From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Oncostatin M promotes biphasic tissue factor expression in smooth muscle cells:
evidence for Erk-1/2 activation
Toshiya Nishibe, Graham Parry, Atsushi Ishida, Salim Aziz, Jacqueline Murray, Yatin Patel, Salman Rahman, Kurt Strand, Keiko Saito,
Yuji Saito, William P. Hammond, Geoffrey F. Savidge, Nigel Mackman, and Errol S. Wijelath
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 sus-
tained pattern of TF messenger RNA
(mRNA). The effect of OSM on TF mRNA
expression was regulated at the transcriptional level as determined by nuclear runoffs and transient transfection of a TF
promoter-reporter gene construct. OSMinduced TF expression was regulated primarily by the transcription factor NF-␬B.
Activation of NF-␬B by OSM did not require I␬B-␣ degradation. Inhibition of MEK
activity by U0126 prevented OSM-induced TF expression by suppressing
NF-␬B DNA binding activity as determined by gel-shift analysis. Further, inhibition of Erk-1/2 protein by antisense treat-
ment resulted in suppression of TF mRNA
expression, indicating a role for Erk-1/2 in
modulating NF-␬B DNA binding activity.
These studies suggest that the induced
expression of TF by OSM is primarily
through the activation of NF-␬B and that
activation of NF-␬B is regulated in part by
the MEK/Erk-1/2 signal transduction pathway. This study indicates that OSM may
play a key role in promoting TF expression in SMCs within atherosclerotic
lesions. (Blood. 2001;97:692-699)
© 2001 by The American Society of Hematology
Introduction
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-␣ (TNF-␣) can induce
TF expression in endothelial cells.12,13 However, in acute arterial
injury and in atherosclerosis, SMCs and macrophages are the most
likely source for TF.14 Recently, SMCs stimulated with plateletderived growth factor (PDGF) and thrombin were shown to express
TF protein on their surface as well as stored intracellularly as a
latent form.14 Given that macrophages and T lymphocytes are
known components of atherosclerotic plaques,15,16 cytokines released by these cells may contribute to the induction of TF within
the lesion. In the present study, we investigated whether oncostatin
M (OSM) is capable of regulating TF expression. OSM is a
macrophage / T-lymphocyte–restricted cytokine and is related to the
family of cytokines that include leukemia inhibitory factor, IL-6,
IL-11, and ciliary neurotrophic factor.17,18 These cytokines share a
common signal transducer receptor component, gp 130, and have
overlapping biological activities.19 OSM has been shown to
stimulate growth of rabbit aortic SMCs and acquired immune
deficiency syndrome–Kaposi sarcoma cells.20-22 In bovine and
human endothelial cells, OSM promotes the expression of urokinase plasminogen activator, basic fibroblast growth factor, granulocyte colony-stimulating factor, and granulocyte-macrophage colony
stimulating factor.23-25 In human fibroblasts, OSM modulates not
only matrix metalloproteinases but also tissue inhibitors of matrix
metalloproteinases.26,27 Further, OSM was demonstrated to stimulate angiogenesis in vivo and was shown to be present in human
aortic aneurysms.28,29 Because OSM is produced by activated
monocytes/macrophages and T lymphocytes, cell types found
frequently at sites of inflammatory wound repair and atherosclerotic lesions, we examined the potential role and mechanism of
OSM in promoting TF expression in SMCs. We report that OSM
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.
lar Biology, 528 18th Ave, Seattle, WA 98122; e-mail: ewijelath@
hopeheart.org.
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.
Submitted March 6, 2000; accepted October 9, 2000.
Reprints: Errol S. Wijelath, The Hope Heart Institute, Department of Molecu-
692
© 2001 by The American Society of Hematology
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
promoted a biphasic pattern of TF expression. This induction is
dependent on activation of NF-␬B that is regulated by the
MEK/Erk-1/2 signaling pathway.
Materials and methods
Materials
Human TF and G3PDH complementary DNAs (cDNAs) were obtained
from American Type Culture Collection (Rockville, MD); S2765 from
Chromogenix (West Chester, OH); Factor VIIa and X from Enzyme
Research Laboratories (South Bend, IN); human recombinant OSM from
R&D Systems (Minneapolis, MN); luciferase assay system, pGL2, pSV-␤galactosidase control vectors, U0126 (ERK-1/2 inhibitor), and Transfast
transfection reagent system from Promega (Madison, WI); NF-␬B inhibitor
peptide and NF-␬B control inhibitor peptide from Biomol (Plymouth
Meeting, PA); NF-␬B and PanErk-1/2 monoclonal antibodies from Signal
Transduction (Lexington, KY); phospho-Erk-1/2 monoclonal antibody
from Biolabs (Beverly, MA); antihuman OSM and I␬B-␣ polyclonal
antibodies from Santa Cruz (Santa Cruz, CA); and polyclonal antihuman TF
antibody from American Diagnostica (Greenwich, CT).
Cell culture
Human SMCs were obtained from Clonetics (San Diego, CA). Canine
SMCs were obtained by enzymatic digestion. Briefly, medial tissue was
minced and digested overnight at 37°C in Dulbecco modified Eagle
(DME)/F12 medium supplemented with 150 U/mL collagenase, 1 U/mL
elastase, 0.25 U/mL DNase I, 2 mM Glutamax-I, and 2% fetal bovine serum
(FBS) for 24 hours. SMCs were cultured in DME/F12 medium supplemented with 10% FBS, 2 mM Glutamax-I, and 50 ␮g/mL gentamicin. Cells
were characterized as SMCs by morphological criteria and by staining with
␣-smooth muscle actin and smooth muscle myosin heavy chain (Sigma, St
Louis, MO). Canine SMCs from passage 1 to 5 and human SMCs from
passage 6 to 8 were used for experiments. Throughout the experiments, the
canine and human cells were incubated in a defined medium, consisting of
DME/F12 with 0.4% Redu-Ser II, 2 mM Glutamax-1, and 50 ␮M
gentamicin for at least 24 hours. All experiments were repeated at least once
in human SMCs to confirm observations made in canine SMCs.
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 ␮L 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 ␮L Tris-buffered saline containing human factor
VIIa and X (5 and 150 nM, respectively) and CaCl2 (5 mM). Then, 250 ␮L
of the supernatant was added to 25 ␮L of a chromogenic substrate for factor
Xa (S2765, 0.2 ␮M final concentration). The chromogenic reaction was
stopped after 3 minutes by addition of 20 ␮L 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.30
TF 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 ␮g) were denatured with dimethyl sulfoxide/glyoxal and
OSM PROMOTES TF EXPRESSION IN SMCs
693
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 ␮g/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 ␮L 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 ␮Ci ␣32P uridine
triphosphate (NEN, Boston, MA) for 30 minutes at 30°C. The reaction was
terminated by adding 1 ␮L proteinase K (20 mg/mL) and 10 ␮L 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 ␮g) 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 ␮g pTF(⫺2106)LUC, or
pGL2 or p19LUC together with 1 ␮g 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
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
694
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
NISHIBE et al
messenger RNA (mRNA) analysis, using a one-step reverse transcriptase–
polymerase chain reaction (RT-PCR) kit (Gibco-BRL). Quantitative RTPCR 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 ␮L 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 at ⫺70°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 ␮L 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 at ⫺70°C. Protein concentrations in both nuclear and cytoplasmic extracts were determined. Nuclear extract (5 ␮g) 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.
Results
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
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
I␬B-␣ 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.
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.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
OSM PROMOTES TF EXPRESSION IN SMCs
695
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
␮g/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 OSMinduced 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 ␮M 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,
Figure 1. OSM induces biphasic TF mRNA expression. (A,B) Kinetics of TF
mRNA induced by OSM and PDGF-BB. SMCs were incubated with OSM (10 ng/mL)
and PDGF-BB (20 ng/mL) for the indicated times, and RNA was extracted from
5 ⫻ 106 cells at the end of each incubation and analyzed as described in “Materials
and methods.” The blots were rehybridized with G3PDH to demonstrate equal RNA
loading. Solid columns represent the aggregate of densitometry analysis from 4
separate experiments. *P ⬍ .05 compared to 0 hour. (C) SMCs cultures were
prestimulated with OSM for 30 minutes, washed 3 times, and incubated in serum-free
DME/F12 medium for the indicated times. RNA was extracted at the end of each
incubation and analyzed for TF mRNA expression by Northern blotting. (D) OSMconditioned medium from 24-hour SMC cultures were used to stimulate SMCs for a
further 24 hours in the presence or absence of a neutralizing monoclonal antibody to
OSM. After 24 hours, RNA was extracted and analyzed for TF expression by Northern
blotting. Results are representative of 3 separate experiments.
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
Table 1. Oncostatin M promotes tissue factor antigen and activity
TF activity
TF antigen
(ng/mL)
Chromogenic assay
(AU)
0
0.41 ⫾ 0.08
2.65 ⫾ 0.80
2.35 ⫾ 0.08
0.5
0.46 ⫾ 0.05
ND
5.53 ⫾ 0.35
1
1.54 ⫾ 0.32
4.07 ⫾ 1.25
5.27 ⫾ 0.09
2
2.39 ⫾ 0.48
ND
5.84 ⫾ 0.05
4
2.37 ⫾ 0.29
7.06 ⫾ 2.21
5.99 ⫾ 0.20
8
2.33 ⫾ 0.23
3.95 ⫾ 1.55
4.76 ⫾ 0.07
Time (h)
Clotting assay
(mU/mL)
Kinetics of tissue factor antigen and activity induced by OSMs in smooth muscle
cells. Cell cultures were incubated with OSM (10 ng/mL), and, at the indicated times,
SMCs were lysed and TF antigen determined by ELISA. Lysates for TF activity were
determined by chromogenic and clotting assays. Results are expressed as
mean ⫾ SD.
TF indicates tissue factor; AU, arbitrary unit; ND, not determined; OSM,
oncostatin M; SMC, smooth muscle cell; ELISA, enzyme-linked immunosorbent
assay.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
696
NISHIBE et al
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
Figure 3. Inhibition of TF expression by NF-␬B and Erk-1/2 inhibitors. (A) SMC
cultures were incubated with the indicated inhibitors for 1 hour prior to stimulation with
OSM (10 ng/mL) for a further hour. Total RNA was extracted at the end of each
incubation and analyzed for TF mRNA expression. The blots were rehybridized with
G3PDH to demonstrate equal RNA loading. (B) SMCs were preincubated for 1 hour
with NF-␬B and Erk-1/2 inhibitors followed by stimulation with OSM (10 ng/mL) for 10
minutes at 37°C. Extracted proteins (30 ␮g) were separated on a 4% to 12% Bis-tris
gradient gel and transferred to a PVDF membrane. Phosphorylated and total Erk-1/2
were visualized by incubating the filters with a monoclonal antibody to p42/44 Erk-1/2
and a pan Erk-1/2 monoclonal antibody. Experiments were repeated twice.
Figure 2. OSM induces an increased rate of TF gene transcription. The
transcription rate of the TF gene was assessed by nuclear run-off assays. (A) Nuclear
extracts were harvested from SMCs treated with or without OSM (10 ng/mL) for 1
hour. Equal amounts of 32P-labeled in vitro transcribed RNA probes were hybridized
to 5 ␮g of denatured TF, G3PDH, and pGEM-4Z cDNA. Results are representative of
2 separate experiments. (B) Activation of TF promoter by OSM was analyzed by
studying the effect of OSM on TF gene promoter fused to luciferase. pTF(⫺2106)LUC,
which contains 2.1 kilobase (kb) of the 5⬘ flanking region of the TF gene promoter, and
pSV–␤-galactosidase control vector were transfected into SMCs. The cells were
treated with OSM (10 ng/mL) for 5 hours before harvesting. Luciferase activity was
determined and normalized for ␤-galactosidase activity. Results are expressed as
mean ⫾ SEM (n ⫽ 4 experiments). *P ⬍ .05 compared to unstimulated SMCs.
(C) SMCs were exposed to vehicle (control, 䡺) or OSM (10 ng/mL, f and F) for 1 or
24 hours before addition of actinomycin D (5 ␮g/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.
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 OSMstimulated 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 activity by the Erk-1/2 pathway
The preceding experiments have demonstrated that both Erk-1/2
and 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
Figure 4. Inhibition of TF mRNA synthesis by Erk-1/2 antisense treatment.
(A) SMCs were transfected with antisense or sense oligonucleotides for 48 hours.
Extracted proteins (30 ␮g) were separated on a 4% to 12% Bis-tris gradient gel and
transferred to a PVDF membrane. Erk-1/2 was visualized by incubating the filters with
a panErk-1/2 monoclonal antibody. (B) SMCs were transfected with antisense or
sense oligonucleotides for 48 hours followed by stimulation with OSM (10 ng/mL) for
1 hour at 37°C. Total RNA was extracted, and TF mRNA expression was analyzed by
quantitative RT-PCR. Solid bars represent the ratio of 32P-labeled TF and s17 PCR
products isolated from 2% agarose gel. Results are representative of 2 separate
experiments.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
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 I␬B-␣ degradation. Western blot analysis was performed to determine whether OSM stimulated I␬B-␣ degradation.
The levels of I␬B-␣ in OSM-treated cells were similar to the
unstimulated control. In contrast, the levels of I␬B-␣ were reduced
in TNF-␣–treated SMCs (Figure 6A). This finding suggests that the
activation of NF-␬B by OSM is independent of I␬B␣ degradation
and further rules out the possibility that U0126 inhibited NF-␬B
nuclear translocation by suppressing I␬B-␣ 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
OSM PROMOTES TF EXPRESSION IN SMCs
697
Figure 6. Assessment of I␬B-␣ and NF-␬B protein levels. (A) Total cellular
proteins from SMC cultures treated for 1 to 30 minutes with OSM (10 ng/mL) and
TNF-␣ (10 ng/mL) were separated on a 4% to 12% Bis-tris gradient gel and
transferred to a PVDF membrane. I␬B-␣ was visualized by incubating the filters with a
polyclonal antibody to I␬B-␣. (B) SMC cultures were stimulated with OSM in the
presence or absence of U0126, NF-␬B inhibitor peptide, or NF-␬B control peptide.
Cytoplasmic and nuclear NF-␬B were isolated as described in “Materials and
methods.” NF-␬B was visualized by incubating the filters with a monoclonal antibody
to NF-␬B. Experiments were repeated twice.
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.
Discussion
Figure 5. OSM induction of NF-␬B and AP-1 nuclear activity. Nuclear extracts of
SMCs were prepared following OSM (10 ng/mL) treatment. EMSA was performed
with the AP-1 (A) or NF-␬B (B) probes. For cold and mutated competition EMSA
experiments, 100-fold molar excess of unlabeled NF-␬B or AP-1 probes were
included in the binding reaction. Experiments were repeated twice.
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-␣ and PDGF.14,37 After the
completion of our study, an independent study showed that OSM
promoted a biphasic induction of IL-6 mRNA expression in
astrocytes.38 Interestingly, induction of IL-6 antigen did not show a
biphasic response. Similarly, in the present study TF antigen levels
remained elevated up to 72 hours (data not shown). The relative
stability of TF antigen39 compared with TF mRNA expression may
explain the biphasic nature of TF mRNA expression and sustained
TF-antigen level. Given that SMCs express the highest number of
OSM receptors,40 it is conceivable that OSM released by macrophages may promote TF expression through interaction with SMCs
within the atherosclerotic lesion and contribute to thrombotic
complications associated with this disease.
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
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
698
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
NISHIBE et al
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-␣ and bacterial
endotoxin (LPS) appear to be regulated primarily by the AP-1,
NF-␬B, and Sp1 transcription factors,37,45,46 whereas the transcriptional factor Egr1 was shown to be important in promoting TF gene
activation in endothelial cells under shear stress conditions.47 The
present study links the Erk-1/2 and NF-␬B pathways in the
induction of TF expression by OSM in SMCs. Although both
Erk-1/2 and NF-␬B inhibition blocked TF expression (Figure 3A),
EMSA studies (Figure 5) showed that, in OSM-stimulated SMCs
treated with U0126 (MEK inhibitor), both AP-1 and NF-␬B
activity were inhibited, whereas inhibition of NF-␬B translocation
using a specific peptide inhibitor suppressed only NF-␬B activity
without affecting AP-1 activity. This finding indicates that NF-␬B
plays a pivotal role in promoting TF expression. However, we
cannot rule out a role for AP-1 in promoting OSM-induced TF
expression. In this regard, several reports have demonstrated that
the interaction of AP-1 and NF-␬B transcriptional factors is
required for maximal induction of TF in endothelial and monocytic
cells.37,45 Thus, the inhibition of TF expression by U1206 may be
due to suppression of both AP-1 and NF-␬B nuclear activity.
The mechanism of NF-␬B activation has only been elucidated
recently.48,49 NF-␬B is a heterodimer composed primarily of a
50-kDa DNA binding subunit and a 65 kDa transactivator (p 65 or
Rel A). NF-␬B is retained in the cytoplasm associated with a family
of inhibitory proteins termed I␬B. This family of I␬Bs includes
I␬B-␣, I␬B-␤, and I␬B-⑀. In response to inflammatory stimuli such
as TNF-␣ and LPS, the I␬Bs are rapidly phosphorylated, releasing
NF-␬B, undergoing ubiquitination and proteolysis by the 26S
proteosome. Released NF-␬B then translocates to the nucleus and
activates transcription of specific genes. In this study, OSM
activated NF-␬B through a mechanism independent of I␬B-␣
degradation. Our observation is similar to reports that phosphorylation of I␬B-␣ at tyrosine 42 promoted NF-␬B mobilization to the
nucleus via a mechanism that does not involve I␬B-␣ degradation.50,51 Further, a recent study in human U937 monocytic cells
demonstrated that treatment with IL-1␤ for 15 minutes caused only
a 15% degradation of I␬B-␣ yet had more than 60% DNA binding,
suggesting the existence of additional mechanisms that regulate
NF-␬B activation.52 The finding that inhibition of Erk-1/2 by
U0126 inhibited NF-␬B binding activity without affecting NF-␬B
nuclear translocation suggests that Erk-1/2 may play a role in
modulating NF-␬B DNA recognition. For instance, Erk-1/2 may
regulate NF-␬B activity by regulating its phosphorylation status.
Indeed, several studies have suggested that the binding of NF-␬B to
its DNA site is dependent on the phosphorylation status of the p65
subunit.53,54 Alternatively, a nuclear protein could prevent DNA
recognition by either interacting with NF-␬B or its DNA binding
site. In summary, this study demonstrates the ability of OSM to
promote prolonged expression of TF in SMCs. Our results indicate
that the induction of TF expression by OSM is through the Erk-1/2
signal transduction pathway that is involved in regulating NF-␬B
DNA recognition.
References
1. Taubman MB, Fallon JT, Schecter AD, et al. Tissue factor in the pathogenesis of atherosclerosis.
Thromb Haemost. 1997;78:200-204.
2. Marmur JD, Rossikhina M, Guha A, et al. Tissue
factor is rapidly induced in arterial smooth muscle
after balloon injury. J Clin Invest. 1993;91:22532259.
3. Schwartz SM, Heimark RL, Majesky MW. Developmental mechanisms underlying pathology of
arteries. Physiol Rev. 1990;70:1177-1209.
4. Speidel CM, Eisenberg PR, Ruf W, Edgington TS,
Abendschein DR. Tissue factor mediates prolonged procoagulant activity on the luminal surface of balloon-injured aortas in rabbits. Circulation. 1995;92:3323-3330.
5. Pawashe AB, Golino P, Ambrosio G, et al. A
monoclonal antibody against rabbit tissue factor
inhibits thrombus formation in stenotic injured
rabbit carotid arteries. Circ Res. 1994;74:56-63.
6. Hatakeyama K, Asada Y, Marutsuka K, Sato Y,
Kamikubo Y, Sumiyoshi A. Localization and activity of tissue factor in human aortic atherosclerotic
lesions. Atherosclerosis. 1997;133:213-219.
7. Marmur JD, Thiruvikraman SV, Fyfe BS, et al.
Identification of active tissue factor in human coronary atheroma. Circulation. 1996;94:1226-1232.
8. Nemerson Y. Tissue factor and hemostasis [published erratum appears in Blood. 1988;71:1178].
Blood. 1988;71:1-8.
9. Edgington TS, Mackman N, Brand K, Ruf W. The
structural biology of expression and function of
tissue factor. Thromb Haemost. 1991;66:67-79.
10. Thiruvikraman SV, Guha A, Roboz J, Taubman
MB, Nemerson Y, Fallon JT. In situ localization of
tissue factor in human atherosclerotic plaques by
binding of digoxigenin-labeled factors VIIa and X
[published erratum appears in Lab Invest. 1997;
76:297-299]. Lab Invest. 1996;75:451-461.
19.
11. Wilcox JN, Smith KM, Schwartz SM, Gordon D.
Localization of tissue factor in the normal vessel
wall and in the atherosclerotic plaque. Proc Natl
Acad Sci U S A. 1989;86:2839-2843.
12. Nawroth PP, Stern DM. Modulation of endothelial
cell hemostatic properties by tumor necrosis factor. J Exp Med. 1986;163:740-745.
13. Bevilacqua MP, Pober JS, Majeau GR, Cotran
RS, Gimbrone MA Jr. Interleukin 1 (IL-1) induces
biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial
cells. J Exp Med. 1984;160:618-623.
14. Schecter AD, Giesen PL, Taby O, et al. Tissue
factor expression in human arterial smooth
muscle cells: TF is present in three cellular pools
after growth factor stimulation. J Clin Invest.
1997;100:2276-2285.
15. Cybulsky MI, Gimbrone MA Jr. Endothelial expression of a mononuclear leukocyte adhesion
molecule during atherogenesis. Science. 1991;
251:788-791.
16. Libby P, Hansson GK. Involvement of the immune
system in human atherogenesis: current knowledge and unanswered questions. Lab Invest.
1991;64:5-15.
17. Bazan JF. Neuropoietic cytokines in the hematopoietic fold. Neuron. 1991;7:197-208.
18. Rose TM, Bruce AG. Oncostatin M is a member
20.
21.
22.
23.
24.
25.
of a cytokine family that includes leukemia-inhibitory factor, granulocyte colony-stimulating factor,
and interleukin 6. Proc Natl Acad Sci U S A. 1991;
88:8641-8645.
Zhang XG, Gu JJ, Lu ZY, et al. Ciliary neurotropic
factor, interleukin 11, leukemia inhibitory factor,
and oncostatin M are growth factors for human
myeloma cell lines using the interleukin 6 signal
transducer gp130. J Exp Med. 1994;179:13371342.
Grove RI, Eberhardt C, Abid S, et al. Oncostatin
M is a mitogen for rabbit vascular smooth muscle
cells. Proc Natl Acad Sci U S A. 1993;90:823-827.
Nair BC, DeVico AL, Nakamura S, et al. Identification of a major growth factor for AIDS-Kaposi’s
sarcoma cells as oncostatin M. Science. 1992;
255:1430-1432.
Miles SA, Martinez-Maza O, Rezai A, et al. Oncostatin M as a potent mitogen for AIDS-Kaposi’s
sarcoma-derived cells. Science. 1992;255:14321434.
Brown TJ, Rowe JM, Liu JW, Shoyab M. Regulation of IL-6 expression by oncostatin M. J Immunol. 1991;147:2175-2180.
Brown TJ, Liu J, Brashem-Stein C, Shoyab M.
Regulation of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor expression by oncostatin M. Blood.
1993;82:33-37.
Hamilton JA, Leizer T, Piccoli DS, Royston KM,
Butler DM, Croatto M. Oncostatin M stimulates
urokinase-type plasminogen activator activity in
human synovial fibroblasts. Biochem Biophys
Res Commun. 1991;180:652-659.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
26. Nemoto O, Yamada H, Mukaida M, Shimmei M.
Stimulation of TIMP-1 production by oncostatin M
in human articular cartilage [see comments]. Arthritis Rheum. 1996;39:560-566.
27. Korzus E, Nagase H, Rydell R, Travis J. The mitogen-activated protein kinase and JAK-STAT
signaling pathways are required for an oncostatin
M-responsive element-mediated activation of matrix metalloproteinase 1 gene expression. J Biol
Chem. 1997;272:1188-1196.
OSM PROMOTES TF EXPRESSION IN SMCs
nificance and therapeutic intervention. Thromb
Haemost. 1997;78:247-255.
36. Ross R. Atherosclerosis—an inflammatory disease [see comments]. N Engl J Med. 1999;340:
115-126.
37. Parry GC, Mackman N. Transcriptional regulation
of tissue factor expression in human endothelial
cells. Arterioscler Thromb Vasc Biol. 1995;15:
612-621.
28. Modur V, Feldhaus MJ, Weyrich AS, et al. Oncostatin M is a proinflammatory mediator: in vivo
effects correlate with endothelial cell expression
of inflammatory cytokines and adhesion molecules. J Clin Invest. 1997;100:158-168.
38. Van Wagoner NJ, Choi C, Repovic P, Benveniste
EN. Oncostatin M regulation of interleukin-6 expression in astrocytes: biphasic regulation involving the mitogen-activated protein kinases ERK1/2
and p38. J Neurochem. 2000;75:563-575.
29. Vasse M, Pourtau J, Trochon V, et al. Oncostatin
M induces angiogenesis in vitro and in vivo. Arterioscler Thromb Vasc Biol. 1999;19:1835-1842.
39. Roussi J, Drouet L, Samama M, Sie P. French
multicentric evaluation of recombinant tissue factor (recombiplastin) for determination of prothrombin time. Thromb Haemost. 1994;72:698704.
30. Morrissey JH, Fair DS, Edgington TS. Monoclonal antibody analysis of purified and cell-associated tissue factor. Thromb Res. 1988;52:247-261.
31. Mackman N, Fowler BJ, Edgington TS, Morrissey
JH. Functional analysis of the human tissue factor promoter and induction by serum. Proc Natl
Acad Sci U S A. 1990;87:2254-2258.
40. Thoma B, Bird TA, Friend DJ, Gearing DP, Dower
SK. Oncostatin M and leukemia inhibitory factor
trigger overlapping and different signals through
partially shared receptor complexes. J Biol Chem.
1994;269:6215-6222.
32. Wolf YG, Rasmussen LM, Ruoslahti E. Antibodies
against transforming growth factor-beta 1 suppress intimal hyperplasia in a rat model. J Clin
Invest. 1994;93:1172-1178.
41. Strand K, Murray J, Aziz S, et al. Induction of the
urokinase plasminogen activator system by oncostatin M promotes endothelial migration. J Cell
Biochem. 2000;79:239-248.
33. Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J. Inhibition of nuclear translocation of transcription factor NF-kappa B by a synthetic peptide containing a cell membrane-permeable motif
and nuclear localization sequence. J Biol Chem.
1995;270:14255-14258.
42. Wijelath ES, Carlsen B, Cole T, Chen J, Kothari
S, Hammond WP. Oncostatin M induces basic
fibroblast growth factor expression in endothelial
cells and promotes endothelial cell proliferation,
migration and spindle morphology. J Cell Sci.
1997;110:871-879.
34. Mackman N, Brand K, Edgington TS. Lipopolysaccharide-mediated transcriptional activation of
the human tissue factor gene in THP-1 monocytic
cells requires both activator protein 1 and nuclear
factor kappa B binding sites. J Exp Med. 1991;
174:1517-1526.
43. Barille S, Akhoundi C, Collette M, et al. Metalloproteinases in multiple myeloma: production of
matrix metalloproteinase-9 (MMP-9), activation of
proMMP-2, and induction of MMP-1 by myeloma
cells. Blood. 1997;90:1649-1655.
35. Fuster V, Fallon JT, Badimon JJ, Nemerson Y.
The unstable atherosclerotic plaque: clinical sig-
44. Mackman N. Regulation of the tissue factor gene.
FASEB J. 1995;9:883-889.
699
45. Oeth P, Parry GC, Mackman N. Regulation of the
tissue factor gene in human monocytic cells: role
of AP-1, NF-kappa B/Rel, and Sp1 proteins in uninduced and lipopolysaccharide-induced expression. Arterioscler Thromb Vasc Biol. 1997;17:365374.
46. Parry GC, Mackman N. A set of inducible genes
expressed by activated human monocytic and
endothelial cells contain kappa B-like sites that
specifically bind c-Rel-p65 heterodimers. J Biol
Chem. 1994;269:20823-20825.
47. Houston P, Dickson MC, Ludbrook V, et al. Fluid
shear stress induction of the tissue factor promoter in vitro and in vivo is mediated by Egr-1.
Arterioscler Thromb Vasc Biol. 1999;19:281-289.
48. Karin M. The beginning of the end: IkappaB kinase (IKK) and NF-kappaB activation. J Biol
Chem. 1999;274:27339-27342.
49. Baldwin AS Jr. The NF-kappa B and I kappa B
proteins: new discoveries and insights. Annu Rev
Immunol. 1996;14:649-683.
50. Abu-Amer Y, Ross FP, McHugh KP, Livolsi A,
Peyron JF, Teitelbaum SL. Tumor necrosis factoralpha activation of nuclear transcription factorkappaB in marrow macrophages is mediated by
c-Src tyrosine phosphorylation of Ikappa Balpha.
J Biol Chem. 1998;273:29417-29423.
51. Imbert V, Rupec RA, Livolsi A, et al. Tyrosine
phosphorylation of I kappa B-alpha activates NFkappa B without proteolytic degradation of I
kappa B-alpha. Cell. 1996;86:787-798.
52. Nasuhara Y, Adcock IM, Catley M, Barnes PJ,
Newton R. Differential IkappaB kinase activation
and IkappaBalpha degradation by interleukin1beta and tumor necrosis factor-alpha in human
U937 monocytic cells: evidence for additional
regulatory steps in kappaB-dependent transcription. J Biol Chem. 1999;274:19965-19972.
53. Finco TS, Baldwin AS. Mechanistic aspects of
NF-kappa B regulation: the emerging role of
phosphorylation and proteolysis. Immunity. 1995;
3:263-272.
54. Naumann M, Scheidereit C. Activation of NFkappa B in vivo is regulated by multiple phosphorylations. EMBO J. 1994;13:4597-4607.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2001 97: 692-699
doi:10.1182/blood.V97.3.692
Oncostatin M promotes biphasic tissue factor expression in smooth
muscle cells: evidence for Erk-1/2 activation
Toshiya Nishibe, Graham Parry, Atsushi Ishida, Salim Aziz, Jacqueline Murray, Yatin Patel, Salman
Rahman, Kurt Strand, Keiko Saito, Yuji Saito, William P. Hammond, Geoffrey F. Savidge, Nigel
Mackman and Errol S. Wijelath
Updated information and services can be found at:
http://www.bloodjournal.org/content/97/3/692.full.html
Articles on similar topics can be found in the following Blood collections
Gene Expression (1086 articles)
Hemostasis, Thrombosis, and Vascular Biology (2485 articles)
Signal Transduction (1930 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.