, Germany
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  • Author Footnotes
    * This work was supported by Deutsche Forschungsgemeinschaft Grant Du 344/1-1.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.
    § To whom all correspondence should be addressed: Franz-Volhard Clinic and Max-Delbrück Center for Molecular Medicine, Wiltbergstraße 50, 13125 Berlin-Buch, Germany. Tel.: 49-30-9417-2451; Fax: 49-30-9417-2453; E-mail: [email protected]
Open AccessPublished:December 15, 2000DOI:https://doi.org/10.1074/jbc.M003626200
      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.
      Jak
      Janus kinase
      Stat
      signal transducers and activators of transcription
      uPA
      urokinase-type plasminogen activator
      uPAR
      uPA receptor
      VSMC
      vascular smooth muscle cells
      PI3-K
      phosphatidylinositol 3-kinase
      SH2
      Src homology 2
      PCR
      polymerase chain reaction
      PBS
      phosphate-buffered saline
      PAGE
      polyacrylamide gel electrophoresis
      A major pathway for signal generation by cytokines, growth factors, and polypeptide hormones involves activation of the Janus tyrosine kinase family (Jak kinases)1 and tyrosine phosphorylation of signal transducers and activators of transcription (Stat) proteins (
      • Ziemiecki A.
      • Harpur A.G.
      • Wilks A.F.
      ,
      • Darnell Jr., J.E.
      • Kerr I.M.
      • Stark G.R.
      ). Jak activation results in tyrosine phosphorylation of several Stat proteins that form homo- and heterodimers and translocate to the nucleus to regulate gene transcription by binding to specific promoter sequences of stimulated genes (). Which members of the Jak and Stat families are activated varies greatly among different agonists and in different cell systems (
      • Schindler C.
      ,
      • Ward A.C.
      • Touw I.
      • Yoshimura A.
      ). We have recently demonstrated that the urokinase receptor (uPAR) utilizes the Jak/Stat pathway for intracellular transmission in human vascular smooth muscle cells (VSMC) and endothelial cells (
      • Dumler I.
      • Weis A.
      • Mayboroda O.A.
      • Maasch C.
      • Jerke U.
      • Haller H.
      • Gulba D.C.
      ,
      • Dumler I.
      • Kopmann A.
      • Weis A.
      • Mayboroda O.A.
      • Wagner K.
      • Gulba D.C.
      • Haller H.
      ). uPA/uPAR is a multifunctional system involved in wound healing, tissue remodeling, immune response, and cancer by affecting cell migration, adhesion, and proliferation (
      • Blasi F.
      ,
      • Chapman H.A.
      ). Some of these functions require uPAR-dependent signal transduction, which remains imperfectly defined. uPAR is associated with two Janus kinases, Jak1 and Tyk2, which become activated in response to uPA and subsequently induce formation and activation of Stat1, Stat2, and Stat4 complexes (
      • Dumler I.
      • Weis A.
      • Mayboroda O.A.
      • Maasch C.
      • Jerke U.
      • Haller H.
      • Gulba D.C.
      ,
      • Dumler I.
      • Kopmann A.
      • Weis A.
      • Mayboroda O.A.
      • Wagner K.
      • Gulba D.C.
      • Haller H.
      ,
      • Dumler I.
      • Kopmann A.
      • Wagner K.
      • Mayboroda O.A.
      • Jerke U.
      • Dietz R.
      • Haller H.
      • Gulba D.C.
      ). Both kinases co-localized with the uPAR to the leading edge of migrating VSMC (
      • Dumler I.
      • Weis A.
      • Mayboroda O.A.
      • Maasch C.
      • Jerke U.
      • Haller H.
      • Gulba D.C.
      ), implying a possible contribution of the Jaks to cell migration, most likely via the interactions with other kinases or signaling molecules. Several reports have shown that Jaks and Stats interfere with multiple signaling cascades, such as Ras/mitogen-activated protein kinase pathway, phosphatidylinositol 3-kinase (PI3-K), Pyk2, and Src kinases. These cascades couple Jak/Stat to pathways with different downstream signaling functions (
      • Ward A.C.
      • Touw I.
      • Yoshimura A.
      ,
      • Chatterjee-Kishore M.
      • Van den Aker F.
      • Stark G.R.
      ).
      We reasoned that PI3-K is a candidate for mediating uPA-induced VSMC migration. PI3-K phosphorylates the D3 position of phosphatidylinositol, and the phosphorylated lipid products of this enzymatic reaction may act on multiple downstream effectors (
      • Kapeller R.
      • Cantley L.C.
      ,
      • Corvera S.
      • Czech M.P.
      ). PI3-K is composed of two subunits, a regulatory p85 subunit and a 110-kDa catalytic subunit. The regulatory p85 subunit contains two Src homology 2 (SH2) domains, which bind to specific phosphotyrosine-containing motifs and have been implicated in mediating protein-protein interactions (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). The ability of p85 SH2 domains to associate with other proteins links PI3-K to distinct signaling cascades required for control of cell growth and proliferation, adhesion and motility, differentiation, and survival (
      • Vanhaesebroeck B.
      • Waterfield M.D.
      ). Moreover, recent reports underscore the importance of PI3-K in cell migration (
      • Vanhaesebroeck B.
      • Jones G.E.
      • Allen W.E.
      • Zicha D.
      • Hooshmand-Rad R.
      • Sawyer C.
      • Wells C.
      • Waterfield M.D.
      • Ridley A.J.
      ,
      • Reiske H.R.
      • Kao S.C.
      • Cary L.A.
      • Guan J.L.
      • Lai J.F.
      • Chen H.C.
      ,
      • Adam L.
      • Vadlamudi R.
      • Kondapaka S.B.
      • Chernoff J.
      • Mendelson J.
      • Kumar R.
      ). In this study, we investigated the extent to which PI3-K and Janus kinases are involved in the uPA-induced cell migration. Our findings demonstrate that in human VSMC, Tyk2, besides activation of Stat proteins in response to uPA, is the main uPA-dependent pathway of PI3-K activation. Tyk2 interacts with PI3-K via the SH2 domains of p85 catalytic subunit that, in turn, leads to p85 tyrosine phosphorylation and to PI3-K activation. The association of both kinases is critical to provide VSMC cytoskeletal reorganization in response to uPA required for cell migration.

      DISCUSSION

      We provide evidence implying novel roles for the Janus kinase Tyk2 and PI3-K in human coronary VSMC. We demonstrate that in addition to known regulation of cell transcription via Stat proteins, Tyk2 associates with PI3-K through the Src homology 2 domains of p85 subunit, thereby providing a crucial link between two signaling pathways. Probably the most important finding is that the revealed Tyk2 and PI3-K association within the cell and their activation are urokinase-responsive and are required for the uPA-related VSMC migration. This observation is the first demonstration that PI3-K is essential for at least some of the uPA/uPAR functions in VSMC and that Tyk2 is the main uPA-dependent pathway of PI3-K activation.
      VSMC proliferation and migration into the intima after vascular injury, as well as their formation of neointima, contribute to vessel narrowing and are pivotal to the atherosclerotic process (
      • Ross R.
      ). uPA and uPAR are active participants in these processes by regulating wound healing, tissue remodeling, and immune responses (
      • Blasi F.
      ). In addition to the effects mediated by proteolysis, uPA and uPAR also display biological functions that are not directly attributable to the formation of plasmin but rather to the induction of cell migration and proliferation control (
      • Dear A.E.
      • Medcalf R.L.
      ). The link between uPA/uPAR and cell motility was established over a decade ago (
      • Gudewicz P.W.
      • Gilboa N.
      ,
      • Fibbi G.
      • Ziche M.
      • Morbidelli L.
      • Magnelli L.
      • Del Rosso M.
      ,
      • Del Rosso M.
      • Anichini E.
      • Pedersen N.
      • Blasi F.
      • Fibbi G.
      • Pucci M.
      • Ruggiero M.
      ). Current data confirm the uPA-dependent cell migration in a wide variety of cell types (
      • Odekon L.
      • Sato Y.
      • Rifkin D.B.
      ,
      • Busso N.
      • Masur S.K.
      • Lazega D.
      • Waxman S.
      • Ossowski L.
      ,
      • Fazioli F.
      • Resnati M.
      • Sidenius N.
      • Higashimoto Y.
      • Appella E.
      • Blasi F.
      ,
      • Nguyen D.H.
      • Hussaini I.M.
      • Gonias S.L.
      ,
      • Nguyen D.H.
      • Catling A.D.
      • Webb D.J.
      • Sankovic M.
      • Walker L.A.
      • Somlyo A.V.
      • Weber M.J.
      • Gonias S.L.
      ,
      • Chiaradonna F.
      • Fontana L.
      • Iavarone C.
      • Carriero M.V.
      • Scholz G.
      • Barone M.V.
      • Stopelli M.P.
      ,
      • Webb D.
      • Nguen H.D.
      • Gonias S.L.
      ). Nevertheless, the proposed molecular mechanisms have been conflicting. The uPA/uPAR-related migratory responses seem to be highly cell specific, implying some structural specificity and diversity of underlying signaling events. Thus, in human breast cancer cells MCF-7 and HT 1080 fibrosacroma cells, uPA-initiated cellular motility required activation of signaling cascade including Ras, mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, and myosin light chain kinase as downstream effectors (
      • Nguyen D.H.
      • Hussaini I.M.
      • Gonias S.L.
      ,
      • Nguyen D.H.
      • Catling A.D.
      • Webb D.J.
      • Sankovic M.
      • Walker L.A.
      • Somlyo A.V.
      • Weber M.J.
      • Gonias S.L.
      ). On the contrary, migration of human epithelial cells seemed to involve the uPA-activated protein kinase C (
      • Busso N.
      • Masur S.K.
      • Lazega D.
      • Waxman S.
      • Ossowski L.
      ), whereas in cells of monocytic lineage these effects were attributed to the activation of protein tyrosine kinases of the Src family (
      • Fazioli F.
      • Resnati M.
      • Sidenius N.
      • Higashimoto Y.
      • Appella E.
      • Blasi F.
      ,
      • Resnati M.
      • Guttinger M.
      • Valcamonica S.
      • Sidenius N.
      • Blasi F.
      • Fazioli F.
      ). In addition, uPA/uPAR-related cell migration is integrin-dependent (
      • Chapman H.A.
      ,
      • Yebra M.
      • Goretzki L.
      • Pfeifer M.
      • Mueller B.M.
      ,
      • Carriero M.
      • Del Vecchio S.
      • Capozzoli M.
      • Franco P.
      • Fontana L.
      • Zannetti A.
      • Botti G.
      • D'Aluto G.
      • Salvatore M.
      • Stoppelli M.P.
      ).
      The molecular machinery of uPA/uPAR-related VSMC migration remains sparsely explored, although the recent generation of transgenic mice deficient in uPA and uPAR demonstrated that VSMC migration is dependent on the fibrinolytic system and is decisive for the severity of vascular damage (
      • Carmeliet P.
      • Moons L.
      • Herbert J.M.
      • Crawley J.
      • Lupu F.
      • Lijnen R.
      • Collen D.
      ,
      • Carmeliet P.
      • Collen D.
      ). Separate recent findings confirm migration of rat VSMC initiated by uPA/uPAR binding and suggest involvement of Src tyrosine kinase and G protein in this process (
      • Degryse B.
      • Resnati M.
      • Rabbani S.A.
      • Villa A.
      • Fazioli F.
      • Blasi F.
      ). We suggested a role for the Janus kinases in VSMC migration and showed that Jak1 and Tyk2 were co-localized with the uPAR to the leading edge of migrating VSMC (
      • Dumler I.
      • Weis A.
      • Mayboroda O.A.
      • Maasch C.
      • Jerke U.
      • Haller H.
      • Gulba D.C.
      ). However, to generate migratory responses, Jaks were expected to signal independently of Stat activation most likely through a link to an additional unknown pathway.
      PI3-K is central to cell migration processes regulated by cytokines and growth factors in diverse cell types including VSMC (
      • Imai Y.
      • Clemmons DR.
      ). Moreover, PI3-K possesses a high capacity to cooperate with other signaling pathways to mediate a required functional response (
      • Vanhaesebroeck B.
      • Waterfield M.D.
      ). Interestingly, several recent reports provide evidence for the interference of PI3-K and Jak/Stat signaling cascade. Thus, the PI3-K p85 regulatory subunit was shown to bind directly to Stat3 protein, which served as an adapter to couple the PI3-K signaling pathway to the interferon receptor in Daudi cells (
      • Pfeffer L.
      • Mullersman J.E.
      • Pfeffer S.R.
      • Murti A.
      • Shi W.
      • Yang C.H.
      ). A similar interaction was demonstrated for the p85 subunit and Stat5 protein in a bone marrow-derived Ba/F3 cell line, where both pathways cooperated to mediate interleukin-3-dependent suppression of apoptosis (
      • Santos S.
      • Dumon S.
      • Mayeux P.
      • Gisselbrecht S.
      • Gouilleux F.
      ). Several other reports provided further evidence for coordinated activation of PI3-K and Stat proteins leading to the functional cooperation of both signaling cascades (
      • Chen R.
      • Chang M.C.
      • Su Y.H.
      • Tsai Y.T.
      • Kuo M.L.
      ,
      • Bao H.
      • Jacobs-Helber S.M.
      • Lawson A.E.
      • Penta K.
      • Wickrema A.
      • Sawyer S.T.
      ). Moreover, recent studies demonstrated the ability of Jaks to associate with and to regulate the p85 subunit of PI3-K, as was shown for Jak3 in human T cells (
      • Sharfe N.
      • Dadi H.K.
      • Roifman C.M.
      ), Jak1 in cardiac myocytes (
      • Oh H.
      • Fujio Y.
      • Kunisada K.
      • Hirota H.
      • Matsu H.
      • Kishimoto T.
      • Yamauchi-Takihara K.
      ), and Jak2 in human neutrophils (
      • Al-Shami A.
      • Naccache P.H.
      ).
      Consistent with these reports, we find that Janus kinase Tyk2, but not Jak1, Jak2, or Jak3, is specifically associated with PI3-K in human VSMC. Moreover, this association is uPA-dependent. The fact that Jak1 was not co-immunoprecipitated with PI3-K is of interest because in our previous studies we did not observe any difference between Tyk2 and Jak1 in terms of uPA stimulation, uPAR association, and polarization to the leading edge of migrating cells (
      • Dumler I.
      • Weis A.
      • Mayboroda O.A.
      • Maasch C.
      • Jerke U.
      • Haller H.
      • Gulba D.C.
      ,
      • Dumler I.
      • Kopmann A.
      • Weis A.
      • Mayboroda O.A.
      • Wagner K.
      • Gulba D.C.
      • Haller H.
      ). The inability to reveal Jak1-PI3-K complexes suggests that this kinase is most likely involved exclusively in the transcriptional regulation via Stat proteins or might perform an additional function in the uPA/uPAR-related signaling. Although we found no association between Jak1 and PI3-K, their cooperation cannot be completely excluded. Nevertheless, two kinases performing the same task would be redundant.
      To address the molecular basis of Tyk2-PI3-K interaction, we performed an adenovirus-mediated Tyk2 overexpression in VSMC. We performed Tyk2 pull-down assays using GST-p85 fusion proteins composed of either the C- or N-terminal SH2 domains of p85. Tyk2 bound to both N- and C-terminal SH2 domains of the p85 regulatory PI3-K subunit. This result is consistent with the findings of others demonstrating that these domains mediate specific protein-protein interactions (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). Tyk2 has a candidate motif, YXXM, which is a potential site for p85 binding. However, determination of whether or not SH2 p85 domains are responsible for the interaction with Tyk2 in the context of above motif requires further verification. Our data are the first to demonstrate that following uPA stimulation, Tyk2 and PI3-K form a specific protein complex in VSMC. Although Tyk2 constitutively associates with p85, uPA induces a strong activation of this interaction. Moreover, uPA induces PI3-K activation within the complex, which we believe is mediated by tyrosine phosphorylation of the PI3-K p85 subunit. This hypothesis, which we are currently pursuing, is supported by the fact that growth factor-promoted p85 phosphorylation increases PI3-K activity (
      • Thakker G.
      • Hajjar D.P.
      • Muller W.A.
      • Rosengart T.K.
      ). However, the increase in PI3-K activity we measured in Tyk2 precipitates in response to uPA was significantly less than the uPA-induced PI3-K activation in whole cell lysates. An intriguing question is raised by these observations, which imply additional still unknown uPA/uPAR-dependent PI3-K functions.
      Our results highlight the importance of PI3-K in the regulation of cell migration in response to uPA. We demonstrate the involvement of PI3-K in uPA-induced signaling leading to cell migration in two in vitro models, the Boyden chamber and the wounding assay. Both techniques allowed us to analyze different parameters of cell migration. Specific PI3-K antagonists, LY294002 and wortmannin, completely abrogated the uPA-responsive increase in cell motility. The observed ability of uPA to initiate sustained PI3-K activation might facilitate the long term migration process.
      One of the issues raised by these observations is whether PI3-K lies upstream or downstream of Tyk2 in the reavealed pathway. Our demonstration that in VSMC expressing the Tyk2 dominant negative form PI3-K activation in response to uPA is completely blocked implicates that PI3-K activation acts broadly downstream of Tyk2 in the signal transduction cascade. Moreover, these data support the concept that in VSMC Tyk2 is central to the PI3-K regulation. This positioning is further supported by our finding that a dominant negative Tyk2 blocks uPA-related cell migration. Upon stimulation, Tyk2, as certain other players in signaling machineries, relocates to the leading edge of the cell membrane that might enhance reaction-limited signal transduction. The results of our study on VSMC morphology reported here imply that polarization of Tyk2 might contribute to the lamellipodial activity required for a spatial asymmetry of migrating cells. However, it appears that the loss of PI3-K activity has similar consequences on lamellipodial activity and migration process as blockage of Tyk2 by its mutant. Overall, these data favor the view that a functional relationship between two kinases is decisive for VSMC cytoskeletal reorganization and migration. Our data are consistent with other reports demonstrating the central role of PI3-K in initiating actin cytoskeletal rearrangements, cell polarization, and cell migration in several cell systems (
      • Vicente-Manzanares M.
      • Rey M.
      • Jones D.R.
      • Sancho D.
      • Mellado M.
      • Rodriguez-Frade J.M.
      • Del Pozo M.A.
      • Yanez-Mo M.
      • De Ana A.M.
      • Martinez-A C.
      • Merida I.
      • Sanchez-Madrid F.
      ,
      • Hooshmand-Rad R.
      • Claesson-Welsh L.
      • Wennström S.
      • Yokote K.
      • Siegbahn A.
      • Heldin C.H.
      ). During these processes, PI3-K is generally translocated from the cytosol to the cytoskeleton-associated fraction presumably via a link to additional proteins (
      • Meng F.
      • Lowell C.A.
      ). It is likely that Tyk2-PI3-K association via the SH2 domains of the p85 subunit is responsible for bringing PI3-K to the cytoskeleton-associated subcellular fraction, since loss of Tyk2 polarization in Ad5Tyk2KE-VSMC correlates with the loss of PI3-K activation and of leading edge formation. However, it is also possible that other adaptor molecules may facilitate translocation of PI3-K. In rat VSMC, Src kinase was presumed to cooperate functionally with uPAR and integrins at the leading edge upon the migration process, since uPA-caused a c-Src redistribution from the cytoplasm to plasma membrane (
      • Degryse B.
      • Resnati M.
      • Rabbani S.A.
      • Villa A.
      • Fazioli F.
      • Blasi F.
      ). These data, coupled with the recent demonstration that the integrin-dependent cell migration requires both Src family kinases and PI3-K (
      • Meng F.
      • Lowell C.A.
      ), as well as with our findings, suggest the existence of a complex functional unit formed at the leading cell membrane in response to uPA. The localized regulation of signaling molecules, such as Src, Tyk2, and PI3-K, by uPA/uPAR and integrins may provide an efficient mechanism for targeting downstream functional effects of these kinases as well as for the balance between these pathways.

      ACKNOWLEDGEMENTS

      We are grateful to Jana Treutler and Steffen Lutter for excellent technical assistance, Cornelia Kirsch for help in the establishment of the PI3-K assay, Karsten Brand for expertise in adenoviral gene transfer, and Prof. Friedrich C. Luft for critical reading of the manuscript.

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