Published online before print January 10, 2005, doi: 10.1161/01.CIR.0000153333.52294.42
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(Circulation. 2005;111:349-355.)
© 2005 American Heart Association, Inc.
Molecular Cardiology |
Tissue Factor Cytoplasmic Domain Stimulates Migration by Activation of the GTPase Rac1 and the Mitogen-Activated Protein Kinase p38
From the Deutsches Herzzentrum und 1. Medizinische Klinik der Technischen Universität München, Munich, Germany.
Correspondence to I. Ott, Deutsches Herzzentrum, Lazarettstrasse 36, 80636 München, Germany. E-mail ott{at}dhm.mhn.de
Received May 27, 2004; revision received May 27, 2004; accepted October 7, 2004.
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Abstract |
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Background— Tissue factor (TF), the surface receptor for the serine protease factor VIIa (FVIIa) and the initiator of the extrinsic coagulation cascade, supports vessel development and tumor metastasis by activation of extracellular, protease-dependent signaling pathways. The molecular mechanisms that do not require proteolytic activity of FVIIa are not yet known. The aim of the study, therefore, was to investigate the effects of active-site–inhibited FVIIa (FFR-FVIIa) on TF-mediated signaling.
Methods and Results— After stimulation with FVIIa and FFR-FVIIa, migration and activation of the GTPase Rac (Rac1) or the mitogen-activated protein kinase p38 (p38) were analyzed in J82 cells. FVIIa and FFR-FVIIa stimulated migration and activation of Rac1 and p38 in a TF-specific, dose- and time-dependent manner. Enhancement of migration required activation of Rac1 and p38, because it was abolished after inhibition with SB203580 or overexpression of dominant negative p38 and Rac1. The cytoplasmic domain of TF was necessary because no effects of FFR-FVIIa could be detected after transfection of a TF deletion mutant lacking the cytoplasmic domain.
Conclusions— We identified a novel signaling pathway through which TF stimulates migration by activation of p38 and Rac1 independent of the proteolytic activity of FVIIa but dependent on the cytoplasmic domain of TF. Binding of FFR-VIIa to TF may stimulate vessel wall remodeling by enhancement of migration through activation of Rac1 and p38. This novel link may provide an insight into the understanding of the nonhemostatic functions of TF.
Key Words: coagulation • endothelium • angiogenesis
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Introduction |
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The protease receptor tissue factor (TF), a 47-kDa transmembrane glycoprotein, is the cellular receptor for the zymogen factor VII (FVII) and the active enzyme form FVIIa. Binding of FVIIa to TF initiates the extrinsic coagulation cascade and supports embryonic vessel development, metastasis, and proinflammatory responses by activation of extracellular, protease-dependent signaling pathways.1 The molecular mechanisms that are independent of the proteolytic activity of FVIIa are not yet known. TF is constitutively expressed in fibroblasts and pericytes surrounding blood vessels but is normally absent from blood cells and vessel wall cells. Under pathophysiological conditions such as sepsis, atherosclerosis, acute myocardial infarction, transplant vasculopathy, or cancer, TF is expressed in monocytes, endothelial cells, smooth muscle cells, and tumor cells.2,3 The contribution of the TF-FVIIa complex to vessel remodeling remains controversial.1 Recent studies suggest that FVIIa mediates cell signaling by 2 mechanisms: one dependent on the TF cytoplasmic domain4,5 and a second that is dependent on the proteolytic activity of FVIIa.6,7 Binding of FVIIa to TF increases intracellular calcium fluxes,8 activates mitogen-activated protein kinases (MAPK),9 and induces expression of proinflammatory cytokines, growth factors, and transcription factors.10 The proteolytic activity of FVIIa but not the cytoplasmic domain of TF is necessary for the activation of protease-activated receptor-2 (PAR-2).7 Independent of the proteolytic activity of its ligand, TF supports cell spreading by binding of the actin-binding protein-280 to its cytoplasmic tail.5 The biological significance and the molecular mechanisms of alternate pathways induced by FVIIa binding to TF remain poorly defined.
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Methods |
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Cells, Transfections, and Plasmids
The human bladder carcinoma cell line J82 (HTB-1) was grown as described previously.5 Human umbilical vein endothelial cells (HUVECs) (Cell Systems) were cultured in endothelial cell growth medium (Cell Systems) and used between passages 2 and 5. Cells were transiently transfected with 2 µg DNA using Effectene (Qiagen). After 24 hours, cells were harvested with cell dissociation buffer (Sigma). Transfection efficiency approximated 74±7% for J82 cells and 33±4% for HUVECs assessed by flow cytometry after transfection with pEGFP-TF (mean±SEM of 5 experiments). Plasmids pRK Rac1, pRK N17Rac1, pRK Cdc42, and pRK N17Cdc42 with myc-tagged and FLAG-tagged dominant negative p38 constructs (pCDN p38 D168A, pCDN p38 T180E Y182A) were kindly provided by E. Lengyel (Frauenklinik der TU München, Munich, Germany).11 Protein expression was verified by immunoblotting using monoclonal anti-myc (Santa Cruz) for pRK Rac1 and pRK N17Rac1 or an anti-FLAG antibody (Stratagene) for pCDN p38 D168A and pCDN p38 T180E Y182A.
Cell Migration
Migration was analyzed with precoated Boyden chambers (QCM-FN Chemicon International). The lower compartment was filled with serum-free media containing FVIIa or FFR-FVIIa (Novo Nordisk). After serum starvation, 4x104 cells/well were added to the upper compartment of the chamber. For inhibitor experiments, antibodies (anti-ß1, Chemicon; anti-TF 6B4 and 5G9, kindly provided by W. Ruf, La Jolla, Calif) or SB203580 (20 µmol/L) were preincubated with the cells for 60 minutes at 37°C and included in both chambers. As a control, we used an irrelevant isotype control antibody. After incubation for 18 hours at 37°C, cells were removed from the upper side of the membrane. The remaining cells on the membrane were stained (Cell Stain, Chemicon) and reported as the mean number of cells per high-power field (x40 magnification). Then, the dye was eluted with extraction buffer for measurement of the absorbance at 570 nm. There was no difference between quantification by optical density and counting of the number of migrated cells. Migration with uncoated membranes was essentially zero and was subtracted from the data.
Surface receptor expression cells were analyzed by flow cytometry after stimulation with 500 nmol/L FFR-FVIIa for 12 hours and staining with anti-TF, anti-ß1, anti-ß3 (Chemicon), anti–PAR-2 (SAM11, Santa Cruz Biotechnologies), and anti–PAR-1 (WEDE, Immunotech) monoclonal antibodies.3
Immunoprecipitation and MAPK p38 Kinase Assay
Inhibitors were preincubated with the resuspended cells for 60 minutes at 37°C; then, stimulation with FVIIa or FFR-FVIIa was performed. Cells were washed and extracted in cold lysis buffer, normalized for protein content, and immunoprecipitated with anti-phospho p38 monoclonal antibody according to the manufacturer’s protocol (Cell Signaling Technology). Phosphorylation of ATF-2 was detected by immunoblotting with rabbit anti–phospho-ATF-2 antibody (1/1000) and quantified by densitometry. The stimulation time for the in vitro kinase assays was 7 minutes if not indicated otherwise.
Rac1 Activation Assay
After stimulation of resuspended cells with FVIIa and FFR-FVIIa, cells were lysed and Rac1 GTP was precipitated with 8 mg PAK Agarose according to the manufacturer’s protocol (Upstate Biotechnology). Precipitated Rac1 and CDC42 were detected by immunoblotting with monoclonal anti-Rac1 (clone 23A8, Upstate Biotechnology) or polyclonal rabbit anti-Cdc42 antibodies (Santa Cruz). The stimulation time for the Rac1 activation assays was 1 minute if not indicated otherwise.
Statistical Analysis
Student’s t test or ANOVA, as appropriate, was used to determine statistical significance between control and treated cells. A value of P<0.05 was considered significant.
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Results |
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Stimulation of Migration by FVIIa and FFR-VIIa
FVIIa activates PAR-2 and may alter migratory responses in some cells. To analyze other TF-dependent pathways, we compared FVIIa and active-site inactivated FVIIa (FFR-FVIIa). Treatment with D-Phe-L-Phe-L-Arg-chloromethyl ketone renders FFR-FVIIa proteolytically inactive but retains its affinity for TF.12 We used the human bladder carcinoma cell line J82, which expresses high levels of TF, low levels of PAR-1, and no PAR-2, as assessed by flow cytometry and polymerase chain reaction (supplemental Table 1 and Figure 1. FFR-FVIIa did not alter migration toward uncoated membranes (results not shown). However, migration of J82 cells toward immobilized fibronectin was dose-dependently enhanced by a chemotactic gradient of FFR-FVIIa in a Boyden chamber assay (Figure 1A). Saturation was achieved at 500 nmol/L because higher concentrations did not increase migration any further (data not shown). FFR-FVIIa in the upper chamber alone or in both chambers did not alter migration compared with migration without FFR-FVIIa (139+15 and 149+14 versus 146+15, number of migrated cells, n=5). Toward FVIIa, a similar increase in the number of migrated cells compared with FFR-FVIIa was observed. This suggests that protease activity is not required. Binding to TF was necessary for enhancement of migration by FFR-FVIIa, because inhibitory anti-TF antibodies abolished the increase in migration by FFR-FVIIa. Enhancement of migration by FFR-FVIIa required fibronectin, because FFR-FVIIa alone did not alter migratory responses, and inhibitory anti-ß1 antibodies abolished the increase in migration (Figure 1B). To evaluate the mechanisms of FFR-FVIIa, we overexpressed TF fused to the N-terminus of enhanced green fluorescent protein (EGFP) in HUVECs, which do not express significant amounts of TF (supplemental Figures and Table). Enhancement of migration by FFR-FVIIa was observed after transfection with TF wild-type but not with a mutant in which the TF cytoplasmic domain was deleted by mutation of codon Lys244 to a termination codon.13 The transfection procedure itself induced only minimal amounts of TF mRNA that were not associated with an increase in TF surface expression or TF activity (supplemental Figures 1 and 2). These results demonstrate the importance of the TF cytoplasmic domain for the stimulation of migration by FFR-FVIIa (Figure 1C).
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cyt), or control vector (pEGFP-C1) were analyzed for migration toward media (open bars) or FFR-FVIIa (500 nmol/L, closed bars). Optical density is expressed as mean±SEM of 6 to 9 experiments. Asterisk indicates P<0.05 compared with unstimulated values.
Various mechanisms may contribute to the effects of FFR-FVIIa on the migratory response: increase in surface expression of adhesion receptors or activation of intracellular signaling pathways. To further define the mechanism, surface expression of TF, ß1-, and 
vß3-integrins was assessed in the presence and absence of FFR-FVIIa in J82 cells. No significant change in TF, ß1-, and vß3-integrin surface expression was found (anti-TF, 45±3 versus 46±3 mean fluorescence; anti-ß1, 301±14 versus 283±10 mean fl.; anti-vß3, 92±14 versus 83±14 mean fl.; mean±SEM, n=8). Hence, TF-mediated increase in migration was not a result of enhanced expression of surface ß1- or vß3-integrin expression.
Stimulation of GTPase Rac1
Because activation of the Rho GTPase Rac1 and Cdc42 is a crucial modulator of chemotaxis,14 we investigated the role of Rac1 and Cdc42 in migration assays after transfection with dominant negative mutants of Rac1 (N17Rac1) or Cdc42 (N17Cdc42). Overexpression of N17Rac1 abolished the enhancement of migration by FFR-FVIIa, whereas transfection of vector alone or Rac1 wild-type did not alter the migration response of J82 cells to FFR-FVIIa (Figure 2A). In contrast, overexpression of dominant negative N17Cdc42 or CdC42 wild-type had no significant effect (results not shown).
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To investigate whether activation of Rac1 by FFR-FVIIa is mediated by engagement of TF, J82 cells were preincubated with inhibitory monoclonal antibodies to TF 6B4 and 5G9 and then stimulated with FFR-FVIIa. Activation of Rac1 induced by FFR-FVIIa was abolished after inhibition of TF (Figure 2, B and C).
If TF acts as a signaling receptor that transmits signals after binding of FVIIa activation, Rac1 may depend on its cytoplasmic domain. To examine the role of the cytoplasmic domain of TF, HUVECs were transfected with a GFP fusion protein of TF and a TF mutant lacking the cytoplasmic domain. Transfection efficiencies were similar, as analyzed by flow cytometry (TF wild-type, 34±3%, and TF mutant, 35±4% GFP-positive cells, n=11). This increase in TF surface expression reflects functional active TF, because it was associated with an increase in TF activity (supplemental Figure 3). Lack of the cytoplasmic domain abrogated FFR-FVIIa–induced Rac1 activation (Figure 2, D and E).
The Rho family GTPases Rac1 and Cdc42 and their immediate downstream effector p21-activated kinase (PAK) have been demonstrated to mediate important effects on the cytoskeleton relevant for cell migration.14 Precipitation of activated Rac1 with PAK was used to determine the amount of activated Rac1. Immunoblotting of PAK-associated proteins with anti-Rac1 antibodies revealed a similar dose-dependent increase in the binding of Rac1 by FVIIa and to a similar extent by FFR-FVIIa (Figure 3, A and B). Because PAK also binds to Cdc42, immunoblotting with anti-Cdc42 antibodies was performed to measure Cdc42 activation. Contrary to Rac1, neither FVIIa nor FFR-FVIIa altered Cdc42 activation (Figure 3B). Time-course experiments revealed that activation of Rac1 was maximal after 1 minute and declined thereafter (Figure 3, C and D)
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Stimulation of the MAPK p38
Activation of p38 may occur in a Rac1-dependent manner. To investigate the roles of MAPK p38 and p42/44, the pharmacological inhibitors SB203580 and PD98059 were used. FFR-FVIIa–induced enhancement of migration was abolished after inhibition of p38 by SB203580 and after transfection of the dominant negative mutants p38 D168A and p38 T180E Y182E (Figure 4, A and B).
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To examine whether FFR-FVIIa and FVIIa alone are sufficient for p38 activation, in vitro kinase assays were performed. Activation of p38 measured by phosphorylation of the p38 substrate ATF-2 induced by FFR-FVIIa and was specific for TF because it was abolished after inhibition of TF (Figure 4, C and D). Furthermore, phosphorylation of ATF-2 required the cytoplasmic domain of TF, because only transfection with wild-type TF, not with the cytoplasmic deleted TF, in HUVECs restored activation of p38 by FFR-FVIIa (Figure 4, E and F).
Additional experiments revealed that FFR-FVIIa induced p38 activation in a dose- and time-dependent manner to an extent similar to proteolytic active FVIIa (Figure 5, A–D). The fact that the proteolytic activity of FVIIa did not alter phosphorylation of ATF-2 compared with FFR-FVIIa supports the concept of an exclusive role of TF in p38 activation.
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Taken together, our results show that FFR-FVIIa enhanced migration of J82 cells toward fibronectin by activation of MAPK p38 and Rac1. This effect occurred as a result of FFR-FVIIa binding to TF and was independent of the proteolytic activity of FVIIa and dependent on the cytoplasmic domain of TF.
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Discussion |
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Major findings of our study are as follows. (1) The TF ligand FVIIa enhanced migration toward fibronectin independent of its proteolytic activity and dependent on the cytoplasmic domain of TF. (2) The cytoplasmic domain of TF was necessary for activation of the MAPK p38 and the GTPase Rac1, which contributed to the enhanced migratory response.
This study identifies a novel function of TF and its potential signaling mechanism. Independent of its proteolytic activity and dependent on the TF cytoplasmic domain, FVIIa enhanced migration and stimulated Rac1 and p38 activation. Inhibition of p38 by SB20358 or overexpression of dominant negative p38 or dominant negative Rac1 abolished TF-induced enhancement of migration. Therefore, activation of p38 or Rac1 was necessary to enhance migration toward fibronectin. It was not sufficient, however, to enhance migration toward FFR-FVIIa alone. Enhancement of migration by FFR-FVIIa required immobilized fibronectin on the lower side of the Boyden chamber as a costimulus. Thus, FFR-FVIIa cannot be considered a chemoattractant by itself but rather a costimulus for integrin-mediated migration. Actin polymerization after activation of p38 and focal adhesion assembly both may allow the actin reorganization required for cell migration.
To date, evidence for signal transduction via the cytoplasmic tail of TF arises from observations of the sequence homology between TF and cytokine receptors,15 phorbol ester–induced phosphorylation of the cytoplasmic tail,16 phosphorylation of the peptide by cell lysate identical to the cytoplasmic domain,17 and interaction of the TF cytoplasmic domain with actin-binding protein 280.5 A role of the cytoplasmic domain of TF on a cellular level has been described in the regulation of vascular endothelial growth factor expression in cancer cell lines18 and calcium fluxes in monocytic cells.4 Although further studies are required for the full understanding of these signaling mechanisms, this study identifies a potential role for the cytoplasmic domain of TF in signaling events in vitro contributing to enhanced cell motility.
Other effects of TF binding to FVIIa include calcium oscillations; activation of p42/44, p38, Rac1, Src, PI(3)K, p70 S6 kinase, and p90 S6 kinase; and induction of gene expression.8,10,19–22 For these signaling events, however, only the extracellular domain of TF, not the cytoplasmic tail, was necessary. Therefore, a dual role for TF evolves: on the one hand, TF serves as a transmembrane receptor; on the other hand, TF provides membrane localization and cofactor activity for stimulation of protease-activated receptors.23 The net effect might depend on receptor distribution. Using polymerase chain reaction and flow cytometry, we could not detect PAR-2 and only low levels of PAR-1 on J82 cells (supplemental Table 1 and Figure 1). Furthermore, PAR-2 was not induced in HUVECs after transfection (supplemental Figure 1). Because PAR-2 is the receptor assumed to be activated by FVIIa10 and inhibition of TF abolished stimulation of migration or p38 and Rac1 activation, FVIIa and FFR-FVIIa may signal through TF. These results stress the importance of cell-type–specific receptor distribution and might explain some of the differences of the effects of FVIIa that have been found with other cell lines. Because FVIIa and FFR-FVIIa bind with different affinities to the inactive pool of TF but with equal affinity to the active pool of TF, the active pool of TF may be required for enhancement of migration.24 This would further explain the similar effects we find with FVIIa and FFR-FVIIa.
Thus, the observed effect of FFR-FVIIa and FVIIa on migration at high concentrations may be because of poorly defined changes in the quaternary structure or subcellular location of TF. Because different pools of TF might be involved, signaling may require higher concentrations of FVIIa than those necessary for initiation of coagulation. It is conceivable, however, that at the site of a thrombus, effective concentrations of FVIIa are achieved. At the site of a ruptured, thrombosed plaque or within a wounded area, these mechanisms may contribute to the migratory capacity of the surrounding cells and may thereby alter vascular remodeling and wound healing. So far, ill-defined costimulating factors may enhance the observed effect of FVIIa and thereby contribute to enhancement of migration in vivo. However, the role of TF-induced migration in vivo remains to be investigated.
The MAPK p38 pathway has been shown to play an important role in mediation of cellular responses to mechanical stress responses, proinflammatory cytokines, and growth factors. Activation of MAPK p38 by cytokines and growth factors promotes chemotaxis in vitro25 and contributes to neointima formation26 and angiogenesis in vivo.27 Phosphorylation of p38 may occur as a result of Rac1 activation, which has been shown to stimulate PAK. PAK regulates the activity of MAPK kinase kinases, which act, in turn, on MAPK kinases and regulate p38.28 Independent of this mechanism, activated Rac1 stimulates PI(3)K, alters actin organization, and thereby enhances motility.14 Possible mechanisms that explain how activation of p38 supports cell motility are phosphorylation of heat-shock protein 27 with subsequent F-actin polymerization.24
In normal vessels, TF is expressed only in adventitial cells. In atherosclerotic lesions, however, TF was found in smooth muscle cells and macrophages and is increased further after balloon injury.2 Because the TF ligand FVIIa is synthesized in smooth muscle cells,29 TF-induced migration might contribute to vascular remodeling and neointima formation, as confirmed by overexpression of TF in the vessel wall.30 Furthermore, angiogenesis requires directed migration of endothelial cells that depends on both cell adhesion to the extracellular matrix and the ability of the cell to detect a chemotactic gradient.31 Because, after stimulation with cytokines and growth factors, TF is induced in endothelial cells32 under inflammatory conditions, stimulation of migration by activation of Rac1 and p38 through the cytoplasmic domain of TF may serve as one mechanism to enhance angiogenic responses. Other scenarios in which TF-induced migration may be of importance are vessel wall remodeling, wound repair, and tumor metastasis.6
Genetic studies provide evidence that the extracellular domain of TF is required for embryogenesis.33 Targeted deletion of the cytoplasmic domain of TF did not affect embryonic development, fertility, or survival in mice.34 Furthermore, low human TF rescues embryonic lethality independent of the cytoplasmic domain of TF.35 The lack of cellular activation by signal transduction via the cytoplasmic domain of TF, therefore, cannot explain the embryonic lethality observed in TF-knockout mice. These findings are consistent with reports indicating an involvement of PAR receptors in FVIIa-induced signaling during physiological development. It is conceivable, however, that signal transduction via the cytoplasmic domain of TF may occur under pathophysiological conditions requiring migration.
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Acknowledgments |
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This study was supported in part by grants from the Deutsche Forschungsgemeinschaft (Ot 158/4-1), the Bayerische Wissenschaftsministerium (Habilitationsförderpreis I.O.), and the Gesellschaft für Thrombose und Hämostase. We are grateful to L.C. Petersen for supplying FVIIa and FFR-FVIIa. We thank W. Ruf for the TF constructs and antibodies and E. Lengyel for the Rac and the p38 plasmids. We also thank B. Campbell, A. Stobbe, and C. Huber for invaluable technical assistance and N. Mackman for helpful discussions.
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Footnotes |
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The online-only Data Supplement, which contains information about Results, can be found with this article at http://www.circulationaha.org.
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References |
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A. Siegbahn, M. Johnell, A. Nordin, M. Aberg, and T. Velling TF/FVIIa Transactivate PDGFR to Regulate PDGF-BB Induced Chemotaxis in Different Cell Types: Involvement of Src And PLC Arterioscler. Thromb. Vasc. Biol., January 1, 2008; 28(1): 135 - 141. [Abstract] [Full Text] [PDF]
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J. S. Palumbo, K. E. Talmage, J. V. Massari, C. M. La Jeunesse, M. J. Flick, K. W. Kombrinck, Z. Hu, K. A. Barney, and J. L. Degen Tumor cell-associated tissue factor and circulating hemostatic factors cooperate to increase metastatic potential through natural killer cell-dependent and-independent mechanisms Blood, July 1, 2007; 110(1): 133 - 141. [Abstract] [Full Text] [PDF]
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S. K. Mandal, U. R. Pendurthi, and L. V. M. Rao Tissue factor trafficking in fibroblasts: involvement of protease-activated receptor-mediated cell signaling Blood, July 1, 2007; 110(1): 161 - 170. [Abstract] [Full Text] [PDF]
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J. Ahamed, F. Niessen, T. Kurokawa, Y. K. Lee, G. Bhattacharjee, J. H. Morrissey, and W. Ruf Regulation of macrophage procoagulant responses by the tissue factor cytoplasmic domain in endotoxemia Blood, June 15, 2007; 109(12): 5251 - 5259. [Abstract] [Full Text] [PDF]
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M. Belting, J. Ahamed, and W. Ruf Signaling of the Tissue Factor Coagulation Pathway in Angiogenesis and Cancer Arterioscler. Thromb. Vasc. Biol., August 1, 2005; 25(8): 1545 - 1550. [Abstract] [Full Text] [PDF]
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