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Factor VIIa/Tissue Factor-induced Signaling via Activation of Src-like Kinases, Phosphatidylinositol 3-Kinase, and Rac*

Open AccessPublished:September 15, 2000DOI:https://doi.org/10.1074/jbc.M907635199
      Tissue factor (TF), apart from activating the extrinsic pathway of the blood coagulation, is a principal regulator of embryonic angiogenesis and oncogenic neoangiogenesis, but also influences inflammation, leukocyte diapedesis and tumor progression. The intracellular domain of TF lacks homology to other classes of receptors and hence the signaling mechanism is poorly understood. Here we demonstrate that factor VIIa (the natural ligand for TF) induces the activation of the Src family members c-Src, Lyn, and Yes, and subsequently phosphatidylinositol 3-kinase (PI3K), followed by stimulation of c-Akt/protein kinase B as well as the small GTPases Rac and Cdc42. In turn Rac mediates p38 mitogen-activated protein (MAP) kinase activation and cytoskeletal reorganization, whereas factor VIIa-induced p42/p44 MAP kinase stimulation required PI3K enzymatic activity but was not inhibited by dominant negative Rac proteins. We propose that this Src family member/PI3K/Rac-dependent signaling pathway is a major mediator of factor VIIa/TF effects in pathophysiology.
      TF
      tissue factor
      MAP kinase
      mitogen-activated protein kinase
      NDGA
      nordihydroguaretic acid
      PAK
      p21-activated kinase
      PI3K
      phosphatidylinositol 3-kinase
      PIP
      phosphatidylinositol phosphate
      PIP2
      phosphatidylinositol bisphosphate
      PIP3
      phosphatidylinositol-3,4,5-trisphosphate
      PKB
      protein kinase B
      TNFα
      tumor necrosis factor α
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      PBS
      phosphate-buffered saline
      EGF
      epidermal growth factor
      PAGE
      polyacrylamide gel electrophoresis
      TLC
      thin layer chromatography
      JNK
      c-Jun N-terminal kinase
      PAR
      protease-activated receptor
      Tissue factor (TF),1 a 47-kDa glycosylated transmembrane protein, is the principal initiator of the extrinsic coagulation pathway, via the binding of factor VII/VIIa, followed by activation of factor X and further hemostasis (
      • Bach R.R.
      ). In addition, TF is critical for blood vessel development, and TF-deficient mice die as a consequence of abnormalities in this development (
      • Bugge T.H.
      • Xiao Q.
      • Kombrinck K.W.
      • Flick M.J.
      • Holmback K.
      • Danton M.J.
      • Colbert M.C.
      • Witte D.P.
      • Fujikawa K.
      • Davie E.W.
      • Degen J.L.
      ,
      • Carmeliet P.
      • Mackman N.
      • Moons L.
      • Luther T.
      • Gressens P.
      • Van Vlaenderen I.
      • Demunck H.
      • Kasper M.
      • Breier G.
      • Evrard P.
      • Muller M.
      • Risau W.
      • Edgington T.
      • Collen D.
      ,
      • Toomey J.R.
      • Kratzer K.E.
      • Lasky N.M.
      • Broze Jr., G.J.
      ). Furthermore, TF regulates inflammation, because inhibition of TF/factor VIIa complex assembly dramatically enhanced survival of baboons to LD100 infusion withEscherichia coli (
      • Creasey A.A.
      • Chang A.C.
      • Feigen L.
      • Wun T.C.
      • Taylor Jr., F.B.
      • Hinshaw L.B.
      ,
      • Taylor F.B.
      • Chang A.C.
      • Peer G.
      • Li A.
      • Ezban M.
      • Hedner U.
      ) and monocyte diapedesis may involve TF (
      • Randolph G.J.
      • Luther T.
      • Albrecht S.
      • Magdolen V.
      • Muller W.A.
      ). Finally TF appears to be important in tumor progression, although this effect may well be dependent on the angiogenic properties of TF (
      • Zhang Y.
      • Deng Y.
      • Luther T.
      • Muller M.
      • Ziegler R.
      • Waldherr R.
      • Stern D.M.
      • Nawroth P.P.
      ,
      • Folkman J.
      ,
      • Folkman J.
      • D'Amore P.A.
      ). The molecular mechanisms, however, by which TF/factor VII or TF/factor VIIa interactions exert these effects, remain poorly defined.
      A first clue as to these mechanisms was provided by Bazan (
      • Bazan J.F.
      ) who demonstrated that TF shows substantial homology with the interferon α/β and γ receptors, and this notion was confirmed by crystallographic studies on TF structure (
      • Harlos K.
      • Martin D.M.
      • O'Brien D.P.
      • Jones E.Y.
      • Stuart D.I.
      • Polikarpov I.
      • Miller A.
      • Tuddenham E.G.
      • Boys C.W.
      ,
      • Muller Y.A.
      • Ultsch M.H.
      • Kelley R.F.
      • de Vos A.M.
      ). Subsequently, it was demonstrated that factor VIIa/TF interaction exerts a variety of cellular changes, including Ca2+ signaling (
      • Rottingen J.A.
      • Enden T.
      • Camerer E.
      • Iversen J.G.
      • Prydz H.
      ,
      • Camerer E.
      • Rottingen J.A.
      • Iversen J.G.
      • Prydz H.
      ), tyrosine phosphorylation in monocytes (
      • Masuda M.
      • Nakamura S.
      • Murakami T.
      • Komiyama Y.
      • Takahashi H.
      ), activation of p42/p44 mitogen-activated protein kinase (MAP kinase) (
      • Poulsen L.K.
      • Jacobsen N.
      • Sorensen B.B.
      • Bergenhem N.C.
      • Kelly J.D.
      • Foster D.C.
      • Thastrup O.
      • Ezban M.
      • Petersen L.C.
      ), and changes in gene expression (
      • Pendurthi U.R.
      • Alok D.
      • Rao L.V.
      ). Hence TF may act as a cellular receptor for factor VII/VIIa, but the molecular details of the subsequent signal transduction as well as how these might relate to the TF action in pathophysiology remain unclear.
      A group of proteins possibly involved in TF action is the Rho family of small GTPases, which have an important function in directing cell motility and migration (
      • Guasch R.M.
      • Scambler P.
      • Jones G.E.
      • Ridley A.J.
      ). Recently, it has become clear that especially Rac is important for angiogenesis, calcium signaling, leukocyte migration, and the control of inflammation and oncogenesis, which are functions associated with TF signaling. Furthermore, a recent study by Timokhina et al. (
      • Timokhina I.
      • Kissel H.
      • Stella G.
      • Besmer P.
      ) has demonstrated that c-Src may act as an upstream regulator of p21 ras, whereas c-Src is a general mediator of signaling of receptors devoid of intrinsic kinase activity like TF. We decided to study, therefore, the role of Rac and the Src-like tyrosine kinases in TF signaling. The results show that TF/factor VIIa interaction stimulates a signaling pathway involving the activation of Src-like family members c-Src, Lyn, and Yes, and subsequently PI3K leading to the stimulation of p42/p44 MAP kinase, c-Akt/PKB, and Rac. In turn, the latter GTPase provokes cytoskeletal reorganization and p38 MAP kinase activation. We propose, that this c-Src-, PI3K-, and p21 rac -dependent signaling pathway is a major mediator of TF effects in pathophysiology.

      DISCUSSION

      TF has important coagulation-dependent and coagulation-independent functions. The latter include the regulation of angiogenesis (
      • Bugge T.H.
      • Xiao Q.
      • Kombrinck K.W.
      • Flick M.J.
      • Holmback K.
      • Danton M.J.
      • Colbert M.C.
      • Witte D.P.
      • Fujikawa K.
      • Davie E.W.
      • Degen J.L.
      ,
      • Carmeliet P.
      • Mackman N.
      • Moons L.
      • Luther T.
      • Gressens P.
      • Van Vlaenderen I.
      • Demunck H.
      • Kasper M.
      • Breier G.
      • Evrard P.
      • Muller M.
      • Risau W.
      • Edgington T.
      • Collen D.
      ,
      • Toomey J.R.
      • Kratzer K.E.
      • Lasky N.M.
      • Broze Jr., G.J.
      ), leukocyte migration (
      • Randolph G.J.
      • Luther T.
      • Albrecht S.
      • Magdolen V.
      • Muller W.A.
      ), and inflammation (
      • Creasey A.A.
      • Chang A.C.
      • Feigen L.
      • Wun T.C.
      • Taylor Jr., F.B.
      • Hinshaw L.B.
      ,
      • Taylor F.B.
      • Chang A.C.
      • Peer G.
      • Li A.
      • Ezban M.
      • Hedner U.
      ). The molecular details of the underlying signaling pathways, induced by TF/factor VIIa interaction, are still obscure. Because many of the cellular effects of TF involve cytoskeletal reorganization, we investigated the role of Cdc42 and Rac proteins, both important regulators of actin remodeling (
      • Nobes C.D.
      • Hall A.
      ,
      • Ridley A.J.
      • Paterson H.F.
      • Johnston C.L.
      • Diekmann D.
      • Hall A.
      ). We demonstrated that addition of factor VIIa to fibroblasts resulted in formation of filopodia and both lamellipodia as well as membrane ruffles, structures that are thought to be highly indicative for Cdc42 and Rac activation, respectively. In agreement, direct activation of both GTPases in response to factor VIIa was observed using an assay relying on the specific interaction of p65PAK with Rac and Cdc42. Interestingly, activation of these GTPases was sensitive to inhibition of Src-like kinases and PI3K, and we investigated the role of these signaling elements in factor VIIa action. We observed activation of the Src-like kinases Yes, Lyn, and c-Src, and we observed stimulation of PI3K. Interestingly, the latter response was sensitive to PP1 and thus Src-like kinases appear to act upstream of PI3K. This suggests that the recently described (c-Kit receptor-induced) signaling pathway involving the sequential activation of c-Src and PI3K leading to the activation of Rac (
      • Timokhina I.
      • Kissel H.
      • Stella G.
      • Besmer P.
      ) also mediates factor VIIa stimulation of the Rac GTPase and that this pathway is also capable of activating Cdc42.
      A role for PI3K upstream of Rac and Cdc42 is in agreement with the insight that this enzyme is a principal regulator of the actin cytoskeleton (
      • Siddhanta U.
      • McIlroy J.
      • Shah A.
      • Zhang Y.
      • Backer J.M.
      ) via the activation of Rac-like proteins (
      • Stephens L.
      • Hawkins P.T.
      • Eguinoa A.
      • Cooke F.
      ,
      • Shaw L.M.
      • Rabinovitz I.
      • Wang H.H.
      • Toker A.
      • Mercurio A.M.
      ). We hypothesize, therefore, that the effects of factor VIIa on angiogenesis and monocyte migration involve a factor VIIa-dependent stimulation of Rac and Cdc42 and subsequent cytoskeletal reorganization. Furthermore, because Rac and Cdc42 are involved in tumorigenesis (
      • Qiu R.G.
      • Chen J.
      • Kirn D.
      • McCormick F.
      • Symons M.
      ,
      • Michiels F.
      • Habets G.G.
      • Stam J.C.
      • van der Kammen R.A.
      • Collard J.G.
      ,
      • Qiu R.G.
      • Abo A.
      • McCormick F.
      • Symons M.
      ,
      • Symons M.
      ), Rac and Cdc42 activation by factor VIIa may be implicated in tumorigenic effects associated with TF. The connection between TF signal transduction and the small GTPases of the Rho family may mediate important aspects of TF action in pathophysiology.
      Next to cytoskeletal rearrangements, Rac is implicated in the activation of p38 MAP kinase (
      • Coso O.A.
      • Chiariello M., Yu, J.C.
      • Teramoto H.
      • Crespo P.
      • Xu N.
      • Miki T.
      • Gutkind J.S.
      ,
      • Minden A.
      • Lin A.
      • Claret F.X.
      • Abo A.
      • Karin M.
      ). We found enhanced p38 MAP kinase phosphorylation upon factor VIIa stimulation, which was sensitive to RacN17. Hence Rac-mediated p38 MAP kinase functions as an effector for factor VIIa, possibly by linking TF action to inflammation.
      TF-mediated activation of p42/p44 MAP kinase was first reported by Poulsen et al. (
      • Poulsen L.K.
      • Jacobsen N.
      • Sorensen B.B.
      • Bergenhem N.C.
      • Kelly J.D.
      • Foster D.C.
      • Thastrup O.
      • Ezban M.
      • Petersen L.C.
      ), using TF-transfected baby hamster kidney cells, a cell type that normally does not express TF. This MAP kinase activation was dependent on MEK and factor VIIa protease activity. In accordance, MAP kinase activation observed in A14 cells was also dependent on MEK activity, whereas the sensitivity of this response forskolin also indicates that c-Raf is involved in MAP kinase stimulation. We observed that PI3K and Src mediated this MAP kinase activation. In addition, the Src-like kinase/PI3K signaling module is responsible for c-Akt/PKB phosphorylation. c-Akt/PKB is a well known downstream target of PI3K (
      • Burgering B.M.
      • Coffer P.J.
      ), and its activation thus further confirms PI3K activation by factor VIIa. Because the Src-like kinase/PI3K signaling module seems essential for stimulation of p42/p44 MAP kinase, the GTPases Rac and Cdc42, and c-Akt/PKB, we propose that this signaling module fulfills a key function in factor VIIa signal transduction.
      The nature of the Src-like kinase mediating PI3K activation remains obscure. We observed factor VIIa-dependent increased enzymatic activity of Lyn, c-Src, and in particular Yes, whereas Fyn was clearly not activated by factor VIIa. However, Yes, c-Src, and Lyn were all sensitive to PP1 in vitro, and this inhibitor may therefore inhibit activation of these kinases in vivo (although inhibition of Yes by PP1 in vivo has not been reported). Therefore, it remains unclear whether only one or possibly more of these kinases are responsible for the activation of the PI3K/Rac/p38 MAP kinase and the PI3K/p42/p44 MAP kinase modules. Moreover, roles for c-Src, Lyn, and Yes upstream of PI3K have all been reported (
      • Ibitayo A.I.
      • Tsunoda Y.
      • Nozu F.
      • Owyang C.
      • Bitar K.N.
      ,
      • Herrera-Velit P.
      • Reiner N.E.
      ,
      • Corey S.
      • Eguinoa A.
      • Puyana-Theall K.
      • Bolen J.B.
      • Cantley L.
      • Mollinedo F.
      • Jackson T.R.
      • Hawkins P.T.
      • Stephens L.R.
      ,
      • Jucker M.
      • Feldman R.A.
      ,
      • Fuhrer D.K.
      • Yang Y.C.
      ,
      • Fukui Y.
      • Saltiel A.R.
      • Hanafusa H.
      ), and the nature of the Src-like kinase upstream of PI3K awaits, therefore, further experimentation.
      Both p42/p44 MAP kinase and p38 MAP kinase show an initial decrease in phosphorylation during the first 5 min of stimulation with factor VIIa, before an increase in phosphorylation occurs after 10 min. We do not currently know which signal transduction components are responsible for this effect, but it is tempting to speculate that a TF/factor VIIa-activated phosphatase is responsible for this effect. As activation of tyrosine phosphatases is well known to be essential for activation of c-Src-like kinases (
      • den Hertog J.
      • Pals C.E.
      • Peppelenbosch M.P.
      • Tertoolen L.G.
      • de Laat S.W.
      • Kruijer W.
      ), stimulation of such phosphatases is not entirely unexpected. It would be interesting to determine the identity and the involvement of such phosphatases, and experiments addressing this issue are currently being performed in our laboratory.
      Recently, Sørensen et al. (
      • Sørensen B.B.
      • Freskgard P.
      • Nielsen L.S.
      • Rao L.V.
      • Ezban M.
      • Petersen L.C.
      ) suggested the involvement of an additional transmembrane protein, involved in factor VIIa/TF-dependent signaling. Factor VIIa uses TF as a docking site rather than as a receptor, and subsequently, it proteolytically cleaves an as yet unknown protease-activated receptor (PAR). So far, only four PARs have been identified (PAR-1 through PAR-4), and three of them, PAR-1, -3, and -4, are activated by thrombin. This might explain the similarities found between thrombin- and factor VIIa/TF-dependent signaling.

      Acknowledgments

      We thank Drs. J. Collard and S. van Delft for kindly providing the glutathione S-transferase-PAK construct. Furthermore, we would like to thank Dr. P. H. Reitsma and R. Doornbos for their helpful suggestions.

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