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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Molecular Biology, The Hope
Heart Institute and Providence Medical Center, Seattle, WA;
Departments of Immunology and Vascular Biology, The Scripps
Research Institute, La Jolla, CA; Division of Cardiothoracic
Surgery, Health Science Center, University of Colorado, Denver, CO;
Coagulation Research Laboratory, Haemophilia Reference Centre,
GKT Medical School, St. Thomas Hospital, London, United Kingdom.
Tissue factor (TF), a transmembrane glycoprotein, initiates the
extrinsic coagulation cascade. TF is known to play a major role in
mediating thrombosis and thrombotic episodes associated with the
progression of atherosclerosis. Macrophages at inflammatory sites, such
as atherosclerotic lesions, release numerous cytokines that are capable
of modulating TF expression. This study examined the role of oncostatin
M (OSM), a macrophage/ T-lymphocyte-restricted cytokine, in the
expression of TF in vascular smooth muscle cells (SMCs). It is reported
here that OSM stimulated a biphasic and sustained pattern of TF
messenger RNA (mRNA). The effect of OSM on TF mRNA expression was
regulated at the transcriptional level as determined by nuclear
run-offs and transient transfection of a TF promoter-reporter gene
construct. OSM-induced TF expression was regulated primarily by the
transcription factor NF- Thrombosis plays an integral role in the
development and progression of atherosclerosis.1 It is
also believed to contribute to neointimal development following acute
arterial injury.2 The thrombus contains growth factors and
cytokines that have been implicated in smooth muscle cell (SMC)
proliferation and migration.3 Data from several studies
have suggested that, in acute vascular injury and in atherosclerosis,
tissue factor (TF) plays a major role in initiating
thrombosis.4-7 TF initiates the clotting cascade by
serving as a cofactor for plasma factor VIIa.8,9 In normal arteries, TF is found predominantly in the adventitia. In experimental animal models, TF is rapidly induced in medial SMCs following balloon
arterial injury and has been shown to accumulate in the neointima.2,5,10 In human atherosclerotic plaques, TF is found associated with SMCs, endothelial cells, macrophages, and extracellular matrix.10,11 With the use of a functional
clotting assay of human coronary atherectomy specimens, it was shown
that the TF present in atherosclerotic plaques was
active.7 Numerous studies have demonstrated that
inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis
factor- Materials
Cell culture
TF procoagulant activity The TF activity expressed by SMCs was measured by a chromogenic assay. Briefly, cells were seeded in 24-well plates at a density of 1 × 105 cells per well in 200 µL culture medium. SMCs were stimulated with OSM (10 ng/mL) for the indicated times. The cells were washed 3 times in Tris-buffered saline (50 mM Tris HCl, 120 mM NaCl, 2.7 mM KCl, 3 mg/mL bovine serum albumin, pH 7.4), followed by incubation for 30 minutes at 37°C with 300 µL Tris-buffered saline containing human factor VIIa and X (5 and 150 nM, respectively) and CaCl2 (5 mM). Then, 250 µL of the supernatant was added to 25 µL of a chromogenic substrate for factor Xa (S2765, 0.2 µM final concentration). The chromogenic reaction was stopped after 3 minutes by addition of 20 µL of 50% acetic acid solution and absorbance was measured at 405 nm with a spectrophotometer. The TF activity was expressed in arbitrary units (AU), using reference curves determined by rabbit brain thromboplastin. The logarithms of the procoagulant activity were linearly related to the absorbance up to 100 AU. TF clotting assay was performed as described previously.30TF enzyme-linked immunosorbent assay Confluent human SMC in 6-well plates were exposed to OSM (10 ng/mL) for the indicated times. SMC were lysed and analyzed for TF antigen, using enzyme-linked immunosorbent assay kits (American Diagnostica) according to the manufacturer's instructions.Total RNA extraction and Northern blot analysis RNA was extracted, using RNeasy kit (Qiagen, Valencia, CA). RNA samples (5 to 10 µg) were denatured with dimethyl sulfoxide/glyoxal and electrophoresed on a 1.2% agarose/10 mM sodium phosphate gel and were transferred onto nylon filters by capillary blotting. The filters were then hybridized in Quickhyb solution (Stratagene, La Jolla, CA) to random primed 32P-labeled cDNA TF probe for 1 hour at 68°C. Filters were washed at 68°C in 0.2 × standard saline citrate/0.2% sodium dodecyl sulfate (SDS) and exposed to Kodak MS films for 48 hours at 70°C. For comparison of RNA loading, filters
were rehybridized with G3PDH probe.
RNA stability analysis SMCs were stimulated for 1 and 24 hours with 10 ng/mL OSM. Actinomycin D (5 µg/mL) was then added to the cultures, and RNA was extracted at the indicated times. Northern analysis was performed, and the membranes were probed with TF and G3PDH. Autoradiographic signals were analyzed on a Macintosh 9600 computer, using the public domain NIH Image analysis program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). The TF signal density was normalized to G3PDH density. The corrected density was then plotted as a percentage of the 0-hour value (log scale) against time.Nuclear run-off analysis Nuclei (2 × 107) from SMCs stimulated for 1 hour with OSM (10 ng/mL) were isolated, and in vitro transcription was carried out in 100 µL of 10 mM Tris-HCl (pH 8.0) buffer, containing 5 mM MgCl2; 300 mM KCl; 0.1 mM EDTA; 1 mM dithiothreitol (DTT); 0.5 mM cytidine triphosphate, guanosine triphosphate, adenosine triphosphate (ATP); and 200 µCi 32P uridine
triphosphate (NEN, Boston, MA) for 30 minutes at 30°C. The reaction
was terminated by adding 1 µL proteinase K (20 mg/mL) and 10 µL
10% SDS followed by incubation at 40°C for 1 hour. Radiolabeled RNA
was precipitated by LiCl after acid phenol/chloroform extraction. Linearized, denatured TF, G3PDH, and pGEM-4Z (Promega) plasmid DNA (5 µg) was vacuum transferred onto nylon membranes (Schleicher and
Schuell, Keene, NH), using a slot blot apparatus. The nylon membranes
were hybridized with radiolabeled RNA (3 × 107 cpm) in
Quickhyb solution for 4 hours. The membranes were then washed with
0.5 × standard saline citrate/0.2% SDS at 60°C before autoradiography for 48 hours at 70°C.
Transfections and luciferase assays The construction of pTF(2106)LUC and p19LUC vectors have been
described previously.31 The pGL2 promoter control and
p19LUC vectors were used as positive and negative controls,
respectively. The pSV- -galactosidase control vector was used to
allow for normalization for transfection efficiencies. SMCs were seeded
at a density of 5 × 105 cells per well in 6-well plates
and grown in the culture medium overnight. Transient transfections were
performed according to the manufacturer's instruction. The cells were
transfected with 1 µg pTF(2106)LUC, or pGL2 or p19LUC together with
1 µg pSV--galactosidase control vector. After transfection, the
cells were incubated in the serum-free medium for 48 hours throughout
experiments. Then, cells were incubated at 37°C for a further 5 hours
either in the presence or absence of 10 ng/mL OSM. -galactosidase
and luciferase activity assay was performed according to the
manufacturer's instruction, and luciferase values were normalized to
-galactosidase levels.
Oligonucleotide transfection and reverse transcriptase-polymerase chain reaction analysis The following phosphorothioated oligonucleotides were used as antisense directed against p42 and p44 Erk: 5'-GCCGCCGCCGCCGCCAT-3'. Control oligonucleotides consisted of scrambled antisense sequence, 5'-CGCGCGCTCGCGCACCC-3' (Biomol). Oligonucleotides were transfected into SMCs in 24-well plates, using Lipofectin reagent (Gibco-BRL, Grand Island, NY) as described by the manufacturer. After 48 hours, the SMC cultures were stimulated for 1 hour with OSM (10 ng/mL). The effect of antisense treatment on Erk-1/2 protein expression was analyzed by preparing total cell protein extract, using sample buffer. RNA was extracted as described above, and 100 ng RNA was used for TF and ribosomal s17 messenger RNA (mRNA) analysis, using a one-step reverse transcriptase-polymerase chain reaction (RT-PCR) kit (Gibco-BRL). Quantitative RT-PCR was performed as previously described with minor modifications.32 Forward primers were end-labeled with 32P- ATP. Preliminary experiments were performed to
determine the number of PCR cycles to ensure that the PCR was done in a
quantitative range. TF and ribosomal s17 primers used were as follows:
TF forward, 5'-CACCTTACCTGGAGACAAACCTC-3'; TF reverse,
5'-TGGGCAACAGAGCAAGACTC-3'; s17 forward, 5'-GAAGGCGGCCCGGGTCATCA-3';
and s17 reverse, 5'-GTAGGCTGA/GGTGACCTG-3'. RT-PCR conditions used were
30 minutes at 45°C (RT step), 2 minutes at 94°C, followed by 18 cycles of 94°C for 15 seconds, 55°C for 20 seconds, and 72°C for
20 seconds. The final extension was carried out at 72°C for 10 minutes. The PCR products, TF (900 base pairs [bp]) and ribosomal s17
(350 bp) were separated on 2% TAE agarose gel. To visualize the
products, gels were exposed to Kodak MS films. The products were
quantified by cutting the bands and counting radioactivity in a
scintillation counter.
Cytoplasmic and nuclear extract Cytoplasmic and nuclear extracts from SMCs were prepared by scraping SMCs into cold phosphate-buffered saline, washed once, and resuspended in 200 µL hypotonic lysis buffer (10 mM HEPES, pH 8, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and protease inhibitors) at 4°C for 15 minutes. NP-40 was added to a final concentration of 0.5%, vortexed for 10 seconds, and centrifuged at 7000g for 5 minutes. The supernatant (cytoplasmic) was stored at70°C. The
nuclei were then solubilized with sample buffer for Western blotting.
Electrophoretic mobility shift assay For electrophoretic mobility shift assay (EMSA) studies, nuclei were extracted with 100 µL cold extraction buffer (20 mM HEPES, pH 8, 20% glycerol, 0.5 M NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT and protease inhibitors) for 30 minutes on ice. After centrifugation, supernatant was frozen at70°C. Protein
concentrations in both nuclear and cytoplasmic extracts were
determined. Nuclear extract (5 µg) was used for the detection of
NF- Bp65 and AP-1 transcription factor, using EMSA kits (Geneka
Biotechnology, Montreal, QC, Canada). The following oligonucleotides
were used: AP-1 site, 5'-CGCTTGATGAGTCAGCCGGAA-3'; mutant
AP-1 site,
5'-CGCTTGATGACCCAGCCGGAA-3'; NF-B site, 5'-AGCTTGGGGTATTTCCAGCCG-3'; and mutant
NF-B site, 5'-AGCTTGGCATAGGTCCAGCCG-3'.
The italicized nucleotides are the consensus binding sequence for the
respective transcription factors. The bold underlined and italicized
nucleotides represent the mutation sites. Binding reactions were
performed according to the manufacturer's protocol.
Western blotting Protein concentrations were determined, and equal amounts of protein were separated on a 4% to 12% Bis-tris polyacrylamide gel. After transferring to nylon filters, the membranes were blocked with 20 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl, 3% gelatin, and 0.5% Tween-20 for 1 hour at room temperature. Membranes were then probed with a polyclonal antibody to IB- or with a monoclonal antibody
to NF-B in 20 mM Tris-HCl (pH 7.5) containing 1% gelatin, 0.15 M
NaCl, and 0.05% Tween-20 for 1 hour at room temperature. Blots were
then washed with 20 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl and
incubated with a peroxidase-linked goat antimouse antibody for 30 minutes. Following washing, bands were developed, using Super Signal
chemiluminescent reagent (Pierce, Rockford, IL).
Statistical analysis Statistical analysis was performed by using StatView 4.51 (Abacus Concepts, Berkeley, CA). Factorial analysis of variance with the Fisher exact test was used as appropriate. All values were expressed as mean ± SEM and P values < .05 were considered statistically significant.
OSM induction of TF expression in SMCs To determine if OSM could increase the expression of TF mRNA in cultured SMCs, cells were treated with 10 ng/mL OSM for the times indicated. Low levels of TF mRNA were detected in unstimulated cells (Figure 1A). OSM induced a biphasic increase in TF mRNA expression (Figure 1A). In the first phase, increased levels of TF mRNA expression were observed within 30 minutes, and maximal levels were observed at 1 hour. In the second phase, TF mRNA levels peaked at 48 hours and remained elevated up to 72 hours. Boiled OSM was used to rule out the effect of lipopolysaccharide (LPS) on TF mRNA induction (data not shown). In contrast, PDGF-BB, a known activator of TF,14 induced only a monophasic pattern of TF expression, peaking at 4 hours and returning to control levels by 24 hours (Figure 1B). Other factors tested (thrombin and FBS) also produced only a monophasic pattern (data not shown).
To test whether the second phase of TF mRNA increase was due to residual OSM, we prestimulated SMC cultures with OSM for 30 minutes. The cultures were then washed and incubated for the indicated times in serum-free medium. At 30 minutes, TF mRNA levels were increased; however, TF mRNA levels were not elevated at 24, 48, and 72 hours (Figure 1C) when compared to SMC cultures incubated throughout with OSM (Figure 1A). When SMC cultures were stimulated with OSM-conditioned medium for 24 hours, an increase in TF mRNA expression was observed, which was neutralized by antibodies to OSM (Figure 1D). This observation indicates that the secondary phase of TF induction was due to residual OSM present in the medium and not through secondary growth factors induced by OSM. The time-course of TF mRNA synthesis in canine aortic SMCs was repeated in human aortic SMCs and was shown to be similar (data not shown). Levels of TF antigen and activity were examined in the first phase of
OSM-induced TF expression (0 to 8 hours). OSM transiently increased TF
antigen and activity with maximal levels between 2 and 4 hours (Table
1). Consistent with our observation that residual OSM promoted the late-phase TF mRNA induction (Figure 1D), TF
antigen and activity levels were still elevated at 72 hours (data not
shown).
OSM promotes activation of TF gene transcription To evaluate whether gene activation, mRNA stability, or a combination of both were responsible for the increased TF mRNA observed following OSM stimulation, nuclear run-off, TF promoter studies, and mRNA stability studies were performed. Having established that both phases of TF induction were due to OSM, the remaining studies were focused primarily on the early phase of TF induction. Exposure to OSM for 1 hour significantly increased TF gene expression over unstimulated cells, whereas the transcription rate of the G3PDH gene was unaffected (Figure 2A). Thus, results from our Northern analysis studies, together with the nuclear run-off experiments, suggest that the increase in TF mRNA induced by OSM reflects specific activation of the TF promoter and enhanced TF gene transcription. To confirm that OSM induced active TF gene transcription, transfection studies were performed, using a 2106-bp fragment of the TF promoter coupled to the luciferase gene (pTF(2106)LUC). Data obtained from transfection
experiments are consistent with nuclear run-off studies (Figure 2B). In
unstimulated cells, pTF(2106)LUC promoter activity
was low. There was a 9-fold increase in pTF(2106)LUC activity when SMCs were stimulated with OSM (10 ng/mL), indicating that
OSM can activate the TF promoter.
To investigate if mRNA stability contributed to TF mRNA accumulation, the levels of TF mRNA were measured in the presence of the transcriptional inhibitor actinomycin D (Figure 2C). The stability of TF mRNA was examined at various intervals following the addition of OSM, at the time points of maximal TF mRNA expression (1 hour and 24 hours) and was compared to unstimulated controls. SMCs were incubated with OSM (10 ng/mL) for 1 hour and 24 hours, followed by actinomycin D (5 µg/mL) to arrest transcription. TF mRNA levels were determined by Northern blot analysis at 0, 30 minutes, 1 hour, and 2 hours. TF mRNA exhibited a similar apparent half-life of 83.6 ± 11.7 minutes for controls (n = 3), 88.5 ± 17.2 minutes for 1 hour postinduction (n = 3), and 97.4 ± 6.1 minutes for 24 hours postinduction (n = 3) (Figure 2C). There were no significant differences between the values. These data indicate that the increase in TF mRNA expression in response to OSM is not dependent on mRNA stability but can be attributed to OSM-induced gene activation. Activation of the MEK/Erk-1/2 pathway is required for OSM-induced TF gene activation To define the signal transduction link between OSM receptor activation and TF gene induction, we investigated the effects of signaling inhibitors on TF expression. SMC cultures were preincubated for 1 hour with either 50 µM U0126 (MEK inhibitor), 50 nM NF-B inhibitor peptide that prevents translocation of the NF-B active complex into the nucleus,33 100 nM FTP-1 (Ras inhibitor)
and 100 nM calphostin (protein kinase C [PKC] inhibitor).
SMCs were then stimulated with OSM (10 ng/mL) for an additional 1 hour. Both MEK and NF-B inhibitors suppressed TF mRNA expression, whereas PKC or Ras inhibition had no effect (Figure
3A). These results indicate that the
downstream signaling events induced by OSM to promote TF mRNA
expression involve MEK/Erk-1/2 and NF-B signaling pathway. Because
U0126 is an inhibitor of the Erk-1/2 pathway that selectively blocks
the Erk-1/2-activating enzyme MEK, we examined the effects of U0126 on
OSM-induced Erk-1/2 activation. With the use of an antibody to
phosphorylated Erk-1/2, we demonstrated that OSM stimulated Erk-1/2
phosphorylation (Figure 3B). This activation of Erk-1/2 by OSM was
inhibited by U0126, whereas the NF-B inhibitor had no effect. We
also examined the effects of OSM on 2 other members of the Erk-1/2
family, p38 MAP kinase and stress-activated-protein kinase-1/c-Jun
NH2-terminal kinase. OSM had no effect on either of these
kinases (data not shown). Thus, these results suggest that activated
Erk-1/2 is required for the induction of TF by OSM.
To confirm the hypothesis that Erk-1/2 mediates TF expression,
experiments were performed to determine whether inhibition of
Erk-1/2 by antisense treatment suppressed OSM-induced TF
mRNA expression. The levels of Erk-1/2 protein in SMCs treated with Erk-1/2-antisense oligonucleotides were significantly reduced (Figure
4A). Quantitative RT-PCR analysis for TF
mRNA in OSM-stimulated cells demonstrated that the addition of Erk-1/2
antisense oligonucleotide suppressed TF mRNA expression (Figure 4B).
These results confirm the view that signaling through the Erk-1/2
pathway is required for OSM-induced TF mRNA expression.
Regulation of NF- B pathways are involved in the induction of TF by OSM. To confirm
whether OSM-induced activation of Erk-1/2 is involved in the activation
of NF-B, we analyzed nuclear extracts from SMC cultures exposed to
OSM in the presence or absence of U0126 for NF-B binding activity by
EMSA. We also investigated whether the AP-1 transcriptional factor was
involved in the induction of TF by OSM because a role for AP-1 in TF
induction has been demonstrated.34 OSM markedly activated
both AP-1 and NF-B binding activity. Unlabeled homologous
oligonucleotides prevented binding of 32P-labeled AP-1
(Figure 5A) and NF-B (Figure 5B)
sequences to nuclear proteins, whereas mutated oligonucleotides had no
effect. U0126 inhibited both AP-1 and NF-B binding.
NF-B-specific inhibitor peptide prevented NF-B but not AP-1
binding. The inhibition of NF-B binding activity by U0126 suggests
that Erk-1/2 activation is linked to NF-B activity (Figure 5B). One
potential mechanism by which U0126 may be exerting its effects is to
inhibit nuclear translocation of NF-B by preventing IB-
degradation. Western blot analysis was performed to determine whether
OSM stimulated IB- degradation. The levels of IB- in
OSM-treated cells were similar to the unstimulated control. In
contrast, the levels of IB- were reduced in TNF--treated SMCs
(Figure 6A). This finding suggests that
the activation of NF-B by OSM is independent of IB degradation
and further rules out the possibility that U0126 inhibited NF-B
nuclear translocation by suppressing IB- degradation. To confirm
this assumption, we examined the effects of U0126 on OSM-induced
NF-B nuclear translocation by Western blot analysis. NF-B protein
was detected in the nuclei of OSM-stimulated SMCs (Figure 6B).
Incubating SMCs with the NF-B peptide inhibitor prevented NF-B
nuclear translocation. However, similar levels of nuclear NF-B
protein were detected in OSM-stimulated SMCs treated with or without
U0126. This finding, together with the observation that U0126 reduced
NF-B nuclear activity (Figure 5), suggests that the inhibitory
effects of U0126 on NF-B nuclear activity are most likely due to
reduced affinity of NF-B for its DNA binding site.
Thrombosis plays an integral role in the development and
progression of atherosclerosis. Accumulation of TF within the
atherosclerotic lesion is believed to play a critical role in
determining its thrombogenicity.35 In the present study,
we have investigated how OSM, a macrophage and T-lymphocyte-restricted
cytokine, regulates TF expression in SMCs. Our results suggest that OSM
expressed in atherosclerotic lesions may contribute to plaque
thrombogenicity by inducing the expression of TF. Because
atherosclerosis is an inflammatory disease, it is believed that
macrophages within the atherosclerotic lesion secrete cytokines that
are capable of promoting TF expression.36 The biphasic and
sustained pattern of TF mRNA expression induced by OSM in SMCs is
unique among cytokines and growth factors known to promote TF
expression such as TNF- We have demonstrated previously that OSM can promote endothelial proliferation and migration through an indirect mechanism that involves basic fibroblast growth factor and the plasminogen activator system.41,42 Consistent with our observation, OSM was shown to promote angiogenesis in vivo and in vitro.29 These studies suggest that OSM found in the atherosclerotic lesion could promote neovascularization within the plaques, a process believed to contribute to plaque instability. In this regard, OSM was also shown to stimulate expression of matrix metalloproteinases, key molecules involved in plaque rupture.27,43 Interestingly, OSM may also contribute to rupturing of aneurysms because it has been shown to be present in human aortic aneurysm specimens obtained from atherosclerotic tissue.28 Our finding that OSM promotes prolonged TF expression, together with the observations of Modur et al,28 suggests that OSM can contribute to all phases of thrombotic complications associated with atherosclerosis (ie, from plaque destabilization to plaque rupture and finally clot formation). The TF promoter is complex and contains numerous binding sites for
transcription factors involved in regulating TF gene
expression.44 For instance, the induction of TF in
endothelial cells and monocytic cells by agents such as TNF- The mechanism of NF-
Submitted March 6, 2000; accepted October 9, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Errol S. Wijelath, The Hope Heart Institute, Department of Molecular Biology, 528 18th Ave, Seattle, WA 98122; e-mail: ewijelath{at}hopeheart.org.
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Abstract
Introduction
) or OSM (10 ng/mL, 
and 
) for 1 or 24 hours before addition of actinomycin D (5 µg/mL). Total RNA was extracted at the indicated times after addition
of actinomycin D. Northern blots were performed and probed with TF and
G3PDH. The signal density of each RNA sample hybridized to TF was
divided by that hybridized to the G3PDH. The corrected density was then
plotted as a percentage of the 0-hour value (log scale) against time.
Results are representative of 3 experiments.
. In response to inflammatory stimuli such as TNF-