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Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration*

Open AccessPublished:September 11, 2013DOI:https://doi.org/10.1074/jbc.M113.463414
      To understand the role of thrombin in inflammation, we tested its effects on migration of THP-1 cells, a human monocytic cell line. Thrombin induced THP-1 cell migration in a dose-dependent manner. Thrombin induced tyrosine phosphorylation of Pyk2, Gab1, and p115 RhoGEF, leading to Rac1- and RhoA-dependent Pak2 activation. Downstream to Pyk2, Gab1 formed a complex with p115 RhoGEF involving their pleckstrin homology domains. Furthermore, inhibition or depletion of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, or Pak2 levels substantially attenuated thrombin-induced THP-1 cell F-actin cytoskeletal remodeling and migration. Inhibition or depletion of PAR1 also blocked thrombin-induced activation of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2, resulting in diminished THP-1 cell F-actin cytoskeletal remodeling and migration. Similarly, depletion of Gα12 negated thrombin-induced Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2 activation, leading to attenuation of THP-1 cell F-actin cytoskeletal remodeling and migration. These novel observations reveal that thrombin induces monocyte/macrophage migration via PAR1-Gα12-dependent Pyk2-mediated Gab1 and p115 RhoGEF interactions, leading to Rac1- and RhoA-targeted Pak2 activation. Thus, these findings provide mechanistic evidence for the role of thrombin and its receptor PAR1 in inflammation.
      Background: The major goal of this study is to test the hypothesis that thrombin plays a role in inflammation.
      Results: Thrombin stimulates monocyte F-actin cytoskeletal remodeling and migration by PAR1, Gα12, Pyk2, Gab1, Rac1, and RhoA-dependent Pak2 activation.
      Conclusion: Pak2 mediates thrombin-PAR1-induced monocyte/macrophage migration.
      Significance: PAR1 could be a potential target for the development of anti-inflammatory drugs.

      Introduction

      Atherosclerosis is the major cause of mortality and morbidity in the world (
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      ). Inflammation appears to be the major factor underlying the atherogenesis, and it may be initiated with endothelial cell dysfunction, leading to leukocyte adhesion, ROS production, and low density lipoprotein (LDL) oxidation (
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      Inflammation, atherosclerosis, and coronary artery disease.
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      ). Several studies have demonstrated that the recruitment of leukocytes and monocytes/macrophages to the site of vascular injury is dependent on the production of cytokines and adhesion molecules by the dysfunctional endothelium, resulting in their adherence to the endothelium and their subsequent transendothelialization (
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      ,
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      ). In addition, the injured/dysfunctional endothelium may expose the underlying vascular matrix to the flowing blood, which can lead to activation of platelets and production of thrombin (
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      Thrombin regulation of platelet interaction with damaged vessel wall and isolated collagen type I at arterial flow conditions in a porcine model. Effects of hirudins, heparin, and calcium chelation.
      ,
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      • Rodríguez C.
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      • Calvayrac O.
      • Badimon L.
      Thrombin and protease-activated receptors (PARs) in atherothrombosis.
      ). Thrombin, which is an extracellular protease, plays an important role in blood clotting by converting fibrinogen to fibrin (
      • Martorell L.
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      • Rodríguez C.
      • Gentile M.
      • Calvayrac O.
      • Badimon L.
      Thrombin and protease-activated receptors (PARs) in atherothrombosis.
      ). A large amount of literature suggests that thrombin, besides its pivotal role in clotting, acts as a mitogen and chemotactic factor to a variety of cell types, including smooth muscle cells (
      • Zigler M.
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      PAR-1 and thrombin. The ties that bind the microenvironment to melanoma metastasis.
      ,
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      • Majumdar M.
      • Kaplan D.D.
      • Brown J.H.
      Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration.
      ). Thrombin mediates its effects via a family of G protein-coupled receptors (GPCRs),
      The abbreviations used are: GPCR
      G protein-coupled receptor
      PAR
      protease-activated receptor
      HASMC
      human aortic smooth muscle cell
      SH2 and SH3
      Src homology 2 and 3, respectively
      PH
      pleckstrin homology
      BM
      binding motif
      ASO
      antisense oligonucleotide
      TRITC
      tetramethylrhodamine isothiocyanate
      GEF
      guanine nucleotide exchange factor
      IB
      immunoblot
      IP
      immunoprecipitation.
      namely, protease-activated receptors (PARs) (
      • Coughlin S.R.
      Protease-activated receptors in hemostasis, thrombosis and vascular biology.
      ,
      • Macfarlane S.R.
      • Seatter M.J.
      • Kanke T.
      • Hunter G.D.
      • Plevin R.
      Proteinase-activated receptors.
      ). PARs are activated by enzymatic cleavage of their extracellular N-terminal end, exposing a new N terminus, which acts as a tethered ligand. The tethered ligand then activates the serpentine receptor by folding back onto the extracellular second loop. Among the four receptors identified to date, thrombin cleaves PAR1, PAR3, and PAR4, with the highest affinity for PAR1 (
      • Coughlin S.R.
      Protease-activated receptors in hemostasis, thrombosis and vascular biology.
      ,
      • Macfarlane S.R.
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      • Kanke T.
      • Hunter G.D.
      • Plevin R.
      Proteinase-activated receptors.
      ,
      • Shi X.
      • Gangadharan B.
      • Brass L.F.
      • Ruf W.
      • Mueller B.M.
      Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis.
      ). PARs mediate blood clotting and mitogenic and chemotactic effects of thrombin via coupling to trimeric G proteins, mostly Gi/o, Gq/11, and G12/13 (
      • Sorensen S.D.
      • Nicole O.
      • Peavy R.D.
      • Montoya L.M.
      • Lee C.J.
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      • Traynelis S.F.
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      Common signaling pathways link activation of murine PAR-1, LPA, and S1P receptors to proliferation of astrocytes.
      ,
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      • Simon M.I.
      Vascular system defects and impaired cell chemokinesis as a result of Gα13 deficiency.
      ). Furthermore, the activation of different G proteins leads to stimulation of various phospholipases, particularly phospholipases Cβ, resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate and the production of inositol 1,4,5-trisphosphate and diacylglycerol, which then causes Ca2+ mobilization and PKC activation (
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      GPCR activation of Ras and PI3Kγ in neutrophils depends on PLCβ2/β3 and the RasGEF RasGRP4.
      ,
      • Joseph S.
      • MacDermot J.
      Thrombin promotes actin polymerization in U937 human monocyte-macrophage cells. Analysis of the signalling mechanisms mediating actin polymerization.
      ). Recently, we have reported that thrombin stimulates Pyk2, a Ca2+-dependent proline-rich tyrosine kinase, in mediating human aortic smooth muscle cell (HASMC) migration and proliferation and that the activation of Pyk2 is essential for balloon injury-induced neointima formation (
      • Gadepalli R.
      • Singh N.K.
      • Kundumani-Sridharan V.
      • Heckle M.R.
      • Rao G.N.
      Novel role of proline-rich nonreceptor tyrosine kinase 2 in vascular wall remodeling after balloon injury.
      ). A role for Pyk2 in atherosclerosis has also been reported previously (
      • Katsume A.
      • Okigaki M.
      • Matsui A.
      • Che J.
      • Adachi Y.
      • Kishita E.
      • Yamaguchi S.
      • Ikeda K.
      • Ueyama T.
      • Matoba S.
      • Yamada H.
      • Matsubara H.
      Early inflammatory reactions in atherosclerosis are induced by proline-rich tyrosine kinase/reactive oxygen species-mediated release of tumor necrosis factor-α and subsequent activation of the p21Cip1/Ets-1/p300 system.
      ). Because thrombin is produced at the site of vascular injury (
      • Badimon L.
      • Badimon J.J.
      • Lassila R.
      • Heras M.
      • Chesebro J.H.
      • Fuster V.
      Thrombin regulation of platelet interaction with damaged vessel wall and isolated collagen type I at arterial flow conditions in a porcine model. Effects of hirudins, heparin, and calcium chelation.
      ,
      • Martorell L.
      • Martínez-González J.
      • Rodríguez C.
      • Gentile M.
      • Calvayrac O.
      • Badimon L.
      Thrombin and protease-activated receptors (PARs) in atherothrombosis.
      ) and it causes endothelial cell barrier disruption (
      • Timmerman I.
      • Hoogenboezem M.
      • Bennett A.M.
      • Geerts D.
      • Hordijk P.L.
      • van Buul J.D.
      The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of β-catenin phosphorylation.
      ) and inflammation (
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      TRAIL/Apo2L mediates the release of procoagulant endothelial microparticles induced by thrombin in vitro. A potential mechanism linking inflammation and coagulation.
      ), we asked the question of whether it also influences the migration of monocytes/macrophages and, if so, whether it requires Pyk2 activation. In the present study, we report that thrombin induces THP-1 cell migration, and this effect requires PAR1-Gα12-mediated Pyk2-dependent Gab1-p115 RhoGEF interactions, leading to Rac1- and RhoA-targeted Pak2 activation.

      DISCUSSION

      Several studies have shown that upon ligand binding and tyrosine phosphorylation, receptor tyrosine kinase receptors, such as c-Met, EGF receptor, and VEGF receptor, recruit and phosphorylate an adaptor protein, Gab1 (
      • Sachs M.
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      Essential role of Gab1 for signaling by the c-Met receptor in vivo.
      ,
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      • Aksamitiene E.
      • Markevich N.I.
      • Borisov N.M.
      • Hoek J.B.
      • Kholodenko B.N.
      Scaffolding protein Grb2-associated binder 1 sustains epidermal growth factor-induced mitogenic and survival signaling by multiple positive feedback loops.
      ,
      • Dance M.
      • Montagner A.
      • Yart A.
      • Masri B.
      • Audigier Y.
      • Perret B.
      • Salles J.P.
      • Raynal P.
      The adaptor protein Gab1 couples the stimulation of vascular endothelial growth factor receptor-2 to the activation of phosphoinositide 3-kinase.
      ,
      • Lock L.S.
      • Maroun C.R.
      • Naujokas M.A.
      • Park M.
      Distinct recruitment and function of Gab1 and Gab2 in Met receptor-mediated epithelial morphogenesis.
      ). Upon tyrosine phosphorylation, Gab1 acts as a docking site for the assembly of multiprotein complexes with the receptor tyrosine kinase receptors. Gab1 contains multiple tyrosyl residues and proline-rich regions that allow its interactions with SH2 and SH3 domain-containing signaling molecules, such as Grb2, PI3K, phospholipase Cγ, SHP2, and Pak4, in the mediation of cellular responses (
      • Sachs M.
      • Brohmann H.
      • Zechner D.
      • Müller T.
      • Hülsken J.
      • Walther I.
      • Schaeper U.
      • Birchmeier C.
      • Birchmeier W.
      Essential role of Gab1 for signaling by the c-Met receptor in vivo.
      ,
      • Kiyatkin A.
      • Aksamitiene E.
      • Markevich N.I.
      • Borisov N.M.
      • Hoek J.B.
      • Kholodenko B.N.
      Scaffolding protein Grb2-associated binder 1 sustains epidermal growth factor-induced mitogenic and survival signaling by multiple positive feedback loops.
      ,
      • Dance M.
      • Montagner A.
      • Yart A.
      • Masri B.
      • Audigier Y.
      • Perret B.
      • Salles J.P.
      • Raynal P.
      The adaptor protein Gab1 couples the stimulation of vascular endothelial growth factor receptor-2 to the activation of phosphoinositide 3-kinase.
      ,
      • Lock L.S.
      • Maroun C.R.
      • Naujokas M.A.
      • Park M.
      Distinct recruitment and function of Gab1 and Gab2 in Met receptor-mediated epithelial morphogenesis.
      ,
      • Okkenhaug K.
      • Rottapel R.
      Grb2 forms an inducible protein complex with CD28 through a Src homology 3 domain-proline interaction.
      ,
      • Paliouras G.N.
      • Naujokas M.A.
      • Park M.
      Pak4, a novel Gab1 binding partner, modulates cell migration and invasion by the Met receptor.
      ). In addition, it has been reported that Gab1 PH domain is required for its membrane translocation and binding to specific membrane lipid molecules, such as phosphatidylinositol 3,4,5-triphosphate (
      • Maroun C.R.
      • Naujokas M.A.
      • Park M.
      Membrane targeting of Grb2-associated binder-1 (Gab1) scaffolding protein through Src myristoylation sequence substitutes for Gab1 pleckstrin homology domain and switches an epidermal growth factor response to an invasive morphogenic program.
      ). In this study, we present evidence that Gab1 is activated by thrombin, a GPCR agonist, in THP-1 cells, mediating their F-actin cytoskeleton reorganization and migration. In addition, the effect of Gab1 on thrombin-induced THP-1 cell F-actin cytoskeleton reorganization and migration appears to be mediated via its interactions with p115 RhoGEF, because both Gab1 and p115 RhoGEF formed a complex with each other involving their PH domains in response to thrombin, and the down-regulation of either one attenuated thrombin-induced THP-1 cell F-actin cytoskeleton formation and migration. GEFs catalyze the exchange of GDP for GTP and mediate activation of small GTPases, such as the Rho family of GTPases (
      • García-Mata R.
      • Burridge K.
      Catching a GEF by its tail.
      ,
      • Rossman K.L.
      • Der C.J.
      • Sondek J.
      GEF means go. Turning on RHO GTPases with guanine nucleotide-exchange factors.
      ). Because RhoGEFs modulate small RhoGTPases activation, we envisioned that Gab1 via p115 RhoGEF might be modulating the stimulation of RhoGTPases, such as Rac1 and RhoA, in the mediation of thrombin-induced THP-1 cell migration. In accordance with this view, we found that down-regulation of either Gab1 or p115 RhoGEF levels attenuated thrombin-induced Rac1 and RhoA activation, resulting in diminished THP-1 cell F-actin cytoskeleton formation and migration. Although our findings demonstrate that the interactions between Gab1 and p115 RhoGEF require their PH domains, it is also possible that, as in the case of Gab1, p115 RhoGEF membrane translocation may also depend on its PH domain, and this may facilitate its interaction with Gab1. Indeed, a previous study from another laboratory (
      • Hart M.J.
      • Sharma S.
      • elMasry N.
      • Qiu R.G.
      • McCabe P.
      • Polakis P.
      • Bollag G.
      Identification of a novel guanine nucleotide exchange factor for the Rho GTPase.
      ) showed that the membrane translocation of p115 RhoGEF requires its PH domain. It was reported that Gab1 PH domain is required for its role in c-Met-mediated epithelial cell-cell contacts (
      • Maroun C.R.
      • Naujokas M.A.
      • Park M.
      Membrane targeting of Grb2-associated binder-1 (Gab1) scaffolding protein through Src myristoylation sequence substitutes for Gab1 pleckstrin homology domain and switches an epidermal growth factor response to an invasive morphogenic program.
      ). The Rho GTPases play an important role in mediating cell-cell contacts by enhancing focal adhesions (
      • Etienne-Manneville S.
      • Hall A.
      Rho GTPases in cell biology.
      ,
      • Burridge K.
      • Wennerberg K.
      Rho and Rac take center stage.
      ). As Gab1 via recruiting RhoGEFs, such as p115 RhoGEF, mediates Rho GTPases activation, there is the possibility that Gab1-induced cell-cell contacts downstream to c-Met may be mediated by Rac1 or RhoA. Some reports showed that both Rac1 and RhoA reciprocate in the stimulus-induced modulation of cell migration and invasion (
      • Mantuano E.
      • Jo M.
      • Gonias S.L.
      • Campana W.M.
      Low density lipoprotein receptor-related protein (LRP1) regulates Rac1 and RhoA reciprocally to control Schwann cell adhesion and migration.
      ). However, in the present study, we showed that Rac1 and RhoA cooperate in the mediation of thrombin-induced F-actin cytoskeleton formation and migration. Other studies have also reported that Rac1 and Cdc42 act upstream to RhoA in the mediation of endothelial cell migration and neutrophil polarization (
      • Zeng H.
      • Zhao D.
      • Mukhopadhyay D.
      KDR stimulates endothelial cell migration through heterotrimeric G protein Gq/11-mediated activation of a small GTPase RhoA.
      ,
      • Van Keymeulen A.
      • Wong K.
      • Knight Z.A.
      • Govaerts C.
      • Hahn K.M.
      • Shokat K.M.
      • Bourne H.R.
      To stabilize neutrophil polarity, PIP3 and Cdc42 augment RhoA activity at the back as well as signals at the front.
      ). Because a similar cascade of signaling events was induced by thrombin in the modulation of HASMC migration, it may be suggested that Gab1-p115 RhoGEF-Rac1-RhoA may be a unifying mechanism in the modulation of cell migration, at least in response to this agonist.
      The Paks (p21-activated kinases) are evolutionarily conserved serine/threonine protein kinases that interact specifically with the Rho family of GTPases, Rac1, and Cdc42 (
      • Hofmann C.
      • Shepelev M.
      • Chernoff J.
      The genetics of Pak.
      ,
      • Bokoch G.M.
      Biology of the p21-activated kinases.
      ). The mammalian Pak family consists of six members, including Pak1 and Pak2. Although the expression of Pak1 appears to be tissue-specific, Pak2 is expressed ubiquitously (
      • Hofmann C.
      • Shepelev M.
      • Chernoff J.
      The genetics of Pak.
      ). Studies have shown that Pak proteins participate in the regulation of actin cytoskeleton reorganization, focal adhesion formation, and cell motility (
      • Parrini M.C.
      • Camonis J.
      • Matsuda M.
      • de Gunzburg J.
      Dissecting activation of the PAK1 kinase at protrusions in living cells.
      ). A large body of evidence also suggests that Rac1 mediates its effects on actin cytoskeleton remodeling via Pak1 (
      • Hofmann C.
      • Shepelev M.
      • Chernoff J.
      The genetics of Pak.
      ,
      • Bokoch G.M.
      Biology of the p21-activated kinases.
      ,
      • Parrini M.C.
      • Camonis J.
      • Matsuda M.
      • de Gunzburg J.
      Dissecting activation of the PAK1 kinase at protrusions in living cells.
      ). In this aspect, we have previously reported that Gab1-p115 RhoGEF-Rac1-RhoA signaling leads to Pak1 activation in thrombin-induced HASMC migration (
      • Gadepalli R.
      • Singh N.K.
      • Kundumani-Sridharan V.
      • Heckle M.R.
      • Rao G.N.
      Novel role of proline-rich nonreceptor tyrosine kinase 2 in vascular wall remodeling after balloon injury.
      ). However, in THP-1 cells, in response to thrombin, we found that only Pak2 and not Pak1 gets activated and mediates THP-1 cell F-actin cytoskeleton formation and migration. Furthermore, Pak2 activation was found to be dependent on activation of Gab1, p115 RhoGEF, Rac1, and RhoA. Therefore, based on our previous and present findings, it may be speculated that the Gab1, p115 RhoGEF, Rac1, and RhoA signaling may be diverging at Pak1/2 levels in the modulation of HASMC versus THP-1 cell migration in response to thrombin (
      • Gadepalli R.
      • Singh N.K.
      • Kundumani-Sridharan V.
      • Heckle M.R.
      • Rao G.N.
      Novel role of proline-rich nonreceptor tyrosine kinase 2 in vascular wall remodeling after balloon injury.
      ). A role for Pak2 in macrophage migration has also been demonstrated previously (
      • Weiss-Haljiti C.
      • Pasquali C.
      • Ji H.
      • Gillieron C.
      • Chabert C.
      • Curchod M.L.
      • Hirsch E.
      • Ridley A.J.
      • Hooft van Huijsduijnen R.
      • Camps M.
      • Rommel C.
      Involvement of phosphoinositide 3-kinase γ, Rac, and PAK signaling in chemokine-induced macrophage migration.
      ). Furthermore, the activation of Pak2 by heterotrimeric G proteins and the adaptor protein NCK may indicate that several signaling events influence Pak2 in the mediation of cell migration (
      • Galisteo M.L.
      • Chernoff J.
      • Su Y.C.
      • Skolnik E.Y.
      • Schlessinger J.
      The adaptor protein Nck links receptor tyrosine kinases with the serine-threonine kinase Pak1.
      ).
      Previously, we showed that Pyk2 mediates Gab1 activation in the modulation of thrombin-induced HASMC migration (
      • Gadepalli R.
      • Singh N.K.
      • Kundumani-Sridharan V.
      • Heckle M.R.
      • Rao G.N.
      Novel role of proline-rich nonreceptor tyrosine kinase 2 in vascular wall remodeling after balloon injury.
      ). Consistent with these observations, thrombin also activated Pyk2 in THP-1 cells mediating their migration. Interestingly, knockdown of Pyk2 levels inhibited thrombin-induced Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2 phosphorylation or activation, suggesting that Pyk2 acts upstream to all of these signaling events in response to thrombin in THP-1 cells. Previously, we have also shown that Pyk2 plays a role in vascular wall remodeling in response to injury (
      • Gadepalli R.
      • Singh N.K.
      • Kundumani-Sridharan V.
      • Heckle M.R.
      • Rao G.N.
      Novel role of proline-rich nonreceptor tyrosine kinase 2 in vascular wall remodeling after balloon injury.
      ). Other studies have reported that Pyk2 is involved in the pathogenesis of atherosclerosis (
      • Katsume A.
      • Okigaki M.
      • Matsui A.
      • Che J.
      • Adachi Y.
      • Kishita E.
      • Yamaguchi S.
      • Ikeda K.
      • Ueyama T.
      • Matoba S.
      • Yamada H.
      • Matsubara H.
      Early inflammatory reactions in atherosclerosis are induced by proline-rich tyrosine kinase/reactive oxygen species-mediated release of tumor necrosis factor-α and subsequent activation of the p21Cip1/Ets-1/p300 system.
      ). Although our previous study demonstrated that Pyk2-mediated neointimal hyperplasia requires activation of Gab1, p115 RhoGEF, Rac1, RhoA, and Pak1, the mechanisms by which Pyk2 mediates atherosclerosis were not clear. Because macrophage migration and foam cell formation are crucial events in the pathogenesis of atherosclerosis (
      • Hansson G.K.
      Inflammation, atherosclerosis, and coronary artery disease.
      ,
      • Libby P.
      • Ridker P.M.
      • Hansson G.K.
      Progress and challenges in translating the biology of atherosclerosis.
      ) and Pyk2 mediates the migration of THP-1 cells, it may be speculated that Pyk2 via facilitating the recruitment of monocytes/macrophages to the sites of endothelial injury may be aiding the disease pathogenesis. However, it remains to be explored whether thrombin stimulates foam cell formation and if Pyk2 has a role in such effects. Macrophages demonstrate an abundance of actin filaments and actin-associated proteins in the cortical cytoplasm. Cortical actin polymerization and the subsequent extension of pseudopodia are important components of the host-defense mechanisms of these cells (
      • Yin H.L.
      • Hartwig J.H.
      The structure of the macrophage actin skeleton.
      ). Based on these observations, one may also anticipate that thrombin via activation of Pyk2-Gab1-p115 RhoGEF-Rac1-RhoA-Pak2 signaling and enhancing actin polymerization may also be involved in the host defense mechanisms of macrophages.
      Thrombin, which is generated at the site of vascular injury, acts through PARs to initiate its coagulant, mitogenic, and/or chemotactic effects. A large body of evidence suggests that thrombin and its receptors (PARs), particularly PAR1, play a role in endothelial barrier disruption, promoting the sticking of leukocytes and monocytes/macrophages to the endothelium and their infiltration into the subendothelial space (
      • Marin V.
      • Farnarier C.
      • Grès S.
      • Kaplanski S.
      • Su M.S.
      • Dinarello C.A.
      • Kaplanski G.
      The p38 mitogen-activated protein kinase pathway plays a critical role in thrombin-induced endothelial chemokine production and leukocyte recruitment.
      ,
      • Badimon L.
      • Badimon J.J.
      • Lassila R.
      • Heras M.
      • Chesebro J.H.
      • Fuster V.
      Thrombin regulation of platelet interaction with damaged vessel wall and isolated collagen type I at arterial flow conditions in a porcine model. Effects of hirudins, heparin, and calcium chelation.
      ,
      • Simoncini S.
      • Njock M.S.
      • Robert S.
      • Camoin-Jau L.
      • Sampol J.
      • Harlé J.R.
      • Nguyen C.
      • Dignat-George F.
      • Anfosso F.
      TRAIL/Apo2L mediates the release of procoagulant endothelial microparticles induced by thrombin in vitro. A potential mechanism linking inflammation and coagulation.
      ,
      • Maroun C.R.
      • Holgado-Madruga M.
      • Royal I.
      • Naujokas M.A.
      • Fournier T.M.
      • Wong A.J.
      • Park M.
      The Gab1 PH domain is required for localization of Gab1 at sites of cell-cell contact and epithelial morphogenesis downstream from the met receptor tyrosine kinase.
      ), which are the hallmarks of atherogenesis (
      • Woollard K.J.
      • Geissmann F.
      Monocytes in atherosclerosis. Subsets and functions.
      ). An enhanced expression of PAR1 in the regions of inflammation associated with macrophage influx, smooth muscle cell proliferation, and mesenchymal-like intimal cells further supports a role for its involvement in atherosclerosis (
      • Nelken N.A.
      • Soifer S.J.
      • O'Keefe J.
      • Vu T.K.
      • Charo I.F.
      • Coughlin S.R.
      Thrombin receptor expression in normal and atherosclerotic human arteries.
      ,
      • Ahn H.S.
      • Chackalamannil S.
      • Boykow G.
      • Graziano M.P.
      • Foster C.
      Development of proteinase-activated receptor 1 antagonists as therapeutic agents for thrombosis, restenosis and inflammatory diseases.
      ,
      • Mansilla S.
      • Boulaftali Y.
      • Venisse L.
      • Arocas V.
      • Meilhac O.
      • Michel J.B.
      • Jandrot-Perrus M.
      • Bouton M.C.
      Macrophages and platelets are the major source of protease nexin-1 in human atherosclerotic plaque.
      ). In line with this assumption, in the present study, we demonstrate that SCH79797, a selective antagonist of PAR1 or PAR1 depletion, inhibits thrombin-induced Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2 activation, leading to diminished THP-1 cell F-actin cytoskeleton formation and migration. This observation infers that PAR1 may be involved in monocyte/macrophage migration to the sites of endothelial injury and perhaps, via disruption of endothelial cell barrier function, may also be playing a role in the transendothelialization of these cells. Many reports have shown that thrombin influences its cellular effects via its PARs coupled to various G proteins, including Go, Gi, Gs, Gq/11, and G12/13 (
      • Trejo J.
      Protease-activated receptors. New concepts in regulation of G protein-coupled receptor signaling and trafficking.
      ,
      • O'Brien P.J.
      • Molino M.
      • Kahn M.
      • Brass L.F.
      Protease activated receptors. Theme and variations.
      ). In this aspect, a recent report showed that thrombin induces embryonic fibroblast migration via Gα12/13-coupled GPCRs that target RhoA activation through LARG, PDZ-RhoGEF, and p115 RhoGEF (
      • Mikelis C.M.
      • Palmby T.R.
      • Simaan M.
      • Li W.
      • Szabo R.
      • Lyons R.
      • Martin D.
      • Yagi H.
      • Fukuhara S.
      • Chikumi H.
      • Galisteo R.
      • Mukouyama Y.S.
      • Bugge T.H.
      • Gutkind J.S.
      PDZ-RhoGEF and LARG are essential for embryo development, and provide a link between thrombin and LPA receptors and Rho activation.
      ). On the other hand, some studies reported that activation of Gα12 inhibits thrombin-induced epithelial cell migration (
      • Kong T.
      • Xu D.
      • Yu W.
      • Takakura A.
      • Boucher I.
      • Tran M.
      • Kreidberg J.A.
      • Shah J.
      • Zhou J.
      • Denker B.M.
      Gα 12 inhibits α2 β1 integrin-mediated Madin-Darby canine kidney cell attachment and migration on collagen-I and blocks tubulogenesis.
      ). However, the present findings indicate that thrombin-induced monocyte/macrophage migration requires the activation of Gα12 but not Gαq/11. Together, these observations reveal that thrombin modulates cell migration via its PARs that are coupled to various G proteins in different cell types. Some studies have reported that Gα12/13 associates with RhoGEFs, such as p115 RhoGEF, involving their RH and DH domains, and enhances their GEF activity (
      • Chen Z.
      • Guo L.
      • Hadas J.
      • Gutowski S.
      • Sprang S.R.
      • Sternweis P.C.
      Activation of p115-RhoGEF requires direct association of Gα13 and the Dbl homology domain.
      ). It was also reported that the enhancement of the RhoGEF activity by Gα12/13 was greater if the RhoGEF is tyrosine-phosphorylated as compared with its non-phosphorylated form (
      • Suzuki N.
      • Nakamura S.
      • Mano H.
      • Kozasa T.
      Gα12 activates Rho GTPase through tyrosine-phosphorylated leukemia-associated RhoGEF.
      ). In this context, in addition to the present study, other reports have also shown that Gα12/13 mediates Pyk2 activation (
      • Shi C.S.
      • Sinnarajah S.
      • Cho H.
      • Kozasa T.
      • Kehrl J.H.
      G13α-mediated PYK2 activation. PYK2 is a mediator of G13α-induced serum response element-dependent transcription.
      ). In addition, we show that Gab1 interacts with p115 RhoGEF, involving their PH domains, and this interaction requires Gα12-Pyk2 activation. Based on all of these observations, it may be speculated that PAR1, upon activation, triggers the formation of a multimeric protein complex comprising Gα12, Pyk2, Gab1, and p115 RhoGEF that leads to activation of Rac1-RhoA-Pak2 signaling in the modulation of monocyte/macrophage migration. A large body of literature suggests that Rac1 and Cdc42 bind and activate Paks (
      • Hofmann C.
      • Shepelev M.
      • Chernoff J.
      The genetics of Pak.
      ,
      • Bokoch G.M.
      Biology of the p21-activated kinases.
      ). However, the present observations reveal that RhoA acts upstream to Pak2 activation. Because RhoA does not bind to Pak2 directly, a possible explanation is that it indirectly mediates Pak2 activation. Indeed, a previous study showed that RhoA in concert with Raf1 mediates Pak1 activation in the modulation of fibroblast transformation (
      • Tang Y.
      • Yu J.
      • Field J.
      Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts.
      ). In summary, as shown in Fig. 11, the present observations reveal that thrombin stimulates monocyte/macrophage migration, and this response requires PAR1-Gα12-dependent Pyk2-mediated Gab1 and p115 RhoGEF interactions, leading to Rac1-RhoA-Pak2 activation and lamellipodia/filopodia formation.
      Figure thumbnail gr11
      FIGURE 11Schematic diagram that shows the potential signaling mechanism by which thrombin induces monocyte/macrophage migration.

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      Linked Article

      • Withdrawal: Novel role for p21-activated kinase 2 in thrombin-induced monocyte migration
        Journal of Biological ChemistryVol. 298Issue 5
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          This article has been withdrawn by the authors. The Journal states that the immunoblots corresponding to ‘Rac1’ in figure 4B and 4H were duplicated. The immunoblots corresponding to ‘beta-Tubulin’ in figure 4B and ‘Pak2’ in figure 5E were duplicated. Possible micrograph reuse for ‘RhoA ASO3 + Thrombin’ panel in figure 4C and ‘Control ASO’ panel in figure 10D. Additional potential reuse in ‘Pak2 ASO2 + Thrombin’ panel in figure 5C and ‘G-alpha-qASO2’ panel in figure 10D. Possible reuse in immunoblot panels for ‘GTP-RhoA’ in figure 4E and ‘GTP-Rac1’ in figure 10J.
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