Download PDF
Thromb Haemost 2003; 89(05): 904-914
DOI: 10.1055/s-0037-1613478
Cellular Proteolysis and Oncology
Schattauer GmbH

Urokinase-induced migration of human vascular smooth muscle cells requires coupling of the small GTPases RhoA and Rac1 to the Tyk2/PI3-K signalling pathway

Ioulia Kiian
1   Charité - Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Humboldt University Berlin, Germany
2   Hanover Medical School, Hanover, Germany
,
Natalia Tkachuk
1   Charité - Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Humboldt University Berlin, Germany
2   Hanover Medical School, Hanover, Germany
,
Hermann Haller
2   Hanover Medical School, Hanover, Germany
,
Inna Dumler
1   Charité - Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Humboldt University Berlin, Germany
2   Hanover Medical School, Hanover, Germany
› Author Affiliations
Further Information

Publication History

Received 30 October 2002

Accepted after revision 15 February 2003

Publication Date:
09 December 2017 (online)

  PDF Download  Permissions and Reprints

Summary

Urokinase-type plasminogen activator (uPA) facilitates cell migration by localizing proteolisys on the cell surface and by inducing intracellular signalling pathways. In human vascular smooth muscle cell (VSMC) uPA stimulates migration via the uPA receptor (uPAR) signalling complex containing the Janus kinase Tyk2 and phosphatidylinositol 3-kinase (PI3-K). We report that active GTP-bound forms of small GTPases RhoA and Rac1, but not Cdc42, are directly associated with Tyk2 and PI3-K in an uPA/uPAR-dependent fashion. Endogenous RhoA, but not Rac1 or Cdc42, was significantly activated in response to uPA. RhoA activation was abolished by cell treatment with two unrelated, structurally distinct, specific inhibitors of PI3-K, wortmannin, and LY294002. Downstream of RhoA, phosphorylation of myosin light chain (MLC) was dramatically upregulated by uPA in a Rho kinase- and PI3-K-dependent manner. Thus, selective Rho kinase inhibitor Y27632 and PI3-K inhibitors wortmannin and LY294002 prevented the uPA-induced stimulation of MLC phosphorylation. Rho kinase inhibition also decreased uPA-stimulated VSMC migration as observed in a Boyden chamber. VSMC immunocytochemical staining demonstrated redistribution of RhoA and Rac1 active forms to the newly formed leading edge of migrating cell. VSMC microinjection with antibodies to either Rho or Rac1 decreased uPA-stimulated cell migration, indicating the involvement of both GTPases in the migration process. Our results provide evidence that the small GTPases RhoA and Rac1, together with Rho kinase, are necessary to mediate the uPA/uPAR-directed migration via the Tyk2/PI3-K signalling complex in human VSMC.

Keywords

Urokinase - urokinase receptor - vascular smooth muscle cells - migration - Rho proteins
 

  • References

  • 1 Blasi F.. The urokinase receptor. A cell surface, regulated chemokine. Acta Pathol Microbiol Scand 1999; 107: 96-101.
    CrossrefPubMedGoogle Scholar
  • 2 Wang Y.. The role and regulation of urokinase-type plasminogen activator receptor gene expression in cancer invasion and metastasis. Med Res Rev 2001; 21: 146-70.
    CrossrefPubMedGoogle Scholar
  • 3 Parfenova YV, Plekhanova OS, Tkachuk VA.. Plasminogen activators in vascular remodeling and angiogenesis. Biochemistry 2002; 67: 119-34.
    PubMedGoogle Scholar
  • 4 Ross R.. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362: 801-9.
    CrossrefPubMedGoogle Scholar
  • 5 Casscells W.. Migration of smooth muscle and endothelial cells. Critical events in restenosis. Circulation 1992; 86: 723-9.
    CrossrefPubMedGoogle Scholar
  • 6 Carmeliet P, Moons L, Herbert JM, Crawley J, Lupu F, Lijnen R, Collen D.. Urokinase but not tissue plasminogen activator mediates arterial neointima formation in mice. Circ Res 1997; 81: 829-39.
    CrossrefPubMedGoogle Scholar
  • 7 Noda-Heiny H, Sobel BE.. Vascular smooth muscle cell migration mediated by thrombin and urokinase receptor. Am J Physiol 1995; 268: C1195-201.
    CrossrefPubMedGoogle Scholar
  • 8 Zhu Y, Bujo H, Yamazaki H, Hirayama S, Kanaki T, Takahashi K, Shibasaki M, Schneider WJ, Yasushi S.. Enhanced expression of the LDL receptor family member LR11 increases migration of smooth muscle cells in vitro. Circulation 2002; 105: 1830-6.
    CrossrefPubMedGoogle Scholar
  • 9 Kush A, Tkachuk S, Lutter S, Haller H, Dietz R, Lipp M, Dumler I.. Monocyte-expressed urokinase regulates human vascular smooth muscle cell migration in a coculture model. Biol Chem 2002; 383: 217-21.
    PubMedGoogle Scholar
  • 10 Blasi F, Carmeliet P.. uPAR: a verastile signalling orchestrator. Nat Rev Mol Cell Biol 2002; 3: 932-43.
    PubMedGoogle Scholar
  • 11 Nguen DH, Webb DJ, Catling AD, Song Q, Dhakephalkar A, Weber MJ, Ravichandran KS, Gonias SL.. Urokinase-type plasminogen activator stimulates the Ras/Extracellular signal-regulated kinase (ERK) signalling pathway and MCF-7 cell migration by a mechanism that requires focal adhesion kinase, Src, and Shc. Rapid dissociation of GRB2/Sps-Shc complex is associated with the transient phosphorylation of ERK in urokinase-treated cells. J Biol Chem 2000; 275: 19382-8.
    CrossrefPubMedGoogle Scholar
  • 12 Konakova M, Hucho F, Schleuning W-D.. Downstream targets of urokinase-type plasminogen-activator-mediated signal transduction. Eur J Biochem 1998; 253: 421-9.
    CrossrefPubMedGoogle Scholar
  • 13 Jo M, Thomas KS, Somlyo AV, Gonias SL.. Cooperativity between the Ras-ERK and RhoRho kinase pathways in urokinase-type plasminogen activator-stimulated cell migration. J Biol Chem 2002; 277: 12479-85.
    CrossrefPubMedGoogle Scholar
  • 14 Kjøller L, Hall AJ.. Rac mediates cytoskeletal rearrangements and increased cell motility induced by urokinase-type plasminogen activator receptor binding to vitronectin. J Cell Biol 2001; 152: 1145-57.
    CrossrefPubMedGoogle Scholar
  • 15 Ma Z, Thomas KS, Webb DJ, Moravec R, Salicioni AM, Mars WM, Gonias SL.. Regulation of Rac1 activation by the low density lipoprotein receptor-related protein. J Cell Biol 2002; 159: 1061-70.
    CrossrefPubMedGoogle Scholar
  • 16 Dumler I, Weis A, Mayboroda OA., Maasch C, Jerke U, Haller H, Gulba DC.. The Jak/Stat pathway and urokinase receptor signalling in human aortic vascular smooth muscle cells. J Biol Chem 1998; 273: 315-21.
    CrossrefPubMedGoogle Scholar
  • 17 Kush A, Tkachuk S, Haller H, Dietz R, Gulba DC, Lipp M, Dumler I.. Urokinase stimulates human vascular smooth muscle cell migration via a phosphatidylinositol 3-kinase-Tyk1 interaction. J Biol Chem 2000; 275: 39466-73.
    CrossrefPubMedGoogle Scholar
  • 18 Ridley AJ.. Rho GTPases and cell migration. J Cell Sci 2001; 114: 2713-22.
    PubMedGoogle Scholar
  • 19 Stoppelli MP, Corti A, Soffientini A, Cassani G, Blasi F, Assoian RK.. Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc Natl Acad Sci U S A 1985; 82: 4939-43.
    CrossrefPubMedGoogle Scholar
  • 20 Ren X-D, Schwartz MA.. Determination of GTP loading on Rho. Methods Enzymol 2000; 325: 264-72.
    PubMedGoogle Scholar
  • 21 Benard V, Bohl BP, Bokoch GM.. Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem 1999; 274: 13198-204.
    CrossrefPubMedGoogle Scholar
  • 22 Miyazaki K, Yano T, Schmidt DJ, Tokui T, Shibata M, Lifshitz LM, Kimura S, Tuft R, Ikebe M.. Rho-dependent agonist-induced spatio-temporal change in myosin phosphorylation in smooth muscle cells. J Biol Chem 2002; 277: 725-34.
    CrossrefPubMedGoogle Scholar
  • 23 Degryse B, Resnati M, Rabbani SA, Villa A, Fazioli F, Blasi F.. Src-dependence and pertussis-toxin sensitivity of urokinase receptor-dependent chemotaxis and cytoskeleton reorganization in rat smooth muscle cells. Blood 1999; 94: 649-62.
    PubMedGoogle Scholar
  • 24 Haller H, Lindschau C, Quass P, Distler A, Luft FC.. Differentiation of vascular smooth muscle cells and the regulation of protein kinase C-alpha. Circ Res 1995; 76: 21-9.
    CrossrefPubMedGoogle Scholar
  • 25 Huang S, Chen L-Y, Zuraw BL, Ye RD, Pan ZK.. Chemoattarctant-stimulated NF-κB activation is dependent on the low molecular weight GTPase RhoA. J Biol Chem 2001; 276: 40977-81.
    CrossrefPubMedGoogle Scholar
  • 26 Seasholtz TM, Zhang T, Morissette MR, Howes AL, Yang AH, Brown JH.. Increased expression and activity of RhoA are associated with increased DNA synthesis and reduced p27Kip1 expression in the vasculature of hyper-tensive rats. Circ Res 2001; 89: 488-95.
    CrossrefPubMedGoogle Scholar
  • 27 Flehming in Elliot CM, Exton JH.. Differential translocation of Rho family GTPases by lysophosphatydic acid, endothelin-1, and platelet-derived growth factor.. Biol Chem 1996; 271: 33067-77.
    CrossrefPubMedGoogle Scholar
  • 28 Kaibuchi K, Kuroda S, Amano M.. Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annu Rev Biochem 1999; 68: 459-86.
    CrossrefPubMedGoogle Scholar
  • 29 Amano M, Fukata Y, Kaibuchi K.. Regulation and functions of Rho-associated kinase. Exp Cell Res 2000; 261: 44-51.
    CrossrefPubMedGoogle Scholar
  • 30 Cho SY, Klemke RL.. Purification of pseudo-podia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold. J Cell Biol 2002; 156: 725-36.
    CrossrefPubMedGoogle Scholar
  • 31 Goncharova EA, Vorotnikov AV, Gracheva EO, Albert WC, Panettieri Jr RA, Stepanova VV, Tkachuk VA.. Activation of p38 MAPkinase and caldesmon phosphorylation are essential for urokinase-induced human smooth muscle cell migration. Biol Chem 2002; 383: 115-26.
    PubMedGoogle Scholar
  • 32 Kjoller L.. The urokinase plasminogen activator receptor in the regulation of the actin cyto-skeleton and cell motility. Biol Chem 2002; 383: 5-19.
    PubMedGoogle Scholar
  • 33 Fazioli F, Resnati M, Sidenius N, Higashimoto Y, Appella E, Blasi F.. A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity. EMBO J 1997; 16: 7279-86.
    CrossrefPubMedGoogle Scholar
  • 34 Resnati M, Guttinger M, Valcamonica S, Sidenius N, Blasi F, Fazioli F.. Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect. EMBO J 1996; 15: 1572-82.
    PubMedGoogle Scholar
  • 35 Busso N, Masur SK, Lazega D, Waxman S, Ossowski L.. Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells. J Cell Biol 1994; 126: 2259-70.
    PubMedGoogle Scholar
  • 36 Resnati M, Pallavicini I, Wang jm, Oppenheim J, Serhan CN, Romano M, Balsi F.. The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/LXA4R. Proc Natl Acad Sci U S A 2002; 99: 1359-64.
    CrossrefPubMedGoogle Scholar
  • 37 Jo MJ, Thomas KS, O’Donnell DM, Gonias SL.. Epidermal growth factor receptor-dependent and –independent cell-signalling pathways originating from the urokinase receptor. J Biol Chem 2003; 278: 1642-6.
    CrossrefPubMedGoogle Scholar
  • 38 Seasholtz TM, Majumdar M, Kaplan DD, Brown J. H.. Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration. Circ Res 1999; 84: 1186-93.
    CrossrefPubMedGoogle Scholar
  • 39 Ai S, Kuzuya M, Koike T, Asai T, Kanda S, Maeda K, Shibata T, Iguchi A.. Rho-Rho kinase is involved in smooth muscle cell migration through myosin light chain phosphorylation-dependent and independent pathways. Atherosclerosis 2001; 155: 321-7.
    CrossrefPubMedGoogle Scholar
  • 40 Chaulet H, Desgranges C, Renault MA, Dupuch F, Ezan G, Peiretti F, Loirand G, Pcaud P, Gadeau AP.. Extracellular nucleotides induce arterial smooth muscle cell migration via osteopontin. Circ Res 2001; 89: 772-8.
    CrossrefPubMedGoogle Scholar
  • 41 Shibata R, Kai H, Seki Y, Kato S, Morimatsu M, Kaibuchi K, Imaizumi T.. Role of Rho-associated kinase in neointima formation after vascular injury. Circulation 2001; 103: 284-9.
    CrossrefPubMedGoogle Scholar
  • 42 Sawada N, Itoh H, Ueyama K, Yamashita J, Doi K, Chun TH, Inoue M, Masatsugu K, Saito T, Fukunaga Y, Sakaguchi S, Arai H, Ohno N, Komeda M, Nakao K.. Inhibition of rho-associated kinase results in suppression of neointimal formation of balloon-injured arteries. Circulation 2000; 101: 2030-3.
    CrossrefPubMedGoogle Scholar
  • 43 Eto Y, Shimokawa H, Morishige K, Hiroki J, Kandabashi T, Matsumoto Y, Amano M, Hoshijima M, Kaibuchi H, Takshita A.. Gene transfer of dominant negative Rho kinase suppresses neointimal formation after balloon injury in pigs. Am J Physiol 2000; 278: H1744-50.
    PubMedGoogle Scholar
  • 44 Graf K, Xi XP, Yang D, Fleck E, Hsueh WA, Law RE.. Mitogen-activated protein kinase activation is involved in platelet-derived growth factor-directed migration by vascular smooth muscle cells. Hypertension 1997; 29: 334-9.
    CrossrefPubMedGoogle Scholar
  • 45 Liu D, Aguirre Ghiso JA, Estrada Y, Ossowski L.. EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma. Cancer Cell 2002; 1: 445-57.
    CrossrefPubMedGoogle Scholar
  • 46 Erickson JW, Zhang C, Kahn RA, Evans T, Cerione RA.. Mammalian Cdc42 is a brefeldin A-sensitive component of the Golgi apparatus. J Biol Chem 1996; 271: 26850-54.
    CrossrefPubMedGoogle Scholar