The putative role of tissue factor (TF) as a
receptor involved in signal transduction is indicated by its sequence
homology to cytokine receptors (Bazan, J. F. (1990) Proc.
Natl. Acad. Sci. U. S. A. 87, 6934-6938). Signal transduction
induced by binding of FVIIa to cells expressing TF was studied with
baby hamster kidney (BHK) cells stably transfected with TF and with a
reporter gene construct encoding a luciferase gene under
transcriptional control of tandem cassettes of signal transducer and
activator of transcription (STAT) elements and one serum response
element (SRE). FVIIa induced a significant luciferase response in cells expressing TF, BHK(+TF), but not in cells without TF. The BHK(+TF) cells responded to the addition of FVIIa in a
dose-dependent manner, whereas no response was observed
with active site-inhibited FVIIa, which also worked as an antagonist to
FVIIa-induced signaling. Activation of the p44/42 MAPK pathway upon
binding of FVIIa to TF was demonstrated by suppression of signaling
with the specific kinase inhibitor PD98059 and demonstration of a
transient p44/42 MAPK phosphorylation. No stimulation of p44/42 MAPK
phosphorylation was observed with catalytically inactive FVIIa
derivatives suggesting that the catalytic activity of FVIIa was
obligatory for activation of the MAPK pathway. Signal transduction
caused by a putative generation of FXa activity was excluded by
experiments showing that FVIIa/TF-induced signaling was not quenched by
tick anticoagulant protein, just as addition of FXa could not induce
phosphorylation of p44/42 MAPK in BHK(+TF) cells. These results suggest
a specific mechanism by which binding of FVIIa to cell surface TF
independent of coagulation can modulate cellular functions and possibly
play a role in angiogenesis and tumor metastasis as indicated by
several recent observations.
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INTRODUCTION |
The extrinsic pathway of blood coagulation is initiated when
FVIIa1 circulating in plasma binds to the integral membrane
protein, tissue factor (TF), exposed to
the blood upon injury of the vessel wall. The biology of TF in blood
coagulation has been studied extensively (see Refs. 1 and 2 for
reviews). Initiation of coagulation as a result of FVII/TF activity is
an extracellular event confined to the outer leaflet of the plasma
membrane on TF-expressing cells. This was firmly established when it
was shown by Paborsky et al. (3) that truncation of the
cytoplasmic C-terminal of TF did not affect its cofactor function in
the coagulation process. Still a number of other observations suggested
an intracellular function of TF. It was found that TF showed sequence
homology to the cytokine receptor superfamily (4). All members of this family are involved in signal transduction. Subclass II, which includes
the receptor for interleukin 10 and receptors for interferon
/
and
, shows the highest homology to TF (5). The crystal structure of
TF (6, 7) further substantiated its structural resemblance to the
cytokine receptors. This homology might imply a possible functional
role for TF as a receptor involved in signal transduction. Studies on a
putative intracellular activity induced by FVII/FVIIa have been
elusive. Two recent studies (8, 9) reported that FVIIa could induce
oscillations in intracellular free calcium in various TF-expressing
cells. The authors proposed that this involved activation of
phospholipase C; however, they failed to demonstrate directly
phosphorylation caused by activation of intracellular kinases. Other
studies have provided evidence that serine residues of the cytoplasmic
domain of TF can be phosphorylated in TF-transfected cells (10) and
that this domain works as a substrate for protein kinase activity when
exposed to cell lysates (11). Signal transduction was also indicated in
studies with cultured human monocytes (12) showing that addition of
FVIIa could induce a transient tyrosine phosphorylation of several
polypeptides. Finally very recent results suggested that FVIIa induced
alteration in gene expression in human fibroblasts (13). With these
diverse observations it was of interest to characterize a putative
FVIIa-induced signal transduction pathway in further detail.
Ligand-induced oligomerization of receptor subunits, which juxtaposes
to engage an intracellular signaling machinery, is a common element in
signal initiation from cytokine receptors (14). This may lead to rapid
phosphorylation of a subset of intracellular receptor-associated
proteins and mitogen-activated (Ser/Thr) kinases (MAPK) (15, 16). These
kinases are arranged in several parallel signaling pathways, the
induction of which may eventually activate the serum response element
(SRE) and lead to transcription (17). Cytokine receptors, which like TF
lack intrinsic kinase catalytic domains, may couple ligand binding to
tyrosine phosphorylation by using noncovalently associated protein
kinases, the Janus kinases, which phosphorylate signal transducers and
activators of transcription (STATs) to induce binding to specific DNA
elements and gene transcription. The JAK-STAT pathway may be engaged in
cross-talk with the MAPK pathway (18). The present study demonstrates
that binding of FVIIa to cell surface TF leads to gene transcription as
a result of signal transduction via the p44/42 MAPK pathway and
activation of SRE and/or STAT elements.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The baby hamster kidney cell line BHK-21
tk
ts13 (ATCC CRL 1632) was cultured in Dulbecco's
modified Eagle's medium containing 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin. A human endothelial cell line ECV-304 (ATCC
CRL-1998) was cultured in Medium 199 with Earle's salts containing
10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin. An
epithelial-like canine kidney cell line MDCK-II was kindly provided by
Professor Bo van Deurs, University of Copenhagen, Denmark and cultured
in Dulbecco's modified medium supplemented with 5% FCS, 100 IU/ml
penicillin, and 100 µg/ml streptomycin. All cell lines were grown in
T-75 or T-175 flasks and subcultured into 24-well tissue culture plates
or 23.8-cm2 single wells.
Proteins--
Human recombinant FVII and FVIIa was expressed and
purified as described (19). FVIIai was obtained by blocking of FVIIa in
the active site with
D-Phe-L-Phe-L-Arg chloromethyl
ketone as described previously (20). FVIIa and FVIIai were iodinated by
the lactoperoxidase/H2O2 method (21) and
purified by size exclusion chromatography on a Pharmacia Biotech Inc.
NAP 5 Sephadex G-25 with 0.1 M
NH4HCO3, 0.5% (w/v) human serum albumin as the mobile phase. FX and FXa were from Enzyme Research (Lafayette, IN).
Recombinant tick anticoagulant protein (rTAP) was kindly provided by
Dr. G. P. Vlasuk, Corvas (San Diego, CA). The inhibitor PD98059
and the phosphospecific antibody against p44/42 MAPK
(Thr202/Tyr204) and Western blot detection kit
were from New England Biolabs (Beverly, MA).
Transfection of BHK Cells with TF and Luciferase Reporter
Constructs--
The complete human TF cDNA was cloned into the
mammalian Zem219b expression vector (22). BHK cells were transfected
with the TF expression plasmid using the calcium phosphate
coprecipitation procedure, essentially as described (23). Cells with
stably integrated constructs were selected with 1 µM
methotrexate. The KZ136 reporter construct contains an inducible
firefly luciferase expression unit and the neomycin resistance gene for
stable selection. Luciferase coding sequences are preceded by TATA and
transcription start sequences of the human c-fos gene
(nucleotides 649-747, GenBankTM accession number K00650).
The luciferase promoter consists of 4 STAT binding elements and a SRE,
which were synthesized as oligonucleotides. The STAT elements used were
those found in the c-fos (M67 version), p21WAF1,
-casein, and the Fcg RI genes. BHK cells with and without
TF at 80% confluence were transfected with KZ136 (40 µg of DNA in a
21-cm2 culture dish), and 24 h later G418 was added to
a final concentration of 1 µg/ml. The selective pressure was changed
to 0.5 µg/ml after 2 weeks.
FVIIa Binding to Cell Surface TF and FX Activation
Assay--
This was performed as described previously (20).
Measurements of Gene Transcription by Stimulation of Luciferase
Activity--
Cells were grown in white view plates (Packard,
Pangbourne, Berks, UK) for approximately 2 days to obtain 95-100%
confluence. Cells were washed and grown for an additional 16-24 h in
medium with FCS reduced to 0.2%. The cells were again washed and
exposed to FCS-free medium (100 µl/well) containing the test
compounds. Following incubation at 37 °C for 6 h, 100 µl of
lysis and luciferase assay buffer (Packard) was added. After an
additional 30-180 min at room temperature, the luciferase activity was
measured in a 1450 MicroBeta Trilux luminescence counter (Wallac,
Finland) using 1 s of integration/well.
Cell Lysis and Western Blot Analysis--
The total amount of
p44/42 MAPK or phosphorylated p44/42 MAPK was detected using a
PhosphoPlusTM MAPK antibody kit (Biolabs) according to the
manufacturer's protocol. BHK(+TF) cells were cultured in medium with
0.1% FCS for 24 h prior to the experiment. The experiment was
performed by adding media with 0.1% FCS containing the various ligands
for the indicated periods of time to the cells. As a positive control
of cell signaling cells were treated with 15% FCS for 15 min in each
experiment. Cells were lysed in 150 µl of SDS sample buffer (9.2%
(w/v) SDS, 25 mM Tris-HCl, 40% (v/v) glycerol, 80 mM EDTA, 1.2% (w/v) bromphenol blue, pH 6.8) supplemented
with 3 mM sodium orthovanadate and 24.2 mM
dithiothreitol. Lysates were loaded on a 12% SDS-polyacrylamide gel. A
biotinylated protein marker was loaded on each gel, and fully
phosphorylated p44/42 MAPK was loaded as a positive control in some
experiments.
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RESULTS AND DISCUSSION |
Characterization of Transfected BHK Cells--
Baby hamster kidney
(BHK) cells without detectable amounts of TF were stably transfected
with the TF expression vector. These BHK(+TF) cells contained
approximately 3 × 106 TF/cell as revealed by a
binding assay with 125I-labeled FVIIa and expressed
functionally active TF working as a cofactor for FVIIa-mediated
activation of FX with an EC50 = 1.0 nM. No
significant activation was observed with untransfected BHK cells
(results not shown). Subsequently the cells were stably transfected
with the KZ136 reporter plasmid encoding a luciferase gene under
transcriptional control of tandem cassettes of STAT1 and -3 and STAT4,
-5, and -6 and one SRE. Serum contains a multitude of growth factors
and activates several of these elements. Addition of 15% serum to
starved cells transfected with the KZ136 reporter construct resulted in
a 5-8-fold increase in luciferase activity independent of coexpression
of TF (results not shown).
Effect of FVIIa, FVII, and FVIIai on BHK(+TF) Cells Transfected
with the KZ136 Signaling Reporter Gene Construct--
Fig.
1A shows that TF-transfected
BHK cells, BHK(+TF), with the reporter construct responded to the
addition of FVIIa, whereas a significant response was not observed when
FVIIa was added to BHK cells without TF. This suggested that FVIIa was
involved in TF-dependent signal transduction resulting in
gene transcription. The FVIIa-induced response was saturable with an
apparent EC50 of approximately 20 nM as
indicated by the results shown in Fig. 1B.

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Fig. 1.
FVIIa-induced stimulation of luciferase
response in BHK cells transfected with TF and the KZ136 reporter
construct. Cells were lysed after a 6-h incubation with FVIIa at
37 °C, and the luciferase activity was measured as described under
"Experimental Procedures." A shows the effect of TF
transfection by comparing the response obtained by addition of 400 nM FVIIa with KZ136-transfected BHK cells with TF,
BHK(+TF), and without TF,
BHK( TF). B shows a dose-response
curve with exposure of BHK(+TF) cells to increasing concentrations of
FVIIa as indicated.
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The mobilization of Ca2+ stores in TF-expressing cells
observed by Rottingen et al. (8), as well as transient
phosphorylation of intracellular monocyte polypeptides observed by
Masuda et al. (12), appeared to require the
participation of catalytically active FVIIa. Hence, we have
investigated whether proteolytically active FVIIa was also mandatory
for TF signal transduction monitored by the luciferase reporter system.
As shown in Fig. 2 the addition of
zymogen, one-chain FVII, induced a luciferase response comparable with
that of the activated protease, FVIIa. However, since FVII bound to
cell surface TF could be activated to FVIIa (24, 25), it was impossible
to exclude a response caused by generation of FVIIa. To circumvent this
possibility we used an inactive FVIIa derivative, FVIIai, blocked in
the active site by FFRck. In contrast to native FVII this variant did
not induce a significant luciferase response.

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Fig. 2.
Effect of FVII derivatives and FXa on the
luciferase response in BHK cells transfected with TF and the KZ136
reporter construct. Cells were lysed after a 6-h incubation with
100 nM FVIIa, 100 nM FVII, 100 nM
FVIIai, 100 nM FXa, 100 nM FXa + 200 nM TAP, and 100 nM FVIIa + 200 nM
TAP at 37 °C, and the luciferase activity was measured as described
under "Experimental Procedures."
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Since FVIIa catalytic activity may thus be required for
TF-dependent signaling it was important to rule out that
signaling occurred indirectly due to generation of FXa in trace amounts catalyzed by FVIIa bound to cell surface TF. If FXa was generated it
might induce signaling through a FXa receptor (26-28) or further downstream activation of the coagulation cascade ultimately resulting in activation of the thrombin receptor (29, 30). In this context it is
important to point out that the cells were exposed to a brief EDTA wash
to remove possible traces of vitamin K-dependent coagulation factors before the addition of FVIIa. Further, Fig. 2 shows
that FVIIa/TF-induced signal transduction was not prevented by TAP
(tick anticoagulant protein) which specifically blocks the active site
in FXa. Moreover, although the addition of FXa was capable of
stimulating the luciferase activity, this response was not
TF-dependent since a similar response was also seen in BHK
cells without TF transfection (results not shown). Additionally, in
contrast to the response induced by FVIIa, the response induced by FXa
was fully suppressed by TAP (Fig. 2). These data strongly suggested
that FVIIa/TF-induced signal transduction did not proceed via FXa or
thrombin.
Since FVIIai did not induce a luciferase response it was of interest to
see whether a FVIIa-induced response could be inhibited by binding of
FVIIai to TF. Fig. 3 shows that addition
of increasing concentrations of FVIIai quenched the signal induced by
20 nM FVIIa in a manner expected when both proteins compete
for the same site(s) on TF.

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Fig. 3.
Effect of FVIIai on the FVIIa-induced
luciferase response in BHK cells transfected with TF and the KZ136
reporter construct. Cells were lysed after a 6-h incubation at
37 °C with 20 nM FVIIa plus various fixed concentrations
of FVIIai as indicated, and the luciferase activity was measured as
described under "Experimental Procedures."
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Test of the Signaling Pathway by Means of the MAPK Inhibitor
PD98059--
Activation of the luciferase reporter construct might
occur via one of the known MAPK pathways. Fig.
4 demonstrates that the FVIIa-induced
luciferase response in BHK(+TF) cells was completely inhibited by the
specific inhibitor PD98059. This inhibitor binds to the inactive form
of MAPKK (MEK) preventing its transformation into an active kinase
(31). Phosphorylation of p44/42 MAPK and further downstream activation
is thereby inhibited. The results shown in Fig. 4 thus suggested that
stimulation of gene transcription by FVIIa/TF might occur via the MAPK
pathway.

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Fig. 4.
Inhibition of the FVIIa-induced luciferase
response by PD98059. Cells were incubated with 100 nM
FVIIa for 6 h at 37 °C in the presence of various
concentrations (0-50 µM) of PD98059. Subsequently the
cells were lysed, and the luciferase activity was measured. The effect
of 50 µM PD98059 on the luciferase response in the
absence of FVIIa is shown for comparison.
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FVIIa-induced Phosphorylation of p44/42 MAPK in BHK(+TF)
Cells--
Using a specific antibody against the phosphorylated
Thr202/Tyr204 residues of p44/42 MAPK it was
possible to confirm this conclusion. BHK(+TF) cells were grown to 90%
confluence and then starved in 0.1% FCS for 24 h. Fig.
5 (panels A and B)
shows a Western blot of cell lysates from BHK(+TF) cells exposed to 100 nM FVIIa for 0, 3, 5, 7, 10, and 40 min (lanes
2-7). Specific antibodies were used to visualize the amount of
activated MAPK (panel A) and of total MAPK (panel
B). Fig. 5A demonstrates that the MAP kinase was
transiently phosphorylated peaking at approximately 10 min, whereas the
amount of total MAPK remained essentially constant (panel
B).

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Fig. 5.
Western blot analysis of FVIIa-induced
phosphorylation of p44/42 MAPK in TF-transfected BHK cells.
Panels A and B show phosphorylated (panel
A) and total p44/42 MAPK (panel B) when cells were
incubated with 100 nM FVIIa for 0, 5, 10, 15, 20, and 40 min (lanes 1-6) or 15% FCS for 15 min (lane 8).
A biotinylated protein marker was loaded in lane 7. Panels C
and D show phosphorylated (panel C) and total
p44/42 MAPK (panel D) when cells were incubated for 15 min
with medium containing 100 nM FVII (lane 2), 100 nM FVIIa (lane 3), 100 nM FVIIai
(lane 4), 100 nM [Ala344]FVII
(lane 5), and 10 nM FXa (lane 6).
Lane 1 shows the medium control without further
additions.
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When a similar experiment was performed with nontransfected BHK cells
the MAPK was not activated by FVIIa, but a phosphorylated MAPK response
was obtained with 15% serum (results not shown).
The results shown in Fig. 5 (panels C and D) were
obtained with BHK(+TF) cells exposed to 100 nM FVII, FVIIa,
FVIIai, [Ala344]FVII, or FXa for 15 min. A profound
activation was seen with FVIIa, less so with FVII, and no significant
activation was induced by FVIIai or an inactive FVII variant,
[Ala344]FVII, in which the active site serine was changed
by site-directed mutagenesis (panel C, lanes
2-5). It was interesting to note that the addition of FXa
(panel C, lane 6) also failed to induce
phosphorylation of p44/42 MAPK. No effect on the total amount of p44/42
MAPK level was observed (panel D). These results strongly
suggested that FVIIa activity was needed for this response and since
FXa did not induce phosphorylation, a putative FVIIa-mediated
generation of FXa could not account for p44/42 MAPK activation with
FVIIa.
Antibodies against phosphorylated SAPK or p38 MAPK were tested in a
similar way in separate experiments; however, none of these pathways
appeared to be stimulated by binding of FVIIa to TF (results not
shown).
FVIIa-induced Phosphorylation of p44/42 MAPK in ECV 304 and MDCK
Cells--
FVIIa/TF-induced stimulation of gene transcription was also
studied in the human immortalized endothelial cell line, ECV 304. After
starvation for 24 h, ECV 304 cells expressed about 25,000 TF
molecules per cell as estimated from the 125I-FVIIa binding
capacity. Cells exposed to 20 nM FVIIa in fresh equilibrated media were lysed and harvested after 0, 5, and 40 min.
Western blots of these lysates are show in Fig.
6, A and B. A clear
enhancement of the phosphorylated p44/42 band was observed after
exposure to FVIIa for 5 min followed by a decreased response at 40 min.

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Fig. 6.
Western blot analysis of FVIIa-induced
phosphorylation of p44/42 MAPK in ECV 304 and MDCK-II cells.
Panels A and B show phosphorylated (A) and
total p44/42 MAPK (B) when ECV 304 cells were incubated with
20 nM FVIIa for 0, 5, and 40 min (lanes 2-4).
Lane 1 was loaded with phosphorylated p44/42 MAPK as a
positive control. Panels C and D show
phosphorylated (C) and total p44/42 MAPK (D) when
MDCK-II cells were incubated with 20 nM FVIIa for 0, 5, 20, and 40 min (lanes 1-4). In both experiments lane
5 shows biotinylated marker proteins and lane 6 the
positive control when cells were exposed to 15% serum for 15 min.
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Camerer et al. (1) showed that FVIIa induced relatively
strong intracellular Ca2+ oscillations in Madin-Darby
canine kidney (MDCK) cells constitutively expressing TF capable of
binding human FVIIa. They failed, however, to observe any
phosphorylation and could not inhibit the oscillations with known
tyrosine kinase inhibitors. It was therefore of interest to see whether
it was possible to observe FVIIa/TF-induced phosphorylation of p44/42
MAPK in these cells. The results shown in Fig. 6, C and
D, suggested that this was in fact the case. When
approximately 80% confluent cells were exposed to 20 nM
FVIIa for various time periods phosphorylation of p44/42 MAPK was
clearly induced but appeared to be delayed compared with BHK(+TF) and
ECV 304 cells. The results obtained with these cells confirmed that
signaling capability was not confined to the somewhat artificial test
system constituted by the transfected BHK cells.
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CONCLUSION |
Several recent studies have provided circumstantial evidence that
FVIIa/TF may be involved in signal transduction and gene transcription.
The present work demonstrates that this is the case and that it occurs
in a FVIIa- and TF-dependent reaction via the p44/42 MAPK
pathway. The results also suggest that the catalytic activity of FVIIa
is mandatory for this process and that an indirect signaling pathway
via FX activation can be excluded. The target substrate for the FVIIa
activity has, however, not been identified, just as it remains to be
shown whether TF as such works as a transmembrane signal transducer
either alone or in combination with a putative
-subunit. The
experiments with the effect of PD98059 on the luciferase response (Fig.
4) showed that this specific inhibitor of the MAPK pathway inhibited
the stimulation induced by FVIIa as well as the background activity. This indicates that MAPK phosphorylation is a probable route to FVIIa-induced gene transcription although the existence of parallel pathways or cross-talk between pathways cannot be excluded. Recent studies have suggested a role for TF in angiogenesis (32, 33), embryo
vascularization (34), tumor metastasis (35-37), and smooth muscle cell
migration (38). FVIIa/TF-induced signal transduction might provide a
common molecular mechanism linking these cellular events together. The
present work has uncovered important details about the signaling
process and provided clues to further elucidate the mechanism.
We thank Dr. L. V. M. Rao, The
University of Texas Health Center at Tyler, Tyler, TX for valuable
suggestions and discussions, Dr. G. P. Vlasuk Corvas, San Diego,
for the generous gift of recombinant TAP, and Grith Hagel, Sanne R. Jensen, Berit Lassen, Lone Sanstrup Langhoff, and Elke Gottfriedsen for
excellent technical assistance.