Identification of Novel Downstream Molecules of Tissue Factor

Article pubs.acs.org/jpr

Identification of Novel Downstream Molecules of Tissue Factor Activation by Comparative Proteomic Analysis Lena Kask,† Anneli Jorsback,‡ Maria Winkvist,‡ Jenny Alfredsson,† Bo Ek,§ Jonas Bergquist,§ and Agneta Siegbahn*,† †

Department of Medical Sciences, Clinical Chemistry and Science for Life Laboratory, Uppsala University, 751 85 Uppsala, Sweden GE Healthcare, Bio-Sciences AB, 753 23 Uppsala, Sweden § Analytical Chemistry, Department of Chemistry − Biomedical Center and Science for Life Laboratory, Uppsala University, PO Box 599, 751 24 Uppsala, Sweden ‡

S Supporting Information *

ABSTRACT: Tissue factor (TF) is both an initiator of blood coagulation and a signaling receptor. Using a proteomic approach, we investigated the role of TF in cell signaling when stimulated by its ligand, activated factor VII (FVIIa). From a 2-D difference gel electrophoresis (DIGE) study we found forty one spots that were differentially regulated over time in FVIIa stimulated cells or in comparison to nonstimulated cells. Mass spectrometry identifies 23 out of these as 13 different proteins. One of them, elongation factor 2 (EF-2), was investigated in greater detail by Western blot, a protein synthesis assay and cell cycle analysis. When tissue factor was stimulated by FVIIa, the phosphorylation of EF-2 increased which inactivates this protein. Analyzing the effect using site inactivated FVIIa (FVIIai), as well as the protease activated receptor 2 (PAR-2) agonist SLIGKV, indicated that the inactivation was not PAR-2 dependent. A panel of tissue factor mutants was analyzed further to try to pinpoint what part of the cytoplasmic domain that is needed for this effect. Performing a protein synthesis assay in two different cell lines we could confirm that protein synthesis decreased upon stimulation by FVIIa. Cell cycle analysis showed that FVIIa also promotes a higher degree of cell proliferation. KEYWORDS: 2-D DIGE, elongation factor 2, intracellular signaling, protein synthesis, tissue factor

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INTRODUCTION Tissue factor (TF), a 47 kDa transmembrane receptor, is the main initiator of blood coagulation.1 TF consists of; a 219 amino acid extracellular domain, a 23 amino acid transmembrane domain, and a 21 amino acid cytoplasmic domain. When a blood vessel is ruptured, TF prevents blood from reaching tissues by initially binding its ligand, activated coagulation factor VII (FVIIa). FVIIa fires off a cascade of events that initiates the coagulation cascade resulting in the activation of a large number of proteins, ending in the formation of a fibrin clot. TF is constitutively expressed in extravascular cells like fibroblasts, smooth muscle cells, and pericytes, but within the vascular system TF expression is strictly regulated. Apart from initiating coagulation, TF is also upregulated during inflammation and has a role in pathological conditions such as atherosclerosis and cancer.2,3 In these processes TF acts as a signaling receptor resulting in for example downstream Ca2+ mobilization4 and phosphorylation of mitogen-activated protein kinases (MAPK).5 TF signaling is often coupled to the protease activated receptor 2 (PAR-2), where the signaling starts off with the TF-FVIIa complex that cleaves off a peptide from the PAR-2.6,7 This process can be mimicked by the PAR-2 agonist peptide SLIGKV. In addition, the platelet derived growth factor β receptor (PDGFRβ) is also © 2013 American Chemical Society

shown to become transactivated when FVIIa binds to TF in cell migration processes.8−10 It has been proposed that TF can stimulate migration by activating p38 and Rac1 independently of the proteolytic activity of FVIIa, but depending on the cytoplasmic domain of TF.11 There have also been suggestions of a yet unknown receptor interacting with the TF-FVIIa signal.12 In the cytoplasmic domain of TF, there are three serines that can potentially be phosphorylated; 253, 258, and 263. Phosphorylation of serines 253 and 258 in the C-terminal domain of TF are proposed to have opposing regulatory roles for the incorporation of TF into microparticles.13 Direct interactions between TF and Akt trigger microvessel formation independent of PAR-2 signaling.14 In this study, they also showed that the cytoplasmic domain was not needed for the effect. With so many functional roles, TF proves that it is a multifunctional protein with numerous signaling pathways to explore. Several microarray studies have been performed showing the upregulation of genes and mRNA, including TF, after FVIIa stimulation. Additionally, one example out of these, IL-8, has been confirmed to increase over time by ELISA.15 To our Received: June 28, 2013 Published: November 25, 2013 477

dx.doi.org/10.1021/pr4006298 | J. Proteome Res. 2014, 13, 477−488

Journal of Proteome Research

Article

Table 1. Identification of Encircled Spots in Figure 1 by Mass Spectrometry Together with Their Mascot Score and Sequence Coveragea spot number

fold change

sequence coverage (%)

Mascot score

1−4 5−8 9−10

1.91 −1.72 −1.27

36−51 23−38 54−55

94−187 68−82 241−246

11 12

1.12 −1.17

40 65

77 228

13

1.16

40

90

14

1.29

50

120

15 16 17−20

−1.8 −1.13 1.22

39 64 32−39

117 188 69−105

21

−1.23

32

96

22

−1.16

46

110

23

−1.21

42

45

protein name elongation factor 2 albumin 78 kDa glucose regulated protein thioredoxin reductase 1 pyruvate kinase isozymes M1/M2 isoform 1 inosine-5′monophosphate dehydrogenase 2 mitochondrial ornithine aminotransferase pyruvate dehydrogenase annexin A2 glyceraldehyde-3-phosphate dehydrogenase L-lactate dehydrogenase B chain proteasome subunit alpha type-6 cofilin-1

a

Spots that differed 10% or more in their intensity ratio between two different treatments and a Student’s t test value below 0.01 were chosen to be of interest. The fold change shows how much the identified protein differed between two treatments, the Mascot score how significant the hit is with the lower limit set to 66, and the sequence coverage shows what percentage of the protein’s sequence is found in the analysis.

Figure 1. 2-D map of PAE/PDGFRβ/TF cells, expressing both PDGFRβ and wild type TF, with the 23 spots of interest encircled and numbered which were further excised from the gel and indentified by mass spectrometry. The identified proteins are shown in Table 1. The proteins were separated by IEF on 3-11 NL IPG strips in the first dimension and on 12.5% DIGE gels in the second dimension. The four spots in the rectangular area were identified as elongation factor 2.

from Sekisui Diagnostics. The FITC labeled PAR-2 antibody (SAM11) was from Santa Cruz Biotechnology and polyclonal neutralizing antibody to PAR-2 (P0067) was a kind gift from Prof. W. Ruf, The Scripps Research Institute, La Jolla, CA.

knowledge, no proteomic study has been done to date. Such a study would elucidate what processes are involved at the protein level, as well as inform more closely on which cellular activities that are ongoing since discrepancies between gene expression and protein production is very common. To study the effects of TF-FVIIa activation on a proteomic level, with a screening perspective, a 2-D DIGE study was performed using TF expressing cells. This technique enables a reliable measurement of significantly induced biological changes when comparing stimulated cells with nonstimulated. Moreover, the use of a 2-D format makes it possible to compare thousands of proteins simultaneously. We hereby report our results from a 2-D DIGE study which has identified several novel proteins found to be significantly regulated when FVIIa is bound to TF. We further show that, for one of these proteins, elongation factor 2 (EF-2), the regulation by the TF-FVIIa complex occurs via TF.

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Cell Culture

Porcine aortic endothelial (PAE) cells previously transfected with human PDGF β-receptor and TF (PAE/PDGFRβ/TF cells) were grown in DMEM F:12 media (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/mL streptomycin. The expression of TF, PAR-2, and PDGFRβ and coagulation activity for TF was measured and controlled beforehand.10 To ensure that the porcine PAR-2 was activated after SLIGKV incubation Src phosphorylation was measured, as well as after FVIIa incubation with similar results as seen before (see Supporting Information, Figure S1A and B).9 The expression of TF in PAE/PDGFRβ/TF cells and cells lacking TF (PAE/ PDGFRβ) was measured by flow cytometry using the FITC conjugated anti-TF 4508 (Figure S1E). Three additional tissue factor variants transfected into PAE cells were also used: one where the cytoplasmic domain of TF is deleted (PAE/PDGFRβ/TFΔcyto), one where the three serines in the cytoplasmic domain are mutated into alanines (PAE/PDGFRβ/TF3SA), and one where only serine 258 is mutated into alanine (PAE/PDGFRβ/S258A). All of these cell variants were grown in the same type of media as described above and were previously tested for FXa generation and FVIIa binding sites. All variants were found to be comparable.10 The percentages of cells positive for TF expression on their surface in the different variants were as follows: PAE/PDGFRβ/TF 40%, PAE/PDGFRβ/TFΔcyto 51%, PAE/PDGFRβ/TF3SA 89%, and PAE/PDGFRβ/S258A 70%. DeltaMFI were PAE/PDGFRβ/TF

EXPERIMENTAL SECTION

Reagents

Recombinant human FVIIa and site inactivated FVIIa (FVIIai, a catalytically inactive version of FVIIa) were a kind gift from Prof. L. C. Petersen, NovoNordisk A/S (Maaloev, Denmark). The PAR-2 agonist peptide, SLIGKV, was purchased from Sigma-Aldrich. All 2-D DIGE consumables and apparatus were from GE Healthcare and the anti-pEF-2 (T56), EF-2, pEF-2K, EF-2K, pSrc (Y416), and β-actin rabbit antibody were purchased from Cell Signaling, and the anti-rabbit antibody (goat IRDye 680) from LICOR. Anti-TF conjugated with FITC (4508) and FVIIa blocking anti-TF (4509) were purchased 478



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Figure 2. PAE/PDGFRβ/TF cells were analyzed by Western blot for pEF-2 and housekeeping protein β-actin after stimulation of FVIIa (A), FVIIai (B), and SLIGKV (C) or EF-2 and β-actin after stimulation of FVIIa (E), FVIIai (F), and SLIGKV (G). The proteins were separated by SDS-PAGE on 4−12% gradient gels before being transferred to a PVDF membrane and then incubated with anti-pEF-2 or anti-EF together with anti-β-actin. A merged graph of the triplicates is shown in panel D for pEF-2 and in panel H for EF-2, where the cells with no addition of substance are set to 100% and the mean ± SEM is shown. Students t test was performed where *p < 0.05 and **p < 0.005.

pH 8.8) supplemented with protease inhibitors (1 mM PMSF, 0.2 mM vanadate, 1 μg/mL leupeptine, 1 μg/mL pepstatin A and phosphoprotease inhibitor cocktails 2 and 3 from SigmaAldrich). The samples were then sonicated with 30 pulses of 1 s with low strength (10% effect) and centrifuged at 12 000 rpm for 12 min after which the supernatant was transferred to a new tube.

3.25 PAE/PDGFRβ/TFΔcyto 4.84, PAE/PDGFRβ/TF3SA 15.05, and PAE/PDGFRβ/S258A 10.08. To ensure that the proteolytic activity was contained in the TF mutants, we analyzed the phosphorylation of Erk after 20 min incubation with FVIIa. All mutants were shown to mediate signaling as seen in Figure S1C and D. PAE cells completely lacking TF (PAE/ PDGFRβ) were used as negative controls both in Western blot and cell cycle analysis. Human foreskin fibroblasts (1137Sk) expressing endogenous TF, verified by flow cytometry (S1F), were grown in IMDM Glutamax media (Invitrogen) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/mL streptomycin.

2-D DIGE

The groups analyzed in the DIGE study were group 1, no stimulation; group 2, FVIIa 20 min; group 3, FVIIa 1 h; group 4, FVIIa 3 h; and group 5, FVIIai 3 h. Four biological replicates were used for each group. The concentration of the cell lysates was determined by using the DC Protein Assay (Bio Rad). Protein samples and the internal standard were each labeled with one CyDye DIGE fluor minimal dye (400 pmol CyDye/ 50 μg protein) according to standard procedure.16 The internal standard is a pooled mixture of all samples in the experiment labeled with Cy2. On each immobilized pH gradient (IPG) strips two samples (50 μg each) are applied together with the pooled internal standard (50 μg) in IPG buffer 3-11 and DTT, final concentrations 2% and 40 mM, respectively. The labeled samples were then run on an isoelectric focusing (IEF) gel in the first dimension, and separated by SDS-PAGE in the second dimension. The first dimension separation was performed on IPGPhor3 according to standard protocol with cup loading on 24 cm 3-11NL IPG strips. Following separation the IPG strips

2-D DIGE Sample Preparation: Stimulation of Cells by FVIIa and FVIIai and Cell Lysis

PAE/PDGFRβ/TF cells that were seeded into 6 cm dishes (1.65 × 106 cells/dish) were washed once with PBS and once with starvation media (DMEM F:12 with 0.1% FBS, 2 mM L-glutamine, and 25 U/ml penicillin) and then incubated with starvation media for 4 h. The cells were then incubated with or without 50 nM FVIIa for different lengths of time (20 min, 1 and 3 h), to enable analysis of both short-term and longterm effects, or with 50 nM FVIIai (3 h), and four biological replicates were conducted. After incubation, the cells were washed three times with 5 mM magnesium acetate and 10 mM Tris-HCl pH 8 (DIGE wash buffer) and then lysed in 2-D lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris-HCl 479



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Figure 3. PAE/PDGFRβ cells were analyzed for pEF-2 or EF-2 together with β-actin by Western blot after stimulation of FVIIa (A, E), FVIIai (B, F), and SLIGKV (C, G). Triplicate results are shown in panel D (pEF-2) and panel H (EF-2) with the mean ± SEM, and the cells with no substance added are set to 100%.

initial MALDI analysis, 20 μL of 0.2% acetic acid was added to all samples that gave a poor MALDI spectra, and/or Mascot score together with a STAGE-filter piece in order to collect eluting peptides.18 The sample was then reanalyzed in the MALDI-MS. Peptide mass fingerprinting was performed manually at first, and if possible recalibrated against tryptic peptides and finally annotated through the use of mMass.19 The output from MASCOT software (Matrix Science) was evaluated and if judged necessary a new slightly refined search was performed. The MALDI analysis was performed on a Bruker Ultraflex II using cinnamic acid as matrix. All samples were analyzed on an Ultraflex II MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) equipped with a smartbeam laser. The instrument was run manually in reflector mode and sets of 200 shots were evaluated and eventually summed.

were equilibrated 15 min in equilibration buffer (EBB, 6 M urea, 75 mM Tris-HCl pH 8.8, 29.3% glycerol, 2% SDS, 0.002% bromphenol blue) with 65 mM DTT and another 15 min in EBB with 135 mM IAA. The second dimension separation was performed according to standard procedure on a DALTsix electrophoresis unit with pre cast DIGE gels (12.5%) followed by scanning using Typhoon FLA 9000. Analysis and evaluation was carried out by the DeCyder 2-D differential analysis software v 7.2. The criteria for picking a spot of interest were a 10% difference between any of the analyzed groups and with a Student’s t test value below 0.01. Preparative Gel for Identification of Proteins

To identify the differently regulated proteins we ran a preparative gel. Rehydration loading was used to apply 750 μg of protein into a 24 cm 3-11 NL IPG strip. A small amount of the protein (50 μg) was prelabeled with Cy5, and the IEF and SDS-PAGE (on a precast DALT gel with plastic backing) was run as described above. The gel was poststained with Deep Purple (GE Healthcare) and scanned in the Cy5 and Cy3 channels of the Typhoon FLA 9000. The 2-D pattern from the Cy5 image was easily matched with the analytical gel set in the DeCyder software and the image from the Deep Purple stain (Cy3 channel) was used for the preparation of a pick list. Gel plugs were automatically picked from the preparative gel by the Spot Picker (GE Healthcare) and subsequently prepared for peptide mass fingerprinting.

Western Blot

Cells (PAE/PDGFRβ/TF, PAE/PDGFRβ, PAE/ PDGFRβ/TFΔcyto, PAE/PDGFRβ/TF3SA, PAE/PDGFRβ/ S258A, and 1137Sk fibroblasts) were seeded into 6-well plates, 0.75 × 106 cells/well for PAE cells and 0.05 × 106 cells/well for fibroblasts. The cells were stimulated with different agents (FVIIa 50 nM, FVIIai 50 nM, or SLIGKV 50 μM) for different lengths of time (20 min, 1 and 3 h). Following incubation, the cells were washed three times with DIGE wash buffer and then lysed in RIPA-buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.1% SDS, 0.5% Na-deoxycholate, and 1% triton X-100) with added protease inhibitors as described above. The lysates were centrifuged for 12 min at 12 000 rpm and the proteins were

Mass Spectrometry Analysis

Gel digestion was performed on selected gel plugs from the DeCyder-platform as described by Havlis et al.17 After the 480



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Figure 4. PAE/PDGFRβ/TFΔcyto cells were analyzed for pEF-2 or EF-2 together with β-actin by Western blot after stimulation of FVIIa (A, E), FVIIai (B, F), and SLIGKV (C, G). Triplicate results are shown in panel D (pEF-2) and panel H (EF-2) with the mean ± SEM, and the cells with no substance added are set to 100%.

Protein Synthesis Assay

separated on 4−12% Tris-Bis gradient gels (Invitrogen) and then transferred to a FL-immobiline PVDF membrane (Millipore). After incubation with Bløk (Millipore) for 1 h the membrane was incubated with one of the following antibodies: anti-phosphorylated EF-2 (T56), anti-EF-2, antiphosphorylated elongation factor 2 kinase (EF-2K), or anti-EF2K rabbit antibody (diluted 1:1000) overnight at 4 °C together with an anti-β actin rabbit antibody as a loading control (diluted 1:2000). The membrane was washed with TBS-T (TBS with 0.1% Tween) and incubated with a secondary antirabbit antibody (diluted 1:10 000) for 90 min before washing the membrane again with TBS-T. The membrane was then scanned by an Odyssey scanner (LICOR) and analyzed by the Odyssey V3.0 software and all the bands were related to the βactin band for loading control. All experiments were performed in triplicates.

Cells (PAE/PDGFRβ/TF, PAE/PDGFRβ/TFΔcyto, PAE/ PDGFRβ/TF3SA and 1137Sk fibroblasts) were seeded into a 96-well plate (Perkin-Elmer) with 20 000 cells per well for PAE cells and 10 000 cells per well for fibroblasts. Cells were washed with PBS and starvation media (media containing 0.1% FBS) and then starved for 4 h (PAE cells) or 24 h (1137Sk). After starvation, the cells were washed three times with methionine free media, containing 0.1% FBS, and then incubated with the same media with the L -azidohomoalanine (AHA) added to function as methionine according to the manufacturer’s instructions. The cells were incubated with or without 50 nM FVIIa or 10 μM cycloheximide for 30 min (PAE cells) or 1 h (1137Sk cells). The Click-iT AHA Alexa Fluor 488 Protein Synthesis HCS Assay (Invitrogen) was then performed according to manufacturer’s protocols and analyzed by using an ArrayScan high content screening system (Cellomics). All experiments were done in triplicate.

Blocking FVIIa Effects with Anti-TF or Anti-PAR-2 Antibody on Human Fibroblasts

1137Sk cells were grown as before in 6-well plates but preincubated with an anti-TF antibody (50 μg/mL, 4509 Sekisui Diagnostics) or anti-PAR-2 (50 μg/mL) 30 min before treatment with FVIIa for 1 h. Cells were lysed and treated as described above before the amount of pEF-2 and EF-2 were analyzed by Western Blot.

Cell Cycle Analysis

PAE/PDGFRβ/TF and PAE/PDGFRβ were seeded into 12-well plates (300 000 cells/well) and starved for 4 h as described above, then treated with FVIIa for 20 min and detached from the plate by trypsin treatment. The cells were washed in PBS and then incubated in PI staining solution (0.1% (v/v) Triton X-100, 10 μg/mL PI, and 100 μg/mL DNase free RNase A in PBS) for 30 min before flow cytometry analysis. The experiments were performed in triplicate.

Blocking FVIIa Effects with EF-2K Inhibitor A484954

PAE cells with wild type TF (PAE/PDGFRβ/TF) were incubated with 100 μM A484954 (R&D systems) for 6 h. FVIIa was then added and the amount of pEF-2 was determined by Western blot after 20 min incubation. 481



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Figure 5. PAE/PDGFRβ/TF3SA cells were analyzed for pEF-2 or EF-2 together with β-actin after stimulation of FVIIa (A, E), FVIIai (B, F), and SLIGKV (C, G). Triplicate results are shown in panel D (pEF-2) and panel H (EF-2) with the mean ± SEM, and the cells with no substance added are set to 100%.

Statistics

matched in each gel. The spots of interest were excised by a spot picker directed from a pick list generated by the DeCyder software from a preparative gel, loaded with 750 μg of total protein. The gel plugs were trypsinized before mass spectrometry analysis, and the peptide pattern that was created was compared to the MASCOT database and evaluated with regards to score values. Out of the 41 spots that were differently regulated, we successfully identified 23. Figure 1 shows the 2-D map of PAE/ PDGFRβ/TF cells with the 23 spots that were identified marked out. The four spots in the rectangular area (1−4) were identified as elongation factor 2. In Table 1, the 23 spots that were identified are presented together with their respective Mascot score and their sequence coverage. The Mascot score indicates the significance of the hit with the limit set to 66 for this analysis, while the sequence coverage shows the percentage of the identified amino acid sequence from the mass spectrometry analysis. The 13 different proteins that we identified are involved in various processes such as metabolism, glycolysis, cell mobility, growth survival, and protein synthesis. With the exception of cofilin-1, all identified proteins had scores ranging from 68 to 246. For example, thioredoxin reductase 1, score 77; pyruvate dehydrogease, score 117; EF-2, score 187; and the 78 kDa glucose-regulated protein (GRP-78) with the score 246. We decided to study EF-2, one of the findings, in further detail, since this protein had a high Mascot score in the mass spectrometry identification and because of its involvement in protein synthesis.

For the 2-D DIGE analysis, the DeCyder software was used and significantly regulated proteins between the groups were determined using the Student’s t test p < 0.01. For Western blot and the protein synthesis assay, Student’s t test p-values were calculated. Results were expressed as a percentage in relation to the control which was set to 100% and with mean ± SEM where p-values < 0.05 were considered to be statistically significant.

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RESULTS AND DISCUSSION

Identification of 23 Spots Regulated by TF-FVIIa in a 2-D DIGE Study

We decided to use a porcine aortic endothelial cell line (PAE) previously transfected with PDGFRβ and TF. The reason for choosing this cell line was that it made it possible to investigate the signaling events in greater detail in the different variants of TF; where the cytoplasmic domain is deleted, where the three serines in the cytoplasmic domain are mutated into alanines, and where only serine 258 is mutated into alanine.10 It also enabled the comparison with cells with no TF transfected. PAE cells transfected with human TF were incubated with FVIIa and FVIIai for different lengths of time. Four biological replicates were used for each time point and the DeCyder software v 7.2 was used both to analyze the significantly induced biological changes, and to identify the regulated proteins. We identified 41 spots of interest that were significantly regulated with respect to FVIIa or FVIIai and time (p < 0.01). In the experiment, a total of 1230 spots could be

Phosphorylation Analysis of EF-2 by Western Blot

PAE cells were stimulated for different lengths of time with FVIIa, FVIIai or SLIGKV after which they were analyzed for 482



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Figure 6. PAE/PDGFRβ/S258A cells were analyzed for pEF-2 or EF-2 together with β-actin after stimulation of FVIIa (A, E), FVIIai (B, F), and SLIGKV (C, G). Triplicate results are shown in panel D (pEF-2) and panel H (EF-2) with the mean ± SEM, and the cells with no substance added are set to 100%.

the amount of phoshorylated EF-2, phosphorylated on threonine 56 (T56). The level of phosphorylation was then compared to that of cells without addition of substance (which was set to 100% in graphs). Different time points were chosen in order to detect both short and long-term effects. β-actin was used as loading control and all experiments were performed in triplicates. The increase of phosphorylation at T56 over time in PAE cells with wild type TF when stimulated with FVIIa and FVIIai was analyzed by Western blot and the results can be seen in Figure 2A and B, and the effect of SLIGKV in Figure 2C. All bands were analyzed using the Odyssey V3.0 software and the results are merged and presented in the graph shown in Figure 2D. Treatment with both FVIIa and FVIIai increase the amount of pEF-2, but SLIGKV only has a minor effect on the degree of phosphorylation. Interestingly, the phosphorylation effect was also seen after incubation with FVIIai, which cannot cleave the PAR-2 receptor, indicating that the effect on pEF-2 is PAR-2 independent. We decided to use 50 nM FVIIa and FVIIai, to ensure a maximal output in signal. This is a concentration much higher than that of circulating FVIIa (∼10 pM), but can mimic local concentrations under certain conditions such as cancer20,21 and inflammation.22 Other studies have used concentrations ranging from 10 to 100 nM, so we therefore ensured we could detect an increased phosphorylation of EF-2 also at 10 nM (119% at 1 h, results not shown). The total amount of EF-2 was analyzed for the three different substances and showed no difference in regulation (Figure 2E−H). As a control, we analyzed cells without any TF transfected (PAE/ PDGFRβ) after incubation with FVIIa or FVIIai with no

increase of pEF-2 detected (Figure 3A and B). SLIGKV did have a slight effect with a decrease in phosphorylation after both 20 min and 1 h (Figure 3C), indicating that PAR-2 activation has a decreasing effect on pEF-2 in itself. The graph with the merged results from the triplicates is shown in Figure 3D. The total amount of EF-2 can be seen in Figure 3E−H. Replicating the same experiment in PAE/PDGFRβ/TFΔcyto cells, that is, cells without the cytoplasmic domain of TF, shows no increase in pEF-2 when either FVIIa or FVIIai is added, Figure 4A and B, respectively. Instead, there is a decrease of pEF-2 after 20 min; however, this is not significant, but at 1 and 3 h no effect is observed. The graph from the triplicates is shown in Figure 4D. On the other hand, addition of SLIGKV leads to significant decrease in the amount of pEF-2 after 20 min of incubation (Figure 4C), indicating that when the TF cytoplasmic domain is missing, PAR-2 only has a negative effect. The total amount of EF-2 is shown in Figure 4E−H. When cells where the three C-terminal serines have been mutated into alanines (PAE/PDGFRβ/TF3SA) were stimulated with FVIIa or FVIIai, we could not detect an increase in pEF-2 either after 1 or 3 h of incubation. In these cells, there was also a decrease in pEF-2 at 20 min, as in cells without the cytoplasmic domain; however, in this case, significant (Figure 5A, B, and D). Again, SLIGKV had a decreasing effect on the phosphorylation that was significant after both 20 min and 1 h of incubation. The total amount of EF-2 is shown in Figure 5E−H. These results indicate that PAR-2 appears to have a negative effect on phosphorylation when stimulated on its own, and for PAR-2 to have a positive effect TF needs to be in close 483



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Figure 7. Human fibroblasts (1137Sk) were analyzed by Western blot for pEF-2 or EF-2 together with β-actin after stimulation of FVIIa (A, E), FVIIai (B, F), and SLIGKV (C, G). Panel D (pEF-2) and panel H (EF-2) show the triplicate results with the mean ± SEM, and the cells where no substance was added are set to 100%. Panels I and K show pEF-2 after blocking TF or PAR-2 with an antibody for 30 min before treatment with FVIIa for 1 h, and total EF-2 is shown in panels J and L.

(PAE/PDGFRβ/S258A), a significant decrease at 20 min (Figure 6A, C, and D). Interestingly, here FVIIai in itself promoted a higher degree of phosphorylation on EF-2 than FVIIa or SLIGKV

proximity as well as containing its full length cytoplasmic domain. The same pattern was seen for FVIIa and SLIGKV in PAE cells where only serine 258 is mutated into alanine 484



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(Figure 6B and D), indicating a direct effect mediated via TF but not involving serine 258. This could be due to a possible negative effect of FVIIa or SLIGKV on PAR-2 activation that FVIIai cannot promote due to its catalytically inactive site, while the possible positive effect is carried out by phosphorylation of either S253 or S263 or both. Total amount of EF-2 can be seen in Figure 6E−H. Analysis of pEF-2 in Human Fibroblasts by Western Blot

To confirm our results seen in the PAE cells, we replicated the same experiment on human fibroblasts (1137Sk) with endogenous TF expression by Western blot. Once again an increase in pEF-2 was observed when cells were stimulated with FVIIa and FVIIai (Figure 7A and B). Although the increase was not as strong, and the effect seems to peak at 1 h rather than at 20 min which was the case in the PAE cells. SLIGKV also increases phosphorylation after 1 and 3 h of incubation (Figure 7C). The triplicates of pEF-2 are shown in Figure 7D, and the total of amount of EF-2 is shown in Figure 7E−H. A possible explanation is a different cell surface receptor distribution of TF and PAR-2 on PAE cells and human fibroblast cells. Since PAE cells overexpress TF, this could explain the stronger effect in these cells compared to the human fibroblasts which express TF at a lower level leading to a different PAR-2 to TF ratio (as seen by flow cytometry in Figure S1). Incubating the cells with an anti-TF antibody 30 min prior to treatment with FVIIa for 1 h abrogates the increasing effect on pEF-2 as seen in Figure 7I with total EF-2 shown in Figure 7J. However, when using a neutralizing PAR-2 antibody, we still observe the effect of increasing amounts of pEF-2 (Figure 7K and total EF-2 in Figure 7L), again indicating PAR-2 independency. Western Blot Analysis of the Phosphorylation of EF-2K

The phosphorylation of EF-2K at serine 366 was determined by Western blot analysis. When EF-2K is phosphorylated at serine 366, the kinase is not able to phosphorylate EF-2, which consequently increases protein synthesis.23 No increase in phosphorylation for EF-2K was found for the PAE cells containing wild type TF (PAE/PDGFRβ/TF), but instead decreased at 20 min incubation (Figure S2A). For the other cell variants and the human fibroblasts, the phosphorylation pattern mirrors the results seen for EF-2; that is, EF-2K phosphorylation is increased at 20 min, correlating well with decreased phosphorylation of EF-2 at 20 min (Figure S2B−D). For the PAE cells lacking TF, we detect no change in phosphorylation for EK-2K (Figure S2E). The results from investigating the phosphorylation of EF-2K are in agreement with our findings that it is not upregulated when protein synthesis is decreased.

Figure 8. Inhibition of EF-2K by A484954. The amount of pEF and EF in PAE/PDGFRβ/TF cells together with β-actin were analyzed by Western blot following stimulation with FVIIa, either with or without the addition of the EF-2K inhibitor A484954 (A, B). Triplicate results are shown in panel C (pEF-2) and panel D (EF-2) with the mean ± SEM, and the cells where no substance was added are set to 100%.

Blocking EF-2K with A484954 Decreased the Phosphorylation of EF-2

To further study the signal transduction pathway, we incubated PAE/PDGFRβ/TF cells with the EF-2K inhibitor A484954 before stimulation with FVIIa. The phosphorylation effect on EF-2 by FVIIa was completely abolished when the EF-2K inhibitor was present (Figure 8), proving that this kinase indeed phosphorylates EF-2 when FVIIa is added.

down to 75% in PAE/PDGFRβ/TF cells already after 30 min of incubation compared to nonstimulated cells (100%) (Figure 9A). The addition of cycloheximide, a well-known cytostatic drug that turns off protein synthesis, reduced the value to 29% compared to the control. Both PAE cells without the TF cytoplasmic domain (PAE/PDGFRβ/TFΔcyto) and cells where TF has three serines mutated into alanines (PAE/PDGFRβ/TF3SA) show a much smaller effect in the decrease in protein synthesis, 89 and 92% respectively (Figure 9A). The human fibroblasts were incubated with FVIIa for 1 h since this time point gave the highest amount of pEF-2 as seen in the Western blot

Decreased Protein Synthesis after Incubation with FVIIa

To investigate if the increase in EF-2 phosphorylation had an effect on protein synthesis we performed a protein synthesis assay where the amount of incorporated L-azidohomoalanine (AHA), an amino acid analog of methionine, indicates the level of protein synthesis. FVIIa decreased the incorporation of AHA 485



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Figure 9. Protein synthesis assay. (A) PAE cells with wild type TF (PAE/PDGFRβ/TF), truncated TF (PAE/PDGFRβ/TFΔcyto), and TF with the three cytoplasmic serines mutated into alanines (PAE/ PDGFRβ/TF3SA) were analyzed using a protein synthesis assay. The incorporation of AHA was measured after stimulation with, or without (control), FVIIa or the cytostatic drug cycloheximide for 30 min. No stimulation (control) was set to 100%. (B) Human fibroblasts (1137Sk) were incubated with, or without (control), FVIIa or cycloheximide for 1 h, and control is shown as 100%. The results are shown as the mean ± SEM. Students t test was performed where *p < 0.05 and **p < 0.005.

Figure 10. Cell cycle analysis. PAE/PDGFRβ/TF cells and PAE/ PDGFRβ cells were incubated with FVIIa for 20 min then incubated with PI-staining solution to measure cell cycle phase (A and B, respectively). The percentage of cells that were in G2/M phase is shown as triplicate in panel C.

experiments. The incorporation of AHA was decreased to 70% in these cells compared to controls shown in the graph in Figure 9B, indicating that the phosphorylation of EF-2 might be coupled to protein synthesis. It has previously been shown that protein synthesis is affected by TF stimulation.24 In that study, the overall production of the elongation factors EF-1α and EF-2 was analyzed after 2 h, but there was no analysis of the phosphorylated form. They suggested that the cytoplasmic domain might, in some way, inhibit protein synthesis which is supporting our theory. The differences in protein synthesis observed in that study compared to ours could be explained by the high degree of variability in the response to FVIIa that exists between various TF-expressing cell lines, as well as the different techniques that were used to perform the analyses.

PDGFRβ cells in Figure 10B. The PAE cells with wild type TF has a larger portion of cells, from 10% to 17%, in the G2/M phase after 20 min incubation with FVIIa, which is not seen for cells lacking TF. Additionally, more cells appear to be apoptotic when not incubated with FVIIa. The triplicates for the cells in G2/M phase for the two different cell lines are shown in Figure 10C. FVIIa antiapoptotic effect has been shown previously,25 and binding of FVIIa to TF seems to trigger the cells into dividing, which is the case in many different cancer cells. The self-produced FVIIa seen in ovarian cancer, for example,20 could trigger cell proliferation to amplify and thereby aiding the invasiveness of the cancer. The same effect, even though smaller but yet significant, was seen for human fibroblast after 1 h incubation with FVIIa (results not shown).

Cell Cycle Analysis

Cell cycle analysis was performed in triplicate using flow cytometry on PAE cells with TF (PAE/PDGFRβ/TF) and without (PAE/PDGFRβ) after 20 min incubation with FVIIa or without (control). Merged results are shown in histograms in Figure 10A for the PAE/PDGFRβ/TF cells, and for PAE/

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CONCLUSIONS We have studied proteins regulation after stimulating TF with FVIIa using the 2-D DIGE technique. The technique has a 486



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major advantage; it eliminates any technical variation, one of the main problems with traditional 2-D electrophoresis, by using a pooled internal standard combined with a software that corrects for the technical variation.16,26,27 In a whole cell system, any small variations can be overshadowed by the major noise from steady-state cellular functions, providing a need for a very sensitive and accurate system. The 2-D DIGE technique takes into consideration that most signals will not change and can thereby screen for the true small variations. We wanted to elucidate the cellular events when TF is stimulated by FVIIa and confirm this at the protein level. In this study, we found 41 differentially regulated spots of which we could successfully identify 13 different proteins by mass spectrometry. Many of the proteins we identified are involved in metabolic processes, protein processing, and export, suggesting that FVIIa triggers a stress response in the cell. GRP78, one of the proteins we found, has been associated with TF before.28 We could also show that phosphorylation of EF-2 increases when FVIIa is bound to TF in both PAE cells and human fibroblasts. EF-2 promotes the step in protein synthesis where the newly synthesized protein chain is transferred from the A to the P site on the ribosome.29 Additionally, it is a component of the mRNA surveillance SURF (SMG1, UPF1, eRF1, eRF3) complex.30 When EF-2 becomes phosphorylated on threonine 56, it becomes completely inactivated and consequently protein synthesis terminates.31,32 Our theory is that the phosphorylation of EF-2, via a string of events, is an effect of FVIIa binding to TF. Accumulated data shows that TF signals via Akt;14 in that study, it is dependent on PAR-2 and might be the first step downstream to EF-2K via p70 S6K. However, in our study, PAR-2 is not needed, but instead the cytoplasmic domain is of importance. The EF-2K could also be affected by increased Ca2+-levels or p38 MAPK downstream of FVIIa activated TF as a stress response from the cell. Moreover, this effect is independent of PAR-2, since FVIIai has the same effect both in PAE cells and human fibroblasts. We also believe that there is no transactivation of the PDGFβR since this is a PAR-2 dependent mechanism as shown previously.9 PAR-2 on the other hand seems to have dual effects on the phosphorylation of EF. PAR-2 may on its own down regulate phosphorylation when the TF cytoplasmic domain is missing, but when the cytoplasmic domain of TF is there, it has an increasing effect, supposedly via TF. The main aim of this investigation was to study intracellular changes when FVIIa binds to TF using 2-D DIGE. We found 13 different proteins regulated by FVIIa, and one of them was EF-2. All the results in this report taken together, the phosphorylation of EF-2 shown by Western blot and the functional readout from the protein synthesis assay, together with the cell cycle analysis, make phosphorylation of EF-2 as a direct effect of FVIIa binding to TF a novel finding in TF signaling.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +46 18611 4251. Fax: +46 18 55 25 62. E-mail: agneta. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants from the Swedish Research Council and the Swedish Heart and Lung Foundation. The authors thank Prof. Wolfram Ruf for the gift of the anti-PAR-2 antibody.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures as described in the text. Characterization of cells and phosphorylation of EF-2K determined by Western blot. This material is available free of charge via the Internet at http://pubs.acs.org 487



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